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Gutierrez-Pajares JL, Iturrieta J, Dulam V, Wang Y, Pavlides S, Malacari G, Lisanti MP, Frank PG. Caveolin-3 Promotes a Vascular Smooth Muscle Contractile Phenotype. Front Cardiovasc Med 2015; 2:27. [PMID: 26664898 PMCID: PMC4671348 DOI: 10.3389/fcvm.2015.00027] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2015] [Accepted: 05/24/2015] [Indexed: 01/12/2023] Open
Abstract
Epidemiological studies have demonstrated the importance of cardiovascular diseases in Western countries. Among the cell types associated with a dysfunctional vasculature, smooth muscle (SM) cells are believed to play an essential role in the development of these illnesses. Vascular SM cells are key regulators of the vascular tone and also have an important function in the development of atherosclerosis and restenosis. While in the normal vasculature, contractile SM cells are predominant, in atherosclerotic vascular lesions, synthetic cells migrate toward the neointima, proliferate, and synthetize extracellular matrix proteins. In the present study, we have examined the role of caveolin-3 in the regulation of SM cell phenotype. Caveolin-3 is expressed in vivo in normal arterial SM cells, but its expression appears to be lost in cultured SM cells. Our data show that caveolin-3 expression in the A7r5 SM cell line is associated with increased expression of contractility markers such as SM α-actin, SM myosin heavy chain but decreased expression of the synthetic phenotype markers such as p-Elk and Klf4. Moreover, we also show that caveolin-3 expression can reduce proliferation upon treatment with LDL or PDGF. Finally, we show that caveolin-3-expressing SM cells are less sensitive to apoptosis than control cells upon treatment with oxidized LDL. Taken together, our data suggest that caveolin-3 can regulate the phenotypic switch between contractile and synthetic SM cells. A better understanding of the factors regulating caveolin-3 expression and function in this cell type will permit the development of a better comprehension of the factors regulating SM function in atherosclerosis and restenosis.
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Affiliation(s)
- Jorge L Gutierrez-Pajares
- Faculté de Médecine, INSERM UMR1069 "Nutrition, Croissance et Cancer", Université François Rabelais de Tours , Tours , France ; Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA
| | - Jeannette Iturrieta
- Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA
| | - Vipin Dulam
- Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA
| | - Yu Wang
- Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA
| | - Stephanos Pavlides
- The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester , Manchester , UK ; The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester , Manchester , UK
| | - Gabriella Malacari
- Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA
| | - Michael P Lisanti
- The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester , Manchester , UK ; The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester , Manchester , UK
| | - Philippe G Frank
- Faculté de Médecine, INSERM UMR1069 "Nutrition, Croissance et Cancer", Université François Rabelais de Tours , Tours , France ; Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA , USA ; Department of Biochemistry and Molecular Biology, Thomas Jefferson University , Philadelphia, PA , USA
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Eddinger TJ. Smooth muscle-protein translocation and tissue function. Anat Rec (Hoboken) 2015; 297:1734-46. [PMID: 25125185 DOI: 10.1002/ar.22970] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2014] [Revised: 03/18/2014] [Accepted: 03/18/2014] [Indexed: 01/25/2023]
Abstract
Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems.
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Affiliation(s)
- Thomas J Eddinger
- Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin
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3
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Guerri-Guttenberg RA, Castilla R, Francos GC, Müller A, Ambrosio G, Milei J. Transforming Growth Factor β1 and Coronary Intimal Hyperplasia in Pediatric Patients With Congenital Heart Disease. Can J Cardiol 2013; 29:849-57. [DOI: 10.1016/j.cjca.2012.11.018] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2012] [Revised: 11/20/2012] [Accepted: 11/23/2012] [Indexed: 11/25/2022] Open
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4
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Sluiter I, van Heijst A, Haasdijk R, Kempen MBV, Boerema-de Munck A, Reiss I, Tibboel D, Rottier RJ. Reversal of pulmonary vascular remodeling in pulmonary hypertensive rats. Exp Mol Pathol 2012; 93:66-73. [PMID: 22472322 DOI: 10.1016/j.yexmp.2012.03.010] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2012] [Accepted: 03/19/2012] [Indexed: 12/27/2022]
Abstract
Pulmonary hypertension is responsible for significant mortality and morbidity among newborns and infants. The pathology is characterized by pulmonary vascular remodeling with medial hypertrophy and adventitial thickening, leading to decreased gas exchange. Since it is unknown if these abnormalities are reversible, we analyzed these vascular changes in pulmonary hypertensive rats. Exposure of rats to hypobaric hypoxia for 4 weeks induced clinical signs of pulmonary hypertension, such as increased right ventricular systolic pressure, increased right ventricular weight and considerable pulmonary vascular remodeling. The vascular changes were associated with the expression of Non -Muscle Myosin Heavy Chain B in the pre-acinar vessels and an increased expression of alpha Smooth Muscle Actin, Smooth Muscle Myosin Heavy Chain 2 and Calponin in the intra-acinar vessels. The right ventricular systolic pressure and right ventricular weight gradually decreased after specific periods of recovery in normoxia, although this reversal did not reach baseline levels after six weeks at normoxia. However, the cellular changes in the pulmonary vasculature were completely reversed. Development of pulmonary hypertension is associated with an increase of synthetic perivascular cells in the pre-acinar arteries and an aberrant differentiation of perivascular cells in the smallest intra-acinar arteries. These cellular and structural changes in the pulmonary vasculature are completely reversible after recovery in normoxia.
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Affiliation(s)
- Ilona Sluiter
- Intensive Care and Department of Pediatric Surgery, Erasmus MC-Sophia, Dr. Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands.
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5
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Guo X, Chen SY. Transforming growth factor-β and smooth muscle differentiation. World J Biol Chem 2012; 3:41-52. [PMID: 22451850 PMCID: PMC3312200 DOI: 10.4331/wjbc.v3.i3.41] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/07/2011] [Revised: 01/19/2012] [Accepted: 01/26/2012] [Indexed: 02/05/2023] Open
Abstract
Transforming growth factor (TGF)-β family members are multifunctional cytokines regulating diverse cellular functions such as growth, adhesion, migration, apoptosis, and differentiation. TGF-βs elicit their effects via specific type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. Knockout mouse models for the different components of the TGF-β signaling pathway have revealed their critical roles in smooth muscle cell (SMC) differentiation. Genetic studies in humans have linked mutations in these signaling components to specific cardiovascular disorders such as aorta aneurysm and congenital heart diseases due to SMC defects. In this review, the current understanding of TGF-β function in SMC differentiation is highlighted, and the role of TGF-β signaling in SMC-related diseases is discussed.
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Affiliation(s)
- Xia Guo
- Xia Guo, Shi-You Chen, Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602, United States
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6
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Sakata N, Fujimitsu K, Jimi S, Takebayashi S. Increased uptake of low density lipoprotein by SV40-transformed smooth muscle cells. Int J Angiol 2011. [DOI: 10.1007/bf02042920] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
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7
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Xiao Y, Huang Z, Yin H, Zhang H, Wang S. Desmuslin gene knockdown causes altered expression of phenotype markers and differentiation of saphenous vein smooth muscle cells. J Vasc Surg 2010; 52:684-90. [PMID: 20573469 DOI: 10.1016/j.jvs.2010.03.069] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2010] [Revised: 03/23/2010] [Accepted: 03/28/2010] [Indexed: 01/12/2023]
Abstract
OBJECTIVE Phenotypic alterations of vascular smooth muscle cells (VSMCs) appear critical to the development of primary varicose veins. Previous study indicated desmuslin, an intermediate filament protein, was differentially expressed in smooth muscle cells (SMCs) isolated from varicose veins; thus, it was naturally hypothesized that altered desmuslin expression might in turn affect the functioning of VSMCs, leading to the phenotypic alterations and varicose vein development. METHODS In this study, expression of desmuslin in normal human saphenous vein SMCs was knocked down using small interfering RNA (siRNA), and control cells were treated with a scrambled siRNA sequence. The levels of several phenotypic markers including smooth muscle (SM) alpha-actin and smooth muscle myosin heavy chain (SM-MHC) were assessed. Collagen formation, matrix metalloproteinase expression (MMP-2), and cytoskeletal and morphological changes were also examined. RESULTS SMCs treated with desmuslin siRNA exhibited significantly increased levels of collagen synthesis and MMP-2 expression and decreased expression levels of SM alpha-actin, SM-MHC, and smoothelin and exhibited disassembly of actin stress fibers when compared with the control cells. Changes in cell morphology and actin fiber networks in VSMCs treated with desmuslin siRNA were consistent with a lower degree of differentiation. CONCLUSIONS These results indicated desmuslin expression is required for the maintenance of VSMC phenotype. Decreased desmuslin expression may affect differentiation of VSMCs and ultimately contribute to the development of varicose veins.
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Affiliation(s)
- Ying Xiao
- Vascular Surgery Institute, Department of Vascular Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People's Republic of China
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8
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Balcells M, Martorell J, Olivé C, Santacana M, Chitalia V, Cardoso AA, Edelman ER. Smooth muscle cells orchestrate the endothelial cell response to flow and injury. Circulation 2010; 121:2192-9. [PMID: 20458015 PMCID: PMC2887340 DOI: 10.1161/circulationaha.109.877282] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
BACKGROUND Local modulation of vascular mammalian target of rapamycin (mTOR) signaling reduces smooth muscle cell (SMC) proliferation after endovascular interventions but may be associated with endothelial cell (EC) toxicity. The trilaminate vascular architecture juxtaposes ECs and SMCs to enable complex paracrine coregulation but shields SMCs from flow. We hypothesized that flow differentially affects mTOR signaling in ECs and SMCs and that SMCs regulate mTOR in ECs. METHODS AND RESULTS SMCs and/or ECs were exposed to coronary artery flow in a perfusion bioreactor. We demonstrated by flow cytometry, immunofluorescence, and immunoblotting that EC expression of phospho-S6 ribosomal protein (p-S6RP), a downstream target of mTOR, was doubled by flow. Conversely, S6RP in SMCs was growth factor but not flow responsive, and SMCs eliminated the flow sensitivity of ECs. Temsirolimus, a sirolimus analog, eliminated the effect of growth factor on SMCs and of flow on ECs, reducing p-S6RP below basal levels and inhibiting endothelial recovery. EC p-S6RP expression in stented porcine arteries confirmed our in vitro findings: Phosphorylation was greatest in ECs farthest from intact SMCs in metal stented arteries and altogether absent after sirolimus stent elution. CONCLUSIONS The mTOR pathway is activated in ECs in response to luminal flow. SMCs inhibit this flow-induced stimulation of endothelial mTOR pathway. Thus, we now define a novel external stimulus regulating phosphorylation of S6RP and another level of EC-SMC crosstalk. These interactions may explain the impact of local antiproliferative delivery that targets SMC proliferation and suggest that future stents integrate design influences on flow and drug effects on their molecular targets.
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MESH Headings
- Animals
- Aorta/physiology
- Arteries/physiology
- Arteries/physiopathology
- Cell Communication/physiology
- Cells, Cultured
- Coronary Vessels/physiology
- Endothelial Cells/metabolism
- Endothelium, Vascular/injuries
- Endothelium, Vascular/pathology
- Endothelium, Vascular/physiopathology
- Humans
- In Vitro Techniques
- Intracellular Signaling Peptides and Proteins/antagonists & inhibitors
- Intracellular Signaling Peptides and Proteins/metabolism
- Muscle, Smooth, Vascular/pathology
- Muscle, Smooth, Vascular/physiopathology
- Myocytes, Smooth Muscle/metabolism
- Phosphorylation
- Protein Serine-Threonine Kinases/antagonists & inhibitors
- Protein Serine-Threonine Kinases/metabolism
- Regional Blood Flow/drug effects
- Regional Blood Flow/physiology
- Ribosomal Protein S6/metabolism
- Signal Transduction
- Sirolimus/analogs & derivatives
- Sirolimus/pharmacology
- Stents/adverse effects
- Swine
- Swine, Miniature
- TOR Serine-Threonine Kinases
- Transcription Factors/metabolism
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Affiliation(s)
- Mercedes Balcells
- Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.
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9
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Perrotta I, Brunelli E, Sciangula A, Zuccalà V, Donato G, Tripepi S, Martinelli GL, Cassese M. Inducible and endothelial nitric oxide synthase expression in human atherogenesis: an immunohistochemical and ultrastructural study. Cardiovasc Pathol 2009; 18:361-8. [DOI: 10.1016/j.carpath.2008.08.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/10/2008] [Revised: 06/25/2008] [Accepted: 08/19/2008] [Indexed: 10/21/2022] Open
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10
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Xiao Y, Huang Z, Yin H, Lin Y, Wang S. In vitro differences between smooth muscle cells derived from varicose veins and normal veins. J Vasc Surg 2009; 50:1149-54. [PMID: 19703751 DOI: 10.1016/j.jvs.2009.06.048] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2009] [Revised: 06/08/2009] [Accepted: 06/21/2009] [Indexed: 10/20/2022]
Abstract
OBJECTIVE The theory of primary venous dilatation leading to secondary valvular incompetence and varicose vein formation has received more attention nowadays. Although many studies have investigated the role of the main components of the venous wall in the development of varicose veins, the leading cause remains unknown. The present study was designed to establish the role of smooth muscle cells (SMCs) of the tunica media on the pathogenesis of varicose veins by analyzing the phenotypic and functional differences between SMCs derived from varicose veins and normal veins. METHODS SMCs were isolated and cultured from saphenous veins of patients with varicose veins and normal veins. Cell proliferation and migration rates were compared. Expression of phenotype-dependent markers and matrix metalloproteinase-2 (MMP) production were analyzed by immunoblotting. Total collagen synthesis was evaluated by measuring the radioactivity of L-[3, 4-(3)H]proline in the media and the cell layer. RESULTS SMCs derived from varicose veins demonstrated increased proliferation (2-fold, P < .01), migration (3-fold, P < .001), MMP-2 production (3-fold, P < .01), and collagen synthesis (>2-fold, P < .001), with decreased expression of phenotype-dependent markers compared with SMCs derived from normal veins (P < .05). CONCLUSION SMCs derived from varicose veins are more dedifferentiated and demonstrate increased proliferative and synthetic capacity than SMCs derived from normal veins. These properties may contribute to the remodeling of the venous wall and the weakening of its antipressure capacity.
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Affiliation(s)
- Ying Xiao
- Research Center of Vascular Surgery, Department of Vascular Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Peoples Republic of China
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11
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12
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Orlandi A, Bochaton-Piallat ML, Gabbiani G, Spagnoli LG. Aging, smooth muscle cells and vascular pathobiology: Implications for atherosclerosis. Atherosclerosis 2006; 188:221-30. [PMID: 16487530 DOI: 10.1016/j.atherosclerosis.2006.01.018] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2005] [Revised: 01/10/2006] [Accepted: 01/18/2006] [Indexed: 10/25/2022]
Abstract
Epidemiological and autopsy studies suggest a close link between aging and the clinical manifestation of atherosclerosis. Several experiments show increased arterial susceptibility to atherogenetic stimuli in aged subjects. All together, these findings support the concept that aging represents an independent atherogenetic risk factor, intimately associated to other parietal, microenvironmental and systemic noxae. Smooth muscle cells (SMCs) represent the major arterial cell population. As aging occurs, SMCs progressively migrate from the tunica media and accumulate into the tunica intima. Myointimal thickening may represent the site where low-grade atherogenic stimuli cause early development and more severe lesion progression. Intimal SMC accumulation is characterized from a switch, from a differentiated to a synthetic phenotype, with reduced myocytic cytoskeletal markers and the expression of new proteins. Aging also associates to changes of SMC proliferative and apoptotic behavior and response to growth factors, such as transforming growth factor-beta1. The alteration of SMC properties represents a crucial event in the pathobiology of arterial wall, since it contributes to the vascular remodeling and decline of function with aging and favors the progression of atherosclerosis. Increased knowledge of biomolecular mechanisms regulating these events helps to develop new strategies aimed at contrasting the adverse effect of vascular aging.
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Affiliation(s)
- Augusto Orlandi
- Anatomic Pathology Institute, Department of Biopathology, Tor Vergata University of Rome, Via Montpellier 1, Rome, Italy.
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13
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Cai WJ, Kocsis E, Wu X, Rodríguez M, Luo X, Schaper W, Schaper J. Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart. Mol Cell Biochem 2004; 264:201-10. [PMID: 15544049 DOI: 10.1023/b:mcbi.0000044389.65590.57] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Previous studies have shown that neointima formation and adventitial remodeling play an important role in the enlargement of collateral vessels (CVs) during coronary arteriogenesis in the dog heart. In this study, we investigated the importance of remodeling of the tunica media in the same model. Basal membrane (BM), contractile and cytoskeletal components of smooth muscle cells (SMCs) were studied in growth of coronary CVs induced by chronic occlusion of the left circumflex (LCX) coronary artery by routine histology, electron microscopy (EM), and immunoconfocal microscopy using antibodies against alpha-smooth actin (alpha-SM actin), calponin, desmin, and laminin. In addition, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) were investigated. The data showed that (1) in normal small arteries (NVs) laminin formed a network in which SMCs were encaged; alpha-SM actin, calponin and desmin were evenly expressed in SMCs; (2) in early (2 weeks) growing CVs the laminin network was disrupted, desmin was significantly reduced in SMCs, but alpha-SM actin and calponin still highly expressed; (3) in actively (6 weeks) growing CVs laminin was still weak in the tunica media (TM), but without network-like structure. Desmin was further reduced in SMCs of TM, whereas alpha-SM actin and calponin showed little changes, although they were significantly decreased in intimal SMCs; (4) in mature CVs, the network-like structure was re-formed, and alpha-SM actin, calponin, and desmin were all similar to that in normal vessels; (5) histology for BM confirmed laminin staining; (6) EM revealed that in NVs the SMCs contained abundant contractile filaments and were surrounded by a layer of BM whereas in growing CVs, BM structure was not observed, but the SMCs in the media still contained many myofilaments; (7) MMP-2 was highly expressed in the media of early growing vessels, but decreased in TM of actively growing vessels where TIMP-1 expression was high. In conclusion, our data revealed features of TM of growing CVs. Disruption and degradation of BM facilitate SMC proliferation, and together with reduction of desmin and fragmentation of the internal elastic lamina enable the vascular wall to expand and enlarge when blood pressure and shear stress increase. MMP2 may be an important player in regulating SMC phenotype, proliferation, migration and maintaining integrity of the vascular wall through governing proteolysis during arteriogenesis.
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Affiliation(s)
- Wei-Jun Cai
- Department of Anatomy, Hunan Medical University, Changsha, Hunan, People's Republic of China.
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14
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Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004; 84:767-801. [PMID: 15269336 DOI: 10.1152/physrev.00041.2003] [Citation(s) in RCA: 2629] [Impact Index Per Article: 125.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms/processes that control differentiation of vascular smooth muscle cells (SMC) during normal development and maturation of the vasculature, as well as how these mechanisms/processes are altered in vascular injury or disease. A major challenge in understanding differentiation of the vascular SMC is that this cell can exhibit a wide range of different phenotypes at different stages of development, and even in adult organisms the cell is not terminally differentiated. Indeed, the SMC is capable of major changes in its phenotype in response to changes in local environmental cues including growth factors/inhibitors, mechanical influences, cell-cell and cell-matrix interactions, and various inflammatory mediators. There has been much progress in recent years to identify mechanisms that control expression of the repertoire of genes that are specific or selective for the vascular SMC and required for its differentiated function. One of the most exciting recent discoveries was the identification of the serum response factor (SRF) coactivator gene myocardin that appears to be required for expression of many SMC differentiation marker genes, and for initial differentiation of SMC during development. However, it is critical to recognize that overall control of SMC differentiation/maturation, and regulation of its responses to changing environmental cues, is extremely complex and involves the cooperative interaction of many factors and signaling pathways that are just beginning to be understood. There is also relatively recent evidence that circulating stem cell populations can give rise to smooth muscle-like cells in association with vascular injury and atherosclerotic lesion development, although the exact role and properties of these cells remain to be clearly elucidated. The goal of this review is to summarize the current state of our knowledge in this area and to attempt to identify some of the key unresolved challenges and questions that require further study.
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MESH Headings
- Aging/metabolism
- Animals
- Arteriosclerosis/genetics
- Cell Differentiation
- Cellular Senescence
- Embryo, Mammalian/cytology
- Embryo, Mammalian/metabolism
- Humans
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/embryology
- Muscle, Smooth, Vascular/metabolism
- Muscle, Smooth, Vascular/pathology
- Myocytes, Smooth Muscle/cytology
- Myocytes, Smooth Muscle/metabolism
- Myocytes, Smooth Muscle/pathology
- Phenotype
- Vascular Diseases/genetics
- Vascular Diseases/metabolism
- Vascular Diseases/pathology
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Affiliation(s)
- Gary K Owens
- Dept. of Molecular Physiology and Biological Physics, Univ. of Virginia School of Medicine, 415 Lane Rd., Medical Research Building 5, Rm. 1220, PO Box 801394, Charlottesville, VA 22908, USA.
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15
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Abstract
The vascular network is a series of linked conduits of blood vessels composed of the endothelium, a monolayer of cells that adorn the vessel lumen and surrounding layer(s) of mesenchymal cells (vascular smooth muscle, pericytes and fibroblasts). In addition to providing structural support, the mesenchymal cells are essential for vessel contractility. The extracellular matrix is a major constituent of blood vessels and provides a framework in which these various cell types are attached and embedded. The composition and organization of vascular extracellular matrix is primarily controlled by the mesenchymal cells, and is also responsible for the mechanical properties of the vessel wall, forming complex networks of structural proteins which are highly regulated. The extracellular matrix also plays a central role in cellular adhesion, differentiation and proliferation. This review examines the cellular and extracellular matrix components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
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Affiliation(s)
- George Bou-Gharios
- Renal Medicine, Imperial College London, Hammersmith Campus, London, W12 ONN, UK
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16
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Abstract
Inflammation plays a critical role in the vascular response to injury. In particular, mechanical injury using techniques such as balloon angioplasty and stenting results in complex inflammatory reactions which influence proliferation of vessel wall constituents such as endothelial cells, smooth muscle cells, and extracellular matrix proteins. Inflammatory cells are recruited to the injured vessel wall initially as a reparative mechanism; however, these same inflammatory processes are also pivotal in the development of restenotic lesions. Leukocytes serve as the primary inflammatory cells but we now know that platelets produce a number of important inflammatory mediators. This review describes the mechanisms that regulate endothelial cell migration, smooth muscle cell activation, and extracellular matrix protein production, all of which are key components in the inflammatory response to vascular injury.
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Affiliation(s)
- C Davis
- Department of Medicine, Cardiovascular Research Center, University of Virginia Health System, Charlottesville, VA 22908-0158, USA
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17
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Heeneman S, Cleutjens JP, Faber BC, Creemers EE, van Suylen RJ, Lutgens E, Cleutjens KB, Daemen MJ. The dynamic extracellular matrix: intervention strategies during heart failure and atherosclerosis. J Pathol 2003; 200:516-25. [PMID: 12845619 DOI: 10.1002/path.1395] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The extracellular matrix is no longer seen as the static embedding in which cells reside; it has been shown to be involved in cell proliferation, migration and cell-cell interactions. Turnover of the different extracellular matrix components is an active process with multiple levels of regulation. Collagen, a major extracellular matrix constituent of the myocardium and the arterial vascular wall, is synthesized by (myo)fibroblasts in the myocardium and smooth muscle cells in the medial arterial vascular wall. Its degradation is controlled by proteinases, which include matrix metalloproteinases. This review will focus on the impact of fibrosis and especially collagen turnover on the progression of heart failure and atherosclerosis, two of the main cardiovascular pathologies. We will discuss data from human studies and animal models, with an emphasis on the effects of interventions on collagen synthesis and degradation. We conclude that there is a dynamic (dis)balance in the rate of collagen synthesis and degradation during heart failure and atherosclerosis, which makes the outcome of interventions not always predictable. Alternative approaches for intervening in collagen metabolism will be discussed as possible therapeutic intervention strategies.
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Affiliation(s)
- Sylvia Heeneman
- Department of Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, 6200 MD Maastricht, The Netherlands
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18
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Hirschi KK, Lai L, Belaguli NS, Dean DA, Schwartz RJ, Zimmer WE. Transforming growth factor-beta induction of smooth muscle cell phenotpye requires transcriptional and post-transcriptional control of serum response factor. J Biol Chem 2002; 277:6287-95. [PMID: 11741973 PMCID: PMC4421896 DOI: 10.1074/jbc.m106649200] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Transforming growth factor-beta induces a smooth muscle cell phenotype in undifferentiated mesenchymal cells. To elucidate the mechanism(s) of this phenotypic induction, we focused on the molecular regulation of smooth muscle-gamma-actin, whose expression is induced at late stages of smooth muscle differentiation and developmentally restricted to this lineage. Transforming growth factor-beta induced smooth muscle-gamma-actin protein, cytoskeletal localization, and mRNA expression in mesenchymal cells. Smooth muscle-gamma-actin promoter-luciferase reporter activity was enhanced by transforming growth factor-beta, and deletion analysis revealed that CArG box 2 in the promoter was necessary for this transcriptional activation. CArG motifs bind transcriptional activator serum response factor; gel shift analyses revealed increased binding of serum response factor-containing complexes to this site in response to transforming growth factor-beta, paralleled by increased serum response factor protein expression. Serum response factor expression was found to be up-regulated by transforming growth factor-beta via transcriptional activation of the gene and post-transcriptional regulation. Using mesenchymal cells stably transfected with wild type or dominant-negative serum response factor, we demonstrated that its expression is sufficient for induction of a smooth muscle phenotype in mesenchymal cells and is necessary for transforming growth factor-beta-mediated smooth muscle induction.
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Affiliation(s)
- Karen K Hirschi
- Department of Pediatrics, Center for Cell and Gene Therapy and Children's Nutrition Research Center, Baylor College of Medicine, Houston, Texas 77030, USA.
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19
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Martínez-González J, Berrozpe M, Varela O, Badimon L. Heterogeneity of smooth muscle cells in advanced human atherosclerotic plaques: intimal smooth muscle cells expressing a fibroblast surface protein are highly activated by platelet-released products. Eur J Clin Invest 2001; 31:939-49. [PMID: 11737236 DOI: 10.1046/j.1365-2362.2001.00920.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND In vascular disease, smooth muscle cells (SMC) undergo phenotypic modulation and may acquire properties resembling those of fibroblasts in tissue wound healing. AIMS We aimed to show the differential expression of a fibroblast surface protein (FSP) by SMC in atherosclerotic lesions. RESULTS In early human coronary atherosclerotic lesions the expression of FSP in the intima was absent. In contrast, 29 of 29 middle/advanced lesions contained intimal SMC expressing high levels of FSP. Fibroblast surface protein positive SMC were negative for desmin but expressed variable levels of alpha-SM actin, SM caldesmon, SM myosin heavy chain and vimentin. Explants from advanced atherosclerotic lesions yielded two main SMC subpopulations. SMC over-expressing FSP exhibited higher in vitro mitogenic response (premitotic DNA synthesis) to sera (2- to 8-fold) and platelet-released products (8- to 26-fold), especially from thrombin-activated platelets, than FSP-negative SMC. CONCLUSIONS Our results suggest that the expression of FSP in SMC could indicate an activated phenotype, and the presence of highly positive FSP cells in the atherosclerotic lesions might be indicative of an increased SMC responsiveness to processes that locally generate thrombin and activate platelets.
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Affiliation(s)
- J Martínez-González
- IIBB/CSIC-Institut de Recerca de l'Hospital de Sant Pau, Avda. Sant Antoni Maria Claret #167, 08025 Barcelona, Spain
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20
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Worth NF, Rolfe BE, Song J, Campbell GR. Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins. CELL MOTILITY AND THE CYTOSKELETON 2001; 49:130-45. [PMID: 11668582 DOI: 10.1002/cm.1027] [Citation(s) in RCA: 154] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta-NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation.
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Affiliation(s)
- N F Worth
- Department of Anatomical Sciences, Centre for Research in Vascular Biology, University of Queensland, Queensland, Australia
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21
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Tamura R, Miyagawa J, Nishida M, Kihara S, Sasada R, Igarashi K, Nakata A, Yamamori K, Kameda-Takemura K, Yamashita S, Matsuzawa Y. Immunohistochemical localization of Betacellulin, a member of epidermal growth factor family, in atherosclerotic plaques of human aorta. Atherosclerosis 2001; 155:413-23. [PMID: 11254912 DOI: 10.1016/s0021-9150(00)00576-1] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Betacellulin (BTC), a new member of the EGF family, has been reported to be a potent mitogen for rat vascular smooth muscle cells (SMCs). BTC mRNA is known to be expressed in several human organs. However, the localization of BTC in human vascular tissues has not yet been clarified. We investigated whether or not BTC protein is involved in the pathogenesis of human atherosclerosis. Recombinant human BTC showed a mitogenic activity on cultured human aortic SMCs by measuring [3H]thymidine incorporation. The immunohistochemical localization of BTC, SMCs, macrophages, EGF receptors and ErbB4 was examined in autopsied human aortas. BTC was detected in both intimal and medial SMCs of the aortic wall. The percentage of BTC-positive medial SMCs in early types of atherosclerotic lesions decreased with age, but in adult, it was significantly higher in advanced types than in early types of atherosclerotic lesions. BTC-positive SMCs were predominantly localized in the medial side of the intima. Furthermore, numerous BTC-positive SMCs and macrophages were observed around the core lesion of atherosclerotic plaques. Receptors for BTC, EGF receptor and ErbB4, were expressed on SMCs, suggesting that BTC is associated with EGF receptor family-mediated signaling. BTC is produced in human aortic tissue and might play important roles in atherogenesis.
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MESH Headings
- Adolescent
- Adult
- Aged
- Aging/metabolism
- Aorta, Thoracic/chemistry
- Aorta, Thoracic/pathology
- Aortic Diseases/metabolism
- Aortic Diseases/pathology
- Arteriosclerosis/metabolism
- Arteriosclerosis/pathology
- Betacellulin
- Cell Division/drug effects
- Cells, Cultured/drug effects
- Child
- Child, Preschool
- ErbB Receptors/analysis
- Female
- Growth Substances/analysis
- Growth Substances/pharmacology
- Humans
- Immunoenzyme Techniques
- Infant
- Infant, Newborn
- Intercellular Signaling Peptides and Proteins
- Macrophages/chemistry
- Male
- Middle Aged
- Muscle, Smooth, Vascular/chemistry
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/pathology
- Receptor, ErbB-4
- Recombinant Fusion Proteins/pharmacology
- Tunica Intima/chemistry
- Tunica Intima/pathology
- Tunica Media/chemistry
- Tunica Media/pathology
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Affiliation(s)
- R Tamura
- Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2, Yamada-oka, Suita, 565-0871, Osaka, Japan
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22
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Carson JA, Fillmore RA, Schwartz RJ, Zimmer WE. The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor. J Biol Chem 2000; 275:39061-72. [PMID: 10993896 DOI: 10.1074/jbc.m006532200] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.
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Affiliation(s)
- J A Carson
- Department of Cellular and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
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23
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Tasker PN, Taylor CW, Nixon GF. Expression and distribution of InsP(3) receptor subtypes in proliferating vascular smooth muscle cells. Biochem Biophys Res Commun 2000; 273:907-12. [PMID: 10891346 DOI: 10.1006/bbrc.2000.3036] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The expression and distribution of types 1, 2, and 3 inositol 1,4, 5-trisphosphate receptor (InsP(3)R) in proliferating, primary cultures of rat aortic smooth muscle were compared to fully developed and differentiated rat aortic smooth muscle. Subtype-specific InsP(3)R antibodies revealed that the expression of type 1 InsP(3)R was similar in cultured aortic cells and aorta homogenate but expression of type 2 and 3 InsP(3)R subtypes was increased 3-fold in cultured aortic cells. The distribution of the type 1 InsP(3)R was located throughout the cytoplasm; type 2 InsP(3)R was found closely associated with the nucleus and at the plasma membrane; type 3 InsP(3)R was distributed predominantly around the nucleus. Alterations in InsP(3)R subtype expression and localization may have important functions in regulating intracellular calcium release around the nucleus when vascular smooth muscle cells switch to a more proliferating phenotype.
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MESH Headings
- Amino Acid Sequence
- Animals
- Blotting, Western
- Calcium Channels/classification
- Calcium Channels/metabolism
- Cell Division
- Cells, Cultured
- Inositol 1,4,5-Trisphosphate Receptors
- Microscopy, Confocal
- Molecular Sequence Data
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/metabolism
- Phenotype
- Rats
- Rats, Sprague-Dawley
- Receptors, Cytoplasmic and Nuclear/classification
- Receptors, Cytoplasmic and Nuclear/metabolism
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Affiliation(s)
- P N Tasker
- Department of Biomedical Sciences, University of Aberdeen, Aberdeen, United Kingdom
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24
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Fitzgerald M, Hayward IP, Thomas AC, Campbell GR, Campbell JH. Matrix metalloproteinase can facilitate the heparanase-induced promotion of phenotype change in vascular smooth muscle cells. Atherosclerosis 1999; 145:97-106. [PMID: 10428300 DOI: 10.1016/s0021-9150(99)00019-2] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Previous studies from this laboratory have shown that degradation of heparan sulphate proteoglycan by both living macrophages and macrophage lysosomal heparanase induces phenotypic change of vascular smooth muscle cells (SMC) from a high volume fraction of myofilaments (V(v)myo) to a low V(v)myo [Campbell et al. Exp Cell Res 1992; 200: 156-167]. The aim of this study was to determine whether matrix metalloproteinase (MMP) activity is also involved in the induction of SMC phenotypic change by macrophages. A specific inhibitor of MMPs (BB94) was able to block macrophage-induced SMC phenotypic change and subsequent DNA synthesis in freshly dispersed SMC seeded in primary culture at confluent density. The inhibitor did not block these SMC changes when SMC were seeded at low density without macrophages nor did it block heparanase activity directly. We also determined whether heparanase and MMP activities are upregulated together in vivo. Artery homogenates were analysed in a heparanase enzyme assay and for MMPs using zymograms. Increased heparanase activity was observed 3-14 days following balloon catheter injury of rabbit carotid arteries, and returned to control levels 6 weeks after injury. Active MMP2 was induced with heparanase after injury. MMP9 induction was also apparent 6 h after injury. Immunohistology on sections of these arteries showed the presence of MMPI1, 2, 3 and 9 with these MMPs being strongly induced in the intima 7 days after balloon catheter injury. Both heparanase and MMP activities were also present in human end-stage complex lesions from coronary arteries, carotid endarterectomies and abdominal aortic aneurysms. Because MMPs and heparanase are expressed at the same time, it is possible that MMPs facilitate heparanase activity in promotion of phenotypic modulation of SMC in vivo during neointimal thickening following injury and in atherosclerotic lesions.
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Affiliation(s)
- M Fitzgerald
- Co-operative Research Centre for Cardiac Technology, Department of Anatomical Sciences, University of Queensland, St. Lucia, Australia
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25
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Tasker PN, Michelangeli F, Nixon GF. Expression and distribution of the type 1 and type 3 inositol 1,4, 5-trisphosphate receptor in developing vascular smooth muscle. Circ Res 1999; 84:536-42. [PMID: 10082475 DOI: 10.1161/01.res.84.5.536] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The recent discoveries of inositol 1,4,5-trisphosphate (IP3) receptor subtypes with different affinities for IP3 and their potential involvement in development has important consequences for vascular smooth muscle. This study has examined the expression and distribution of the type 1 and type 3 IP3 receptor subtypes in developing rat vascular smooth muscles. Immunoblotting of portal vein and aorta from neonatal (2 to 4 days) and fully developed (6 weeks) rats revealed significantly higher levels of the type 3 IP3 receptor expression in neonatal, compared with developed, vascular smooth muscles. In contrast, expression of the type 1 IP3 receptor in neonates was lower compared with developed vascular smooth muscles. Immunolocalization of the type 3 IP3 receptors in neonatal tissues revealed that staining corresponded to the distribution of the sarcoplasmic reticulum (visualized by osmium ferricyanide staining of thin tissue sections), which suggested localization of the type 3 IP3 receptor throughout the sarcoplasmic reticulum network. We conclude that type 3 IP3 receptors are the predominant subtype in the development of vascular smooth muscle and are distributed throughout the sarcoplasmic reticulum in these cells. The switch in isoforms of the IP3 receptor during development from the type 3 with low affinity for IP3 to the higher-affinity type 1 receptor may play a role in calcium-mediated regulation of developing vascular smooth muscle.
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MESH Headings
- Actins/metabolism
- Animals
- Animals, Newborn
- Calcium/metabolism
- Calcium Channels/metabolism
- Calcium-Binding Proteins/metabolism
- Inositol 1,4,5-Trisphosphate/metabolism
- Inositol 1,4,5-Trisphosphate Receptors
- Microfilament Proteins
- Microscopy, Confocal
- Microscopy, Electron
- Muscle Development
- Muscle, Smooth, Vascular/growth & development
- Muscle, Smooth, Vascular/metabolism
- Rats
- Receptors, Cytoplasmic and Nuclear/metabolism
- Sarcoplasmic Reticulum/metabolism
- Calponins
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Affiliation(s)
- P N Tasker
- Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK
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26
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Porto LC, Ferreira MA, Costa AM, da Silveira PR. Immunolabeling of type IV collagen, laminin, and alpha-smooth muscle actin cells in the intima of normal and varicose saphenous veins. Angiology 1998; 49:391-8. [PMID: 9591531 DOI: 10.1177/000331979804900508] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Smooth muscle cells (SMC) of normal and varicose human saphenous intima were studied on cryostat sections by immunohistochemistry with alpha-smooth muscle actin (ASMA), type IV collagen, and laminin antibodies and also by transmission electron microscopy. The findings suggest two structurally distinct subtypes of smooth muscle cells with thin and thicker external lamina. Thin external lamina SMC were characterized by laminin, type IV collagen, weaker external lamina reactivity, and intense cytoplasmic alpha-smooth muscle actin immunoreactivity. Ultrastructurally, they exhibited abundant cytoplasmic microfilaments and thin external lamina. These cells were found isolated or, more frequently, clustered in fascicles close to the subendothelium in focal or zonal cushions, or in diffuse enlargement of the intima. In contrast, thicker external lamina smooth muscle cells were intensely immunolabeled for laminin and collagen IV, showing irregular cytoplasmic ASMA reaction. Single or clustered thicker external lamina SMC were seen predominantly in zonal cushions and in intima diffuse enlargement. It is very likely that these cells secrete these matrices in a nonpolarized fashion. The thicker external lamina of these SMCs showed a fine granular amorphous aspect sometimes intermingled with microfibrils. These external lamina were interposed between neighboring cells and exposed to collagen fibrils and elastic fibers. The cells also exhibited rarefaction of the cytoplasmic filaments. Intermediary cells exhibiting both features were rarely seen. Thicker external lamina SMC should be discussed in the context of an adaptive/proliferative response leading to dysfunction of the fibroelastic properties of the vein wall.
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Affiliation(s)
- L C Porto
- Departamento de Histologia e Embriologia, Instituto de Biologia, Rio de Janeiro, Brazil
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27
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Zheng B, Duan C, Clemmons DR. The effect of extracellular matrix proteins on porcine smooth muscle cell insulin-like growth factor (IGF) binding protein-5 synthesis and responsiveness to IGF-I. J Biol Chem 1998; 273:8994-9000. [PMID: 9535886 DOI: 10.1074/jbc.273.15.8994] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The aim of this study was to determine if cultured porcine vascular smooth muscle cells (pSMCs) that had been maintained on different extracellular matrix proteins had an alteration in their expression of insulin-like growth factor binding protein-5 (IGFBP-5) and their responsiveness to insulin-like growth factor-I (IGF-I). When pSMCs were plated on fibronectin, they synthesized 6.0 +/- 1.2-fold more IGFBP-5 than did cells maintained on laminin and type IV collagen. IGF-I increased IGFBP-5 gene expression 3-fold in the cells plated on fibronectin. The addition of an RGD peptide and echistatin to pSMC cultures that had been plated on fibronectin inhibited IGFBP-5 mRNA expression. The addition of an antibody against alpha2beta1 integrin partially reversed the inhibitory effects of laminin and type IV collagen on IGFBP-5 expression. Cells maintained on fibronectin had a 5.0 +/- 1.1-fold greater DNA synthesis response to IGF-I compared with those maintained on laminin/type IV collagen, and echistatin significantly inhibited the DNA synthesis response of the fibronectin-maintained cells to IGF-I. The anti-alpha2beta1 antibody partially reversed the inhibitory effect of laminin and type IV collagen on IGF-I-stimulated DNA synthesis. The addition of IGFBP-5 to cultures plated on laminin and type IV collagen significantly increased their response to IGF-I. Atherosclerotic plaques from pig aorta contained abundant fibronectin and had increased IGFBP-5 mRNA (4.5 +/- 1.5-fold) compared with tissue from the normal vessel wall that had a low fibronectin content. These results indicate that fibronectin, laminin, and type IV collagen have major effects on IGFBP-5 expression and on IGF-I-stimulated pSMC responses and that these effects are mediated by their respective integrins.
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MESH Headings
- Animals
- Antibodies, Monoclonal/pharmacology
- Aorta, Thoracic/cytology
- Aorta, Thoracic/drug effects
- Aorta, Thoracic/metabolism
- Arteriosclerosis/metabolism
- Arteriosclerosis/pathology
- Cell Division/drug effects
- Cells, Cultured
- Collagen/pharmacology
- DNA/biosynthesis
- Extracellular Matrix Proteins/pharmacology
- Fibronectins/pharmacology
- Gene Expression Regulation/drug effects
- Humans
- Insulin-Like Growth Factor Binding Protein 2/biosynthesis
- Insulin-Like Growth Factor Binding Protein 5/biosynthesis
- Insulin-Like Growth Factor I/pharmacology
- Integrins/antagonists & inhibitors
- Integrins/physiology
- Intercellular Signaling Peptides and Proteins
- Kinetics
- Laminin/pharmacology
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/metabolism
- Oligopeptides/pharmacology
- Peptides/pharmacology
- Platelet Aggregation Inhibitors/pharmacology
- RNA, Messenger/biosynthesis
- Receptors, Collagen
- Recombinant Proteins/pharmacology
- Swine
- Transcription, Genetic/drug effects
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Affiliation(s)
- B Zheng
- Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7170, USA
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28
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Campbell JH, Campbell GR. The cell biology of atherosclerosis--new developments. AUSTRALIAN AND NEW ZEALAND JOURNAL OF MEDICINE 1997; 27:497-500. [PMID: 9448904 DOI: 10.1111/j.1445-5994.1997.tb02225.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
In the development of atherosclerotic lesions, three basic processes occur: 1) invasion of the artery wall by leucocytes, particularly monocytes and T-lymphocytes; 2) smooth muscle phenotypic modulation, proliferation, and synthesis of extracellular matrix; and 3) intracellular (macrophage and smooth muscle) lipoprotein uptake and lipid accumulation. Invasion of the vessel wall by leucocytes is mediated through the expression of adhesion molecules on both leucocytes and the endothelium making them 'sticky'. The adhesion molecules are induced by inflammatory mediators released from leucocytes and endothelium, and these in turn are induced by high serum cholesterol levels or complement fragments. Leucocytes which have adhered to the endothelium are chemo-attracted into the vessel wall by cytokines produced by early arriving leucocytes or by low density lipoprotein which has passively passed into the wall, in the process being trapped and oxidised. The oxidised low density lipoprotein is taken up by scavenger receptors (which are not subject to down-regulation) on both macrophages and smooth muscle cells. The overaccumulation of lipid is toxic to the cells and they die contributing to the central necrotic core. The macrophages and T-lymphocytes produce substances which induce smooth muscle cells of the artery wall to change from a 'contractile' (high volume fraction of myofilaments [Vvmyo]) to a 'synthetic' (low Vvmyo) phenotype. In this altered state they respond to growth factors released from macrophages, platelets, regenerating endothelial cells and smooth muscle cells; produce large amounts of matrix; express lipoprotein scavenger receptors; express adhesion molecules for leucocytes; and express HLA-DR following exposure to the T-lymphocyte product, IFN-delta, suggesting that they can become involved in a generalised immune reaction.
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Affiliation(s)
- J H Campbell
- Centre for Research in Vascular Biology, University of Queensland, Brisbane
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29
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Kim S, Ip HS, Lu MM, Clendenin C, Parmacek MS. A serum response factor-dependent transcriptional regulatory program identifies distinct smooth muscle cell sublineages. Mol Cell Biol 1997; 17:2266-78. [PMID: 9121477 PMCID: PMC232076 DOI: 10.1128/mcb.17.4.2266] [Citation(s) in RCA: 184] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
The SM22alpha promoter has been used as a model system to define the molecular mechanisms that regulate smooth muscle cell (SMC) specific gene expression during mammalian development. The SM22alpha gene is expressed exclusively in vascular and visceral SMCs during postnatal development and is transiently expressed in the heart and somites during embryogenesis. Analysis of the SM22alpha promoter in transgenic mice revealed that 280 bp of 5' flanking sequence is sufficient to restrict expression of the lacZ reporter gene to arterial SMCs and the myotomal component of the somites. DNase I footprint and electrophoretic mobility shift analyses revealed that the SM22alpha promoter contains six nuclear protein binding sites (designated smooth muscle elements [SMEs] -1 to -6, respectively), two of which bind serum response factor (SRF) (SME-1 and SME-4). Mutational analyses demonstrated that a two-nucleotide substitution that selectively eliminates SRF binding to SME-4 decreases SM22alpha promoter activity in arterial SMCs by approximately 90%. Moreover, mutations that abolish binding of SRF to SME-1 and SME-4 or mutations that eliminate each SME-3 binding activity totally abolished SM22alpha promoter activity in the arterial SMCs and somites of transgenic mice. Finally, we have shown that a multimerized copy of SME-4 (bp -190 to -110) when linked to the minimal SM22alpha promoter (bp -90 to +41) is necessary and sufficient to direct high-level transcription in an SMC lineage-restricted fashion. Taken together, these data demonstrate that distinct transcriptional regulatory programs control SM22alpha gene expression in arterial versus visceral SMCs. Moreover, these data are consistent with a model in which combinatorial interactions between SRF and other transcription factors that bind to SME-4 (and that bind directly to SRF) activate transcription of the SM22alpha gene in arterial SMCs.
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MESH Headings
- Animals
- Base Sequence
- Binding Sites/genetics
- Cells, Cultured
- DNA/genetics
- DNA/metabolism
- DNA-Binding Proteins/metabolism
- Gene Expression Regulation, Developmental
- Genes, Reporter
- Lac Operon
- Mice
- Mice, Transgenic
- Microfilament Proteins
- Molecular Sequence Data
- Muscle Development
- Muscle Proteins/genetics
- Muscle, Smooth/cytology
- Muscle, Smooth/growth & development
- Muscle, Smooth/metabolism
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/growth & development
- Muscle, Smooth, Vascular/metabolism
- Mutation
- Nuclear Proteins/metabolism
- Promoter Regions, Genetic
- Rats
- Serum Response Factor
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Affiliation(s)
- S Kim
- Department of Medicine, University of Chicago, Illinois 60637, USA
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30
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Chen YH, Chen YL, Lin SJ, Chou CY, Mar GY, Chang MS, Wang SP. Electron microscopic studies of phenotypic modulation of smooth muscle cells in coronary arteries of patients with unstable angina pectoris and postangioplasty restenosis. Circulation 1997; 95:1169-75. [PMID: 9054846 DOI: 10.1161/01.cir.95.5.1169] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
BACKGROUND Proliferation and matrix protein secretion of coronary smooth muscle cells (SMCs) have been suggested as one of the mechanisms responsible for the development of postangioplasty restenosis and an alternative cause of unstable angina. Phenotypic modulation of SMCs may produce a pool of cells potentially responsive to growth stimulation that can synthesize abundant extracellular matrix. This study tested the hypothesis that phenotypic modulation of SMCs occurred during the evolution of postangioplasty restenosis and unstable angina. METHODS AND RESULTS The SMCs of coronary atherectomy specimens from 24 patients were identified under electron microscope. Volume fractions of synthetic organelles (VFSOs) and other features related to phenotypic modulation of SMCs were measured. The results showed that the VFSO in SMCs from 5 patients with unstable angina (group 2) resembled those from 9 patients with postangioplasty restenosis (group 3; 0.42 +/- 0.13 versus 0.36 +/- 0.10; P = NS), and both were significantly higher than those from 6 patients with stable angina (group 1; 0.21 +/- 0.11). Four patients with restenosis lesions who underwent angioplasty > 6 months ago (group 4) also had a low VFSO in SMCs (0.19 +/- 0.05). This value was significantly less than those in groups 2 and 3 (P < .05) but similar to that in group 1. CONCLUSIONS The coronary lesions from patients with unstable angina resembled those from patients with postangioplasty restenosis in terms of the phenotypic modulation and VFSO in SMCs. Our findings therefore suggest that after phenotypic modulation, the SMCs may become responsive to growth stimulation, with an ability to massively proliferate and synthesize abundant extracellular matrix. These processes may lead to plaque expansion and eventually to the development of unstable angina and restenosis.
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Affiliation(s)
- Y H Chen
- Department of Medicine, Veterans General Hospital-Taipei, Taiwan
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31
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Augier T, Charpiot P, Chareyre C, Remusat M, Rolland PH, Garçon D. Medial elastic structure alterations in atherosclerotic arteries in minipigs: plaque proximity and arterial site specificity. Matrix Biol 1997; 15:455-67. [PMID: 9106157 DOI: 10.1016/s0945-053x(97)90019-6] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Using a model of atherosclerosis in minipigs, we analyzed changes in elastic structure within the medial sections of the abdominal aorta and left interventricular coronary artery both in the vicinity of and distal to atheromatous plaques. Twenty-four animals, divided into three groups, were fed either a control diet or a hypercholesterolemic and hyperhomocysteinic atherogenic diet, alone or in association with an antihypertensor, namely isosorbide dinitrate (Risordan). The atherogenic diet, administered for a period of four months, induced in the minipig advanced noncalcified atherosclerotic lesions that were histologically similar to those found in humans. A morphodensitometric analysis of the medial elastic structures was carried out on images obtained from specifically stained transverse arterial sections examined under a light microscope. The volume density of the elastic structures was diminished in the arterial media of the atherosclerotic animals due to opening and widening of the fenestrae in the elastic laminate and increased communication between the interlamellar spaces. Whereas this elastolytic process was uniform and independent of the proximity of atheromatous plaques in the left interventricular coronary artery, it was intensified in the vicinity of the plaques in the abdominal aorta. Overall elastolytic activity was increased in the walls of atheromatous artery in both arterial sites, and metalloproteinases were implied in this increase of activity. We previously reported that treatment with isosorbide dinitrate significantly reduced the moderate systolic hypertension and the increase in transparietal stress observed in the abdominal aorta of atheromatous animals. We report here that isosorbide dinitrate prevented the atherogenic-diet-induced deterioration of the elastic structure in these arteries; complete inhibition of changes to the elastic laminae was evident in areas remote from plaque formation, but only partial inhibition in the vicinity of such plaques. It did not, however, prevent structural damage in the left interventricular coronary artery or modify the increase in parietal elastolytic activity in either of the two arteries. This suggests that damage to the elastic structure in atheromatous arteries is dependent not only on overall elastolytic activity but also on localized factors, possibly related to parietal stresses, affected by the presence of atheromatous plaques.
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Affiliation(s)
- T Augier
- C.J.F. INSERM 94-01 Biochemistry Lab, School of Pharmacy, Marseille, France
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32
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Owens GK, Wise G. Regulation of differentiation/maturation in vascular smooth muscle cells by hormones and growth factors. AGENTS AND ACTIONS. SUPPLEMENTS 1997; 48:3-24. [PMID: 9177097 DOI: 10.1007/978-3-0348-7352-9_1] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Smooth muscle cells (SMC) within atherosclerotic lesions show marked alterations in their differentiated properties as compared to normal medial SMC. This process of de-differentiation of SMC has been referred to as "phenotypic modulation", and is characterized by increased growth responsiveness, altered lipid metabolism, increased matrix production, and loss of contractile proteins, all of which can contribute to the development and/or progression of atherosclerotic disease. As such there has been much interest in understanding mechanisms and factors that control the differentiation of the vascular SMC. This paper reviews the effects of growth factors, growth inhibitors, and other extrinsic factors on differentiation/maturation of SMC, with a particular emphasis on consideration of factors that may contribute to abnormal control of SMC differentiation in vascular disease. In addition, we will briefly summarize what is currently known regarding molecular mechanisms that control the coordinate expression of genes encoding for SMC-selective/specific proteins that are required for the differentiated function of the vascular SMC.
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Affiliation(s)
- G K Owens
- Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, School of Medicine, Charlottesville 22906-0011, USA
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33
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Samaha FF, Ip HS, Morrisey EE, Seltzer J, Tang Z, Solway J, Parmacek MS. Developmental pattern of expression and genomic organization of the calponin-h1 gene. A contractile smooth muscle cell marker. J Biol Chem 1996; 271:395-403. [PMID: 8550594 DOI: 10.1074/jbc.271.1.395] [Citation(s) in RCA: 85] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Calponin-h1 is a 34-kDa myofibrillar thin filament, actin-binding protein that is expressed exclusively in smooth muscle cells (SMCs) in adult animals. To examine the molecular mechanisms that regulate SMC-specific gene expression, we have examined the temporal, spatial, and cell cycle-regulated patterns of expression of calponin-h1 gene expression and isolated and structurally characterized the murine calponin-h1 gene. Calponin-h1 mRNA is expressed exclusively in SMC-containing tissues in adult animals. During murine embryonic development, calponin-h1 gene expression is (i) detectable in E9.5 embryos in the dorsal aorta, cardiac outflow tract, and tubular heart, (ii) sequentially up-regulated in SMC-containing tissues, and (iii) down-regulated to non-detectable levels in the heart during late fetal development. In addition, the gene is expressed in resting rat aortic SMCs, but its expression is rapidly down-regulated when growth-arrested cells reenter phase G1 of the cell cycle and proliferate. Calponin-h1 is encoded by a 10.7-kilobase single copy gene composed of seven exons, which is part of a multigene family. Transient transfection analyses demonstrated that 1.5 kilobases of calponin-h1 5'-flanking sequence is sufficient to program high level transcription of a luciferase reporter gene in cultured primary rat aortic SMCs and the smooth muscle cell line, A7r5. Taken together, these data suggest that the calponin-h1 gene will serve as an excellent model system with which to examine the molecular mechanisms that regulate SMC lineage specification, differentiation, and phenotypic modulation.
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MESH Headings
- Amino Acid Sequence
- Animals
- Base Sequence
- Biological Evolution
- Calcium-Binding Proteins/genetics
- Calcium-Binding Proteins/metabolism
- Cell Line
- Cloning, Molecular
- DNA, Complementary
- Gene Expression Regulation, Developmental
- Humans
- Mice
- Microfilament Proteins
- Molecular Sequence Data
- Multigene Family
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/metabolism
- Muscle, Smooth, Vascular/physiology
- Phenotype
- Promoter Regions, Genetic
- Rats
- Sequence Homology, Amino Acid
- Transcription, Genetic
- Calponins
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Affiliation(s)
- F F Samaha
- Department of Medicine, University of Chicago, Illinois 60637, USA
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34
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Li H, Freeman MW, Libby P. Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines. J Clin Invest 1995; 95:122-33. [PMID: 7814605 PMCID: PMC295387 DOI: 10.1172/jci117628] [Citation(s) in RCA: 142] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Scavenger receptor (ScR)-mediated uptake of modified lipoproteins may contribute to the transformation of smooth muscle cells into lipid-laden foam cells during atherogenesis. This study examined the in vivo expression of ScRs in aortas, with or without balloon injury, taken from hypercholesterolemic or normocholesterolemic rabbits. Numerous intimal cells in the rabbit aortic lesions expressed ScRs as detected by immunocytochemical staining with a goat anti-rabbit ScR antibody. Single immunostaining for cell identification markers in serial sections, as well as double staining, confirmed the expression of ScRs by both intimal smooth muscle cells and macrophages. To explore potential inducers of ScR expression by smooth muscle cells in vivo, we studied the regulation of ScR expression in vitro by cytokines known to be present in atherosclerotic lesions. Tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) increased ScR mRNA levels, protein expression, and AcLDL degradative activity in cultured rabbit aortic smooth muscle cells. The induction of ScR expression in intimal smooth muscle cells in vivo could be a useful marker of smooth muscle cell activation during atherogenesis and may contribute to foam cell formation by this cell type following balloon injury and/or hypercholesterolemia. Cytokines, such as TNF-alpha or IFN-gamma, may stimulate some of the phenotypic changes that characterize the alteration in gene expression of intimal smooth muscle cells in rabbit atherosclerotic lesions.
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MESH Headings
- Animals
- Aorta/injuries
- Aorta/metabolism
- Aorta/pathology
- Arteriosclerosis/metabolism
- Cell Differentiation
- Cells, Cultured
- Cytokines/pharmacology
- Diet, Atherogenic
- Foam Cells
- Gene Expression Regulation
- Hypercholesterolemia/metabolism
- Immunohistochemistry
- Interferon-gamma/pharmacology
- Lipoproteins, LDL/metabolism
- Male
- Membrane Proteins
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/metabolism
- Rabbits
- Receptors, Immunologic/isolation & purification
- Receptors, Immunologic/metabolism
- Receptors, Lipoprotein
- Receptors, Scavenger
- Scavenger Receptors, Class B
- Tumor Necrosis Factor-alpha/pharmacology
- Tunica Intima/metabolism
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Affiliation(s)
- H Li
- Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115
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35
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Epstein SE, Speir E, Unger EF, Guzman RJ, Finkel T. The basis of molecular strategies for treating coronary restenosis after angioplasty. J Am Coll Cardiol 1994; 23:1278-88. [PMID: 8176084 DOI: 10.1016/0735-1097(94)90368-9] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Excessive smooth muscle cell proliferation significantly contributes to restenosis, which occurs in 25% to 50% of patients within 6 months of coronary angioplasty. Because successful treatment will probably depend on our acquiring a comprehensive knowledge of the molecular and cellular mechanisms involved, this report reviews 1) information relevant to the molecular and cellular mechanisms responsible for the smooth muscle cell(s) response to vascular injury, and 2) several molecular-based therapeutic strategies currently being explored as possible approaches to the control of restenosis, including recombinant DNA technology to target delivery of cytotoxic molecules to proliferating smooth muscle cell(s), antisense strategies to inhibit expression of gene products necessary for cell proliferation and gene therapy.
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Affiliation(s)
- S E Epstein
- Cardiology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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36
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Rennick RE, Campbell JH, Campbell GR. Macrophages enhance binding of beta-VLDL and cholesterol ester accumulation in cultured aortic smooth muscle cells. Heart Vessels 1994; 9:19-29. [PMID: 8113154 DOI: 10.1007/bf01744492] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The effect of macrophages on the uptake of beta-very low-density lipoprotein (beta-VLDL) by smooth muscle cells (SMC) expressing different morphological phenotypes was examined in culture. The SMC were grown alone and in co-culture with macrophages for four days, then incubated with different concentrations of 125I-beta-VLDL for 3 h at 4 degrees C or with 75 ug/ml beta-VLDL for 24 h at 37 degrees C. The binding of beta-VLDL to SMC at 4 degrees C was enhanced in the presence of macrophages irrespective of the phenotype expressed by SMC. This occurred through modification of the lipoprotein, since binding of re-isolated macrophage-conditioned beta-VLDL to SMC was 12.5 times that of fresh beta-VLDL. This modified form of beta-VLDL competed with fresh beta-VLDL for binding to SMC. Binding was inhibited in the presence of probucol, suggesting that an oxidative mechanism may be involved. The presence of macrophages also enhanced the accumulation of beta-VLDL-derived cholesterol in SMC. While most of this is a consequence of the enhanced binding, macrophages may also act directly on SMC to increase cholesterol accumulation, since the activity of acid cholesterol ester hydrolase and neutral cholesterol ester hydrolase in SMC was reduced in the presence of macrophages.
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Affiliation(s)
- R E Rennick
- Baker Medical Research Institute, Prahran, Victoria, Australia
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37
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Abstract
Atherosclerosis is a complex disease of uncertain cause. Its pathobiology is believed to represent an abnormal expression of the processes of vascular healing. Etiologic models derive from a 'response to injury' paradigm and can be divided into three separate disease stages: endothelial dysfunction, smooth muscle proliferation and architectural disruption. The initiating event of endothelial dysfunction is unknown, but is believed to be related to low-density lipoproteins and/or their oxidized derivatives. Endothelial injury is signalled to the smooth muscle cells of the media by three routes: direct cell-cell interaction, secretion of soluble growth factors and monocyte-derived cytokines. Monocytes are recruited by the endothelium and invade the subintimal space by a complex interaction of a variety of adhesion proteins and receptors on both cell types. Smooth muscle cell proliferation is initiated by a change in phenotype expression from 'contractile' to 'synthetic' resulting from the binding of fibronectin to specific integrin receptors. Three functionally distinct activities may represent separate subtypes of the 'synthetic phenotype': migration from the media to the intima, increased proliferation and inappropriate extracellular matrix synthesis. The loss of normal regulatory control and anchorage independence of proliferation suggest a relationship to oncogenic transformation. Both migration and proliferation result from the binding of platelet-derived growth factor-like factors to smooth muscle cell receptors, which initiates a cascade of intracellular molecular events leading either to cytoskeletal locomotory restructuring or cell cycle activation. Both pathways also appear to be coregulated by integrin receptors and both depend upon phosphorylation of cell membrane, cytosolic and nuclear regulatory proteins. Clinical expression of atherosclerosis may follow sudden loss of architectural integrity of the intimal plaque by three different mechanisms: plaque fissuring, intraluminal plaque rupture or intramural hemorrhage related to abnormal vessel wall stress and/or biochemistry.
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Affiliation(s)
- M Sanders
- Department of Molecular Genetics and Microbiology, U.M.D.N.J.-Robert Wood Johnson Medical School, Piscataway 08854-5635
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38
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Kockx MM, De Meyer GR, Jacob WA, Bult H, Herman AG. Triphasic sequence of neointimal formation in the cuffed carotid artery of the rabbit. ACTA ACUST UNITED AC 1993; 12:1447-57. [PMID: 1360258 DOI: 10.1161/01.atv.12.12.1447] [Citation(s) in RCA: 72] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90 +/- 0.36% of the medial smooth muscle cells (SMCs) were replicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for alpha-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76 +/- 0.22% at day 7). In the third phase the number of subendothelial cells increased (104 +/- 15 SMC nuclei per section at day 7, of which 8.89 +/- 2.26% were PCNA-positive) and was associated with deposition of collagen type IV and fibronectin. At 14 days a complete, circular neointima was present and contained 2.13 +/- 0.28% replicating SMCs. The media showed 0.44 +/- 0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage.
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Affiliation(s)
- M M Kockx
- Department of Pathology, General Hospital Middleheim, Antwerp, Belgium
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39
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van Neck JW, Bloemers HP. Molecular aspects of pathological processes in the artery wall. Mol Biol Rep 1992; 17:1-15. [PMID: 1287474 DOI: 10.1007/bf01006394] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Affiliation(s)
- J W van Neck
- Department of Biochemistry, University of Nijmegen, The Netherlands
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40
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Campbell JH, Rennick RE, Kalevitch SG, Campbell GR. Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype. Exp Cell Res 1992; 200:156-67. [PMID: 1563484 DOI: 10.1016/s0014-4827(05)80084-9] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.
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Affiliation(s)
- J H Campbell
- Department of Anatomy, University of Queensland, St. Lucia, Australia
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41
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Epstein SE, Siegall CB, Biro S, Fu YM, FitzGerald D, Pastan I. Cytotoxic effects of a recombinant chimeric toxin on rapidly proliferating vascular smooth muscle cells. Circulation 1991; 84:778-87. [PMID: 1860221 DOI: 10.1161/01.cir.84.2.778] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
BACKGROUND Restenosis after percutaneous transluminal coronary angioplasty is associated with activation of medial smooth muscle cells (SMCs); they proliferate, migrate to the subintima, and narrow the vessel lumen. Cancer cells often express more cell surface receptors than do normal cells. This has allowed tumor cells to be specifically targeted using cytotoxic agents. We have examined whether a similar concept can be applied to rapidly proliferating but nontransformed SMCs. Pseudomonas exotoxin (PE; MW, 66 kDa) is a potent toxin that kills cells by inhibiting protein synthesis; its toxicity is diminished when its cell recognition domain is deleted to produce a 40-kDa protein (PE40). METHODS AND RESULTS A complementary DNA encoding transforming growth factor alpha (TGF alpha) was ligated to that encoding PE40 and the chimeric toxin TGF alpha-PE40, which is cytotoxic to cancer cells displaying epidermal growth factor (EGF) receptors, was expressed in Escherichia coli. The ability of this toxin to kill proliferating SMCs was tested. When cells were seeded at low density (2,500 cells/cm2) and grown in medium supplemented with 10% fetal bovine serum, they were found to be rapidly proliferating; these cells were very sensitive to the cytotoxic effects of TGF alpha-PE40 (ID50, 4.0 +/- 0.17 ng/ml). In contrast, cytotoxicity was 30-fold less (ID50, 125 +/- 23 ng/ml; p less than 0.0004) when cells were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum). CONCLUSIONS Competition studies using excess EGF indicated that the cytotoxic effects of TGF alpha-PE40 are specifically mediated by the EGF receptor. EGF receptor binding analysis demonstrated that rapidly proliferating SMCs display 10-fold more EGF receptors than do quiescent SMCs in vitro. Thus, a chimeric toxin targeted toward the EGF receptor can selectively kill rapidly proliferating SMCs. Whether this toxin or other chimeric toxins directed against other cell surface receptors will effectively inhibit SMCs proliferating in vivo or be useful in preventing restenosis remains to be determined.
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Affiliation(s)
- S E Epstein
- Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
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42
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Hultgårdh-Nilsson A, Krondahl U, Querol-Ferrer V, Ringertz NR. Differences in growth factor response in smooth muscle cells isolated from adult and neonatal rat arteries. Differentiation 1991; 47:99-105. [PMID: 1955111 DOI: 10.1111/j.1432-0436.1991.tb00227.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
The development of atherosclerosis includes an abnormal proliferation of smooth muscle cells (SMCs) in the arterial intima. The factors responsible for this process remain to be identified, but earlier studies have suggested that age-related changes in growth-regulatory mechanisms may be involved. In the present study growth-regulatory mechanisms of neonatal and adult rat SMCs have been compared both in early passage and after subcultivation. Neonatal SMCs in early passage were found to have a high rate of spontaneous DNA synthesis and showed little response to stimulation with growth factors. Early passage adult SMCs showed a lower rate of spontaneous DNA synthesis but responded well to exogenous growth factors. There was no difference in the gene or surface expression of receptors for platelet-derived growth factor (PDGF) between neonatal and adult cells, and there was no significant difference in the amount of inositol phosphate formed in the cells after stimulation with PDGF BB. However, there was increased expression of PDGF A chain mRNA in serum-starved neonatal cells as compared to adult serum-starved SMCs. After subcultivation (seven to nine passages) neonatal SMCs started to become senescent, had a low rate of spontaneous DNA synthesis and were more sensitive to growth factor stimulation than in early passage. Adult SMCs did not demonstrate signs of senescence after subcultivation. The results demonstrate marked differences in the mechanisms regulating growth of neonatal and adult rat SMCs and suggest that the increased sensitivity of adult cells to exogenous growth factors and the inability of these cells to become senescent may be important factors in atherogenesis.
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Affiliation(s)
- A Hultgårdh-Nilsson
- Department of Medical Cell Genetics, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden
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43
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Horrigan S, Campbell JH, Campbell GR. Oxidation of beta-very low density lipoprotein by endothelial cells enhances its metabolism by smooth muscle cells in culture. ARTERIOSCLEROSIS AND THROMBOSIS : A JOURNAL OF VASCULAR BIOLOGY 1991; 11:279-89. [PMID: 1900191 DOI: 10.1161/01.atv.11.2.279] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
We have previously shown that beta-very low density lipoprotein (beta-VLDL) incubated with bovine aortic endothelial cells (ECs) is bound and internalized more readily by cultured rabbit aortic smooth muscle cells (SMCs) than is beta-VLDL incubated in the absence of ECs, resulting in enhanced accumulation of cholesterol. To investigate the mechanism by which this occurs, beta-VLDL from hypercholesterolemic rabbit serum was incubated with cultured bovine aortic ECs. This resulted in the formation of thiobarbituric acid (TBA)-reactive material indicating extensive lipid peroxidation. The formation of TBA-reactive material, the increased metabolism of beta-VLDL by rabbit aortic SMCs, and the increased accumulation of cholesterol were prevented by superoxide dismutase, EDTA, several antioxidants, and, to a lesser extent, by 5,8,11,14-eicosatetraynoic acid, but not by acetylsalicylic acid, suggesting that potential oxidizing agents were the superoxide anion, metal ions, and lipoxygenase derivatives, but not cyclooxygenase derivatives. The percentage composition of phospholipid, protein, triglyceride, and free and esterified cholesterol of EC-modified beta-VLDL did not differ significantly from the unmodified lipoprotein. Displacement studies showed that only part of the interaction of both EC-beta-VLDL and unmodified beta-VLDL occurred through the B/E receptor and that the EC-beta-VLDL displaced 125I-beta-VLDL to a greater extent than did unmodified beta-VLDL. This indicated that the EC-beta-VLDL interacted more strongly with receptors on SMCs.
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Affiliation(s)
- S Horrigan
- Cell Biology Laboratory, Baker Medical Research Institute, Prahran, Australia
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44
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Abstract
1. Heparan sulfate proteoglycan in the basal lamina of smooth muscle cells is important in the maintenance of the 'contractile', high volume fraction of myofilaments (Vvmyo) phenotype. The mechanism by which this occurs may involve the continuous internalization of heparan sulfate by the smooth muscle cells themselves. 2. One macrophage can degrade all the heparan sulfate from three smooth muscle cells by the action of heparan sulfate-degrading enzymes in their lysosomes, thus leaving none available for internalization by the smooth muscle cell until it has synthesized more, and leading to the induction of smooth muscle phenotypic change from a high Vvmyo to a low Vvmyo. 3. In this altered phenotype the smooth muscle cells proliferate in response to mitogens, synthesize large amounts of extracellular matrix and accumulate lipid, all characteristics of the smooth muscle cell in developing atheroma.
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Affiliation(s)
- J H Campbell
- Cell Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia
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45
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Thyberg J, Hedin U, Sjölund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. ARTERIOSCLEROSIS (DALLAS, TEX.) 1990; 10:966-90. [PMID: 2244864 DOI: 10.1161/01.atv.10.6.966] [Citation(s) in RCA: 421] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- J Thyberg
- Department of Medical Cell Biology, Karolinska Institute, Stockholm, Sweden
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46
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Affiliation(s)
- G R Campbell
- Department of Anatomy, University of Melbourne, Parkville, Victoria, Australia
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47
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TACHAS GEORGE, CAMPBELL JULIEH, CAMPBELL GORDONR. Genes Expressed during Vascular Smooth Muscle Phenotypic Modulation. Ann N Y Acad Sci 1990. [DOI: 10.1111/j.1749-6632.1990.tb42339.x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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48
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Merrilees MJ, Campbell JH, Spanidis E, Campbell GR. Glycosaminoglycan synthesis by smooth muscle cells of differing phenotype and their response to endothelial cell conditioned medium. Atherosclerosis 1990; 81:245-54. [PMID: 2350373 DOI: 10.1016/0021-9150(90)90072-q] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The synthesis of glycosaminoglycans (GAG) by contractile and irreversible synthetic phenotypes of vascular smooth muscle cells (SMC), and their response to endothelial cell conditioned medium (ECCM), has been investigated. Contractile SMC, (with a high volume fraction of myofilaments) were obtained by culturing freshly isolated rabbit aortic SMC for 3 days in primary culture. Irreversible synthetic SMC (with a low volume fraction of myofilaments) were obtained by serially passaging SMC to achieve more than 5 cumulative population doublings. In fresh medium both phenotypes produced significant amounts of GAG, but irreversible synthetic cells were more than twice as active on a per cell and cell volume basis. The proportions of individual GAG also changed with change in phenotype. Hyaluronic acid (HA) was the predominant GAG (78%) synthesised by contractile SMC but was significantly reduced (47%) in the irreversible synthetic cells with a corresponding increase in sulphated GAG (SGAG). The changed levels in GAG synthesis were independent of SMC growth. Both phenotypes responded to ECCM from bovine endothelial cells (EC) and significantly increased their synthesis of GAG and by the same relative amounts (50-100%). This response was density dependent, with ECCM from low and high density cultures of EC producing maximal responses and EC of intermediate densities producing minimal increases. Furthermore, dense cultures of EC preferentially stimulated SGAG. These findings show that an increase in synthesis of SMC GAG, and especially sulphated GAG as is found in atherosclerosis, may occur either through a change in phenotype or through endothelial mediated stimulation of GAG synthesis by either phenotype.
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Affiliation(s)
- M J Merrilees
- Department of Anatomy, School of Medicine, University of Auckland, New Zealand
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Mayberg MR, Okada T, Bark DH. The significance of morphological changes in cerebral arteries after subarachnoid hemorrhage. J Neurosurg 1990; 72:626-33. [PMID: 2319321 DOI: 10.3171/jns.1990.72.4.0626] [Citation(s) in RCA: 111] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
A porcine model was developed to allow quantitative assessment of morphological changes in cerebral arteries after subarachnoid hemorrhage and to determine the significance of structural changes in producing arterial narrowing. Whole blood was selectively applied to the middle cerebral artery (MCA) of seven pigs. After 10 days, vessels were perfusion-fixed and examined by light and transmission electron microscopy and immunohistochemistry. The MCA's exposed to whole blood for 10 days showed prominent luminal narrowing associated with profound ultrastructural changes affecting all layers of the vessel wall. Morphometric analysis, however, demonstrated that significant reductions in the luminal cross-sectional area (-55.8% +/- 12.5%, p less than 0.005) and increases in radial wall thickness (75.1% +/- 10.5%, p less than 0.005) were associated with only minimal increase in the cross-sectional area of the vessel wall (12.5% +/- 15%, p less than 0.025). By stereological analysis, the volume density of individual components of the arterial wall was unchanged in MCA's exposed to blood. Vessels exposed to blood showed a 44% reduction in smooth-muscle cell immunoreactive actin and increased collagen in the extracellular matrix of the vessel wall. These data suggest that structural changes in cerebral arteries after subarachnoid hemorrhage do not directly contribute to vessel narrowing through increases in wall mass. Nevertheless, such changes may reflect pathological mechanisms which act to augment prolonged vasoconstriction or inhibit the maintenance of normal vascular tone.
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Affiliation(s)
- M R Mayberg
- Department of Neurosurgery, University of Washington, Seattle
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50
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Mayberg MR, Okada T, Bark DH. Morphologic Changes in Cerebral Arteries after Subarachnoid Hemorrhage. Neurosurg Clin N Am 1990. [DOI: 10.1016/s1042-3680(18)30816-7] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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