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Angiogenic Potential of Bone Marrow Derived CD133 + and CD271 + Intramyocardial Stem Cell Trans- Plantation Post MI. Cells 2019; 9:cells9010078. [PMID: 31892273 PMCID: PMC7016579 DOI: 10.3390/cells9010078] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Revised: 12/16/2019] [Accepted: 12/24/2019] [Indexed: 01/09/2023] Open
Abstract
Background: Bone marrow (BM)-derived stem cells with their various functions and characteristics have become a well-recognized source for the cell-based therapies. However, knowledge on their therapeutic potential and the shortage for a cross-link between distinct BM-derived stem cells, primed after the onset of myocardial infarction (MI), seems to be still rudimentary. Therefore, the post-examination of the therapeutic characteristics of such primed hematopoietic CD133+ and mesenchymal CD271+ stem cells was the object of the present study. Methods and Results: The effects of respective CD133+ and CD271+ mononuclear cells alone as well as in the co-culture model have been explored with focus on their angiogenic potential. The phenotypic analysis revealed a small percentage of isolated cells expressing both surface markers. Moreover, target stem cells isolated with our standardized immunomagnetic isolation procedure did not show any negative alterations following BM storage in regard to cell numbers and/or quality. In vitro network formation relied predominantly on CD271+ stem cells when compared with single CD133+ culture. Interestingly, CD133+ cells contributed in the tube formation, only if they were cultivated in combination with CD271+ cells. Additional to the in vitro examination, therapeutic effects of the primed stem cells were investigated 48 h post MI in a murine model. Hence, we have found a lower expression of transforming growth factor βeta 3 (TGFβ3) as well as an increase of the proangiogenic factors after CD133+ cell treatment in contrast to CD271+ cell treatment. On the other hand, the CD271+ cell therapy led to a lower expression of the inflammatory cytokines. Conclusion: The interactions between CD271+ and CD133+ subpopulations the extent to which the combination may enhance cardiac regeneration has still not been investigated so far. We expect that the multiple characteristics and various regenerative effects of CD271+ cells alone as well as in combination with CD133+ will result in an improved therapeutic impact on ischemic heart disease.
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The neuropeptide galanin promotes an anti-thrombotic phenotype on endocardial endothelial cells from heart failure patients. Auton Neurosci 2017; 206:35-42. [PMID: 28720509 DOI: 10.1016/j.autneu.2017.07.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2017] [Revised: 06/06/2017] [Accepted: 07/09/2017] [Indexed: 02/07/2023]
Abstract
Thromboembolic complications are a significant cause of mortality and re-hospitalization in heart failure (HF) patients. One source of thrombi is the ventricular endocardial surface that becomes increasingly pro-thrombotic as HF progresses. Anticoagulation comes with bleeding risks so identifying therapeutic agents for improving cardiac endothelial health are of critical clinical importance. Endocardial endothelial cells are closely apposed to cardiac sympathetic nerves. In HF, cardiac sympathetic nerves are dysregulated and promote disease progression. Whether endocardial endothelial health and function is impacted by sympathetic dysregulation in HF is unknown. Also unexplored is the impact of neuropeptides, such as galanin and neuropeptide Y (NPY), co-released from sympathetic nerve terminals, on endothelial health. In this study we examined the effect of sympathetic nerve-released neurotransmitters and neuropeptides on the procoagulant phenotype of cultured human endocardial endothelial cells from HF patients. As a functional readout of procoagulant state we examined thrombin-mediated von Willebrand factor (vWF) extrusion and multimer expression. We demonstrate that vWF extrusion and multimer expression is promoted by thrombin, that isoproterenol (a beta-adrenergic receptor agonist) augments this effect, whereas co-treatment with the beta-blockers propranolol and carvedilol blocks this effect. We also show that vWF extrusion and multimer expression is attenuated by treatment with the neuropeptide galanin, but not with NPY. Our results are consistent with a protective role of beta-blockers and galanin on endocardial endothelial health in heart failure. Improving endothelial health through galanin therapy is a future clinical application of this study.
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Geron M, Kumar R, Matzner H, Lahiani A, Gincberg G, Cohen G, Lazarovici P, Priel A. Protein toxins of the Echis coloratus viper venom directly activate TRPV1. Biochim Biophys Acta Gen Subj 2017; 1861:615-623. [PMID: 28063984 DOI: 10.1016/j.bbagen.2017.01.004] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2016] [Revised: 12/13/2016] [Accepted: 01/03/2017] [Indexed: 12/23/2022]
Abstract
BACKGROUND Peptide and protein toxins are essential tools to dissect and probe the biology of their target receptors. Venoms target vital physiological processes to evoke pain. Snake venoms contain various factors with the ability to evoke, enhance and sustain pain sensation. While a number of venom-derived toxins were shown to directly target TRPV1 channels expressed on somatosensory nerve terminals to evoke pain response, such toxins were yet to be identified in snake venoms. METHODS We screened Echis coloratus saw-scaled viper venom's protein fractions isolated by reversed phase HPLC for their ability to activate TRPV1 channels. To this end, we employed heterologous systems to analyze TRPV1 and NGF pathways by imaging and electrophysiology, combined with molecular biology, biochemical, and pharmacological tools. RESULTS We identified TRPV1 activating proteins in the venom of Echis coloratus that produce a channel-dependent increase in intracellular calcium and outwardly rectifying currents in neurons and heterologous systems. Interestingly, channel activation was not mediated by any of its known toxin binding sites. Moreover, although NGF neurotropic activity was detected in this venom, TRPV1 activation was independent of NGF receptors. CONCLUSIONS Echis coloratus venom contains proteins with the ability to directly activate TRPV1. This activity is independent of the NGF pathway and is not mediated by known TRPV1 toxins' binding sites. GENERAL SIGNIFICANCE Our results could facilitate the discovery of new toxins targeting TRPV1 to enhance current understanding of this receptor activation mechanism. Furthermore, the findings of this study provide insight into the mechanism through which snakes' venom elicit pain.
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Affiliation(s)
- Matan Geron
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Rakesh Kumar
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Henry Matzner
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Adi Lahiani
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Galit Gincberg
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Gadi Cohen
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Philip Lazarovici
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel
| | - Avi Priel
- Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel.
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Strande JL, Routhu KV, Lecht S, Lazarovici P. Nerve growth factor reduces myocardial ischemia/reperfusion injury in rat hearts. J Basic Clin Physiol Pharmacol 2013; 24:81-4. [PMID: 23314533 DOI: 10.1515/jbcpp-2012-0045] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2012] [Accepted: 12/05/2012] [Indexed: 12/20/2022]
Abstract
BACKGROUND Nerve growth factor (NGF) is a neurotrophin that supports the survival and differentiation of sympathetic neurons, and its increased expression after myocardial infarct was correlated with cardiac sympathetic hyperinnervation and arrhythmias. However, it is unclear whether NGF protects the heart during infarct. In this study, we sought to address this issue in rat heart exposed to ischemia/reperfusion injury (IRI). METHODS NGF was administered intravenously (IV), 15 min before ischemia, at different concentrations in the absence or presence of inhibitors of phosphatidylinositol-3 kinase (PI3K) or nitric oxide synthase (NOS) in different groups of rats (n=6) with left coronary occlusion for 30 min followed by 120-min reperfusion. The area at risk and infarct to risk ratios were determined from sections stained with 1% 2,3,5-triphenylterazolium chloride. RESULTS NGF treatment at doses of 0.015-15 μg/kg, with an optimal dose of 0.15 μg/kg given IV before ischemia, reduced the infarct size from about 60% at the area of risk to about 25%, indicating cardioprotection by about 60%. The infarct-sparing effects of NGF were partially abolished by the inhibition of PI3K and NOS using wortmannin and N(G)-monomethyl-l-arginine, respectively. CONCLUSIONS We have demonstrated for the first time that NGF attenuates myocardial infarct damage in an in vivo rat model of myocardial regional IRI. This cardioprotective effect is proposed to be related to the activities of PI3K and NOS. This suggests that NGF has a potential therapeutic role in the treatment of IRI.
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Affiliation(s)
- Jennifer L Strande
- Division of Cardiovascular Medicine, Medical College of Wisconsin, Milwaukee, WI, USA
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Ettinger K, Lecht S, Arien-Zakay H, Cohen G, Aga-Mizrachi S, Yanay N, Saragovi HU, Nedev H, Marcinkiewicz C, Nevo Y, Lazarovici P. Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model. Cell Signal 2012; 24:2378-88. [PMID: 22960610 DOI: 10.1016/j.cellsig.2012.08.008] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2012] [Revised: 08/06/2012] [Accepted: 08/23/2012] [Indexed: 01/11/2023]
Abstract
The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.
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Affiliation(s)
- Keren Ettinger
- Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
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Angioneural crosstalk in scaffolds with oriented microchannels for regenerative spinal cord injury repair. J Mol Neurosci 2012; 49:334-46. [PMID: 22878912 DOI: 10.1007/s12031-012-9863-9] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2012] [Accepted: 07/23/2012] [Indexed: 01/15/2023]
Abstract
The aim of our work is to utilize the crosstalk between the vascular and the neuronal system to enhance directed neuritogenesis in uniaxial guidance scaffolds for the repair of spinal cord injury. In this study, we describe a method for angioneural regenerative engineering, i.e., for generating biodegradable scaffolds, produced by a combination of controlled freezing (freeze-casting) and lyophilization, which contain longitudinally oriented channels, and provide uniaxial directionality to support and guide neuritogenesis from neuronal cells in the presence of endothelial cells. The optimized scaffolds, composed of 2.5 % gelatin and 1 % genipin crosslinked, were characterized by an elastic modulus of ~51 kPa and longitudinal channels of ~50 μm diameter. The scaffolds support the growth of endothelial cells, undifferentiated or NGF-differentiated PC12 cells, and primary cultures of fetal chick forebrain neurons. The angioneural crosstalk, as generated by first forming endothelial cell monolayers in the scaffolds followed by injection of neuronal cells, leads to the outgrowth of long aligned neurites in the PC12/endothelial cell co-cultures also in the absence of exogenously added nerve growth factor. Neuritogenesis was not observed in the scaffolds in the absence of the endothelial cells. This methodology is a promising approach for neural tissue engineering and may be applicable for regenerative spinal cord injury repair.
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AngiotensinII preconditioning promotes angiogenesis in vitro via ERKs phosphorylation. J Biomed Biotechnol 2012; 2012:737134. [PMID: 22500105 PMCID: PMC3303689 DOI: 10.1155/2012/737134] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2011] [Revised: 11/03/2011] [Accepted: 11/21/2011] [Indexed: 01/19/2023] Open
Abstract
AngiotensinII (AngII) is involved in not only the formation of cardiac hypertrophy but also the development of cardiac remodeling both of which are associated with myocardial angiogenesis. This study was therefore performed to clarify the effects of AngII on the formation of vasculatures by cultured cardiac microvascular endothelial cells (CMVECs) after a long-period stimulation with or without the AngII preconditioning. Incubation with AngII for 18 hrs significantly impaired the formation of capillary-like tubes comparing to that without AngII. CMVECs with AngII pretreatment for 5 and 10 min formed more capillary-like tubes than those without AngII pretreatment, suggesting that preconditioning with AngII at a lower dose for a short period could prevent the further damage of CMVECs by a higher concentration of AngII. Moreover, AngII (10(-7) M) stimulation for 5 and 10 min significantly induced the increase in extracellular signal-regulated protein kinases (ERKs) phosphorylation, and an ERKs inhibitor, PD98059, abrogated the increase in the formation of capillary-like tubes induced by the AngII-pretreatment. In conclusion, preconditioning with a lower concentration of AngII for a short period prevents the subsequent impairment of CMVECs by a higher dose of AngII, at least in part, through the increase in ERKs phosphorylation.
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Lecht S, Arien-Zakay H, Wagenstein Y, Inoue S, Marcinkiewicz C, Lelkes PI, Lazarovici P. Transient signaling of Erk1/2, Akt and PLCgamma induced by nerve growth factor in brain capillary endothelial cells. Vascul Pharmacol 2010; 53:107-14. [PMID: 20434587 DOI: 10.1016/j.vph.2010.04.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2009] [Revised: 03/15/2010] [Accepted: 04/22/2010] [Indexed: 11/26/2022]
Abstract
Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as proliferation and migration of endothelial cells (ECs) from different vascular beds, induces capillary sprouting in chorioallantoic membrane and improves in vivo vascularization in a hind-limb ischemic model. In the present study, we sought to investigate the signaling properties of NGF in a microcapillary ECs model compared to those of a neuronal model. NGF-induced phosphorylation of signaling molecules Erk1/2, Akt and PLCgamma were measured using Western blotting and compared between mouse NGF (mNGF) and snake venom NGF analogues. NGFs-induced signaling was TrkA mediated as evident by inhibition with the TrkA antagonist K252a. NGF and its analogues-induced signaling in ECs were characterized by a transient effect in contrast to a prolonged stimulation in neuronal cells. The potency of mouse, cobra and viper NGFs to induce Erk1/2 phosphorylation in ECs was higher than in neurons. In ECs, mNGF exhibited the highest efficacy of stimulation of Erk1/2 phosphorylation, followed by viper and cobra NGFs. The efficacy of stimulation of Erk1/2 phosphorylation measured with neurons was opposite from that in ECs. NGF-induced temporal signaling differences between ECs and neurons may explain the dual vascular and neurotrophic effects of this growth factor.
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Affiliation(s)
- Shimon Lecht
- Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
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Lecht S, Arien-Zakay H, Kohan M, Lelkes PI, Lazarovici P. Angiostatic effects of K252a, a Trk inhibitor, in murine brain capillary endothelial cells. Mol Cell Biochem 2010; 339:201-13. [PMID: 20148355 DOI: 10.1007/s11010-010-0386-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2009] [Accepted: 01/25/2010] [Indexed: 12/16/2022]
Abstract
Nerve growth factor (NGF) supports the survival and differentiation of sympathetic and sensory neurons and is also mitogenic for a variety of tumors. K252a, an antagonist of NGF receptor TrkA, was previously used as a pharmacological tool to study NGF actions and as a lead compound for developing anti-tumor drugs. Since recently, NGF was characterized as an angiogenic factor, we sought to investigate the angiostatic properties of K252a on endothelial cells (ECs). For this purpose, we used a murine brain microcapillary ECs model in which we found autocrine release of NGF in the culture medium and activation of TrkA receptor-induced downstream signaling molecules Erk1/2, Akt, and PLCgamma. In this model, we demonstrated the angiostatic property of K252a based on its ability to affect several important angiogenic steps. K252a, but not its cell membrane impermeable analogue K252b at 100 nM: (i) inhibited the proliferation of the ECs by 45 +/- 9%; (ii) reduced by 70 +/- 4% the migration of the ECs measured in a wound-closure model; (iii) reduced by 29 +/- 9% the formation of tube-like structures of the ECs cultured on Matrigel; (iv) stimulated by 100 +/- 25% the collagen deposition by the ECs, a process responsible for the increased endothelial barrier functions expressed by 22 +/- 5% reduction of paracellular permeability and by 17 +/- 3% elevation of transendothelial electrical resistance. These data suggest that NGF/TrkA may represent a target for the development of novel, K252a-derived multikinase inhibitors drugs with anti-tumor and angiostatic dual activities.
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Affiliation(s)
- Shimon Lecht
- School of Pharmacy-Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel
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