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Rufaihah AJ, Chen CK, Yap CH, Mattar CNZ. Mending a broken heart: In vitro, in vivo and in silico models of congenital heart disease. Dis Model Mech 2021; 14:dmm047522. [PMID: 33787508 PMCID: PMC8033415 DOI: 10.1242/dmm.047522] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Birth defects contribute to ∼0.3% of global infant mortality in the first month of life, and congenital heart disease (CHD) is the most common birth defect among newborns worldwide. Despite the significant impact on human health, most treatments available for this heterogenous group of disorders are palliative at best. For this reason, the complex process of cardiogenesis, governed by multiple interlinked and dose-dependent pathways, is well investigated. Tissue, animal and, more recently, computerized models of the developing heart have facilitated important discoveries that are helping us to understand the genetic, epigenetic and mechanobiological contributors to CHD aetiology. In this Review, we discuss the strengths and limitations of different models of normal and abnormal cardiogenesis, ranging from single-cell systems and 3D cardiac organoids, to small and large animals and organ-level computational models. These investigative tools have revealed a diversity of pathogenic mechanisms that contribute to CHD, including genetic pathways, epigenetic regulators and shear wall stresses, paving the way for new strategies for screening and non-surgical treatment of CHD. As we discuss in this Review, one of the most-valuable advances in recent years has been the creation of highly personalized platforms with which to study individual diseases in clinically relevant settings.
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Affiliation(s)
- Abdul Jalil Rufaihah
- Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228
| | - Ching Kit Chen
- Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228
- Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228
| | - Choon Hwai Yap
- Division of Cardiology, Department of Paediatrics, Khoo Teck Puat -National University Children's Medical Institute, National University Health System, Singapore 119228
- Department of Bioengineering, Imperial College London, London, UK
| | - Citra N Z Mattar
- Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228
- Department of Obstetrics and Gynaecology, National University Health System, Singapore 119228
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Sgarra L, Bortone AS, Potenza MA, Nacci C, De Salvia MA, Acquaviva T, De Cillis E, Ciccone MM, Grimaldi M, Montagnani M. Endothelial Dysfunction May Link Interatrial Septal Abnormalities and MTHFR-Inherited Defects to Cryptogenic Stroke Predisposition. Biomolecules 2020; 10:861. [PMID: 32512924 PMCID: PMC7355772 DOI: 10.3390/biom10060861] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2020] [Revised: 05/22/2020] [Accepted: 06/02/2020] [Indexed: 12/21/2022] Open
Abstract
We explored the significance of the L-Arginine/asymmetric dimethylarginine (L-Arg/ADMA) ratio as a biomarker of endothelial dysfunction in stroke patients. To this aim, we evaluated the correlation, in terms of severity, between the degree of endothelial dysfunction (by L-Arg/ADMA ratio), the methylene tetrahydrofolate reductase (MTHFR) genotype, and the interatrial septum (IAS) phenotype in subject with a history of stroke. Methods and Results: L-Arg, ADMA, and MTHFR genotypes were evaluated; the IAS phenotype was assessed by transesophageal echocardiography. Patients were grouped according to the severity of IAS defects and the residual enzymatic activity of MTHFR-mutated variants, and values of L-Arg/ADMA ratio were measured in each subgroup. Of 57 patients, 10 had a septum integrum (SI), 38 a patent foramen ovale (PFO), and 9 an ostium secundum (OS). The L-Arg/ADMA ratio differed across septum phenotypes (p ≤ 0.01), and was higher in SI than in PFO or OS patients (p ≤ 0.05, p ≤ 0.01, respectively). In the PFO subgroup a negative correlation was found between the L-Arg/ADMA ratio and PFO tunnel length/height ratio (p ≤ 0.05; r = - 0.37; R2 = 0.14). Interestingly, the L-Arg/ADMA ratio varied across MTHFR genotypes (p ≤ 0.0001) and was lower in subgroups carrying the most impaired enzyme with respect to patients carrying the conservative MTHFR (p ≤ 0.0001, p ≤ 0.05, respectively). Consistently, OS patients carried the most dysfunctional MTHFR genotypes, whereas SI patients the least ones. Conclusions: A low L-Arg/ADMA ratio correlates with impaired activity of MTHFR and with the jeopardized IAS phenotype along a severity spectrum encompassing OS, PFO with long/tight tunnel, PFO with short/large tunnel, and SI. This infers that genetic MTHFR defects may underlie endothelial dysfunction-related IAS abnormalities, and predispose to a cryptogenic stroke. Our findings emphasize the role of the L-Arg/ADMA ratio as a reliable marker of stroke susceptibility in carriers of IAS abnormalities, and suggest its potential use both as a diagnostic tool and as a decision aid for therapy.
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Affiliation(s)
- Luca Sgarra
- Department of Biomedical Sciences and Human Oncology—Section of Pharmacology, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (L.S.); (M.A.P.); (C.N.); (M.A.D.S.)
| | - Alessandro Santo Bortone
- Department of Emergency and Organ Transplantation—Section of Cardiovascular Diseases, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (A.S.B.); (T.A.); (E.D.C.); (M.M.C.)
| | - Maria Assunta Potenza
- Department of Biomedical Sciences and Human Oncology—Section of Pharmacology, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (L.S.); (M.A.P.); (C.N.); (M.A.D.S.)
| | - Carmela Nacci
- Department of Biomedical Sciences and Human Oncology—Section of Pharmacology, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (L.S.); (M.A.P.); (C.N.); (M.A.D.S.)
| | - Maria Antonietta De Salvia
- Department of Biomedical Sciences and Human Oncology—Section of Pharmacology, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (L.S.); (M.A.P.); (C.N.); (M.A.D.S.)
| | - Tommaso Acquaviva
- Department of Emergency and Organ Transplantation—Section of Cardiovascular Diseases, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (A.S.B.); (T.A.); (E.D.C.); (M.M.C.)
| | - Emanuela De Cillis
- Department of Emergency and Organ Transplantation—Section of Cardiovascular Diseases, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (A.S.B.); (T.A.); (E.D.C.); (M.M.C.)
| | - Marco Matteo Ciccone
- Department of Emergency and Organ Transplantation—Section of Cardiovascular Diseases, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (A.S.B.); (T.A.); (E.D.C.); (M.M.C.)
| | - Massimo Grimaldi
- General Hospital “F. Miulli” Acquaviva delle Fonti, 70021 Bari, Italy;
| | - Monica Montagnani
- Department of Biomedical Sciences and Human Oncology—Section of Pharmacology, Medical School, University of Bari “Aldo Moro”, 70124 Bari, Italy; (L.S.); (M.A.P.); (C.N.); (M.A.D.S.)
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Wang Y, Yao F, Wang L, Li Z, Ren Z, Li D, Zhang M, Han L, Wang SQ, Zhou B, Wang L. Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation. Nat Commun 2020; 11:2585. [PMID: 32444791 PMCID: PMC7244751 DOI: 10.1038/s41467-020-16204-w] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Accepted: 04/20/2020] [Indexed: 12/21/2022] Open
Abstract
Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon co-culture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration.
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Affiliation(s)
- Yin Wang
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Fang Yao
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Lipeng Wang
- State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing, 100871, China
| | - Zheng Li
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Zongna Ren
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Dandan Li
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Mingzhi Zhang
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Leng Han
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX, 77030, USA
| | - Shi-Qiang Wang
- State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing, 100871, China
| | - Bingying Zhou
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China
| | - Li Wang
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, China.
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Peters MM, Sampaio-Pinto V, da Costa Martins PA. Non-coding RNAs in endothelial cell signalling and hypoxia during cardiac regeneration. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2020; 1867:118515. [DOI: 10.1016/j.bbamcr.2019.07.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/29/2019] [Revised: 06/19/2019] [Accepted: 07/18/2019] [Indexed: 01/08/2023]
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Engineer A, Saiyin T, Greco ER, Feng Q. Say NO to ROS: Their Roles in Embryonic Heart Development and Pathogenesis of Congenital Heart Defects in Maternal Diabetes. Antioxidants (Basel) 2019; 8:antiox8100436. [PMID: 31581464 PMCID: PMC6826639 DOI: 10.3390/antiox8100436] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 09/09/2019] [Accepted: 09/18/2019] [Indexed: 12/12/2022] Open
Abstract
Congenital heart defects (CHDs) are the most prevalent and serious birth defect, occurring in 1% of all live births. Pregestational maternal diabetes is a known risk factor for the development of CHDs, elevating the risk in the child by more than four-fold. As the prevalence of diabetes rapidly rises among women of childbearing age, there is a need to investigate the mechanisms and potential preventative strategies for these defects. In experimental animal models of pregestational diabetes induced-CHDs, upwards of 50% of offspring display congenital malformations of the heart, including septal, valvular, and outflow tract defects. Specifically, the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) signaling is a major driver of the development of CHDs in offspring of mice with pregestational diabetes. NO from endothelial nitric oxide synthase (eNOS) is crucial to cardiogenesis, regulating various cellular and molecular processes. In fact, deficiency in eNOS results in CHDs and coronary artery malformation. Embryonic hearts from diabetic dams exhibit eNOS uncoupling and oxidative stress. Maternal treatment with sapropterin, a cofactor of eNOS, and antioxidants such as N-acetylcysteine, vitamin E, and glutathione as well as maternal exercise have been shown to improve eNOS function, reduce oxidative stress, and lower the incidence CHDs in the offspring of mice with pregestational diabetes. This review summarizes recent data on pregestational diabetes-induced CHDs, and offers insights into the important roles of NO and ROS in embryonic heart development and pathogenesis of CHDs in maternal diabetes.
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Affiliation(s)
- Anish Engineer
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, ON, N6A 5C1, Canada.
| | - Tana Saiyin
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, ON, N6A 5C1, Canada.
| | - Elizabeth R Greco
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, ON, N6A 5C1, Canada.
| | - Qingping Feng
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, ON, N6A 5C1, Canada.
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Determination of dynamic thiol/disulphide homeostasis in children with tetralogy of Fallot and ventricular septal defect. Cardiol Young 2019; 29:499-504. [PMID: 30932800 DOI: 10.1017/s104795111900012x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Oxidative stress may contribute to the pathogenesis of congenital heart defects, but the role of dynamic thiol/disulphide homeostasis has not been evaluated. The objective of this study was to assess whether there are changes in thiol/disulphide homeostasis and nitric oxide levels in children with tetralogy of Fallot (TOF) and ventricular septal defect (VSD). A total of 47 children with congenital heart defects (24 TOF and 23 VSD) and 47 healthy age- and sex-matched controls were included in this study. Serum total thiol and native thiol levels were measured using a novel automatic spectrophotometric method. The amount of dynamic disulphide bonds and related ratios were calculated from these values. Serum nitric oxide levels were detected using a chemiluminescence assay. We found that the average native thiol, total thiol, and disulphide levels were decreased in patients with VSD when compared with healthy individuals (p < 0.001, p < 0.001, and p < 0.01, respectively). While native thiol levels were decreased (p < 0.01), disulphide levels were elevated in the TOF group (p < 0.05). We observed marked augmentation of disulphide/native thiol (p < 0.001) and disulphide/total thiol ratios (p < 0.01) in the TOF group. However, there was a significant decrease in native thiol/total thiol ratio in patients with TOF. No significant changes in these ratios were noted in the VSD group. We detected significant elevations in serum nitric oxide levels in children with TOF and VSD (p < 0.001 for all). These results are the first to demonstrate that thiol/disulphide homeostasis and nitric oxide are associated with TOF and VSD in children.
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Engineer A, Saiyin T, Lu X, Kucey AS, Urquhart BL, Drysdale TA, Norozi K, Feng Q. Sapropterin Treatment Prevents Congenital Heart Defects Induced by Pregestational Diabetes Mellitus in Mice. J Am Heart Assoc 2018; 7:e009624. [PMID: 30608180 PMCID: PMC6404194 DOI: 10.1161/jaha.118.009624] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/05/2018] [Accepted: 09/17/2018] [Indexed: 01/05/2023]
Abstract
Background Tetrahydrobiopterin is a cofactor of endothelial NO synthase ( eNOS ), which is critical to embryonic heart development. We aimed to study the effects of sapropterin (Kuvan), an orally active synthetic form of tetrahydrobiopterin on eNOS uncoupling and congenital heart defects ( CHD s) induced by pregestational diabetes mellitus in mice. Methods and Results Adult female mice were induced to pregestational diabetes mellitus by streptozotocin and bred with normal male mice to produce offspring. Pregnant mice were treated with sapropterin or vehicle during gestation. CHD s were identified by histological analysis. Cell proliferation, eNOS dimerization, and reactive oxygen species production were assessed in the fetal heart. Pregestational diabetes mellitus results in a spectrum of CHD s in their offspring. Oral treatment with sapropterin in the diabetic dams significantly decreased the incidence of CHD s from 59% to 27%, and major abnormalities, such as atrioventricular septal defect and double-outlet right ventricle, were absent in the sapropterin-treated group. Lineage tracing reveals that pregestational diabetes mellitus results in decreased commitment of second heart field progenitors to the outflow tract, endocardial cushions, and ventricular myocardium of the fetal heart. Notably, decreased cell proliferation and cardiac transcription factor expression induced by maternal diabetes mellitus were normalized with sapropterin treatment. Furthermore, sapropterin administration in the diabetic dams increased eNOS dimerization and lowered reactive oxygen species levels in the fetal heart. Conclusions Sapropterin treatment in the diabetic mothers improves eNOS coupling, increases cell proliferation, and prevents the development of CHD s in the offspring. Thus, sapropterin may have therapeutic potential in preventing CHD s in pregestational diabetes mellitus.
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Affiliation(s)
- Anish Engineer
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
| | - Tana Saiyin
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
| | - Xiangru Lu
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
| | - Andrew S. Kucey
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
| | - Brad L. Urquhart
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
| | - Thomas A. Drysdale
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
- Department of PediatricsSchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
- Children's Health Research InstituteLondonOntarioCanada
| | - Kambiz Norozi
- Department of PediatricsSchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
- Children's Health Research InstituteLondonOntarioCanada
- Department of Paediatric Cardiology and Intensive Care MedicineHannover Medical SchoolHannoverGermany
- Department of Paediatric Cardiology and Intensive Care MedicineUniversity of GöttingenGermany
| | - Qingping Feng
- Department of Physiology and PharmacologySchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
- Department of MedicineSchulich School of Medicine and DentistryUniversity of Western OntarioLondonOntarioCanada
- Children's Health Research InstituteLondonOntarioCanada
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Ogonowski N, Piro G, Pessah D, Arreche N, Puchulu B, Balaszczuk AM, Fellet AL. Thyroid disorders and nitric oxide in cardiovascular adaptation to hypovolemia. J Endocrinol 2016; 230:185-95. [PMID: 27270898 DOI: 10.1530/joe-16-0203] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/09/2016] [Accepted: 06/03/2016] [Indexed: 11/08/2022]
Abstract
This study aimed to investigate whether nitric oxide participates in the cardiovascular function and haemodynamic adaptation to acute haemorrhage in animals with thyroid disorders. Sprague-Dawley rats aged 2months old treated with T3 (hyper, 20μg/100g body weight) or 0.02% methimazole (hypo, w/v) during 28days were pre-treated with N(G) nitro-l-arginine methyl ester (L-NAME) and submitted to 20% blood loss. Heart function was evaluated by echocardiography. Measurements of arterial blood pressure, heart rate, nitric oxide synthase activity and protein levels were performed. We found that hypo decreased fractional shortening and ejection fraction and increased left ventricle internal diameter. Hyper decreased ventricle diameter and no changes in cardiac contractility. Haemorrhage elicited a hypotension of similar magnitude within 10min. Then, this parameter was stabilized at about 30-40min and maintained until finalized, 120min. L-NAME rats showed that the immediate hypotension would be independent of nitric oxide. Nitric oxide synthase inhibition blunted the changes of heart rate induced by blood loss. Hyper and hypo had lower atrial enzyme activity associated with a decreased enzyme isoform in hypo. In ventricle, hyper and hypo had a higher enzyme activity, which was not correlated with changes in protein levels. Haemorrhage induced an increased heart nitric oxide production. We concluded that thyroid disorders were associated with hypertrophic remodelling which impacted differently on cardiac function and its adaptation to a hypovolemia. Hypovolemia triggered a nitric oxide synthase activation modulating the heart function to maintain haemodynamic homeostasis. This involvement depends on a specific enzyme isoform, cardiac chamber and thyroid state.
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Affiliation(s)
- Natalia Ogonowski
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Giselle Piro
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Déborah Pessah
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Noelia Arreche
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Bernardita Puchulu
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Ana M Balaszczuk
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Andrea L Fellet
- Department of PhysiologySchool of Pharmacy and Biochemistry, IQUIMEFA-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
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England J, Pang KL, Parnall M, Haig MI, Loughna S. Cardiac troponin T is necessary for normal development in the embryonic chick heart. J Anat 2016; 229:436-49. [PMID: 27194630 PMCID: PMC4974548 DOI: 10.1111/joa.12486] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/17/2016] [Indexed: 12/30/2022] Open
Abstract
The heart is the first functioning organ to develop during embryogenesis. The formation of the heart is a tightly regulated and complex process, and alterations to its development can result in congenital heart defects. Mutations in sarcomeric proteins, such as alpha myosin heavy chain and cardiac alpha actin, have now been associated with congenital heart defects in humans, often with atrial septal defects. However, cardiac troponin T (cTNT encoded by gene TNNT2) has not. Using gene‐specific antisense oligonucleotides, we have investigated the role of cTNT in chick cardiogenesis. TNNT2 is expressed throughout heart development and in the postnatal heart. TNNT2‐morpholino treatment resulted in abnormal atrial septal growth and a reduction in the number of trabeculae in the developing primitive ventricular chamber. External analysis revealed the development of diverticula from the ventricular myocardial wall which showed no evidence of fibrosis and still retained a myocardial phenotype. Sarcomeric assembly appeared normal in these treated hearts. In humans, congenital ventricular diverticulum is a rare condition, which has not yet been genetically associated. However, abnormal haemodynamics is known to cause structural defects in the heart. Further, structural defects, including atrial septal defects and congenital diverticula, have previously been associated with conduction anomalies. Therefore, to provide mechanistic insights into the effect that cTNT knockdown has on the developing heart, quantitative PCR was performed to determine the expression of the shear stress responsive gene NOS3 and the conduction gene TBX3. Both genes were differentially expressed compared to controls. Therefore, a reduction in cTNT in the developing heart results in abnormal atrial septal formation and aberrant ventricular morphogenesis. We hypothesize that alterations to the haemodynamics, indicated by differential NOS3 expression, causes these abnormalities in growth in cTNT knockdown hearts. In addition, the muscular diverticula reported here suggest a novel role for mutations of structural sarcomeric proteins in the pathogenesis of congenital cardiac diverticula. From these studies, we suggest TNNT2 is a gene worthy of screening for those with a congenital heart defect, particularly atrial septal defects and ventricular diverticula.
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Affiliation(s)
- Jennifer England
- School of Life Sciences, Medical School, University of Nottingham, Nottingham, UK
| | - Kar Lai Pang
- School of Life Sciences, Medical School, University of Nottingham, Nottingham, UK
| | - Matthew Parnall
- School of Life Sciences, Medical School, University of Nottingham, Nottingham, UK
| | - Maria Isabel Haig
- School of Life Sciences, Medical School, University of Nottingham, Nottingham, UK
| | - Siobhan Loughna
- School of Life Sciences, Medical School, University of Nottingham, Nottingham, UK
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Roberts MA, Tran D, Coulombe KL, Razumova M, Regnier M, Murry CE, Zheng Y. Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue. Tissue Eng Part A 2016; 22:633-44. [PMID: 26955856 PMCID: PMC4840925 DOI: 10.1089/ten.tea.2015.0482] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2015] [Accepted: 02/29/2016] [Indexed: 01/08/2023] Open
Abstract
Cardiac tissue engineering is a strategy to replace damaged contractile tissue and model cardiac diseases to discover therapies. Current cardiac and vascular engineering approaches independently create aligned contractile tissue or perfusable vasculature, but a combined vascularized cardiac tissue remains to be achieved. Here, we sought to incorporate a patterned microvasculature into engineered heart tissue, which balances the competing demands from cardiomyocytes to contract the matrix versus the vascular lumens that need structural support. Low-density collagen hydrogels (1.25 mg/mL) permit human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to form a dense contractile tissue but cannot support a patterned microvasculature. Conversely, high collagen concentrations (density ≥6 mg/mL) support a patterned microvasculature, but the hESC-CMs lack cell-cell contact, limiting their electrical communication, structural maturation, and tissue-level contractile function. When cocultured with matrix remodeling stromal cells, however, hESC-CMs structurally mature and form anisotropic constructs in high-density collagen. Remodeling requires the stromal cells to be in proximity with hESC-CMs. In addition, cocultured cardiac constructs in dense collagen generate measurable active contractions (on the order of 0.1 mN/mm(2)) and can be paced up to 2 Hz. Patterned microvascular networks in these high-density cocultured cardiac constructs remain patent through 2 weeks of culture, and hESC-CMs show electrical synchronization. The ability to maintain microstructural control within engineered heart tissue enables generation of more complex features, such as cellular alignment and a vasculature. Successful incorporation of these features paves the way for the use of large scale engineered tissues for myocardial regeneration and cardiac disease modeling.
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Affiliation(s)
- Meredith A. Roberts
- Department of Bioengineering, University of Washington, Seattle, Washington
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington
| | - Dominic Tran
- Department of Bioengineering, University of Washington, Seattle, Washington
| | - Kareen L.K. Coulombe
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington
- Department of Pathology, University of Washington, Seattle, Washington
| | - Maria Razumova
- Department of Bioengineering, University of Washington, Seattle, Washington
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington
| | - Michael Regnier
- Department of Bioengineering, University of Washington, Seattle, Washington
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington
| | - Charles E. Murry
- Department of Bioengineering, University of Washington, Seattle, Washington
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington
- Department of Pathology, University of Washington, Seattle, Washington
- Department of Medicine/Cardiology, University of Washington, Seattle, Washington
| | - Ying Zheng
- Department of Bioengineering, University of Washington, Seattle, Washington
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington
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11
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Wang W, Lee Y, Lee CH. Effects of nitric oxide on stem cell therapy. Biotechnol Adv 2015; 33:1685-96. [PMID: 26394194 DOI: 10.1016/j.biotechadv.2015.09.004] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Revised: 09/14/2015] [Accepted: 09/18/2015] [Indexed: 12/27/2022]
Abstract
The use of stem cells as a research tool and a therapeutic vehicle has demonstrated their great potential in the treatment of various diseases. With unveiling of nitric oxide synthase (NOS) universally present at various levels in nearly all types of body tissues, the potential therapeutic implication of nitric oxide (NO) has been magnified, and thus scientists have explored new treatment strategies involved with stem cells and NO against various diseases. As the functionality of NO encompasses cardiovascular, neuronal and immune systems, NO is involved in stem cell differentiation, epigenetic regulation and immune suppression. Stem cells trigger cellular responses to external signals on the basis of both NO specific pathways and concerted action with endogenous compounds including stem cell regulators. As potency and interaction of NO with stem cells generally depend on the concentrations of NO and the presence of the cofactors at the active site, the suitable carriers for NO delivery is integral for exerting maximal efficacy of stem cells. The innovative utilization of NO functionality and involved mechanisms would invariably alter the paradigm of therapeutic application of stem cells. Future prospects in NO-involved stem cell research which promises to enhance drug discovery efforts by opening new era to improve drug efficacy, reduce drug toxicity and understand disease mechanisms and pathways, were also addressed.
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Affiliation(s)
- Wuchen Wang
- School of Pharmacy University of Missouri, Kansas City, USA
| | - Yugyung Lee
- School of Computing and Engineering, University of Missouri, Kansas City, USA
| | - Chi H Lee
- School of Pharmacy University of Missouri, Kansas City, USA.
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12
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Campbell KA, Li X, Biendarra SM, Terzic A, Nelson TJ. Nos3-/- iPSCs model concordant signatures of in utero cardiac pathogenesis. J Mol Cell Cardiol 2015; 87:228-36. [PMID: 26344701 DOI: 10.1016/j.yjmcc.2015.08.021] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Revised: 08/28/2015] [Accepted: 08/29/2015] [Indexed: 01/17/2023]
Abstract
BACKGROUND Through genome-wide transcriptional comparisons, this study interrogates the capacity of in vitro differentiation of induced pluripotent stem cells (iPSCs) to accurately model pathogenic signatures of developmental cardiac defects. METHODS AND RESULTS Herein, we studied the molecular etiology of cardiac defects in Nos3(-/-) mice via transcriptional analysis of stage-matched embryonic tissues and iPSC-derived cells. In vitro comparisons of differentiated cells were calibrated to in utero benchmarks of health and disease. Integrated systems biology analysis of WT and Nos3(-/-) transcriptional profiles revealed 50% concordant expression patterns between in utero embryonic tissues and ex vivo iPSC-derived cells. In particular, up-regulation of glucose metabolism (p-value=3.95×10(-12)) and down-regulation of fatty acid metabolism (p-value=6.71×10(-12)) highlight a bioenergetic signature of early Nos3 deficiency during cardiogenesis that can be recapitulated in iPSC-derived differentiated cells. CONCLUSIONS The in vitro concordance of early Nos3(-/-) disease signatures supports the utility of iPSCs as a cellular model of developmental heart defects. Moreover, this study supports the use of iPSCs as a platform to pinpoint initial stages of congenital cardiac pathogenesis.
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Affiliation(s)
- Katherine A Campbell
- Department of Molecular Pharmacology and Experimental Therapeutics, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Center for Regenerative Medicine, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Division of General Internal Medicine, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA
| | - Xing Li
- Department of Health Sciences Research, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Division of Biomedical Statistics and Informatics, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA
| | - Sherri M Biendarra
- Department of Molecular Pharmacology and Experimental Therapeutics, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA
| | - Andre Terzic
- Department of Molecular Pharmacology and Experimental Therapeutics, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Center for Regenerative Medicine, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Division of Cardiovascular Diseases, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Department of Medical Genetics, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA
| | - Timothy J Nelson
- Department of Molecular Pharmacology and Experimental Therapeutics, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Center for Regenerative Medicine, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Division of Cardiovascular Diseases, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Division of General Internal Medicine, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Center for Transplantation and Clinical Regeneration, 200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA; Division of Pediatric Cardiology,200 First Street SW, Mayo Clinic, Rochester, MN 55905, USA.
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13
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Beltran-Povea A, Caballano-Infantes E, Salguero-Aranda C, Martín F, Soria B, Bedoya FJ, Tejedo JR, Cahuana GM. Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells. World J Stem Cells 2015; 7:605-617. [PMID: 25914767 PMCID: PMC4404395 DOI: 10.4252/wjsc.v7.i3.605] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2014] [Revised: 11/13/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide (NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemness.
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14
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Netti VA, Iovane AN, Vatrella MC, Magnani ND, Evelson PA, Zotta E, Fellet AL, Balaszczuk AM. Dehydration affects cardiovascular nitric oxide synthases and caveolins in growing rats. Eur J Nutr 2014; 55:33-43. [DOI: 10.1007/s00394-014-0820-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2014] [Accepted: 12/10/2014] [Indexed: 01/08/2023]
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15
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Zhou L, Zang G, Zhang G, Wang H, Zhang X, Johnston N, Min W, Luke P, Jevnikar A, Haig A, Zheng X. MicroRNA and mRNA signatures in ischemia reperfusion injury in heart transplantation. PLoS One 2013; 8:e79805. [PMID: 24278182 PMCID: PMC3835872 DOI: 10.1371/journal.pone.0079805] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2013] [Accepted: 10/04/2013] [Indexed: 11/19/2022] Open
Abstract
Ischemia reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation, leading to graft failures and lower long-term survival rate of the recipient. Several studies have demonstrated that microRNAs (miRNAs) are vital regulators of signalling pathways involved in I/R injury. The present study aims to quantify the altered expression levels of miRNA and mRNA upon I/R injury in a mouse heart transplantation model, and to investigate whether these miRNA can regulate genes involved in I/R injury. We performed heterotopic heart transplantation on mouse models to generate heart tissue samples with I/R and non-I/R (control). The expression levels of miRNAs as well as genes were measured in heart grafts by microarray and real time RT-PCR. miRNA alteration in cardiomyocytes exposed to hypoxia was also detected by qRT-PCR. We observed significant alterations in miRNA and gene expression profile after I/R injury. There were 39 miRNAs significantly downregulated and 20 upregulated up to 1.5 fold in heart grafts with I/R injury compared with the grafts without I/R. 48 genes were observed with 3 fold change and p<0.05 and 18 signalling pathways were enriched using Keggs pathway library. Additionally, hypoxia/reperfusion induced primary cardiomyocyte apoptosis and altered miRNA expression profiles. In conclusion, this is the first report on miRNA expression profile for heart transplantation associated with I/R injury. These findings provide us with an insight into the role of miRNA in I/R injury in heart transplantation.
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Affiliation(s)
- Liangyi Zhou
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
| | - Guoyao Zang
- Sir Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
| | - Guangfeng Zhang
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
| | - Hansong Wang
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
| | - Xusheng Zhang
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
| | - Nathan Johnston
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
| | - Weiping Min
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
- Lawson Health Research Institute, London Ontario, Canada
- Multiple Organ Transplant Program, London Ontario, Canada
| | - Patrick Luke
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
- Multiple Organ Transplant Program, London Ontario, Canada
| | - Anthony Jevnikar
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
- Lawson Health Research Institute, London Ontario, Canada
- Multiple Organ Transplant Program, London Ontario, Canada
| | - Aaron Haig
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
| | - Xiufen Zheng
- Department of Pathology, Surgery, Medicine, and Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
- Lawson Health Research Institute, London Ontario, Canada
- * E-mail:
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16
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Haplotypes of NOS3 gene polymorphisms in dilated cardiomyopathy. PLoS One 2013; 8:e70523. [PMID: 23923002 PMCID: PMC3726655 DOI: 10.1371/journal.pone.0070523] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2012] [Accepted: 06/19/2013] [Indexed: 11/19/2022] Open
Abstract
Dilated Cardiomyopathy (DCM) is characterized by systolic dysfunction, followed by heart failure necessitating cardiac transplantation. The genetic basis is well established by the identification of mutations in sarcomere and cytoskeleton gene/s. Modifier genes and environmental factors are also considered to play a significant role in the variable expression of the disease, hence various mechanisms are implicated and one such mechanism is oxidative stress. Nitric Oxide (NO), a primary physiological transmitter derived from endothelium seems to play a composite role with diverse anti-atherogenic effects as vasodilator. Three functional polymorphisms of endothelial nitric oxide synthase (NOS3) gene viz., T-786C of the 5′ flanking region, 27bp VNTR in intron4 and G894T of exon 7 were genotyped to identify their role in DCM. A total of 115 DCM samples and 454 controls were included. Genotyping was carried out by PCR -RFLP method. Allelic and genotypic frequencies were computed in both control & patient groups and appropriate statistical tests were employed. A significant association of TC genotype (T-786C) with an odds ratio of 1.74, (95% CI 1.14 - 2.67, p = 0.01) was observed in DCM. Likewise the GT genotypic frequency of G894T polymorphism was found to be statistically significant (OR 2.10, 95% CI 1.34–3.27, p = 0.0011), with the recessive allele T being significantly associated with DCM (OR 1.64, 95% CI 1.18 - 2.30, p = 0.003). The haplotype carrying the recessive alleles of G894T and T-786C, C4bT was found to exhibit 7 folds increased risk for DCM compared to the controls. Hence C4bT haplotype could be the risk haplotype for DCM. Our findings suggest the possible implication of NOS3 gene in the disease phenotype, wherein NOS3 may be synergistically functioning in DCM associated heart failure via the excessive production of NO in cardiomyocytes resulting in decreased myocardial contractility and systolic dysfunction, a common feature of DCM phenotype.
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17
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Napoli C, Paolisso G, Casamassimi A, Al-Omran M, Barbieri M, Sommese L, Infante T, Ignarro LJ. Effects of nitric oxide on cell proliferation: novel insights. J Am Coll Cardiol 2013; 62:89-95. [PMID: 23665095 DOI: 10.1016/j.jacc.2013.03.070] [Citation(s) in RCA: 214] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/15/2013] [Accepted: 03/19/2013] [Indexed: 12/13/2022]
Abstract
Nitric oxide (NO) has been suggested to be a pathophysiological modulator of cell proliferation, cell cycle arrest, and apoptosis. In this context, NO can exert opposite effects under diverse conditions. Indeed, several studies have indicated that low relative concentrations of NO seem to favor cell proliferation and antiapoptotic responses and higher levels of NO favor pathways inducing cell cycle arrest, mitochondria respiration, senescence, or apoptosis. Here we report the effects of NO on both promotion and inhibition of cell proliferation, in particular in regard to cardiovascular disease, diabetes, and stem cells. Moreover, we focus on molecular mechanisms of action involved in the control of cell cycle progression, which include both cyclic guanosine monophosphate-dependent and -independent pathways. This growing field may lead to broad and novel targeted therapies against cardiovascular diseases, especially concomitant type 2 diabetes, as well as novel bioimaging NO-based diagnostic tools.
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Affiliation(s)
- Claudio Napoli
- Department of General Pathology, Excellence Research Centre on Cardiovascular Diseases, U.O.C. Immunohematology, Second University of Naples, Naples, Italy; Fondazione SDN, IRCCS, Naples, Italy.
| | - Giuseppe Paolisso
- Division of Geriatrics, 1st School of Medicine, Second University of Naples, Naples, Italy
| | - Amelia Casamassimi
- Department of General Pathology, Excellence Research Centre on Cardiovascular Diseases, U.O.C. Immunohematology, Second University of Naples, Naples, Italy
| | - Mohammed Al-Omran
- College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia
| | - Michelangela Barbieri
- Division of Geriatrics, 1st School of Medicine, Second University of Naples, Naples, Italy
| | - Linda Sommese
- Department of General Pathology, Excellence Research Centre on Cardiovascular Diseases, U.O.C. Immunohematology, Second University of Naples, Naples, Italy
| | | | - Louis J Ignarro
- Department of Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California
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18
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NOing the heart: role of nitric oxide synthase-3 in heart development. Differentiation 2012; 84:54-61. [PMID: 22579300 DOI: 10.1016/j.diff.2012.04.004] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2012] [Revised: 04/03/2012] [Accepted: 04/10/2012] [Indexed: 01/30/2023]
Abstract
Congenital heart disease is the most common birth defect in humans. Identifying factors that are critical to embryonic heart development could further our understanding of the disease and lead to new strategies of its prevention and treatment. Nitric oxide synthase-3 (NOS3) or endothelial nitric oxide synthase (eNOS) is known for many important biological functions including vasodilation, vascular homeostasis and angiogenesis. Over the past decade, studies from our lab and others have shown that NOS3 is required during heart development. More specifically, deficiency in NOS3 results in congenital septal defects, cardiac hypertrophy and postnatal heart failure. In addition, NOS3 is pivotal to the morphogenesis of major coronary arteries and myocardial capillary development. Interestingly, these effects of NOS3 are mediated through induction of transcription and growth factors that are crucial in the formation of coronary arteries. Finally, deficiency in NOS3 results in high incidences of bicuspid aortic valves, a disease in humans that often leads to complications with age including aortic valve stenosis or regurgitation, endocarditis, aortic aneurysm formation, and aortic dissection. In summary, these data suggest NOS3 plays a critical role in embryonic heart development and morphogenesis of coronary arteries and aortic valves.
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19
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Jiang R, Wang S, Takahashi K, Fujita H, Fruci CR, Breyer MD, Harris RC, Takahashi T. Generation of a conditional allele for the mouse endothelial nitric oxide synthase gene. Genesis 2012; 50:685-92. [PMID: 22467476 DOI: 10.1002/dvg.22026] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2011] [Revised: 03/16/2012] [Accepted: 03/18/2012] [Indexed: 01/21/2023]
Abstract
Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here, we have generated a floxed eNOS allele in which exons 9-12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre-deleted alleles displayed truncated eNOS protein, lack of vascular NO production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scarring. These findings demonstrate that the floxed allele is normal and it can be converted to a non-functional eNOS allele through Cre recombination. This mouse will allow time- and cell-specific eNOS deletion.
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Affiliation(s)
- Rosie Jiang
- Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
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20
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Lv L, Jiang SS, Xu J, Gong JB, Cheng Y. Protective effect of ligustrazine against myocardial ischaemia reperfusion in rats: the role of endothelial nitric oxide synthase. Clin Exp Pharmacol Physiol 2012; 39:20-7. [PMID: 22004361 DOI: 10.1111/j.1440-1681.2011.05628.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
1. The aim of the present study was to determine whether ligustrazine (2,3,5,6-tetramethylpyrazine; TMP) exerts a cardioprotective effect during myocardial ischaemia reperfusion (IR), and to investigate the underlying mechanisms and the role of endothelial nitric oxide synthase (eNOS) in cardioprotection. 2. Sprague-Dawley rats were divided into a sham group and five IR groups: IR control, TMP pretreated, TMP + wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor), N(G) -nitro-L-arginine methyl ester (L-NAME; a NOS inhibitor) and TMP + L-NAME. IR was produced by 35 min of regional ischaemia followed by 120 min of reperfusion. Myocardial infarct size, oxidative stress, myocardial apoptosis, nitric oxide (NO) production, and expression of phosphorylated protein kinase B (Akt) and eNOS were measured. 3. TMP markedly decreased infarct size and attenuated myocardial apoptosis, as evidenced by a decrease in the apoptotic index and reduced caspase-3 activity. TMP treatment caused a marked increase in NO production. Cotreatment with wortmannin or L-NAME completely blocked the TMP-induced NO increase. TMP induced phosphorylation of Akt at Ser 473 (1.61 ± 0.18 vs 0.79 ± 0.10 in the IR control group) and phosphorylation of eNOS at Ser1177 (1.87 ± 0.33 vs 0.94 ± 0.22 in the IR control group). Wortmannin abrogated the phosphorylation of Akt and eNOS induced by TMP. 4. These data suggest that ligustrazine has anti-apoptotic and cardioprotective effects against myocardial IR injury and that it acts through the PI3K/Akt pathway. In addition, the phosphorylation of eNOS with subsequent NO production was found to be an important downstream effector that contributes significantly to the cardioprotective effect of TMP.
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Affiliation(s)
- Lei Lv
- Department of Cardiology, School of Medicine, Nanjing University, Jinling Hospital, Nanjing, Jiangsu, China
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Zhu H, Yang Y, Wang Y, Li J, Schiller PW, Peng T. MicroRNA-195 promotes palmitate-induced apoptosis in cardiomyocytes by down-regulating Sirt1. Cardiovasc Res 2011; 92:75-84. [PMID: 21622680 DOI: 10.1093/cvr/cvr145] [Citation(s) in RCA: 154] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
AIMS Free fatty acids induce apoptosis in cardiomyocytes, which is implicated in lipotoxic cardiomyopathy. However, the underlying mechanisms remain not fully understood. MicroRNAs (miRNAs) are non-coding small RNAs that control gene expression at the post-transcriptional level. Dysregulated miRNAs have been shown to be involved in heart diseases. This study was to examine whether miR-195 regulates palmitate-induced cardiomyocyte apoptosis by targeting Sirt1, a known anti-apoptotic protein. METHODS AND RESULTS In cultured neonatal mouse cardiomyocytes, palmitate up-regulated miR-195 expression, increased reactive oxygen species (ROS) production, and induced apoptosis as determined by up-regulation of caspase-3 activity and DNA fragmentation. Inhibition of miR-195 decreased ROS production and apoptosis in palmitate-stimulated cardiomyocytes. In contrast, a miR-195 mimic enhanced palmitate-induced ROS production and apoptosis. The induction of miR-195 correlated with a reduction in Sirt1 and Bcl-2. We further showed that miR-195 targeted and inhibited Sirt1 expression through two target sites located in the 3' un-translational region of Sirt1 mRNA. In concordance, inhibition of miR-195 increased Sirt1 protein in cardiomyocytes whereas the miR-195 mimic reduced it. Activation of Sirt1 or overexpression of Bcl-2 inhibited palmitate-induced apoptosis. On the other hand, inhibition of Sirt1 enhanced apoptosis. The inhibitory effect of Sirt1 on apoptosis was associated with a reduction in ROS. CONCLUSIONS This study demonstrates a pro-apoptotic role of miR-195 in cardiomyocytes and identifies Sirt1 as a direct target of miR-195. The effect of miR-195 on apoptosis is mediated through down-regulation of Sirt1 and Bcl-2 and ROS production. Thus, miR-195 may be a new therapeutic target for lipotoxic cardiomyopathy.
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Affiliation(s)
- Huaqing Zhu
- Critical Illness Research, Lawson Health Research Institute, VRL 6th Floor, A6-140, 800 Commissioners Road, London, Ontario, Canada N6A 4G5
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Xaymardan M, Cimini M, Fazel S, Weisel RD, Lu WY, Martin U, Harvey RP, Li RK. c-Kit function is necessary for in vitro myogenic differentiation of bone marrow hematopoietic cells. Stem Cells 2010; 27:1911-20. [PMID: 19544423 DOI: 10.1002/stem.106] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
In recent years, the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated, but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs, and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor, Gleevec (imatinib mesylate; Novartis International, Basel, Switzerland, http://www.novartis.com), to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5, sarcomeric proteins beta-MHC and MLC-2v, and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy, the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating, myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters.
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Affiliation(s)
- Munira Xaymardan
- Division of Cardiovascular Surgery, Toronto General Research Institute, University of Toronto, Toronto, Ontario, Canada
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Hammoud L, Burger DE, Lu X, Feng Q. Tissue inhibitor of metalloproteinase-3 inhibits neonatal mouse cardiomyocyte proliferation via EGFR/JNK/SP-1 signaling. Am J Physiol Cell Physiol 2009; 296:C735-45. [DOI: 10.1152/ajpcell.00246.2008] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We have recently demonstrated that tissue inhibitor of metalloproteinase-3 (TIMP-3) decreases neonatal cardiomyocyte proliferation (Hammoud L, Xiang F, Lu X, Brunner F, Leco K, Feng Q. Cardiovasc Res 75: 359–368, 2007). The aim of the present study was to delineate a pathway through which TIMP-3 exerts its antiproliferative effect. Experiments were conducted on neonatal cardiomyocyte cultures and heart tissues isolated from wild-type (WT) and TIMP-3−/− mice. Deficiency in TIMP-3 decreased p27 expression and increased cardiomyocyte proliferation in cardiomyocytes and neonatal hearts. A TIMP-3/epidermal growth factor (EGF) receptor (EGFR)/c-Jun NH2-terminal kinase (JNK)/SP-1/p27 pathway was investigated. JNK phosphorylation and EGFR protein levels were increased in TIMP-3−/− cardiomyocytes and heart tissues. Treatment with recombinant TIMP-3 decreased JNK phosphorylation and EGFR expression/phosphorylation. Inhibition of JNK activity using SP-600125 decreased SP-1 phosphorylation, increased p27 expression, and decreased cardiomyocyte proliferation. Furthermore, treatment with the EGFR specific inhibitor PD-168393 or the EGF-neutralizing antibody decreased cardiomyocyte proliferation as well as phosphorylation of JNK and SP-1 in both WT and TIMP-3−/− cardiomyocytes. We conclude that TIMP-3 inhibits neonatal mouse cardiomyocyte proliferation by upregulating p27 expression. The effects of TIMP-3 are mediated via inhibition of EGFR expression/phosphorylation, and decreases in JNK and SP-1 signaling.
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Vascular endothelial growth factor, left ventricular dysfunction and mortality in hemodialysis patients. J Hypertens 2008; 26:1875-82. [PMID: 18698224 DOI: 10.1097/hjh.0b013e328307c3d2] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
OBJECTIVES Vascular endothelial growth factor induces nitric oxide-dependent angiogenic effects and participates in the inflammatory response. This cytokine is over-expressed in the myocardium in experimental models of pressure overload and renal mass ablation, and vascular endothelial growth factor is increased in end-stage renal disease. We investigated the relationship between vascular endothelial growth factor, left ventricular function (by midwall fractional shortening) and mortality in a prospective cohort study in 228 hemodialysis patients. RESULTS Serum vascular endothelial growth factor concentration was associated directly with interleukin-6 and tumor necrosis factor-alpha (P < 0.01) and inversely with albumin (P = 0.007) but was independent of the endogenous inhibitor of nitric oxide synthesis, asymmetric dimethylarginine. Vascular endothelial growth factor was inversely related with midwall fractional shortening (P = 0.002) and predicted mortality (P = 0.02). In multivariate analyses testing the involvement of this angiogenic cytokine in left ventricular dysfunction and death, these links remained substantially unmodified after adjustment for Framingham risk factors, risk factors peculiar to end-stage renal disease (Hb, Ca, P) and previous cardiovascular complications. However, these links became weaker and not significant when biomarkers of inflammation and asymmetric dimethylarginine were sequentially introduced into the multivariate models. In crude and adjusted analyses, left ventricular function was lowest in patients who displayed both high vascular endothelial growth factor and high asymmetric dimethylarginine, intermediate in patients with either high vascular endothelial growth factor or high asymmetric dimethylarginine and highest in those with low asymmetric dimethylarginine and low vascular endothelial growth factor (P = 0.001). CONCLUSION Vascular endothelial growth factor is associated with left ventricular systolic dysfunction and mortality in hemodialysis patients. Vascular endothelial growth factor appears to be in the pathway whereby inflammation and nitric oxide inhibition lead to cardiomyopathy and death in hemodialysis patients.
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Ionova IA, Vásquez-Vivar J, Whitsett J, Herrnreiter A, Medhora M, Cooley BC, Pieper GM. Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes. Am J Physiol Heart Circ Physiol 2008; 295:H2178-87. [PMID: 18835915 PMCID: PMC2614582 DOI: 10.1152/ajpheart.00748.2008] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2008] [Accepted: 09/25/2008] [Indexed: 12/25/2022]
Abstract
Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.
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Affiliation(s)
- Irina A Ionova
- Department of Surgery (Transplant Surgery), Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA
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Pimiento JM, Maloney SP, Tang PCY, Muto A, Westvik TS, Fitzgerald TN, Fancher TT, Tellides G, Dardik A. Endothelial nitric oxide synthase stimulates aneurysm growth in aged mice. J Vasc Res 2008; 45:251-8. [PMID: 18182824 DOI: 10.1159/000112940] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2007] [Accepted: 10/20/2007] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND/AIMS Age-associated changes in endothelial nitric oxide synthase (eNOS) expression have not been definitively linked to the pathophysiology of aortic aneurysms. We examined the role of eNOS in human patients and an age-appropriate mouse model. METHODS eNOS transcripts and immunodetectable protein were assessed by quantitative PCR and immunohistochemistry in human ascending thoracic aneurysms (n = 29) and referent aortae (n = 31). Carotid aneurysms were induced with CaCl2 in young adult (3 months) and aged (18 months) C57BL/6 and eNOS-knockout (eNOS-KO) mice. RESULTS eNOS transcripts and protein were reduced in human aneurysms compared with controls, although aortic eNOS expression also decreased with patient age. Aged wild-type mice had significantly larger aneurysm diameter than young adult mice. Aged wild-type mice had reduced eNOS transcripts and protein compared with young adult mice. Aged eNOS-KO mice had smaller aneurysms compared with aged wild-type mice but similar size aneurysms compared with young eNOS-KO and young wild-type mice. CONCLUSION eNOS expression is reduced in both aged human and aged mouse endothelium and eNOS expression is linked to aneurysm expansion in aged but not young adult mice. These findings support the relevance of age-associated changes in eNOS expression in clinical aneurysmal disease.
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Affiliation(s)
- Jose M Pimiento
- Department of Surgery, Yale University School of Medicine, New Haven, Conn 06520-8089, USA
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Lucitti JL, Jones EAV, Huang C, Chen J, Fraser SE, Dickinson ME. Vascular remodeling of the mouse yolk sac requires hemodynamic force. Development 2007; 134:3317-26. [PMID: 17720695 PMCID: PMC4260474 DOI: 10.1242/dev.02883] [Citation(s) in RCA: 374] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a(-/-)). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.
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Affiliation(s)
- Jennifer L. Lucitti
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Elizabeth A. V. Jones
- Department of Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Chengqun Huang
- Department of Medicine, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0641, USA
| | - Ju Chen
- Department of Medicine, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0641, USA
| | - Scott E. Fraser
- Biological Imaging Center, Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA
| | - Mary E. Dickinson
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
- Biological Imaging Center, Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA
- Author for correspondence ()
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