Role In Cardiovascular Development Research Articles

AJAP1 Regulates Human Epithelial Cell Behaviors Required for Epicardium and EPDC Formation During Cardiac Development

Introduction: The epicardium forms the outermost layer of the heart. It is composed of a single layer of specialized epithelial cells that make important cellular contributions to cardiovascular development. During embryonic development, the epicardium is a source of multipotent progenitor cells, growth factors and extracellular matrix (ECM) components. The epicardium gives rise to multipotent epicardial-derived cells (EPDCs) which undergo epithelial-to-mesenchymal transition and differentiate into non-myocyte cardiac lineages. It is also a source of paracrine signals essential for myocardial growth and coronary vessel formation that become dormant after gestation. Adult epicardial cells can be reactivated in response to myocardial injury or ischemia to express a fetal gene program for regenerative cardiac repair. The drive to harness epicardial cells for cardiac regeneration is tethered to its roles in cardiovascular development. Thus, it is necessary to identify molecules that regulate epicardial cell behaviors required for epicardium formation and EPDC differentiation. Using our Tbx5-deficient mouse model of defective epicardium formation ( Tbx5epi−/−), we identified a transcript that is novel to the heart. Adherens junction associated protein 1 is a cell adhesion molecule that is encoded by the AJAP1 transcript which was downregulated in Tbx5epi−/− mice with impaired epicardium formation. Our preliminary studies revealed that AJAP is expressed in epithelial cells of the epicardium during heart development. Silencing of AJAP1 in primary cultured human epithelial cells reduces their ability to migrate. A role for AJAP1 in the epicardium during cardiac development remains to be identified. Study Objective: This study was designed to investigate the impact of AJAP1 on epithelial cell functions that are required for formation of the epicardium and differentiation of EPDCs. Methods: Studies were conducted in a primary human epithelial cell line, HMEpiC, which served as an in vitro model system to examine the contributions of AJAP1 to epithelial cell behaviors. HMEpiCs were transfected with small interfering RNAs (siRNAs) targeting either human AJAP1 to knock down its endogenous expression or a non-targeting negative control siRNA. AJAP1 knockdown was confirmed by quantitative PCR and immunofluorescence. We conducted assays on control and AJAP1 knockdown (KD) cells to examine the effects on epithelial cell behaviors contributing to epicardium formation and EPDC differentiation. This included analysis of cell proliferation, apoptosis, and adhesion to extracellular matrices. Results: Endogenous AJAP1 mRNA expression was reduced significantly by siRNA-mediated knockdown in human epithelial cells. We observed changes in cell morphology that correlated with a significant increase in apoptosis in AJAP1 KD cells compared to negative control cells. Proliferation of HMEpiCs was unchanged by AJAP1 knockdown. Also, we observed a significant increase in the adhesion of AJAP1 KD cells to certain extracellular matrices versus control cells. Conclusion: These findings provide evidence for the AJAP1 cell adhesion molecule to regulate epithelial cell behaviors that are crucial to formation of the epicardium and differentiation of EPDCs during cardiac development. PCOM Center for Chronic Disorders of Aging; PCOM Division of Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TBX5 genetic variants and SCD-CAD susceptibility: insights from Chinese Han cohorts.

The prevention and prediction of sudden cardiac death (SCD) present persistent challenges, prompting exploration into common genetic variations for potential insights. T-box 5 (TBX5), a critical cardiac transcription factor, plays a pivotal role in cardiovascular development and function. This study systematically examined variants within the 500-bp region downstream of the TBX5 gene, focusing on their potential impact on susceptibility to SCD associated with coronary artery disease (SCD-CAD) in four different Chinese Han populations. In a comprehensive case-control analysis, we explored the association between rs11278315 and SCD-CAD susceptibility using a cohort of 553 controls and 201 SCD-CAD cases. Dual luciferase reporter assays and genotype-phenotype correlation studies using human cardiac tissue samples as well as integrated in silicon analysis were applied to explore the underlining mechanism. Binary logistic regression results underscored a significantly reduced risk of SCD-CAD in individuals harboring the deletion allele (odds ratio = 0.70, 95% CI [0.55-0.88], p = 0.0019). Consistent with the lower transcriptional activity of the deletion allele observed in dual luciferase reporter assays, genotype-phenotype correlation studies on human cardiac tissue samples affirmed lower expression levels associated with the deletion allele at both mRNA and protein levels. Furthermore, our investigation revealed intriguing insights into the role of rs11278315 in TBX5 alternative splicing, which may contribute to alterations in its ultimate functional effects, as suggested by sQTL analysis. Gene ontology analysis and functional annotation further underscored the potential involvement of TBX5 in alternative splicing and cardiac-related transcriptional regulation. In summary, our current dataset points to a plausible correlation between rs11278315 and susceptibility to SCD-CAD, emphasizing the potential of rs11278315 as a genetic risk marker for aiding in molecular diagnosis and risk stratification of SCD-CAD.

Transcription Factor 21: A Transcription Factor That Plays an Important Role in Cardiovascular Disease.

According to the World Health Organisation's Health Report 2019, approximately 17.18 million people die from cardiovascular disease each year, accounting for more than 30% of all global deaths. Therefore, the occurrence of cardiovascular disease is still a global concern. The transcription factor 21 (TCF21) plays an important role in cardiovascular diseases. This article reviews the regulation mechanism of TCF21 expression and activity and focuses on its important role in atherosclerosis in order to contribute to the development of diagnosis and treatment of cardiovascular diseases. TCF21 is involved in the phenotypic regulation of vascular smooth muscle cells (VSMCs), promotes the proliferation and migration of VSMCs, and participates in the activation of inflammatory sequences. Increased proliferation and migration of VSMCs can lead to neointimal hyperplasia after vascular injury. Abnormal hyperplasia of neointima and inflammation are one of the main features of atherosclerosis. Therefore, targeting TCF21 may become a potential treatment for relieving atherosclerosis. TCF21 as a member of basic helix-loop-helix transcription factors regulates cell growth and differentiation by modulating gene expression during the development of different organs and plays an important role in cardiovascular development and disease. VSMCs and cells derived from VSMCs constitute the majority of plaques in atherosclerosis. TCF21 plays a key role in regulation of VSMCs' phenotype, thus accelerating atherogenesis in the early stage. However, TCF21 enhances plaque stability in late-stage atherosclerosis. The dual role of TCF21 should be considered in the translational medicine.

Effects of pericyte and smooth muscle specific knock-out of CXCL12 on cardiac function after myocardial ischemia in mice

Abstract Introduction Ischemic cardiomyopathy as a result of myocardial infarction represents one of the most common causes of heart failure (1). The C-X-C motif chemokine ligand 12 (CXCL12) and its receptor CXCR4 facilitate cardiac repair after myocardial ischemia (MI) and play fundamental roles in cardiovascular development (2). However, the cell- and tissue-specific actions of CXCL12 are poorly understood, limiting the introduction of targeted therapies. Here, we aimed to examine the role of pericyte and smooth muscle cell-specific CXCL12 expression on myocardial function after MI. Methods We generated a smooth muscle protein 22-alpha-Cre and a pericyte protein NG2-Cre driven mouse model to ablate CXCL12 specifically in smooth muscle cells (SM22-Cre x CXCL12flox/flox) and in pericytes (NG2-Cre x CXCL12flox/flox). Genotyping was performed using ear hole biopsy by PCR. Hearts were analysed morphologically by histology and immunofluorescence. Cardiac function and dimensions of the wall thicknesses and cardiac cavities were determined by echocardiography. Myocardial infarction was induced in the animals and cardiac function was further investigated 28 days after MI. Results In immunofluorescence, we found that CXCL12 is highly expressed in SM22+ smooth muscle cells and in cells surrounding arterial blood vessels (3). NG2-CXCL12-/- mice did not show increased mortality, they behaved inconspicuously and no obvious developmental defects could be detected. Echocardiographic analysis of cardiac function in NG2-Cre x CXCL12flox/flox mice without infarction showed no significant difference in left ventricular (LV) performance and no evidence of abnormal geometry of the ventricles compared with controls. Assessment of SM22-Cre x CXCL12flox/flox mice after MI revealed reduced ejection fraction (EF) (29.0%±9.7 vs. 40.0%±8.1; n=10; p=0.027) and stroke volume (SV) (19.0µl±3.3 vs. 32.7µl±10.3; n=10; p=0.002) compared to non-transgenic controls. In contrast NG2-Cre x CXCL12flox/flox mice showed no significant changes in EF (34.6%±11.4 vs. 40.0%±8.1; n=10; p=0.368) and SV (27.7µl±7.3 vs. 32.7µl±10.3; n=10; p=0.303). Conclusion Our findings suggest that pericyte specific CXCL12 plays no major role in cardiac development and function after MI. In contrast, KO of CXCL12 in smooth muscle seems to deteriorate cardiac function after ischemia.

Association of Circulating Plasma Secreted Frizzled-Related Protein 5 (Sfrp5) Levels with Cardiac Function.

Secreted frizzled-related protein 5 (SFRP5) is a novel anti-inflammatory adipokine that may play a role in cardiovascular development and disease. However, there is yet to be a comprehensive investigation into whether circulating SFRP5 can be a biomarker for cardiac function. Plasma SFRP5 levels were measured via ELISA in 262 patients admitted to a cardiology unit. Plasma SFRP5 levels were significantly lower in patients with a history of heart failure (HF), coronary artery disease (CAD), and atrial fibrillation (AF; p = 0.001). In univariate analyses, SFRP5 levels were also significantly positively correlated with left ventricular ejection fraction (LVEF) (r = 0.52, p < 0.001) and negatively correlated with E/E' (r = -0.30, p < 0.001). Patients with HF, CAD, low LVEF, low triglycerides, high CRP, and high eGFR were associated with lower SFRP5 levels independent of age, BMI, or diabetes after multivariate analysis (overall model r = 0.729, SE = 0.638). Our results show that low plasma SFRP5 levels are independently associated with the presence of HF, CAD, and, importantly, impaired LV function. These results suggest a potential role of SFRP5 as a biomarker, as well as a mediator of cardiac dysfunction independent of obesity and metabolic regulation.

Leucine zipper protein 1 prevents doxorubicin-induced cardiotoxicity in mice

ObjectiveDoxorubicin (DOX) is commonly used for chemotherapy; however, its clinical value is extremely dampened because of the fatal cardiotoxicity. Leucine zipper protein 1 (LUZP1) plays critical roles in cardiovascular development, and this study is designed for determining its function and mechanism in DOX-induced cardiotoxicity. MethodsCardiac-specific Luzp1 knockout (cKO) and transgenic (cTG) mice received a single or repeated DOX injections to establish acute and chronic cardiotoxicity. Biomarkers of inflammation, oxidative damage and cell apoptosis were evaluated. Transcriptome and co-immunoprecipitation analysis were used to screen the underlying molecular pathways. Meanwhile, primary cardiomyocytes were applied to confirm the beneficial effects of LUZP1 in depth. ResultsLUZP1 was upregulated in DOX-injured hearts and cardiomyocytes. Cardiac-specific LUZP1 deficiency aggravated, while cardiac-specific LUZP1 overexpression attenuated DOX-associated inflammation, oxidative damage, cell apoptosis and acute cardiac injury. Mechanistic studies revealed that LUZP1 ameliorated DOX-induced cardiotoxicity through activating 5′-AMP-activated protein kinase (AMPK) pathway, and AMPK deficiency abolished the cardioprotection of LUZP1. Further findings suggested that LUZP1 interacted with protein phosphatase 1 to activate AMPK pathway. Moreover, we determined that cardiac-specific LUZP1 overexpression could also attenuate DOX-associated chronic cardiac injury in mice. ConclusionLUZP1 attenuates DOX-induced inflammation, oxidative damage, cell apoptosis and ventricular impairment through regulating AMPK pathway, and gene therapy targeting LUZP1 may provide novel therapeutic approached to treat DOX-induced cardiotoxicity.

VGLL4-TEAD1 promotes vascular smooth muscle cell differentiation from human pluripotent stem cells via TET2

The Hippo signaling pathway plays a critical role in cardiovascular development and stem cell differentiation. Using microarray profiling, we found that the Hippo pathway components vestigial-like family member 4 (VGLL4) and TEA domain transcription factor 1 (TEAD1) were upregulated during vascular smooth muscle cell (VSMC) differentiation from H1 ESCs (H1 embryonic stem cells). To further explore the role and molecular mechanisms of VGLL4 in regulating VSMC differentiation, we generated a VGLL4-knockdown H1 ESC line (heterozygous knockout) using the CRISPR/Cas9 system and found that VGLL4 knockdown inhibited VSMC specification. In contrast, overexpression of VGLL4 using the PiggyBac transposon system facilitated VSMC differentiation. We confirmed that this effect was mediated via TEAD1 and VGLL4 interaction. In addition, bioinformatics analysis revealed that Ten-eleven-translocation 2 (TET2), a DNA dioxygenase, is a target of TEAD1, and a luciferase assay further verified that TET2 is the target of the VGLL4-TEAD1 complex. Indeed, TET2 overexpression promoted VSMC marker gene expression and countered the VGLL4 knockdown-mediated inhibitory effects on VSMC differentiation. In summary, we revealed a novel role of VGLL4 in promoting VSMC differentiation from hESCs and identified TET2 as a new target of the VGLL4-TEAD1 complex, which may demethylate VSMC marker genes and facilitate VSMC differentiation. This study provides new insights into the VGLL4-TEAD1-TET2 axis in VSMC differentiation and vascular development.

Ablation of CXCR4 expression in cardiomyocytes exacerbates isoproterenol‑induced cell death and heart failure.

CXCR4 is a seven‑transmembrane‑spanning Gi‑coupled receptor for the SDF‑1 chemokine and plays a critical role in cardiovascular development and post‑injury repair. However, the specific role of CXCR4 in cardiomyocytes is incompletely understood. It was hypothesized that CXCR4 activation in cardiomyocytes antagonizes β‑adrenoceptor/Gs signaling‑induced cardiac dysfunction. Cardiomyocyte‑specific CXCR4 knockout (CXCR4‑CMKO) mice were generated by crossing CXCR4fl/fl and MHC‑Cre+/‑ mice. Their cardiac structure and function in the basal state are equivalent to that of the control MHC‑Cre+/‑ littermates until at least 4months old. However, following continuous subcutaneous administration of isoproterenol (Iso) via an osmotic mini‑pump, the ventricular myocardial contractility, dilation, cardiomyocyte apoptosis, and interstitial fibrosis are worse in CXCR4‑CMKO mice than in MHC‑Cre+/‑ littermates. In the cultured H9C2 cardiomyocytes, SDF‑1 treatment markedly attenuated Iso‑induced apoptosis and reduction in phospho‑Akt, and this protective effect was lost by knockdown of CXCR4 or by co‑treatment with Gi inhibitors. In conclusion, CXCR4 promotes cardiomyocyte survival and heart function during β‑adrenergic stress.

Elabela: novel perspectives on vascular physiology and disease

Recently, a novel endogenous ligand of APJ called elabela has been identified to play an important role in cardiovascular development. Elabela and its G-protein-coupled receptor APJ are widely expressed throughout the human body. These have been identified to play a significant role in diverse biological processes, especially in the normal and pathological cardiovascular systems. In addition, increasing evidence indicates that elabela is also intimately associated with a large number of vascular physiological processes in adulthood, such as angiogenesis, vascular tone and atherosclerosis. However, a comprehensive summary of elabela in the vascular system has not been reported to date. In this review, we provide an overview of elabela in vascular physiology and diseases. Collectively, elabela, a potential biomarker and therapeutic peptide, exerts diverse biological vascular effects in adults.

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment.

It is well known that multiple microRNAs play crucial roles in cardiovascular development, including miR-133a. Additionally, retinoic acid regulates atrial marker expression. In order to analyse the role of miR-133a as a modulator of retinoic acid signalling during the posterior segment of heart tube formation, we performed functional experiments with miR-133a and retinoic acid by means of microinjections into the posterior cardiac precursors of both primitive endocardial tubes in chick embryos. Subsequently, we subjected embryos to whole mount in situ hybridisation, immunohistochemistry and qPCR analysis. Our results demonstrate that miR-133a represses RhoA and Cdc42, as well as Raldh2/Aldh1a2, and the specific atrial markers Tbx5 and AMHC1, which play a key role during differentiation. Furthermore, we observed that miR-133a upregulates p21 and downregulates cyclin A by repressing RhoA and Cdc42, respectively, thus functioning as a cell proliferation inhibitor. Additionally, retinoic acid represses miR-133a, while it increases Raldh2, Tbx5 and AMHC1. Given that RhoA and Cdc42 are involved in Raldh2 expression and that they are modulated by miR-133a, which is influenced by retinoic acid signalling, our results suggest the presence of a negative feedback mechanism between miR-133a and retinoic acid during early development of the posterior cardiac tube segment. Despite additional unexplored factors being possible contributors to this negative feedback mechanism, miR-133a might also be considered as a potential therapeutic tool for the diagnosis, therapy and prognosis of cardiac diseases.

Predisposition to atrioventricular septal defects may be caused by SOX7 variants that impair interaction with GATA4.

Atrioventricular septal defects (AVSD) are a complicated subtype of congenital heart defects for which the genetic basis is poorly understood. Many studies have demonstrated that the transcription factor SOX7 plays a pivotal role in cardiovascular development. However, whether SOX7 single nucleotide variants are involved in AVSD pathogenesis is unclear. To explore the potential pathogenic role of SOX7 variants, we recruited a total of 100 sporadic non-syndromic AVSD Chinese Han patients and screened SOX7 variants in the patient cohort by targeted sequencing. Functional assays were performed to evaluate pathogenicity of nonsynonymous variants of SOX7. We identified three rare SOX7 variants, c.40C > G, c.542G > A, and c.743C > T, in the patient cohort, all of which were found to be highly conserved in mammals. Compared to the wild type, these SOX7 variants had increased mRNA expression and decreased protein expression. In developing hearts, SOX7 and GATA4 were highly expressed in the region of atrioventricular cushions. Moreover, SOX7 overexpression promoted the expression of GATA4 in human umbilical vein endothelial cells. A chromatin immunoprecipitation assay revealed that SOX7 could directly bind to the GATA4 promoter and luciferase assays demonstrated that SOX7 activated the GATA4 promoter. The SOX7 variants had impaired transcriptional activity relative to wild-type SOX7. Furthermore, the SOX7 variants altered the ability of GATA4 to regulate its target genes. In conclusion, our findings showed that deleterious SOX7 variants potentially contribute to human AVSD by impairing its interaction with GATA4. This study provides novel insights into the etiology of AVSD and contributes new strategies to the prenatal diagnosis of AVSD.

Endothelial MEKK3-KLF2/4 signaling integrates inflammatory and hemodynamic signals during definitive hematopoiesis

AbstractThe hematopoietic stem cells (HSCs) that produce blood for the lifetime of an animal arise from RUNX1+ hemogenic endothelial cells (HECs) in the embryonic vasculature through a process of endothelial-to-hematopoietic transition (EHT). Studies have identified inflammatory mediators and fluid shear forces as critical environmental stimuli for EHT, raising the question of how such diverse inputs are integrated to drive HEC specification. Endothelial cell MEKK3-KLF2/4 signaling can be activated by both fluid shear forces and inflammatory mediators, and it plays roles in cardiovascular development and disease that have been linked to both stimuli. Here we demonstrate that MEKK3 and KLF2/4 are required in endothelial cells for the specification of RUNX1+ HECs in both the yolk sac and dorsal aorta of the mouse embryo and for their transition to intraaortic hematopoietic cluster (IAHC) cells. The inflammatory mediators lipopolysaccharide and interferon-γ increase RUNX1+ HECs in an MEKK3-dependent manner. Maternal administration of catecholamines that stimulate embryo cardiac function and accelerate yolk sac vascular remodeling increases EHT by wild-type but not MEKK3-deficient endothelium. These findings identify MEKK-KLF2/4 signaling as an essential pathway for EHT and provide a molecular basis for the integration of diverse environmental inputs, such as inflammatory mediators and hemodynamic forces, during definitive hematopoiesis.

Precise Dose of Folic Acid Supplementation Is Essential for Embryonic Heart Development in Zebrafish

Simple SummaryFolic acid is an essential vitamin for human beings. It has become a consensus to supplement folic acid during pregnancy. It is reported that 15~20% of people in the world supplement folic acid excessively. We found that excessive folic acid supplementation or insufficient folic acid intake could lead to abnormal heart development in zebrafish embryos. We elucidated the mechanism of folic acid on early cardiac development for the first time. These results provide a scientific basis for the important reasonable supplement dose of folic acid. At the same time, we constructed zebrafish mutants with abnormal folate metabolism, which provide a novel biological model for the study of folate acid metabolism.Folic acid, one of the 13 essential vitamins, plays an important role in cardiovascular development. Mutations in folic acid synthesis gene 5,10-methylenetetrahydrofolate reductase (MTHFR) is associated with the occurrence of congenital heart disease. However, the mechanisms underlying the regulation of cardiac development by mthfr gene are poorly understood. Here, we exposed zebrafish embryos to excessive folate or folate metabolism inhibitors. Moreover, we established a knock-out mutant of mthfr gene in zebrafish by using CRISPR/Cas9. The zebrafish embryos of insufficient or excessive folic acid and mthfr−/− mutant all gave rise to early pericardial edema and cardiac defect at 3 days post fertilization (dpf). Furthermore, the folic acid treated embryos showed abnormal movement at 5 dpf. The expression levels of cardiac marker genes hand2, gata4, and nppa changed in the abnormality of folate metabolism embryos and mthfr−/− mutant, and there is evidence that they are related to the change of methylation level caused by the change of folate metabolism. In conclusion, our study provides a novel model for the in-depth study of MTHFR gene and folate metabolism. Furthermore, our results reveal that folic acid has a dose-dependent effect on early cardiac development. Precise dosage of folic acid supplementation is crucial for the embryonic development of organisms.

Spotlight on Isl1: A Key Player in Cardiovascular Development and Diseases.

Cardiac transcription factors orchestrate a regulatory network controlling cardiovascular development. Isl1, a LIM-homeodomain transcription factor, acts as a key player in multiple organs during embryonic development. Its crucial roles in cardiovascular development have been elucidated by extensive studies, especially as a marker gene for the second heart field progenitors. Here, we summarize the roles of Isl1 in cardiovascular development and function, and outline its cellular and molecular modes of action, thus providing insights for the molecular basis of cardiovascular diseases.

Smooth muscle cell specific ablation of CXCL12 downregulates endothelial CXCR7 leading to defective coronary arteries and cardiac hypertrophy

Abstract Aims The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12−/−). Methods and results SM-CXCL12−/− mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12−/− mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12−/− mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Conclusion Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): FWF-Austria

Bioengineering Systems for Modulating Notch Signaling in Cardiovascular Development, Disease, and Regeneration.

The Notch intercellular signaling pathways play significant roles in cardiovascular development, disease, and regeneration through modulating cardiovascular cell specification, proliferation, differentiation, and morphogenesis. The dysregulation of Notch signaling leads to malfunction and maldevelopment of the cardiovascular system. Currently, most findings on Notch signaling rely on animal models and a few clinical studies, which significantly bottleneck the understanding of Notch signaling-associated human cardiovascular development and disease. Recent advances in the bioengineering systems and human pluripotent stem cell-derived cardiovascular cells pave the way to decipher the role of Notch signaling in cardiovascular-related cells (endothelial cells, cardiomyocytes, smooth muscle cells, fibroblasts, and immune cells), and intercellular crosstalk in the physiological, pathological, and regenerative context of the complex human cardiovascular system. In this review, we first summarize the significant roles of Notch signaling in individual cardiac cell types. We then cover the bioengineering systems of microfluidics, hydrogel, spheroid, and 3D bioprinting, which are currently being used for modeling and studying Notch signaling in the cardiovascular system. At last, we provide insights into ancillary supports of bioengineering systems, varied types of cardiovascular cells, and advanced characterization approaches in further refining Notch signaling in cardiovascular development, disease, and regeneration.

Regulation of the Muscarinic M3 Receptor by Myocardin-Related Transcription Factors.

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) are co-factors of serum response factor (SRF) that activate the smooth muscle cell (SMC) gene program and that play roles in cardiovascular development and mechanobiology. Gain and loss of function experiments have defined the SMC gene program under control of MRTFs, yet full understanding of their impact is lacking. In the present study, we tested the hypothesis that the muscarinic M3 receptor (CHRM3) is regulated by MRTFs together with SRF. Forced expression of MYOCD (8d) in human coronary artery (SMC) followed by RNA-sequencing showed increased levels of M2, M3, and M5 receptors (CHRM2: 2-fold, CHRM3: 16-fold, and CHRM5: 2-fold). The effect of MYOCD on M3 was confirmed by RT-qPCR using both coronary artery and urinary bladder SMCs, and correlation analyses using human transcriptomic datasets suggested that M3 may also be regulated by MRTF-B. Head-to-head comparisons of MYOCD, MRTF-A and MRTF-B, argued that while all MRTFs are effective, MRTF-B is the most powerful transactivator of CHRM3, causing a 600-fold increase at 120h. Accordingly, MRTF-B conferred responsiveness to the muscarinic agonist carbachol in Ca2+ imaging experiments. M3 was suppressed on treatment with the MRTF-SRF inhibitor CCG-1423 using SMCs transduced with either MRTF-A or MRTF-B and using intact mouse esophagus in culture (by 92±2%). Moreover, silencing of SRF with a short hairpin reduced CHRM3 (by >60%) in parallel with α-actin (ACTA2). Tamoxifen inducible knockout of Srf in smooth muscle reduced Srf (by 54±4%) and Chrm3 (by 41±6%) in the urinary bladder at 10days, but Srf was much less reduced or unchanged in aorta, ileum, colon, trachea, and esophagus. Longer induction (21d) further accentuated the reduction of Chrm3 in the bladder and ileum, but no change was seen in the aorta. Single cell RNA-sequencing revealed that Mrtfb dominates in ECs, while Myocd dominates in SMCs, raising the possibility that Chrm3 may be driven by Mrtfb-Srf in the endothelium and by Myocd-Srf in SMCs. These findings define a novel transcriptional control mechanism for muscarinic M3 receptors in human cells, and in mice, that could be targeted for therapy.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.

Smooth Muscle Specific Ablation of CXCL12 in Mice Downregulates CXCR7 Associated with Defective Coronary Arteries and Cardiac Hypertrophy.

The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12−/−). SM-CXCL12−/− mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12−/− mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12−/− mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling.

Cell Cycle Withdrawal Limit the Regenerative Potential of Neonatal Cardiomyocytes.

The neonatal mouse possesses a transient capacity for cardiac regeneration during the first few days of life. The regenerative response of neonatal mouse is primarily mediated by pre-existing cardiomyocyte (CM) proliferation, which has been identified as the primary source of myocardial regeneration. Postnatal 4-day-old (P4) mouse CMs appear to undergo a rapid transition from hyperplastic to hypertrophic growth and binucleation. By 7 days following birth this regenerative potential is lost which coincidently correspond with CM cell cycle arrest and binucleation. CCM2-like (Ccm2l) plays pivotal roles in cardiovascular development and cardiac growth, indicating a potential function in heart regeneration postnatally. The aim of this study was to determine the cardiac regeneration ability of P4 neonatal mouse using a novel and more reproducible injury model and to determine whether Ccm2l has any functional roles in heart repair following ischemic injury. We performed a modified left anterior descending artery (LAD) ligation procedure on P4 mice to examine cardiac regenerative responses at different time points. Additionally, we generated an endothelial-specific Ccm2l gain-of-function transgenic mouse to determine the role of Ccm2l in neonatal cardiac regeneration. We found that the P4 mouse heart harbor a robust regenerative response after injury that was through the proliferation of pre-existing CMs but cardiac hypertrophy and subsequent remodeling was still evident 60 days after LAD ligation. Furthermore, we show that endothelial-specific overexpression of Ccm2l does not promote CM proliferation and heart repair after LAD ligation. The neonatal heart at P4 harbors a robust but incomplete capacity for cardiac regeneration. Endothelial overexpression of Ccm2l has no effect on cardiac regeneration.