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Objective: Atherosclerosis is accumulation of lipids and extracellular matrix in the arterial wall. TIMPs (tissue inhibitor of metalloproteinases) can impact plaque deposition by regulating ECM (extracellular matrix) turnover. TIMP4 also influences lipid metabolism and smooth muscle cell (SMC) proliferation. We investigated the role of TIMP4 in atherosclerosis. Approach and Results: Mice lacking low-density lipoprotein receptor ( Ldlr −/− ) and Timp4 ( Timp4 −/− / Ldlr −/− ) were fed high-fat diet (HFD) or regular laboratory diet. After 3 or 6 months, HFD-fed male and female Timp4 −/− / Ldlr −/− mice exhibited higher plaque density in the abdominal aorta (but not in aortic valves, arch, thoracic aorta) compared with Ldlr −/− mice. Although plasma lipid and cholesterol levels were lower in Timp4 −/− / Ldlr −/− -HFD, cholesterol content in the abdominal aorta was higher along with elevated inflammatory cytokines, MMP (matrix metalloproteinase) activities, CD68 + /calponin + macrophage-like SMCs in Timp4 −/− / Ldlr −/− -HFD compared with Ldlr −/− -HFD mice. In vitro, oxidized LDL (low-density lipoprotein) markedly increased CD68 expression, reduced SMC markers, increased lipid uptake, and reduced cholesterol efflux protein ABCA1 (ATP-binding cassette transporter A1) in Timp4 −/− / Ldlr −/− compared with Ldlr −/− primary SMCs from abdominal, but not thoracic aorta. TIMP4 expression in the abdominal aorta (in vivo) and its corresponding SMCs (in vitro) was ≈2-fold higher than in the thoracic aorta and SMCs; TIMP4 levels decreased following HFD. Timp4 -deficiency in bone marrow–derived macrophages did not alter their foam cell formation capacity. Conclusions: TIMP4 protects against plaque deposition in the abdominal aorta independent of plasma cholesterol levels. TIMP4 prevents proteolytic degradation of ABCA1 in SMCs, hindering cholesterol accumulation and transdifferentiation to macrophage-like foam cells, representing a novel negative regulator of atherosclerosis.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Although model organisms have provided insight into the earliest stages of cardiac vascularization, we know very little about this process in humans. Here we show that spatially micropatterned human pluripotent stem cells (hPSCs) enablein vitromodeling of this process, corresponding to the first three weeks ofin vivohuman development. Using four hPSC fluorescent reporter lines, we create cardiac vascular organoids (cVOs) by identifying conditions that simultaneously give rise to spatially organized and branched vascular networks within endocardial, myocardial, and epicardial cells. Using single-cell transcriptomics, we show that the cellular composition of cVOs resembles that of a 6.5 post-conception week (PCW) human heart. We find that NOTCH and BMP pathways are upregulated in cVOs, and their inhibition disrupts vascularization. Finally, using the same vascular-inducing factors to create cVOs, we produce hepatic vascular organoids (hVOs). This suggests there is a conserved developmental program for creating vasculature within different organ systems.Graphic Abstract

Vascular smooth muscle cells (VSMCs), a highly mosaic tissue, arise from multiple distinct embryonic origins and populate different regions of our vascular network with defined boundaries. Accumulating evidence has revealed that the heterogeneity of VSMC origins contributes to region-specific vascular diseases such as atherosclerosis and aortic aneurysm. These findings highlight the necessity of taking into account lineage-dependent responses of VSMCs to common vascular risk factors when studying vascular diseases. This chapter describes a reproducible, stepwise protocol for the generation of isogenic VSMC subtypes originated from proepicardium, second heart field, cardiac neural crest, and ventral somite using human induced pluripotent stem cells. By leveraging this robust induction protocol, patient-derived VSMC subtypes of desired embryonic origins can be generated for disease modeling as well as drug screening and development for vasculopathies with regional susceptibility.

Aortic aneurysm is an asymptomatic disease with dire outcomes if undiagnosed. Aortic aneurysm rupture is a significant cause of death worldwide. To date, surgical repair or endovascular repair (EVAR) is the only effective treatment for aortic aneurysm, as no pharmacological treatment has been found effective. Aortic aneurysm, a focal dilation of the aorta, can be formed in the thoracic (TAA) or the abdominal (AAA) region; however, our understanding as to what determines the site of aneurysm formation remains quite limited. The extracellular matrix (ECM) is the noncellular component of the aortic wall, that in addition to providing structural support, regulates bioavailability of an array of growth factors and cytokines, thereby influencing cell function and behavior that ultimately determine physiological or pathological remodeling of the aortic wall. Here, we provide an overview of the ECM proteins that have been reported to be involved in aortic aneurysm formation in humans or animal models, and the experimental models for TAA and AAA and the link to ECM manipulations. We also provide a comparative analysis, where data available, between TAA and AAA, and how aberrant ECM proteolysis versus disrupted synthesis may determine the site of aneurysm formation., Aortic aneurysm: Different locations, different causes A review of aneurysm formation, swelling in blood vessel, in the aorta, examines distinctions between two forms of the condition and the role of proteins in the extracellular matrix which surrounds cells of the arterial wall. Rupture of aneurysms in the aorta, the body’s main artery, is a major cause of death. Researchers led by Zamaneh Kassiri at the University of Alberta, Edmonton, Canada, emphasize that aneurysms in the thoracic and abdominal regions of the aorta are distinct conditions with crucial differences in their causes. Disrupted production and assembly of the extracellular matrix and its proteins may underlie thoracic aneurysm formation. Factors triggering the degradation of extracellular matrix proteins may be more significant in abdominal aneurysms. Understanding the differing molecular mechanisms involved could help address the current lack of effective drug treatments for these dangerous conditions.

SummaryCongenital heart defects, the most common birth disorders, are the clinical manifestation of anomalies in fetal heart development - a complex process involving dynamic spatiotemporal coordination among various precursor cell lineages. This complexity underlies the incomplete understanding of the genetic architecture of congenital heart diseases (CHDs). To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We identified similarities and differences of regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts. We interpreted deep learning models that predict cell-type resolved, base-resolution chromatin accessibility profiles from DNA sequence to decipher underlying TF motif lexicons and infer the regulatory impact of non-coding variants. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in CHD cases versus controls. We used CRISPR-based perturbations to validate an enhancer harboring a nominated regulatory CHD mutation, linking it to effects on the expression of a known CHD gene JARID2. Together, this work defines the cell-type resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements as a component of the genetic etiology of CHD.

Introduction and Hypothesis: Statins, a class of HMG-CoA reductase inhibitors that repress the mevalonate pathway, have been increasingly recognized to reduce cardiovascular risks in a pleiotropic manner independent of their lipid-lowering effects. Yet, the precise molecular mechanisms underlying their cardiovascular protection effects remain elusive. As an unlimited alternate source of human primary cells, we sought to use human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) to tackle this question. Methods: We treated iPSC-ECs with or without statins in both baseline and prodiabetic conditions, and evaluated their biological functions specifically at the transcriptional and epigenetic levels using an array of state-of-the-art technologies, such as transcriptome profiling, ChIP-seq, ATAC-seq, and CRISPR interference (CRISPRi). Results: We observed that, compared to vehicles, statins significantly improved endothelial functions in both baseline and prodiabetic conditions in iPSC-ECs. Mechanistically, statins could reduce chromatin accessibility at TEAD elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Interestingly, inhibition of geranylgeranyltransferase I (GGTase I), a mevalonate pathway intermediate, was able to block YAP nuclear translocation and thereby YAP activity by suppressing RhoA signaling. Based on these observations, we further confirmed that inhibition of any component of the GGTase-RhoA-YAP/TEAD signaling axis using either genetic or pharmacological approaches was effective to rescue EndMT-associated endothelial dysfunction, especially under prodiabetic conditions. Conclusions: Taken together, our study identifies a novel protective role of statins in endothelial dysfunction by epigenetically repressing EndMT, and proposes that GGTase, RhoA, and YAP inhibitors may be tested as potential therapeutic candidates in addition to statins to prevent or treat endothelial dysfunction-associated cardiovascular diseases.

Significance Vascular diseases remain a major health burden, and AAAs lack effective medical therapy. We demonstrate a seminal role for APLN in AAA pathogenesis based on loss-of-function and gain-of-function approaches and included human vascular SMCs and AA tissue obtained from patients. We identified NEP as a dominant inactivating enzyme for native APLN-17. This allowed us to design and synthesize a stable and bioactive APLN analog resistant to NEP degradation that showed profound therapeutic effects against AAA. Our study clearly defines the APLN pathway as a central node in the pathogenesis of AAA and elucidate a therapeutic strategy of enhancing the APLN pathway by using a stable APLN analog to treat AAA., Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln−/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln−/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln−/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr−/− mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

The developmental origin of vascular smooth muscle cells (VSMCs) has been increasingly recognized as a major determinant for regional susceptibility or resistance to vascular diseases. As a human material-based complement to animal models and human primary cultures, patient induced pluripotent stem cell iPSC-derived VSMCs have been leveraged to conduct basic research and develop therapeutic applications in vascular diseases. However, iPSC-VSMCs (induced pluripotent stem cell VSMCs) derived by most existing induction protocols are heterogeneous in developmental origins. In this review, we summarize signaling networks that govern in vivo cell fate decisions and in vitro derivation of distinct VSMC progenitors, as well as key regulators that terminally specify lineage-specific VSMCs. We then highlight the significance of leveraging patient-derived iPSC-VSMCs for vascular disease modeling, drug discovery, and vascular tissue engineering and discuss several obstacles that need to be circumvented to fully unleash the potential of induced pluripotent stem cells for precision vascular medicine.

Background: Myocardial infarction (MI) occurs when blood flow to a region of the myocardium is interrupted. Over time, the damaged myocardium is replaced with scar tissue through activation of an inflammatory phase, followed by a reparative and a maturation phase. A disintegrin and metalloproteinase-15 (ADAM15) is a membrane-bound enzyme that is expressed in inflammatory cells and the heart, and its expression in the heart is increased following myocardial infarction. However, its function in heart disease has not yet been explored. We hypothesized that ADAM15 deficiency will impede the inflammatory response post-MI, which could reduce inflammation but also hinder scar formation causing adverse remodeling of the surviving myocardium. Methods: MI was induced in adult male wildtype (WT) and ADAM15-deficient ( Adam15 -/- ) mice by permanent ligation of the left anterior descending artery. LV structure, systolic and diastolic functions were assessed by echocardiography. Hearts were excised at 3-days or 1 week post-MI, processed for histological and molecular analyses. Fibrillar collagen organization was assessed by Second Harmonic Generation. Cardiac fibroblasts (cFBs) were isolated from WT or Adam15 -/- hearts, and subjected to ischemia (hypoxia + nutrient deletion) in vitro . Results: Adam15 -/- mice exhibited significantly compromised survival post-MI, mainly due to LV rupture. Cardiac contractility was reduced in Adam15 -/- compared to WT-MI mice. Collagen fibers in the scar tissue were distorted and scarce in Adam15 -/- MI hearts, associated with reduced levels of a major cross-linking enzyme, lysyl oxidase. In vitro, Adam15 -/- cFBs showed impaired myofibroblast transformation under ischemic conditions, suggesting attenuated fibroblast activation. Conclusion: Adam15 -deficiency impairs the wound healing process post-MI, leading to deleterious remodelling and LV rupture.

Background: Cardiovascular diseases (CVD) are the leading cause of death in the United States. Statins, a class of hydroxy-methylglutaryl-coenzyme A reductase (HMGCR) inhibitors, have been proven to be effectively prevent and treat CVD by improving vascular functions independent of their cholesterol-lowering effect. However, the molecular mechanisms by which statins improve cardiovascular functions remain elusive. In this study, we used human induced pluripotent stem cells-derived endothelial cells (hiPSC-ECs) to explore the protective role of statins in the vascular system. Methods and Results: hiPSCs were generated from 3 healthy individuals and differentiated into endothelial cells using two different iPSC clones and two batches (4 biological replicates for each individual). hiPSC-ECs of each individual were treated with simvastatin or a vehicle control. The RNA-sequencing analysis was performed on 12 control and 12 statin-treated hiPSC-ECs. A total number of 2,580 differentially expressed genes (DEGs) were found in simvastatin-treated hiPSC-ECs. Gene enrichment analysis revealed that statin-upregulated DEGs were highly enriched in angiogenesis and anti-inflammation pathways. Interestingly, statin-downregulated DEGs were significantly enriched in epigenetic regulation and nucleosome assembly pathways, suggesting an epigenetic regulatory role of stains in vascular gene expression. To test this hypothesis, we further performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis on vehicle or simvastatin treated hiPSC-ECs. Transcription factor (TF) binding motif analyses of ATAC-seq peaks using HOMER revealed that YAP/TEAD binding was the most significantly downregulated TF after statin treatment. Furthermore, we observed that statin can improve endothelial functions (e.g., angiogenesis and nitric oxide production) after the YAP activity was inhibited. The ChIP-seq analysis showed that statin downregulated the expression levels of genes associated with the endothelial-to-mesenchymal transition (EndoMT) by attenuating the binding capacity of YAP. As such, simvastatin effectively rescued diabetic vascular dysfunction mainly through inhibiting EndoMT. Conclusion: We found that statins can improve endothelial functions through attenuating the chromatin accessibility of EndoMT-related genes, a process tightly regulated by the activity of YAP. These findings will provide novel insights into the protective mechanisms of statins in the cardiovascular system beyond their cholesterol-lowering effects.

Myocardial fibrosis is a hallmark of cardiac remodeling, which can progressively lead to heart failure, a leading cause of death worldwide. The effector cells of fibrosis in the heart are cardiac fibroblasts (CFs). There is currently no effective therapeutic strategy clinically available to specifically attenuate maladaptive responses of CFs. Large-scale applications such as high-throughput drug screening are difficult due to the limited availability of human primary CFs, thus limiting the development of future treatments. Here, we describe a robust induction protocol that can be used to generate a scalable, consistent, genetically defined source of quiescent CFs from human induced pluripotent stem cells for cardiac fibrosis modeling, drug discovery, and tissue engineering.

Chronic iron overload results in heart and liver diseases and is a common cause of morbidity and mortality in patients with genetic hemochromatosis and secondary iron overload. We investigated the role of tissue inhibitor of metalloproteinase 3 (TIMP3) in iron overload-mediated tissue injury by subjecting male mice lacking Timp3 ( Timp3−/−) and wild-type (WT) mice to 12 wk of chronic iron overload. Whereas WT mice with iron overload developed diastolic dysfunction, iron-overloaded Timp3−/− mice showed worsened cardiac dysfunction coupled with systolic dysfunction. In the heart, loss of Timp3 was associated with increased myocardial fibrosis, greater Timp1, matrix metalloproteinase ( Mmp) 2, and Mmp9 expression, increased active MMP-2 levels, and gelatinase activity. Iron overload in Timp3−/− mice showed twofold higher iron accumulation in the liver compared with WT mice because of constituently lower levels of ferroportin. Loss of Timp3 enhanced the hepatic inflammatory response to iron overload, leading to greater neutrophil and macrophage infiltration and increased hepatic fibrosis. Expression of inflammation-related MMPs (MMP-12 and MMP-13) and inflammatory cytokines (IL-1β and monocyte chemoattractant protein-1) was elevated to a greater extent in iron-overloaded Timp3−/− livers. Gelatin zymography demonstrated equivalent increases in MMP-2 and MMP-9 levels in WT and Timp3−/− iron-overloaded livers. Loss of Timp3 enhanced the susceptibility to iron overload-mediated heart and liver injury, suggesting that Timp3 is a key protective molecule against iron-mediated pathology. NEW & NOTEWORTHY In mice, loss of tissue inhibitor of metalloproteinase 3 ( Timp3) was associated with systolic and diastolic dysfunctions, twofold higher hepatic iron accumulation (attributable to constituently lower levels of ferroportin), and increased hepatic inflammation. Loss of Timp3 enhanced the susceptibility to iron overload-mediated injury, suggesting that Timp3 plays a key protective role against iron-mediated pathology.

Hypertrophic cardiomyopathy (HCM) is a common inherited heart disease with a prevalence of about 0.2%. HCM is typically caused by mutations in genes encoding sarcomere or sarcomere-associated proteins. Here, we characterized induced pluripotent stem cell (iPSC) lines generated from the peripheral blood mononuclear cells of three HCM patients each carrying c.433C > T, c.610C > T, or c.235C > T mutation in the TNNI3 gene by non-integrated Sendai virus. All of the three lines exhibited normal morphology, expression of pluripotent markers, stable karyotype, and the potential of trilineage differentiation. The cardiomyocytes differentiated from these iPSC lines can serve as useful tools to model HCM in vitro.

Hypertension is associated with hypertrophy and hyperplasia of smooth muscle cells (SMCs). Disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme reported to mediate SMC hypertrophy through activation of epidermal growth factor receptor (EGFR). We investigated the role of ADAM17 in Ang II-induced hypertension and end-organ damage. VSMC was isolated from mice with intact ADAM17 expression (Adam17f/f) or lacking ADAM17 in the SMC (Adam17f/f/CreSm22). Human VSMCs were isolated from the aorta of donors, and ADAM17 deletion was achieved by siRNA transfection. Ang II suppressed proliferation and migration of Adam17-deficient SMCs, but did not affect apoptosis (mouse and human), this was associated with reduced activation of EGFR and Erk1/2 signaling. Adam17f/f/CreSm22 and littermate Adam17f/f mice received saline or Ang II (Alzet pumps, 1.5mg/kg/d; 2 or 4weeks). Daily blood pressure measurement in conscious mice (telemetry) showed suppressed hypertension in Adam17f/f/CreSm22 mice during the first week of Ang II infusion, but by the second week, it become comparable to that in Adam17f/f mice. EGFR activation remained suppressed in Adam17f/f/CreSm22-Ang II arteries. Ex vivo vascular function and compliance assessed in mesenteric arteries were comparable between genotypes. Consistent with the transient protection against Ang II-induced hypertension, Adam17f/f/CreSm22 mice exhibited significantly lower cardiac hypertrophy and fibrosis, and renal fibrosis at 2weeks post-Ang II, however this protection was abolished by the fourth week of Ang II infusion. In conclusion, while Adam17-deficiency suppresses Ang II-induced SMC remodeling in vitro, in vivo Adam17-deficiency provides only a transient protective effect against Ang II-mediated hypertension and end-organ damage.

Rationale: Activated fibroblasts are the major cell type that secretes excessive extracellular matrix in response to injury, contributing to pathological fibrosis and leading to organ failure. Effective anti-fibrotic therapeutic solutions, however, are not available due to the poorly defined characteristics and unavailability of tissue-specific fibroblasts. Recent advances in single-cell RNA-sequencing fill such gaps of knowledge by enabling delineation of the developmental trajectories and identification of regulatory pathways of tissue-specific fibroblasts among different organs. Objective: This study aims to define the transcriptome profiles of tissue-specific fibroblasts using recently reported mouse single-cell RNA-sequencing atlas and to develop a robust chemically defined protocol to derive cardiac fibroblasts (CFs) from human induced pluripotent stem cells for in vitro modeling of cardiac fibrosis and drug screening. Methods and Results: By analyzing the single-cell transcriptome profiles of fibroblasts from 10 selected mouse tissues, we identified distinct tissue-specific signature genes, including transcription factors that define the identities of fibroblasts in the heart, lungs, trachea, and bladder. We also determined that CFs in large are of the epicardial lineage. We thus developed a robust chemically defined protocol that generates CFs from human induced pluripotent stem cells. Functional studies confirmed that iPSC-derived CFs preserved a quiescent phenotype and highly resembled primary CFs at the transcriptional, cellular, and functional levels. We demonstrated that this cell-based platform is sensitive to both pro- and anti-fibrosis drugs. Finally, we showed that crosstalk between human induced pluripotent stem cell-derived cardiomyocytes and CFs via the atrial/brain natriuretic peptide-natriuretic peptide receptor-1 pathway is implicated in suppressing fibrogenesis. Conclusions: This study uncovers unique gene signatures that define tissue-specific identities of fibroblasts. The bona fide quiescent CFs derived from human induced pluripotent stem cells can serve as a faithful in vitro platform to better understand the underlying mechanisms of cardiac fibrosis and to screen anti-fibrotic drugs.

A disintegrin and metalloprotease-17 (ADAM17) belongs to a family of transmembrane enzymes, and it can mediate ectodomain shedding of several membrane-bound molecules. ADAM17 levels are elevated in patients with hypertrophic and dilated cardiomyopathy; however, its direct role in hypertrophic cardiomyopathy is unknown. Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17 flox/flox /αMHC-Cre; ADAM17 f/f /Cre) and littermates with intact ADAM17 levels (ADAM17 f/f ) were subjected to cardiac pressure–overload by transverse aortic constriction. Cardiac function/architecture was assessed by echocardiography at 2 and 5 weeks post transverse aortic constriction. ADAM17 knockdown enhanced myocardial hypertrophy, fibrosis, more severe left ventricular dilation, and systolic dysfunction at 5 weeks post transverse aortic constriction. Pressure overload–induced upregulation of integrin β1 was much greater with ADAM17 knockdown, concomitant with the greater activation of the focal adhesion kinase pathway, suggesting that integrin β1 could be a substrate for ADAM17. ADAM17 knockdown did not alter other cardiomyocyte integrins, integrin α5 or α7, and HB-EGF (heparin-bound epidermal growth factor), another potential substrate for ADAM17, remained unaltered after pressure overload. ADAM17-mediated cleavage of integrin β1 was confirmed by an in vitro assay. Intriguingly, ADAM17 knockdown did not affect the myocardial hypertrophy induced by a subpressor dose of angiotensin II, which occurs independent from the integrin β1–mediated pathway. ADAM17-knockdown enhanced the hypertrophic response to cyclic mechanical stretching in neonatal rat cardiomyocytes. This study reports a novel cardioprotective function for ADAM17 in pressure overload cardiomyopathy, where loss of ADAM17 promotes hypertrophy by reducing the cleavage of cardiac integrin β1, a novel substrate for ADAM17. This function of ADAM17 is selective for pressure overload–induced myocardial hypertrophy and dysfunction, and not agonist-induced hypertrophy.

A disintegrin and metalloproteinases (ADAMs) are a family of membrane-bound proteases. ADAM-TSs (ADAMs with thrombospondin domains) are a close relative of ADAMs that are present in soluble form in the extracellular space. Dysregulated production or function of these enzymes has been associated with pathologies such as cancer, asthma, Alzheimer's and cardiovascular diseases. ADAMs contribute to angiogenesis, hypertrophy and apoptosis in a stimulus- and cell type-dependent manner. Among the ADAMs identified so far (34 in mouse, 21 in human), ADAMs 8, 9, 10, 12, 17 and 19 have been shown to be involved in cardiovascular development or cardiomyopathies; and among the 19 ADAM-TSs, ADAM-TS1, 5, 7 and 9 are important in development of the cardiovascular system, while ADAM-TS13 can contribute to vascular disorders. Meanwhile, there remain a number of ADAMs and ADAM-TSs whose function in the cardiovascular system has not been yet explored. The current knowledge about the role of ADAMs and ADAM-TSs in the cardiovascular pathologies is still quite limited. The most detailed studies have been performed in other cell types (e.g. cancer cells) and organs (nervous system) which can provide valuable insight into the potential functions of ADAMs and ADAM-TSs, their mechanism of action and therapeutic potentials in cardiomyopathies. Here, we review what is currently known about the structure and function of ADAMs and ADAM-TSs, and their roles in development, physiology and pathology of the cardiovascular system.

Rationale: ADAM17 (a disintegrin and metalloproteinase-17) is a membrane-bound enzyme that regulates bioavailability of multiple transmembrane proteins by proteolytic processing. ADAM17 has been linked to several pathologies, but its role in thoracic aortic aneurysm (TAA) has not been determined. Objective: The objective of this study was to explore the cell-specific functions of vascular ADAM17 in the pathogenesis and progression of TAA. Methods and Results: In aneurysmal thoracic aorta from patients, ADAM17 was increased in tunica media and intima. To determine the function of ADAM17 in the major cells types within these regions, we generated mice lacking ADAM17 in smooth muscle cells (SMC; Adam17 f/f /Sm22 Cre/+ ) or endothelial cells ( Adam17 f/f /Tie2 Cre/+ ). ADAM17 deficiency in either cell type was sufficient to suppress TAA dilation markedly and adverse remodeling in males and females (in vivo) although through different mechanisms. ADAM17 deficiency in SMCs prevented the contractile-to-synthetic phenotypic switching in these cells after TAA induction, preventing perivascular fibrosis, inflammation, and adverse aortic remodeling. Loss of ADAM17 in endothelial cells protected the integrity of the intimal barrier by preserving the adherens junction (vascular endothelial-cadherin) and tight junctions (junctional adhesion molecule-A and claudin). In vitro studies on primary mouse thoracic SMCs and human primary aortic SMCs and endothelial cells (± ADAM17 small interfering RNA) confirmed the cell-specific functions of ADAM17 and demonstrated the cross-species validity of these findings. To determine the impact of ADAM17 inhibition in treating TAA, we used an ADAM17-selective inhibitor (PF-548) before or 3 days after TAA induction. In both cases, ADAM17 inhibition prevented progression of aneurysmal growth. Conclusions: We have identified distinct cell-specific functions of ADAM17 in TAA progression, promoting pathological remodeling of SMC and impairing integrity of the intimal endothelial cell barrier. The dual impact of ADAM17 deficiency (or inhibition) in protecting 2 major cell types in the aortic wall highlights the unique position of this proteinase as a critical treatment target for TAA.

An aortic aneurysm is characterized by widening of the aortic lumen diameter with adverse remodelling of the vascular extracellular matrix. A thoracic aortic aneurysm (TAA) is highly prevalent in patients with a bicuspid aortic valve (BAV). We investigated the structural remodelling in the aneurysmal ascending aorta in correlation to molecular alterations in male versus female BAV-TAA patients. Aneurysmal aortic samples (diameter >4.4 cm) from male and female patients were compared to non-aneurysmal non-BAV samples. The diameter of the aneurysmal aorta was smaller in females but comparable to that of male patients when normalized to body mass index. Disorganized elastin fibres and reduced elastin protein were found in samples from males and females with BAV-TAA. However, disarrayed collagen fibres and reduced protein were detected only in aortas from males with BAV-TAA. Elastin and collagen I messenger RNA (mRNA)levelswerecomparableintheBAV-TAAandcontrol groups of both genders, suggesting post-translation degradation. Total elastase activity was elevated similarly in both genders. The activity of MT1-MMP, a potent collagenase, was increased more in aortic samples from males thanfemales with BAV-TAA samples. MMP8 and MMP13 were lower whereas MMP2 was higher in female compared to samples from male BAV-TAA group. TIMP3 and TIMP4 decreased similarly in both genders, while TIMP2 increased in the female BAV-TAA group. Lower smooth muscle cell density in the medial layer of aortas from males with BAV-TAA samples corresponded to the increased caspase-3 cleavage compared to that of females. Conclusion :W e report a gender-dependent MMP/TIMP axis, collagen remodelling and smooth muscle cell (SMC) survival in the BAV-TAA aorta. The elevated TIMP2 could protect against the collagenolytic activity of MT1-MMP, leading to reduced collagen disarray and degradation in female BAV. Key message & More collagen degradation/disarray and smooth muscle cell loss in male BAV-TAA patients. & Similar elastin degradation/disarray in the aneurysmal aorta of both genders. & Reduced collagen and elastin in BAV-TAA due to enhanced degradation, not reduced synthesis. & Elevated TIMP2 in female BAV-TAA aortas protects against collagen degradation by MT1-MMP.

Objective— Aortic aneurysm, focal dilation of the aorta, results from impaired integrity of aortic extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are traditionally known as ECM-degrading enzymes. MMP2 has been associated with aneurysm in patients and in animal models. We investigated the role of MMP2 in thoracic aortic aneurysm using 2 models of aortic remodeling and aneurysm. Approach and Results— Male 10-week-old MMP2-deficient (MMP2 −/− ) and wild-type mice received angiotensin II (Ang II, 1.5 mg/kg/day) or saline (Alzet pump) for 4 weeks. Although both genotypes exhibited dilation of the ascending aorta after Ang II infusion, MMP2 −/− mice showed more severe dilation of the thoracic aorta and thoracic aortic aneurysm. The Ang II–induced increase in elastin and collagen (mRNA and protein) was markedly suppressed in MMP2 −/− thoracic aorta and smooth muscle cells, whereas only mRNA levels were reduced in MMP2 −/− -Ang II abdominal aorta. Consistent with the absence of MMP2, proteolytic activities were lower in MMP2 −/− -Ang II compared with wild-type-Ang II thoracic and abdominal aorta. MMP2-deficiency suppressed the activation of latent transforming growth factor-β and the Smad2/3 pathway in vivo and in vitro. Intriguingly, MMP2 −/− mice were protected against CaCl 2 -induced thoracic aortic aneurysm, which triggered ECM degradation but not synthesis. Conclusions— This study reveals the dual role of MMP2 in ECM degradation, as well as ECM synthesis. Moreover, the greater susceptibility of the thoracic aorta to impaired ECM synthesis, compared with vulnerability of the abdominal aorta to aberrant ECM degradation, provides an insight into the regional susceptibility of the aorta to aneurysm development.

The extracellular matrix (ECM) serves a number of functions in every tissue including the myocardium. While the function of ECM as a structural scaffold is well established, it has become increasingly recognized that it plays a number of additional functions including providing a reservoir for growth factors and cytokines allowing their rapid release and activation in response to environmental cues. In addition, components of the ECM are critical in the interstitial transport of numerous molecules and drugs. Therefore, impaired integrity of the ECM would influence multiple aspects of an organ’s structural and function. In the myocardium, the primary component of the ECM network structure is the fibrillar collagens I and III. Multiple steps and various enzymes are involved from collagen mRNA synthesis to collagen fibre formation. Alterations in each step can impact collagen fibre production resulting in an uncoupling between collagen mRNA and protein levels. In this chapter, we will provide an overview of the mechanisms involved in myocardial fibrosis, the disease-dependent nature and consequence of different types of fibrosis, clinical biomarkers of collagen turnover, and potential therapeutic approaches in managing myocardial fibrosis.

Background— A disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme that mediates shedding of many membrane-bound molecules, thereby regulating multiple cellular responses. We investigated the role of cardiomyocyte ADAM17 in myocardial infarction (MI). Methods and Results— Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17 flox/flox /α-MHC-Cre; f/f/Cre) and parallel controls (ADAM17 flox/flox ; f/f) were subjected to MI by ligation of the left anterior descending artery. Post MI, f/f/Cre mice showed compromised survival, higher rates of cardiac rupture, more severe left ventricular dilation, and suppressed ejection fraction compared with parallel f/f-MI mice. Ex vivo ischemic injury (isolated hearts) resulted in comparable recovery in both genotypes. Myocardial vascular density (fluorescent-labeled lectin perfusion and CD31 immunofluorescence staining) was significantly lower in the infarct areas of f/f/Cre-MI compared with f/f-MI mice. Activation of vascular endothelial growth factor receptor 2 (VEGFR2), its mRNA, and total protein levels were reduced in infarcted myocardium in ADAM17 knockdown mice. Transcriptional regulation of VEGFR2 by ADAM17 was confirmed in cocultured cardiomyocyte–fibroblast as ischemia-induced VEGFR2 expression was blocked by ADAM17-siRNA. Meanwhile, ADAM17-siRNA did not alter VEGF A bioavailability in the conditioned media. ADAM17 knockdown mice (f/f/Cre-MI) exhibited reduced nuclear factor-κB activation (DNA binding) in the infarcted myocardium, which could underlie the suppressed VEGFR2 expression in these hearts. Post MI, inflammatory response was not altered by ADAM17 downregulation. Conclusions— This study highlights the key role of cardiomyocyte ADAM17 in post-MI recovery by regulating VEGFR2 transcription and angiogenesis, thereby limiting left ventricular dilation and dysfunction. Therefore, ADAM17 upregulation, within the physiological range, could provide protective effects in ischemic cardiomyopathy.

Background— Myocardial reperfusion after ischemia (I/R), although an effective approach in rescuing the ischemic myocardium, can itself trigger several adverse effects including aberrant remodeling of the myocardium and its extracellular matrix. Tissue inhibitor of metalloproteinases (TIMPs) protect the extracellular matrix against excess degradation by matrix metalloproteinases (MMPs). TIMP4 levels are reduced in myocardial infarction; however, its causal role in progression of post-I/R injury has not been explored. Methods and Results— In vivo I/R (20-minute ischemia, 1-week reperfusion) resulted in more severe systolic and diastolic dysfunction in TIMP4 −/− mice with enhanced inflammation, oxidative stress (1 day post-I/R), hypertrophy, and interstitial fibrosis (1 week). After an initial increase in TIMP4 (1 day post-I/R), TIMP4 mRNA and protein decreased in the ischemic myocardium from wild-type mice by 1 week post-I/R and in tissue samples from patients with myocardial infarction, which correlated with enhanced activity of membrane-bound MMP, membrane-type 1 MMP. By 4 weeks post-I/R, wild-type mice showed no cardiac dysfunction, elevated TIMP4 levels (to baseline), and normalized membrane-type 1 MMP activity. TIMP4-deficient mice, however, showed exacerbated diastolic dysfunction, sustained elevation of membrane-type 1 MMP activity, and worsened myocardial hypertrophy and fibrosis. Ex vivo I/R (20- or 30-minute ischemia, 45-minute reperfusion) resulted in comparable cardiac dysfunction in wild-type and TIMP4 −/− mice. Conclusions— TIMP4 is essential for recovery from myocardial I/R in vivo, primarily because of its membrane-type 1 MMP inhibitory function. TIMP4 deficiency does not increase susceptibility to ex vivo I/R injury. Replenishment of myocardial TIMP4 could serve as an effective therapy in post-I/R recovery for patients with reduced TIMP4.