Statins improve endothelial function via suppression of epigenetic-driven EndMT.

The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.

Recent advances in regulating the proliferation or maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

In the last decade, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM)-based cell therapy has drawn broad attention as a potential therapy for treating injured hearts. However, mass production of hiPSC-CMs remains challenging, limiting their translational potential in regenerative medicine. Therefore, multiple strategies including cell cycle regulators, small molecules, co-culture systems, and epigenetic modifiers have been used to improve the proliferation of hiPSC-CMs. On the other hand, the immaturity of these proliferative hiPSC-CMs could lead to lethal arrhythmias due to their limited ability to functionally couple with resident cardiomyocytes. To achieve functional maturity, numerous methods such as prolonged culture, biochemical or biophysical stimulation, in vivo transplantation, and 3D culture approaches have been employed. In this review, we summarize recent approaches used to promote hiPSC-CM proliferation, and thoroughly review recent advances in promoting hiPSC-CM maturation, which will serve as the foundation for large-scale production of mature hiPSC-CMs for future clinical applications.

Protocol to generate cardiac pericytes from human induced pluripotent stem cells.

Cardiac pericytes are a critical yet enigmatic cell type within the coronary microvasculature. Since primary human cardiac pericytes are not readily accessible, we present a protocol to generate them from human induced pluripotent stem cells (hiPSCs). Our protocol involves several steps, including the generation of intermediate cell types such as mid-primitive streak, lateral plate mesoderm, splanchnic mesoderm, septum transversum, and epicardium, before deriving cardiac pericytes. With hiPSC-derived cardiac pericytes, researchers can decipher the mechanisms underlying coronary microvascular dysfunction. For complete details on the use and execution of this protocol, please refer to Shen et al.1.

Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease.

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 contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

Generation of two induced pluripotent stem cell lines from dilated cardiomyopathy patients carrying TTN mutations.

Dilated cardiomyopathy (DCM) is a common heart disease that can lead to heart failure and sudden cardiac death. Mutations in the TTN gene are the most frequent cause of DCM. Here, we generated two human induced pluripotent stem cell (iPSC) lines from the peripheral blood mononuclear cells (PBMCs) of two DCM patients carrying c.94816C>T and c.104188A>G mutations in TTN, respectively. The two lines exhibited a normal morphology, full expression of pluripotency markers, a normal karyotype and the ability of trilineage differentiation. The two lines can serve as useful tools for drug screening and mechanism studies on DCM.

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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.

Generation of Embryonic Origin-Specific Vascular Smooth Muscle Cells from Human Induced Pluripotent Stem Cells.

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.

Generation of Quiescent Cardiac Fibroblasts Derived from Human Induced Pluripotent Stem Cells.

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.

Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying TNNI3 mutations.

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.

Loss of TIMP4 (Tissue Inhibitor of Metalloproteinase 4) Promotes Atherosclerotic Plaque Deposition in the Abdominal Aorta Despite Suppressed Plasma Cholesterol Levels.

[Figure: see text].

Generation of Vascular Smooth Muscle Cells From Induced Pluripotent Stem Cells: Methods, Applications, and Considerations.

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.

Extracellular matrix, regional heterogeneity of the aorta, and aortic aneurysm.

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.

Generation of Quiescent Cardiac Fibroblasts From Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis.

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.

Apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs.

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.

Cell-Specific Functions of ADAM17 Regulate the Progression of Thoracic Aortic Aneurysm.

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; Adam17f/f/Sm22Cre/+ ) or endothelial cells (Adam17f/f/Tie2Cre/+ ). 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.

TIMP3 deficiency exacerbates iron overload-mediated cardiomyopathy and liver disease.

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-1ß 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.

Loss of smooth muscle cell disintegrin and metalloproteinase 17 transiently suppresses angiotensin II-induced hypertension and end-organ damage.

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.

A Disintegrin and Metalloprotease-17 Regulates Pressure Overload-Induced Myocardial Hypertrophy and Dysfunction Through Proteolytic Processing of Integrin ß1.

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 ß1 was much greater with ADAM17 knockdown, concomitant with the greater activation of the focal adhesion kinase pathway, suggesting that integrin ß1 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 ß1 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 ß1-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 ß1, a novel substrate for ADAM17. This function of ADAM17 is selective for pressure overload-induced myocardial hypertrophy and dysfunction, and not agonist-induced hypertrophy.