Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.

In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These phenotypic switches confound the interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we used poly(ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic hearts, respectively. Low hydrogel stiffness converted activated CFs into a quiescent state with a reduced abundance of α-smooth muscle actin (α-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated ß-galactosidase (SA-ß-Gal) activity and increased expression of p16 and p21, which are antiproliferative markers of senescence. Using dynamically stiffening hydrogels with phototunable cross-linking capabilities, we demonstrate that premature, substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low-stiffness hydrogels, with a reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-κB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment in which they reside.NEW & NOTEWORTHY Our findings highlight the advantages and pitfalls associated with culturing cardiac fibroblasts on hydrogels of varying stiffness. The findings also define stiffness-dependent signaling and transcriptional networks in cardiac fibroblasts.

Transcatheter aortic valve replacements alter circulating serum factors to mediate myofibroblast deactivation.

The transcatheter aortic valve replacement (TAVR) procedure has emerged as a minimally invasive treatment for patients with aortic valve stenosis (AVS). However, alterations in serum factor composition and biological activity after TAVR remain unknown. Here, we quantified the systemic inflammatory effects of the TAVR procedure and hypothesized that alterations in serum factor composition would modulate valve and cardiac fibrosis. Serum samples were obtained from patients with AVS immediately before their TAVR procedure (pre-TAVR) and about 1 month afterward (post-TAVR). Aptamer-based proteomic profiling revealed alterations in post-TAVR serum composition, and ontological analysis identified inflammatory macrophage factors implicated in myofibroblast activation and deactivation. Hydrogel biomaterials used as valve matrix mimics demonstrated that post-TAVR serum reduced myofibroblast activation of valvular interstitial cells relative to pre-TAVR serum from the same patient. Transcriptomics and curated network analysis revealed a shift in myofibroblast phenotype from pre-TAVR to post-TAVR and identified p38 MAPK signaling as one pathway involved in pre-TAVR-mediated myofibroblast activation. Post-TAVR serum deactivated valve and cardiac myofibroblasts initially exposed to pre-TAVR serum to a quiescent fibroblast phenotype. Our in vitro deactivation data correlated with patient disease severity measured via echocardiography and multimorbidity scores, and correlations were dependent on hydrogel stiffness. Sex differences in cellular responses to male and female sera were also observed and may corroborate clinical observations regarding sex-specific TAVR outcomes. Together, alterations in serum composition after TAVR may lead to an antifibrotic fibroblast phenotype, which suggests earlier interventions may be beneficial for patients with advanced AVS to prevent further disease progression.

Contemporary Use of Embolic Protection Devices During Saphenous Vein Graft Intervention.

BACKGROUND: Guidelines recommend use of embolic protection devices during percutaneous coronary intervention of saphenous vein grafts, but the use of these devices in contemporary practice is unclear. We thus sought to evaluate the patient characteristics and clinical outcomes associated with embolic protection device use during contemporary saphenous vein graft percutaneous coronary intervention. METHODS AND RESULTS: We identified patients undergoing isolated saphenous vein graft percutaneous coronary intervention in the Veterans Affairs Healthcare System from January 2008 to June 2017. Patient and procedural characteristics associated with embolic protection device use were assessed, as well as unmeasured site variation. A propensity-matched cohort was constructed to compare outcomes at 30 days, including unsuccessful intervention, periprocedural myocardial infarction, and death. We identified 7266 vein graft interventions, and embolic protection was used in 37.9% of cases, with a significant decline over time ( P=0.001) that was most pronounced from 2014 to 2017 ( P<0.001). There was significant institutional variation in the use of embolic protection, with a 15.50 (95% credible interval, 9.21-29.71)-fold difference in odds of device use by changing facilities independent of patient or procedural factors. Use of embolic protection was associated with reduced risk of unsuccessful intervention (odds ratio, 0.27; 95% credible interval, 0.17-0.42) and 30-day mortality (odds ratio, 0.56; 95% credible interval, 0.36-0.87). CONCLUSIONS: Use of embolic protection is decreasing with time and occurs in less than half of vein graft interventions. There is significant site variation in the use of embolic protection independent of patient characteristics, suggesting opportunities for the development of uniform practices to improve outcomes among those undergoing saphenous vein graft percutaneous coronary intervention.

Overlapping and Divergent Actions of Structurally Distinct Histone Deacetylase Inhibitors in Cardiac Fibroblasts.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development.

Reversal of severe angioproliferative pulmonary arterial hypertension and right ventricular hypertrophy by combined phosphodiesterase-5 and endothelin receptor inhibition.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) are treated with vasodilators, including endothelin receptor antagonists (ERAs), phosphodiesterase-5 (PDE-5) inhibitors, soluble guanylyl cyclase activators, and prostacyclin. Despite recent advances in pharmacotherapy for individuals with PAH, morbidity and mortality rates in this patient population remain unacceptably high. Here, we tested the hypothesis that combination therapy with two PAH drugs that target distinct biochemical pathways will provide superior efficacy relative to monotherapy in the rat SU5416 plus hypoxia (SU-Hx) model of severe angioproliferative PAH, which closely mimics the human condition. METHODS: Male Sprague Dawley rats were injected with a single dose of SU5416, which is a VEGF receptor antagonist, and exposed to hypobaric hypoxia for three weeks. Rats were subsequently housed at Denver altitude and treated daily with the PDE-5 inhibitor, tadalafil (TAD), the type A endothelin receptor (ETA) antagonist, ambrisentan (AMB), or a combination of TAD and AMB for four additional weeks. RESULTS: Monotherapy with TAD or AMB led to modest reductions in pulmonary arterial pressure (PAP) and right ventricular (RV) hypertrophy. In contrast, echocardiography and invasive hemodynamic measurements revealed that combined TAD/AMB nearly completely reversed pulmonary hemodynamic impairment, RV hypertrophy, and RV functional deficit in SU-Hx rats. Efficacy of TAD/AMB was associated with dramatic reductions in pulmonary vascular remodeling, including suppression of endothelial cell plexiform lesions, which are common in human PAH. CONCLUSIONS: Combined therapy with two vasodilators that are approved for the treatment of human PAH provides unprecedented efficacy in the rat SU-Hx preclinical model of severe, angioproliferative PAH.

HDAC6 contributes to pathological responses of heart and skeletal muscle to chronic angiotensin-II signaling.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

Targeting cardiac fibroblasts to treat fibrosis of the heart: focus on HDACs.

Cardiac fibrosis is implicated in numerous physiologic and pathologic conditions, including scar formation, heart failure and cardiac arrhythmias. However the specific cells and signaling pathways mediating this process are poorly understood. Lysine acetylation of nucleosomal histone tails is an important mechanism for the regulation of gene expression. Additionally, proteomic studies have revealed that thousands of proteins in all cellular compartments are subject to reversible lysine acetylation, and thus it is becoming clear that this post-translational modification will rival phosphorylation in terms of biological import. Acetyl groups are conjugated to lysine by histone acetyltransferases (HATs) and removed from lysine by histone deacetylases (HDACs). Recent studies have shown that pharmacologic agents that alter lysine acetylation by targeting HDACs have the remarkable ability to block pathological fibrosis. Here, we review the current understanding of cardiac fibroblasts and the fibrogenic process with respect to the roles of lysine acetylation in the control of disease-related cardiac fibrosis. Potential for small molecule HDAC inhibitors as anti-fibrotic therapeutics that target cardiac fibroblasts is highlighted. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium."

Class I HDACs regulate angiotensin II-dependent cardiac fibrosis via fibroblasts and circulating fibrocytes.

Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells.