Clint L. Miller

CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or ‘efferocytosis’. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

Role of Ca2+/Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase 1 in Mediating Cardiomyocyte Hypertrophy

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/calmodulin (CaM)–activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca2+/CaM–stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca2+/CaM–stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG–dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Regulation of Phosphodiesterase 3 and Inducible cAMP Early Repressor in the Heart

Growing evidence suggests that multiple spatially, temporally, and functionally distinct pools of cyclic nucleotides exist and regulate cardiac performance, from acute myocardial contractility to chronic gene expression and cardiac structural remodeling. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cyclic GMP, regulate the amplitude, duration, and compartmentation of cyclic nucleotide–mediated signaling. In particular, PDE3 enzymes play a major role in regulating cAMP metabolism in the cardiovascular system. PDE3 inhibitors, by raising cAMP content, have acute inotropic and vasodilatory effects in treating congestive heart failure but have increased mortality in long-term therapy. PDE3A expression is downregulated in human and animal failing hearts. In vitro, inhibition of PDE3A function is associated with myocyte apoptosis through sustained induction of a transcriptional repressor ICER (inducible cAMP early repressor) and thereby inhibition of antiapoptotic molecule Bcl-2 expression. Sustained induction of ICER may also cause the change of other protein expression implicated in human and animal failing hearts. These data suggest that the downregulation of PDE3A observed in failing hearts may play a causative role in the progression of heart failure, in part, by inducing ICER and promoting cardiac myocyte dysfunction. Hence, strategies that maintain PDE3A function may represent an attractive approach to circumvent myocyte apoptosis and cardiac dysfunction.

Loss of CDKN2B Promotes p53-Dependent Smooth Muscle Cell Apoptosis and Aneurysm Formation

Objective—Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. Methods and Results—Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow–derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. Conclusion—These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis

Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.

Cyclic nucleotide phosphodiesterase 1A: a key regulator of cardiac fibroblast activation and extracellular matrix remodeling in the heart

Cardiac fibroblasts become activated and differentiate to smooth muscle-like myofibroblasts in response to hypertension and myocardial infarction (MI), resulting in extracellular matrix (ECM) remodeling, scar formation and impaired cardiac function. cAMP and cGMP-dependent signaling have been implicated in cardiac fibroblast activation and ECM synthesis. Dysregulation of cyclic nucleotide phosphodiesterase (PDE) activity/expression is also associated with various diseases and several PDE inhibitors are currently available or in development for treating these pathological conditions. The objective of this study is to define and characterize the specific PDE isoform that is altered during cardiac fibroblast activation and functionally important for regulating myofibroblast activation and ECM synthesis. We have found that Ca2+/calmodulin-stimulated PDE1A isoform is specifically induced in activated cardiac myofibroblasts stimulated by Ang II and TGF-β in vitro as well as in vivo within fibrotic regions of mouse, rat, and human diseased hearts. Inhibition of PDE1A function via PDE1-selective inhibitor or PDE1A shRNA significantly reduced Ang II or TGF-β-induced myofibroblast activation, ECM synthesis, and pro-fibrotic gene expression in rat cardiac fibroblasts. Moreover, the PDE1 inhibitor attenuated isoproterenol-induced interstitial fibrosis in mice. Mechanistic studies revealed that PDE1A modulates unique pools of cAMP and cGMP, predominantly in perinuclear and nuclear regions of cardiac fibroblasts. Further, both cAMP-Epac-Rap1 and cGMP-PKG signaling was involved in PDE1A-mediated regulation of collagen synthesis. These results suggest that induction of PDE1A plays a critical role in cardiac fibroblast activation and cardiac fibrosis, and targeting PDE1A may lead to regression of the adverse cardiac remodeling associated with various cardiac diseases.

Targeting Cyclic Nucleotide Phosphodiesterase in the Heart: Therapeutic Implications

The second messengers, cAMP and cGMP, regulate a number of physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression and cardiac structural remodeling. Emerging evidence suggests that multiple spatiotemporally distinct pools of cyclic nucleotides can discriminate specific cellular functions from a given cyclic nucleotide-mediated signal. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic AMP and/or cyclic GMP, control the amplitude, duration, and compartmentation of cyclic nucleotide signaling. To date, more than 60 different isoforms have been described and grouped into 11 broad families (PDE1–PDE11) based on differences in their structure, kinetic and regulatory properties, as well as sensitivity to chemical inhibitors. In the heart, PDE isozymes from at least six families have been investigated. Studies using selective PDE inhibitors and/or genetically manipulated animals have demonstrated that individual PDE isozymes play distinct roles in the heart by regulating unique cyclic nucleotide signaling microdomains. Alterations of PDE activity and/or expression have also been observed in various cardiac disease models, which may contribute to disease progression. Several family-selective PDE inhibitors have been used clinically or pre-clinically for the treatment of cardiac or vascular-related diseases. In this review, we will highlight both recent advances and discrepancies relevant to cardiovascular PDE expression, pathophysiological function, and regulation. In particular, we will emphasize how these properties influence current and future development of PDE inhibitors for the treatment of pathological cardiac remodeling and dysfunction.

Coronary Heart Disease-Associated Variation in TCF21 Disrupts a miR-224 Binding Site and miRNA-Mediated Regulation

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3′ untranslated region (3′-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3′ UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.

Disease-related growth factor and embryonic signaling pathways modulate an enhancer of TCF21 expression at the 6q23.2 coronary heart disease locus

Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor TCF21 gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a cis-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered TCF21 expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at TCF21.

Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci

Coronary artery disease (CAD) is the leading cause of mortality and morbidity, driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies have identified >150 loci associated with CAD and myocardial infarction susceptibility in humans. A majority of these variants reside in non-coding regions and are co-inherited with hundreds of candidate regulatory variants, presenting a challenge to elucidate their functions. Herein, we use integrative genomic, epigenomic and transcriptomic profiling of perturbed human coronary artery smooth muscle cells and tissues to begin to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps, we prioritize 64 candidate variants and perform allele-specific binding and expression analyses at seven top candidate loci: 9p21.3, SMAD3, PDGFD, IL6R, BMP1, CCDC97/TGFB1 and LMOD1. We validate our findings in expression quantitative trait loci cohorts, which together reveal new links between CAD associations and regulatory function in the appropriate disease context.