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Objective: Reported associations between kidney risk variants (G1 and G2) in APOL1 (apolipoprotein L1), encoding APOL1, and cardiovascular disease have been conflicting. We sought to explore associations of APOL1 risk variants with cause of sudden death using the CVPath Sudden Death Autopsy Registry. Approach and Results: APOL1 haplotypes and causes of sudden death, as determined through autopsy and histopathology, were obtained for 764 Black subjects. Genotyping revealed APOL1 risk alleles in 452 of 764 (59%) subjects with 347 (77%) subjects carrying one risk allele and 105 (23%) subjects harboring 2 risk alleles. APOL1 risk allele carrier status was associated with a significantly increased risk of coronary thrombosis due to plaque rupture, versus noncarriers (odds ratio for rupture, 1.655 [95% CI, 1.079–2.539]; P =0.021). Histological examinations showed coronary plaques in carriers of 2 APOL1 risk alleles had larger necrotic cores compared with noncarriers (necrotic core area/total plaque area: 46.79%±6.47% versus 20.57%±5.11%; P =0.0343 in ruptured plaques, and 41.48%±7.49% versus 18.93%±3.97%; P =0.0342 in nonruptured plaques), and immunohistochemical and immunofluorescent staining revealed APOL1-positive areas localized primarily to the necrotic core. Conclusions: APOL1 risk alleles were independently associated with an increased risk of thrombotic coronary death due to plaque rupture. Our results suggest that carriers of both 1 and 2 APOL1 risk alleles have greater accumulation of APOL1 protein within culprit plaques and greater necrotic core sizes than noncarriers. These findings suggest that APOL1 plays a role in determining plaque stability.

In this second of a 5-part Focus Seminar series, we focus on precision medicine in the context of vascular disease. The most common vascular disease worldwide is atherosclerosis, which is the primary cause of coronary artery disease, peripheral vascular disease, and a large proportion of strokes and other disorders. Atherosclerosis is a complex genetic disease that likely involves many hundreds to thousands of single nucleotide polymorphisms, each with a relatively modest effect for causing disease. Conversely, although less prevalent, there are many vascular disorders that typically involve only a single genetic change, but these changes can often have a profound effect that is sufficient to cause disease. These are termed "Mendelian vascular diseases," which include Marfan and Loeys-Dietz syndromes. Given the very different genetic basis of atherosclerosis versus Mendelian vascular diseases, this article was divided into 2 parts to cover the most promising precision medicine approaches for these disease types.

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub- phenotyping. We hypothesized that unbiased clustering of plaques based on gene expression results in an alternative categorization of late-stage atherosclerotic lesions.We analyzed the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These novel plaque phenotypes associated with clinical presentation (pIn conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in- depth transcriptomic based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to differential underlying biology of symptomatic lesions.

Significance Here, we present unequivocal evidence of physical association of AMPK holoenzymes with mitochondrial reticulum (mitoAMPK) across multiple mouse tissues with evidence of conservation in human skeletal muscle and heart. We demonstrate that mitoAMPK is activated heterogeneously across the mitochondrial reticulum by mitochondrial energetic stress. Finally, we present evidence that suggests activation of mitoAMPK in skeletal muscle is required for mitophagy. We propose that mitoAMPK responds to mitochondrial microenvironment cues to maintain energetic homeostasis through mitochondrial quality control., Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Aims De-differentiation and activation of pro-inflammatory pathways are key transitions vascular smooth muscle cells (SMCs) make during atherogenesis. Here, we explored the upstream regulators of this 'atherogenic transition'. Methods and results Genome-wide sequencing studies, including ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq, were performed on cells isolated from both murine SMC-lineage tracing models of atherosclerosis and human atherosclerotic lesions. At the bulk level, alterations in chromatin accessibility were associated with the atherogenic transitioning of lesional SMCs, especially in relation to genes that govern differentiation status and complement-dependent inflammation. Using computational biology, we observed that a transcription factor previously related to coronary artery disease, ATF3 (Activating transcription factor 3), was predicted to be an upstream regulator of genes altered during the transition. At the single-cell level, our results indicated that ATF3 is a key repressor of SMC transitioning towards the subset of cells that promote vascular inflammation by activating the complement cascade. The expression of ATF3 and complement component C3 were negatively correlated in SMCs from human atherosclerotic lesions, suggesting translational relevance. Phenome-wide association studies indicated that genetic variation that results in reduced expression of ATF3 is correlated with an increased risk for atherosclerosis, and the expression of ATF3 was significantly downregulated in humans with advanced vascular disease. Conclusion Our study indicates that the plasticity of atherosclerotic SMCs may in part be explained by dynamic changes in their chromatin architecture, which in turn may contribute to their maladaptive response to inflammation-induced stress. Translational perspective The recent CANTOS and COLCOT trials have shown that targeting inflammatory pathways lowers the risk of major adverse cardiovascular events. However, more specific targets are needed to avoid immunosuppressive side effects. Our data identify an upstream regulator of pro-inflammatory SMCs, ATF3, which is involved in the initial atherogenic transitioning of lesional SMCs. Restoring ATF3 activity may prevent the de-differentiation of SMCs and offer a novel translational approach for the suppression of complement-dependent inflammation in atherosclerotic lesions.

In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed 'fibromyocytes', rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21-a causal CAD gene-markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.

Background: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM. Methods: We used patient-derived RAF1 S257L/+ and CRISPR-Cas9–generated isogenic control inducible pluripotent stem cell (iPSC)–derived cardiomyocytes to model NS RAF1 -associated HCM and to further delineate the molecular mechanisms underlying the disease. Results: We show that mutant iPSC–derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC–derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC–derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM. Conclusions: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders.

Aims While most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation. Methods and results We carried out a genome-wide association study for MI in the UK Biobank (n∼472 000), followed by a meta-analysis with summary statistics from the CARDIoGRAMplusC4D Consortium (n∼167 000). Multiple independent replication analyses and functional approaches were used to prioritize loci and evaluate positional candidate genes. Eight novel regions were identified for MI at the genome wide significance level, of which effect sizes at six loci were more robust for MI than for CAD without the presence of MI. Confirmatory evidence for association of a locus on chromosome 1p21.3 harbouring choline-like transporter 3 (SLC44A3) with MI in the context of CAD, but not with coronary atherosclerosis itself, was obtained in Biobank Japan (n∼165 000) and 16 independent angiography-based cohorts (n∼27 000). Follow-up analyses did not reveal association of the SLC44A3 locus with CAD risk factors, biomarkers of coagulation, other thrombotic diseases, or plasma levels of a broad array of metabolites, including choline, trimethylamine N-oxide, and betaine. However, aortic expression of SLC44A3 was increased in carriers of the MI risk allele at chromosome 1p21.3, increased in ischaemic (vs. non-diseased) coronary arteries, up-regulated in human aortic endothelial cells treated with interleukin-1β (vs. vehicle), and associated with smooth muscle cell migration in vitro. Conclusions A large-scale analysis comprising ∼831 000 subjects revealed novel genetic determinants of MI and implicated SLC44A3 in the pathophysiology of vulnerable plaques.

Summary Despite the emerging evidence implying early vascular contributions to neurodegenerative syndromes, the role of vascular smooth muscle cells (VSMCs) in the pathogenesis of Alzheimer disease (AD) is still not well understood. Herein, we show that VSMCs in brains of patients with AD and animal models of the disease are deficient in multiple VSMC contractile markers which correlated with Tau accumulation in brain arterioles. Ex vivo and in vitro experiments demonstrated that VSMCs undergo dramatic phenotypic transitions under AD-like conditions, adopting pro-inflammatory phenotypes. Notably, these changes coincided with Tau hyperphosphorylation at residues Y18, T205, and S262. We also observed that VSMC dysfunction occurred in an age-dependent manner and that expression of Sm22α protein was inversely correlated with CD68 and Tau expression in brain arterioles of the 3xTg-AD and 5xFAD mice. Together, these findings further support the contribution of dysfunctional VSMCs in AD pathogenesis and nominate VSMCs as a potential therapeutic target in AD., Graphical abstract, Highlights • Loss of VSMC contractile phenotypes correlates with Tau accumulation in brain arterioles • VSMC dysfunction promotes the hyperphosphorylation of Tau protein at multiple residues • VSMC dysfunction occurs in an age-dependent manner in brain arterioles of patients with AD • Vascular smooth muscle cell is a promising therapeutic target in AD, Molecular neuroscience; Immunology; Omics

Background and AimsThe atherosclerotic plaque microenvironment is highly complex, and selective agents that modulate plaque stability or other plaque phenotypes are not yet available. We sought to investigate the human atherosclerotic cellular environment using scRNA-seq to uncover potential therapeutic approaches. We aimed to make our workflow user-friendly, reproducible, and applicable to other disease-specific scRNA-seq datasets.MethodsHere we incorporate automated cell labeling, pseudotemporal ordering, ligand-receptor evaluation, and drug-gene interaction analysis into an enhanced and reproducible scRNA-seq analysis workflow. Notably, we also developed an R Shiny based interactive web application to enable further exploration and analysis of the scRNA dataset.ResultsWe applied this analysis workflow to a human coronary artery scRNA dataset and revealed distinct derivations of chondrocyte-like and fibroblast-like cells from smooth muscle cells (SMCs), and show the key changes in gene expression along their de-differentiation path. We highlighted several key ligand-receptor interactions within the atherosclerotic environment through functional expression profiling and revealed several attractive avenues for future pharmacological repurposing in precision medicine. Further, our interactive web application, PlaqView (www.plaqview.com), allows other researchers to easily explore this dataset and benchmark applicable scRNA-seq analysis tools without prior coding knowledge.ConclusionsThese results suggest novel effects of chemotherapeutics on the atherosclerotic cellular environment and provide future avenues of studies in precision medicine. This publicly available workflow will also allow for more systematic and user-friendly analysis of scRNA datasets in other disease and developmental systems. PlaqView allows for rapid visualization and analysis of atherosclerosis scRNA-seq datasets without the need of prior coding experience. Future releases of PlaqView will feature additional larger scRNA-seq and scATAC-seq atherosclerosis-related datasets, thus providing a critical resource for the field by promoting data harmonization and biological interpretation.

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. Methods and Results: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3 . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of “dedifferentiated” vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don’t eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Rationale: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Objective: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Methods and Results: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6 −/− versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6 −/− mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. Conclusions: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.

Nonalcoholic steatohepatitis is a common disease involving chronic accumulation of fat and inflammation in the liver, often leading to advanced fibrosis, cirrhosis, and cancer. It is known that nonalcoholic steatohepatitis shares many features with atherosclerosis; however, there are still no effective therapeutics. In a recent study published in Nature, investigators demonstrated that mice lacking a high-density lipoprotein–associated gene were surprisingly protected from both steatohepatitis and atherosclerosis through the stabilization of the liver X receptor. This work reveals a timely candidate target for 2 highly prevalent cardiovascular diseases.

Coronary artery disease is a complex cardiovascular disease involving an interplay of genetic and environmental influences over a lifetime. Although considerable progress has been made in understanding lifestyle risk factors, genetic factors identified from genome-wide association studies may capture additional hidden risk undetected by traditional clinical tests. These genetic discoveries have highlighted many candidate genes and pathways dysregulated in the vessel wall, including those involving smooth muscle cell phenotypic modulation and injury responses. Here, we summarize experimental evidence for a few genome-wide significant loci supporting their roles in smooth muscle cell biology and disease. We also discuss molecular quantitative trait locus mapping as a powerful discovery and fine-mapping approach applied to smooth muscle cell and coronary artery disease-relevant tissues. We emphasize the critical need for alternative genetic strategies, including cis/trans-regulatory network analysis, genome editing, and perturbations, as well as single-cell sequencing in smooth muscle cell tissues and model organisms, under both normal and disease states. By integrating multiple experimental and analytical modalities, these multidimensional datasets should improve the interpretation of coronary artery disease genome-wide association studies and molecular quantitative trait locus signals and inform candidate targets for therapeutic intervention or risk prediction.

Objective— Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. Approach and Results— Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7 , and PLN expression positively correlated to typical SMC markers in plaques (Pearson r >0.6, P r >0.8, P PDLIM7, PLN , and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. Conclusions— We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

Background The genetic determinants of heart failure (HF) and response to medical therapy remain unknown. We hypothesized that identifying genetic variants of HF that associate with response to medical therapy would elucidate the genetic basis of cardiac function. Objectives This study sought to identify genetic variations associated with response to HF therapy. Methods This study compared extremes of response to medical therapy in 866 HF patients using a genome-wide approach that informed the systems-based design of a customized single nucleotide variant array. The effect of genotype on gene expression was measured using allele-specific luciferase reporter assays. Candidate gene transcription-deficient mice underwent echocardiography and treadmill exercise. The ability of the target gene agonist to rescue mice from chemically-induced HF was assessed with echocardiography. Results Of 866 HF patients, 136 had an ejection fraction improvement of 20% attributed to resynchronization (n = 83), revascularization (n = 7), tachycardia resolution (n = 2), alcohol cessation (n = 1), or medications (n = 43). Those with the minor allele for rs7767652, upstream of hypocretin (orexin) receptor-2 (HCRTR2), were less likely to have improved left ventricular function (odds ratio: 0.40 per minor allele; p = 3.29 × 10−5). In a replication cohort of 798 patients, those with a minor allele for rs7767652 had a lower prevalence of ejection fraction >35% (odds ratio: 0.769 per minor allele; p = 0.021). In an HF model, HCRTR2-deficient mice exhibited poorer cardiac function, worse treadmill exercise capacity, and greater myocardial scarring. Orexin, an HCRTR2 agonist, rescued function in this HF mouse model. Conclusions A systems approach identified a novel genetic contribution to human HF and a promising therapeutic agent efficacious in an HF model.

The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection., Author summary Chronic, or non-healing, wounds represent a costly burden to patients, and bacterial infection of wounds is an important driver of chronicity. A variety of bacterial species often occur in chronic wounds, but it is unknown why certain species are observed in some wound infections and not others. In this study, genetic variation of wound clinic patients was compared to the bacteria observed in their infected wounds. Through these comparisons, genetic variation in the TLN2 and ZNF521 genes was found to be associated with both the number of bacteria observed in wounds and the abundance of common pathogens (primarily Pseudomonas aeruginosa and Staphylococcus epidermidis). Moreover, Pseudomonas infected wounds were found to have fewer species present and wounds with fewer species were slower to heal. Furthermore, patient genes associated with microbiomes commonly encode proteins known to be important for cellular structures important to healing and to which bacteria directly interact. Experimental investigation of one such gene, TLN2, identified genotype-dependent differences in the expression of functionally different versions of TLN2 that is hypothesized to shape differences in cellular adhesion structures. Finally, a new statistical approach is presented in which patient biomarkers are used to predict the number of species observed during infection. Overall, our results describe how patient genetic variation influence the types of bacteria likely to infect an individual as well as influence healing.

Coronary artery disease (CAD) is the leading cause of mortality worldwide and poses a considerable public health burden. Recent genome-wide association studies (GWAS) have revealed >100 genetic loci associated with CAD susceptibility in humans. While a number of these loci harbor gene targets of currently approved therapies, such as statins and PCSK9 inhibitors, the majority of the annotated genes at these loci encode for proteins involved in vessel wall function with no known drugs available. Importantly many of the associated genes linked to vascular (smooth muscle, endothelial, and macrophage) cell processes are now organized into distinct functional pathways, e.g., vasodilation, growth factor responses, extracellular matrix and plaque remodeling, and inflammation. In this mini-review, we highlight the most recently identified loci that have predicted roles in the vessel wall and provide genetic context for pre-existing therapies as well as new drug targets informed from GWAS. With the development of new modalities to target these pathways, (e.g., antisense oligonucleotides, CRISPR/Cas9, and RNA interference) as well as the computational frameworks to prioritize or reposition therapeutics, there is great opportunity to close the gap from initial genetic discovery to clinical translation for many patients affected by this common disease.

Aims Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD. Methods and results By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques. Conclusion The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.

Atherosclerotic coronary artery disease (CAD) continues to be the leading cause of mortality and morbidity worldwide, with an estimated 40% of one’s lifetime risk attributed to genetic factors. Meta-analysis of genome-wide association studies implemented to identify this risk in human populations, has now identified rs2820315 (P=7.7E-10; OR=1.05), in the smooth muscle cell-restricted gene, Leiomodin 1 (LMOD1), as the leading genetic polymorphism associated with CAD. However, the causal mechanism by which this polymorphism is responsible for predisposition to CAD remains to be identified. Expression quantitative trait loci (eQTL) mapping in GTEx and STARNET databases revealed that carriers of the risk allele, rs34091558, which is in tight linkage disequilibrium with rs2820315, display significantly attenuated LMOD1 expression in vascular tissues than carriers of the ancestral allele. Allelic expression imbalance analyses in heterozygous HCASMC donors further demonstrated cis-acting effects of the polymorphism on LMOD1 gene expression. To determine the mechanism responsible for this reduction in LMOD1 expression, we performed position weight matrix (PWM) motif analyses and found that rs34091558 disrupts the binding site of a transcription factor called forkhead box O3 (FOXO3). Subsequent chromatin immunoprecipitation and reporter assays demonstrated reduced FOXO3 binding and transcriptional activity by the risk allele in cultured HCASMCs. Platelet-derived growth factor BB (PDGF-BB) stimulation also significantly reduced LMOD1 expression coincident with FOXO3 knockdown. Finally, both gain and loss-of-function for FOXO3 and LMOD1 in HCASMC delineated a regulatory circuit by which LMOD1 regulates SMC proliferation, migration and contraction, characteristic features of atherosclerotic lesion progression. Taken together, these results provide compelling functional evidence that: 1) rs3091558 is associated with reduced LMOD1 expression, 2) this reduction appears to be mediated through the inhibition of FOXO3 binding and 3) changes in vessel wall processes through LMOD1 dysregulation may partially explain the heritable risk for CAD.

Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-β-stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance.

Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5 ± 7.8% vs. 83.9 ± 4.7%) as well as compensatory expansion of left ventricular diameter (4.19 ± 0.19 mm vs. 3.10 ± 0.18 mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0 ± 1.8% vs. 24.6 ± 3.8%) and apoptotic cell number (1.3 ± 1.0% vs. 5.6 ± 1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.

Coronary artery disease (or coronary heart disease), is the leading cause of mortality in many of the developing as well as the developed countries of the world. Cholesterol-enriched plaques in the heart's blood vessels combined with inflammation lead to the lesion expansion, narrowing of blood vessels, reduced blood flow, and may subsequently cause lesion rupture and a heart attack. Even though several environmental risk factors have been established, such as high LDL-cholesterol, diabetes, and high blood pressure, the underlying genetic composition may substantially modify the disease risk; hence, genome composition and gene-environment interactions may be critical for disease progression. Ongoing scientific efforts have seen substantial advancements related to the fields of genetics and genomics, with the major breakthroughs yet to come. As genomics is the most rapidly advancing field in the life sciences, it is important to present a comprehensive overview of current efforts. Here, we present a summary of various genetic and genomics assays and approaches applied to coronary artery disease research.

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 Ca(2+)/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.

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., Coronary heart disease is the leading cause of death worldwide with multiple environmental and genetic risk factors. Here the authors integrate genomic, epigenomic and transcriptomic mapping to elucidate causal variation and mechanisms of known genetic associations.

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.

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall., Author Summary Coronary artery disease (CAD) is responsible for the majority of deaths in the Western world, and is due in part to environmental and metabolic factors. However, half of the risk for developing heart disease is genetically predetermined. Genome-wide association studies in human populations have identified over 100 sites in the genome that appear to be associated with CAD, however, the mechanisms by which variation in these regions are responsible for predisposition to CAD remain largely unknown. We have begun to study a gene that contributes to CAD risk, the TCF21 gene. Through genomic studies we show that this gene is involved in processes related to alterations in vascular gene expression, and in particular those related to the smooth muscle cell biology. With cell culture models, we show that TCF21 regulates the differentiation state of this cell type, which is believed critical for vascular disease. Using mouse genetic models of atherosclerotic vascular disease we provide evidence that this gene is expressed in precursor cells that migrate into the disease lesions and contribute to the formation of the fibrous cap that is believed to stabilize these lesions and prevent heart attacks.

The cyclic nucleotides cAMP and cGMP play important roles in mediating both protective and detrimental signaling in heart failure. By acting as regulators of cAMP and cGMP, the phosphodiesterases play important roles in modulating this signaling. A comparison of PDE expression between control mice and mice given transverse aortic constriction revealed that expression of phosphodiesterase 1C (PDE1C) was increased significantly by TAC, both on the mRNA and protein levels. To determine whether this was protective or maladaptive, we performed TAC on mice with a genetic deletion of PDE1C. While TAC-operated WT mice experienced significant overall cardiac hypertrophy and cardiomyocyte hypertrophy, these were reduced in PDE1C KO mice. Cardiac function, as assessed by echocardiography, was also reduced significantly in WT TAC mice, but was preserved in PDE1C KO TAC mice. Histological analysis indicated that TAC-operated PDE1C KO mice also experienced reduced cardiomyocyte apoptosis compared to WT mice, indicating a potential cardioprotective mechanism for PDE1C deletion. Cardiomyocytes isolated from PDE1C KO mice experienced reduced Ang II or Iso-induced cell death compared to WT myocytes, indicating that this was a cardiomyocytes-specific effect of PDE1C deletion. Ang II-induced cardiomyocyte cell death and apoptosis were also blocked via pharmacological PDE1 inhibition. PDE1C is able to hydrolyze either cAMP or cGMP; therefore, it seemed possible that this protective mechanism was dependent on either PKA- or PKG-mediated signaling. While PKG inhibition did not alter the protective effect of PDE1 inhibition in isolated cardiomyocytes, PKA inhibition blocked it. Overexpression studies also indicated that PDE1C is localized to the cell membrane in cardiomyocytes. Therefore, we propose that by modulating a novel, membrane-localized, anti-apoptotic, cAMP/PKA-dependent pathway in cardiomyocytes, PDE1C potentially represents a novel therapeutic target in heart failure.

Large-scale genome-wide association studies (GWAS) have identified 46 loci that are associated with coronary heart disease (CHD). Additionally, 104 independent candidate variants (false discovery rate of 5 %) have been identified (Schunkert H, Konig IR, Kathiresan S, Reilly MP, Assimes TL, Holm H et al. Nat Genet 43:333-8, 2011; Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR et al. Nat Genet 45:25-33, 2012; C4D Genetics Consortium. Nat Genet 43:339-44, 2011). The majority of the causal genes in these loci function independently of conventional risk factors. It is postulated that a number of the CHD-associated genes regulate basic processes in the vascular cells involved in atherosclerosis, and that study of the signaling pathways that are modulated in this cell type by causal regulatory variation will provide critical new insights for targeting the initiation and progression of disease. In this review, we will discuss the types of experimental approaches and data that are critical to understanding the molecular processes that underlie the disease risk at 9p21.3, TCF21, SORT1, and other CHD-associated loci.

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., Author Summary As much as half of the risk of developing coronary heart disease is genetically predetermined. Genome-wide association studies in human populations have now uncovered multiple sites of common genetic variation associated with heart disease. However, the biological mechanisms responsible for linking the disease associations with changes in gene expression are still underexplored. One of these variants occurs within the vascular developmental factor, TCF21, leading to dysregulated gene expression. Using various in silico and molecular approaches, we identify an intricate allele-specific regulatory mechanism underlying altered expression of TCF21. Notably, we observe that two apparently independent risk alleles identified in distinct populations function through a similar regulatory mechanism. Together these data suggest that conserved upstream pathways may organize the complex genetic etiology of coronary heart disease and potentially lead to new treatment opportunities.

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.

Genome-wide association studies (GWAS) to date have discovered thousands of genetic variants linked to human diseases and traits, which hold the potential to unravel the mechanisms of complex phenotypes. However, given that the majority of these associated variants reside in non-coding genomic regions, their predicted cis and trans-regulatory functions remain largely undefined. Here we show that correlation between human diseases and traits can follow geographical distribution of human populations, and that the underlying mechanism is at least partly genetically based. We report two Type 2 Diabetes (T2D) GWAS variants (rs7903146 and rs12255372) in the TCF7L2 locus that regulate expression in skin tissues but not lymphoblastoid or adipose tissues, of the KITLG gene that encodes an important regulator of melanogenesis and light hair color in European populations. We also report extensive binding events of TCF7L2 protein in the promoter region, immediate upstream region and first intron of the KITLG gene, which supports a trans-interaction between TCF7L2 and KITLG. We further show that both light hair color and T2D genetic variants are correlated with geographic latitude. Taken together, our observations suggest that natural variation in transcription factor loci in European human populations may be an underlying and confounding factor for the geographical correlation between human phenotypes, such as type 2 diabetes and light hair color. We postulate that transcription factor regulation may confound the correlation between seemingly diverse human traits. Furthermore, our findings demonstrate the importance of dissecting the genomic architecture of GWAS loci using multiple genetic and genomic datasets.

Objective— The phenotypic modulation of vascular smooth muscle cells (VSMCs) to a synthetic phenotype is vital during pathological vascular remodeling and the development of various vascular diseases. An increase in type I collagen (collagen I) has been implicated in synthetic VSMCs, and cyclic nucleotide signaling is critical in collagen I regulation. Herein, we investigate the role and underlying mechanism of cyclic nucleotide phosphodiesterase 1 (PDE1) in regulating collagen I in synthetic VSMCs. Methods and Results— The PDE1 inhibitor IC86340 significantly reduced collagen I in human saphenous vein explants undergoing spontaneous remodeling via ex vivo culture. In synthetic VSMCs, high basal levels of intracellular and extracellular collagen I protein were markedly decreased by IC86340. This attenuation was due to diminished protein but not mRNA. Inhibition of lysosome function abolished the effect of IC86340 on collagen I protein expression. PDE1C but not PDE1A is the major isoform responsible for mediating the effects of IC86340. Bicarbonate-sensitive soluble adenylyl cyclase/cAMP signaling was modulated by PDE1C, which is critical in collagen I degradation in VSMCs. Conclusion— These data demonstrate that PDE1C regulates soluble adenylyl cyclase/cAMP signaling and lysosome-mediated collagen I protein degradation, and they suggest that PDE1C plays a critical role in regulating collagen homeostasis during pathological vascular remodeling.

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2+ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2+ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2+ /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 Ca 2+ /CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca 2+ and cGMP signaling cross-talk during cardiac hypertrophy.