No abstract

BACKGROUND For more than a decade, risk stratification for hypertrophic cardiomyopathy has been enhanced by targeted genetic testing. Using sequencing results, clinicians routinely assess the risk of hypertrophic cardiomyopathy in a patient’s relatives and diagnose the condition in patients who have ambiguous clinical presentations. However, the benefits of genetic testing come with the risk that variants may be misclassified. METHODS Using publicly accessible exome data, we identified variants that have previously been considered causal in hypertrophic cardiomyopathy and that are overrepresented in the general population. We studied these variants in diverse populations and reevaluated their initial ascertainments in the medical literature. We reviewed patient records at a leading genetic-testing laboratory for occurrences of these variants during the near-decade-long history of the laboratory. RESULTS Multiple patients, all of whom were of African or unspecified ancestry, received positive reports, with variants misclassified as pathogenic on the basis of the understanding at the time of testing. Subsequently, all reported variants were recategorized as benign. The mutations that were most common in the general population were significantly more common among black Americans than among white Americans (P<0.001). Simulations showed that the inclusion of even small numbers of black Americans in control cohorts probably would have prevented these misclassifications. We identified methodologic shortcomings that contributed to these errors in the medical literature. CONCLUSIONS The misclassification of benign variants as pathogenic that we found in our study shows the need for sequencing the genomes of diverse populations, both in asymptomatic controls and the tested patient population. These results expand on current guidelines, which recommend the use of ancestry-matched controls to interpret variants. As additional populations of different ancestry backgrounds are sequenced, we expect variant reclassifications to increase, particularly for ancestry groups that have historically been less well studied. (Funded by the National Institutes of Health.)

Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanisms by which aldosterone promotes endothelial dysfunction remain unknown. Glucose-6-phosphate dehydrogenase (G6PD) modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO(*)). Here we show that aldosterone (10(-9)-;10(-7) mol/l) decreased endothelial G6PD expression and activity in vitro, resulting in increased oxidant stress and decreased NO(*) levels-similar to what is observed in G6PD-deficient endothelial cells. Aldosterone decreased G6PD expression by increasing expression of the cyclic AMP-response element modulator (CREM) to inhibit cyclic AMP-response element binding protein (CREB)-mediated G6PD transcription. In vivo, infusion of aldosterone decreased vascular G6PD expression and impaired vascular reactivity. These effects were abrogated by spironolactone or vascular gene transfer of G6pd. These findings demonstrate that aldosterone induces a G6PD-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6PD activity.

Background Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO•) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ETB), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species (ROS) generation and decreasing NO• levels. We hypothesized that aldosterone modulates PAH by disrupting ETB-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress. Methods and Results In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO• metabolites in the absence of left heart failure. In human pulmonary artery endothelial cells (HPAECs), endothelin-1 increased aldosterone levels via PGC-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased ROS production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ETB to decrease endothelin-1-stimulated eNOS activity. Substitution of ETB-Cys405 with alanine improved ETB-dependent NO• synthesis under conditions of oxidant stress, confirming that Cys405 is a redox sensitive thiol that is necessary for ETB-eNOS signaling. In HPAECs, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated ROS generation and restored ETB-dependent NO• production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in two animal models of PAH in vivo. Conclusions Our findings demonstrate that aldosterone modulates an ETB cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO• and promote PAH.

Background Pulmonary hypertension (PH) is associated with increased morbidity across the cardiopulmonary disease spectrum. Based largely on expert consensus opinion, PH is defined by a mean pulmonary artery pressure (mPAP) ≥25 mmHg. Although mPAP levels below this threshold are common among populations at risk for PH, the relevance of mPAP <25 mmHg to clinical outcome is unknown. Methods and Results We analyzed retrospectively all US veterans undergoing right heart catheterization (RHC)(2007–2012) in the Veterans Affairs health care system (N=21,727; 908 day median follow-up). Cox proportional hazards models were used to evaluate the association between mPAP and outcomes of all-cause mortality and hospitalization, adjusted for clinical covariates. When treating mPAP as a continuous variable, the mortality hazard increased beginning at 19 mmHg (HR=1.183, 95% CI [1.004–1.393]) relative to 10 mmHg. Therefore, patients were stratified into three groups: referent (≤18 mmHg; N=4,207), borderline PH (19–24 mmHg; N=5,030), and PH (≥25 mmHg; N=12,490). The adjusted mortality hazard was increased for borderline PH (HR=1.23, 95% CI [1.12–1.36], P<0.0001) and PH (HR=2.16, 95% CI [1.96–2.38], P<0.0001) compared to the referent group. The adjusted hazard for hospitalization was also increased in borderline PH (HR=1.07, 95% CI [1.01–1.12], P=0.0149) and PH (HR=1.15, 95% CI [1.09–1.22], P<0.0001). The borderline PH cohort remained at increased risk for mortality after excluding the following high-risk subgroups: patients with pulmonary artery wedge pressure >15 mmHg, pulmonary vascular resistance ≥3.0 Wood units, or inpatient status at the time of RHC. Conclusions These data illustrate a continuum of risk according to mPAP level, and that borderline PH is associated with increased mortality and hospitalization. Future investigations are needed to test the generalizability of our findings to other populations and study the effect of treatment on outcome in borderline PH.

Background Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA (miRNA) are potential candidates for coordinated regulation of these disease stimuli. Methods and Results Using a network biology approach, we identify miRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying miRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haplo insufficiency of the BMP Receptor Type 2 (BMPRII). To validate these predictions, we have found that hypoxia and BMPRII signaling independently up-regulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 down-regulates BMPRII expression. Furthermore, miR-21 directly represses RhoB expression and Rho kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is up-regulated in pulmonary tissue from several rodent models of PH and in humans with PH. Upon induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. Conclusions A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying miRNA in PH.

The unique biochemical profile of homocysteine is characterized by chemical reactivity supporting a wide range of molecular effects, and a tendency to promote oxidant stress-induced cellular toxicity. Numerous epidemiological reports have established hyperhomocysteinemia as an independent risk factor for cardiovascular disease, cerebrovascular disease, dementia-type disorders, and osteoporosis-associated fractures. Although combined folic acid and B-vitamin therapy substantially reduces homocysteine levels, results from randomized placebo-controlled clinical trials testing the effect of vitamin therapy on outcome in these diseases are mixed, but have generally fallen short of expectations. These results have led some to abandon homocysteine monitoring in the management of patients with cardiovascular or cognitive disorders. These trials, however, have generally included patients with only mildly elevated homocysteine levels, and have not addressed several clinical scenarios in which homocysteine level reduction may be effective, including the primary prevention of atherothrombotic disease in individuals at low-or intermediate-risk, or those with severe hyperhomocysteinemia.

Background Pulmonary arterial hypertension (PAH) is characterized by dysregulated proliferation of pulmonary artery smooth muscle cells leading to (mal)adaptive vascular remodeling. In the systemic circulation, vascular injury is associated with downregulation of sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and alterations in Ca2+ homeostasis in vascular smooth muscle cells that stimulate proliferation. We, therefore, hypothesized that downregulation of SERCA2a is permissive for pulmonary vascular remodeling and the development of PAH. Methods and Results SERCA2a expression was decreased significantly in remodeled pulmonary arteries from patients with PAH and the rat monocrotaline model of PAH in comparison with controls. In human pulmonary artery smooth muscle cells in vitro, SERCA2a overexpression by gene transfer decreased proliferation and migration significantly by inhibiting NFAT/STAT3. Overexpresion of SERCA2a in human pulmonary artery endothelial cells in vitro increased endothelial nitric oxide synthase expression and activation. In monocrotaline rats with established PAH, gene transfer of SERCA2a via intratracheal delivery of aerosolized adeno-associated virus serotype 1 (AAV1) carrying the human SERCA2a gene (AAV1.SERCA2a) decreased pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and fibrosis in comparison with monocrotaline-PAH rats treated with a control AAV1 carrying β-galactosidase or saline. In a prevention protocol, aerosolized AAV1.SERCA2a delivered at the time of monocrotaline administration limited adverse hemodynamic profiles and indices of pulmonary and cardiac remodeling in comparison with rats administered AAV1 carrying β-galactosidase or saline. Conclusions Downregulation of SERCA2a plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PAH. Selective pulmonary SERCA2a gene transfer may offer benefit as a therapeutic intervention in PAH.