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Peroxynitrite is a damaging agent of oxidative stress that has been difficult to monitor in living cells. Here, an isatin-based chemiluminescent probe for peroxynitrite is reported.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure–activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL (22) and 1 μg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Azanone (HNO) is a reactive nitrogen species with pronounced biological activity and high therapeutic potential for cardiovascular dysfunction. A critical barrier to understanding the biology of HNO and furthering clinical development is the quantification and real‐time monitoring of its delivery in living systems. Herein, we describe the design and synthesis of the first chemiluminescent probe for HNO, HNOCL‐1, which can detect HNO generated from concentrations of Angeli's salt as low as 138 nm with high selectivity based on the reaction with a phosphine group to form a self‐cleavable azaylide intermediate. We have capitalized on this high sensitivity to develop a generalizable kinetics‐based approach, which provides real‐time quantitative measurements of HNO concentration at the picomolar level. HNOCL‐1 can monitor dynamics of HNO delivery in living cells and tissues, demonstrating the versatility of this method for tracking HNO in living systems.

Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD risk.

BackgroundMicroRNA miR‐214 has been implicated in many biological cellular functions, but the impact of miR‐214 and its target genes on vascular smooth muscle cell (VSMC) proliferation, migration, and neointima smooth muscle cell hyperplasia is unknown.Methods and ResultsExpression of miR‐214 was closely regulated by different pathogenic stimuli in VSMCs through a transcriptional mechanism and decreased in response to vascular injury. Overexpression of miR‐214 in serum‐starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miR‐214 dramatically increased VSMC proliferation and migration. Gene and protein biochemical assays, including proteomic analyses, showed that NCK associated protein 1 (NCKAP1)—a major component of the WAVE complex that regulates lamellipodia formation and cell motility—was negatively regulated by miR‐214 in VSMCs. Luciferase assays showed that miR‐214 substantially repressed wild‐type but not the miR‐214 binding site mutated version of NCKAP1 3′ untranslated region luciferase activity in VSMCs. This result confirmed that NCKAP1 is the functional target of miR‐214 in VSMCs. NCKAP1 knockdown in VSMCs recapitulates the inhibitory effects of miR‐214 overexpression on actin polymerization, cell migration, and proliferation. Data from cotransfection experiments also revealed that inhibition of NCKAP1 is required for miR‐214–mediated lamellipodia formation, cell motility, and growth. Importantly, locally enforced expression of miR‐214 in the injured vessels significantly reduced NCKAP1 expression levels, inhibited VSMC proliferation, and prevented neointima smooth muscle cell hyperplasia after injury.ConclusionsWe uncovered an important role of miR‐214 and its target gene NCKAP1 in modulating VSMC functions and neointima hyperplasia. Our findings suggest that miR‐214 represents a potential therapeutic target for vascular diseases.

BackgroundTo investigate whether neutrophil elastase (NE) plays a causal role in atherosclerosis, and the molecular mechanisms involved.Methods and Results NE genetic–deficient mice (Apolipoprotein E−/−/NE −/− mice), bone marrow transplantation, and a specific NE inhibitor (GW311616A) were employed in this study to establish the causal role of NE in atherosclerosis. Aortic expression of NE mRNA and plasma NE activity was significantly increased in high‐fat diet (HFD)–fed wild‐type (WT) (Apolipoprotein E−/−) mice but, as expected, not in NE‐deficient mice. Selective NE knockout markedly reduced HFD‐induced atherosclerosis and significantly increased indicators of atherosclerotic plaque stability. While plasma lipid profiles were not affected by NE deficiency, decreased levels of circulating proinflammatory cytokines and inflammatory monocytes (Ly6Chi/CD11b+) were observed in NE‐deficient mice fed with an HFD for 12 weeks as compared with WT. Bone marrow reconstitution of WT mice with NE −/− bone marrow cells significantly reduced HFD‐induced atherosclerosis, while bone marrow reconstitution of NE −/− mice with WT bone marrow cells restored the pathological features of atherosclerotic plaques induced by HFD in NE‐deficient mice. In line with these findings, pharmacological inhibition of NE in WT mice through oral administration of NE inhibitor GW311616A also significantly reduced atherosclerosis. Mechanistically, we demonstrated that NE promotes foam cell formation by increasing ATP‐binding cassette transporter ABCA1 protein degradation and inhibiting macrophage cholesterol efflux.ConclusionsWe outlined a pathogenic role for NE in foam cell formation and atherosclerosis development. Consequently, inhibition of NE may represent a potential therapeutic approach to treating cardiovascular disease.

Cbx3 modulates VSMC contractile and collagen gene expression, as well as VSMC proliferation, migration, and apoptosis via a Notch3 pathway, and plays an important role in controlling injury-induced neointima formation.