Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca²⁺/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis.

Reduction of oxidized methionines is emerging as a major protein repair pathway. The lack of methionine sulfoxide reductase A (MsrA) exacerbates cardiovascular disease phenotypes driven by increased oxidative stress. However, the role of MsrA on maintaining cellular homeostasis in the absence of excessive oxidative stress is less well understood.

The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. However, the mechanisms by which statins protect the vasculature from irradiation injury remain poorly understood.

Multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMCs), but its function in experimental hypertension has not been explored. The aim of this study was to determine the impact of CaMKII inhibition selectively in VSMCs on Ang II hypertension.

The essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function.

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While in vitro data regarding the role of CaMKII in the regulation of endothelial nitric oxide synthase (eNOS) are contradictory, its role in endothelial function in vivo remains unknown. Using two novel transgenic models to express CaMKII inhibitor peptides selectively in endothelium, we examined the effect of CaMKII on eNOS activation, NO production, vasomotor tone and blood pressure. Under baseline conditions, CaMKII activation was low in the aortic wall. Consistently, systolic and diastolic blood pressure, heart rate and plasma NO levels were unaltered by endothelial CaMKII inhibition. Moreover, endothelial CaMKII inhibition had no significant effect on NO-dependent vasodilation. These results were confirmed in studies of aortic rings transduced with adenovirus expressing a CaMKII inhibitor peptide. In cultured endothelial cells, bradykinin treatment produced the anticipated rapid influx of Ca2+ and transient CaMKII and eNOS activation, whereas CaMKII inhibition blocked eNOS phosphorylation on Ser-1179 and dephosphorylation at Thr-497. Ca2+/CaM binding to eNOS and resultant NO production in vitro were decreased under CaMKII inhibition. Our results demonstrate that CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro, but does not regulate NO production, vasorelaxation or blood pressure in vivo under baseline conditions.

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect, resulting from the nonspecific cytoablative actions of chemoagents, including 5-fluorouracil (5-FU) and irinotecan (CPT-11). Preventive strategies are urgently needed for the predictable CIM. Previously, we have demonstrated an important role of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) in the prevention of cyclophosphamide-induced mucositis in mice. In this study, the preventive role of rhIL-1Ra was further evaluated in 5-FU- and CPT-11-induced mucositis mouse models. rhIL-1Ra pretreatment reduced the incidence, severity, and duration of chemotherapy-induced diarrhea, through attenuating crypt apoptosis and improving crypt survival in wild-type mice, but not in IL-1RI(-/-), p53(-/-), and p21(-/-) mice. Further studies demonstrated that rhIL-1Ra promoted the cell cycle arrest of intestinal crypt epithelia (ICE) through elevating the cellular level of p21(WAF1) and p27(KIP1), which was abolished in IL-1RI(-/-) and p53(-/-) mice, and in p21(WAF1) and p27(KIP1) silenced IEC-6 cells. Importantly, the tumor growth and sensitivity to chemotherapy were not affected by rhIL-1Ra in cultures of tumor cell lines and in a syngeneic tumor-transplantation mouse model. The present study demonstrated that rhIL-1Ra effectively and specifically protected ICE from chemotoxicity through reversible reduction of the basal level of IL-1 signaling to promote normal cell cycle arrest, but not tumor cells. Our findings support the clinical development of rhIL-1Ra in the prevention of CIM.

An improved method for detecting coronary heart disease (CHD) could have substantial clinical impact. Building on the idea that systemic effects of CHD risk factors are a conglomeration of genetic and environmental factors, we use machine learning techniques and integrate genetic, epigenetic and phenotype data from the Framingham Heart Study to build and test a Random Forest classification model for symptomatic CHD. Our classifier was trained on n = 1,545 individuals and consisted of four DNA methylation sites, two SNPs, age and gender. The methylation sites and SNPs were selected during the training phase. The final trained model was then tested on n = 142 individuals. The test data comprised of individuals removed based on relatedness to those in the training dataset. This integrated classifier was capable of classifying symptomatic CHD status of those in the test set with an accuracy, sensitivity and specificity of 78%, 0.75 and 0.80, respectively. In contrast, a model using only conventional CHD risk factors as predictors had an accuracy and sensitivity of only 65% and 0.42, respectively, but with a specificity of 0.89 in the test set. Regression analyses of the methylation signatures illustrate our ability to map these signatures to known risk factors in CHD pathogenesis. These results demonstrate the capability of an integrated approach to effectively model symptomatic CHD status. These results also suggest that future studies of biomaterial collected from longitudinally informative cohorts that are specifically characterized for cardiac disease at follow-up could lead to the introduction of sensitive, readily employable integrated genetic-epigenetic algorithms for predicting onset of future symptomatic CHD.

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.

Type 2 diabetes (T2D) is associated with a strongly increased risk for restenosis after angioplasty driven by proliferation of vascular smooth muscle cells (VSMCs). Here, we sought to determine whether and how mitochondrial dysfunction in T2D drives VSMC proliferation with a focus on ROS and intracellular [Ca 2+ ] that both drive cell proliferation, occur in T2D and are regulated by mitochondrial activity.

Type 2 diabetes (T2D) is associated with increased risk of atherosclerotic vascular disease due to excessive vascular smooth muscle cell (VSMC) proliferation. Here, we investigated the role of mitochondrial dysfunction and Ca2+ levels in VSMC proliferation in T2D. VSMCs were isolated from normoglycemic and T2D-like mice induced by diet. The effects of mitochondrial Ca2+ uptake were studied using mice with selectively inhibited mitochondrial Ca2+/calmodulin-dependent kinase II (mtCaMKII) in VSMCs. Mitochondrial transition pore (mPTP) was blocked using ER-000444793. VSMCs from T2D compared to normoglycemic mice exhibited increased proliferation and baseline cytosolic Ca2+ levels ([Ca2+]cyto). T2D cells displayed lower endoplasmic reticulum Ca2+ levels, reduced mitochondrial Ca2+ entry, and increased Ca2+ leakage through the mPTP. Mitochondrial and cytosolic Ca2+ transients were diminished in T2D cells upon platelet-derived growth factor (PDGF) administration. Inhibiting mitochondrial Ca2+ uptake or the mPTP reduced VSMC proliferation in T2D, but had contrasting effects on [Ca2+]cyto. In T2D VSMCs, enhanced activation of Erk1/2 and its upstream regulators was observed, driven by elevated [Ca2+]cyto. Inhibiting mtCaMKII worsened the Ca2+ imbalance by blocking mitochondrial Ca2+ entry, leading to further increases in [Ca2+]cyto and Erk1/2 hyperactivation. Under these conditions, PDGF had no effect on VSMC proliferation. Inhibiting Ca2+-dependent signaling in the cytosol reduced excessive Erk1/2 activation and VSMC proliferation. Our findings suggest that altered Ca2+ handling drives enhanced VSMC proliferation in T2D, with mitochondrial dysfunction contributing to this process.

Two-pore K(+) channels have emerged as potential targets to selectively regulate cardiac cell membrane excitability; however, lack of specific inhibitors and relevant animal models has impeded the effort to understand the role of 2-pore K(+) channels in the heart and their potential as a therapeutic target. The objective of this study was to determine the role of mechanosensitive 2-pore K(+) channel family member TREK-1 in control of cardiac excitability.

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.

Radiation therapy strongly increases the risk of atherosclerotic vascular disease, such as carotid stenosis. Radiation induces DNA damage, in particular in mitochondria, but the upstream and downstream signaling events are poorly understood. The objective of this study was to define such mechanisms.

Staphylococcus aureus infective endocarditis (IE) is a fast-progressing and tissue-destructive infection of the cardiac endothelium. The superantigens (SAgs) toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin C (SEC), and the toxins encoded by the enterotoxin gene cluster (egc) play a novel and essential role in the etiology of S. aureus IE. Recent studies indicate that SAgs act at the infection site to cause tissue pathology and promote vegetation growth. The underlying mechanism of SAg involvement has not been clearly defined. In SAg-mediated responses, immune cell priming is considered a primary triggering event leading to endothelial cell activation and altered function. Utilizing immortalized human aortic endothelial cells (iHAECs), we demonstrated that TSST-1 directly activates iHAECs, as documented by upregulation of vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1). TSST-1-mediated activation results in increased monolayer permeability and defects in vascular reendothelialization. Yet stimulation of iHAECs with TSST-1 fails to induce interleukin-8 (IL-8) and IL-6 production. Furthermore, simultaneous stimulation of iHAECs with TSST-1 and lipopolysaccharide (LPS) inhibits LPS-mediated IL-8 and IL-6 secretion, even after pretreatment with either of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β. IL-8 suppression is not mediated by TSST-1 binding to its canonical receptor major histocompatibility complex class II (MHC-II), supporting current evidence for a nonhematopoietic interacting site on SAgs. Together, the data suggest that TSST-1 differentially regulates cell-bound and secreted markers of endothelial cell activation that may result in dysregulated innate immune responses during S. aureus IE. Endothelial changes resulting from the action of SAgs can therefore directly contribute to the aggressive nature of S. aureus IE and development of life-threatening complications.

The purpose of this study was to investigate the applicability of laser speckle flowgraphy (LSFG) for a longitudinal study of blood flow parameters in mice before, during, and after continuous infusion of angiotensin-II.

Background Sorbin and SH3 domain containing 2 (Sorbs2) protein is a cytoskeletal adaptor with an emerging role in cardiac biology and disease; yet, its potential relevance to adult-onset cardiomyopathies remains underexplored. Sorbs2 global knockout mice display lethal arrhythmogenic cardiomyopathy; however, the causative mechanisms remain unclear. Herein, we examine Sorbs2 dysregulation in heart failure, characterize novel Sorbs2 cardiomyocyte-specific knockout mice (Sorbs2-cKO), and explore associations between Sorbs2 genetic variations and human cardiovascular disease. Methods and Results Bioinformatic analyses show myocardial Sorbs2 mRNA is consistently upregulated in humans with adult-onset cardiomyopathies and in heart failure models. We generated Sorbs2-cKO mice and report that they develop progressive systolic dysfunction and enlarged cardiac chambers, and they die with congestive heart failure at about 1 year old. After 3 months, Sorbs2-cKO mice begin to show atrial enlargement and P-wave anomalies, without dysregulation of action potential-associated ion channel and gap junction protein expressions. After 6 months, Sorbs2-cKO mice exhibit impaired contractility in dobutamine-treated hearts and skinned myofibers, without dysregulation of contractile protein expressions. From our comprehensive survey of potential mechanisms, we found that within 4 months, Sorbs2-cKO hearts have defective microtubule polymerization and compensatory upregulation of structural cytoskeletal and adapter proteins, suggesting that this early intracellular structural remodeling is responsible for contractile dysfunction. Finally, we identified genetic variants that associate with decreased Sorbs2 expression and human cardiac phenotypes, including conduction abnormalities, atrial enlargement, and dilated cardiomyopathy, consistent with Sorbs2-cKO mice phenotypes. Conclusions Our studies show that Sorbs2 is essential for maintaining structural integrity in cardiomyocytes, likely through strengthening the interactions between microtubules and other cytoskeletal proteins at cross-link sites.

Background Sex-specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries. Methods and Results Enhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura-2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production. Conclusions Our data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.

The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease.