Arpeeta Sharma

A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice.

Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A-Cav-1 and a mutant cell-permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1-deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A-Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release.

Derivative of bardoxolone methyl, dh404, in an inverse dose-dependent manner lessens diabetes-associated atherosclerosis and improves diabetic kidney disease.

Oxidative stress and inflammation are inextricably linked and play essential roles in the initiation and progression of diabetes complications such as diabetes-associated atherosclerosis and nephropathy. Bolstering antioxidant defenses is an important mechanism to lessen oxidative stress and inflammation. In this study, we have used a novel analog of the NFE2-related factor 2 (Nrf2) agonist bardoxolone methyl, dh404, to investigate its effects on diabetic macrovascular and renal injury in streptozotocin-induced diabetic apolipoprotein E−/− mice. We show that dh404, at lower but not higher doses, significantly lessens diabetes-associated atherosclerosis with reductions in oxidative stress (in plasma, urine, and vascular tissue) and proinflammatory mediators tumor necrosis factor-α, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemotactic protein-1 (MCP-1). We demonstrate that dh404 attenuates functional (urinary albumin-to-creatinine ratio) and structural (mesangial expansion) glomerular injury and improves renal tubular injury. Liver functional and structural studies showed that dh404 is well tolerated. Complementary in vitro studies in normal rat kidney cells showed that dh404 significantly upregulates Nrf2-responsive genes, heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and glutathione-S transferase, with inhibition of transforming growth factor-β–mediated profibrotic fibronectin, collagen I, and proinflammatory interleukin-6. Higher doses of dh404 were associated with increased expression of proinflammatory mediators MCP-1 and nuclear factor-κB. These findings suggest that this class of compound is worthy of further study to lessen diabetes complications but that dosage needs consideration.

Myoferlin is critical for endocytosis in endothelial cells.

Myoferlin is a member of the ferlin family of proteins that promotes endomembrane fusion with the plasma membrane in muscle cells and endothelial cells. In addition, myoferlin is necessary for the surface expression of vascular endothelial growth factor receptor 2 through the formation of a protein complex with dynamin-2 (Dyn-2). Since Dyn-2 is necessary for the fission of endocytic vesicles from the plasma membrane, we tested the hypothesis that myoferlin may regulates aspects of receptor-dependent endocytosis. Here we show that myoferlin gene silencing decreases both clathrin and caveolae/raft-dependent endocytosis, whereas ectopic myoferlin expression in COS-7 cells increases endocytosis by up to 125%. Interestingly, we have observed that inhibition of Dyn-2 activity or caveolin-1 (Cav-1) expression impairs endocytosis as well as membrane resealing after injury, indicating that Dyn-2 and Cav-1 also participate in both membrane fission and fusion processes. Mechanistically, myoferlin partially colocalizes with Dyn-2 and Cav-1 and forms a protein complex with Cav-1 solubilized from tissue extracts. Together, these data describe a new role for myoferlin in receptor-dependent endocytosis and an overlapping role for myoferlin-Dyn-2-Cav-1 protein complexes in membrane fusion and fission events.

Targeting Endothelial Dysfunction in Vascular Complications Associated with Diabetes

Cardiovascular complications associated with diabetes remain a significant health issue in westernized societies. Overwhelming evidence from clinical and laboratory investigations have demonstrated that these cardiovascular complications are initiated by a dysfunctional vascular endothelium. Indeed, endothelial dysfunction is one of the key events that occur during diabetes, leading to the acceleration of cardiovascular mortality and morbidity. In a diabetic milieu, endothelial dysfunction occurs as a result of attenuated production of endothelial derived nitric oxide (EDNO) and augmented levels of reactive oxygen species (ROS). Thus, in this review, we discuss novel therapeutic targets that either upregulate EDNO production or increase antioxidant enzyme capacity in an effort to limit oxidative stress and restore endothelial function. In particular, endogenous signaling molecules that positively modulate EDNO synthesis and mimetics of endogenous antioxidant enzymes will be highlighted. Consequently, manipulation of these unique targets, either alone or in combination, may represent a novel strategy to confer vascular protection, with the ultimate goal of improved outcomes for diabetes-associated vascular complications.

A New Role for the Muscle Repair Protein Dysferlin in Endothelial Cell Adhesion and Angiogenesis

Objective—Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC. Methods and Results—EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing. Conclusion—Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.

Are reactive oxygen species still the basis for diabetic complications

Despite the wealth of pre-clinical support for a role for reactive oxygen and nitrogen species (ROS/RNS) in the aetiology of diabetic complications, enthusiasm for antioxidant therapeutic approaches has been dampened by less favourable outcomes in large clinical trials. This has necessitated a re-evaluation of pre-clinical evidence and a more rational approach to antioxidant therapy. The present review considers current evidence, from both pre-clinical and clinical studies, to address the benefits of antioxidant therapy. The main focus of the present review is on the effects of direct targeting of ROS-producing enzymes, the bolstering of antioxidant defences and mechanisms to improve nitric oxide availability. Current evidence suggests that a more nuanced approach to antioxidant therapy is more likely to yield positive reductions in end-organ injury, with considerations required for the types of ROS/RNS involved, the timing and dosage of antioxidant therapy, and the selective targeting of cell populations. This is likely to influence future strategies to lessen the burden of diabetic complications such as diabetes-associated atherosclerosis, diabetic nephropathy and diabetic retinopathy.

Deciphering the Binding of Caveolin-1 to Client Protein Endothelial Nitric-oxide Synthase (eNOS) SCAFFOLDING SUBDOMAIN IDENTIFICATION, INTERACTION MODELING, AND BIOLOGICAL SIGNIFICANCE

Background: One of the most significant client proteins of Cav-1 is the endothelial nitric-oxide synthase (eNOS), but their specific binding site is unknown. Results: We describe how Cav-1 binds to eNOS and how biologically active NO can be increased. Conclusion: We provide the most detailed characterization of eNOS binding to Cav-1. Significance: Our data provide a deeper understanding of Cav-1 signaling and NO generation in physiological processes. Caveolin-1 (Cav-1) gene inactivation interferes with caveolae formation and causes a range of cardiovascular and pulmonary complications in vivo. Recent evidence suggests that blunted Cav-1/endothelial nitric-oxide synthase (eNOS) interaction, which occurs specifically in vascular endothelial cells, is responsible for the multiple phenotypes observed in Cav-1-null animals. Under basal conditions, Cav-1 binds eNOS and inhibits nitric oxide (NO) production via the Cav-1 scaffolding domain (CAV; amino acids 82–101). Although we have recently shown that CAV residue Phe-92 is responsible for eNOS inhibition, the “inactive” F92A Cav-1 mutant unexpectedly retains its eNOS binding ability and can increase NO release, indicating the presence of a distinct eNOS binding domain within CAV. Herein, we identified and characterized a small 10-amino acid CAV subsequence (90–99) that accounted for the majority of eNOS association with Cav-1 (Kd = 49 nm), and computer modeling of CAV(90–99) docking to eNOS provides a rationale for the mechanism of eNOS inhibition by Phe-92. Finally, using gene silencing and reconstituted cell systems, we show that intracellular delivery of a F92A CAV(90–99) peptide can promote NO bioavailability in eNOS- and Cav-1-dependent fashions. To our knowledge, these data provide the first detailed analysis of Cav-1 binding to one of its most significant client proteins, eNOS.

Direct Endothelial Nitric Oxide Synthase Activation Provides Atheroprotection in Diabetes-Accelerated Atherosclerosis

Patients with diabetes have an increased risk of developing atherosclerosis. Endothelial dysfunction, characterized by the lowered bioavailability of endothelial NO synthase (eNOS)–derived NO, is a critical inducer of atherosclerosis. However, the protective aspect of eNOS in diabetes-associated atherosclerosis remains controversial, a likely consequence of its capacity to release both protective NO or deleterious oxygen radicals in normal and disease settings, respectively. Harnessing the atheroprotective activity of eNOS in diabetic settings remains elusive, in part due to the lack of endogenous eNOS-specific NO release activators. We have recently shown in vitro that eNOS-derived NO release can be increased by blocking its binding to Caveolin-1, the main coat protein of caveolae, using a highly specific peptide, CavNOxin. However, whether targeting eNOS using this peptide can attenuate diabetes-associated atherosclerosis is unknown. In this study, we show that CavNOxin can attenuate atherosclerotic burden by ∼84% in vivo. In contrast, mice lacking eNOS show resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, CavNOxin lowered oxidative stress markers, inhibited the expression of proatherogenic mediators, and blocked leukocyte-endothelial interactions. These data are the first to show that endogenous eNOS activation can provide atheroprotection in diabetes and suggest that CavNOxin is a viable strategy for the development of antiatherosclerotic compounds.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-β (TGF-β) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-β-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.

Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo

Angiogenesis consists in the growth of new blood vessels from pre-existing ones. Although anti-angiogenesis interventions have been shown to have therapeutic properties in human diseases such as cancer, their effect is only partial and the identification of novel modulators of angiogenesis is warranted. Recently, we reported the unexpected proteomic identification in endothelial cells (EC) of Myoferlin, a member of the Ferlin family of transmembrane proteins. Ferlins are well known to regulate the fusion of lipid vesicles at the plasma membrane in muscle cells, and we showed that Myoferlin gene knockdown not only decreases lipid vesicle fusion in EC but also attenuates Vascular Endothelial Growth Factor (VEGF) Receptor-2 (VEGFR-2) expression. Herein, we show that Myoferlin gene silencing in cultured EC also results in attenuated expression of a second tyrosine kinase receptor, Tie-2, which is another well-described angiogenic receptor. Most importantly, we provide evidence that delivery of a low-volume Myoferlin siRNA preparation in mouse tissues results in attenuated angiogenesis and edema formation. This provides the first evidence that acute Myoferlin knockdown has anti-angiogenic effects and validates Myoferlin as an anti-angiogenesis target. Furthermore, this supports the unexpected but increasingly accepted concept that proper tyrosine kinase receptors expression at the plasma membrane requires Myoferlin.