Andy Trane

Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition.

The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

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.

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.

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.

Caveolin as a potential drug target for cardiovascular protection

Caveolae and caveolin are key players in a number of disease processes. Current research indicates that caveolins play a significant role in cardiovascular disease and dysfunction. The far-reaching roles of caveolins in disease and dysfunction make them particularly notable therapeutic targets. In particular, caveolin-1 (Cav-1) and caveolin-3 (Cav-3) have been identified as potential regulators of vascular dysfunction and heart disease and might even confer cardiac protection in certain settings. Such a central role in vascular health therefore makes manipulation of Cav-1/3 function or expression levels clear therapeutic targets in a variety of cardiovascular related disease states. Here, we highlight the role of Cav-1 and Cav-3 in cardiovascular health and explore the potential of Cav-1 and Cav-3 derived experimental therapeutics.

Amlodipine increases endothelial nitric oxide release by modulating binding of native eNOS protein complex to caveolin-1

Amongst calcium channel blockers, amlodipine is known to have unique cardioprotective activities likely attributable to its capacity to increase nitric oxide (NO) release from endothelial cells (EC). Because endothelial NO synthase (eNOS), the main source of NO in EC is known to be inhibited by caveolin-1 (Cav-1), the purpose of this study is to investigate the possibility that amlodipine can modulate eNOS interaction with Cav-1. Using cultured EC, we confirm that amlodipine potentiates vascular endothelial growth factor (VEGF)-induced NO release. eNOS trafficking to specialized plasma membrane microdomains, which is essential to eNOS signaling, is unaffected by amlodipine. However, glutathione s-transferase (GST) pulldown assays reveal that amlodipine can prevent binding of native, acylated eNOS complexes to the active domain of Cav-1 in a concentration-dependent fashion, suggesting that amlodipine has an antagonistic effect on the native eNOS/Cav-1 signaling complex. Moreover, experiments performed in a reconstituted cell line confirm that amlodipines effect on NO release is highly selective for the eNOS/Cav-1 interaction. To our knowledge, these data are the first to demonstrate a direct effect of amlodipine on the eNOS/Cav-1 protein complex and support the concept of developing novel therapies specifically aimed at modulating the eNOS/Cav-1 interaction to improve endothelial function in cardiovascular diseases.

Tropoelastin enhances nitric oxide production by endothelial cells.

AIMS This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells. MATERIALS AND METHODS Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin. RESULTS Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W. CONCLUSION Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.

Caveolin-1 scaffolding domain residue phenylalanine 92 modulates Akt signaling

Caveolin-1 (Cav-1), the homo-oligomeric coat protein of cholesterol-rich caveolae signalosomes, regulates signaling proteins including endothelial nitric oxide synthase (eNOS). The Cav-1 scaffolding domain (a.a. 82-101) inhibits activated eNOS from producing vascular protective nitric oxide (NO), an enzymatic process involving trafficking and phosphorylation. However, we demonstrated that Cav-1 proteins and peptides bearing F92A substitution (CAV(F92A)) could promote cardioprotective NO, most likely by preventing inhibition of eNOS by Cav-1. Herein, we showed that wild-type CAV sequence could, similar to CAV(F92A), stimulate basal NO release, indicating a need to better characterize the importance of F92 in the regulation of eNOS by Cav-1/CAV. To reduce uptake sequence-associated effects, we conjugated a wild-type CAV derivative (CAV(WT)) or a F92A variant (CAV(F92A)) to antennapedia peptide (AP) or lipophilic myristic acid (Myr) and compared their effect on eNOS regulation in endothelial cells. We observed that both CAV(WT) and CAV(F92A) could increase basal NO release, although F92A substitution potentiates this response. We show that F92A substitution does not influence peptide uptake, endogenous Cav-1 oligomerization status and Cav-1 and eNOS distribution to cholesterol-enriched subcellular fractions. Instead, F92A substitution in CAV(WT) influences Akt activation and downstream eNOS phosphorylation status. Furthermore, we show that the cell permeabilization sequence could alter subcellular localization of endogenous proteins, an unexpected way to target different protein signaling cascades. Taken together, this suggests that we have identified the basis for two different pharmacophores to promote NO release; furthermore, there is a need to better characterize the effect of uptake sequences on the cellular trafficking of pharmacophores.

Endothelial dysfunction in systemic hypertension

Hypertension, defined as a rise in arterial blood pressure in the absence of a specific cause, leads to a myriad of cardiovascular complications that account for a high number of deaths globally. Progress in understanding hypertension comes from pioneering studies of the vasculature in both in vivo and in vitro settings. A common factor in hypertension and cardiovascular diseases is a decrease in the bioavailability of nitric oxide (NO), a potent endogenous vasodilator and a direct marker of endothelial function. This phenomenon, called endothelial dysfunction (ED), has increasingly gained importance in cardiovascular pathogenesis. Indeed, clinical evidence supports strong associations between ED and cardiovascular disease arising from common risk factors such as smoking, diet, lack of exercise, aging and genetic determinants. Besides NO, other factors that are produced in the endothelium, such as prostacyclin and endothelium-derived hyperpolarizing factor, can also be involved with, or act synergistically in, ED. This chapter focuses on the importance of NO in settings of ED and hypertension, and discusses the arguments around ED as a cause or consequence of hypertension by providing experimental and clinical evidence. We also aim to highlight current therapeutic strategies to improve vascular health by targeting levels of NO synthesis and raise questions emphasizing the need to further our knowledge in the molecular determinants in ED and hypertension.