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

Serum Amyloid A Stimulates Vascular and Renal Dysfunction in Apolipoprotein E-Deficient Mice Fed a Normal Chow Diet

Elevated serum amyloid A (SAA) levels may promote endothelial dysfunction, which is linked to cardiovascular and renal pathologies. We investigated the effect of SAA on vascular and renal function in apolipoprotein E-deficient (ApoE−/−) mice. Male ApoE−/− mice received vehicle (control), low-level lipopolysaccharide (LPS), or recombinant human SAA by i.p. injection every third day for 2 weeks. Heart, aorta and kidney were harvested between 3 days and 18 weeks after treatment. SAA administration increased vascular cell adhesion molecule (VCAM)-1 expression and circulating monocyte chemotactic protein (MCP)-1 and decreased aortic cyclic guanosine monophosphate (cGMP), consistent with SAA inhibiting nitric oxide bioactivity. In addition, binding of labeled leukocytes to excised aorta increased as monitored using an ex vivo leukocyte adhesion assay. Renal injury was evident 4 weeks after commencement of SAA treatment, manifesting as increased plasma urea, urinary protein, oxidized lipids, urinary kidney injury molecule (KIM)-1 and multiple cytokines and chemokines in kidney tissue, relative to controls. Phosphorylation of nuclear-factor-kappa-beta (NFκB-p-P65), tissue factor (TF), and macrophage recruitment increased in kidneys from ApoE−/− mice 4 weeks after SAA treatment, confirming that SAA elicited a pro-inflammatory and pro-thrombotic phenotype. These data indicate that SAA impairs endothelial and renal function in ApoE−/− mice in the absence of a high-fat diet

Molecular and pharmacological characterization of mutant (F92A) caveolin-1 : a direction towards increasing nitric oxide bioavailability

Nitric Oxide (NO) produced by the endothelium is a critical mediator of vascular function and plays an important role in the protection against various cardiovascular diseases1. In fact, a central feature of most cardiovascular diseases is reduced bioavailability of NO resulting from impaired endothelial function. Consequently, therapies that improve NO synthesis and availability in disease settings are relevant. Endothelial nitric oxide synthase (eNOS) is a membrane enzyme expressed exclusively in vascular endothelial cells and is responsible for NO production. Improper regulation of the enzyme results in production of eNOS-derived superoxide anion (O₂₋) instead of NO. O₂₋ is an oxidative stress mediator and scavenges NO, thereby contributing to lowered NO bioavailability. Extensive research has demonstrated a number of factors involved in positively regulating eNOS activity. However, one of the few proteins that bind to eNOS under basal conditions and inhibit NO release is Caveolin-1 (Cav-1), the major coat protein of plasma membrane lipid-enriched invaginations known as caveolae². Recently, it was demonstrated that a single amino acid substitution of the Cav-1 protein, mutant known as F92A Cav-1, is unable to inhibit eNOS³. Furthermore, preliminary data indicates that high expression of F92A Cav-1 can increase basal NO release. Due to the significance of NO in vascular function, the current work explores the possible mechanisms by which F92A Cav-1 potentiates eNOS activity and NO release. We report that F92A Cav-1 preserves the unique properties of Cav-1, including targeting to caveolae and forming high molecular weight oligomers, which are essential for caveolae organelle biogenesis. Moreover, F92A Cav-1 still retains the ability to bind to eNOS without altering its subcellular localization, indicating that F92A Cav-1 can prevent eNOS binding to endogenous Cav-1, which could rationalize the increased NO release observed. Lastly, we provide evidence that over-expression of F92A Cav-1 reduces the release of basal O₂₋ in endothelial cells as compared to WT Cav-1, revealing another potential positive effect of the mutant Cav-1. Hence, this report compares the biological properties of WT and F92A Cav-1 and the data collected is aimed at describing a therapeutically relevant pharmacological target to increase NO bioavailability in cardiovascular disease settings.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Therapeutic use of a mutant Caveolin-1 peptide to reduce atherosclerosis induced by hypercholesterolemia and diabetes

Endothelial dysfunction is a well-established response to cardiovascular risk factors, such as hypercholesterolemia and diabetes, and is the critical first step of atherogenesis. Nitric oxide (NO), the key regulator of endothelial function, is greatly diminished in atherosclerotic disease settings resulting in augmented oxidative stress and endothelial activation, a process that involves upregulation of endothelial adhesion molecules and increased leukocyte-endothelial interactions, all of which are major steps in the pathogenesis of atherosclerosis. Pharmacological inhibition of endothelial nitric oxide synthase (eNOS), the main vascular source of protective NO, induces endothelial dysfunction and promotes atherosclerosis. While there is little doubt that endothelial dysfunction is directly linked to atherosclerosis and cardiovascular disease in patients, whether the endogenous pool of eNOS and associated NO release can be considered a direct, therapeutically relevant primary target for atheroprotection is unknown. Caveolin-1 (Cav-1), the major coat protein of plasma membrane caveolae, binds to and inhibits endogenous eNOS. Previously, we have reported that a Cav-1-derived cell permeable peptide with an inactivated eNOS inhibitory domain, known as CavNOxin is able to increase basal NO release without interfering with the biological activities of Cav-1. Herein the current thesis, I hypothesize that ‘antagonizing’ the eNOS/Cav-1 interaction to specifically relieve eNOS from the inhibitory clamp of Cav-1, through the intracellular delivery of CavNOxin, is a potentially novel and unexplored anti-atherosclerotic therapeutic strategy. I show that CavNOxin is able to significantly attenuate hypercholesterolemia- and diabetes-induced atherosclerosis. In contrast, mice lacking eNOS showed resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, I show that CavNOxin reduces oxidative stress, expression of pro-atherogenic mediators (in particular VCAM-1) and leukocyte-endothelial interactions. These data are the first to document the use of an eNOS-specific activator to directly reduce oxidative stress and increase atheroprotective endothelial function specifically through endogenous eNOS. In addition, this study provides target validation for the eNOS/Cav-1 interaction, which is highly endothelium-specific, as a strategy for the development of anti-atherosclerotic compounds.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

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