Hilda Vargas-Robles

The role of actin-binding proteins in the control of endothelial barrier integrity

The endothelial barrier of the vasculature is of utmost importance for separating the blood stream from underlying tissues. This barrier is formed by tight and adherens junctions (TJ and AJ) that form intercellular endothelial contacts. TJ and AJ are integral membrane structures that are connected to the actin cytoskeleton via various adaptor molecules. Consequently, the actin cytoskeleton plays a crucial role in regulating the stability of endothelial cell contacts and vascular permeability. While a circumferential cortical actin ring stabilises junctions, the formation of contractile stress fibres, e. g. under inflammatory conditions, can contribute to junction destabilisation. However, the role of actin-binding proteins (ABP) in the control of vascular permeability has long been underestimated. Naturally, ABP regulate permeability via regulation of actin remodelling but some actin-binding molecules can also act independently of actin and control vascular permeability via various signalling mechanisms such as activation of small GTPases. Several studies have recently been published highlighting the importance of actin-binding molecules such as cortactin, ezrin/radixin/moesin, Arp2/3, VASP or WASP for the control of vascular permeability by various mechanisms. These proteins have been described to regulate vascular permeability under various pathophysiological conditions and are thus of clinical relevance as targets for the development of treatment strategies for disorders that are characterised by vascular hyperpermeability such as sepsis. This review highlights recent advances in determining the role of ABP in the control of endothelial cell contacts and vascular permeability.

Small GTPases of the Ras superfamily regulate intestinal epithelial homeostasis and barrier function via common and unique mechanisms

The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.

Correlation between Circulating Adhesion Molecule Levels and Albuminuria in Type-2 Diabetic Hypertensive Patients

Background/Aims: Endothelial dysfunction, a common feature among hypertensive and type-2 diabetic patients, is associated with inflammation, increased levels of circulating soluble adhesion molecules (SAM), and urinary albumin excretion. The aim of this study was to evaluate the role of circulating SAM levels in the development of albuminuria in hypertensive type-2 diabetic patients. Methods: We studied 30 hypertensive type-2 diabetic patients and 30 non-diabetic normotensive subjects, and measured their VCAM-1, ICAM-1 and E-selectin levels by ELISA, and their 24-hour urinary albumin excretion by nephelometry; the levels of circulating adhesion molecules and albuminuria were correlated with the Spearman correlation coefficient. Results: We found that the diabetic patients had significantly (p < 0.001) higher levels of circulating SAM than control subjects. When levels of circulating SAM were correlated with albuminuria, we found a significant correlation between VCAM-1 levels and 24-hour urinary albumin excretion (r = 0.4, p < 0.02). Conclusion: Our results suggest that VCAM-1 may be a marker of nephropathy in hypertensive type-2 diabetic patients.

Cyclooxygenase-2 and kidney failure.

Cyclooxygenase (COX)-dependent prostaglandins are necessary for normal kidney function. These prostaglandins are associated with inflammation, maintenance of sodium and water homeostasis, control of renin release, renal vasodilation, vasoconstriction attenuation, and prenatal renal development. COX-2 expression is regulated by the renin-angiotensin system, glucocorticoids or mineralcorticoids, and aldosterone, supporting a role for COX-2 in kidney function. Indeed, COX-2 mRNA and protein levels as well as enzyme activity are increased, along with PGE2, during kidney failure. In addition, changes in COX-2 expression are associated with increased blood pressure, urinary volume, sodium and protein and decreased urinary osmolarity. Intrarenal mechanisms such as angiotensin II (Ang II) production, increased sodium delivery, glomerular hypertension, and renal tubular inflammation have been suggested to be responsible for the increase in COX-2 expression. Although, specific COX-2 pharmacological inhibition has been related to the prevention of kidney damage, clinical studies have reported that COX-2 inhibition may cause side effects such as edema or a modest elevation in blood pressure and could possibly interfere with antihypertensive drugs and increase the risk of cardiovascular complications. Thus, administration of COX-2 inhibitors requires caution, especially in the presence of underlying cardiovascular disease.

Correlation between Levels of Circulating Adipokines and Adiponectin/Resistin Index with Carotid Intima-Media Thickness in Hypertensive Type 2 Diabetic Patients

Background: Hypoadiponectinemia and hyperresistinemia are associated with cardiovascular disease. The increase in the carotid intima-media thickness (CIMT) assessed by B-mode ultrasound has been directly associated with increased risk of myocardial infarction and stroke. Objective: To evaluate the correlation between adipokine levels with CIMT in hypertensive type 2 diabetic patients. Methods: Serum levels of adiponectin and resistin levels were measured by ELISA in 30 type 2 diabetic patients with never-treated hypertension and in age-matched healthy controls. The CIMT (B-mode color imaging of extracranial carotid arteries using high-resolution ultrasound) was also obtained. The relationship between adipokine levels and the adiponectin/resistin index with the CIMT was assessed by the Pearson correlation coefficient test. Results: Adiponectin was lower (p < 0.05), and resistin higher (p < 0.01) in patients than in controls, CIMT correlated positively with resistin (R = 0.45, p < 0.02) and the adiponectin/resistin index (R = 0.58, p < 0.001), but not with adiponectin levels (r = -0.11, p > 0.1) in patients. Whereas only adiponectin levels correlated - negatively - with CIMT (r = -0.39, p < 0.02) in controls. Conclusion: Our results shown that the adiponectin/resistin index seems to be more strongly associated with atherosclerosis than adipokine levels, and may be used as a reliable marker of cardiovascular risk in type 2 diabetic hypertensive patients.

Oxidative Stress-Dependent Coronary Endothelial Dysfunction in Obese Mice

Obesity is involved in several cardiovascular diseases including coronary artery disease and endothelial dysfunction. Endothelial Endothelium vasodilator and vasoconstrictor agonists play a key role in regulation of vascular tone. In this study, we evaluated coronary vascular response in an 8 weeks diet-induced obese C57BL/6 mice model. Coronary perfusion pressure in response to acetylcholine in isolated hearts from obese mice showed increased vasoconstriction and reduced vasodilation responses compared with control mice. Vascular nitric oxide assessed in situ with DAF-2 DA showed diminished levels in coronary arteries from obese mice in both basal and acetylcholine-stimulated conditions. Also, released prostacyclin was decreased in heart perfusates from obese mice, along with plasma tetrahydrobiopterin level and endothelium nitric oxide synthase dimer/monomer ratio. Obesity increased thromboxane A2 synthesis and oxidative stress evaluated by superoxide and peroxynitrite levels, compared with control mice. Obese mice treated with apocynin, a NADPH oxidase inhibitor, reversed all parameters to normal levels. These results suggest that after 8 weeks on a high-fat diet, the increase in oxidative stress lead to imbalance in vasoactive substances and consequently to endothelial dysfunction in coronary arteries.

Prevention of Renal Injury and Endothelial Dysfunction by Chronic l-Arginine and Antioxidant Treatment

We evaluated the effects of vitamins with antioxidant properties (a combination of vitamins C and E) and l-arginine treatment on renal failure in mice by measuring survival rate. The molecular changes were elucidated by determining endothelial tetrahydrobiopterin (BH4) levels and nitric oxide synthase (eNOS) mRNA expression in mice with renal ablation. Previous studies have shown that endothelial dysfunction in 5/6 nephrectomized mice is associated with decreased nitric oxide (NO) bioavailability and increased vascular superoxide production. WTC57 mice were divided into three groups: Group 1 was the sham-operated group (C); Group 2 was the 5/6 nephrectomized group (Nfx); and Group 3 was a group of 5/6 nephrectomized mice, treated with l-arginine and vitamins with antioxidant properties (NfxTx; 200 mg/kg l-arginine, 83 mg/kg vitamin C, and 46.6 mg/kg vitamin E). After 20 weeks of treatment, urinary protein excretion, blood pressure, BH4 and dihydrobiopterin (BH2) levels, eNOS mRNA, oxidative stress, and survival rate were determined. An increase in urinary protein excretion, blood pressure, and oxidative stress was prevented in the NfxTx group, but not in the Nfx group. BH4 and eNOS mRNA expression was increased by 32% and 78%, respectively, in the NfxTx group. Furthermore, the treatment increased the survival rate by 33%. Our results indicate that under normal conditions, NO appears to protect renal function. However, this NO-dependent protection is lost during kidney failure, probably due to increased reactive oxygen species synthesis. The treatment restores the viability of NO and prevents the BH4 oxidation. Therefore, this treatment may represent a therapeutic approach for the management of kidney disease.

Correlation Between the Levels of Circulating Adhesion Molecules and Atherosclerosis in Hypertensive Type-2 Diabetic Patients

Endothelial dysfunction is a common feature in type-2 diabetic patients and in hypertension, and is associated with inflammation, increased levels of circulating soluble adhesion molecules, and atherosclerosis. The aim of this study was to evaluate the relationship between the levels of circulating soluble adhesion molecules and the degree of atherosclerosis in hypertensive type-2 diabetic patients. We studied 30 hypertensive type-2 diabetic patients in whom VCAM-1, ICAM-1, and E-selectin were measured by ELISA. Additionally, the intimal-medial thickness of both the common and internal carotid arteries was measured (B-mode ultrasound). The levels of circulating adhesion molecules and maximal carotid artery intimal-medial thicknesses were correlated using the Spearman correlation coefficient test. Statistical analysis was performed with ANOVA. We found significant correlations between ICAM-1 (r = 0.5) levels and maximal carotid artery intimal-medial thickness these patients. No correlation was observed with E-selectin and VCAM-1. Our results suggest that ICAM-1 is associated and correlated with the degree of atherosclerosis in type-2 diabetic hypertensive patients.

The Effect of Trandolapril and Its Fixed-Dose Combination with Verapamil on Circulating Adhesion Molecules Levels in Hypertensive Patients with Type 2 Diabetes

Background and Aim. Endothelial dysfunction in hypertensive type-2 diabetic patients is associated with increased levels of circulating soluble adhesion molecules (SAM). SAM participate in the development of diabetic macroangiopathy and microangiopathy. The aim of this study was to compare the effect of trandolapril (T) and its fixed-dose combination with verapamil (FDTV) on SAM levels in hypertensive type-2 diabetic patients. Methods. Forty type-2 diabetic patients with never-treated hypertension were randomly assigned to two groups. One group (FDTV) received 2/180 mg once a day; the other group received T 2 mg once a day. Study drugs were administered for three months in both groups. VCAM-1, ICAM, and E-selectin were measured by ELISA at the beginning and end of the study. Patients were evaluated monthly for blood pressure, fasting serum glucose, and adverse events. Statistical analysis was performed with ANOVA. Results. Both therapeutics regimens reduced significantly the levels of the SAM tested. When both groups were compared, we did not find a significant difference in ICAM and E-selectin reduction. However, VCAM-1 presented a significantly greater reduction (p = 0.022) in the trandolapril-verapamil group. No patient suffered adverse events. Conclusion. Our results show that FDTV produces a greater reduction of VCAM-1 circulating levels than trandolapril alone. This may explain some of the beneficial effects of this fixed dosed combination that are non-related to its antihypertensive effects.

L-Arginine and Antioxidant Diet Supplementation Partially Restores Nitric Oxide-Dependent Regulation of Phenylephrine Renal Vasoconstriction in Diabetics Rats

OBJECTIVE The increase of reactive oxygen species (ROS) in diabetes potentiates the vascular effects of phenylephrine through nitric oxide (NO) impairment, facilitating the development of diabetic nephropathy. We propose that the combination of an antioxidant and L-arginine as diet supplements could prevent the increased vascular response to phenylephrine in diabetic animals. DESIGN Changes in the adrenergic system play an important role in the development of vascular complications in the prediabetic condition. The vasoconstrictor effects of phenylephrine are regulated by NO, and the impairment of endothelium-dependent vasodilation in diabetes is associated with ROS. SETTING Diabetes was induced with a low dose (55 mg/kg body weight) of streptozotocin in 7-week-old rats. Diabetic rats were fed with a diet supplement containing a combination of vitamin E, vitamin C, eicosapentaenoic acid, docosahexaenoic acid, and L-arginine, and the effects on phenylephrine-induced renal vascular responses were evaluated. RESULTS Phenylephrine increased the renal perfusion pressure of isolated perfused kidneys from diabetic rats compared with nondiabetic rats. This effect was associated with reduced nitrite release as well as reduced plasma tetrahydrobiopterin and increased superoxide anions in the renal tissue. Diet supplementation with a combination of L-arginine and vitamins in diabetic rats partially prevented the generation of superoxide associated with recovery of the renal release of NO and decreased phenylephrine-induced vasoconstrictor effects, compared with untreated diabetic rats. However, the administration of L-arginine or vitamins alone did not affect phenylephrine-induced vasoconstriction. Vitamin treatment alone did decrease superoxide generation. CONCLUSION The protective mechanism of NO on the vasoconstrictor effects of phenylephrine in the kidney is lost during the development of diabetes, probably via the actions of ROS through a decrease in tetrahydrobiopterin, thus contributing to the pathogenesis of diabetic nephropathy. Restoration of this protective NO mechanism can be achieved by simultaneously stimulating NO synthesis and preventing the effects of ROS through the use of L-arginine and a combination of vitamins E and C as diet supplementation.