Fernando Pérez-Barriocanal

Endoglin regulates nitric oxide-dependent vasodilatation

Endoglin is a membrane glycoprotein that plays an important role in cardiovascular development and angiogenesis. We examined the role of endoglin in the control of vascular tone by measuring nitric oxide (NO)‐dependent vasodilation in haploinsufficient mice (Eng+/−) and their Eng+/+ littermates. The vasodilatory effect of acetylcholine, bradykinin, and sodium nitroprusside was assessed in anesthetized mice; in isolated, perfused hindlimbs; and in aortic rings. The substantial hypotensive and vasodilatory response induced by acetylcholine and bradykinin in Eng+/+ was markedly reduced in Eng+/− mice. Both kinds of animals had similar responses to sodium nitroprusside, suggesting that the deficient vasodilatory effect is not due to a NO response impairment. Urinary and plasma concentrations of nitrites, a NO metabolite, were lower in Eng+/− than in Eng+/+ mice. The levels of endothelial nitric oxide synthase (eNOS) in kidneys and femoral arteries were about half in Eng+/− than in Eng+/+ mice and were also reduced in primary cultures of aortic endothelial cells from Eng+/− compared with those from Eng+/+ mice. Furthermore, overexpression or suppression of endoglin in cultured cells induced a marked increase or decrease in the protein levels of eNOS, respectively. Thus, our results in vivo and in vitro demonstrate a relationship between endoglin and NO‐dependent vasodilation mediated by the regulation of eNOS expression.

Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2‐dependent TGF‐β signaling

The endothelial nitric oxide synthase (eNOS) is a critical regulator of cardiovascular homeostasis, whose dysregulation leads to different vascular pathologies. Endoglin is a component of the transforming growth factor beta (TGF‐β) receptor complex present in endothelial cells that is involved in angiogenesis, cardiovascular development, and vascular homeostasis. Haploinsufficient expression of endoglin has been shown to downregulate endothelium‐derived nitric oxide in endoglin+/− (Eng+/−) mice and cultured endothelial cells. Here, we find that TGF‐β1 leads to an increased vasodilatation in Eng+/+ mice that is severely impaired in Eng+/− mice, suggesting the involvement of endoglin in the TGF‐β regulated vascular homeostasis. The endoglin‐dependent induction of eNOS occurs at the transcriptional level and is mediated by the type I TGF‐β receptor ALK5 and its downstream substrate Smad2. In addition, Smad2‐specific signaling is upregulated in endoglin‐induced endothelial cells, whereas it is downregulated upon endoglin gene suppression with small interference RNA (siRNA). The endoglin‐dependent upregulation of Smad2 was confirmed using eNOS and pARE promoters, whose activities are known to be Smad2 dependent, as well as with the interference of Smad2 with siRNA, Smurf2, or a dominant negative form of Smad2. Furthermore, increased expression of endoglin in endoglin‐inducible endothelial cells or in transfectants resulted in increased levels of Smad2 protein without affecting the levels of Smad2 mRNA. The increased levels of Smad2 appear to be due to a decreased ubiquitination and proteasome‐dependent degradation leading to stabilization of Smad2. These results suggest that endoglin enhances Smad2 protein levels potentiating TGF‐β signaling, and leading to an increased eNOS expression in endothelial cells. J. Cell. Physiol. 210: 456–468, 2007.

Role of inflammation in túbulo-interstitial damage associated to obstructive nephropathy.

Obstructive nephropathy is characterized by an inflammatory state in the kidney, that is promoted by cytokines and growth factors produced by damaged tubular cells, infiltrated macrophages and accumulated myofibroblasts. This inflammatory state contributes to tubular atrophy and interstitial fibrosis characteristic of obstructive nephropathy. Accumulation of leukocytes, especially macrophages and T lymphocytes, in the renal interstitium is strongly associated to the progression of renal injury. Proinflammatory cytokines, NF-κB activation, adhesion molecules, chemokines, growth factors, NO and oxidative stress contribute in different ways to progressive renal damage induced by obstructive nephropathy, as they induce leukocytes recruitment, tubular cell apoptosis and interstitial fibrosis. Increased angiotensin II production, increased oxidative stress and high levels of proinflammatory cytokines contribute to NF-κB activation which in turn induce the expression of adhesion molecules and chemokines responsible for leukocyte recruitment and iNOS and cytokines overexpression, which aggravates the inflammatory response in the damaged kidney. In this manuscript we revise the different events and regulatory mechanisms involved in inflammation associated to obstructive nephropathy.

Quercetin reduces cisplatin nephrotoxicity in rats without compromising its anti-tumour activity

BACKGROUND Nephrotoxicity is the major limitation for the clinical use of cisplatin as an anti-tumoural drug. Our aim was to investigate the protective effect of quercetin on cisplatin nephrotoxicity in a rat tumour model in vivo and to examine the mechanisms of renal protection. METHODS Breast adenocarcinoma (13762 Mat B-III) cells were inoculated subcutaneously in male Fischer rats and 7 days later, the rats were administered daily with quercetin [50 mg/kg/day, intraperitoneally (i.p.)] or vehicle. Four days after that, the rats were given a single dose of cisplatin (4 mg/kg, i.p.) or vehicle. Tumour growth and renal function were monitored throughout the experiment. Two or 6 days after cisplatin administration, the rats were killed and the kidneys and tumours were removed to examine renal function and toxicity markers in both tissues. RESULTS In the kidney, cisplatin treatment induced: (i) a decrease in renal blood flow and glomerular filtration rate, (ii) tubular necrosis/apoptosis, (iii) increased lipid peroxidation and decreased endogenous antioxidant systems, (iv) increased expression of inflammation markers and (v) increased activity of the apoptosis executioner caspase-3. Cisplatin effectively reduced tumour size and weight. CONCLUSIONS Co-treatment with quercetin partially prevented all the renal effects of cisplatin, whereas it did not impair its anti-tumour activity. In conclusion, in a model of tumour-bearing rats, quercetin prevents the nephrotoxic effect of cisplatin without affecting its anti-tumour activity.

Effect of the flavonoid quercetin on cadmium-induced hepatotoxicity.

The present study was designed to evaluate whether treatment with quercetin exerts any beneficial effect on cadmium (Cd)-induced hepatotoxicity in order to establish the possible protective mechanisms of quercetin. Wistar rats were distributed in four experimental groups: control, Cd, quercetin, and Cd+quercetin. Hepatic toxicity was evaluated by measuring plasma concentrations of markers of hepatic injury. The activity of antioxidant enzymes in liver was also measured. Hepatic expression of metallothioneins (MT), and endothelial nitric oxide synthase (eNOS) was assayed by Western and Northern blot. Our results demonstrated that Cd administration induced an increased marker enzyme activity in plasma. This effect was not inhibited by quercetin. However, the administration of quercetin softened Cd-induced oxidative damage. MT levels in liver were substantially increased when the animals received Cd and quercetin. Hepatic eNOS expression was significantly increased after treatment with Cd and quercetin, being this increase higher than in animals receiving Cd alone. In conclusion, in this experimental model, quercetin was not able to prevent the Cd-induced liver damage although the animals that received both, Cd and quercetin showed a marked improvement in oxidative stress and an increase in the MT and eNOS expression. These results suggest that other mechanisms different to oxidative stress could be involved in hepatic damage.

Endoglin Upregulation During Experimental Renal Interstitial Fibrosis in Mice

Abstract—The goal of the present study was to evaluate the role of endoglin, a transforming growth factor-&bgr;1 (TGF-&bgr;1) accessory receptor, in the pathogenesis of renal fibrosis. This was achieved by testing a model of tubulo-interstitial fibrosis induced by unilateral ureteral obstruction in endoglin heterozygous (Eng+/−) mice. Northern and Western blot analysis revealed that endoglin expression in kidneys of these mice was significantly reduced compared with Eng+/+ littermates. Pronounced interstitial fibrosis induced by ureteral obstruction was confirmed histologically by Masson’s trichromic staining and by increased immunostaining for fibronectin and laminin without significant differences between Eng+/− and Eng+/+ mice. Ureteral obstruction induced significant increases in &agr;2(I) and &agr;1(IV) collagen, fibronectin, and TGF-&bgr;1 mRNA levels, as well as in total kidney collagen but changes were similar in Eng+/− and Eng+/+ mouse kidneys. Ureteral obstruction also induced a 2-fold increase in endoglin mRNA levels in both Eng+/+ mice and Eng+/− mice, which was confirmed by Western blot analysis. Thus, the present study provides clear evidence that endoglin is upregulated in the kidneys of mice with interstitial fibrosis induced by unilateral ureteral ligation. However, Eng+/− mice do not show any changes in the severity of renal disease induced in this model when compared with normal mice, suggesting that the absolute level of endoglin is not critical for the effects of TGF-&bgr;1 in the renal fibrosis process.

Nephrotoxicity of Uranium: Pathophysiological, Diagnostic and Therapeutic Perspectives

As in the case of other heavy metals, a considerable body of evidence suggests that overexposure to uranium may cause pathological alterations to the kidneys in both humans and animals. In the present work, our aim was to analyze the available data from a critical perspective that should provide a view of the real danger of the nephrotoxicity of this metal for human beings. A further aim was to elaborate a comparative compilation of the renal pathophysiological data obtained in humans and experimental animals with a view to gaining more insight into our knowledge of the mechanisms of action and renal damage. Finally, we address the existing perspectives for the improvement of diagnostic methods and the treatment of intoxications by uranium, performing an integrated analysis of all these aspects.

Protective effect of trans-resveratrol on gentamicin-induced nephrotoxicity.

Reactive oxygen species (ROS) have been involved in glomerular filtration rate (GFR) reduction observed after gentamicin treatment. trans-Resveratrol (TR), a natural hydroxystilbene, has been identified to be a potent inhibitor of ROS production. The aim of this work has been to study whether TR has a protective effect on gentamicin-induced nephrotoxicity in vivo and the effect of TR on lipid peroxidation and the oxidative stress induced by gentamicin. Animals that received a daily intraperitoneal injection of gentamicin (100 mg/kg body weight) showed lower GFR and renal blood flow (RBF) and higher urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) than control rats. Rats receiving TR together with gentamicin showed higher GFR and RBF and lower NAG urinary excretion than rats receiving gentamicin alone. Moreover, renal lipid peroxidation increased in rats receiving gentamicin alone, and this increase was prevented by the administration of TR. The concentration in plasma of antioxidants was higher in the group that received TR with gentamicin than in the gentamicin and control groups. The activities of lactate dehydrogenase and alkaline phosphatase were higher in rats treated with gentamicin than in control rats and were reduced by the treatment with TR. This study demonstrates an improvement in renal function in response to the administration of TR in gentamicin-induced nephrotoxicity. At least a part of this effect of TR could be based on its antioxidant activity.

Vascular gene transfer driven by endoglin and ICAM-2 endothelial-specific promoters

The involvement of the vascular endothelium in a large number of diseases supports the importance of vascular-specific gene delivery for their treatment. The hereditary hemorrhagic telangiectasia type 1 is an example of a vascular inherited disease (OMIM 187300). This is an autosomal dominant vascular disorder originated by mutations in the endoglin gene and associated with frequent epistaxis, telangiectases, gastrointestinal bleedings, and arteriovenous malformations in brain, lung and liver. Here, we address for the first time the possibility of using in vivo gene transfer to target endoglin expression to the vasculature. The promoter of the endothelial gene, ICAM-2, was used to generate transgenic animals which demonstrated endothelial expression of endoglin. Next, the promoters of the human endothelial genes, endoglin and ICAM-2, were inserted upstream of the human endoglin cDNA, and the resulting constructs were systemically or locally delivered, demonstrating endoglin expression in the vessel walls of liver, lung and skin. These gene transfer experiments represent an initial step in the treatment of the hereditary hemorrhagic telangiectasia type 1 by gene therapy, and suggest that endoglin and ICAM-2 promoters can be used to deliver other genes to the endothelium specifically.

Endoglin Regulates Cyclooxygenase-2 Expression and Activity

The endoglin heterozygous (Eng+/−) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng+/− mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E2 were observed in the Eng+/− mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng+/− but not in Eng+/+ mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N&ohgr;-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng+/+ mice. N&ohgr;-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-β1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng+/− mice.