Protective effect of new nitrosothiols on the early inflammatory response to kidney ischemia/reperfusion and transplantation in rats.

Renal ischemia/reperfusion (I/R) is characterized by severe inflammatory damage. We assessed the effect of administrating recently developed nitrosothiol compounds acting as nitric oxide (NO) donors on the production of cytokines and other markers of acute inflammatory reaction in an experimental model of warm (I/R), and in a model of cold ischemia and transplant in rats. Warm ischemia was achieved by ligation of left renal pedicle for 60 min, followed by contralateral nephrectomy. NO-donors LA-803, LA-807, LA-810 were administered i.v. (1.8 micromol/kg) during 30 min before reperfusion. Cold ischemia was achieved by preservating the kidney for 24 h in Euro Collins and grafting it in consanguineous Fisher 344/Ico rats. LA-803 was administered in the preservation fluid and in the recipient rat. Reperfusion time was 4 h in warm ischemia and 3 h in cold ischemia + transplantation. Administration of LA-803, LA-807 and, in a lower proportion, LA-810 prevented from the enhanced production of tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), and interleukin-1beta (IL-1beta), the decrease in interleukin-6 (IL-6) and interleukin-10 (IL-10), the increase in tissue level of superoxide anion (SOA) and superoxide dismutase (SOD), and the increase in neutrophil infiltration induced by warm I/R. Treatment with LA-803 in animals with renal transplantation after cold ischemia was also associated with reduced plasma levels of TNF, IFN-gamma, and IL-1beta, increased plasma levels of IL-6 and IL-10, reduced renal levels of SOA and SOD, and reduced neutrophil infiltration. These data demonstrate that systemic administration of new NO-donors with nitrosothiol structure diminished inflammatory responses in a kidney subjected to warm I/R or cold ischemia and transplantation.

Mice deficient in telomerase activity develop hypertension because of an excess of endothelin production.

BACKGROUND: Telomere shortening has been related to vascular dysfunction and hypertension. In the present study, we analyzed the influence of telomerase deficiency and telomere shortening on arterial pressure (AP). METHODS AND RESULTS: AP was evaluated in 6-month-old mice lacking the RNA component of the telomerase (terc-/-) at the first generation and third generation (G3). First generation and G3 mice showed higher AP than wild-type (WT) mice. To analyze the mechanisms involved, mean AP and vascular resistance in response to vasoactive substances were measured in G3 and WT mice. These mice showed similar responses to acetylcholine, N(G)-nitro-L-arginine methyl ester, angiotensin II, and losartan administration. Mean AP did not increase after endothelin-1 (ET-1) administration in G3 mice, but it did in WT animals. Bosentan treatment decreased mean AP only in G3 mice. Serum and urine concentrations of ET-1 were higher in terc-/- than in WT mice. Endothelin-converting enzyme (ECE-1) mRNA expression was higher in terc-/- animals than in the WT group. FR901533, an ECE antagonist, decreased blood pressure in conscious G3 mice. Studies in mouse embryonic fibroblasts from G3 mice suggest that ECE-1 overexpression could be mediated by reactive oxygen species in an AP-1-dependent mechanism, in which some kinases such as PI3-kinase, Akt, erk1/2, and Jun Kinase could be involved. An increased activity of nicotinamide adenine dinucleotide phosphate oxidase seems to be the main source of reactive oxygen species. CONCLUSIONS: Mice lacking telomerase activity show hypertension as a result of an increase in plasma ET-1 levels, which is a consequence of ECE-1 overexpression. A direct link between telomerase activity and hypertension is reported.

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 E(2) 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(omega)-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng(+/+) mice. N(omega)-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-beta1 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.

Vav3 proto-oncogene deficiency leads to sympathetic hyperactivity and cardiovascular dysfunction.

Although much is known about environmental factors that predispose individuals to hypertension and cardiovascular disease, little information is available regarding the genetic and signaling events involved. Indeed, few genes associated with the progression of these pathologies have been discovered despite intensive research in animal models and human populations. Here we identify Vav3, a GDP-GTP exchange factor that stimulates Rho and Rac GTPases, as an essential factor regulating the homeostasis of the cardiovascular system. Vav3-deficient mice exhibited tachycardia, systemic arterial hypertension and extensive cardiovascular remodeling. These mice also showed hyperactivity of sympathetic neurons from the time of birth. The high catecholamine levels associated with this condition led to the activation of the renin-angiotensin system, increased levels of kidney-related hormones and the progressive loss of cardiovascular and renal homeostasis. Pharmacological studies with drugs targeting sympathetic and renin-angiotensin responses confirmed the causative role and hierarchy of these events in the development of the Vav3-null mouse phenotype. These observations uncover the crucial role of Vav3 in the regulation of the sympathetic nervous system (SNS) and cardiovascular physiology, and reveal a signaling pathway that could be involved in the pathophysiology of human disease states involving tachycardia and sympathetic hyperactivity with unknown etiologies.

Reduced angiogenic responses in adult Endoglin heterozygous mice.

OBJECTIVE: To determine if angiogenesis is altered in adult Endoglin heterozygous (Eng(+/-)) mice, the animal model for the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). METHODS: Primary cultures of endothelial cells were generated from Eng(+/-) and Eng(+/+) mice and analyzed for proliferation, migration, and ability to form capillary-like tubes. Endothelial cells derived from umbilical veins of newborns (HUVEC) with an HHT1 genotype were also tested for capillary formation. Two in vivo models of angiogenesis were tested in the Eng(+/-) and Eng(+/+) mice: Matrigel implant-dependent angiogenesis and reperfusion following hindlimb ischemia. RESULTS: The Eng(+/-) endothelial cells displayed significantly reduced proliferation and migration, increased collagen production, and decreased NO synthase expression and vascular endothelial growth factor (VEGF) secretion. They also showed impaired capillary tube formation in vitro, as did the HHT1 HUVEC. These endothelial cell-specific abnormalities were associated with impaired Matrigel-dependent capillary tube formation in vivo and delayed reperfusion following hindlimb ischemia. CONCLUSIONS: Although vascular development is normal in Eng(+/-) mice, angiogenic abnormalities were observed in the adult mice and their isolated endothelial cells. These results suggest that a normal level of endoglin is required for full angiogenic activity.

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.