Dopamine D4 receptor subtype activation reduces the rat cardiac parasympathetic discharge.

The dopaminergic system influences the heart rhythm by inhibiting the rat cardiac sympathetic and parasympathetic neurotransmissions through activation of D2-like receptors (encompassing the D2, D3, and D4 subtypes). Whereas D2 receptor subtype activation results in cardiac sympatho-inhibition, the dopamine receptor subtypes involved in rat cardiac vago-inhibition remain unknown. Hence, this study investigated the specific functional role of the D2-like receptor subtypes (D2, D3, and/or D4) inhibiting the rat heart cholinergic drive. For this purpose, male Wistar rats were pithed and prepared for cardiac vagal stimulation. Bradycardic responses were obtained by electrical stimulation of vagal fibres (3, 6, 9 Hz; n = 100) or i.v. acetylcholine (ACh; 1, 5, 10 µg/kg; n = 15). Expression of D2, D3, and D4 receptors was studied in left and right atrium samples by PCR (n = 4). Intravenous injections of quinpirole (D2-like agonist; 1-30 µg/kg), but not of SFK-38393 (D1-like agonist; 1-30 µg/kg), dose-dependently inhibited the vagally induced bradycardia. The vago-inhibition induced by quinpirole (which failed to affect the bradycardia to i.v. ACh) was unchanged after i.v. injections of the antagonists L-741,626 (D2; 100 µg/kg) or SB-277011-A (D3; 100 µg/kg), but it was abolished by L-745,870 (D4; 100 µg/kg). mRNA levels of D2, D3, and D4 receptor subtype were detected in the left and right rat atria. Our results suggest that the quinpirole-induced vagolytic effect involves prejunctional D4 receptor subtypes, located in the left and right atria. This provides new evidence on the relevance of D4 receptor modulating the heart parasympathetic control.

Continuous endoglin (CD105) overexpression disrupts angiogenesis and facilitates tumor cell metastasis.

Endoglin (CD105) is an auxiliary receptor for members of the TFG-ß superfamily. Whereas it has been demonstrated that the deficiency of endoglin leads to minor and defective angiogenesis, little is known about the effect of its increased expression, characteristic of several types of cancer. Angiogenesis is essential for tumor growth, so high levels of proangiogenic molecules, such as endoglin, are supposed to be related to greater tumor growth leading to a poor cancer prognosis. However, we demonstrate here that endoglin overexpression do not stimulate sprouting or vascularization in several in vitro and in vivo models. Instead, steady endoglin overexpression keep endothelial cells in an active phenotype that results in an impairment of the correct stabilization of the endothelium and the recruitment of mural cells. In a context of continuous enhanced angiogenesis, such as in tumors, endoglin overexpression gives rise to altered vessels with an incomplete mural coverage that permit the extravasation of blood. Moreover, these alterations allow the intravasation of tumor cells, the subsequent development of metastases and, thus, a worse cancer prognosis.

Pregnancy-Induced High Plasma Levels of Soluble Endoglin in Mice Lead to Preeclampsia Symptoms and Placental Abnormalities.

Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (fWT(hsEng+)) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, fWT(hsEng+) mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.

5-HT modulates the rat mesenteric vasopressor outflow by 5-HT1D sympatholytic receptors.

5-hydroxytryptamine (5-HT) modulates noradrenergic activity in different cardiovascular territories, but its effect on the mesenteric vasopressor outflow has not yet been clarified. This study investigated the in vivo serotonergic influence, characterizing 5-HT receptors implicated, in sympathetic innervation of mesenteric vasculature. Wistar rats were anaesthetised and prepared for the in situ autoperfused rat mesentery, monitoring systemic blood pressure (SBP), heart rate (HR) and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency-dependent increases in MPP (9 ± 1.6, 25.7 ± 3.9 and 60.2 ± 5 mmHg for 2, 4 and 8 Hz, respectively), without altering SBP or HR. 5-HT (1-25 µg/kg), 5-carboxamidotryptamine (5-HT1/7 agonist; 25 µg/kg) or L-694,247 (5-HT1D agonist; 1-25 µg/kg) i.a. bolus inhibited vasopressor responses by mesenteric nerves electrical stimulation, unlike i.a. bolus of agonists 8-OH-DPAT (5-HT1A ), CGS-12066B (5-HT1B ), BRL 54443 (5-HT1e/1F ), α-methyl-5-HT (5-HT2 ), 1-PBG (5-HT3 ), cisapride (5-HT4 ) or AS-19 (5-HT7 ) (25 µg/kg each). Interestingly, i.a. L-694,247 (25 µg/kg) also reduced the exogenous norepinephrine-induced vasoconstrictions. Pretreatment with selective 5-HT1D receptor antagonist, LY310762 (1 mg/kg, i.v.), completely abolished L-694,247- and 5-HT-induced mesenteric sympathoinhibition. Furthermore, ELISA analysis confirmed 5-HT1D receptors expression in mesenteric artery. These findings suggest that serotonergic mechanisms-induced sympathoinhibition of mesenteric noradrenergic outflow is mediated by pre and/or postjunctional 5-HT1D receptors.

Endoglin and Activin Receptor-like Kinase 1 (Alk1) Modify Adrenomedullin Expression in an Organ-Specific Manner in Mice.

Hereditary hemorrhagic telangiectasia (HHT) is a rare disease characterized by vascular malformations and profuse bleeding. The disease is caused by mutations in the components of the BMP-9 receptor: endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1) genes. Recently, we reported that HHT patients expressed higher serum levels of adrenomedullin (AM) than healthy volunteers; thus, we studied the expression of AM (by enzyme immunoassay, qRT-PCR, immunohistochemistry, and Western blotting) in mice deficient in either one of the receptor components to investigate whether these defects may be the cause of that elevated AM in patients. We found that AM expression is not affected by these mutations in a consistent pattern. On the contrary, in some organs (blood, lungs, stomach, pancreas, heart, kidneys, ovaries, brain cortex, hippocampus, foot skin, and microvessels), there were no significant changes, whereas in others we found either a reduced expression (fat, skin, and adrenals) or an enhanced production of AM (cerebellum and colon). These results contradict our initial hypothesis that the increased AM expression found in HHT patients may be due directly to the mutations, but open intriguing questions about the potential phenotypic manifestations of Eng and Acvrl1 mutants that have not yet been studied and that may offer, in the future, a new focus for research on HHT.

Functional Alterations Involved in Increased Bleeding in Hereditary Hemorrhagic Telangiectasia Mouse Models.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal-dominant genetic disorder involving defects in two predominant genes known as endoglin (ENG; HHT-1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT-2). It is characterized by mucocutaneous telangiectases that, due to their fragility, frequently break causing recurrent epistaxis and gastrointestinal bleeding. Because of the severity of hemorrhages, the study of the hemostasis involved in these vascular ruptures is critical to find therapies for this disease. Our results demonstrate that HHT patients with high bleeding, as determined by a high Epistaxis Severity Score (ESS), do not have prolonged clotting times or alterations in clotting factors. Considering that coagulation is only one of the processes involved in hemostasis, the main objective of this study was to investigate the overall mechanisms of hemostasis in HHT-1 (Eng +/-) and HHT-2 (Alk1 +/-) mouse models, which do not show HHT vascular phenotypes in the meaning of spontaneous bleeding. In Eng +/- mice, the results of in vivo and in vitro assays suggest deficient platelet-endothelium interactions that impair a robust and stable thrombus formation. Consequently, the thrombus could be torn off and dragged by the mechanical force exerted by the bloodstream, leading to the reappearance of hemorrhages. In Alk1 +/- mice, an overactivation of the fibrinolysis system was observed. These results support the idea that endoglin and Alk1 haploinsufficiency leads to a common phenotype of impaired hemostasis, but through different mechanisms. This contribution opens new therapeutic approaches to HHT patients' epistaxis.

Oral fluoxetine treatment changes serotonergic sympatho-regulation in experimental type 1 diabetes.

AIMS: This study investigated whether fluoxetine treatment changes the 5-HT regulation on vascular sympathetic neurotransmission in type 1 diabetes. MAIN METHODS: Four-week diabetes was obtained by a single alloxan s.c. administration in male Wistar rats, administering fluoxetine for 14 days (10 mg/kg/day; p.o.). Systolic blood pressure, heart rate, glycaemia, body weight (BW) evolution, creatinine, and blood urea nitrogen (BUN) were monitored. Afterward, rats were pithed to perform the vascular sympathetic stimulation. 5-HT1A/1D/2A receptors expression was analysed by Western blot in thoracic aorta. Both i.v. norepinephrine and the electrical stimulation of the spinal sympathetic drive evoked vasoconstrictor responses. KEY FINDINGS: Fluoxetine treatment significantly reduced the BW gain, hyperglycaemia, creatinine, and BUN in diabetic rats. The electrical-produced vasopressor responses were greater in untreated than in fluoxetine-treated diabetic rats. 5-HT decreased the sympathetic-produced vasopressor responses. While 5-CT, 8-OH-DPAT and L-694,247 (5-HT1/7, 5-HT1A and 5-HT1D agonists, respectively) reproduced 5-HT-evoked inhibition, the 5-HT2 activation by α-methyl-5-HT augmented the vasoconstrictions. The 5-CT sympatho-inhibition was reversed by 5-HT1A plus 5-HT1D antagonists (WAY-100,635 and LY310762, respectively), whereas ritanserin (5-HT2A antagonist) blocked the α-methyl-5-HT potentiating effect. Norepinephrine-generated vasoconstrictions were increased or diminished by α-methyl-5-HT or 5-CT, respectively. 5-HT1A/1D/2A receptors were expressed at vascular level, being 5-HT1A expression increased by fluoxetine in diabetic rats. SIGNIFICANCE: Our findings suggest that fluoxetine improves metabolic and renal profiles, changes the vasopressor responses, and 5-HT receptors modulating sympathetic activity in diabetic rats: 5-HT1A/1D are involved in the sympatho-inhibition, while 5-HT2A is implicated in the sympatho-potentiation, being both effects pre and/or postjunctional in nature.

Influence of elevated sleep-time blood pressure on vascular risk and hypertension-mediated organ damage.

We report the analysis of 252 hypertensive patients whose blood pressure (BP) was assessed by around-the-clock ambulatory BP monitoring compared to office BP measurement during a follow-up investigation of 8.7 y (SD: 2.43 y) that evaluated the added value of measuring sleep-time BP values. We found that 37.3% of the patients had mismatched diagnoses between the two techniques of BP assessment, with 11.5% of the patients showing white-coat hypertension and 25.8% masked hypertension. Only 12.3% of the diagnosed and treated patients presented normal BP values. Nocturnal (sleep-time) hypertension was present in 70.63%. The sleep-time systolic BP mean was found to be an independent vascular risk factor (F = 9.005, p < .001), indirectly measured through the 10-year risk of morbidity and mortality. Additionally, the elevated sleep-time systolic BP mean was a better marker of subclinical hypertension-mediated organ damage (ρ = 0.19, p < .01) than either the awake (ρ = 0.168, p < .01) or 24 (ρ = 0.184, p < .01) systolic BP means. In conclusion, the accuracy and sleep-time measurements provided by ambulatory BP make it particularly relevant in hypertension diagnosis and management. The use of the ambulatory BP measurement method could end up modifying current therapeutic targets, with sleep-time systolic BP mean becoming a main one, in order to optimize hypertension control and reduce hypertension-related organ pathology and cardiovascular disease morbidity and mortality.

S-endoglin expression is induced in senescent endothelial cells and contributes to vascular pathology.

Senescence of endothelial cells (ECs) may contribute to age-associated cardiovascular diseases, including atherosclerosis and hypertension. The functional and gene expression changes associated with cellular senescence are poorly understood. Here, we have analyzed the expression, during EC senescence, of 2 different isoforms (L, long; S, short) of endoglin, an auxiliary transforming growth factor (TGF)-beta receptor involved in vascular remodeling and angiogenesis. As evidenced by RT-PCR, the S/L ratio of endoglin isoforms was increased during senescence of human ECs in vitro, as well as during aging of mice in vascularized tissues. Next, the effect of S-endoglin protein on the TGF-beta receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-beta type I receptors, ALK5 and ALK1, although the interaction with ALK5 was stronger than with ALK1. S-endoglin conferred a lower proliferation rate to ECs and behaved differently from L-endoglin in relation to TGF-beta-responsive reporters with ALK1 or ALK5 specificities, mimicking the behavior of the endothelial senescence markers Id1 and plasminogen activator inhibitor-1. In situ hybridization studies demonstrated the expression of S-endoglin in the endothelium from human arteries. Transgenic mice overexpressing S-endoglin in ECs showed hypertension, decreased hypertensive response to NO inhibition, decreased vasodilatory response to TGF-beta(1) administration, and decreased endothelial nitric oxide synthase expression in lungs and kidneys, supporting the involvement of S-endoglin in the NO-dependent vascular homeostasis. Taken together, these results suggest that S-endoglin is induced during endothelial senescence and may contribute to age-dependent vascular pathology.

Hypertension exhibits 5-HT4 receptor as a modulator of sympathetic neurotransmission in the rat mesenteric vasculature.

Sympathetic overdrive is a key player in hypertension, where the mesenteric vasculature plays a relevant role in modulating blood pressure. Although 5-HT inhibits noradrenergic mesenteric neurotransmission in normotensive rats, its effect on the mesenteric sympathetic drive in hypertensive rats has not been studied. We investigated the influence of in vivo 5-HT by characterizing the implicated serotonergic receptors on the mesenteric sympathetic outflow in rats with N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Hypertension was induced in male Wistar rats by L-NAME administration (30 mg/kg per day; 21 days) in drinking water. The rats were anesthetized (sodium pentobarbital; 60 mg/kg, i.p.), prepared for the in situ autoperfused rat mesentery, and subjected for monitoring their systemic blood pressure (SBP), heart rate (HR), and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency-dependent increases in MPP without altering SBP or HR. The 5-HT and cisapride (5-HT4 agonist) i.a. bolus (1-25 µg/kg) inhibited vasopressor responses by electrical stimulation of the mesenteric nerves, unlike an i.a. bolus (25 µg/kg each) of the agonist 5-carboxamidotryptamine (5-HT1/7 agonist), α-methyl-5-HT (5-HT2), or 1-PBG (5-HT3). However, i.a. cisapride (25 µg/kg) did not affect the noradrenaline-induced vasoconstriction in the mesenteric vasculature. Administration of the selective 5-HT4 receptor antagonist GR 125487 (1 mg/kg, i.v.) completely abolished cisapride- and 5-HT-evoked mesenteric sympatholytic effects. Additionally, ELISA analysis demonstrated higher 5-HT4 receptor expression in mesenteric arteries from L-NAME-hypertensive compared with normotensive rats. Our findings suggest that L-NAME-induced hypertension modifies the 5-HT modulation of the rat mesenteric sympathetic drive: prejunctional 5-HT4 receptors are involved in the serotonergic sympathoinhibitory effect.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms.

BACKGROUND: Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells. METHODS: The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls. RESULTS: First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections. CONCLUSIONS: The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

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.

Characterization of contractile 5-hydroxytryptamine receptor subtypes in the in situ autoperfused kidney in the anaesthetized rat.

Using several 5-hydroxytryptamine (5-HT) agonists and antagonists, we attempted to characterize the receptor subtypes involved in the contractile response to 5-HT in the in situ autoperfused rat kidney. An intra-arterial (i.a.) bolus injection of 5-HT (0.00000125 to 0.1 microg/kg) increased renal perfusion pressure in a dose-dependent way but did not affect the systemic blood pressure. The selective 5-HT2 receptor agonist alpha-methyl-5-HT (alpha-methyl-5-hydroxytryptamine) and the non-selective 5-HT2C receptor agonist (1-(3-chlorophenyl)piperazine), m-CPP, caused a local vasoconstrictor effect in the autoperfused rat kidney, whereas BW723C86, a selective 5-HT2B receptor agonist, the 5-HT1 receptor agonist 5-carboxamidotryptamine, 5-CT, and the selective 5-HT3 receptor agonist m-CPBG (1-(m-chlorophenyl)-biguanide) did not modify the renal perfusion pressure. The vasoconstrictor effect elicited by alpha-methyl-5-HT and m-CPP was significantly decreased by ritanserin (a 5-HT2 receptor antagonist), SB 206553 (3,5-Dihydro-5-methyl-N-3pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), a selective 5-HT2B/2C receptor antagonist and enalapril, but was not modified by pretreatment with spiperone (a 5-HT2A receptor antagonist). The results of protein expression analyses allow us to postulate that 5HT-SRC (a 5-HT2C receptor protein) is expressed in renal tissue and differentially expressed in renal artery. Our data suggest also that the serotonergic vasoconstrictor response induced in the in situ autoperfused rat kidney would be mediated by local 5-HT2C receptor activation.

The mitogen-activated protein kinase Erk5 mediates human mesangial cell activation.

BACKGROUND: Mesangial activation occurs in many forms of renal disease that progress to renal failure. Mitogen-activated protein kinases (MAPKs) are important mediators involved in the intracellular network of interacting proteins that transduce extracellular stimuli to intracellular responses. The extracellular signal-regulated kinases 5 (Erk5) MAPK pathway has been involved in regulating several cellular responses. Thus, we examined the expression of Erk5 in human renal tissue and the function of Erk5 in cultured human mesangial cells. METHODS: Erk5 was visualized in human renal tissue by immunohistochemistry and in mesangial cells by immunofluorescence microscopy using the anti-Erk5 C-terminus antibody. Erk5 expression and activation, and collagen I expression were determined by western blot. To generate a dominant-negative form of the Erk5 in human mesangial cells, an EcoRI fragment from wild-type pCEFL-HA-Erk5 was subcloned into the EcoRI site of pCDNA3. Cell proliferation was analysed by an MTT-based assay. Cell contraction was analysed by studying the changes in the planar cell surface area. RESULTS: Erk5 was expressed in the kidney, mainly localized at the glomerular mesangium. In cultured human mesangial cells, Erk5 was activated by foetal calf serum (FCS), high glucose, endothelin-1, platelet-activating factor (PAF), epidermal growth factor (EGF) and transforming growth factor beta-1 (TGF-beta1). The expression of a dominant-negative form of Erk5 in human mesangial cells resulted in a significant decrease in proliferation, EGF-induced cell contraction and TGF-beta1-induced collagen I expression. CONCLUSIONS: These results suggest that Erk5 is involved in agonist-induced mesangial cell contraction, proliferation and ECM accumulation and point to a multifunctional role of Erk5 in the pathophysiology of glomerular mesangial cells.

Human recombinant erythropoietic agents do not induce changes in circulating levels of endoglin and vascular endothelial growth factor in anemic cancer patients.

The correlation of erythropoietin (EPO) receptor levels with angiogenesis and progression in some cancers has suggested that EPO could acts directly as an angiogenic factor. The purpose of this study was to assess the effect of treatment with human recombinant erythropoietic (rHuEPO) agents in cancer patients with chemotherapy-induced anaemia on endoglin and vascular endothelial growth factor (VEGF) circulating levels as a possible marker of angiogenesis. Endoglin and VEGF were measured in serum samples from 25 cancer patients with chemotherapy-induced anemia before and after 3-4 weeks of treatment with rHuEPO. A group of 28 healthy voluntaries was used as control. VEGF serum levels were significantly higher in cancer patients than in controls. For endoglin, higher levels were observed without reaching statistical significance. No statistically significant differences in endoglin and VEGF serum levels were found between samples obtained before and after treatment with rHuEPO agents. In conclusion, our result do not support that rHuEpo treatment in anaemic cancer patients induce angiogenesis.

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.

Circulating soluble endoglin modifies the inflammatory response in mice.

Inflammation is associated with every health condition, and is an important component of many pathologies such as cardiovascular diseases. Circulating levels of soluble endoglin have been shown to be higher in the serum of patients with cardiovascular diseases with a significant inflammatory component. The aim of this study was to evaluate the implication of circulating soluble endoglin in the inflammatory response. For this purpose, a transgenic mouse expressing human soluble endoglin (sEng+) was employed, and three different inflammatory approaches were used to mimic inflammatory conditions in different tissues. This study shows that control sEng+ mice have a normal inflammatory state. The lung and kidney injury induced by the inflammatory agents was reduced in sEng+ mice, especially the intra-alveolar and kidney infiltrates, suggesting a possible reduction in inflammation induced by soluble endoglin. To deepen into this possible effect, the leukocyte number in the bronchoalveolar lavage and air pouch lavage was evaluated and a significant reduction of neutrophil infiltration in LPS-treated lungs and ischemic kidneys from sEng+ with respect to WT mice was observed. Additionally, the mechanisms through which soluble endoglin prevents inflammation were studied. We found that in sEng+ animals the increment of proinflammatory cytokines, TNFα, IL1ß and IL6, induced by the inflammatory stimulus was reduced. Soluble endoglin also prevents the augmented adhesion molecules, ICAM, VCAM and E-selectin induced by the inflammatory stimulus. In addition, vascular permeability increased by inflammatory agents was also reduced by soluble endoglin. These results suggest that soluble endoglin modulates inflammatory-related diseases and open new perspectives leading to the development of novel and targeted approaches for the prevention and treatment of cardiovascular diseases.

Resveratrol inhibits gentamicin-induced mesangial cell contraction.

Gentamicin is an aminoglycoside antibiotic that is very effective in treating different gram negative infections, however, one of its main side effects is nephrotoxicity. Gentamicin-induced decreases in glomerular filtration rate could be mediated by mesangial cell contraction. Resveratrol, a natural hydroxystilbene, has been identified to be a potent antioxidant with many biological activities including protection against kidney diseases. As we have previously demonstrated that gentamicin induced a reduction of planar surface area of cultured rat mesangial cells, and that resveratrol has a protective effect on gentamicin-induced nephrotoxicity in vivo, the aim of this study was to investigate the effect of resveratrol on gentamicin-induced mesangial cell contraction. This study demonstrates that the contractile effect of gentamicin on mesangial cells can be prevented by incubation with resveratrol at an optimal dose of 10 microM, as it blunted the gentamicin-induced reduction in planar cell surface area and the number of contracted cells. Besides, the preincubation with 10(-5)M diphenylene iodinium (DPI), an inhibitor of the NADP(H) oxidase, also blunted gentamicin-induced cell contraction. This preventive effect was higher when cells were incubated with both substances together. These results strongly suggest that the protective effect resveratrol against gentamicin-induced reduction in renal function in vivo could be mediated by inhibiting gentamicin-induced mesangial cells contraction.

Expression of endoglin in human mesangial cells: modulation of extracellular matrix synthesis.

Transforming growth factor-beta (TGF-beta) has been identified as a key mediator of glomerulosclerosis in kidney diseases. Endoglin is a component of the TGF-beta receptor system that is upregulated during glomerulosclerosis, suggesting a role during progression of renal diseases characterized by extracellular matrix (ECM) synthesis and accumulation. The expression of endoglin was demonstrated in cultured human mesangial cells (HMC) by flow cytometry, Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analyses. TGF-beta upregulated not only the expression of endoglin, but also that of TGF-beta itself, TGF-beta receptor type II, collagen I, collagen IV, and fibronectin. To study the role of endoglin in TGF-beta responses, transfectant fibroblasts overexpressing endoglin were analyzed. Untreated and TGF-beta-treated endoglin(+) cells showed significantly lower levels of collagens than those in control cells, indicating that endoglin negatively regulates ECM levels of collagens. These findings may have important implications in the pathological states associated with renal fibrosis.

Role of vascular nitric oxide in experimental liver cirrhosis.

One of the most important features of liver cirrhosis is the splanchnic and systemic arterial vasodilation, related to an increase in vascular capacity and an active vasodilation. This arterial vasodilation seems to be the consequence of the excessive generation of vasodilating substances, which also contributes to a lower than normal pressor response to circulating nervous or humoral substances. The following review analyzes the mechanisms responsible for the vascular hyporesponse to vasoconstrictors observed in the experimental models of liver cirrhosis. It has become increasingly clear that, among the great variety of substances studied, nitric oxide (NO) seems to be one of the main contributors to this vascular alteration, since elimination of the endothelium or inhibition of its synthesis corrects it. The mechanism by which NO interferes with the contractile apparatus in smooth muscle cells seems to be related to a direct effect on calcium entry from the extracellular space and release from the internal stores.