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.

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.

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.

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.

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.

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.

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.

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.

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.

Peripheral 5-HT1D and 5-HT7 serotonergic receptors modulate sympathetic neurotransmission in chronic sarpogrelate treated rats.

5-HT2 receptor activation induces vasoconstriction, hypertension and platelet aggregation; therefore, its blocking may be useful in cardiovascular diseases, probably due to alterations in the modulation of serotonergic system. The aim of this study was to evaluate whether 5-HT2 receptor blockade changes serotonergic modulation of sympathetic neurotransmission in pithed rats. Serotonergic modulation of sympathetic neurotransmission was investigated in Wistar rats treated with sarpogrelate, a 5-HT2 receptor antagonist, during 14 days (30 mg/kg/day). After central nervous system destruction, we conducted electrical stimulation throughout the spinal cord flow to study the 5-HT-related products action on adrenergic system. 5-Hydroxytryptamine exerted inhibition of sympathetic outflow in sarpogrelate-treated pithed rats. This effect was mimicked and enhanced by 5-CT (5-HT1/7 receptor agonist). L-694,247 and AS-19, 5-HT1D and 5-HT7 receptor agonists respectively, reproduced this action. Pretreatment with LY310762+SB258719 (5-HT1D and 5-HT7 receptor antagonists, respectively) completely abolished 5-CT inhibitory action. The nature of this action was prejunctional since these agonists did not modify the pressor responses induced by exogenous noradrenaline. Western Blot analysis confirmed a higher expression of 5-HT1D receptors in sarpogrelate-treated rats. Experimental 5-HT2 receptor blockade induces changes in the 5-HT receptors involved in the serotonergic inhibition of sympathetic-induced pressor responses. Prejunctional activation of 5-HT1D and 5-HT7 receptors induces a significantly higher serotonergic inhibition on adrenergic neurotransmission in sarpogrelate-treated pithed rats. The antagonism of 5-HT2 receptors produces an enhancement of serotonergic sympathoinhibitory effect, which may explain the beneficial effects of this blockade in cardiovascular disorders where 5-hydroxytryptamine plays a crucial role.

Alteration in endoglin-related angiogenesis in refractory cytopenia with multilineage dysplasia.

The functional mechanisms involved in angiogenesis and the potential role of endoglin (ENG), recently described as a new marker for this process, have not been explored in Myelodysplastic Syndromes (MDS). In order to gain insight in MDS angiogenesis a combined analysis in bone marrow (BM) of gene expression levels, angiogenesis-related soluble factors and functional angiogenesis-related studies was carried out. Ninety-seven MDS patients and forty-two normal BM samples were studied. The morphology of the capillary-like structures originated by two endothelial cells lines in the BM environment of patients with refractory cytopenia with multilineage dysplasia (RCMD) was different from those of the remaining MDS. In addition, the BM mononuclear cells from RCMD patients displayed over-expression of VEGF, HIF and FN1 while they showed reduced expression of ENG in contrast to the normal ENG expression of the remaining low-risk MDS and the high expression of ENG in high-risk MDS subtype. Moreover, higher soluble ENG and soluble FLT-1 levels in BM microenvironment were observed in RCMD cases, which distinguished them from other individuals. Therefore, the present study suggests that the patterns of angiogenesis are different between the MDS subtypes. The differences in angiogenesis observed in RCMD patients could be related to ENG abnormalities.

Endoglin haploinsufficiency promotes fibroblast accumulation during wound healing through Akt activation.

Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng(+/-)) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng(+/-) MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng(+/-) mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng(+/-) mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.

Participation of cyclooxygenase pathway in the vasoconstriction induced by 5-HT in the in situ autoperfused kidney of long-term diabetic rats.

We attempted to characterize the 5-hydroxytryptamine (5-HT) receptor type/subtype and mediator mechanisms involved in the contractile effects of 5-HT in the in situ autoperfused kidney of long-term diabetic rats. Diabetes was induced in male Wistar rats by a single subcutaneous injection of alloxan. Intra-arterial (i.a.) bolus injection of 5-HT (0.00000125 to 0.1µg/kg) increased renal perfusion pressure in a dose dependent way but did not affect the systemic blood pressure in long-term diabetic rats. The selective 5-HT2 receptor agonist, α-methyl-5-HT, caused a local vasoconstrictor effect in the in situ autoperfused rat kidney similar to 5-HT. However, BW723C86, a selective 5-HT2B receptor agonist, m-CPP (1-(3-chlorophenyl)piperazine), a selective 5-HT2B/2C receptor agonist, the 5-HT1 receptor agonist, 5-carboxamidotryptamine (5-CT), and the selective 5-HT3 receptor agonist, 1-phenylbiguanide did not modify the renal perfusion pressure. In long-term diabetic rats, vasoconstriction elicited by 5-HT and α-methyl-5-HT was significantly decreased by ritanserin (a 5-HT2 receptor antagonist), spiperone (a 5-HT2A receptor antagonist), and the cyclooxygenase (COX) inhibitors, indomethacin (non-selective COX inhibitor), FR 122047 or nimesulide (selective COX-1 and COX-2 inhibitors, respectively), but was not modified by pretreatment with SB 206553 (3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), a non-selective 5-HT2C receptor antagonist, prazosin, propranolol, enalapril or losartan. The results of protein expression support these results: COX-1 and COX-2 are expressed in renal tissue. Inducible COX (COX-2) is overexpressed in long-term diabetes. Our data suggest that, in the in-situ autoperfused kidney of long-term diabetic rats, 5-HT vasoconstrictor action is mediated, through cyclooxygenase pathway, by local activation of 5-HT2A receptors.

Reduced plasma levels of Ang-2 and sEng as novel biomarkers in hereditary hemorrhagic telangiectasia (HHT).

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT; OMIM 187300) is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations caused by mutations in certain elements of the TGF-beta receptor complex. In the case of HHT1 mutations in the endoglin gene are responsible, whereas mutations in the ALK1 gene (an activin receptor-like kinase 1), lead to HHT2. Another two loci found at chromosome 5 and chromosome 7, whose target genes remain unidentified, lead to types 3 and 4 of the disease, respectively. Mutations in the MADH4/SMAD4 gene, another member of the TGF-beta signalling pathway, lead to a combined syndrome of familial juvenile polyposis associated with HHT. METHODS: In an attempt to identify some soluble components differentially expressed in the plasma of HHT patients, angiopoietin-2 and soluble endoglin concentrations were analyzed with standard quantitative sandwich ELISA. RESULTS: Angiopoietin-2 and soluble endoglin levels are reduced in plasma of HHT patients compared to control individuals, and a diagnostic algorithm for HHT based on these protein levels is proposed. CONCLUSIONS: Down-regulated protein levels of angiopoietin-2 and soluble endoglin in plasma represent novel HHT biomarkers that could be useful in the biochemical diagnosis of HHT facilitating the rapid identification of potential HHT patients.

Characterization of the contractile 5-hydroxytryptamine receptor in the autoperfused kidney of L-NAME hypertensive rats.

We evaluated the renal vascular effects of serotonin in Nomega-Nitro-l-arginine-hypertensive rats (L-NAME, 30 mg/kg/day, administered over 21 days in drinking water), a model of systemic hypertension with glomerular capillary hypertension and renal disease due to chronic blockade of endogenous synthesis of nitric oxide (NO) and compared with those obtained in normotensive rats. Using several agonists and antagonists of 5-hydroxytryptamine (5-HT), we characterized 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 (0.00000125-0.1 microg/kg) increased renal perfusion pressure in a dose-dependent manner, but did not affect systemic blood pressure. The selective 5-HT(2) receptor agonist alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) caused a local vasoconstrictor effect in the autoperfused rat kidney. The selective 5-HT(2B) receptor agonist BW723C86, the non-selective 5-HT(2C) receptor agonist (1-(3-chlorophenyl) piperazine), m-CPP, the 5-HT(1) receptor agonist 5-carboxamidotryptamine (5-CT) and the selective 5-HT(3) receptor agonist (1-(m-chlorophenyl)-biguanide), m-CPBG, did not modify renal perfusion pressure. The vasoconstrictor effect elicited by alpha-methyl-5-HT was significantly decreased by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2A) receptor antagonist spiperone, but was not modified by pretreatment with the selective 5-HT(2B/2C) receptor antagonist (3,5-dihydro-5-methyl-N-3pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), SB206553. The results of protein expression analyses allow us to postulate that the 5-HT(2A) receptor protein 5HT-SRC is expressed in renal tissue and differentially expressed in the renal artery of hypertensive (L-NAME) rats. Our data suggest that the serotonergic vasoconstrictor response induced in the in-situ autoperfused hypertensive rat kidney would be mediated by local activation of the 5-HT(2A) receptor.

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.

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.