Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model.

Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease, caused by Dirofilaria immitis, causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction, and hypoxia. The aim of this study was to analyze whether the excretory/secretory and surface-associated antigens of adult worms interact and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEFG-A (Vascular Endothelial Growth Factor A), as well as DiES+VEFG-A and Cut+VEFG-A were used. The production of VEFG-A and other proangiogenic [soluble VEFGR-2 (sVEFGR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEFGR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non­stimulated cultures were used as controls. DiES+VEFG-A and Cut+VEFG-A significantly increased the production of VEFG-A and sVEFGR-2, and only Cut+VEFG-A significantly increased the production of VEFGR-1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEFG-A produced a significant increase in viable cell number and significant decrease cell migration, as well as in the organization and number of cell connections. Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbß3 integrin.

BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.

Effect of somatic antigens of Dirofilaria repens adult worms on angiogenesis, cell proliferation and migration and pseudo-capillary formation in human endothelial cells.

BACKGROUND: Angiogenesis is defined as the formation of new vessels by sprouting of endothelial cells from pre-existing vessels in response to stimuli, such as hypoxia or inflammation. Subcutaneous dirofilariasis, caused by Dirofilaria repens, is a zoonotic disease characterized by the formation of subcutaneous nodules with the presence of at least one encapsulated worm, showing perivascular vascularization around it. The aim of this study is to analyze whether the somatic antigen of adult D. repens worms interacts with and modulates the angiogenic mechanism, cell proliferation and migration, and formation of pseudo-capillaries. METHODS: The expression of VEGF-A, VEGFR-1/sFlt, VEGFR-2, mEnd and sEnd in cultures of human vascular endothelial cells stimulated with somatic antigen of adult worms of D. repens (DrSA), vascular endothelial growth factor (VEGF) and DrSA + VEGF were evaluated by using ELISA commercial kits. Cellular viability was analyzed by live cell count, cytotoxicity assays by using a commercial kit, cell proliferation by MTT-based assay, cell migration by wound-healing assay carried out by scratching wounds and capacity of formation of pseudo-capillaries analyzing cell connections and cell groups in Matrigel cell cultures. In all cases unstimulated cultures were used as controls. RESULTS: DrSA + VEGF significantly increased the expression of VEGF-A, VEGFR-2 and mEndoglin compared to other groups and unstimulated cultures. Moreover, DrSA + VEGF produced cell proliferation and migration and increased the formation of pseudo-capillaries. CONCLUSIONS: Somatic antigen of adult D. repens worms activated the proangiogenic mechanism, cell proliferation and cell migration as well as formation of pseudo-capillaries in this in vitro human endothelial cell model. These processes could be related to the survival of adult D. repens in subcutaneous nodules in infected hosts.

Labetalol and soluble endoglin aggravate bile acid retention in mice with ethinylestradiol-induced cholestasis.

Labetalol is used for the therapy of hypertension in preeclampsia. Preeclampsia is characterized by high soluble endoglin (sEng) concentration in plasma and coincides with intrahepatic cholestasis during pregnancy (ICP), which threatens the fetus with the toxicity of cumulating bile acids (BA). Therefore, we hypothesized that both labetalol and increased sEng levels worsen BA cumulation in estrogen-induced cholestasis. C57BL/6J, transgenic mice overexpressing human sEng, and their wild-type littermates were administrated with ethinylestradiol (EE, 10 mg/kg s.c., the mice model of ICP) and labetalol (10 mg/kg s.c.) for 5 days with sample collection and analysis. Plasma was also taken from healthy pregnant women and patients with ICP. Administration of labetalol to mice with EE cholestasis aggravated the increase in BA plasma concentrations by induction of hepatic Mrp4 efflux transporter. Labetalol potentiated the increment of sEng plasma levels induced by estrogen. Increased plasma levels of sEng were also observed in patients with ICP. Moreover, increased plasma levels of human sEng in transgenic mice aggravated estrogen-induced cholestasis in labetalol-treated mice and increased BA concentration in plasma via enhanced reabsorption of BAs in the ileum due to the upregulation of the Asbt transporter. In conclusion, we demonstrated that labetalol increases plasma concentrations of BAs in estrogen-induced cholestasis, and sEng aggravates this retention. Importantly, increased sEng levels in experimental and clinical forms of ICPs might present a novel mechanism explaining the coincidence of ICP with preeclampsia. Our data encourage BA monitoring in the plasma of pregnant women with preeclampsia and labetalol therapy.

Mesenchymal Stromal Cells Combined With Elastin-Like Recombinamers Increase Angiogenesis In Vivo After Hindlimb Ischemia.

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)-based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti-human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti-CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.

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 Other Angiogenesis Markers in Recurrent Varicose Veins.

BACKGROUND: Surgery on varicose veins (crossectomy and stripping) may lead to recurrence, with clinical and socioeconomic repercussions. The etiopathogenesis of varicose veins has yet to be fully understood. OBJECTIVE: Study the expression of endoglin and other molecules involved in the neovascularisation process in patients suffering from this disease. METHODS: Total of 43 patients that have undergone surgery for varicose veins (24 primary and 19 recurrent). Endoglin and other molecules were identified on the venous wall (proximal -saphenofemoral junction- and distal), via real-time RT-PCR, and in serum, via ELISA: endoglin (Eng), vascular endothelial growth factor (VEGF-A), its receptors 1 and 2 (VEGFR1 or FLT1), (VEGFR2 or FLK), and the hypoxia-inducible factor (HIF-1A). All the patients signed a consent form. RESULTS: The recurrent group recorded a higher expression of Eng, VEGF-A, VEGFR1, and VEGFR2 at the level of proximal venous wall compared to the primary group. HIF-1A did not record any differences. As regards the determination of the distal venous wall, no markers recorded differences between the groups. Among the serum determinations, only sFLT1 recorded a significant drop among the patients with recurrent varicose veins. CONCLUSIONS: Patients with recurrent varicose veins record a higher expression of endoglin and other markers of angiogenesis in proximal veins. Endoglin in the blood (sEng) serves no apparent purpose in recurrent varicose veins.

High Soluble Endoglin Levels Affect Aortic Vascular Function during Mice Aging.

Endoglin is a 180 kDa transmembrane glycoprotein that was demonstrated to be present in two different endoglin forms, namely membrane endoglin (Eng) and soluble endoglin (sEng). Increased sEng levels in the circulation have been detected in atherosclerosis, arterial hypertension, and type II diabetes mellitus. Moreover, sEng was shown to aggravate endothelial dysfunction when combined with a high-fat diet, suggesting it might be a risk factor for the development of endothelial dysfunction in combination with other risk factors. Therefore, this study hypothesized that high sEng levels exposure for 12 months combined with aging (an essential risk factor of atherosclerosis development) would aggravate vascular function in mouse aorta. Male transgenic mice with high levels of human sEng in plasma (Sol-Eng+) and their age-matched male transgenic littermates that do not develop high soluble endoglin (Control) on a chow diet were used. The aging process was initiated to contribute to endothelial dysfunction/atherosclerosis development, and it lasted 12 months. Wire myograph analysis showed impairment contractility in the Sol-Eng+ group when compared to the control group after KCl and PGF2α administration. Endothelium-dependent responsiveness to Ach was not significantly different between these groups. Western blot analysis revealed significantly decreased protein expression of Eng, p-eNOS, and ID1 expression in the Sol-Eng+ group compared to the control group suggesting reduced Eng signaling. In conclusion, we demonstrated for the first time that long-term exposure to high levels of sEng during aging results in alteration of vasoconstriction properties of the aorta, reduced eNOS phosphorylation, decreased Eng expression, and altered Eng signaling. These findings suggest that sEng can be considered a risk factor for the development of vascular dysfunction during aging and a potential therapeutical target for pharmacological intervention.

The Dual Effect of the BMP9-ALK1 Pathway in Blood Vessels: An Opportunity for Cancer Therapy Improvement?

The improvement of cancer therapy efficacy, the extension of patient survival and the reduction of adverse side effects are major challenges in cancer research. Targeting blood vessels has been considered a promising strategy in cancer therapy. Since the tumor vasculature is disorganized, leaky and triggers immunosuppression and tumor hypoxia, several strategies have been studied to modify tumor vasculature for cancer therapy improvement. Anti-angiogenesis was first described as a mechanism to prevent the formation of new blood vessels and prevent the oxygen supply to tumor cells, showing numerous limitations. Vascular normalization using low doses of anti-angiogenic drugs was purposed to overcome the limitations of anti-angiogenic therapies. Other strategies such as vascular promotion or the induction of high endothelial venules are being studied now to improve cancer therapy. Bone morphogenetic protein 9 (BMP9) exerts a dual effect through the activin receptor-like kinase 1 (ALK1) receptor in blood vessel maturation or activation phase of angiogenesis. Thus, it is an interesting pathway to target in combination with chemotherapies or immunotherapies. This review manuscript explores the effect of the BMP9-ALK1 pathway in tumor angiogenesis and the possible usefulness of targeting this pathway in anti-angiogenesis, vascular normalization or vascular promotion therapies.

Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

Hot and Cold Tumors: Is Endoglin (CD105) a Potential Target for Vessel Normalization?

Tumors are complex masses formed by malignant but also by normal cells. The interaction between these cells via cytokines, chemokines, growth factors, and enzymes that remodel the extracellular matrix (ECM) constitutes the tumor microenvironment (TME). This TME can be determinant in the prognosis and the response to some treatments such as immunotherapy. Depending on their TME, two types of tumors can be defined: hot tumors, characterized by an immunosupportive TME and a good response to immunotherapy; and cold tumors, which respond poorly to this therapy and are characterized by an immunosuppressive TME. A therapeutic strategy that has been shown to be useful for the conversion of cold tumors into hot tumors is vascular normalization. In this review we propose that endoglin (CD105) may be a useful target of this strategy since it is involved in the three main processes involved in the generation of the TME: angiogenesis, inflammation, and cancer-associated fibroblast (CAF) accumulation. Moreover, the analysis of endoglin expression in tumors, which is already used in the clinic to study the microvascular density and that is associated with worse prognosis, could be used to predict a patient's response to immunotherapy.

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.

Soluble Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Participating in Aggravation of NASH-Related Changes in Mouse Liver.

Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis with inflammation and fibrosis. Membrane endoglin (Eng) expression is shown to participate in fibrosis, and plasma concentrations of soluble endoglin (sEng) are increased in patients with hypercholesterolemia and type 2 diabetes mellitus. We hypothesize that NASH increases both hepatic Eng expression and sEng in blood and that high levels of sEng modulate cholesterol and bile acid (BA) metabolism and affect NASH progression. Three-month-old transgenic male mice overexpressing human sEng and their wild type littermates are fed for six months with either a high-saturated fat, high-fructose high-cholesterol (FFC) diet or a chow diet. Evaluation of NASH, Liquid chromatography-mass spectrometry (LC/MS) analysis of BA, hepatic expression of Eng, inflammation, fibrosis markers, enzymes and transporters involved in hepatic cholesterol and BA metabolism are assessed using Real-Time Quantitative Reverse Transcription Polymerase Chain reaction (qRT-PCR) and Western blot. The FFC diet significantly increases mouse sEng levels and increases hepatic expression of Eng. High levels of human sEng results in increased hepatic deposition of cholesterol due to reduced conversion into BA, as well as redirects the metabolism of triglycerides (TAG) to its accumulation in the liver, via reduced TAG elimination by ß-oxidation combined with reduced hepatic efflux. We propose that sEng might be a biomarker of NASH development, and the presence of high levels of sEng might support NASH aggravation by impairing the essential defensive mechanism protecting NASH liver against excessive TAG and cholesterol accumulation, suggesting the importance of high sEng levels in patients prone to develop NASH.

Pathophysiological mechanisms underlying a rat model of triple whammy acute kidney injury.

Simultaneous administration of certain antihypertensive (renin-angiotensin system inhibitors and diuretics) and nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a renal toxicity syndrome known as "triple whammy" acute kidney injury (TW-AKI), yet poorly characterized at the pathophysiological level, as no specific experimental model exists on which to conduct preclinical research. Herein, we generated and characterized a rat model of TW-AKI (0.7 mg/kg/day trandolapril +400 mg/kg/day ibuprofen +20 mg/kg/day furosemide). Double treatments involving the NSAID caused a subclinical acute kidney injury, as they reduced glomerular filtration rate to a significant but not sufficient extent to increase Crpl concentration. Only the triple treatment generated an overt AKI with increased Crpl provided that animals were under partial water ingestion restriction. Histological examination revealed no evidence of tissue renal injury, and no proteinuria or makers of renal damage were detected in the urine. These findings, along with a normal fractional excretion of sodium and glucose, indicated that these drug combinations produce a prerenal type of AKI. In fact, blood pressure and renal blood flow were also reduced (most markedly following the triple combination), although renal dysfunction was more pronounced than expected for the corresponding pressure drop, supporting a key pathological role of the interference with renal autoregulation mechanisms. In summary, prerenal TW-AKI only occurs when volemia is challenged (i.e., by furosemide in partially water-deprived animals) under the effects of renin-angiotensin system inhibitors and NSAIDs. This model will facilitate further pathophysiological knowledge for a better diagnosis and clinical handling of this syndrome.

Long term effects of soluble endoglin and mild hypercholesterolemia in mice hearts.

Soluble endoglin (sEng) released into the circulation was suggested to be related to cardiovascular based pathologies. It was demonstrated that a combination of high sEng levels and long-term exposure (six months) to high fat diet (HFD) resulted in aggravation of endothelial dysfunction in the aorta. Thus, in this study, we hypothesized that a similar experimental design would affect the heart morphology, TGFß signaling, inflammation, fibrosis, oxidative stress and eNOS signaling in myocardium in transgenic mice overexpressing human sEng. Three-month-old female transgenic mice overexpressing human sEng in plasma (Sol-Eng+ high) and their age-matched littermates with low levels of human sEng (Sol-Eng+ low) were fed a high-fat diet containing 1.25% of cholesterol and 40% of fat for six months. A blood analysis was performed, and the heart samples were analyzed by qRT-PCR and Western blot. The results of this study showed no effects of sEng and HFD on myocardial morphology/hypertrophy/fibrosis. However, the expression of pSmad2/3 and p-eNOS was reduced in Sol-Eng+ high mice. On the other hand, sEng and HFD did not significantly affect the expression of selected members of TGFß signaling (membrane endoglin, TGFßRII, ALK-5, ALK-1, Id-1, PAI-1), inflammation (VCAM-1, ICAM-1), oxidative stress (NQO1, HO-1) and heart remodeling (PDGFß, COL1A1, ß-MHC). In conclusion, the results of this study confirmed that sEng, even combined with a high-fat diet inducing hypercholesterolemia administered for six months, does not affect the structure of the heart with respect to hypertrophy, fibrosis, inflammation and oxidative stress. Interestingly, pSmad2/3/p-eNOS signaling was reduced in both the heart in this study and the aorta in the previous study, suggesting a possible alteration of NO metabolism caused by six months exposure to high sEng levels and HFD. Thus, we might conclude that sEng combined with a high-fat diet might be related to the alteration of NO production due to altered pSmad2/3/p-eNOS signaling in the heart and aorta.

Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels (sEng+) showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male sEng+ mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in sEng+ mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.

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.

High soluble endoglin levels regulate cholesterol homeostasis and bile acids turnover in the liver of transgenic mice.

AIMS: Increased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. MAIN METHODS: Nine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. KEY FINDINGS: sEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. SIGNIFICANCE: For the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.

Soluble endoglin and hypercholesterolemia aggravate endothelial and vessel wall dysfunction in mouse aorta.

BACKGROUND AND AIMS: Increased plasma levels of soluble endoglin (sEng) were detected in patients with endothelial dysfunction-related disorders and hypercholesterolemia. In this study, we hypothesized that high levels of sEng accompanied by mild hypercholesterolemia could aggravate endothelial and vessel wall dysfunction and affect endoglin/eNOS signaling in mouse aorta. METHODS: Three-month-old female transgenic mice on CBAxC57BL/6J background, with high levels of sEng (Sol-Eng+high HFD), and their littermates with low levels of sEng (Sol-Eng+low HFD), were fed a high fat diet for six months. Plasma samples were used for biochemical, ELISA and Luminex analyses of total cholesterol, sEng and inflammatory markers. Functional parameters of aorta were assessed with wire myograph 620M. Western blot analyses of membrane endoglin/eNOS signaling and endothelial dysfunction/inflammation markers in aorta were performed. RESULTS: Functional analysis of aorta showed impaired KCl induced vasoconstriction, endothelial-dependent relaxation after the administration of acetylcholine as well as endothelial-independent relaxation induced by sodium nitroprusside in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. Ach-induced vasodilation after administration of l-NAME was significantly higher in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. The expression of endoglin, p-eNOS/eNOS, pSmad2/3/Smad2/3 signaling pathway was significantly lower in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. CONCLUSIONS: The results indicate that long-term hypercholesterolemia combined with high levels of sEng leads to the aggravation of endothelial and vessel wall dysfunction in aorta, with possible alterations of the membrane endoglin/eNOS signaling, suggesting that high levels of soluble endoglin might be considered as a risk factor of cardiovascular diseases.

Human endoglin as a potential new partner involved in platelet-endothelium interactions.

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbß3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann's thrombasthenia patients lacking the αIIbß3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng +/-) mice compared to Eng +/+ animals. These results suggest a new role for endoglin in αIIbß3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.