Alicia Rodríguez-Barbero

Endoglin regulates nitric oxide-dependent vasodilatation

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

L- and S-endoglin differentially modulate TGFβ1 signaling mediated by ALK1 and ALK5 in L6E9 myoblasts

TGFβ regulates cellular processes by binding to type I and type II TGFβ receptors (TβRI and TβRII, respectively). In addition to these signaling receptors, endoglin is an accessory TGFβ receptor that regulates TGFβ signaling. Although there are two different alternatively spliced isoforms of endoglin, L-endoglin (L, long) and S-endoglin (S, short), little is known about the effects of S-endoglin isoform on TGFβ signaling. Here, we have analyzed the TGFβ1 signaling pathways and the effects of L- and S-endoglin in endoglin-deficient L6E9 cells. We found that TGFβ activates two distinct TβRI-Smad signaling pathways: ALK1-Smad1-Id1 and ALK5-Smad2-PAI1, in these cells. Interestingly, L-endoglin enhanced the ALK1-Id1 pathway, while S-endoglin promoted the ALK5-PAI1 route. These effects on signaling are supported by biological effects on TGFβ1-induced collagen I expression and inhibition of cell proliferation. Thus, while L-endoglin decreased TGFβ1-induced collagen I and CTGF expression and increased TGFβ1-induced proliferation, S-endoglin strongly increased TGFβ1-induced collagen I and CTGF expression, and reduced TGFβ1-induced cell proliferation.

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.

Transforming growth factor-β1 induces collagen synthesis and accumulation via p38 mitogen-activated protein kinase (MAPK) pathway in cultured L6E9 myoblasts

Transforming growth factor‐β (TGF‐β) plays a pivotal role in the extracellular matrix accumulation observed in chronic progressive tissue fibrosis, but the intracellular signaling mechanism by which TGF‐β stimulates this process remains poorly understood. We examined whether mitogen‐activated protein kinase (MAPK) routes were involved in TGF‐β1‐induced collagen expression in L6E9 myoblasts. TGF‐β1 induced p38 and extracellular signal‐regulated kinase (ERK) 1/2 phosphorylation whereas no effect on Jun N‐terminal kinase phosphorylation was observed. Biochemical blockade of p38 but not of the ERK MAPK pathway abolished TGF‐β1‐induced α2(I) collagen mRNA expression and accumulation. These data indicate that TGF‐β1‐induced p38 activation is involved in TGF‐β1‐stimulated collagen synthesis.

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)-&bgr; 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-&bgr; receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-&bgr; 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-&bgr;–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-&bgr;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.

Potential use of isolated glomeruli and cultured mesangial cells as in vitro models to assess nephrotoxicity

The purpose of this short review is to present the potential of using isolated glomeruli and cultured mesangial cells as two differentin vitro models to assess the glomerular effect of molecules with nephrotoxic properties. The advantage of using isolated renal glomeruli is that they conserve the architecture of this anatomical region of the kidney; moreover, they are free of any vascular, nervous or humoral influences derived from other regions of the kidney. Mesangial cells are perivascular pericytes located within the central portion of the glomerular tuft between capillary loops. Mesangial cells have a variety of functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, and control of glomerular hemodynamics, mainly the ultrafiltration coefficient Kf, via mesangial cell contraction or release of vasoactive hormones. Most authors agree that mesangial cells play a major role in glomerular contraction, filtration surface area, and Kf regulation. One of the major effects of toxicants on glomerular structures is contraction. We can assess quantitatively the degree of toxicant-induced mesangial cell contraction or glomerular contraction by measuring the changes in planar cell surface area or apparent glomerular cross-sectional area after exposition to the toxicant. Thesein vitro models can also reveal glomerular effects of xenobiotics that are difficult or impossible to observe in vivo. In addition, these studies permit a fundamental examination of the mechanism of action of xenobiotics on glomerular cells, including the possibility that at least a part of their effects are mediated by local mediators released by glomerular cells. We review the effects and the mechanisms of action of several toxicants such as gentamicin, cyclosporin, cisplatin, and cadmium on isolated glomeruli or cultured mesangial cells. As suchin vitro results confirmin vivo renal hemodynamic changes caused by toxicants, we conclude that these models are fruitful tools for the study of renal toxicity. Thesein vitro systems might also serve as a predictive tool in the evaluation of drugs inducing changes in glomerular filtration rate and as a way to propose protective agents against these dramatic hemodynamic effects.

Endoglin modulation of TGF-beta1-induced collagen synthesis is dependent on ERK1/2 MAPK activation

Background/Aims: Transforming growth factor-β1 (TGF-β1) plays a pivotal role in the extracellular matrix accumulation observed in fibrotic diseases. Endoglin is an important component of the TGF-β receptor complex highly expressed in tissues undergoing fibrotic processes. Endoglin expression regulates the effect of TGF-β on extracellular matrix synthesis. The purpose of our study has been to understand the molecular mechanism by which endoglin exerts its effects on fibrosis and the possible role of MAP kinases in these effects. Methods: We have assessed in mock and in endoglin-transfected L6E9 myoblasts the effect of TGF-β1 on collagen mRNA by Northern blot and effect of TGF-β1 on collagen content in the cultured medium by [3H]-Proline incorporation into collagen proteins. Total and activated MAPK and their role on collagen synthesis were assessed by Western blot. Results: TGF-β1 induced an increase on α2 (I) collagen mRNA expression and collagen accumulation in mock-transfected myoblasts, whereas the response was much lower in endoglintransfected cells. TGF-β1 activated the ERK1/2 and p38 MAPK pathways but not the JNK pathway in L6E9 myoblasts. TGF-β1-induced α2 (I) collagen mRNA expression and collagen accumulation were completely inhibited by SB203580, in either mock or endoglintransfected myoblasts. PD98059 increased TGF-β1 induced-collagen synthesis and accumulation in endoglin-transfected myoblasts but not in mock cells. Conclusion: Our studies demonstrate that TGF-β1- induced collagen synthesis is mediated by p38 MAPK activation in L6E9 myoblasts. Furthermore, endoglin expression reduces basal and TGF-β1 induced collagen synthesis when ERK1/2 pathway is operating.

Endoglin expression regulates basal and TGF-β1-induced extracellular matrix synthesis in cultured L6E9 myoblasts

Background/aims: TGF-β1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-β) receptor complex. Endoglin is upregulated by TGF-β1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-β1. Methods: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. Results: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased α 2 (I) procollagen mRNA expression in endoglin transfectants. TGF-β1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. Conclusion: These results demonstrate that endoglin expression negatively regulates basal and TGF-β1-induced CTGF and collagen expression and synthesis.

Endoglin Regulates Cyclooxygenase-2 Expression and Activity

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

Identification of serum endoglin as a novel prognostic marker after acute myocardial infarction.

Endoglin is a proliferation‐associated and hypoxia‐inducible protein expressed in endothelial cells. The levels of soluble circulating endoglin and their prognostic significance in patients with acute myocardial infarction (AMI) are not known. In this observational prospective study serum endoglin levels were measured by ELISA in 183 AMI patients upon admission to hospital and 48 hrs later and in 72 healthy controls. Endoglin levels in AMI patients on admission were significantly lower than in healthy controls (4.25 ± 0.99 ng/ml versus 4.59 ± 0.87 ng/ml; P= 0.013), and decreased further in the first 48 hours (3.65 ± 0.76 ng/ml, P < 0.001). Upon follow‐up (median 319 days), patients who died had a significantly greater decrease in serum endoglin level over the first 48 hrs than those who survived (1.03 ± 0.91 versus 0.54 ± 0.55 ng/ml; P= 0.025). Endoglin decrease was an independent predictor of short‐term (30 days) (hazard ratio 2.33;95% CI = 1.27–4.23; P= 0.006) cardiovascular mortality, and also predicts overall cardiovascular mortality during the follow‐up (median 319 days) in AMI patients (hazard ratio 2.13;95% CI = 1.20–3.78; P= 0.01). In conclusion, early changes in serum endoglin may predict mortality after AMI.