Published online before print October 30, 2008, doi: 10.1161/CIRCRESAHA.108.176552
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© 2008 American Heart Association, Inc.
Molecular Medicine |
S-Endoglin Expression Is Induced in Senescent Endothelial Cells and Contributes to Vascular Pathology
From the Centro de Investigaciones Biologicas (F.J.B., C.L., C.B.), Consejo Superior de Investigaciones Cientificas, Madrid; CIBER de Enfermedades Raras (F.J.B., C.L., C.B.), Instituto de Salud Carlos III, Madrid; Instituto "Reina Sofía" de Investigación Nefrológica (M.T.G., B.O., S.V., A.R.-B, J.M.L.-N.), Departamento de Fisiologia & Farmacologia, Universidad de Salamanca and Red de Investigacion Renal; and Instituto de Investigaciones Biomedicas Alberto Sols (M.Q., E.P-G.), Consejo Superior de Investigaciones Cientificas–Universidad Autonoma de Madrid, Spain.
Correspondence to Carmelo Bernabeu, Centro de Investigaciones Biologicas, CSIC, Ramiro de Maetzu 9, 28040 Madrid, Spain. E-mail bernabeu.c{at}cib.csic.es
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Abstract |
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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)-β 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-β receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-β 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-β–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-β1 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.
Key Words: endothelial cells • hypertension • TGF-β receptors • aging • endoglin
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Introduction |
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Cardiovascular repair mechanisms become progressively impaired with age, and advanced age is itself a significant risk factor for cardiovascular disease.1 Defects in age-associated remodeling of the vascular wall are, in part, attributable to the declining of endothelial function.2,3 Thus, vascular processes such as angiogenesis, nutrient trafficking, vascular repair, and homeostasis are impaired because of attenuation of endothelial cell (EC) proliferation, migration, or dilator responses. EC proliferation diminishes with age, entering in an irreversible senescent state.4 Senescent cells undergo growth arrest in the G1 phase and a change in morphology and metabolism. Some of the senescence-associated changes include cellular enlargement, altered response to growth factors such as transforming growth factor (TGF)-β1, and expression of senescence-associated β-galactosidase (SA-β-gal).5 Also, alterations in the expression and/or activity of the endothelial nitric oxide synthase (eNOS) are critical for the attenuation of the endothelium-dependent dilatory responses with age.6,7 Unfortunately, most of the functional and gene expression changes associated with EC senescence remain largely unknown.2,4
TGF-β is a family of multifunctional cytokines that regulate EC growth, differentiation, migration, senescence, extracellular matrix production, and angiogenesis.8 These functions are mediated through binding to a TGF-β–specific membrane receptor complex in ECs that contains 3 types of receptors: types I (TβRI) and II (TβRII), with cytoplasmic Ser/Thr-kinase activity8; and an auxiliary receptor named endoglin.9 Two different TβRIs, named ALK5 and ALK1, have been shown to balance TGF-β signaling by regulating opposite functions in ECs.8 Activation of the receptor complex triggers the phosphorylation by TβRI of the Smad family of proteins, which translocate to the nucleus to regulate gene expression. The activated TβRI determines the specificity of the downstream signaling. Thus, ALK5 phosphorylates Smad2/Smad3, whereas ALK1 activates Smad1/Smad5.8
Endoglin is a 180-kDa homodimer predominantly expressed in ECs that plays an important role in angiogenesis, vascular remodeling, and vascular pathology.9 Mutations in endoglin and ALK1 genes are responsible for an autosomal dominant vascular dysplasia termed hereditary hemorrhagic telangiectasia, characterized by recurrent hemorrhages and cerebral, hepatic, and pulmonary arteriovenous malformations.8 The in vivo expression of 2 different alternatively spliced isoforms, long (L)-endoglin and short (S)-endoglin, has been demonstrated in human and mouse tissues.10–12 L-endoglin, the predominant isoform in ECs, directly interacts with ALK1, ALK5, and TβRII13,14 and modulates EC responses to TGF-β.13,15–17 Whereas L-endoglin represses the TGF-β/ALK5/Smad3 pathway involved in plasminogen activator inhibitor (PAI)-1 expression,18,19 it activates that of TGF-β/ALK5/Smad2 to enhance eNOS expression20,21 and triggers the TGF-β/ALK1/Smad1 route to increase Id1 expression.13,15 Although, the role of S-endoglin in TGF-β signaling has been recently addressed in keratinocytes12 and myoblasts,22 little is known about the expression and function of S-endoglin in ECs. Here, we have analyzed the enhanced expression of S-endoglin during EC senescence and the functions affected by this isoform.
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Materials and Methods |
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Cell culture13,19,23; senescence-associated β-gal staining5; semiquantitative and quantitative RT-PCR11,24; in situ hybridization and expression vectors12,14; reporter assays13; transgenic mice expressing human S-endoglin in ECs (S-Eng+ mice)25; transfections, immunoprecipitation, and Western blot analyses13,20,26; urinary nitrite measurements20; and radiotelemetry monitoring of blood pressure27 are described in the expanded Materials and Methods section in the online data supplement, available online at http://circres.ahajournals.org.
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Results |
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S-Endoglin Expression Increases in Senescent ECs
Human umbilical vein endothelial cell (HUVEC) primary cultures showed increased senescence morphology along the number of passages, as evidenced by the blue SA-β-gal activity staining of their cytoplasm (Figure 1A). The size of SA-β-gal–positive cells was significantly larger than that of SA-β-gal negative cells, as expected for senescent cells (Figure I in the online data supplement). At the 14th passage, cells stopped proliferating, in agreement with the high percentage of senescent cells. At each passage, total RNA from HUVECs was isolated and subjected to RT-PCR using specific primers to detect TβRII and endoglin transcripts (Figure 1B). Along the different passages, no significant differences were obtained in the expression levels of total endoglin or TβRII (Figure 1C and 1D). Interestingly, S-endoglin began to be expressed at the 12th passage and displayed the highest expression level at the 14th passage (Figure 1C and 1E), sharing expression with its longer counterpart in senescent cells. Next, the expression of mouse S-endoglin during endothelial senescence in vivo was assessed. As determined by quantitative RT-PCR using specific primers (Figure 1F), transcript levels of S-endoglin were significantly increased in lungs from 32- and 60-week-old mice as compared with young animals (4 weeks old) (Figure 1G). To assess whether S-endoglin is expressed by ECs in vivo, in situ hybridization experiments with human coronary arteries were carried out (Figure 2). The antisense S-endoglin–specific riboprobe clearly hybridized with the endothelium monolayer and with some ECs in the subendothelial zone and tunica media that likely belong to the network of small vessels that vascularize the large coronary artery. Some scattered smooth muscle cells were also stained in the tunica media, which is compatible with the expression of endoglin protein in smooth muscle cells from human atherosclerotic plaques.28 As a negative control, the sense S-endoglin–specific riboprobe did not yield any signal above background levels. The endothelial labeling of S-endoglin was reproduced in human veins and lungs (supplemental Figure II).
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90% confluence. Blue cytoplasmic staining is indicative of SA-β-gal activity. Representative phase contrast microscopy fields from young (second passage) and senescent (13th passage) cells are included (left). Cells positive for SA-β-gal were counted from 4 random fields, and the percentage of senescent cells was represented as the means±SEM (right). B, Schematic representation of alternative splicing in the human endoglin gene (ENG). A 135-bp intron between exons E13 and E14 is retained, and a premature stop codon (
) arrests translation, giving rise to S-endoglin. L-endoglin is synthesized when the same 135-bp intron is spliced and translation stops in E14 (
). Endoglin primers that simultaneously amplify both isoforms are indicated with red arrows. Amino acid sequences from S- and L-endoglin cytoplasmic tails are shown. Sequences that differ between L and S isoforms are in blue. C through E, RT-PCR. C, Total RNA was isolated from HUVECs and submitted to semiquantitative RT-PCR using specific primers for S- and L-endoglin, TβRII, and GAPDH as an internal control. D, Total endoglin and TβRII levels. E, S-endoglin/L-endoglin ratio. F and G, Effect of aging on S-endoglin expression. F, Schematic representation of alternative splicing in mouse endoglin gene (eng). Primers that specifically amplify S-endoglin are indicated with red arrows. Cytoplasmic amino acid sequences that differ between L and S isoforms are in blue. G, Total RNA was isolated from lungs of mice of different ages and submitted to quantitative RT-PCR. The amount of S-endoglin amplificate (2–
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Interaction of S-Endoglin With ALK5 and ALK1
The extracellular and cytoplasmic domains of L-endoglin contribute to the physical and functional interaction with ALK5 and ALK1.13,14 Thus, it was of interest to investigate the behavior of S-endoglin in this context. Because both isoforms share a common extracellular domain, we assessed whether the S-endoglin cytoplasmic domain was able to physically interact with both TGF-β type I receptors. COS-7 cells were cotransfected with hemagglutinin (HA)-tagged ALK5 or ALK1 plus the HA-tagged vectors TMCT-EndoL or TMCT-EndoS, containing the transmembrane and cytoplasmic domains from L- or S-endoglin, respectively. We found that antibodies against either ALK5 or ALK1 can coimmunoprecipitate the cytoplasmic tails from both endoglin isoforms (Figure 3A). Interestingly, the amount of TMCT-EndoS or TMCT-EndoL coimmunoprecipitated with ALK5 was similar, whereas the amount of TMCT-EndoS coimmunoprecipitated with ALK1 was much lower than that of TMCT-EndoL (Figure 3C). These results suggest that the S-endoglin cytoplasmic tail interacts with ALK5 and ALK1 but shows a lower affinity for ALK1 than for ALK5. The specificity of these interactions was confirmed by using an HA-tagged irrelevant protein that yielded negative results. As a control, a Western blot analysis of the total cellular extracts is included (Figure 3B).
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We next assayed the interacting ability of the full-length isoform by cotransfecting Flag-tagged S-endoglin with HA-tagged ALK5 or ALK1 in COS-7 cells. Antibodies against the Flag epitope in S-endoglin were able to coimmunoprecipitate ALK5 with high yield (Figure 4A). By contrast, the S-endoglin/ALK1 interaction appeared to be much weaker (<50-fold; Figure 4C). A Western blot analysis of total cellular extracts was included to monitor the recombinant protein expression (Figure 4B).
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S-Endoglin and L-Endoglin Modulate the ALK5 and ALK1 Signaling Pathways Differently
The different behaviors of S-endoglin versus L-endoglin, with respect to the interaction with the type I receptors, suggest a specific functional role for S-endoglin in the endothelial TGF-β system. To address this issue, ECs were transfected with S-endoglin expression vector and the effect on the ALK5/ALK1 pathways was assessed (Figure 5). In HMEC-1 cells, the presence of S-endoglin enhanced the TGF-β–induced activity of the ALK5/Smad3 specific reporter p(CAGA)12-Luc compared with control cells (Figure 5A). In the same experiment, L-endoglin overexpression led to the inhibition of the p(CAGA)12-Luc activity in response to TGF-β, as reported.13 Next, the endothelial system GM7372-EL, engineered to express human L-endoglin in the presence of doxycycline,23 was used. Again, TGF-β–induced activity of the p(CAGA)12-Luc reporter was significantly increased on S-endoglin expression (Figure 5B). By contrast, L-endoglin expression, induced with doxycycline, not only inhibited the basal TGF-β–induced activity of the ALK5 reporter but also counteracted its S-endoglin–dependent enhancement. These results indicate that S-endoglin and L-endoglin have distinct effects on the ALK5/Smad3 signaling pathway. Because the extracellular domain is common to both endoglin variants,11 we assessed whether the isoform-specific cytoplasmic tails were responsible for their divergent behavior. Dissection of the independent functional role of each isoform is not easy to address in ECs because these cells show high levels of the predominant L-endoglin. The parental myoblast cell line L6E9 has the advantage of providing an endoglin-free background, and stable transfectants expressing either L-endoglin or S-endoglin19 somehow mimic the independent contribution of each isoform in young or senescent cells, respectively. Thus, myoblasts stably expressing S-endoglin or L-endoglin were transfected with TMCT-EndoL or TMCT-EndoS constructs, respectively. Ectopic expression of the cytoplasmic region of L-endoglin markedly reduced the TGF-β–induced activity of the p(CAGA)12-Luc reporter in S-endoglin–expressing cells (Figure 5C). Conversely, the cytoplasmic region of S-endoglin clearly enhanced the TGF-β–induced activity of the same reporter in L-endoglin–expressing cells, suggesting that the modulator effect of endoglin isoforms on the ALK5 pathway depends on their cytoplasmic region.
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The differential effect of S-endoglin versus L-endoglin was also assessed using the p(BRE)2-Luc reporter vector as a sensor for the ALK1/Smad1 pathway. S-endoglin expression did not affect the basal reporter activity, whereas the presence of L-endoglin markedly induced the p(BRE)2-Luc activity (Figure 5D). As expected, high doses of TGF-β1 inhibited p(BRE)2-Luc activity.
The results shown above indicate that S-endoglin and L-endoglin isoforms have distinct functional roles on the ALK5 and ALK1 signaling pathways. Because upregulation of PAI-1 and Id1 is specifically dependent on the TGF-β/ALK5 or TGF-β/ALK1 pathway, respectively,15,29 we assessed the effect of S-endoglin on pId1-Luc and p800-Luc reporters driven by the promoters of these genes. On transfection of HMEC-1, the Id1 promoter activity, both in the absence and in the presence of TGF-β1, decreased in response to increasing amounts of S-endoglin (Figure 5E). As a control, L-endoglin induced the transcriptional activity of pId1-Luc vector. On the other hand, increasing amounts of S-endoglin augmented in a dose-dependent fashion the activity of the PAI-1 reporter (Figure 5F). By contrast, L-endoglin inhibited the activity of PAI-1 reporter, as expected. Moreover, the effect of S-endoglin on the expression of the senescence associated markers PAI-1 and Id1,4,30 was assessed in senescent ECs whose S-endoglin levels are upregulated (Figure 1). As shown by semiquantitative RT-PCR experiments, senescent HUVECs display higher transcript levels of PAI-1 and lower transcript levels of Id1 than control cells (Figure 5G). In the same experiment, no significant differences in the levels of ALK1 or ALK5 were observed. As expected, the transcript levels of telomerase, a marker of senescence, were clearly downregulated. To address the role of S-endoglin in EC proliferation, murine lung ECs derived from S-Eng+ transgenic animals were analyzed. As shown in Figure 5H, S-Eng+ ECs displayed a lower proliferation rate than ECs from wild-type animals. Because cellular senescence is associated with decreased cell proliferation, this result supports the contribution of S-endoglin to EC senescence.
Taken together, these results suggest that, at variance with L-endoglin, S-endoglin potentiates the ALK5 pathway, leading to increased PAI-1, whereas it inhibits the ALK1 route, leading to decreased Id1 expression, thus mimicking the phenotype of senescent ECs.
Hemodynamics and eNOS Expression in Transgenic Mice Expressing S-Endoglin
The prevalence of cardiovascular disease increases with age and is associated with increased arterial pressure and decreased expression and/or activity of eNOS. To address the potential contribution of S-endoglin to the vascular pathology, S-Eng+ transgenic mice25 were analyzed. The weight was similar for S-Eng+ mice and control littermates (supplemental Figure III). Systolic, diastolic and mean arterial pressure values were higher in S-Eng+ than in control littermates (Figure 6A through 6C), whereas no significant differences were observed in heart rate (Figure 6D). Because endoglin regulates the expression of eNOS and thereby the NO-dependent vascular homeostasis,20 the role of NO was next addressed. Intravenous infusion of the nonspecific nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) increased blood pressure in both S-Eng+ and control littermate mice (Figure 6E and 6F), while reducing the heart rate (Figure 6G). Interestingly, L-NAME infusion resulted in a smaller increase in systolic and diastolic (33% less) blood pressure in S-Eng+ as compared with control mice (Figure 6E and 6F), suggesting less biologically available NO in the setting of S-Eng+ overexpression. Furthermore, acetylcholine-induced hypotension, an effect that is also mediated by NO, was significantly reduced in S-Eng+ as compared with control mice, whereas no significant differences were observed with sodium nitroprusside (an NO synthesis–independent, cGMP-dependent vasodilator) or with hydralazine (an NO synthesis–independent, cGMP-independent vasodilator) (supplemental Table II), again suggesting a lower NO production in S-Eng+ as compared with control mice. In fact, lower urinary nitrite excretion in S-Eng+ mice compared with control littermates was observed (Figure 6H), thus reflecting a reduced renal NO synthesis. In addition, eNOS expression was significantly lower, whereas cyclooxygenase (COX)-2 expression was significantly higher, in either kidney or lungs of S-Eng+ mice compared with control littermates (Figure 7A and 7B). Because TGF-β1 leads to an increased vasodilatation in Eng+/+ mice that is severely impaired in Eng+/– mice,21 the possible involvement of S-endoglin in the TGF-β-regulated vascular homeostasis was addressed next. S-Eng+ mice and control littermates showed a decrease in arterial pressure (diastolic and systolic) after TGF-β1 injection, but the decrease was significantly lower in S-Eng+ mice than in control littermates (Figure 7C and 7D). As a control, no significant differences were observed in the effects of TGF-β1 on heart rate between S-Eng+ mice and control littermates (Figure 7E).
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Discussion |
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Senescence of endothelial cells plays a critical role in cardiovascular diseases that are associated with aging. However, the underlying molecular mechanisms that involve the senescent ECs are poorly understood. Here, we report, for the first time, that expression of S-endoglin is induced during senescence of ECs (Figure 8). We provide evidence that in vivo and in vitro expression of S-endoglin contributes to the cardiovascular pathology associated with age, although a limitation of this study is the lack of functional analysis on blocking S-endoglin. Our present data support that upregulation of S-endoglin expression is part of the senescent program of ECs rather than being the sole responsible for senescence.
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To date, most of the studies on endoglin have focused on the predominant L-endoglin isoform, although significant levels of S-endoglin mRNA are coexpressed with L-endoglin in vivo.11,12 These isoforms are generated by alternative splicing, and the only difference between human S-endoglin and L-endoglin transcripts is an intronic sequence of 135-bp that is retained in S-endoglin.11 Unfortunately, no consensus small interfering RNA motifs could be identified in this sequence. Consequently, the analysis of functional effects of S-endoglin silencing is not feasible, and this represents a technical limitation of this study. On the other hand, S-endoglin and L-endoglin proteins vary from each other in their cytoplasmic tails that contain 14 and 47 amino acids, respectively, with a sequence of only 7 residues being specific for S-endoglin. This small structural difference explains the lack of appropriate tools to clearly distinguish between L-endoglin and S-endoglin in tissues and primary cultured cells. Thus, analysis by ectopic expression of cDNAs encoding human L- and S-endoglin in cell transfectants and transgenic mice represents an excellent model system to understand the role of these isoforms. In this study, we demonstrate that the functional role of L-endoglin is different from that of S-endoglin in ECs. This comparative study has been addressed by analyzing the TGF-β signaling pathway in ECs, where the TGF-β type I receptors ALK1 and ALK5 appear to play an active role in regulating the activation/resolution phases of angiogenesis.8,15 We show that S-endoglin interacts with both ALK5 and ALK1, although the interaction with ALK5 was stronger than with ALK1. This is at variance with L-endoglin that shows a higher affinity for ALK1 versus ALK5.13 Because the extracellular domain of endoglin is common in both isoforms, their cytoplasmic tails appear to be responsible for these differences. Thus, whereas ALK5 interacts with both endoglin cytoplasmic tails with comparable affinity, the interaction between ALK1 and the cytoplasmic tail of S-endoglin was significantly lower than with the long counterpart. Moreover, S-endoglin behaved differently than L-endoglin in relation to several TGF-β-responsive reporters with different specificities. Thus, S-endoglin expression increases the ALK5 signaling pathway, whereas L-endoglin inhibits the same pathway in ECs. Interestingly, S-endoglin also favors ALK5/Smad3 signaling in mouse carcinoma cells and rat myoblasts.22,24 On the other hand, L-endoglin, but not S-endoglin, stimulated the ALK1 signaling pathway. These results are in agreement with reports showing that L-endoglin promotes endothelial cell proliferation via TGF-β/ALK1 signaling, whereas it interferes with the TGF-β/ALK5 pathway,9,13,15 and suggest that S-endoglin acts as an antagonist of L-endoglin. In fact, S-endoglin appears to have a negative effect on EC proliferation. This is compatible with the reported antiangiogenic effect of S-endoglin in contrast to the proangiogenic role attributed to L-endoglin.12,26,31 Interestingly, angiogenic function is progressively impaired with increasing age,32 which fits well with the upregulation of S-endoglin in senescent ECs and its antiangiogenic effect. This short endoglin isoform is able to exert opposite effects to that of the predominantly expressed L-endoglin on the TGF-β signal, including the positive and negative cooperation with ALK5 and ALK1, respectively. Because both endoglin isoforms are coexpressed in senescent ECs, this raises the question about the molecular mechanism by which L-endoglin and S-endoglin antagonize with each other. The reported heterodimeric association between S-endoglin and L-endoglin12 could possibly account for a mutually interfering effect. Of note, ALK5 and ALK1 levels are not significantly altered between young and senescent ECs. This suggests that the S-endoglin/L-endoglin ratio in ECs may contribute to balancing the TGF-β signal through ALK5 or ALK1 and their important roles in vascular pathophysiology.8 In this context, increased synthesis of extracellular matrix is a pathological process widely related to high circulating levels of TGF-β and with the activation of the ALK5/Smad3 signaling pathway.33,34 Whereas S-endoglin highly increased the ALK5 signaling pathway, L-endoglin inhibited the same route, in agreement with a number of reports describing the role of L-endoglin as an antagonist of ALK5-mediated TGF-β induction of extracellular matrix synthesis.18,19,35 The positive contribution of S-endoglin to the profibrotic TGF-β/ALK5 pathway suggests its involvement in the increased fibrosis associated with vascular senescence.36
Further support for the involvement of S-endoglin in the cardiovascular pathology associated with senescence was obtained using S-endoglin transgenic animals. S-Eng+ mice show hypertension, a defective hypertensive response to the inhibition of NO synthesis with L-NAME, and a decreased urinary nitrite excretion, thus suggesting a defective NO synthesis. This interpretation is supported by the decreased eNOS expression in lungs and kidneys of these animals, as well as the positive relationship between levels of L-endoglin and eNOS reported in both mice and cultured ECs.20,26 In addition, endoglin-deficient mice show a defective endothelium-dependent vasorelaxation and decreased NO production,20 similarly to S-endoglin transgenic mice. Of note, NO inhibits EC senescence,37 thus supporting the involvement of S-endoglin in this process. Furthermore, in both cultured ECs and endoglin-deficient mice, low levels of L-endoglin are associated with high COX-2 expression,26 a correlation also found in S-Eng+ mice. Thus, in vivo overexpression of S-endoglin appears to result in the same phenotype as L-endoglin deficiency, suggesting opposing functional effects of both isoforms on the NO and COX-2 systems. On the other hand, S-Eng+ transgenic mice also show a reduced hypotensive response to TGF-β1 administration, in agreement with the finding that TGF-β1 regulates eNOS expression38 and induces vasodilatation in wild-type mice, which is severely impaired in endoglin-deficient animals.21 Thus, S-endoglin seems to have the opposite behavior than L-endoglin in these vascular parameters.
Taken together, these findings suggest that the alternatively spliced S-endoglin contributes to the cardiovascular pathology associated with aging. Further studies on the triggering mechanisms that control the alternative splicing of endoglin gene will help to better understand the genetic and physiological programs under which S-endoglin exert its function.
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Acknowledgments |
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We acknowledge the technical assistance of Annette Düwel in the care and genotyping of the S-Eng+ mice.
Sources of Funding
This work was supported by Ministerio de Ciencia e Innovacion grants SAF2007-61827 (to C.B.), SAF2007-63893 (to J.M.L.-N.), and SAF2007-63821 (to M.Q.); Junta de Castilla y Leon grant SA001/C05 (to J.M.L.-N.); CIBER de Enfermedades Raras, ISCIII-CB06/07/0038 (to C.B.); Red de Investigacion en Enfermedades Renales, ISCIII-RD06/0016 (to J.M.L.-N.); and support from the European Regional Development Fund to the Cardiovascular phenotyping unit, which includes the telemetry equipment. F.J.B. is a CIBER de Enfermedades Raras postdoctoral researcher.
Disclosures
None.
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Footnotes |
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Original received March 31, 2008; revision received October 20, 2008; accepted October 21, 2008.
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