Published online before print November 11, 2004, doi: 10.1161/01.RES.0000150369.68826.2f
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Transforming Growth Factor ß Receptor Endoglin Is Expressed in Cardiac Fibroblasts and Modulates Profibrogenic Actions of Angiotensin II
From the Departments of Medicine (K.C., J.L.M., D.L.), Physiology and Biophysics (J.L.M.), University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock; and Departments of Medicine (J.J.) and Pathology (L.J.), VA Boston Healthcare System and Boston University School of Medicine, Boston, Mass.
Correspondence to Jacob Joseph, MD, Cardiology Section (111), VA Boston Healthcare System, 1400 VFW Pkwy, West Roxbury, MA 02132. E-mail jacob.joseph{at}med.va.gov
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Abstract |
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Angiotensin II (Ang II) is a powerful mediator of adverse cardiac remodeling and fibrosis. However, the mechanisms of Ang II–induced myocardial fibrosis remain to be clarified. We postulated that Ang II alters transforming growth factor ß (TGF-ß) receptor expression, specifically that of endoglin, and thereby modulates cardiac fibroblast (CF) collagen metabolism. Experiments were conducted using CF from adult Sprague Dawley rats to determine the expression of TGF-ß1 receptors including endoglin, and the role of Ang II type 1 (AT1) and type 2 (AT2) receptors, and MAPK p42/44 in this process. The functional role of endoglin in modulating Ang II effects on matrix metalloproteinase-1 (MMP-1) and type I collagen expression was also analyzed. Endoglin gene and protein expression were consistently identified in quiescent CFs. Ang II increased the expression of endoglin mRNA and protein in a concentration and time-dependent manner, with no effect on TGF-ß receptors I and II expression. This effect was AT1 receptor mediated, because AT1 receptor antagonists valsartan, candesartan, and losartan inhibited Ang II–induced endoglin expression, whereas the AT2 receptor antagonist PD123319 had no effect. MAPKp42/44 inhibition attenuated Ang II–induced endoglin expression. Ang II–induced decrease in MMP-1 protein expression and increase in type I collagen protein expression were both blocked by a specific endoglin antibody. Hence, our results indicate that endoglin is upregulated in CFs by Ang II via the AT1 receptor and modulates profibrotic effects of Ang II. These findings provide novel insights into Ang II–induced cardiac remodeling.
Key Words: angiotensin II • collagen • endoglin • fibroblasts • remodeling
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Introduction |
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Activation of the renin-angiotensin system (RAS) is a central mediator of progressive cardiac remodeling.1 Cardiac remodeling denotes alterations in the structural components of the myocardium involving both myocyte and nonmyocyte compartments, a process which underlies progressive heart failure.2 Angiotensin II (Ang II), the main effector hormone of the RAS, modulates cardiac remodeling by causing myocyte hypertrophy and myocardial fibrosis.3,4 Myocardial interstitial changes, characterized by increases in total fibrillar collagen (types I and III), changes in the ratio of types I/III collagen, and alterations in collagen cross-linking may adversely affect cardiac diastolic and systolic function.1,5,6 Various models of cardiac remodeling and failure have demonstrated that Ang II is a powerful mediator of myocardial fibrosis.1–4 Several in vitro studies have shown that Ang II increases collagen production by cultured cardiac fibroblasts.7–10 In addition to these earlier studies demonstrating a direct effect of Ang II on cardiac fibroblast function, recent data also indicate an indirect effect of Ang II on cardiac fibroblasts mediated through myocytes.11
Transforming growth factor ß (TGF-ß1) modulates tissue fibrosis by direct effects on fibroblast function.12 This powerful fibrogenic cytokine has been demonstrated to mediate myocardial fibrotic response in various models of progressive cardiac remodeling.13 TGF-ß1 directly increases cardiac fibroblast collagen production in vitro, possibly by converting cardiac fibroblasts to the more synthetic myofibroblast phenotype.14,15 Interestingly, Ang II increases TGF-ß1 production by adult cardiac fibroblasts, indicating that its effects on cardiac fibrosis may be mediated by autocrine production of TGF-ß1.16 Animal models of progressive cardiac fibrosis have indicated that Ang II acts by upregulating expression of TGF-ß1 to produce myocardial fibrosis.17 However, increased expression of TGF-ß1 alone does not explain the cardiac effects of Ang II, and recent data suggests that other factors in addition to TGF-ß1 expression may be involved, such as receptor expression.18
Endoglin (CD 105) is a homodimeric membrane glycoprotein that is a type III TGF-ß1 receptor.19 This receptor is highly expressed in vascular endothelial cells, and is critical for angiogenesis in vivo.20,21 Studies have shown that endoglin modulates the function of TGF-ß1 by binding to and modulating signal transduction by the major TGF-ß1 receptors, type I and II.22,23 Recent reports have shown that endoglin is expressed in stromal tissues, and may modulate fibrosis in progressive renal disease, scleroderma, and postradiotherapy breast fibrosis.24–28 This study was designed to identify the expression of the TGF-ß1 receptors, specifically endoglin, in cardiac fibroblasts, and to study the interaction between Ang II and endoglin in modulating collagen metabolism. Our results demonstrate that endoglin is expressed in cardiac fibroblasts, and that it is upregulated by Ang II via the AT1 receptor. In addition, our data demonstrate that endoglin is a potent mediator of profibrotic effects of Ang II on cardiac fibroblasts.
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Materials and Methods |
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Cell Culture
Cardiac fibroblasts were prepared by the methods of Meszaros et al.29 Briefly, the ventricles of 2 to 3 hearts from adult male Sprague-Dawley rats (250 to 300 g; Jackson Laboratories, Bar Harbor, Me) were minced, pooled, and placed in a collagenase/protease (Sigma) digestion solution. Cells dissociated in the first treatment were discarded. After three digestions, cells were pooled, and debris and myocytes were removed by unit gravity sedimentation. Fibroblasts were isolated and suspended in DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum. After a 60-minute period of attachment to uncoated culture plates, cells that were weakly attached or unattached were rinsed free and discarded. Cells in passages 2 and 3 were used for all studies. Each study comprised 5 independent experiments. Because data from experiments using cells in passage 2 and 3 were similar, they were combined for statistical analysis. The purity of these cultures was >95% cardiac fibroblasts as measured by vimentin expression and negative by desmin (myocytes), smooth muscle
-actin (VSMCs), and von Willebrand factor (endothelial cells). All animals received humane care according to the guidelines stated in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985).
Study Design
First, we identified the basal expression of endoglin (mRNA and protein) in cardiac fibroblasts (CF). Next, the CF were incubated with Ang II (10–10 to 10–6 mol/L) for 3 to 24 hours to determine the regulation of endoglin (mRNA and protein). The concentration and time point for maximal effect of Ang II were used in subsequent experiments.
To examine the receptor specificity of Ang II action, CF were pretreated with or without 3 different Ang II type1 receptor blockers (losartan, candesartan, or valsartan, each 10–6 mol/L) or the AT2 receptor blocker PD123319 (10–6 mol/L) and then exposed to Ang II. The harvested cells were used to measure the expression of endoglin, and of TGF-ß1 receptors I and II (time course only).
To explore the molecular basis of the action of Ang II, we studied mitogen-activated protein kinase (MAPKp42/44) signaling pathway. For this purpose, CF were pretreated with the MAPKp42/44 inhibitor PD98059 (10–6 mol/L) for 30 minutes and then the cells were exposed to Ang II. The harvested cells were used to measure endoglin expression and MAPKp42/44 activity.
To examine the specific role of upregulation of endoglin, the expression of type I collagen and matrix metalloproteinases-1 (MMP-1) were examined in parallel experiments. Fibroblasts were pretreated with a monoclonal rat endoglin-blocking antibody (DAKO; 10 µg/mL) and then the cells were exposed to Ang II. The harvested cells were used to measure expression of type I collagen and MMP-1.
Concentrations of all reagents and the duration of incubation were chosen based on our previous studies30–33 on endothelial cells.
Total RNA (5 µg) extracted from cultured CF was reverse-transcribed with Oligo dT (Promega) and M-MLV reverse transcriptase (Promega) at 42°C for 1 hour, and 1.5 µL of the reverse-transcribed material was amplified with Taq DNA polymerase (Promega) using a primer pair specific to rat endoglin (upper primer, ATCCAACACCATAGAGCTAG; lower primer, TGGCTGAGGGGACAAGTTC).28 The RT-PCR product was 614 base pairs (position from 7 to 569). For PCR, 28 cycles were used at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. Rat ß-actin was amplified as a reference for semiquantitation of endoglin mRNA.
Western Blot Analysis
Fibroblast lysates from each experiment were separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes. After incubation in blocking solution (4% nonfat milk, Sigma), membranes were incubated with 1:1000 dilution primary antibody to endoglin (DAKO), TGF-ß1 type I or II receptor (Santa Cruz Biotechnology), MMP-1 (Oncogene Science), or type I collagen (Santa Cruz Biotechnology) overnight at 4°C. Membranes were washed and then incubated with 1:2000 dilution second antibody (Amersham) for 1 hour, the membranes were detected with the ECL system, and relative intensities of protein bands were analyzed by MSF-300G Scanner. For assessing MAPK phosphorylation, the membranes were incubated overnight with rabbit polyclonal phosphospecific MAPK antibodies (Calbiochem Co) that detect MAPKp42/44 only when catalytically activated by phosphorylation at Tyr-204. Membranes were detected with the ECL system, subsequently stripped and reprobed with MAPK antibody (Calbiochem Co.), and relative intensities of protein bands were analyzed.33
Data Analysis
All data (mean±SD) represent mean of at least five independent experiments. Data were evaluated by ANOVA with a Student-Newman-Keuls post hoc test using Sigmastat (SPSS Inc). A value of P<0.05 was considered significant.
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Results |
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Endoglin Expression in Cardiac Fibroblasts and its Upregulation by Ang II
Cardiac fibroblasts consistently expressed small amounts of endoglin; both mRNA and protein were detected in cultured CF. Interestingly, Ang II had a significant stimulatory effect on both transcription and translation of endoglin. Incubation of CF with Ang II induced the expression of both endoglin mRNA and protein in a concentration-dependent manner (10–10 to 10–6 mol/L) (Figure 1). The effect of Ang II on endoglin expression was also time dependent, as shown in Figure 2. This effect of Ang II (10–6 mol/L) on endoglin mRNA expression reached a peak at 12 hours and slightly decreased by 24 hours, but was still higher than the control value. A similar time course was seen with endoglin protein expression with a peak at 12 hours.
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Ang II Type 1 Receptor Mediates Ang II–Induced Endoglin Expression
Figure 3 shows the results of experiments examining the role of Ang II types 1 (AT1) and 2 (AT2) receptors in inducing endoglin expression. CF were pretreated for 30 minutes with AT1 receptor blockers (ARBs) Losartan, Candesartan, or Valsartan (10–6 mol/L), or the Ang II type 2 receptor blocker PD 123319, and then exposed to Ang II (10–6 mol/L) for 12 hours. All three ARBs abrogated Ang II–induced expression of both endoglin mRNA and protein, whereas the type 2 receptor blocker had no effect. There were no significant differences between the three ARBs in terms of blocking endoglin expression, although valsartan seemed to have a slightly greater effect. Because the MAP kinase pathway is a known signal transduction mechanism of Ang II effects on endoglin expression in endothelial cells,33 we also examined the role of this pathway in Ang II–induced endoglin expression in cardiac fibroblasts. As shown in Figure 4, the MAPK inhibitor PD 98059 inhibited expression of endoglin protein, concomitant with a decrease in expression of the active phosphorylated p42/44 MAPK.
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Endoglin Modulates Ang II Effects on Collagen Metabolism
We used a monoclonal antibody to endoglin to examine the role of endoglin in modulating Ang II effects on CF. Cardiac fibroblasts were pretreated with endoglin antibody (10 µg/mL) for 30 minutes and then exposed to Ang II (10–6 mol/L) for 12 hours (time of peak Ang II effect on endoglin expression). As shown in Figure 5, Ang II increased expression of type I collagen protein, whereas it decreased the expression of the collagen degrading enzyme matrix metalloproteinase (MMP)-1. Both of these effects were antagonized by the endoglin antibody, indicating that endoglin may play an important role in the autocrine loop of Ang II induced TGF-ß1 secretion and modulation of CF function.
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Ang II Effect on TGF-ß Type I and II Receptor Expression in Cardiac Fibroblasts
We examined the effect of Ang II on TGF-ß1 type I and II receptor protein expression. Both receptor proteins were expressed at baseline in CF. Ang II at 10–6 mol/L (concentration producing peak effect on endoglin expression) did not alter expression of either receptor at all time points tested up to 48 hours (Figure 6).
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Discussion |
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Progressive myocardial fibrosis is an important structural component of both diastolic and systolic heart failure from diverse cardiovascular pathologies, and Ang II plays a significant role in promoting myocardial fibrosis. In spite of compelling in vivo data connecting Ang II to myocardial fibrosis, the precise mechanisms of Ang II–induced myocardial fibrosis have not been elucidated. We examined whether endoglin, which modulates the actions of TGF-ß1, is a mediator of Ang II effects on collagen metabolism in CF. Our results indicate that Ang II promotes endoglin expression in cultured CF through its type 1 receptor. Endoglin receptor expression in response to Ang II alters the net balance of collagen metabolism in CF toward collagen deposition. These results indicate a novel mechanism for Ang II induced myocardial fibrosis and a potential mechanism of benefit of ARBs.
Cardiac remodeling in response to hemodynamic stress and/or injury includes changes in the cardiomyocyte compartment, which accounts for one third of cells in the myocardium,34,35 as well as the nonmyocyte compartment (endothelial cells, vascular smooth muscle cells, macrophages, and cardiac fibroblasts), which accounts for two thirds of cardiac cells. Pathological changes in cardiac remodeling (reviewed in detail36) includes myocyte hypertrophy, myocyte death, and dynamic changes in the interstitium. Interstitial changes involve qualitative and quantitative alterations in the fibrillar collagen network (composed mainly of types I and III fibrillar collagen), including changes in collagen cross linking.2,6,36 A phenotypic change of the cardiac fibroblast to the more synthetic myofibroblast phenotype has been proposed to occur under the influence of cytokines such as TGF-ß and to promote deposition of collagen.37,38 Myocardial collagen content is tightly regulated by a balance between collagen production and degradation. Extracellular degradation of collagen is the major rate limiting step in collagen metabolism and is effected by matrix metalloproteinases (MMPs).39 Collagenases (MMP types 1, 8, and 13) initiate degradation of collagen, whereas gelatinases (MMP-2 and MMP-9) effect digestion of collagen breakdown products (gelatins). Among the collagenases, MMP-13 is predominantly expressed in rodents, although MMP-1 is also expressed in rat hearts and its expression is altered in pathological states.40–42 The intramyocardial coronary arterioles also undergo changes in cardiac remodeling resulting in medial thickening and perivascular fibrosis. Collectively these changes progressively impair cardiac function.
Mammalian TGF-ß exists in three isoforms, TGF-ß1, -ß2, and -ß3, and TGF-ß1 has an important role in cardiac development and in adverse cardiac remodeling in response to injury such as myocardial infarction.12 TGF-ß1 induces cardiac fibrosis possibly by direct effects on cardiac fibroblast function, especially the phenotypic transformation of CF to the more synthetic myofibroblast.14,15 TGF-ß1 antagonism has been shown to ameliorate cardiac remodeling and fibrosis.13 The effects of the TGF-ß family are mediated through several receptors, principally the types I, II, and III receptors.43 Types I and II receptors have been studied in detail; binding of TGF-ß1 to these receptors promote signal transduction and cellular effects. Endoglin or the type III receptor was initially described as crucial for vasculogenesis, and its defect to be responsible for hereditary hemorrhagic telangiectasia.44 The function of this receptor has not been fully elucidated; however, it interacts with TGF-ß1 receptors I and II to modulate signal transduction.19–22 Hence, endoglin may play a major role in the response of tissues to TGF-ß1. Recent reports indicate that endoglin is overexpressed in renal tissues during progressive renal disease, and may modulate the remodeling process.24,25 Interestingly, studies in renal disease, scleroderma, and postradiation breast fibrosis indicate that endoglin may downregulate fibrotic mechanisms.26–28 Hence, endoglin may play an important role in the tissue remodeling process; however, specific mechanisms need to be elucidated.
Angiotensin II is a powerful stimulus to progressive cardiac remodeling and heart failure.1–4 Myocardial fibrosis is a prominent feature of cardiac remodeling secondary to Ang II. Even though Ang II has direct effects on cardiac fibroblast function, paracrine mechanisms, including those involving the myocyte, have been invoked to explain the pathogenesis of Ang II–induced cardiac fibrosis. Pathak and coworkers11 examined adult CF in culture and demonstrated that paracrine factors produced by myocytes are necessary for collagen production by CF in response to Ang II. Lee and coworkers21 have shown that Ang II increases TGF-ß1 production by CF, and this autocrine mechanism has been proposed to promote Ang II induced myocardial fibrosis in vivo. For example, Kupfahl and coworkers45 have shown that Ang II increases TGF-ß1 expression in human atrial myocardium. Tomita and coworkers46 studied the relationship of Ang II, TGF-ß1 and cardiac fibrosis in an in vivo rat model of cardiac remodeling induced by inhibition of nitric oxide synthesis. In this study, an AT1 receptor antagonist prevented the early induction of TGF-ß1 and later development of cardiac fibrosis, indicating that Ang II was acting indirectly through expression of TGF-ß1 to induce cardiac fibrosis. Our premise for these experiments was based on the postulate that in addition to the autocrine production of TGF-ß1, Ang II may modulate cardiac fibroblast function by upregulating endoglin expression in CFs.
Our results demonstrate that CFs express endoglin, and that Ang II stimulates this expression. All three angiotensin receptor blocking agents tested abrogated Ang II induced endoglin expression, indicating that the Ang II type 1 receptor mediated this effect. The Ang II type 2 receptor blocker had no effect on Ang II induced endoglin expression. These results are in accordance with current literature indicating that most of the adverse effects on cardiac structure and function attributable to Ang II are mediated through its type 1 receptor.1–4
Various signal transduction pathways have been implicated in Ang II effects on cardiac fibroblast function including activation of protein kinase C, activation of MAP-kinases, Raf-1 hyperphosphorylation, and expression of early (c-fos and c-jun) and intermediate response (c-myc and c-myb) genes (reviewed in detail47). Tharaux and coworkers,48 in their study of molecular pathways involved in Ang II–induced collagen I gene activation, showed that the MAPK/ERK cascade and TGF-ß1 were involved in the activation of type I collagen gene. In contrast, P38 kinase pathway or NF-
B did not play a role in Ang II–induced collagen gene activation. A prior study from our laboratory demonstrating Ang II–induced upregulation of endoglin in endothelial cells also showed that the MAPK pathway was involved in this AT1 receptor dependent process.33 Our results in cardiac fibroblasts also show that MAPK p42/44 inhibition decreased Ang II induced endoglin expression, indicating that the MAPK pathway may modulate this process in CF. Ang II did not alter the expression of TGF-ß1 receptors I and II in cardiac fibroblasts, unlike in vascular smooth muscle cells,49 where it has been shown to upregulate TGF-ß1 type I receptor expression. Our prior study in endothelial cells also demonstrated that Ang II upregulated the expression of TGF-ß1 receptors types I and II, mechanisms which do not seem to operate in cardiac fibroblasts.
We also analyzed the functional role of Ang II–induced endoglin expression in CF. Ang II increased type I collagen protein expression, whereas it decreased MMP-1 protein expression, changes which could promote collagen deposition in vivo. These effects are consistent with in vitro studies done by several investigators demonstrating that Ang II increased type I collagen production by cultured CF.7–10 Both endoglin blockade and ARBs decreased Ang II induced type I collagen protein expression, whereas they prevented Ang II–mediated decrease in MMP-1 expression. Because we studied only MMP-1 expression as opposed to expression and activity of the major rat collagenase MMP-13, and gelatinases MMP-2 and -9, it is possible that our in vitro results may not correlate with a significant effect of endoglin on collagen degradation in vivo. Overall these results indicate that expression of the endoglin receptor may be an important mediator of Ang II effects on CF, although in vivo relevance needs to be proven by further experiments in various models of cardiac remodeling.
Conclusions
Our studies demonstrate that endoglin, the type III TGF-ß1 receptor, modulates the profibrogenic effects of Ang II in cardiac fibroblasts. This effect of Ang II is mediated by the Ang II type 1 receptor and is blocked by ARBs. The net effect of Ang II induced endoglin expression is to alter collagen metabolism in the direction of collagen deposition. These findings indicate a novel link between Ang II and myocardial fibrosis. Further studies will delineate the specific mechanisms of interaction between Ang II and TGF-ß1 signaling in cardiac fibroblasts, the role of endoglin in this process, and the in vivo relevance of this interaction.
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Acknowledgments |
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The studies described were supported in part by grants from Novartis Pharmaceuticals and Astra-Zeneca Pharmaceuticals (J.J.).
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Footnotes |
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Original received August 6, 2004; revision received October 21, 2004; accepted November 1, 2004.
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