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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2546.)
© 2000 American Heart Association, Inc.

Vascular Biology

Endoglin Is Overexpressed After Arterial Injury and Is Required for Transforming Growth Factor-ß–Induced Inhibition of Smooth Muscle Cell Migration

Xiaoli Ma; Marino Labinaz; Jason Goldstein; Harvey Miller; Wilbert J. Keon; Michelle Letarte; Edward O’Brien

From the Division of Cardiology (X.M., M.Lab., J.G., H.M., E.O.) and the Division of Cardiovascular Surgery (W.J.K.), University of Ottawa Heart Institute, Ottawa, Ontario, Canada, and the Cancer and Blood Program (M.Let.), The Hospital for Sick Children and the Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

Correspondence to Edward R. O’Brien, MD, FRCPC, FACC, Division of Cardiology, Vascular Biology Laboratory, University of Ottawa Heart Institute, 40 Ruskin St, Ottawa, Ontario, Canada K1Y 4W7. E-mail eobrien{at}ottawaheart.ca

	Abstract

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Abstract—Endoglin is a homodimeric membrane glycoprotein primarily expressed on endothelial cells. In association with transforming growth factor (TGF)-ß receptors I and II, it can bind TGF-ß1 and -ß3 and form a functional receptor complex. There is increasing evidence that endoglin can modulate the cellular response to TGF-ß, a factor implicated in vascular lesion formation in human and experimental models. The purpose of this study was to analyze the expression of endoglin in normal and balloon-injured porcine coronary arteries and in normal and atherosclerotic human coronary arteries and to determine its ability to mediate the effects of TGF-ß on the migration of vascular smooth muscle cells (SMCs). In normal porcine coronary arteries, endoglin was of low abundance and was found primarily on endothelial cells and adventitial fibroblasts, as well as on a minority of medial SMCs. On days 3, 7, and 14 after angioplasty, endoglin was present not only on endothelial cells but also on adventitial myofibroblasts and medial SMCs of porcine coronary arteries. By day 28, few or no cells expressed endoglin. In situ hybridization revealed that endoglin mRNA expression appeared to be highest in endothelial cells on days 3, 7, and 14 days after injury and absent thereafter. With a second balloon injury, a similar pattern of endoglin protein and mRNA expression was observed. In human vascular tissue, endoglin immunolabeling was higher in endarterectomy specimens removed from diseased coronary arteries than in normal internal mammary arteries. In vitro, antisense oligonucleotides to endoglin decreased its expression and antagonized the TGF-ß–mediated inhibition of human and porcine SMC migration. In summary, upregulation of endoglin occurs during arterial repair and in established atherosclerotic plaques and may be required for modulation of SMC migration by TGF-ß.

Key Words: endoglin • transforming growth factor-ß • receptors • smooth muscle cells • endothelial cells

	Introduction

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The transforming growth factor (TGF)-ß family of proteins plays an important role in atherogenesis and restenosis. For example, TGF-ß1 and the TGF-ß1 inducible gene h3 are overexpressed in atherosclerotic and restenotic human lesions.^{1 2 3} In various animal models of arterial repair, TGF-ß1 expression is upregulated after injury.^{4 5 6} Moreover, the infusion of TGF-ß1 polypeptide or transfection of TGF-ß1 cDNA into injured arteries increases extracellular matrix production and accelerates lesion formation, whereas antibodies to TGF-ß reduce intimal hyperplasia.^{7 8 9 10} The ability of vascular cells to respond to TGF-ß depends on specific transmembrane receptors known as type I (TßR-I) and type II (TßR-II), which are serine/threonine kinases. Subsequent to ligand binding, TßR-II recruits TßR-I, induces its phosphorylation, and thereby initiates the signaling pathway.¹¹ Whereas smooth muscle cells (SMCs) from normal arteries are growth-inhibited by TGF-ß1 in vitro, those derived from atherosclerotic and restenotic lesions are resistant to the antiproliferative effect of TGF-ß.^{12 13} The latter phenomenon may be due to an alteration in the distribution of TGF-ß receptors in atherosclerotic plaques.^{2 13}

Endoglin (or CD105) is a homodimeric membrane glycoprotein that (in association with TGF-ß receptors) binds TGF-ß1 and -ß3 isoforms in human endothelial cells (ECs).^{14 15 16} It is composed of an extracellular domain of 561 amino acid residues, a single transmembrane domain of 25 amino acid residues, and a short intracellular domain of 47 amino acids that does not include a signal transduction domain.¹⁷ Initially identified in human pre-B leukemic cells, endoglin has subsequently been shown to be expressed by ECs and by activated monocytes/macrophages, as well as by various mesenchymal cells, including fibroblasts and vascular SMCs.^{18 19 20}

Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1), an autosomal-dominant disorder characterized by the presence of dilated postcapillary venules and large arteriovenous malformations.^{21 22 23} Although a number of endoglin mutations have been identified in patients with HHT, it is now recognized that reduced levels of functional endoglin (haplo insufficiency) rather than a dominant-negative effect of the mutated protein is the underlying cause of HHT1.^{24 25} However, increased endoglin expression is observed in ECs of microvessels from pathological skin lesions and in the neovessels of tumors, suggesting a role for endoglin during EC proliferation.^{26 27 28} More recently, Adam et al²⁹ have documented endoglin mRNA and protein in human SMCs, suggesting that endoglin expression in the artery wall is not restricted to the endothelium and may be involved in other disease processes, including atherosclerosis and restenosis.

Endoglin plays a regulatory role in several TGF-ß pathways. Studies using antibodies specific for endoglin or TßR-II demonstrate coimmunoprecipitation of endoglin with TßR-I and TßR-II in the presence of TGF-ß.^{15 16} Endoglin alone does not bind TGF-ß1 or -ß3 but requires the presence of TßR-II.³⁰ Endoglin can also interact with other members of the TGF-ß superfamily (eg, activin A and bone morphogenic protein-7 and -2) via its association with their respective ligand binding kinase receptors.³⁰ Overexpression of endoglin in murine fibroblasts leads to decreased migration in chemotaxis and wound-healing assays.³¹ In contrast, antagonism of endoglin (with antibodies or antisense oligonucleotides) attenuates TGF-ß–induced inhibition of human extravillous trophoblast differentiation and placental migration in vitro.³² Therefore, it is possible that endoglin may be required for TGF-ß signaling. The purpose of the present study was to examine the expression pattern of endoglin in normal and diseased arteries as well as its role in mediating the biological effect of TGF-ß in vascular SMCs. As will be described, endoglin is overexpressed by SMCs, myofibroblasts, and ECs of diseased arteries and may play an important role in regulating TGF-ß–induced inhibition of SMC migration.

	Methods

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The following is an abridged version of Methods. An expanded methodology section is available online at http://atvb.ahajournals.org.

Porcine Balloon Injury Model
A previously reported porcine model of coronary artery injury was used in the present study.^{33 34} All studies were carried out with approval of the University of Ottawa Animal Care Committee and followed the guidelines of the Canadian Council on Animal Care.

Human Arterial Tissue
Three normal internal mammary arteries and 3 coronary endarterectomy samples were obtained from patients undergoing bypass surgery at the University of Ottawa Heart Institute. The Ethics Review Committee at the Ottawa Civic Hospital approved the use of this tissue for research purposes, and all patients gave informed consent before tissue harvesting. All tissues were placed in OCT compound (Miles Laboratories) immediately after harvesting and were frozen and stored at -80°C. A cryostat was later used to cut 5-µm sections, which were placed on glass slides.

Antibodies
Commercially available monoclonal antibodies (Mabs) recognizing smooth muscle -actin and bromodeoxyuridine (BrdU) were used to immunolabel SMCs and proliferating cells, respectively. The Mabs GRE and 29G8 obtained from the V and VI International Leukocyte Workshops were used to immunolabel human endoglin.^{35 36} In addition, the Mabs 44G4, P3D1, and P4A4, which are specific for human endoglin, were used for flow cytometric analysis and immunoprecipitation of radiolabeled endoglin.^{32 36 37}

Immunohistochemistry
Immunohistochemistry was carried out as previously described.³⁸ Negative controls were carried out with nonimmune IgG1 or IgM antibodies, depending on the nature of the primary antibody.

In Situ Hybridization
In situ hybridization was performed as previously described.³ Briefly, ³⁵S-UTP–labeled antisense and sense riboprobes were generated from a porcine endoglin cDNA construct and hybridized with the tissue slides.¹⁵ After overnight drying, the slides were dipped in Kodak NTB-2 emulsion and stored for 3 weeks before being developed and counterstained with hematoxylin.

Endoglin Antagonism and SMC Migration
To evaluate the role of endoglin in mediating the biological effects of TGF-ß, human coronary artery SMCs were processed, and the following steps were taken: (1) endoglin expression in SMCs was confirmed by use of flow cytometric analysis, (2) antagonism of endoglin expression by antisense oligonucleotides to endoglin was demonstrated by use of metabolic labeling studies, and (3) a Boyden chamber assay was used to examine the effect of these same oligonucleotides on mediating the effects of TGF-ß on SMC migration.³⁹

Statistical Analysis
All results are expressed as the mean±1 SD. For comparison of multigroup variables, the variance of means was analyzed by a 1-way ANOVA. Differences were considered significant at P<0.05.

	Results

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Pattern of Endoglin Expression in Porcine Coronary Arteries
Twenty pigs were studied, with 5 pigs euthanized at each of the 4 time points. For each pig, 1 artery subjected to single injury (SI), 1 artery subjected to double injury (DI), and 1 control artery were harvested and examined in detail.

Normal Coronary Arteries
In normal arteries, the intima consisted of an intact endothelial layer (Figure 1A). Medial SMCs were immunolabeled with the anti–smooth muscle -actin antibody, whereas cells in the adventitia were not (Figure 1B). A low level of endoglin expression was found in ECs lining the central arterial lumen, adventitial fibroblasts, and a minority of medial SMCs (Figure 1C and 1D). No endoglin mRNA expression was detected in normal arteries by in situ hybridization (data not shown).

View larger version (86K): [in this window] [in a new window]   Figure 1. Normal porcine coronary artery. A, Normal porcine coronary artery is shown (Movat pentachrome stain, original magnification x40). B, Immunolabeling with anti–smooth muscle -actin antibody demonstrates presence of SMCs in media (m) and absence of smooth muscle -actin immunopositive cells in the adventitia (a) (hematoxylin nuclear counterstain, original magnification x200). C and D, Immunolabeling with anti-endoglin antibody (29G8) shows that many ECs express endoglin, although low levels of expression were also found in some medial SMCs and adventitial fibroblasts (hematoxylin nuclear counterstain, original magnification x200 [C] and x1000 [D]).

3 Days After SI
Three days after SI, the central lumen area appeared larger because of overstretching of the artery by the balloon catheter and the creation of medial dissection planes. The external elastic lamina remained intact (Figure 2A). Smooth muscle -actin immunopositive SMCs were found in the media. In the adventitia, there was an increase in the number of cells and the extracellular matrix content in injured compared with normal arteries.³³ A minority of the adventitial cells expressed smooth muscle -actin protein at this point (Figure 2B). Anti-BrdU immunolabeling showed that the proliferative activity of the adventitial fibroblasts exceeded that of medial SMCs (Figure 2C). Endoglin was immunolocalized to lumenal ECs and medial SMCs as well as adventitial fibroblasts (Figure 2D). Omission of the primary anti-endoglin antibody resulted in an absence of the color reaction product (Figure 2E). Endoglin mRNA expression was abundant in lumenal ECs and much lower in SMCs and fibroblasts elsewhere in the arterial wall. (Figure 2F)

View larger version (148K): [in this window] [in a new window]   Figure 2. Porcine coronary artery 3 days after single injury. A, Large dissection has resulted in separation of the media; however, external elastic lamina is intact. Neointima has not yet developed (Movat pentachrome stain, original magnification x40). B, This panel and all subsequent panels are high-power photomicrographs of the area in panel A that is adjacent to the medial dissection (hematoxylin nuclear counterstain, original magnification x100). Immunolabeling with antibody to smooth muscle -actin demonstrates the presence of SMCs in the media (m) and the emergence of smooth muscle -actin immunopositive myofibroblasts in the adventitia (a). C, BrdU immunolabeling demonstrates the high proliferative profile of adventitial cells, which are primarily myofibroblasts. Inset shows BrdU immunopositive proliferating cells (original magnification x400). D, Immunolabeling with Mab 29G8 demonstrates the expression of endoglin in ECs lining the central lumen and adventitial vasa vasorum as well as adventitial myofibroblasts. Some medial SMCs also showed a lower abundance of immunolabeling for endoglin. E, Immunolabeling with nonspecific antibody IgM failed to demonstrate a color reaction product (negative control). F, In situ hybridization with radiolabeled endoglin riboprobe demonstrates that endoglin mRNA was abundantly expressed by lumenal ECs (upper inset, original magnification x400) although medial SMCs and adventitial myofibroblasts also expressed endoglin mRNA (lower inset, original magnification x400).

7 and 14 Days After SI
Please refer to Figure I (published online at http://atvb.ahajournals.org) for a graphic presentation of these data. By 7 days after SI, a modest neointima had formed, especially in areas with disruption of the media. Similarly, the expansion in the adventitia (hereafter referred to as the neoadventitia) was more pronounced in areas with medial disruption. There was abundant expression of smooth muscle -actin by adventitial fibroblasts. As previously characterized by Shi et al⁴⁰ and Scott et al,⁴¹ who used the same porcine model, these adventitial cells can be considered myofibroblasts. In contrast to arteries 7 days after SI, the neointima and neoadventitia were larger 14 days after SI, and as previously described, lumenal narrowing also occurred.^{33 34} By 7 and 14 days after SI, endoglin expression was evident in the lumenal endothelium, neointima (particularly in the subendothelial layers), media, and neoadventitial myofibroblasts. In situ hybridization demonstrated upregulated endoglin mRNA expression, principally by ECs lining the central arterial lumen but also by ECs of the neoadventitial vasa vasorum.

28 Days After SI and DI
By 28 days after SI, lumenal narrowing had progressed and was primarily due to the constrictive effect of fibrotic scar tissue in the neoadventitia.³³ A modest nonobstructive neointima was also present. Endoglin immunolabeling was essentially negative at this late stage after balloon injury. Similarly, endoglin mRNA expression was not detected by in situ hybridization. In the DI arteries, the pattern of endoglin mRNA and protein expression was similar to the temporal and spatial pattern observed after SI, with the exception of very modest endoglin protein expression in lumenal ECs and intimal SMCs 28 days after DI. Please refer to Figure II (published online at http://atvb.ahajournals.org) for a graphic presentation of these data.

Expression of Endoglin in Human Arterial Tissue
In normal human internal mammary arteries, endoglin was detected in ECs and in a minority of medial SMCs. In contrast, diseased vascular tissue in the form of coronary endarterectomy specimens demonstrated more abundant immunolabeling for endoglin in ECs and intimal SMCs (Figure 3).

View larger version (110K): [in this window] [in a new window]   Figure 3. Endoglin protein expression in normal and diseased human arteries. Panels A and B show normal internal mammary artery and endarterectomy specimen from a diseased coronary artery, respectively, that were immunolabeled by the 44G4 anti-endoglin antibody. Panels C and D show adjacent tissue sections corresponding to panels A and B, respectively, that have been immunolabeled with a nonspecific IgG antibody (negative control). In the normal internal mammary artery, endoglin was expressed in ECs (lumenal to the internal elastic lamina, designated by arrows) and some medial SMCs (lumenal to the external elastic lamina, designated by arrowheads). Although there is a brownish-orange reaction product with the nonspecific IgG antibody in panel C, this is limited to the fibrofatty tissues in the adventitia and is unrelated to the specific immunolabeling seen in panel A. In the human coronary endarterectomy sample, there is specific immunolabeling of SMCs within the plaque that is not seen with the nonspecific IgG antibody in panel D. In all panels, hematoxylin nuclear counterstain was used (original magnification x200).

Role of Endoglin in SMC Migration
Before initiating migration studies, we confirmed that vascular SMCs in culture express endoglin and that antisense oligonucleotides to endoglin reduce the expression of endoglin (see Figure III, published online at http://atvb.ahajournals.org for a graphic presentation of these data). Human coronary artery SMCs were grown in culture. Their identity was confirmed by typical morphology (eg, spindle shape and hill-and-valley pattern) and positive immunolabeling with an antibody to smooth muscle -actin. Using flow cytometry and the Mab P3D1, we then demonstrated that >95% of human SMCs in culture express cell surface endoglin. Similar profiles were also observed when Mabs 44G4 and GRE were used (data not shown). The level of endoglin expression, estimated from the mean fluorescence intensity, was approximately equivalent to 10% of that observed on human umbilical vein endothelial cells in culture (data not shown). Next, we used metabolic labeling to demonstrate that antisense oligonucleotides to endoglin reduce endoglin expression in vitro. SMCs synthesize endoglin as a partially processed intracellular dimeric precursor (160 kDa) that is converted to a fully processed dimeric glycoprotein of 180 kDa. When cells were treated for 24 hours with 5 µmol/L of antisense phosphorothioate oligonucleotides to endoglin, synthesis of this protein was partially inhibited relative to the sense oligonucleotide–treated cells. PhosphorImager (Molecular Dynamics) quantification of 8 different gel lanes, fractionated under reducing and nonreducing conditions, yielded a mean protein level in the antisense-treated SMCs that was 66% of that observed in the sense or scrambled cells. As will be shown below, this reduced level of endoglin expression is proportional to the reduction in the effect of TGF-ß on SMC migration that is observed when the same antisense oligonucleotides are included in a migration assay.

A modified Boyden chamber assay was used to examine the potential role of endoglin in mediating the biological effect of TGF-ß1. TGF-ß1 is known to inhibit platelet-derived growth factor (PDGF)-AB–stimulated SMC migration in a concentration-dependent manner.⁴² Using human coronary artery SMCs, we observed a 6.8-fold increase in SMC migration when PDGF-AB was added to the medium (P<0.001 versus DMEM alone). Addition of TGF-ß reduced the PDGF-AB–stimulated migration to 1.9 times that of baseline (DMEM) levels (P<0.001). Low concentrations (eg, 0.05 µmol/L and 0.5 µmol/L) of antisense oligonucleotides to endoglin had no effect on the ability of TGF-ß1 to inhibit PDGF-AB–induced SMC migration. Antisense oligonucleotides to endoglin at a concentration of 5 µmol/L reversed the TGF-ß1 effect to 3.6 times that of baseline levels (P<0.03 versus absence of oligonucleotides). However, this effect was not reproduced with equivalent concentrations of sense and scrambled oligonucleotides (eg, compared with antisense, P=0.002 and 0.004, respectively; Figure 4A). Similar migration results were observed when porcine SMCs were exposed to antisense endoglin oligonucleotides (Figure 4B). With porcine vascular SMCs, a 9.1-fold increase in SMC migration occurred when PDGF-AB was added to DMEM (P<0.001). Addition of TGF-ß1 reduced the PDGF-AB–stimulated migration to 1.7 times that of baseline levels (P<0.001). Low concentrations (eg, 0.2 µmol/L and 1.0 µmol/L) of antisense oligonucleotides to endoglin had no effect on the ability of TGF-ß1 to inhibit PDGF-AB–induced SMC migration. However, antisense oligonucleotides to endoglin at a concentration of 10 µmol/L reversed the TGF-ß1 effect to 7.6 times that of baseline (DMEM) levels (P<0.001). Again, this effect was not reproduced with equivalent concentrations of sense and scrambled oligonucleotides (eg, compared with antisense, P<0.001 for both).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effect of antisense endoglin oligonucleotides on SMC migration. SMCs were preincubated in a suspension containing oligonucleotides for 24 hours before seeding in the upper wells of a modified Boyden chamber apparatus. PDGF-AB (5 ng/mL) was added to DMEM in the lower wells to stimulate migration. TGF-ß1 (10 ng/mL) was added, as well as escalating concentrations of antisense, sense, or scrambled oligonucleotides. Cells migrating to the lower side of the membrane were counted 8 hours after incubation. The number of migrating SMCs was expressed as a mean per high-power field (HPF). Panels A and B show migration results obtained with human and porcine SMCs, respectively. Antisense oligonucleotides specific for endoglin antagonized the effect of TGF-ß1 on PDGF-AB–stimulated human and porcine SMC migration. The results obtained in the presence versus absence of oligonucleotides were compared by ANOVA. *P<0.03 and *P<0.001 for experiments involving human and porcine SMCs, respectively. For both cell types, the migration results obtained with the maximum concentration of antisense oligonucleotide were different from those obtained with the same concentration of either sense or scrambled oligonucleotides (P<0.005).

	Discussion

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In the present study, we documented the expression pattern of endoglin in diseased arteries. Endoglin was present in ECs, adventitial fibroblasts, and some medial SMCs of normal arteries. However, 3, 7, and 14 days after balloon injury of porcine coronary arteries, endoglin was overexpressed in ECs, adventitial (myo)fibroblasts, and medial SMCs. By 28 days after SI or DI, endoglin expression was very low or absent. In human vascular tissue, endoglin immunolabeling was more abundant in endarterectomy specimens resected from coronary arteries with complex atherosclerotic plaques than in normal internal mammary arteries. Our data indicate that during arterial repair and in established atherosclerotic lesions, endoglin is overexpressed by ECs, SMCs, and myofibroblasts. In vitro, we also demonstrated a role for endoglin in TGF-ß signaling in SMCs. Antisense oligonucleotides to endoglin reduced endoglin expression and antagonized TGF-ß–mediated attenuation of PDGF-induced SMC migration. Hence, endoglin may be important in mediating the effects of TGF-ß during the response to arterial injury.

Balloon injury of porcine coronary arteries results in a repair process that is akin to the healing of a classic skin wound.^{33 34} TGF-ß is known to participate in many facets of this repair process.⁴³ For example, Wysocki et al⁴ demonstrated in pig coronary arteries that TGF-ß mRNA expression is upregulated within hours of balloon injury. Furthermore, Shi et al⁵ showed that the early upregulation of TGF-ß in pig coronary arteries occurs primarily in the adventitia and is associated with the phenotypic differentiation of fibroblasts into myofibroblasts. Recently, we and others have demonstrated that expansion of the neoadventitia is a fibroproliferative process that plays a central role in neointimal formation and arterial contracture (or "negative remodeling") after balloon injury.^{33 40 41} The neoadventitia consists of myofibroblasts that proliferate, synthesize extracellular matrix proteins, and migrate inward to expand the neointima.⁴⁰

The expression of endoglin on ECs is also upregulated after injury, when granulation tissue is a major component of the vascular healing response. This is consistent with the data indicating that endoglin is expressed at higher levels on proliferating tumor ECs and that antibodies to endoglin can produce shrinkage of experimental tumors in mice, which is presumably due to the inhibition of angiogenesis.^{28 45} In our porcine model, we recently documented the kinetics of EC proliferation after balloon injury and observed EC labeling indices similar to those of tumor neovessels (eg, 3% to 15% within 14 days after injury).³⁴ Because the vasa vasorum are intimately associated with atherosclerotic plaques, it would therefore be of interest to test the efficacy of endoglin antagonism as a strategy to limit vascular lesion formation.⁴⁶

Regulation of the relative abundance of ligands and receptors may play an important role in mediating the biological actions of TGF-ß in atherosclerotic lesions. McCaffrey et al¹³ demonstrated that there are differences in TGF-ß receptor expression patterns in vascular SMCs derived from normal versus diseased arteries. In vitro, SMCs derived from atherosclerotic lesions are reported to have decreased TßR-II mRNA synthesis and fewer TßR-II at the cell surface compared with normal arteries. Recently, however, O’Brien et al³ and Ward et al⁴⁷ demonstrated opposing results. In injured animal arteries and in atherosclerotic human aortas, these investigators demonstrated increased expression of not only TGF-ß but also its receptors, and they surmised that TGF-ß is unlikely to be protective against lesion formation but that it more likely participates in the pathogenesis of lipid-rich atherosclerotic lesions. In the present study, the upregulated expression of endoglin by ECs, SMCs, and adventitial myofibroblasts of diseased arteries suggests that the abundance of endoglin relative to TßR-I and TßR-II may also be altered in these cell types during the development of vascular disease. The human endoglin promoter has recently been cloned and is shown to have strong activity in ECs but not in epithelial cells and fibroblasts.⁴⁸ Therefore, cell type may be crucial in determining not only the expression pattern of endoglin and TGF-ß receptors but also the vascular activity of TGF-ß.

In the porcine model of coronary artery injury and repair, TGF-ß and endoglin expression are upregulated after balloon injury, which may result in abnormal TGF-ß signaling and vascular repair.⁴ Two important pieces of data support this hypothesis. First, in vitro, heterotypic coculture experiments demonstrate that endothelial cells induce SMC differentiation through a TGF-ß pathway.⁴⁹ Second, endoglin knockout mice have recently been found to die by gestational day 10.5 to 11.5 because of defective vascular development.^{50 51} Loss of endoglin was accompanied by poor SMC development and the absence of supporting cells, presumably mesenchymally derived SMC precursors around the endothelium of capillary networks.⁵⁰ Hence, endoglin may play a critical role, not only in arterial repair but also in vascular development and the maintenance of vessel integrity.

Although the expression pattern of endoglin in porcine coronary arteries was limited to the early interval after balloon injury (ie, days 3 to 14), we were surprised to observe overexpression of endoglin in diseased human coronary arteries with chronic lesions. The differences in endoglin expression between balloon-injured porcine coronary arteries and atherosclerotic human endarterectomy tissue most likely relate to differences in the histology of these specimens. Many of the early pig lesions retain features of a normal artery, whereas the endarterectomy specimens consist of complex fibrotic plaques with necrotic cores, inflammatory cells, lipid deposits, and a different milieu of growth factors. However, it is interesting to note that by 28 days after DI, low levels of endoglin protein expression persisted in these relatively mature porcine lesions.

The results of the in vitro migration assays show that antisense oligonucleotides to endoglin partially reverse the inhibitory effect of TGF-ß1 on SMC migration. It is somewhat surprising that the antisense oligonucleotides had such a rapid effect in vitro; however, others have also observed this phenomena. Biro et al³⁹ used the same protocol and showed that antisense oligonucleotides targeting c-myc have an inhibitory effect on not only SMC migration but also proliferation. In the present study, the antisense oligonucleotides targeted the initiation codon of endoglin, and as Bennett and Schwartz⁵² have shown, there is cytoplasmic localization of oligonucleotides in vascular SMCs after 1 or 2 hours. However, because it is likely that there is already endoglin present on the cell membrane (and present for the duration of this experiment), the predesigned antisense effect may not be the only mechanism of inhibition. For example, we cannot rule out the possibility that these oligonucleotides may interact with endoglin protein or other proteins to cause aberrant TGF-ß1 signaling. The binding of single-stranded DNA aptamers to proteins is a well-documented phenomenon.⁵³ Nonetheless, our data parallel the observations that overexpression of endoglin in murine fibroblasts leads to the attenuation of migration, whereas antagonism of endoglin in human trophoblasts enhances migration.^{31 32}

In conclusion, our data demonstrate that endoglin is expressed at low levels in normal porcine coronary arteries and overexpressed at early intervals after balloon injury not only in ECs and fibroblasts but also transiently in SMCs and (myo)fibroblasts. In atherosclerotic human coronary artery tissue, endoglin was overexpressed relative to normal control arteries. Our in vitro studies suggest that endoglin may play an important role in mediating the biological effect of TGF-ß during SMC migration. Future studies involving the modulation of endoglin expression in vivo may provide more mechanistic insights into the role of endoglin in maintaining vascular homeostasis as well as repair.

	Acknowledgments

 
This work was supported by grants-in-aid to E.O. from the Heart and Stroke Foundation of Ontario and the Medical Research Council of Canada. M.L. is a Terry Fox Research Scientist of the National Cancer Institute of Canada. E.O. is a Research Scholar of the Heart and Stroke Foundation of Canada. The authors gratefully acknowledge the expert secretarial assistance of Valerie Duffin, Kathleen Lydon-Hassen, and Lizanne Curtin, RN, for recruiting surgical patients for donation of tissue and Sonia Vera for performing the metabolic studies.

Received August 26, 1999; accepted September 20, 2000.

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