Human Connective Tissue Growth Factor Is Expressed in Advanced Atherosclerotic Lesions
Background Atherosclerosis affects certain but not all vascular beds of the human circulation. Its molecular mechanisms are only partially understood. Human connective tissue growth factor (hCTGF) is a novel cysteine-rich, secreted polypeptide. hCTGF is implicated in connective tissue formation, which may play an important role in atherosclerosis.
Methods and Results By using a differential cloning technique, we isolated a cDNA clone from a human aorta cDNA library, which is identical to hCTGF. Northern analysis shows that hCTGF mRNA was expressed at 50- to 100-fold higher levels in atherosclerotic blood vessels compared with normal arteries. In vascular smooth muscle cells, high-level expression of hCTGF mRNA was induced by transforming growth factor-β1. Using in situ hybridization and immunohistochemistry, we found that all advanced atherosclerotic lesions of human carotid arteries (eight patients; mean age, 69; age range, 57 to 85 years) and femoral arteries (two patients; mean age, 71.5 years) that we tested expressed high levels of both hCTGF mRNA and protein. hCTGF expression was localized mainly to smooth muscle cells in the plaque lesions that are negative for proliferating cell nuclear antigen staining. In addition, some CD-31–positive endothelial cells of plaque vessels expressed high levels of hCTGF mRNA and protein. hCTGF-positive cells were found predominantly in areas with extracellular matrix accumulation and fibrosis. In contrast, in normal arteries, we were unable to detect either hCTGF mRNA or immunoreactive hCTGF protein.
Conclusions In the present study, we have shown for the first time that both hCTGF mRNA and protein are expressed in human arteries in vivo and that hCTGF may represent a novel factor expressed at high levels specifically in advanced lesions and may play a role in the development and progression of atherosclerosis.
Despite the recent progress in the discovery and understanding of the factors that may be involved in the development and progression of atherosclerotic lesions, the pathogenesis of atherosclerosis is far from elucidated.1 2 Depending on the anatomic location, however, the development and progression of atherosclerotic lesions are highly heterogeneous. For example, atherosclerotic lesions are typically located in the aorta and its major branches, such as the carotid and coronary arteries. In contrast, atherosclerotic lesions and intimal thickening of the IMA are found in <5% of these patients.3 The IMA has been shown to have little if any evidence of atherosclerosis in all age groups at postmortem examination.4 When used as a bypass graft, IMA has been found to be superior to all other alternative treatments for coronary atherosclerosis, including vein grafts and angioplasty.5 6 It is not clear, however, why the IMA is more resistant to the development of atherosclerosis than other blood vessels, but different biological properties of endothelial cells and VSMC may play a role.7 8 9 Possibly, some factors that control the susceptibility of the blood vessel to atherosclerosis are present at higher levels in atherosclerosis-prone vessels than in atherosclerosis-resistant arteries. We therefore designed a differential cloning strategy in which we used a human aorta cDNA library and an IMA cDNA library to identify and characterize genes that may be involved in the development and progression of atherosclerosis.
In the present study, we report the isolation of a cDNA clone, designated as CL59, that is found in the aorta cDNA library but not in the IMA cDNA library. Sequence analysis of CL59 cDNA revealed that it is identical to hCTGF.10 hCTGF belongs to a new family of cysteine-rich, secreted growth regulators. We found high-level expression of hCTGF mRNA and peptide only in advanced atherosclerotic lesions but not in normal arteries, suggesting that hCTGF may play a role in atherosclerosis.
Tissue Preparation and Cell Culture
Human abdominal aorta distal to the renal arteries was obtained from donors, and IMA was obtained from patients who had undergone coronary artery bypass graft surgery patients or from donors. Carotid arteries and femoral arteries were collected from patients undergoing endarterectomy and directional atherectomy, respectively. Tissue samples were either fixed in 4% buffered paraformaldehyde and embedded in paraffin or deep frozen in liquid nitrogen and stored at −70°C until use. Primary cultures of VSMC were prepared from aortas obtained at surgery. After the removal of endothelium and adventitia, blood vessels were minced into 0.5- to 1-mm-diameter pieces, placed in petri dishes coated with 0.1% gelatin (Sigma), and grown in DMEM (GIBCO BRL) supplemented with 20% FCS (GIBCO BRL), 2 mmol/L l-glutamine (GIBCO BRL), 100 U/mL penicillin, 50 μg/mL streptomycin (GIBCO BRL), and 10 mmol/L HEPES buffer, pH 7.4 (GIBCO BRL). VSMC of passages 3 through 8 were used for experiments. Before each experiment, 80% confluent cells grown in either 10- or 15-cm petri dishes were incubated in serum-free media (DMEM) supplemented with 0.1% BSA (Sigma) for 48 hours to obtain quiescent nondividing cells. VSMC were characterized by indirect immunofluorescence staining using specific anti-smooth muscle α-actin antibodies as previously described.8
Preparation of RNA
Total RNA was extracted from IMA or aorta through homogenization of the frozen tissue in 4 mol/L guanidine isothiocyanate buffer, layering over a cushion of 5.6 mol/L CsCl/25 mmol/L sodium acetate, and centrifugation at 175 000g for 24 to 36 hours.11 Poly(A)+ RNA was purified through two cycles of selection on an oligo(dT) cellulose column (Pharmacia).
cDNA Library Construction and Differential Hybridization
Differential cloning was performed starting with two cDNA libraries: one from the aortic blood vessel obtained from a 38-year-old white man and one from a pool of IMAs obtained during bypass surgery. First-strand cDNA synthesis was performed using random six-base primers (GIBCO BRL) and Superscript reverse transcriptase (GIBCO BRL). Second-strand synthesis was catalyzed with Escherichia coli DNA polymerase (GIBCO BRL) and size fractionated with agarose gel electrophoresis. High-molecular-weight cDNAs were ligated to EcoRI adaptors and cloned into λ ZAP II vector (Stratagene).
Differential screening of the cDNA library was performed as described previously.12 The aorta cDNA library was plated at low density, and duplicate lifts were hybridized with 32P-labeled cDNA probes from either IMA or aorta libraries. The autoradiographs were compared to identify plaques expressed at relatively higher levels in the aorta. Differentially expressed plaques were purified by two additional rounds of screening. Approximately 20 000 primary plaques were screened and 60 differentially expressed clones were identified. Ten were selected after two additional rounds of differential hybridization screening, all of which were verified to be more abundant in the aorta cDNA library.
DNA Sequencing and Analysis
Partial nucleotide sequence of differentially expressed cDNA clones and full-length nucleotide sequences were determined by the dideoxy chain–termination technique13 using Sequenase (United States Biochemical Co) version 2.0 and [α-35S]dATP (Amersham). Synthetic oligonucleotides were obtained from Appligene Inc and used as sequencing primers. Sequence data were analyzed on the VAX mainframe of the Basel University biocomputing facility with use of the GCG software package. For comparison with known sequences, the FASTA14 and BLAST15 programs were used to search the GenBank and EMBL nucleotide sequence (GenEMBL) data bases.
Northern Blot Analysis
RNAs were prepared from aorta or IMA or from aortic VSMC stimulated with 2.5 ng/mL TGF-β1 (Boehringer-Mannheim) or a mixture of 2 ng/mL PDGF-AA and 2 ng/mL PDGF-BB (Upstate Biotechnology Inc) for 30 minutes or 1, 3, 5, or 7 hours. Then, 20 μg total RNA (cultured cells) or 5 μg poly(A)+ RNA (aorta or IMA) was subjected to electrophoresis on 1% formaldehyde-agarose gels and transferred to Hybond-N filters (Amersham). The filters were hybridized with the indicated probe prepared by random prime labeling and washed at 65°C. Filters were hybridized with a 32P-labeled 0.6-kb cDNA fragment contained within the open reading frame of CL59, a 1.4-kb Xba I/Pst I fragment of osteopontin cDNA (American Type Culture Collection; No. 6105216 ) and a 1.9-kb EcoRI fragment of β-actin or a 1.4-kb Pst I fragment of the MHC (HLA-B) (the generous gift of Dr A.W.A. Hahn, Basel University). Membranes were exposed to Kodak XRP x-ray film using an intensifying screen at −70°C for 24 hours. To study the human tissue distribution of hCTGF, an MTN blot (Clontech Inc) was obtained. Autoradiographs were quantified using the image analysis program NIH Image version 1.54 (public domain program from NIH).
In Situ Hybridization
In situ hybridization was carried out as described previously.17 Tissues were either deep frozen (femoral arteries) or fixed in 4% buffered paraformaldehyde and embedded in paraffin (carotid arteries). Serial 10-μm cryosections of femoral arteries and 4-μm paraffin sections of carotid arteries were mounted on 2% 3-aminopropyl-triethoxysilan (Sigma)−coated slides, dewaxed, rehydrated, and pretreated with 50 μg/mL proteinase K (Boehringer-Mannheim) for 30 minutes. Sections were again fixed in 4% paraformaldehyde and acetylated with 100 mmol/L acetic anhydride in triethanolamine buffer, pH 8.0. Prehybridization was performed for 2 hours at 55°C. Sections were hybridized with 1×106 cpm of 35S-UTP–labeled antisense hCTGF or sense control riboprobes at 55°C overnight. Riboprobes were prepared from pT3/T7 (GIBCO BRL) plasmid containing a 1.5-kb EcoRI/Kpn I fragment of CL59 cDNA using the Megascript in vitro transcription system (Ambion). After a high stringency wash, tissue sections were dehydrated and dried. For autoradiography, the slides were dipped in Ilford K5 emulsion, dried, and exposed for 1 week at 4°C. Slides were developed with Kodak D19 developer, washed and counterstained with cresyl violet, dehydrated, and mounted with Depex. Alternatively, a nonradioactive in situ hybridization protocol was performed using DIG-labeled hCTGF riboprobes (Boehringer-Mannheim). Prehybridization, hybridization, and stringency washes were performed exactly as described above. hCTGF mRNA was detected with the use of a immunological detection method according to the manufacturer's instructions; sections were counterstained with methylene green for 10 minutes at 37°C and mounted with Kaiser's solution (Merck). For visualization of connective tissue distribution, adjacent sections were also stained with Goldner-Weigert-Elastica trichrome stain.
Production of Site-Specific Antipeptide Antibodies for hCTGF
Site-specific polyclonal antibodies were raised in chicken for hCTGF using the following peptide sequences: N′-Cys-Glu-Ala-Asp-Leu-Glu-Glu-Asn-Ile-Lys-C′. Chicken IgY were collected and purified by affinity column chromatography with the use of the synthetic peptide coupled to agarose (Kementec). Antibody was characterized with the use of ELISA and Western blot (data not shown).
Serial paraformaldehyde-fixed paraffin sections were mounted onto silan-coated slides, dewaxed and rehydrated, washed in PBS, and incubated in 1 μg/mL affinity-purified site-specific antipeptide antibodies for hCTGF for 2 hours at room temperature. Antibody specificity could be demonstrated by preincubation with 5 μg/mL hCTGF peptide that completely abolished the hCTGF staining. Adjacent sections were incubated in 1:100 dilution of primary monoclonal antibodies specific for smooth muscle α-actin (1A4; Dako), the endothelial cell marker CD-31 (JC/70A; Dako), the macrophage-specific marker CD-68 (KP1; Dako), or the PCNA (Novo Castra Laboratories) for 2 hours at room temperature; washed in PBS for 10 minutes; incubated in biotinylated secondary antibody for 30 minutes at room temperature; and finally visualized with the use of the avidin-biotin-peroxidase–labeling system (ABC Elite Kit, Vector). Sections were counterstained with hematoxylin (Merck) and mounted with Kaiser's solution (Merck).
CL59 Is Identical to hCTGF
Sequence analysis revealed that cDNA clone CL59, which was isolated from the aorta cDNA library through differential cloning, contained a 2312–base-pair insert with 99% homology to a recently identified hCTGF.10 Genomic Southern analysis shows that hCTGF is encoded by a single copy gene that spans <6.0 kb of the human genome (data not shown). In addition, a single 2.4-kb hCTGF transcript is detectable in Northern analysis of all adult human tissues tested, including heart, brain, placenta, lung, liver, muscle, kidney, and pancreas; it is most abundant in the kidney (30-fold higher than in brain) (Fig 1⇓).
hCTGF Is Differentially Expressed in Human Blood Vessels
To confirm that hCTGF is differentially expressed in human vascular tissue in a single individual, we performed Northern blot analysis using poly(A)+ mRNA isolated from both IMA and aorta tissue from two renal donors (Fig 2A⇓). In both a 31-year-old and a 68-year-old patient, hCTGF expression was 10-fold higher in the aorta than the IMA of the same person (when normalized to β-actin). In addition, hCTGF expression in the IMA of the 68-year-old patient was eightfold higher than that in the IMA of the 31-year-old patient (Fig 2A⇓). Furthermore, a pool of poly(A)+ mRNA (lane 1) collected from IMA of 16 coronary bypass patients with severe coronary atherosclerosis (mean age, 63 years; age range, 53 to 72 years) also expressed higher levels of hCTGF compared with the 31-year-old patient. Interestingly, hCTGF mRNA levels in the aorta of the 68-year-old patient were 70-fold higher than that in the IMA of the 31-year-old patient (Fig 2⇓).
The Northern blot in Fig 2A⇑ was rehybridized with a cDNA probe to osteopontin, which has been shown to be expressed at high levels in atherosclerotic plaques.2 18 19 20 High levels of osteopontin mRNA were found in the aorta of the elderly patients only (Fig 2A⇑).
Northern blot analysis of poly(A)+ RNA from intact aorta of four patients of different ages (Fig 2B⇑) revealed at least 50-fold higher hCTGF expression in the 66- and 71-year-old patients compared with the 31- and 43-year-old patients.
In Situ Detection and Localization of hCTGF mRNA and Protein
To confirm the high-level expression of hCTGF in arteries with atherosclerotic lesions in vivo, we performed in situ hybridization and immunohistochemistry with hCTGF riboprobe and anti-hCTGF peptide antibody. A total of 10 atherosclerotic lesions from carotid and femoral arteries of patients (mean age, 69 years; age range, 57 to 85 years) with symptomatic atherosclerotic disease were studied. In addition, three IMAs, five normal aortas, and one normal iliac artery from young renal donors (mean age, 39 years; age range, 27 to 55 years) were used for comparison (Table⇓). Macroscopic examination of carotid and femoral arteries showed that all lesions were of the complicated type with thrombus formation, fissures, and ulceration of luminal surface. Microscopic examination also showed the presence of a lipid/necrotic core and calcification (Fig 3⇓). Five of eight patients with carotid lesions had recurrent cerebral ischemic episodes and stroke. All patients showed either stenotic or occluded internal and/or external carotid arteries on angiography.
With the use of in situ hybridization and immunohistochemistry, hCTGF mRNA and protein could not be detected in normal blood vessels (Table⇑ and Fig 4⇓). In contrast, all plaque lesions expressed both hCTGF mRNA and protein (Table⇑ and Figs 3 and 5 through 8⇑⇓⇓⇓⇓). We found high-level expression of hCTGF mRNA predominantly at the shoulder of fibrous caps and at margins along a lipid core or a necrotic core of the hyperplastic intima (Fig 3A⇑). High-level expression was also prominent in intimal cells located next to the media layer (Fig 5A⇓). In general, these cells were embedded in extracellular matrix consisting mainly of collagen fibers (Fig 3D⇑). Immunohistochemistry of consecutive sections revealed that cells expressing hCTGF were predominantly VSMC that stained positive for α-actin and negative for CD-68 (Fig 5A through 5F⇓). However, we also found cells expressing hCTGF in areas in which staining for smooth muscle cell α-actin was negative (Figs 5D, 5F, 7A, and 7C⇓⇓⇓⇓). These cells were also negative for CD-68 staining (Fig 5B and 5E⇓⇓), a macrophage-specific marker, and for CD-31, an endothelial cell–specific marker (data not shown).
To investigate whether VSMC expressing hCTGF mRNA also produce hCTGF protein, we performed immunohistochemistry using a site-specific anti-hCTGF peptide antibody (1 μg/mL final concentration). The pattern of staining for both hCTGF mRNA and protein in consecutive sections of an atherosclerotic plaque was similar (Fig 6A and 6C⇑⇑). hCTGF protein staining was abolished by preincubation of anti-hCTGF antibody with 5 μg/mL hCTGF peptide (Fig 7B⇑). In addition, staining of adjacent sections with anti-PCNA antibody showed that none of the cells in the same area expressed PCNA protein (Figs 6D and 7D⇑⇑). Only a few cells in atherosclerotic lesions stained positive for PCNA, and almost all PCNA-positive cells were localized to areas with accumulations of CD-68–positive macrophages (data not shown). CD-68–positive macrophages were found mainly in areas around calcification, where hCTGF and α-actin staining was mostly negative.
We found that CD-68–positive macrophages in atherosclerotic plaques did not express detectable levels of hCTGF mRNA or protein. hCTGF mRNA expression was also undetectable in Northern blot analysis with the use of circulating macrophages as well as lung macrophages obtained from bronchial lavage (data not shown).
hCTGF mRNA and protein expression was also found in CD-31–positive endothelial cells in the atherosclerotic lesions. hCTGF-positive endothelial cells were found at the luminal site of the plaques (Fig 5A and 5D⇑⇑) and in vasa vasorum inside the plaque lesions (Fig 8⇑). hCTGF expression in endothelial cells, however, was highly heterogeneous. High-level expression of hCTGF occurred in plaque vessels where the neighboring capillaries in the same area were negative for hCTGF expression (data not shown). The reason for this heterogeneity is not clear; however, because VSMC in the same area stained positive for hCTGF, lack of staining was not the result of a technical failure.
Regulation of hCTGF Expression in VSMC
To gain insight into the regulation of hCTGF by known growth factors implicated in atherosclerosis, quiescent human VSMC from aorta were stimulated with either 2.5 ng/mL TGF-β or a mixture of 2 ng/mL PDGF-AA and 2 ng/mL PDGF-BB (Fig 9⇓). hCTGF mRNA expression increased ≤20-fold in the presence of TGF-β, reaching a maximum after 7 hours (Fig 9⇓, top). PDGF, however, had less effect on hCTGF mRNA expression; after 7 hours of stimulation, transcript levels increased maximally by threefold compared with untreated controls (Fig 9⇓, bottom).
In the present study, we report the isolation of CL59 cDNA through use of a differential cloning approach. This cDNA was isolated from a cDNA library constructed from human aortic tissue, which contains higher levels of CL59 mRNA compared with IMA. Sequence analysis of CL59 cDNA revealed that it is identical to the recently cloned hCTGF.10
Our data suggest a potential role for hCTGF in human atherosclerosis based on the following observations: (1) hCTGF mRNA is expressed at much higher levels in arteries with atherosclerotic lesions than in normal arteries, as determined with Northern analysis. (2) In situ hybridization and immunohistological analysis indicate that both hCTGF mRNA and protein are expressed at high levels in atherosclerotic lesions but not in normal arteries. (3) High-level expression of hCTGF in intimal smooth muscle cells is found predominantly in areas with extracellular matrix accumulation and fibrosis.
In situ hybridization of atherosclerotic and normal arteries using either 35S- or DIG-labeled hCTGF riboprobe revealed hCTGF mRNA expression only in advanced atherosclerotic lesions, not in normal tissue. In the Northern analysis, however, we found low but detectable levels of hCTGF mRNA expression in the normal arteries. Similar observations were made with osteopontin; low levels of mRNA were detected in normal human media through the use of Northern analysis but not in the same tissue through the use of in situ hybridization.18 20 An obvious explanation is that the local tissue concentrations of hCTGF mRNA in normal vessels are below the detection threshold of the in situ hybridization technique, whereas after tissue extraction, the total amount of poly(A)+ mRNA is sufficient for detection by Northern blotting. The sensitivity of two detection methods for in situ hybridization was different (ie, the DIG-labeled riboprobe was less sensitive than the radioactive 35S-labeled riboprobe). Nevertheless, even with DIG-labeled probes, we found high-level hCTGF mRNA expression in all atherosclerotic lesions from carotid and femoral arteries. In contrast, we were unable to detect hCTGF mRNA expression in normal arteries of young renal donors, even with the more sensitive 35S-labeled probe.
In atherosclerotic lesions, hCTGF was expressed in both α-actin–positive and –negative intimal VSMC. Because PCNA staining of both cell types was also negative, cell replication with a loss of α-actin expression can be ruled out. In fact, we found that only a few cells in atherosclerotic plaques stained positive for PCNA. These results are consistent with the results of O'Brien et al,21 who also found that <1% of intimal cells of primary and restenotic coronary atherectomy specimens stained positive for PCNA.
The physiological function of hCTGF in vivo is not yet clear. However, Igarashi et al22 found increased expression of CTGF mRNA in regenerating skin that is preceded by increases in TGF-β mRNA expression. In addition, TGF-β, but not PDGF, epidermal growth factor, or basic fibroblast growth factor, stimulated hCTGF mRNA expression in human skin fibroblasts.22 Interestingly, hCTGF mRNA has been found in sclerotic lesions but not in the normal skin of patients with systemic sclerosis.23 In these patients, hCTGF mRNA expression correlates with extracellular matrix accumulation, connective tissue formation, and skin sclerosis.23 Therefore, hCTGF is thought to have a regulatory function for extracellular matrix production in skin fibroblasts.
hCTGF may also play a role in atherosclerosis. Early atherosclerotic lesions are characterized by the accumulation of inflammatory cells and by intimal smooth muscle cell proliferation, migration, and extracellular matrix deposition.1 2 Serum- or blood cell–derived factors such as TGF-β have also been implicated in the pathogenesis of atherosclerosis.1 2 24 25 26 TGF-β1 can induce overproduction of extracellular matrix proteins in intimal VSMC.25 Importantly, direct transfer of an expression vector carrying the TGF-β1 gene into arteries stimulated extracellular matrix production accompanied by intimal and medial hyperplasia.27 We also found that TGF-β induces sustained high-level expression of hCTGF in cultured aorta smooth muscle cells. During the development of atherosclerotic lesions, for example, TGF-β may act directly on endothelial and smooth muscle cells to stimulate hCTGF expression and secretion, which in turn may induce extracellular matrix production in VSMC. The in situ hybridization and immunohistochemistry data that we present appear to support this hypothesis.
We have established that smooth muscle cells in advanced atherosclerotic lesions express high levels of hCTGF and that these cells are frequently localized to areas with accumulation of extracellular matrix and fibrosis. In contrast, hCTGF mRNA and protein were undetectable in normal blood vessels. We cannot yet conclude that hCTGF is directly responsible for the progression of atherosclerotic lesions; however, further elucidation of the function of hCTGF in VSMC will be possible when recombinant or purified CTGF protein is available in sufficient quantity. Nevertheless, hCTGF appears to represent a new marker for advanced atherosclerotic lesions and therefore merits a closer look as a potential participant in atherosclerotic lesion development.
Selected Abbreviations and Acronyms
|hCTGF||=||human connective tissue growth factor|
|MHC||=||major histocompatibility antigen|
|PCNA||=||proliferating cell nuclear antigen|
|PDGF||=||platelet-derived growth factor|
|TGF-β1||=||transforming growth factor-β1|
|VSMC||=||vascular smooth muscle cell(s)|
This work was supported by Swiss National Science Foundation Grants (32-32541.91 and 3100-047119.96/1), the Hochschulstiftung der Universita¨t Bern, and by the Swiss Cardiology Foundation. We are grateful for the excellent technical assistance of P. Reiser. We also thank Drs J. Fingerle and D. Kling (Roche Basel) for help with the histology; Drs G. Noll, U. Arnett, and Z. Yang for help in collecting the human tissues; Drs P. Basset, P. Bouillet, and A.W.A. Hahn for helpful discussion; Drs D. Hartman, I. Park, and E. Battegay for critical reading and discussion of the manuscript; and B. Wessner for help with the color photography. The sequence of hCTGF reported in this article has been deposited in the GenBank/EMBL database (accession No. X78947).
- Received July 10, 1996.
- Revision received September 23, 1996.
- Accepted October 5, 1996.
- Copyright © 1997 by American Heart Association
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