Heat shock protein (HSP) 47 and collagen are upregulated during neointimal formation in the balloon-injured rat carotid artery

Atherosclerosis 157 (2001) 361– 368 www.elsevier.com/locate/atherosclerosis

Heat shock protein (HSP) 47 and collagen are upregulated during neointimal formation in the balloon-injured rat carotid artery Shigeru Murakami a,*, Yoshihisa Toda a, Takayuki Seki a, Eiji Munetomo a, Yukiko Kondo a, Takanobu Sakurai a, Yoko Furukawa a, Mototaka Matsuyama a, Takatoshi Nagate a, Nobuko Hosokawa b, Kazuhiro Nagata b a

b

Medicinal Research Laboratories, Taisho Pharmaceutical Co. Ltd., Ohmiya 330 -8530, Japan Institute for Frontier Medical Sciences, Kyoto Uni6ersity, and CREST, JST, Kyoto 606 -8397, Japan Received 13 June 2000; received in revised form 11 October 2000; accepted 19 October 2000

Abstract Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is thought to be essential for the proper processing and secretion of procollagen molecules. We investigated the time course and localization of HSP47 and collagen expression after balloon catheter angioplasty in the rat carotid artery, based on the premise that accumulation of extracellular matrix components is a main feature of intimal hyperplasia in humans and in laboratory animals. Low levels of HSP47 expression were evident in uninjured carotid arteries. Northern blot analysis revealed that HSP47 mRNA expression was markedly stimulated 1 – 3 days after the induced injury and a high level was maintained for 7 days, followed by a gradual decline for up to 21 days after the injury. These changes in HSP47 expression paralleled changes in a1(I) collagen expression. Immunohistochemical staining revealed colocalization of HSP47 and collagen in smooth muscle cells (SMCs) of the media and intima. In situ hybridization analysis showed that activated SMCs, which proliferated and migrated into the intima, expressed high levels of HSP47. In cultured human aortic SMCs, similar upregulation of HSP47 and a1(I) collagen by TGF-b was noted. These results show that SMCs activated after balloon injury express high levels of HSP47 and collagen during cell proliferation and migration, hence an overproduction of collagen and development of intimal thickening. Thus, HSP47 plays a role in the formation and progression of neointima after angioplasty. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Heat shock protein 47; Collagen; Balloon injury; Carotid artery; Smooth muscle cells

1. Introduction The accumulation of collagen and other extracellular matrix and subsequent intimal thickening are common characteristics of advanced atherosclerotic lesions. Formation of the neointima following injury is believed to be important for the initial steps and progression of atherosclerotic lesions as well as the rapid restenosis after angioplasty [1,2]. Steps involved in formation of neointima have been extensively studied both in human and experimental situations [3 – 6]. Balloon catheter deendothelialization is a popular experimental model for studying atherogenesis and restenosis after percuta* Corresponding author. Tel.: + 81-48-6631111; fax: +81-486527254. E-mail address: [email protected] (S. Murakami).

neous transluminal angioplasty [3–9]. In this model, following de-endothelialization there occurs the proliferation of intimal cells to form a thickened neointima. Heat shock proteins (HSPs), or molecular chaperones, are a family of proteins synthesized by prokaryotic and eukaryotic cells in response to a heat shock or to other environmental stresses [10,11]. The activation of heat shock gene transcription during the stress response is mediated by heat shock transcription factor (HSF), which binds to heat shock elements (HSE) in the promoters of the heat shock gene [12,13]. HSP47 is of particular interest, because it binds specifically to procollagens and collagens [14]. HSP47 resides in the endoplasmic reticulum as a collagen-specific molecular chaperone, and is essential for the proper processing and secretion of procollagen molecules [15 –19]. Recently, mice homozygous for the disrupted HSP47 gene

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were obtained. The disruption resulted in embryonic lethality by 11.5 days post-coitus and caused a molecular abnormality in the following procollagens (unpublished data). Mature collagen chains, normally processed, were rare in HSP47-deficient mice. Coexpression of HSP47 with collagen is seen in various types of cells and tissues, even under different conditions. In fibroblasts, the synthesis of both HSP47 and type I collagen decreases after malignant transformation by Rous sarcoma virus [14], simian virus 40 [20], and the activated c-Ha-ras oncogene [21]. Furthermore, such a coexpression of HSP47 with collagen has also been noted in experimental models of fibrotic diseases [22– 25]. Marked increases in the synthesis of collagen and HSP47 occurred in a time dependent manner during the progression of carbon tetrachlorideinduced liver fibrosis in rats [22]. Expression of HSP47 mRNA was detected in Ito cells where collagen was mainly synthesized. In renal fibrosis models, synthesis of HSP47 correlated with that of collagen [23– 25]. The expression of HSP47 mRNA decreased in the rat kidney treated with angiotensin converting enzyme inhibitor or angiotensin II receptor antagonist, an event accompanied by a decrease in collagen mRNA expression [26]. Studies done using antisense oligonucleotides against HSP47 showed that reduction of HSP47 expression results in decreased collagen production in an experimental glomerulonephritis model [27]. As a marked increase in collagen production is a central event in atherosclerotic and restenotic vascular diseases, we investigated the expression of HSP47 in a rat model of vascular disease.

2. Materials and methods

2.1. Carotid arterial injury Male Wistar rats (250– 300 g) purchased from SLC (Hamamatsu, Japan) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) The endothelium of the left common artery was removed by passage of a 2F Fogarty catheter (Baxter). This we inserted into the external carotid artery, passed down to the aortic arch, inflated, then drew up with a twisting motion to the carotid bifurcation. The balloon was then deflated. This procedure was done three times to ensure complete denudation and the external carotid artery was tied off and blood flow through the internal carotid artery was maintained.

2.2. Cell culture Human aortic smooth muscle cells (SMCs) were obtained from Kurabo (Osaka, Japan). SMCs were seeded in a 75 cm2 flask (Corning) at 2×105 cells and were

incubated in Humedia-SG2 (Kurabo), supplemented with 5% (v/v) FBS, EGF (0.5 mg/ml), fibroblast growth factor basic (2 mg/ml), gentamycin (50 mg/ml), and amphotericin B (50 mg/ml) for 8 days at 37°C in 5% CO2/95% air. The cells were provided fresh media every 2 days. Prior to the experiment, the nearly confluent cells were made quiescent by exposure to culture Humedia-SB2 (Kurabo) without FBS for 24 h. TGF-b (Sigma Chemical Co.) at 15 ng/ml was then added to the culture medium in order to stimulate collagen synthesis, followed by incubation for 24 h.

2.3. DNA probes We isolated total RNA from rat liver using Isogen (Nippon Gene) and reverse transcribed 3 mg total RNA at 42°C for 1 h using Superscript II RT (Gibco). PCR was performed using a pair of rat a1(I) collagen specific primers (sense, 5%-ACCTCAAGATGTGCCACTCTGAC-3%; antisense, 5%-AATCGACTGTTGCCTTCGCC-3%), corresponding to nucleotides 1304–1326 and 1720–1739, respectively. The cycling parameters were 94°C for 9 min, 94°C for 30 s, and extension at 60°C for 30 s for 30 cycles with a final extension period of 10 min at 60°C using Ready-To-Go PCR Beads (Amersham Pharmacia Biotech). The PCR products were labeled with PCR DIG Labeling Mix (Roche Diagnostics) using the above stated pair of rat a1(I) collagen specific primers. As a control, digoxigenin (DIG)-labeled GAPDH DNA probe was prepared, using a GAPDH Control Amplimer Set (Clontech). For the DIG-labeled rat HSP47 DNA probe, PCR was done using a pair of rat HSP47 specific primers (sense, 5%-AGAGGTCACCAAGGATGTGGAG-3%; antisense, 5%-TGGGGCATGAGGATGATGAG-3%), corresponding to nucleotides 676–697 and 917–936, respectively, using pYL43 as a template.

2.4. Northern blot analysis Carotid arteries were rapidly excised from the rats at the indicated time (0, 1, 3, 7, 14, and 21 days after balloon injury), rinsed in chilled PBS, and flash-frozen in liquid nitrogen. Uninjured (0 day) or injured carotid arteries of seven–15 rats in each group were pooled and total RNA was prepared by the single-step guanidium thiocyanate procedure using Isogen [28]. mRNA was then amplified, using mRNA Kit Oligo [dT]30 (Bio101). For RNA isolation from SMCs, the cells were trypsinized, harvested, and total RNA was isolated using Isogen. For northern blot analysis, 0.5 mg mRNA (carotid arteries) or 15 mg total RNA (SMCs) were subjected to electrophoresis, using 1% agarose gels containing folmaldehyde then transferred onto Hybond N nylon membranes (Amersham Pharmacia Biotech). mRNA was then cross-linked to the filter by UV illumi-

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nation at 1200 mw/cm2 using FUNA-UV-Linker (Funakoshi, Japan). For a1(I) collagen, the filters were pre-hybridized for 1 h at 55°C, then hybridized in DIG Easy Hyb solution containing DIG-labeled DNA probe (15 ng/ml) for 16 h at 55°C. After hybridization, filters were washed in 0.1× SSC – 0.1% SDS at 60°C. For HSP47, the filters were pre-hybridized for 1 h at 42°C, then hybridized in DIG Easy Hyb solution containing the DIG-labeled DNA probe (15 ng/ml) for 16 h at 55°C. After hybridization, filters were washed in 0.1× SSC – 0.1% SDS at 47°C. GAPDH was used as a control for equivalent transfer of the samples. The filters were pre-hybridized for 1 h at 37°C, then hybridized in DIG Easy Hyb solution containing DIG-labeled DNA probe (25 ng/ml) for 16 h at 37°C. After hybridization, filters were washed in 0.1×SSC – 0.1% SDS at 42°C. For immunological detection, we used a DIG Wash and Block Buffer Set, Anti-digoxigenin-AP Fab fragment, and CDP-Star (Roche Diagnostics). Filters were exposed for several minutes using Hyperfilm-ECL screens (Amersham Pharmacia Biotech).

2.5. In situ hybridization In situ hybridization was done using standard protocols and an in situ hybridization kit (Nippon Gene). Briefly, each section was deparaffinized and dehydrated in a graded series of ethanol. The sections were digested by proteinase K (10 mg/ml) in 10 mM Tris– HCl (pH 8.0), containing 1 mM EDTA for 30 min at 37°C. After washing in Tris-buffered saline (TBS) pH 7.4, the sections were dehydrated in ethanol, and equilibrated in prehybridization solution for 2 h at 37°C. After washing in TBS, the sections were dehydrated in ethanol. The digixigenin (DIG)-labeled probes were heated for 5 min at 100°C and chilled on ice, then mixed in prehybridization solution. The mixture was applied to each section and the sections incubated overnight at 37°C. Sections were washed three times in TBS at 37°C, and washed twice in TBS at room temperature. The sections were then incubated with alkaline phosphatase conjugated anti-digoxigenin for 30 min at room temperature. After washing three times in TBS, a mixture of nitroblue tetrazolium solution and 5-bromo-4-chloro-3-indolyl phosphate solution was added for color development. To generate a riboprobe, a 1.5 kb mouse HSP47 cDNA and 0.6 kb human collagen a1(I) cDNA fragment were subcloned into pBluescript (Toyobo). An antisense riboprobe of HSP47 was labeled with DIG-labeled UTP using a DIG RNA Labeling Kit (Boehringer Mannheim) according to the manufacture’s protocol. As nagative control, sense riboprobe was used.

2.6. Histochemical studies The rat carotid artery of each rat was fixed with

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buffered 4% paraformaldehyde, and embedded in paraffin and specimens were cut at a thickness of 4 mm, deparaffinized in xylene and immersed in a graded series of ethanol for rehydration. Sections were stained with hemotoxylin– eosin (HE) and sirius red staining. After endogenous peroxidase activity was blocked with a 0.3% hydrogen peroxide complex for 30 min at room temperature, the sections were treated with antibody against HSP47 (StressGen Biotechnologies Corp) at a dilution of 1:500 for 12 h at 4°C, followed by incubation with biotinylated anti-mouse monoclonal antibody and avidin–biotin peroxidase complex (Dako A/S). After rinsing with PBS, the tissue sections were reacted with a solution of 0.2 mg/ml 3,3%-diaminobezidine and H2O2, and counterstained with Mayer’s hematoxylin. For double immunostaining, antibody recognizing a-smooth muscle actin, 1A4/EPOS-HRP (Dako A/S) and antimouse HSP47 monoclonal antibody (StressGen Biotechnologies Corp) were used. After blocking endogenous peroxidase, the sections of carotid artery specimens were treated with normal horse serum for 1 h at room temperature, then treated overnight with the first non diluted antibody 1A4/EPOS-HRP at 4°C. The peroxidase reaction was developed with 3,3%-diaminobenzidine and H2O2. The sections were incubated with avidin D blocking solution and biotin blocking solution (Vector Laboratories Inc) each for 15 min. The specimens were then incubated overnight at 4°C with the second antibody against HSP47 in a dilution of 1:500, followed by a biotinylated second-step antibody and then avidin– biotin–alkaline phosphatase complex using standard methods, Vectastain ABC-AP kit (Vector Laboratories Inc). Alkaline phosphatase activity was visualized using alkaline phosphatase substrate kit, Vector Blue (Vector Laboratories Inc).

2.7. Immunoelectron microscopy The carotid artery was excised and fixed by immersion in 2% paraformaldehyde and 0.25% glutaraldehyde containing 0.05% CaCl2 in 0.1 M cacodylate buffer pH 7.3 at 4°C for 1 h. The fixed samples were rinsed in the same buffer at 4°C for 1 h, then dehydrated in a graded series of dimethylformamide and infiltrated with LR White resin. Polymerization was performed under ultraviolet light at − 20°C for 24 h and then at room temperature for 24 h. Ultrathin sections were cut and picked up on collodion-coated 300 mesh nickel grids. The grids were floated on 0.1% gelatin–phosphate-buffered saline at room temperature for 5 min, rinsed in PBS and incubated with mouse monoclonal antibody against HSP47 (0.5 mg/ml) at room temperature for 2 h. After washing with PBS, the grids were further incubated with goat antimouse IgG conjugated 10 nm-gold (British BioCell International) at room temperature for 1 h. The sections were washed with distilled water, stained with uranyl

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acetate for 10 min and observed under a Hitachi H7500 electron microscope.

3. Results

3.1. Changes in mRNA expression of HSP47 and h1(I) collagen A representative series of northern blots to detect the temporal expression of HSP47 and a1(I) collagen mRNA in the carotid arteries after balloon injury is shown in Fig. 1a, and the corresponding quantitative data after normalizing to the internal control, GAPDH, are illustrated in Fig. 1b. As shown in Fig. 1, the basal mRNA levels (uninjured artery) for HSP47 and a1(I)

Fig. 2. In situ analysis of HSP47 and a1(I) collagen expression in rat carotid arteries after balloon injury. HSP47 mRNA was assayed with antisense riboprobe in uninjured arteries (a) and arteries 5 (b), 7 (c), and 14 days (d) after injury. Corresponding sense experiment was also performed in uninjured arteries (e) and arteries 5 (f), 7 (g), and 14 days (h) after injury. mRNA for a1(I) collagen was assayed with antisense riboprobe in uninjured arteries (i) and arteries 5 (j), 7 (k), and 14 days (l) after injury. Arrow heads indicate the internal elastic lamina. Original magnification × 400.

collagen were low. The expression of HSP47 was increased from 1 day after the induced injury, a high level was maintained for 3–7 days after the injury, then the level decreased. The expression of a1(I) collagen decreased on day 1, and then markedly increased 3 days after the injury and a high level was maintained for 3–7 days. Although time course of mRNA expression of HSP47 was similar to that of a1(I) collagen, upregulation of HSP47 mRNA expression proceeded that of a1(I) collagen mRNA expression.

3.2. Localization of HSP47 and h1(I) collagen mRNA expression

Fig. 1. Time course of HSP47 and a1(I) collagen mRNA expression in rat carotid arteries. (a) mRNA was purified from arteries 1, 3, 5, 7, 14, and 21 days after injury or from uninjured arteries (Normal) of 7 –15 rats. Northern blot analysis was performed with 0.5 mg of mRNA. After electrophoresis, mRNA was transferred to filters and hybridized with the digoxigenin-labeled rat DNA probe for HSP47 and a1(I) collagen. (b) The relative amount of HSP47 and a1(I) collagen were quantified by scanning densitometry and normalized to that of GAPDH. The data are based on two –three independent experiments for each time point.

Tissue expression of HSP47 was investigated by in situ hybridization on cross sections of rat carotid arteries. For each antisense experiment with a HSP47 riboprobe (Fig. 2, a–d), a corresponding sense experiment was done (Fig. 2, e–h). HSP47 was detected in endothelium of uninjured arteries (Fig. 2a). Five days after injury, a time when SMCs in the media actively replicate, the luminal surface was free of endothelial cells due to balloon de-endothelialization. SMCs in the media, particularly SMCs closest to the lumen exhibited

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a high expression of HSP47 (Fig. 2b). Formation of neointima was apparent and the intima consisted of a layer of three or four SMCs at 7 days after injury (Fig. 2c). Almost all SMCs in the neointima strongly expressed HSP47. At 14 days after injury, HSP47 expression was visible throughout the entire expanding neointima (Fig. 2d). Tissue expression of a1(I) collagen was also analyzed by in situ hybridization (Fig. 2, i–l), together with corresponding sense experiment (data not shown). Distribution of a1(I) collagen and HSP47 expression were similar. The in situ analysis suggests colocalization of HSP47 and collagen mRNA expression in balloon-injured arteries.

3.3. Localization of HSP47 and collagen protein expression We performed immunohistochemical analysis using a monoclonal antibody against HSP47 (Fig. 3, a–d) and sirius red staining (Fig. 3, e– h) to examine tissue local-

Fig. 3. Immunohistochemical analysis of HSP47 and collagen in rat arteries after injury. Uninjured arteries (a) and arteries 5 (b), 7 (c), and 14 days (d) after injury were stained for HSP47 using monoclonal antibody against HSP47. Collagen was stained with sirius red staining in uninjured arteries (e) and arteries 5 (f), 7 (g), and 14 days (h) after injury. Arrow heads indicate the internal elastic lamina. Original magnification × 400.

Fig. 4. Double immunohistochemical staining of injured arteries 14 days after injury using antibodies against HSP47 and a-smooth muscle actin (a). Blue indicates HSP47 and brown indicates a-smooth muscle actin. Single staining of HSP47 are also shown (b). Arrows indicate the internal elastic lamina. Original magnification ×400. A higher-power view of (a), demonstrating the presence of HSP47 in smooth muscle cells (C), × 1000.

ization of HSP47 and collagen, respectively. HSP47 was positive in the endothelium and adventitia in uninjured arteries (Fig. 3a). Scattered staining of HSP47 was visible in the upper region of the media. Concerning collagen, the adventitia was positive to sirius red staining and the media was weakly positive to sirius red staining (Fig. 3e). At 5 days after injury, SMCs in upper media were stained for HSP47 (Fig. 3b). HSP47 was strongly positive in SMCs of neointima at 7 days after injury (Fig. 3c), which was consistent with the expression of HSP47 mRNA (Fig. 2c). In the neointima, weak sirius red staining was visible (Fig. 3g). At 14 days after injury, syrius red stained collagen was visible over the entire intima (Fig. 3h). HSP47 protein was distributed throughout the expanding neointima (Fig. 3d), in line with HSP47 mRNA experssion. Double immunohistochemical staining using antibodies for HSP47 and a-smooth muscle actin clearly demonstrated the presence of HSP47 protein in the intimal SMCs (Fig. 4). We further examined the localization of HSP47 in the neointima, using immunoelectron microscopy. Ultrathin sections were labeled with antimouse IgG conjugated gold. As shown in Fig. 5, gold particles showing the presence of HSP47 were detected in dilated cisternal spaces of the rough endoplasmic reticulum of intimal SMCs. There were few gold particles outside the cells. In medial SMCs of uninjured arteries, HSP47 was also observed in the cisternal spaces of rough endoplasmic reticulum.

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3.4. Expression of HSP47 and collagen mRNA in cultured SMCs The above findings indicate that coexpression of HSP47 and collagen was stimulated in vascular SMCs during the development of restenotic change. As TGFb is a major factor directly related to the progression of atherosclerotic and restenotic lesions, we examined the expression of HSP47 and a1(I) collagen in cultured human aortic SMCs. When SMCs were incubated with TGF-b, the induction of HSP47 and collagen was in parallel (Fig. 6).

4. Discussion Previous studies have shown a marked increase in the expression of HSP47 and collagen in liver and kidney during the development of fibrosis [22– 25]. The current study provides the evidence that similar coexpression of HSP47 and collagen is upregulated during the progression of vascular neointimal formation. Immunohistochemical and immunoelectron microscopical analysis revealed that smooth muscle cells (SMCs) are major cells in expressing and synthesizing HSP47.

Fig. 5. Detection of HSP47 in smooth muscle cells of the rat carotid artery by immunoelectron microscopy. Ultrathin sections were prepared from injured carotid artery 14 days after injury. HSP47 is detected as gold particles in dialated cisternal spaces of the rough endoplasmic reticulum of smooth muscle cells.

Fig. 6. Effect of TGF-b on HSP47 and a1(I) collagen mRNA expression in cultured smooth muscle cells. Cultures of quiescent human smooth muscle cells were treated with 15 ng/ml of TGF-b. After 24, total RNA (15 mg) was isolated and analyzed by Northern blot.

The balloon catheter de-endothelialization model used here is characterized by intimal and medial remodeling, endothelial cell regrowth, medial and intimal cell proliferation and production of the extracellular matrix. There is a sustained change to a synthetic phenotype from contractile phenotype in the SMC response to paracrine and autocrine stimuli following balloon injury. Synthetic SMCs migrate through breaks in the internal elastic membrane from the tunica media and proliferate within the intima. These SMCs generate an abundant and increasingly collagen-rich extracellular matrix leading to the development of intimal thickening. Following catheterization and de-endothelialization of the carotid artery, SMC migrate within 2–5 days and 3–4 weeks for maximal stenosis to develop. The resultant lesions are extensive, composed almost entirely of SMCs and matrix. Zeymer et al. examined the time course of SMC proliferation after balloon injury, using thymidine, bromodeoxyuridine and proliferating cell nuclear antigen in rats [29]. DNA synthesis was maximal 3 days after injury and returned to normal level in the media 14 days after injury. In the intima, it was maximal 7 days after injury and decreased 14 days after injury. Fingerle et al. reported similar results using in vivo thymidine labeling, in which labeling of medial SMCs reached a peak 4 days after injury; this peak was 7 days after injury in the intima [30]. Using this same rat balloon model, mRNA expression of HSP47 and type I collagen was enhanced at 1–3 days after injury, a time when cell proliferation of medial SMCs reaches maximal [29,30]. The elevated expression of HSP47 and

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collagen continued for 7 days after the injury. During this period, formation of a neointima starts and proliferation of neointimal SMCs reaches a peak. In situ hybridization analysis showed that actively proliferating SMCs in the media expressed high levels of HSP47 5 days after injury. By 7 days after injury, migration and further proliferation of medial SMCs resulted in the formation of the apparent neointima, and SMCs exhibited high levels of HSP47. These SMCs synthesize and secrete abundant extracellular matrix including collagen, and further neointimal thickening occurs. At 14 days after injury, SMCs expressing HSP47 and collagen were noted throughout the intima. These observations suggest that activated SMCs begin to synthesize collagen when proliferation and migration of SMCs are stimulated following balloon injury, which results in the development of intimal thickening. From 3 days after injury, parallel changes occurred in the expression of HSP47 and collagen in SMCs. However, findings that upregulation of HSP47 mRNA expression preceded that of collagen for up to 3 days after injury implies that HSP47 may be essential for synthesis, processing and secretion of procollagen molecules. Since endothelial cells [31] and adventitial fibroblasts [32] synthesize and secrete collagen, the coexpression of HSP47 and collagen suggest that HSP47 has a role in these cells as a collagen-specific chaperone, albeit the level of expression being considerably lower than those of the intima. Since our data show that synthetic SMCs are likely to be the main cells expressing HSP47 in the course of neointimal development, expression of HSP47 and collagen was further studied in cultured aortic SMCs. The mRNA expression of collagen and HSP47 was similarly upregulated by TGF-b, which means that HSP47 is involved in TGF-b-induced production of collagen in SMCs. Recently, Yamamura et al. found two regions of the HSP47 promoter responsible for TGF-b response and they suggested that coexpression of HSP47 and collagen may be regulated by TGF-b1, at the transcriptional level [33]. We found that TGF-b stimulated the expression of both HSP47 and collagen in cultured SMCs. Rasmussen et al. showed that autocrine TGF-b activating in vascular SMCs was associated with the accumulation of extracellular matrix in neointimal lesions of balloon injury rat model [34]. In addition, TGF-b was seen to regulate transcription of the collagen gene through several cis-element of promoters [35,36]. These observations indicate that TGF-b mediates the overproduction of collagen in vascular lesions. Indeed, there are several lines of evidence to show that TGF-b plays a role in the progression of vascular disease: TGF-b accelerated intimal formation in a rabbit balloon model [37], and neutralization of TGF-b by TGF-b antibodies reduced intimal lesions in a rat carotid artery balloon model [38].

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Coexpression of HSP47 and collagens was noted in the liver [22] and kidney [23–25]. Moriyama et al. have shown parallel expression of HSP47 and collagen in a unilateral ureteral obstruction model [24], and that an angiotensin receptor antagonist and an angiotensin converting enzyme inhibitor suppressed the progression of fibrotic changes in this model [26]. Suppression of fibrosis was accompanied by decreased mRNA experssion of HSP47 and collagen type I. In experimental liver fibrosis, parallel induction of HSP47 with collagen was noted [22]. Sunamoto et al. recently reported that antisense oligonucleotides against HSP47 suppressed collagen accumulation in an experimental glomerulonephritis model induced by an anti-Thy-1 antibody [27]. Thus, expression of HSP47 and collagen is coordinately regulated during the fibrotic process in various tissues including vascular hyperplasia. These findings suggest that control of HSP47 expression leads to effective treatment of subjects with fibrotic diseases. In summary, our results suggest the possible involvement of HSP47 in the pathogenesis of vascular neointimal thickening. Migration and proliferation of SMCs in response to injury are initial key event in the development of intimal thickening after balloon injury, and SMCs produce large amounts of collagen, which results in the accumulation of extracellular matrix and formation of neointima. In this process, HSP47 may play a crucial role in synthesis, processing, and secretion of collagen molecules as a collagen-specific molecular chaperone.

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