Levels Of Heat Shock Protein 47 Research Articles

Effectiveness of conjunctival bleb scarring by knock-down of heat shock protein 47 in rat model.

To evaluate the effectiveness of knock-down of heat shock protein 47 (HSP47) on conjunctival bleb scarring in a rat model and its possible mechanism. Male Sprague-Dawley rats were used for glaucoma filtration surgery (GFS) and were treated with either phosphate buffered solution, shControl, mitomycin C, or sh-HSP47 using a microsyringe immediately after GFS. The morphology of filtering blebs was observed postoperatively. The levels of HSP47 were analyzed at 2, 5, 8, and 11d after GFS via real-time quantitative polymerase chain reaction (PCR) and Western blot. The silencing effect of HSP47, the expression of collagen I and III, and the potential signaling pathways of HSP47 during scarification were explored 11d post GFS. The protein levels of transforming growth factor-β1 (TGF-β1), phospho-Smad2 (pSmad2), phospho-Smad3 (p-Smad3), and phospho-p38 (p-p38) were also analyzed using Western blot. Sh-HSP47 treatment significantly prolonged the functional filtration bleb retention. The levels of HSP47 were increased significantly at 5, 8, and 11d postoperatively compared to the control group (P<0.05, P<0.01, and P<0.001). The levels of HSP47 protein at day 11 postoperatively were significantly down-regulated after HSP47 silencing using sh-HSP47 adenovirus transfection (P<0.01). Expression levels of collagen I and III within the blebs were significantly reduced in the absence of HSP47 (P<0.01). Moreover, the protein levels of TGF-β1, p-Smad2/3, and p-p38 were dramatically inhibited after treatment with sh-HSP47 (P<0.01). The inhibitory effects of HSP47 knock-down on scarring after GFS have the potential to be an efficacious therapeutic option for the treatment of conjunctival bleb scarring.

Inhibition of heat shock protein 47 suppressed collagen production in Tenon's capsule fibroblasts.

Glaucoma is the leading cause of irreversible blindness worldwide, and conjunctival bleb scarring remains the most frequent reason for the failure of glaucoma filtration surgery. Excessive proliferation of fibroblasts from Tenon's capsule and excessive deposition of collagen contribute to the scarification of the conjunctival bleb. Heat shock protein 47 (HSP47) is assumed to act as a collagen-specific molecular chaperone, and thereby involved in the pathogenesis of fibrotic diseases. Therefore, we investigated the effect of HSP47 knockout against collagen type I (COLI) production in rat tenon's fibroblasts. Newborn rat tenon's fibroblasts were cultured and verified by anti-vimentin antibody. Transfection efficiency of small interference RNA targeted against HSP47 was confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) at 48 h after siRNA transfection and by western blot at 72 h after transfection. The mRNA and protein expression of HSP 47 and COLI were detected by RT-qPCR and western blot. The proliferation of cells was measured by cell counting kit-8 assay. HSP47 siRNA down-regulated the mRNA and protein levels of HSP47 in rat Tenon's fibroblasts, and suppressed the mRNA and protein expression of COLI. Moreover, HSP47 siRNA had no significant effect on proliferation of rat Tenon's fibroblasts. HSP47 siRNA inhibits the production of COLI in rat Tenon's fibroblasts, and may be the potential therapeutic method in bleb scarring after glaucoma filtration surgery.

High Levels of Heat Shock Protein 47 in the Aqueous Humor of Patients with Acute Primary Angle Closure

Introduction: Acute primary angle closure (APAC) is often characterized by acute elevation of intraocular pressure accompanied by severe ocular and systemic symptoms. Excessive collagen accumulation, which can be caused by upregulated heat shock protein 47 (HSP47) expression, can produce scarring in rat conjunctival blebs. Meanwhile, the presence of HSP47 in human aqueous humor and its levels are yet to be determined. Methods: We examined 32 consecutive patients with APAC and 16 age-matched participants without APAC scheduled for cataract surgery who were enrolled as a control group. Aqueous humor samples were collected from all subjects at the time of surgery and compared between the subjects with and without APAC. Results: The levels of HSP47 in the aqueous humor of patients with APAC (1,210.4 ± 450.2 pg/mL) were found to be significantly increased (p = 0.001) compared with those in the control group (863.4 ± 240.0 pg/mL). Notably, the levels of HSP47 negatively correlated with the age of patients with APAC (p = 0.023). Conclusion: HSP47 was upregulated in the aqueous humor of patients with APAC and may play a role in scarring after trabeculectomy for APAC.

Investigating the role of heat shock protein 47 in fibrosis in Crohn’s disease

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract that share similar genetic risk factors. However, while fibrotic stricture of the intestine is a major characteristic of CD; it is rarely observed in UC. Deposition of collagen in the extracellular matrix contributes to the formation of fibrotic strictures in CD, but the underlying mechanisms are unknown. In the present study, we found that heat shock protein 47 (HSP47), a stress-response protein that acts as a molecular chaperone during the processing and secretion of collagen, expressed in the intestinal tissue from patients with CD. Serum HSP47 levels and anti-HSP47 antibody titers were significantly higher in patients with CD than in those with UC. Furthermore, anti-HSP47 antibody levels correlated significantly with fibrosis in CD. In addition, HSP47 inhibition significantly suppressed collagen production in fibroblasts in vitro. These findings suggest that HSP47 is a biomarker for differentiating fibrotic from non-fibrotic forms of CD. Additionally, we propose that HSP47 could be a potential target for treating fibrosis in patients with CD.

Roles of transforming growth factor - β1 and heat shock protein 47 in progression of Schistosoma japonicum-induced hepatic fibrosis

To investigate the dynamic expression of transforming growth factor-β1 (TGF-β1) and heat shock protein 47 (HSP47) and explore their roles in the progression of hepatic fibrosis induced by Schistosoma japonicum infection. Fifty female mice of the ICR strain were randomly divided into the infection group and the normal control group, of 25 mice in each group. Each mouse in the infection group was infected with 20 ± 1 cercariae of S. japonicum via the abdominal skin, while uninfected animals served as normal control. Five mice were sacrificed 4, 6, 8, 10 and 12 weeks post-infection and liver tissues were sampled. Serum HSP47 and TGF-β1 was determined using enzyme-linked immunosorbent assay (ELISA), and the pathological changes of liver specimens were observed with hematoxylin & eosin (HE) staining. In addition, the synthesis of alpha 1 chain of type I collagen (COL1A1) was measured using Masson staining, and the mRNA expression of TGF-β1, HSP47 and COL1A1 was determined using real-time fluorescent quantitative PCR (qPCR) assay. During the period of S. japonicum-induced hepatic fibrosis, the serum HSP47 and TGF-β1 levels and the mRNA expression of TGF - β1, HSP47 and COL1A1 gradually increased with the progression of hepatic fibrosis. The serum levels of HSP47 and TGF-β1 were (179.26 ± 29.87) pg/mL and (22.37 ± 5.21) ng/mL 6 weeks post-infection, respectively, which were significantly greater than those [(150.29 ± 34.91) pg/mL and (18.54 ± 7.78) ng/mL, respectively] in the normal control group (both P values < 0.05). In addition, the mRNA expression of HSP47, COL1A1 and TGF-β1 was (0.86 ± 0.04), (1.17 ± 0.06) and (0.64 ± 0.13) in mouse liver specimens, which was significantly higher than that (0.23 ± 0.03, 0.20 ± 0.02 and 0.38 ± 0.02) in the normal control group (all P values < 0.01). The expression of TGF-β1 and HSP47 during the period of S. japonicum-induced hepatic fibrosis is consistent with the progression of the hepatic fibrosis, and exhibits the same tendency with type I collagen expression. HSP47 is a novel promising diagnosis marker and therapeutic target for S. japonicum-induced hepatic fibrosis.

The heat shock protein 47 as a potential biomarker and a therapeutic agent in cancer research

Heat shock protein 47 (HSP47) is an important chaperone required for the correct folding and secretion of collagen. Several studies revealed that HSP47 has a role in numerous steps of collagen synthesis, preventing procollagen aggregation and inducing hydroxylation of proline and lysine residues. HSP47 is encoded by the SERPINH1 gene, which is located on chromosome 11q13.5, one of the most frequently amplified regions in human cancer. The altered expression levels of HSP47 have been correlated with several types of cancer, such as cervical, breast, pancreatic and gastric cancers. Studies have shown that HSP47 promotes tumor angiogenesis, growth, migration and metastatic capacity. In this review, we highlight the fundamental aspects of the interaction between HSP47 and collagen and the recent discoveries of the role of this chaperone in different types of malignant neoplasias. We also discuss recent treatments using HSP47 as a therapeutic target, and present evidences that HSP47 is an essential protein for cancer biology and a potential molecular target for chemotherapy.

MicroRNA-29b ameliorates ileocolonic anastomosis fibrosis in interleukin-10-knockout mice via targeting heat shock protein 47

Objective The purpose of this study is to investigate the effect of microRNA-29b (miR-29b) and heat shock protein 47 (HSP47) in the fibrosis of ileocolonic anastomosis of interleukin (IL)-10 knockout (IL-10KO) mice undergoing ileocecal resection and anastomosis (ICA). Methods A new model of ICA in IL-10KO mice was used to investigate the postsurgical fibrosis of anastomosis. 12 IL-10KO mice were randomized to IL-10KO and IL-10KO-ICA group. Aged-matched wild-type (WT) mice were assigned to WT and WT-ICA group. AgomiR-29b was used to up-regulate the expression of miR-29b. Crohn’s disease activity index (CDAI) was employed to evaluate the general condition of mice 8 weeks after the beginning of the experiment. Effects of miR-29b treatment on ileocolonic anastomosis were determined by histopathological and Western blotting analysis. Levels of miR-29b and HSP47 was measured by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting. Western blotting was also used to assess the level of Collagen-Ⅰ (Col-Ⅰ) and Collagen-Ⅲ (Col-Ⅲ). Results Mean CDAI (WT group: 0.833±0.753 vs. WT-ICA group: 3.167±1.169 vs. IL-10KO group: 2.333±1.033 vs. IL-10KO-ICA group: 8.833±1.471, all P=0.000 when compare IL-10-ICA group to other groups) and fibrosis score (WT group: 0.333±0.516 vs. WT-ICA group: 0.833±0.408 vs. IL-10KO group: 1.667±0.817 vs. IL-10KO-ICA group: 4.000±0.632, all P=0.000, when compare IL-10-ICA group to other groups) of mice in IL-10KO-ICA group was significantly upregulated compared with other groups 8 weeks after the beginning of the experiment. Western blotting showed that the production of HSP47 was significantly inhibited by overexpression of miR-29b (A value: agomiR-NC group: 0.631±0.084 vs. agomiR-29b group: 0.268±0.069, P=0.000). Futhermore, overexpression of miR-29b significantly reduced the level of Col-Ⅰ (A value: agomiR-NC group: 0.588±0.900 vs. agomiR-29b group: 0.380±0.150, P=0.037) and Col-Ⅲ (A value: agomiR-NC group: 0.610±0.130 vs. agomiR-29b group: 0.412±0.094, P=0.017) of anastomosis of mice in IL-10KO-ICA group. Conclusion Upregulation of miR-29b may be a therapeutic strategy against postoperative anastomotic fibrosis in CD patients throughinhibition of HSP47. Key words: Crohn’s disease; Fibrosis; MicroRNA-29b; Heat shock protein 47

Proteomics analysis of differential protein expression identifies heat shock protein 47 as a predictive marker for lymph node metastasis in patients with colorectal cancer.

The discovery of biomarkers to predict the potential for lymph node (LN) metastasis in patients with colorectal cancer (CRC) is essential for developing improved strategies for treating CRC. In the present study, they used isobaric tags for relative and absolute quantitation to conduct a proteomic analysis designed to identify novel biomarkers for predicting LN metastasis in patients with CRC. They identified 60 differentially expressed proteins specifically associated with LN metastasis in CRC patients and classified the molecular and functional characteristics of these proteins by bioinformatic approaches. A literature search led them to select heat shock protein 47 (HSP47) as the most suitable candidate biomarker for predicting LN metastasis. Validation analysis by immunohistochemistry showed that HSP47 expression in patients with CRC and the number of HSP47-positive spindle cells in the tumor stroma were significantly higher compared with those in adjacent normal colonic mucosa, and the number of the latter cells increased with tumor progression. Further, the number of HSP47-positive spindle cells in stroma was a more informative marker for identifying LN metastasis than HSP47expression. Multivariate analysis identified spindle cells that expressed elevated levels of HSP47 as an independent predictive biomarker for CRC with LN metastasis. Moreover, these cells served as an independent marker of disease-free and overall survival of patients with CRC. Their data indicate that the number of HSP47-positive spindle cells in the stroma of CRC may serve as a novel predictive biomarker of LN metastasis, early recurrence and poor prognosis.

Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis

Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and studied the efficacy of multiple putative antifibrotic compounds.Our results demonstrated that human PCLS remained viable for 48h and the early onset of fibrosis was observed during culture, as demonstrated by an increased gene expression of Heat Shock Protein 47 (HSP47) and Pro-Collagen 1A1 (PCOL1A1) as well as increased collagen 1 protein levels. SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK) showed a marked decrease in HSP47 and PCOL1A1 gene expression, whereas specific inhibitors of Smad 3 and Rac-1 showed no or only minor effects. Regarding the studied antifibrotics, gene levels of HSP47 and PCOL1A1 could be down-regulated with sunitinib and valproic acid, while PCOL1A1 expression was reduced following treatment with rosmarinic acid, tetrandrine and pirfenidone. These results are in contrast with prior data obtained in rat PCLS, indicating that antifibrotic drug efficacy is clearly species-specific.Thus, human PCLS is a promising model for liver fibrosis. Moreover, MAPK signaling plays an important role in the onset of fibrosis in this model and transforming growth factor beta pathway inhibitors appear to be more effective than platelet-derived growth factor pathway inhibitors in halting fibrogenesis in PCLS.

Involvement of collagen-binding heat shock protein 47 in scleroderma-associated fibrosis.

Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.

Heat shock protein 47 is up-regulated in conjunctival bleb scarring after filtration surgery in rats

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is involved in the biosynthesis and secretion of procollagen. Recent studies have shown a close association between increased HSP47 and excessive accumulation of collagens in various fibrotic diseases. This study was conducted to evaluate whether HSP47 plays a role in conjunctival bleb scarring after filtration surgery in rats. Trabeculectomy of the right eye was performed in Sprague-Dawley (SD) rats. Eight rats were euthanized at 2, 5, 8 and 11 days after surgery. Four rats were used to extract mRNA and the other four were used to extract protein. Blebs and normal control conjunctival tissues were collected. Real-time PCR (RT-PCR) and Western blot methods were used to evaluate alterations in HSP47 levels and type I and type III collagen. Bleb formation was observed in all eyes. Both the expression of HSP47 mRNA and protein in conjunctival blebs increased at 2, 5, 8 and 11 days postoperatively compared with that in normal control conjunctival tissues. The differences of both the mean mRNA and protein levels of HSP47 in blebs at each time point and in the normal control conjunctiva were statistically significant (mRNA level: F = 175.811, p < 0.001; protein level: F = 68.356, p < 0.001). Type I and type III collagen levels in blebs were raised at different time points both at mRNA and at protein levels. The differences between mean mRNA and protein levels of both type I and type III collagen in blebs at 2, 5, 8 and 11 days after surgery and in the normal control conjunctiva were statistically significant (mRNA level: FI = 182.210, p I < 0.001; FIII = 125.490, p III < 0.001; protein level: FI = 160.092, p I < 0.001; FIII = 62.374, p III < 0.001 ). The amount of HSP47 in bleb positively correlated with that of both type I and type III collagens (mRNA level: rsI = 0.688, p I = 0.003; rsIII = 0.900, p III < 0.001; protein level: rsI = 0.688, p I = 0.003; rsIII = 0.832, p III < 0.001). HSP47 is likely to be involved in the pathogenesis of conjunctival bleb scarring and may play a role in the process of conjunctival bleb fibrosis.

Serum heat shock protein 47 levels are elevated in acute exacerbation of idiopathic pulmonary fibrosis

Little is known about the pathophysiology of acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF). Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules. Previous studies in experimental animal fibrosis models have shown that downregulation of HSP47 expression reduces collagen production and diminishes fibrosis progression. In this study, serum HSP47 levels were evaluated to elucidate pathogenic differences involving HSP47 between AE-IPF and stable (S)-IPF. Subjects comprised 20 AE-IPF and 33 S-IPF patients. Serum levels of HSP47, Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-A, SP-D, and lactate dehydrogenase (LDH) were measured. Immunohistochemical analysis of lung HSP47 expression was determined in biopsy and autopsy tissues diagnosed as diffuse alveolar damage (DAD) and usual interstitial pneumonia (UIP). Serum levels of HSP47 were significantly higher in AE-IPF than in S-IPF patients, whereas serum levels of KL-6, SP-A, and SP-D did not differ significantly. Receiver operating characteristic curves revealed that HSP47 was superior for discriminating AE-IPF and S-IPF. The cutoff for HSP47 resulting in the highest diagnostic accuracy was 559.4 pg/mL; sensitivity, specificity, and diagnostic accuracy were 100.0%, 93.9%, and 96.2%, respectively. Immunohistochemical analysis revealed that pulmonary HSP47 expression was greater in DAD than UIP tissues. Serum HSP47 was significantly higher in AE-IPF than in S-IPF patients, suggesting that underlying fibrogenic mechanisms involving HSP47 differ in the two conditions.

Production and degradation of extracellular matrix in reversible glomerular lesions in rat model of habu snake venom-induced glomerulonephritis

We investigated the mechanism of development and repair process of glomerular injury in a rat model of habu snake (Trimeresurus flavoviridis) venom (HSV)-induced glomerulonephritis. Glomerulonephritis was induced in rats by intravenously injecting HSV at 3 mg/kg. Renal tissue was isolated and subjected to immunohistochemical analysis for expression levels of type IV collagen, heat shock protein 47 (HSP47), transforming growth factor-β (TGF-β), and matrix metalloproteinase-3 (MMP-3), as well as its transcription factor Ets-1. Expression levels of HSP47, TGF-β, and type IV collagen began to increase in the mesangial area starting from day 14 and peaked on day 21, followed by a gradual decrease. Expression levels of MMP-3 and Ets-1 started to increase coinciding with peak production of mesangial matrix on day 21, peaking on day 35, followed by gradual decrease. Expression of MMP-3 and Ets-1 persisted until day 63, whereas that of HSP47 and type IV collagen returned to baseline level at this time point. Time-course changes of extracellular matrix (ECM) accumulation in glomeruli in the HSV-induced glomerulonephritis model were correlated with those of factors involved in both ECM production and degradation systems. Continued expression of factors in the degradation system seems particularly important for the repair process. These findings might lead to new therapies that prevent and repair glomerular injury.

Tavşan Erken Ateroskleroz Modelinde HSP47 ile Kolajen Arasındaki Endotelin Reseptöründen Bağımsız İlişki

Objective: Collagen in the extracellular matrix (ECM) plays an important role in modulation of response to the vascular injury during the progression of atherosclerosis and restenosis. Collagen can regulate smooth muscle cell (SMC) proliferation, migration and matrix metalloproteinase (MMP) production. Therefore, collagen turnover in the arteries is an important determinant of intimal thickening. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is thought to be essential for the processing and secretion of procollagen molecules. Endothelin (ET) is a strong chemoatractant and mitogen promoting SMC proliferation and migration. The aim of this study was to investigate the possible role of HSP47 and its relation to collagen synthesis, and the effects of a nonselective ETA/ETB receptor antagonist, TAK-044 in collar-induced early atherosclerosis model. Material and Methods: New Zealand white rabbits were divided into two groups. Both groups received vehicle (0.9% NaCl 0.8 ml/kg/day, s.c.) or TAK-044 (5 mg/kg/day, s.c.) for three weeks. On 8th day, a non-occlusive silicon collar was placed around the left carotid artery. The right carotid artery was sham-operated. HSP47 expression in carotid arteries were determined immunohistochemically. Furthermore, total collagen levels, collagen expression and type I procollagen expression were established. Results: HSP47 expression correlated with collagen expression did not change in collared arteries. TAK-044 treatment did not affect HSP47 and collagen levels. Conclusion: There was a correlation between HSP47 expression and collagen expression in carotid arteries. However, intimal thickening did not induce HSP47 expression and early collagen development. The ineffectivenes of TAK-044 suggests that ET-1 signaling may not be implicated in HSP47 and collagen in this model.

Heat Shock Protein 47: A Novel Biomarker of Phenotypically Altered Collagen-Producing Cells

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that helps the molecular maturation of various types of collagens. A close association between increased expression of HSP47 and the excessive accumulation of collagens is found in various human and experimental fibrotic diseases. Increased levels of HSP47 in fibrotic diseases are thought to assist in the increased assembly of procollagen, and thereby contribute to the excessive deposition of collagens in fibrotic areas. Currently, there is not a good universal histological marker to identify collagen-producing cells. Identifying phenotypically altered collagen-producing cells is essential for the development of cell-based therapies to reduce the progression of fibrotic diseases. Since HSP47 has a single substrate, which is collagen, the HSP47 cellular expression provides a novel universal biomarker to identify phenotypically altered collagen-producing cells during wound healing and fibrosis. In this brief article, we explained why HSP47 could be used as a universal marker for identifying phenotypically altered collagen-producing cells.

Effect of heat shock protein 47 on collagen synthesis of keloid in vivo

Keloid is regarded as a fibroproliferative disorder with excessive accumulation of extracellular matrix. However, the molecular mechanism of keloid formation is not well understood and no treatment modality is consistently effective. Heat shock protein 47 (HSP47) is known as a collagen-specific molecular chaperone which plays a critical role in collagen biosynthesis. Results of our previous in vitro experiments demonstrated that HSP47 might be an important reason for excessive collagen accumulation in regard to keloid formation. Our objective is to investigate whether HSP47 has an influence on collagen metabolism in animal keloid models. The constructed plasmids, carrying HSP47-small hairpin RNA (shRNA), were transfected into animal keloid models, in comparison with the control groups. After transfection, the mRNA and protein expression of HSP47 and collage type I were detected by quantitative real-time PCR and Western blot. Both the mRNA and protein levels of HSP47 in animal keloid models were decreased dramatically after transfection of the HSP47- shRNA plasmid, in comparison with the control group. Following the down-regulation of HSP47, we found that the volume of animal keloid models and the major collagen expression were reduced correspondingly. Combining the results of our previous in vitro experiment results, we suggest that overexpression of HSP47 in keloid fibroblast cells could induce excessive collagen accumulation by enhancing collagen synthesis, which not only presents a possible mechanism of keloid formation, but also offers a therapeutic potential of RNA interference to HSP47 for the treatment of keloids and other fibroproliferative disorders.

Effect of HSP47 expression levels on heterotrimer formation among type IV collagen α3, α4 and α5 chains

We previously established stable transformants of the human embryonic kidney 293 (HEK293) cell line that express type IV collagen α3, α4 and wild-type or mutant-type α5 chains. Using these cell lines, we confirmed that these three chains form a heterotrimer and that α5 chains containing mutations seen in Alport syndrome are defective in heterotrimerization. In these studies, the amount of heterotrimer that formed was much less than expected relative to the amount of α(IV) chains expressed. The aim of the present study was to determine the effect of the collagen-specific molecular chaperone heat shock protein 47 (HSP47), whose expression is low in HEK293 cells, on the heterotrimerization of α3(IV), α4(IV) and α5(IV) chains. Reduction of HSP47 levels by siRNA resulted in defects of heterotrimerization among the three chains, indicating that HSP47 plays a critical role in the heterotrimerization. On the other hand, overexpression of HSP47 did not influence heterotrimerization. Since many enzymes and molecular chaperons assist correct folding and trimerization of collagens, one or more enzymes and/or molecular chaperones, other than HSP47, might be deficient in HEK293 cells. Overexpression of HSP47 decreased the secretion of heterotrimers containing the mutant α5(IV) chain, suggesting that HSP47 overexpression might enhance the quality control mechanisms of collagen synthesis by inhibiting the secretion of incorrectly structured heterotrimers.

Suppression of Renal Tubulointerstitial Fibrosis by Small Interfering RNA Targeting Heat Shock Protein 47

Background/Aim: Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model. Methods: The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting. Results: Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA. Conclusions: Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.

The collagen-specific molecular chaperone HSP47: is there a role in fibrosis?

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. Recent studies have shown a close association between increased expression of HSP47 and excessive accumulation of collagens in scar tissues of various human and experimental fibrotic diseases. It is presumed that the increased levels of HSP47 in fibrotic diseases assist in excessive assembly and intracellular processing of procollagen molecules and, thereby, contribute to the formation of fibrotic lesions. Studies have also shown that suppression of HSP47 expression can reduce accumulation of collagens to delay the progression of fibrotic diseases in experimental animal models. Because HSP47 is a specific chaperone for collagen synthesis, it provides a selective target to manipulate collagen production, a phenomenon that might have enormous clinical impact in controlling a wide range of fibrotic diseases. Here, we outline the fibrogenic role of HSP47 and discuss the potential usefulness of HSP47 as an anti-fibrotic therapeutic target.

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats

Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG). HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry. In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels. Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.