Functional role of inflammation in the surgical injury induced vascular remodeling of male albino rats.

Our present study investigates the cellular and molecular inflammatory events in male albino rat arterial injury. Male albino rats were subjected to longitudinal incision and carotid artery clamping for the duration of 45 days. Heat shock protein (HSP) 27, HSP70, HSP47 and Nuclear Factor kappa B (NF-κB) expressions were determined by qPCR and Western blot method. The morphology of vessel wall alteration was studied by the light microscopy. The expression of NF-κB was found to be increased after ten days of carotid artery injury. The qPCR and Western blot analysis showed elevation in HSP47, HSP27, and HSP70 expression, ten days following the surgical injury. The neointima-formation and the media layer discontinuity were evidenced by light microscopy. The dendritic-like cells were in close contact with the lymphocytes. Our study reports that the surgical injury induces an inflammatory response through the increased NF-κB and HSPs expression.

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.

Changes in Testicular Interstitial Connective Tissue of Hamsters (Mesocricetus auratus) During Ageing and After Exposure to Short Photoperiod.

The testicular interstitium of Syrian hamster (Mesocricetus auratus) was studied during ageing and in testicular regression after exposure to a short photoperiod, in relation to the interstitial cells and their connective tissue. This tissue was assessed histochemically using Masson's trichrome technique and the expression of Heat Shock Protein 47 (HSP-47) and collagen IV (α5) was assessed in Leydig cells. Finally, an ultrastructural analysis of some cells of the testicular interstitium was made. Leydig cells were positive for HSP-47 and collagen IV (α5). Ageing did not change the parameters studied while the short photoperiod altered the synthetic activity of Leydig cells. The positivity index of these cells for HSP-47 was significantly higher in the regressed testis, but was lower for collagen IV (α5). During ageing no change were observed. Ultrastructural Leydig cells showed a discontinuous basal lamina that did not change during ageing. The basal lamina was not identified in Leydig cells regressed by exposure to a short photoperiod. In conclusion; the intertubular connective tissue suffers little change with age. By contrast, in the testis regressed after exposure to a short photoperiod the studied parameters related to the intertubular connective tissue were altered. These changes are probably related with the low synthetic activity of regressed Leydig cell.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

The imbalance between proliferation and apoptosis contributes to degeneration of aortic valves and bioprostheses.

BACKGROUND: The pathomechanisms underlying aortic valve degeneration are incompletely understood. Therefore, the aim of our work was to assess the quantitative changes of proliferation and apoptosis accompanied by cellular phenotype alternations and matrix secretionin aortic sclerotic and stenotic valves and degenerative bioprostheses, as well as to detect the expression pattern of the rapamycin receptor FKBP12 across these three valve types. METHODS: Mild-to-moderate sclerotic and stenotic valves and degenerative bioprostheses from 30 patients (n = 10 per group) were collected at autopsy or by surgery. Ki67+, FKBP12+, alpha-actin+, HSP47+ and TUNEL+ apoptotic cells were analyzed by immunohistochemistry. RESULTS: The main finding was the reduced proliferation and increased apoptosis in stenotic valves (ST) compared to the sclerotic ones (SC) (proliferation: ST: 20.8 ± 2.0% vs. SC: 30.1 ±2.2%, apoptosis: ST: 40.7 ± 5.0% vs. SC: 28.0 ± 5.1%, p < 0.05, respectively). Analogical alternations were found in degenerative bioprostheses (BP) (proliferation: 4.8 ± 2.3%; apoptosis: 13.1 ± 6.8%). Corresponding changes were observed in the valve cellularity (ST: 893 ± 168, SC: 1034 ± 284, BP: 385 ± 179 cells/mm2, p < 0.05, respectively). The FKBP12 signaling was reduced in diseased valves and bioprostheses (ST: 28.1 ± 3.6%, SC: 42.2 ± 3.8%, BP: 5.8 ± 1.9%, p < 0.05, respectively). Further, the augmented alpha-actin expressionwas observed as the degenerative process progressed (ST: 30.3 ± 5.0%, SC: 22.6 ± 2.7%, BP:8.7 ± 4.0%, p < 0.05, respectively), followed by the upregulation of HSP47 (ST: 22.6 ± 2.8%,SC: 15.4 ± 2.1%, BP: 3.4 ± 1.0%, p < 0.05, respectively) and consecutive matrix accumulation. CONCLUSIONS: The imbalance between proliferation and apoptosis with cellular phenotypical shift and subsequent matrix secretion may contribute to aortic valve stenosis and bioprosthesis degeneration. The identification of FKBP12 expression may implicate potential therapeutic strategies.

Immunoexpression of á2-integrin and Hsp47 in hereditary gingival fibromatosis and gingival fibromatosis-associated dental abnormalities

Objective: The purpose of the present study was to investigate the expression of the α2-integrin subunit and heat shock protein 47 (Hsp47) in two families with isolated gingival fibromatosis (GF) form and one family with GF associated with dental abnormalities and normal gingiva (NG). Study Design: Immunohistochemistry was performed with antibodies against α2-integrin and Hsp47 in specimens from two unrelated families with hereditary gingival fibromatosis (Families 1 and 2) and from one family with a gingival fibromatosis-associated dental abnormality (Family 3); NG samples were used for comparison. The results were analysed statistically. Results: Immunoreactivity for α2-integrin and Hsp47 was observed in the nucleus of epithelial cells of both the basal and suprabasal layer and a more discreet signal was noted in connective tissue in all study samples. Hsp47 showed higher immunoreactivity in Family 2 compared with the other families (p≤0.05). Despite the markup α2-integrin was higher in Family 3 there was no statistically significant difference between the families studied (p≥0.05). Conclusions: Our results confirmed the heterogeneity of GF, such that similar patterns of expression of the condition may show differences in the expression of proteins such as Hsp47. Although no difference in α2-integrin expression was observed between GF and NG groups, future studies are necessary to determine the exact role of this protein in the various forms of GF and whether it contributes to GF pathogenesis (AU)

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Tumor budding, myofibroblast proliferation, and fibrosis in obstructing colon carcinoma: the roles of Hsp47 and basic fibroblast growth factor.

This study was designed to assess the mechanism of obstruction in obstructing colorectal carcinomas. Thirty-five cases of obstructing colorectal carcinoma and 34 cases of non-obstructing carcinoma were studied. The lesions were immunohistochemically analyzed using antibodies for pan-cytokeratin, α-smooth muscle actin, matrix metalloproteinase-7, 47-kDa heat shock protein (Hsp47), basic fibroblast growth factor (bFGF), myeloperoxidase, and CD68. Compared with non-obstructing cases, obstructing carcinoma cases included lesions of poorer differentiation. A higher value of tumor budding was observed in obstructing than in non-obstructing carcinoma. A higher number of α-smooth muscle actin-positive myofibroblasts, a higher expression of Hsp47 in stromal spindle cells, and a higher expression of bFGF in inflammatory cells were also significant in obstructing carcinoma. Therefore, obstructing colon carcinomas were characterized by poorer differentiation of cancer cells, a high level of tumor budding, and stromal myofibroblast proliferation resulting in fibrosis. Correlative Hsp47 expression in fibroblasts with bFGF in inflammatory cells may contribute to stromal fibrosis.

Immunolocalization of heat shock proteins 27 and 47 during repair of induced oral ulcers.

Heat shock proteins (Hsps) 27 and 47 are involved in the control of apoptosis, cell migration, and collagen synthesis. There is some understanding of the immunolocalization of these proteins during the repair process in skin and gastrointestinal mucosa, but their expressions in normal and injured oral mucosa are unknown. The aim of this study was to analyze the immunolocalization and intensity of these proteins in oral ulcers induced in rats and to compare these expression levels with those reported in skin and gastric mucosa. Ulcers were induced on the ventral surface of the tongues of rats. The rats were then euthanized at 0, 24, 48, 72, and 120 h. Hsp27 expression remained low in the first hours of repair, but was higher at 72 h, mainly in the migrating epithelium. Expression of Hsp47 was high at 48 h, mainly in fibroblasts, cells of the vascular wall, and basal keratinocytes of migrating epithelium. In the control group, expressions of these proteins were low, which indicates that these Hsps are constitutive proteins in oral mucosa. Expression levels were similar to those reported in the healing of skin lesions and gastric ulcer, suggesting a common mechanism of Hsp activation in the repair of these tissues.

Molecular targets for diabetes mellitus-associated erectile dysfunction.

Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCore suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-gamma, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-gamma, and Hsp47 confirmed increased p53 and 14-3-3-gamma and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a approximately 3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment.

The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine A.

BACKGROUND AND OBJECTIVE: Heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. Heat shock protein 47 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare Hsp47 expression in normal gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of Hsp47 expression. MATERIAL AND METHODS: Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of cyclosporine A on the expression of Hsp47 in human gingival fibroblasts. In addition, Aggregatibacter actinomycetemcomitans, interleukin-1 alpha (IL-1 alpha) and mitogen-activated protein kinase kinase (MEK) inhibitor U0126 were added to seek the possible regulatory mechanisms of Hsp47 expression. RESULTS: A significantly higher percentage of cells positively stained for Hsp47 was noted in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). Expression of Hsp47 was observed mainly in the cytoplasm of fibroblasts, endothelial cells, epithelial cells and inflammatory cells. Expression of Hsp47 was significantly higher in cyclosporine A-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). Cyclosporine A upregulated Hsp47 expression in human gingival fibroblasts in a dose-dependent manner (p < 0.05). The addition of A. actinomycetemcomitans or interleukin-1 alpha significantly increased Hsp47 expression compared with cyclosporine A alone (p < 0.05). The MEK inhibitor U0126 was found to inhibit cyclosporine A-induced Hsp47 expression (p < 0.05). CONCLUSION: Expression of Hsp47 is significantly upregulated in cyclosporine A-induced gingival overgrowth specimens, and Hsp47 expression induced by cyclosporine A in fibroblasts may be mediated by the MEK signal transduction pathway. The expression of Hsp47 could be significantly enhanced by A. actinomycetemcomitans and interleukin-1 alpha.

Carbon dioxide laser irradiation stimulates mineralization in rat dental pulp cells.

AIM: To examine the effect of carbon dioxide laser irradiation on mineralization in dental pulp cells. METHODOLOGY: Rat dental pulp cells were irradiated with a carbon dioxide laser at 2 W output power for 20, 40 and 60 s, and were cultured in ascorbic acid and beta-glycerophosphate containing media. Cell viability was examined 24 h after laser irradiation by a modified MTT assay. Alizarin Red S staining was performed 10 days after laser irradiation. The amounts of secreted collagen from the cells after irradiation were quantified following Sirius Red staining. The expression levels of collagen type I and HSP47, collagen-binding stress protein, were analysed by real-time PCR. HSP47 protein expression was examined by Western blotting. Statistical analysis was performed using one-way analysis of variance (anova) followed by the Tukey's multiple comparison test. RESULTS: The cell viability was not affected by laser irradiation at 2 W for up to 40 s. However, it was significantly decreased by 20% at 60 s (P < 0.05). The amount of mineralization after 10 days of irradiation at 2 W for 40 s was significantly increased in comparison to the other conditions (P < 0.05). The extracellular collagen production was significantly increased by 73% on day 2 and 38% on day 4 after laser irradiation (P < 0.05). Although collagen type I gene expression was not changed by laser irradiation, HSP47 gene and protein expression was induced within 12 and 24 h, respectively. CONCLUSIONS: These results suggested that carbon dioxide laser irradiation stimulated mineralization in dental pulp cells. The laser irradiation also increased HSP47 expression but not collagen gene expression.

High expression of HSP47 in ulcerative colitis-associated carcinomas: proteomic approach.

BACKGROUND: Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease, known to be associated with a markedly increased risk of colorectal carcinoma development. METHODS: Using proteomic analysis with two-dimensional gel electrophoresis and mass spectrometry, differentially expressed proteins were assessed between UC-associated cancer and sporadic colon cancer cell lines. Western blot and immunostaining were performed for confirming the expression. RESULTS: Heat-shock protein of 47 kDa (HSP47) was identified as one of the proteins expressed more highly in UC-associated cancer cell lines, and an immunohistochemical examination confirmed significantly higher levels of HSP47 in UC-associated colon cancers than in sporadic counterparts, the expression increasing with a progression of neoplastic lesions. Heat-shock protein of 47 kDa was further found to be coexpressed with type I collagen in the cytoplasm, and both HSP47 and type I collagen were released from cultured cells into the culture medium. CONCLUSION: These results suggest that overexpression of HSP47 is a unique characteristic of UC-associated carcinoma related to type I collagen synthesis, with possible clinical applications.

Heat shock proteins 47 and 70 expression in rodent skin model as a function of contact cooling temperature: Are we overcooling our target?

BACKGROUND AND OBJECTIVES: The degree of protective cooling required during laser therapy to achieve an optimal result is unknown. The expression of heat shock proteins, Hsp47 and Hsp70, were examined in the epidermis and dermis as biomarkers to quantify the degree and depth of tissue affected by non-ablative laser treatment using variable protective cooling parameters. STUDY DESIGN/MATERIALS AND METHODS: Twenty-one male Sprague-Dawley rats were treated with a 1,319 nm Nd:YAG laser using a sapphire cooling plate attached to the hand piece. A 4 cmx4 cm area on each side of the rat was treated with the same energy and pulse settings, with variable contact cooling. Protective cooling parameters, for each degree increment, ranging from 0 to 25 degrees C were studied. Immunohistochemistry (IHC), Western blot and PCR were performed to evaluate the effects of superficial cooling on Hsp47, and Hsp70 expressions. RESULTS: Our data showed the extent of topical cooling needed to produce a thermal effect at different depths in the dermis, quantified by the expression of Hsp47 and Hsp70. Significant Hsp expression was observed with cooling of 13 degrees C and warmer; no identifiable cellular reaction was observed when cooling below 5 degrees C. There was no evidence of epidermal injury when treating the skin with any protective cooling ranging from 0 to 25 degrees C. CONCLUSION: Our data would suggest contact cooling temperatures 5 degrees C and below completely protects through the entire dermis. There was no evidence of epidermal injury with protective cooling at any temperature between 0 and 25 degrees C. Warmer temperatures are safe and adequately protect the epidermis in this model.

Exogenous nitric oxide enhances the synthesis of type I collagen and heat shock protein 47 by normal human dermal fibroblasts.

BACKGROUND: It is well established that the alterations of dermal matrix contributes to skin aging characterized by wrinkles. On the other hand, physiological NO is useful to maintain skin homeostasis such as a vasodilatation. However, a role of NO on production of dermal matrix has been clarified. OBJECTIVE: In this study, we have attempted to analyze the role of NO on type I collagen synthesis of normal human dermal fibroblasts including expression of procollagen alphaI S(1) mRNA/protein and heat shock protein 47 (HSP47). METHODS: The effects of NO which was generated by two types of NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), on type I collagen and HSP47 and their related mRNA expression were examined with ELISA and RT-PCR. RESULTS: NO was significantly accelerated the production of type I collagen by fibroblasts corresponding with up-regulation of procollagen alphaI (1) mRNA. Furthermore, NO increased both levels of HSP47 protein and mRNA in fibroblasts in a dose-dependent manner. CONCLUSIONS: These results suggest that NO has dual effects on collagen synthesis by fibroblasts as follows; one is the direct stimulation of collagen synthesis due to the up-regulation of procollagen alphaI(1) mRNA, and the other is an indirect effect through the increase of HSP47 mRNA expression. This is the first report that exogenous NO stimulates HSP47 production by dermal fibroblasts.

The receptor for advanced glycation end products is highly expressed in the skin and upregulated by advanced glycation end products and tumor necrosis factor-alpha.

Advanced glycation end products (AGEs) form non-enzymatically from reactions of proteins with reducing sugars. In the skin, AGEs were reported to accumulate in dermal elastin and collagens and to interact nonspecifically with the cell membrane of dermal fibroblasts. Therefore, AGEs may influence the process of skin aging. We investigated the presence of the AGE receptor RAGE in skin and the influence of AGEs on receptor expression and the formation of extracellular matrix (ECM). Sections of sun-protected and sun-exposed skin were analyzed with monoclonal antibodies against (RAGE), heat-shock protein 47, factor XIIIa, CD31, and CD45. RAGE was mainly expressed in fibroblasts, dendrocytes, and keratinocytes and to a minor extent in endothelial and mononuclear cells. Human foreskin fibroblasts (HFFs) highly expressed RAGE on the protein and mRNA level when analyzed by quantitative Western blotting and real-time PCR. Incubation of HFFs with the specific RAGE ligand Nepsilon-(carboxymethyl)lysine-modified BSA (CML-BSA) and tumor necrosis factor-alpha resulted in significant upregulation of RAGE expression. CML-BSA induced a mildly profibrogenic pattern, increasing connective tissue growth factor, transforming growth factor-beta (TGF-beta) 1, and procollagen-alpha1(I) mRNA, whereas expression of matrix metalloproteinase (MMP)-1, -2, -3, and -12 was unaffected. We conclude that in HFFs, AGE-RAGE interactions may influence the process of skin aging through mild stimulation of ECM gene expression.