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Type I and V collagens are the major components of fibrillogenic proteins in fish skin, and their hydrolysis products possess hyaluronidase inhibitory activity. In this study, for the first time, type I and V collagens were isolated from the skin of shortbill spearfish and striped marlin. Type I (2α1[I]α2[I]) and type V (α1[V]α3[V]α2[V]) collagens composed of distinct α-peptide chains with comparable structures were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometric chromatography. After enzymatic digestion, the collagen peptides were purified by using ultrafiltration (30 KDa) and high-performance liquid chromatography (RP-HPLC) to yield CPI-F3 and CPV-F4 fractions with strong hyaluronidase inhibition rates (42.17% and 30.09%, respectively). Based on the results of simulated gastrointestinal fluid, temperature, and pH stability assays, CPI-F3 and CPV-F4 exhibited stability in gastric fluid and showed no significant changes under the temperature range from 50 to 70 °C (p > 0.05). The results of this first research on the bioactivity of type V collagen peptides provide valuable information for the biomedical industry and show the potential for future bioactivity investigations of type V collagen and its peptides.

Previous studies have reported a close relationship between type V collagen (Col V) and tumor invasion and motility in both breast cancer (BC) and lung cancer (LC). The present work aims to determine whether the extracellular-matrix (ECM)-defined microenvironment influences patient clinical outcome and investigate to which extent histological patterns of Col V expression in malignant cells have a prognostic effect in patients. To that end, we examined the expression of Col V in the tissues of 174 primary tumors (MM, N = 82; LC, N = 41; and BC, N = 46) by immunohistochemistry. We found: (1) diffuse strong green birefringence in membrane and cytoplasm individualizing malignant cells in MM; (2) a focal and weak birefringence mainly in cytoplasmic membrane involving groups of malignant cells in LC and BC; (3) higher average H-score of Col V in MM than in LC and BC samples; (4) a direct correlation between Col V histologic pattern and TNM stage IV, status and median overall survival; (5) patients with LC in TNM stage I, and Col V ≤ 41.7 IOD/mm2 had a low risk of death and a median survival time more than 20 months; (6) patients with MM in TNM stage IV and Col V > 41.7 IOD/mm2 presented a high risk of death and a median survival time of just 20 months. These findings suggest that high levels of Col V individualizing malignant cells, as observed in MM, and low levels grouping malignant cells, as observed in LC and BC, confers different immune-privileged tissue microenvironment for tumor invasion with impact on prognosis of the patients., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The mammary gland structurally and functionally remodels during pregnancy, during lactation and after weaning. There are three types of fibrillar collagens, types I, III, and V, in mammary stromal tissue. While the importance of the fibrillar structure of collagens for mammary morphogenesis has been suggested, the expression patterns of each type of fibrillar collagen in conjunction with mammary remodeling remain unclear. In this study, we investigated their expression patterns during pregnancy, parturition, lactation and involution. Type I collagen showed a well-developed fibril structure during pregnancy, but the fibrillar structure of type I collagen then became sparse at parturition and during lactation, which was concurrent with the downregulation of its mRNA and protein levels. The well-developed fibrillar structure of type I collagen reappeared after weaning. On the other hand, type V collagen showed a well-developed fibrillar structure and upregulation in the lactation period but not in the periods of pregnancy and involution. Type III collagen transiently developed a dense fibrillar network at the time of parturition and exhibited drastic increases in mRNA expression. These results indicate that each type of fibrillar collagen is distinctly involved in structural and functional remodeling in mammary glands during pregnancy, parturition, lactation, and involution after weaning. Furthermore, in vitro studies of mammary epithelial cells showed regulatory effects of type I collagen on cell adhesion, cell proliferation, ductal branching, and β-casein secretion. Each type of fibrillar collagen may have different roles in defining the cellular microenvironment in conjunction with structural and functional mammary gland remodeling.

Objective: Previous studies have reported a close relationship between malignant mesothelioma (MM) and the immune matricial microenvironment (IMM). One of the major problems in these studies is the lack of adequate adjustment for potential confounders. Therefore, the aim of this study was to identify and quantify risk factors such as IMM and various tumor characteristics and their association with the subtype of MM and survival., Methods: We examined IMM and other tumor markers in tumor tissues from 82 patients with MM. These markers were evaluated by histochemistry, immunohistochemistry, immunofluorescence, and morphometry. Logistic regression analysis, cluster analysis, and Cox regression analysis were performed., Results: Hierarchical cluster analysis revealed two clusters of MM that were independent of clinicopathologic features. The high-risk cluster included MM with high tumor cellularity, high type V collagen (Col V) fiber density, and low CD8 + T lymphocyte density in the IMM. Our results showed that the risk of death was increased for patients with MM with high tumor cellularity (OR = 1.63, 95% CI = 1.29-2.89, P = .02), overexpression of Col V (OR = 2.60, 95% CI = 0.98-6.84, P = .04), and decreased CD8 T lymphocytes (OR = 1.001, 95% CI = 0.995-1.007, P = .008). The hazard ratio for the high-risk cluster was 2.19 (95% CI = 0.54-3.03, P < .01) for mortality from MM at 40 months., Conclusion: Morphometric analysis of Col V, CD8 + T lymphocytes, and tumor cellularity can be used to identify patients with high risk of death from MM., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Recent reports in rat models have shown that fibroblasts in the epiligament, an enveloping tissue of the ligament, are not static cells and play an important role during the early ligament healing of isolated grade III injury of the collateral ligaments of the knee. Fibroblasts produce collagen types I, III and V and infiltrate within the ligament body via the endoligament. In addition, similarities have been reported between the structure of the epiligament of the medial collateral ligament and anterior cruciate ligament of the knee in rat and in human. In line with the ascribed role of the epiligament tissue and the synthesis of these collagens and their role in ligament healing, the aim of this study was to determine their presence in the normal epiligament of the aforementioned ligaments in humans, to compare their differential expression and to present a novel hypothesis about the failure of healing of the anterior cruciate ligament in contrast to the medial collateral ligament., Materials and Methods: We used samples from the mid-substance of the medial collateral and the anterior cruciate ligament of the knee joint, acquired from 12 fresh knee joints. Routine histological analysis was performed through hematoxylin and eosin stain, Mallory's trichrome stain and Van Gieson's stain. The immunohistochemical analysis was conducted using monoclonal antibodies against collagen type I and V and procollagen type III. The number of cells in the epiligament, endoligament and the ligament tissue was assessed quantitatively through a computerized system for image analysis NIS-Elements Advanced Research and Statistica software., Results: Our observations revealed certain differences in the morphology of the epiligament, as well as variations in the expression of the investigated molecules. Expression of collagen type I was mostly low-positive (1+) in the epiligament and positive (2+) in the ligament tissue of both ligaments. Expression of procollagen type III was mostly positive (2+) in the epiligament and ligament tissue of the medial collateral ligament, low-positive (1+) in the epiligament and negative (0) in ligament tissue of the anterior cruciate ligament. Expression of collagen type V was predominantly low-positive (1+) in the epiligament and negative (0) in the ligament tissue of both ligaments. The immunoreactivity for all three molecules was always higher in the epiligament of the medial collateral ligament than that of the anterior cruciate ligament., Conclusions: The results of our study illustrate for the first time that fibroblasts in the human epiligament are indeed responsible for the synthesis of the main types of collagen participating in the early ligament healing, thus corresponding to previous data of the medial collateral ligament healing in animal models. The differences between the epiligament of the investigated ligaments could add a novel explanation for the failed anterior cruciate ligament healing., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Objective: To evaluate the frequency of anti-collagen type V in humans with early systemic sclerosis (SSc) compared to defined SSc patients and healthy controls, since collagen type V was shown to be overexpressed in early SSc patients' skin and there is no data concerning the presence of this antibody in early stages of human SSc. Experimental studies showed that animal models immunized with collagen type V developed a disease similar to human systemic sclerosis (SSc), with antibodies production, mainly in early stages post-immunization., Methods: Eighty-one female SSc patients were included and divided into two groups: early-SSc (18 patients-EULAR Preliminary Criteria) and defined-SSc (63 patients-ACR Criteria 1980). The control group consisted of 19 healthy women age-matched to Early-SSc group. Anti-collagen type V was performed by ELISA. Data was analyzed by appropriate tests., Results: The prevalence of anti-collagen type V in early-SSc, defined-SSc and control groups was respectively 33, 17 and 5% (p = 0.07). SSc patients with anti-collagen type V had shorter disease duration compared to those without this antibody (8.8 ± 5.1 vs. 14.7 ± 8.9, p = 0.006). Likewise, early-SSc patients with anti-collagen V also had a shorter disease duration than patients negative for this antibody (4.6 ± 2.2 vs. 9.7 ± 5.2, p = 0.04). No association with clinical subsets or scleroderma antibodies specificities was observed (p > 0.05)., Conclusion: The production of anti-collagen type V in SSc seems to be an early event independent of other antibodies specificities. Further studies are necessary to determine if the underlying mechanism for this chronology involves a primary immune response to abnormal expression of collagen type V.

Background: The greatest challenge to long-term graft survival is the development of chronic lung allograft dysfunction. Th17 responses to collagen type V (colV) predispose lung transplant patients to the severe obstructive form of chronic lung allograft dysfunction, known as bronchiolitis obliterans syndrome (BOS). In a previous study cohort (n = 54), pretransplant colV responses were increased in recipients expressing HLA-DR15, consistent with the high binding avidity of colV (α1) peptides for HLA-DR15, whereas BOS incidence, which was known to be strongly associated with posttransplant autoimmunity to colV, was higher in patients who themselves lacked HLA-DR15, but whose lung donor expressed it., Methods: To determine if this DR-restricted effect on BOS incidence could be validated in a larger cohort, we performed a retrospective analysis of outcomes for 351 lung transplant recipients transplanted between 1988 and 2008 at the University of Wisconsin. All subjects were followed until graft loss, death, loss to follow-up, or through 2014, with an average follow-up of 7 years. Comparisons were made between recipients who did or did not develop BOS. Grading of BOS followed the recommendations of the international society for heart and lung transplantation., Results: Donor HLA-DR15 was indeed associated with increased susceptibility to severe BOS in this population. We also discovered that HLA-DR7 expression by the donor or HLA-DR17 expression by the recipient decreased susceptibility., Conclusions: We show in this retrospective study that specific donor HLA class II types are important in lung transplantation, because they are associated with either protection from or susceptibility to development of severe BOS.

The present study was aimed to investigate the relationship between the expression of collagen type V alpha 2 chain (COL5A2) and clinical outcomes of patients with bladder cancer.Chi-square test and log-rank-based survival analysis were performed to assess the correlation of COL5A2 expression with clinical characteristics and survivals of patients with bladder cancer using GSE13507. Gene set enrichment analysis was conducted to study the relevant mechanisms.Bladder cancer patients in COL5A2 low expression group were associated with better invasiveness (P < .0001), tumor grade (P = .001), T staging (P < .0001), N staging (P = .002), cancer specific survival (P < .0001), overall survival (P < .0001), and a trend of better M staging (P = .053) than those in COL5A2 high expression group.COL5A2 might affect the progression of bladder cancer through "Coagulation," "Hypoxia," "Apical junction," "Ultraviolet response," "Epithelial mesenchymal transition," "Angiogenesis," "KRAS (KRAS proto-oncogene, GTPase) signaling,""Complement,""IL2-STAT5-signaling," "Inflammatory response," "IL6-JAK-STAT3-signaling," "Myogenesis," "TNF α signaling," "Apoptosis," and "Hedgehog-signaling."Our results demonstrated that COL5A2 was correlated with poor clinical outcomes and survivals of patients with bladder cancer, suggesting that it could be regarded as a biomarker of bladder cancer.

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro., (Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

Background and Aim: Infliximab is effective in the induction and maintenance of remission in Crohn's disease. Whether, the perioperative administration of anti-TNF-a compromises intestinal healing leading to anastomotic failure and increased risk of postoperative complications, remains controversial. The aim of the study was to evaluate the effect of Infliximab on intestinal anastomosis healing., Methods: Fifty six wistar rats were divided into 4 groups: (a) 20 rats were subjected to excision of part of the terminal ileum followed by anastomosis which was evaluated on the 3rd or 7th postoperative day; (b) 20 rats received Infliximab and thereafter, the same surgical protocol as group (a) was followed; (c) 8 rats received Infliximab and served as relative control group; and (d) 8 served as absolute control group. Bursting pressure was used for testing intestinal healing. Additionally, the anastomoses were examined macroscopically, histologically and immunohistochemically for TGFb1, MMP1, MMP2 and Collagen V. The results were confirmed by Western blot analysis., Results: There were no significant differences in bursting pressures and septic intra-abdominal events among non-Infliximab (a) and Infliximab-treated (b) groups. Infliximab-treated (b) group showed mild to moderate inflammation, whereas the non-Infliximab (a) group exhibited severe inflammation. Expression of TGFb1, MMP2 and collagen V was significantly higher in the Infliximab-treated (b) group., Conclusion: Infliximab seems to influence intestinal healing in terms of less inflammatory activity and higher tissue remodeling activity., (Copyright © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Although the presence of renal cysts has been reported to be associated with aortic aneurysm or dissection by imaging studies, an autopsy study has not been performed. Therefore, in our institute, recent consecutive adult autopsy cases (n=108, 64 males and 44 females) were reviewed. The circumferences and atherosclerosis ratios of both thoracic and abdominal aorta were individually measured and graded. The number of renal cysts was scored and graded. Age of subjects along with histories of smoking, hypertension, and diabetes mellitus were confirmed. Multiple linear regression analyses demonstrated that severity of atherosclerosis and the number of renal cysts were correlated with thoracic aortic circumference, while only the number of renal cysts was correlated with abdominal aortic circumference (p<0.05), which was more predominant in female subjects (p<0.05). Microscopically, significantly more dilated renal tubules (by Student's t-test, p<0.05) along with decreased stainability of basement membrane by Periodic acid-Schiff staining and immunostaining of type IV collagen were noted in background renal tissues in cases with numerous renal cysts than in age- and sex-matched controls without renal cysts (n=10 vs. 10). The present study suggests that a syndrome that affects both aorta and renal tubules may exist., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Radiotherapy can lead to a reduction of bone density with an increased risk of pathological fractures. Bisphosphonates may represent a preventive treatment option by increasing the density of anorganic bone mineral. Yet it is unknown how bisphosphonates act on irradiated collagen cross-links, which play an essential role for the mechanical stability of bone. The aim of this study was to evaluate the effects of zoledronate on bone collagens and their cross-links after irradiation. The right femur of 37 rats was irradiated with a single dose of 9.5 Gy at a high dose rate using an afterloading machine. Half of the rats (n=18) received additionally a single dose zoledronate (0.1 mg/kg body weight). Fourteen and 100 days after irradiation the femora were collected for histologic evaluation and determination of the collagen cross-links lysylpyridinoline, hydroxylysylpyridinoline, and hydroxyproline. The collagen types were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fourteen days after treatment the lysylpyridinoline levels of all treatment groups were significantly lower compared to the untreated control. After 100 days, in the combined radiotherapy+zoledronate group significantly lower lysylpyridinoline values were determined (p=0.009). Radiotherapy and/or zoledronate did not change significantly the level of hydroxylysylpyridinoline. The concentration of hydroxyproline was 14 days after irradiation significantly higher in the combined treatment group compared to the control. No significant differences were observed 100 days after treatment. Zoledronate does not have the ability to restore the physiological bone collagen cross-link levels after radiotherapy. However, this would be necessary for regaining the physiological mechanical stability of bone after irradiation and therefore to prevent effectively radiation-induced fractures.

In the search for an ideal minimally-invasive multipotent postnatal stem cells' source, the aim of the present study was to isolate and characterize multipotent postnatal stem/progenitor cells from the human alveolar bone proper tissue of the oral cavity. Cells were isolated from human alveolar bone parts, immunomagnetically sorted using STRO-1 antibodies and characterized flow cytometrically for the expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1 surface markers. Colony-formation and multilineage differentiation potential were tested. Mineralized tissue marker expression was examined using real time polymerase chain reaction (PCR). The cells were plastic-adherent and showed colony-formation. Cells expressed the surface markers CD73, CD90, CD105, STRO-1 and CD146/MUC18, while lacking the expression of the hematopoietic markers CD14, CD34 and CD45. Cells could be differentiated into osteoblastic, adipocytic and chondroblastic lineages. Unstimulated cells expressed alkaline phosphatase (ALP), type I, III and V collagens, osteonectin and osteocalcin in a very distinctive pattern. This study presents a practical and minimally-invasive scheme for the isolation of multipotent postnatal stem/progenitor cells from the human alveolar bone tissue of the oral cavity., (Copyright © 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Introduction: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions., Methods: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio)., Results: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001)., Conclusions: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: The importance of type V collagen and its relationships with other types of collagen and with vascular and epithelial apoptosis were studied in a model of chemical carcinogenesis in the mouse lung., Methods: TWO GROUPS OF MALE BALB/C MICE WERE STUDIED: a) animals that received two intraperitoneal doses of 3 g/kg urethane carcinogen (urethane group = 24); and b) animals submitted to a sham procedure, comparable to the test group (control group = 7). Both groups were sacrificed after 120 days. In situ detection of apoptosis, immunohistochemistry, immunofluorescence and histomorphometry were used to evaluate the fraction occupied by the tumor, vascular and epithelial apoptosis, and type V, III and I collagen fibers in the lung parenchyma from both groups., Results: The lung parenchyma from the urethane group showed low fractions of vascular and epithelial apoptosis as well as reduced type V collagen fibers when compared to the control group. A significant direct association was found between type V and III collagen fibers and epithelial apoptosis, type V collagen fibers and vascular apoptosis, and type V and type I collagen fibers., Conclusion: The results show that a direct link between low amounts of type V collagen and decreased cell apoptosis may favor cancer cell growth in the mouse lung after chemical carcinogenesis, suggesting that strategies aimed at preventing decreased type V collagen synthesis or local responses to reduced apoptosis may have a greater impact in lung cancer control.

The extracellular matrix of peripheral nerve plays a vital role in terms of normal nerve fibre function and also in the regenerative response following nerve injury. Nerve fibre loss is a major feature of diabetic neuropathy; however, the regenerative response is limited and this may be associated with changes in the composition of the extracellular matrix. Glycoproteins and collagens are major components of the extracellular matrix and are known to be important in terms of axonal growth. This work has therefore examined whether changes in the expression of two major glycoproteins, laminin and tenascin, and three collagen types (IV, V and VI) occur in the endoneurial and perineurial connective tissue compartments of human diabetic nerve. Despite being known to have a positive effect in terms of axonal growth, laminin levels were not elevated in the diabetic nerves. However, the pattern of tenascin expression did differ between the two groups being found in association with axon myelin units in the diabetic samples only. The pattern of collagen IV expression was the same in both tissue groups and was not found to be up-regulated. However, levels of collagen V and VI were both significantly increased in the endoneurium and for collagen VI also in the perineurium.

This study investigated the contents and distribution of collagen V (Col V) in skin lesions of the patients with systemic sclerosis (SSc) and its roles in the pathogenesis. The contents and distribution for alpha1 chain of collagen type I, III and V [alpha1 (I), alpha1 (III) and alpha1 (V)] in skin lesions of 36 patients with SSc (9 cases of mild fibrosis, 14 moderate, and 13 severe) were detected by using immunohistochemical SP method. Six cases of normal skin tissues served as controls. The results showed that there was diffuse distribution for three kinds of collagens in dermis. The deep staining alpha1 (I) and alpha1 (III) masses or bands were seen in reticular layer, while alpha1 (V) was distributed more homogeneously. From control to weak, moderate and severe fibrosis stages, alpha1 (I), alpha1 (III) and alpha1 (V) showed a gradually increased trend in skin lesions (P<0.05). alpha1 (V) was obviously elevated in skin lesions at early stage and persisted in whole fibrotic process and risen in greater contents, while alpha1 (I) and alpha1 (III) were to go higher late and were apparently elevated at moderate and late stages. Compared with alpha1 (I), alpha1 (V) took leading increase at early stage in skin lesions (P<0.01), and had more elevated contents than alpha1 (III) at moderate and late stages. The fibrotic changes in dermal reticular layer occurred earlier than those in papillary layer, and the abnormalities of alpha1 (V)/alpha1 (I) ratio appeared before alpha1 (III)/alpha1 (I) ratio. It was concluded that a lot of alpha1 (V) began to deposit in greater contents prior to alpha1 (I) and alpha1 (III) at early stage in SSc and persisted in whole fibrotic process. The changes of alpha1 (V) contents in reticular layer occurred earlier than those in papillary layer, and it suggested that the fibrosis in reticular layer appeared earlier.

The development of a sarcomatoid morphotype is recognized as an extreme form of dedifferentiation in renal cell carcinoma and is associated with a poor prognosis. Although sarcomatoid renal cell carcinoma shows pronounced spindle cell morphology, clear cell renal cell carcinoma may show early spindle cell change with cellular elongation, and the prognostic significance of this is debated. To determine the relationship between sarcomatoid renal cell carcinoma and clear cell renal cell carcinoma showing early spindle cell change, we have investigated collagen expression using immunohistochemistry in these 2 tumor types. Both sarcomatoid renal cell carcinoma and early spindle cell change tumors showed pericellular interstitial expression of collagen types I and III, whereas sarcomatoid renal cell carcinoma also showed cytoplasmic expression of these collagen types. Expression of these collagen types in typical clear cell renal cell carcinoma was, in occasional cases, limited to faint and patchy staining in a pericellular interstitial distribution. Tumor cells did not stain for collagen type IV in sarcomatoid renal cell carcinoma, early spindle cell change, or typical clear cell renal cell carcinoma. In sarcomatoid renal cell carcinoma, there was diffuse pericellular expression of collagen type V and patchy pericellular expression of collagen type VI, whereas early spindle cell change tumors showed patchy pericellular staining with antibodies to collagen type V. Collagen type VI expression in early spindle cell change was largely confined to the vascular adventitia and areas of scarring, although very occasional foci of faint interstitial staining were also seen. In typical clear cell renal cell carcinoma, staining of collagen types V and VI was limited to the vascular adventitia and foci of desmoplasia, whereas no staining of tumor cell cytoplasm were seen. This study has shown that collagen expression of sarcomatoid renal cell carcinoma differs from that of early spindle cell change and provides validating evidence that these 2 morphotypes should not be considered together for classification purposes.

Recent evidence suggests that type V collagen plays a role in organizing collagen fibrils, thus maintaining fibril size and spatial organization uniform. In this study we sought to characterize the importance of type V collagen morphological disorganization and to study the relationship between type V collagen, active remodeling of the pulmonary vascular/parenchyma (fibroblastic foci), and other collagen types in usual interstitial pneumonia (UIP). We examined type V collagen and several other collagens in 24 open lung biopsies with histological pattern of UIP from patients with idiopathic pulmonary fibrosis (IPF). We used immunofluorescence, morphometry, and three-dimensional reconstruction to evaluate the amount of collagen V and its interaction with the active remodeling progression in UIP, as well as types I and III collagen fibers. Active remodeling progression was significantly related to type V collagen density (p<0.05), showing a gradual and direct increase to minimal, moderate, and severe fibrosis degree in UIP and in the three different areas: normal, intervening, and mural-organizing fibrosis in UIP. Parenchymal changes were characterized by morphological disorganization of fibrillar collagen with diverse disarray and thickness when observed by three-dimensional reconstruction. We concluded that in the different temporal stages of UIP, vascular/parenchyma collagen type V is increased, in disarray, and is the most important predictor of survival.

Type V and VI collagen were capable to joint each other and with type I and IV collagen, as well as mucopolysaccharides. This capability suggested that these collagens function for cohesion of fibrillar tissue components of dermis. This study demonstrated the locality of these types of collagen in dermis. Fresh specimens of normal skin were fixed in 2% paraformaldehyde in phosphate-buffered saline, overnight. Besides, in order to loosen the twist of collagen fibril, some pieces of the skin specimens were treated by citrate buffer pH 3.0, prior to fixation. The specimens were embedded in Technovit 4100 and the ultrathin sections were stained by antibody to type V collagen and followed by antibody to type I, III, IV and VI collagen. The immune reactant was visualized by gold particles for electron microscopic observation. Type V and VI collagen formed networks in dermis and jointed to collagen fibrils, elastic fibre and basal lamina. Type V collagen was found inside collagen fibrils, broad elastic fibres and junctions. Dermo-epidermal junction showed type V collagen on the dermal aspects of basal lamina and at the sites where anchoring filaments joint to basal lamina, while in junction of mesenchymal tissues, no precise structural components for type V collagen were identified. Type VI collagen wove with type V collagen in dermis and associated with mucopolysaccharides. In conclusion, type V collagen formed networks in dermal interfibrillar space and participated in assembling collagen fibrils and forming broad elastic fibres. Epithelial and mesenchymal cells cohered to the underlying dermal matrix in the junction by type V collagen. Type VI collagen interwove with type V collagen in the interfibrous space and associated with mucopolysaccharides. Types V and VI collagen preserved architecture of dermal matrix.

Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate-protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm(-2)) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm(-2) of biotinylated heparin (a stoichiometric ratio of 1:6 heparin-streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm(-2) of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV-heparin complex ( approximately 1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin-HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.

Cartilage exerts many functions in different tissues and parts of the body. Specific requirements presumably also account for a specific biochemical composition. In this study, we investigated the presence and distribution pattern of matrix components, in particular collagen types in the major human cartilages (hyaline, fibrous, and elastic cartilage) by histochemical and immunohistochemical means. Macroscopically normal articular cartilages, menisci, disci (lumbar spine), epiglottal, and tracheal tissues were obtained from donors at autopsy. Aurical and nasal cartilages were part of routine biopsy samples from tumor resection specimens. Conventional histology and immunohistochemical stainings with collagen types I, II, III, IV, V, VI, and X and S-100 protein antibodies were performed on paraformaldehyde-fixed and paraffin-embedded specimens. The extracellular matrix is the functional component of all cartilages as indicated by the low cell densities. In particular major scaffold forming collagen types I (in fibrous cartilage) and II (in hyaline and elastic cartilages) as well as collagen type X (in the calcified layer of articular cartilages, the inner part of tracheal clips, and epiglottis cartilage) showed a specific distribution. In contrast, the "minor" collagen types III, V, and VI were found in all, collagen type IV in none of the cartilage subtypes. In this study, we present a biochemical profile of the major cartilage types of the human body which is important for understanding the physiology and the pathophysiology of cartilages.

Porcine small intestinal submucosa (SIS) was previously shown to enhance the mechanical properties of healing medial collateral ligaments (MCL), and the histomorphological appearance and collagen type V/I ratio were found to be close to those of normal MCL. We hypothesized that at a longer term, 26 weeks, SIS could guide a better organized neo-ligament formation, increasing mechanical properties and increasing collagen fibril diameters mediated by a reduction in collagen type V. A 6 mm gap injury in the right MCL was surgically created in 38 rabbits, while the contralateral intact MCL served as a sham-operated control. In half the animals, a strip of SIS was sutured onto the severed ends. In the other half, no SIS was applied. The cross-sectional area (CSA) was determined with a laser micrometer system. The femur-MCL-tibia complex was mechanically tested in uniaxial tension. Histomorphology was determined through H&E and immunofluorescent staining and transmission electron microscopy (TEM). Sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine collagen type V/I ratio. SIS-treated MCLs displayed a 28% reduction in CSA, a 33% increase in tangent modulus, and a 50% increase in tensile strength compared with the nontreated group (p < 0.05). TEM showed groups of collagen fibrils with larger diameters in the SIS-treated ligaments in comparison with uniformly small fibrils for the nontreated group. H&E staining showed more densely stained collagen fibers in the SIS-treated group aligned along the longitudinal axis with more interspersed spindle-shaped cells. Immunofluorescent staining showed less collagen type V signals, confirmed by a 5% lower ratio of collagen type V/I compared with the nontreated controls (p < 0.05). The findings extend the shorter term 12-week results, and support the potential of porcine SIS as a bioscaffold to enhance ligament healing., (Copyright 2006 Orthopaedic Research Society)

Large cell calcifying Sertoli cell tumor is a rare type of testicular tumor. Reported herein is a Japanese patient with this tumor not associated with Carney's complex. An 11-year-old boy was admitted to hospital because of left testicular enlargement, and radical orchiectomy was performed. Macroscopically, the tumor was well circumscribed and had a maximum diameter of approximately 2 cm. The cut surface showed a yellow-white solid mass. Histologically, the tumor was composed of large neoplastic cells with abundant eosinophilic cytoplasm with a tubular, trabecular, and solid arrangement and loose myxoid stroma with irregularly shaped calcification. Immunohistochemically, the tumor cells were positive for vimentin, S-100 protein, calretinin, inhibin-alpha, melan-A, and CD10, and type IV collagen and laminin were observed in the extracellular matrix around the tumor cells. The distributions of melan-A, CD10, and mitochondria were characteristically patchy; in contrast, they were diffusely distributed in the cytoplasm in a control case of Leydig cell tumor. The differences in immunostaining patterns for melan-A, CD10, and mitochondria as well as positivity for S-100 protein-beta might be useful diagnostic hallmarks of large cell calcifying Sertoli cell tumor for discrimination from Leydig cell tumor.

Objective: To explore the mechanism of the bronchial asthma and to study the treating effects of Zhichuan Capsule on the airway remodeling of asthmatic model rats., Methods: The rat model was established by being sensitized and activated with different density of ovalbumin through prolonged and repeated exposure for 8 weeks. The rats were randomly divided into model group, Zhichuan Capsule treated group, dexameson treated group, and Zhichuan Capsule and dexameson treated group. Another group of normal rats were taken as control. General histological changes were observed by hematoxylin and eosin stained sections. Being standardized by internal perimeter (Pi), the wall thickness (d), internal area (Ai), outer area (Ao) and wall area (WA) of the airway were quantified by computer-assisted image analysis system. The express of MMP-9, TIMP-1, Col I, Col III and ColV in the airway were examined by immunocytochemical methods. During the course of airway remodeling, the dynamic changes of model rats were observed at different time points (2, 4, 6 and 8 weeks after the activating). Statistical comparison was performed by ANOVA followed by Fisher LSD test., Results: (1) Histologic examination showed eosinophil infiltration within the airway walls, epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways in model rats, and that the collagen deposition increased accompanied by increasing of TIMP-1. In the model rats, MMP-9 increased at the time point of 2 weeks, but it decreased in the late stage (8 weeks after activating) of airway remodeling. And the level of TIMP-1 was far higher than MMP-9 at the time point of 8 weeks. (2) Zhichuan Capsule could down-regulate the level of TIMP-1 in the airway wall, as well as the thickness of airway wall and the collagen deposition. And there were progressing effects when it was used together with dexameson., Conclusion: (1) The early increase of MMP-9 is a key point to start remodeling; and the increase of TIMP-1 in the late stage, which inhibits collagenase activity, may play an important role in developing airway fibrosis. Imbalance between MMP-9 and TIMP-1 is a marker of airway remodeling. (2) Zhichuan Capsule can decrease the deposition of collagen and suppress the airway remodeling by inhibiting the TIMP-1 expression.

Background: Repeated attacks of bronchial asthma lead to different degrees of airway remodeling, the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin II is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma, we observed the effects of an angiotensin II type 1 receptor antagonist (valsartan) on the expression of collagen III, collagen V, and transforming growth factor beta1 (TGF-beta1) mRNA and protein in the airway walls of sensitized rats., Methods: Forty Wistar rats were randomly divided into 5 groups: control group, sensitized group, and valsartan groups 1, 2, and 3. The rats in the sensitized group and in valsartan groups 1, 2, and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1, 2, and 3 were drenched with valsartan (10 microg, 20 microg, or 30 microg, respectively) at the time of the ovalbumin challenges. The expression of collagen III, collagen V, and TGF-beta1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-beta1 mRNA was detected by in situ hybridization., Results: The expression in the airways of collagen III and collagen V was significantly higher in rats from the sensitized group (7.73 +/- 0.81, 1.34 +/- 0.28) and from valsartan groups 1, 2, and 3 (5.73 +/- 0.64, 1.13 +/- 0.15; 4.96 +/- 0.51, 0.98 +/- 0.08; 4.43 +/- 0.35, 0.93 +/- 0.06, respectively) than those in the control group (2.65 +/- 0.38, 0.67 +/- 0.08, P < 0.05). In addition, collagen levels were significantly lower in valsartan groups 1, 2, and 3 than those from the sensitized group (P < 0.05). The expression of TGF-beta1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49% +/- 3.46%, 29.73% +/- 3.25%) and from valsartan groups 1, 2, and 3 (16.47% +/- 1.94%, 19.41% +/- 1.87%; 14.38% +/- 1.58%, 18.29% +/- 1.43%; 12.96% +/- 1.73%, 18.63% +/- 1.11%, respectively) than that from the control group (7.84% +/- 1.61%, 5.63% +/- 1.07%, P < 0.05). TGF-beta1 mRNA and protein levels were significantly lower in valsartan groups 1, 2, and 3 than that in the sensitized group (P < 0.05)., Conclusions: Angiotensin II receptor antagonist valsartan can suppress synthesis of collagen III and collagen V by downregulating TGF-beta1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy.

Objective: The objective of this study was to measure the amount of viable bone cells present in different types of bone graft., Material and Methods: Bone chips were harvested from the trabecular or cortical bone of the mandible or the iliac crest and either milled or not. The average size of unmilled bone particles was 5 x 5 x 5 mm and that of milled was 2 x 2 x 2 mm. Drill sludge was obtained using either a ball reamer, a diamond ball or an implant drill (the latter from mandibular bone and of average dimension 1 x 1 x 1 mm). A measure of 0.5 g of each category was cultured in Dulbecco's modified Eagle's medium with additives for four weeks. Cell counts were performed. An analysis of the osteocalcin synthesis, the alkaline phosphatase (ALP) activity, the collagen types and the concentration of bone-specific collagen cross-links in medium supernatants was performed., Results: Cells stained positively for osteocalcin and ALP in all groups. Bone-specific collagen cross-links could be quantified and collagen of types I and V was present with no difference in all groups. Unmilled spongy bone chips revealed greater cell counts than milled (P<0.05). Spongy bone chips revealed greater cell counts than cortical bone chips (P<0.05). Drill sludge obtained by hard alloy ball reamer showed the least amount of viable cells (P<0.05)., Conclusions: Bone milling reduces the quantity of osteoblasts. Bone obtained by the ball reamer supplies a smaller number of cells than bone obtained by other methods. Unmilled spongy bone chips appear to offer the greatest amount of viable osteoblasts.

Injuries to soft tissues such as tendons are becoming ever more frequent among the elderly. While increasing levels of activity likely contribute to these injuries, age-related declines in tendon strength may also be important. Whether these declines in biomechanical properties are associated with changes in fibril diameter or collagen type remains in question. In this study, age-related changes were investigated in patellar tendons from young adult rabbits (1-year old, n = 17) and from rabbits at the onset of senescence (4-year old, n = 33). Patellar tendon biomechanics was correlated with both collagen fibril diameter and with the presence of type V collagen, a known regulator of collagen fibril diameter. We hypothesize that (a) aging from I to 4 years results in significant reductions in patellar tendon biomechanical properties, and (b) these age-related declines are associated with smaller fibril diameters and with the presence of type V collagen. Maximum stress declined 25% between I and 4 years of age (100.7 +/- 5.6 MPa and 74.3 +/- 3.4 MPa, respectively, p < 0.0003) (mean +/- SEM) and strain energy density declined 40% (p < 0.001). The distribution of collagen fibrils from 4-year old rabbits was skewed significantly towards smaller diameters compared to fibrils from 1-year old rabbits (p < 0.001). Type V collagen was observed only in the 4-year old rabbit tendons. These correlations suggest that with increasing age after skeletal maturity, type V collagen may help to regulate the assembly and thus diameter of collagen fibrils and thereby adversely affect patellar tendon strength.

This study was designed to study wound strength at the dentine/connective tissue interface and at the bone/connective tissue interface following full thickness flap surgery. Flaps of uniform dimension were outlined in four young adult beagle dogs using a standardised double bladed knife and vertical incisions 10 mm apart, which extended 8 mm apical to the gingival margin. Bone was removed from half the sites (eight sites in each dog), giving 32 flaps replaced on dentine and 32 sites on bone. A tensile force was applied using a microprocessor force gauge at 1, 2, 3, 7, 10, 14, 21, and 28 days. Mean tensile strengths were markedly weaker for the dentine/flap interface. At 7 days the value for flaps to dentine was 1.82 N, in contrast to 5.08 N for flaps replaced on bone. Inflammatory cell counts tended to fall markedly at 3 days for both modalities, but were higher for the dentine/flap modality at all time points. Fibroblast density peaked at 7-14 days but did not vary with type of flap over the time points studied. The amounts of fibrin were greater for the dentine/flap interface at all time points but decreased for both flap types as time progressed. Collagen type V was localised to the basement membrane and blood vessels and tended to show more foci for flaps replaced on dentine. Procollagen levels showed little change over the healing interval for both flap/bone and flap/dentine interfaces. Type III collagen synthesis was at peak levels during the first week. These findings would support efforts to stabilise periodontal flaps at early time points, especially those on dentine.

The contents of type I, type III and type V collagen and the collagen type specific distributions in liver under normal and cirrhotic conditions were examined. In CCl4 injected rat, the increasing amount of type V collagen was a specific event during the progression of cirrhosis. In normal liver, immunohistochemical observation showed that type V collagen was localized on the fine fibrils, while type I was localized on the thick fibril. Type V collagen was partially colocalized with type IV collagen. In the cirrhotic liver, type V collagen was localized on the margin of the thick fibrous septa along with type IV collagen. Type I collagen existed in the core region of fibrous septa where the stellate cells were prominent. To elucidate the mechanism of the type specific deposition of collagen in the liver, we constructed a coculture system using both stellate cells and hepatocytes. In this system, type V collagen was mainly deposited on hepatocyte colonies not on stellate cells, while type I collagen fibrils were localized on stellate cells. The spatial positioning of type I and type V collagens in vitro was similar to that in the liver. In the cell adhesion assay, the adhesion of stellate cells to type V collagen was poorer than that of the hepatocytes. The collagen type-specific affinity of the stellate cells and hepatocytes may explain the specific localization of type V collagen in the liver and coculture system. These results suggested that the functions of type V collagen are not only to connect type IV collagen with type I collagen fibril, but also to protect the parenchyma from excess type I collagen deposition produced by stellate cells under pathological conditions.

Collagen is one of the chief components of the extracellular matrix of gingival connective tissue, where five different types have been identified to date. The molecular mechanism of collagen loss in periodontitis still needs to be explored. In the present study total collagen content was investigated in gingival connective tissue of adult periodontitis (AP) as well as early onset periodontitis patients (EOP) and clinically healthy subjects. Furthermore, collagen type I, III, IV, V andVI content was evaluated in gingival biopsies obtained from periodontitis patients. There was a statistically significant difference between AP (25.1 +/- 8.1 microg/mg) and EOP (15.6 +/- 4.0microg/mg) groups with regard to the total collagen content (P < 0.05). In the clinically healthy control group the total collagen content was 20.7 +/- 4.6microg/mg. Moreover, the distribution of collagen types exhibited variations in pooled homogenates of each periodontitis group. The total collagen loss seemed to be greater in the EOP patients than in the AP patients. When the ratio of fibril forming collagens to nonfibrillar collagens was evaluated, it seems to be decreased in AP patients in comparison to EOP patients. The findings of the present study suggest that different collagen types present in various periodontitis categories may be related with diverse pathogenic mechanisms acting in these diseases.