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Alpha-1 Type Ⅲ Collagen (COL3A1) encodes the Collagen alpha-1(Ⅲ) chain, which is a fibrillar collagen that exists in extensile connective tissues. Few studies have reported its role in tumorigenicity. In the present study, we identified that COL3A1 protein and mRNA expression levels were considerably up-regulated in esophageal squamous cell carcinoma (ESCC) cells in comparison with normal esophageal squamous epithelial cells (P < 0.05). Immunohistochemical (IHC) analysis of 114 paraffin-embedded archived ESCC tissues demonstrated that COL3A1 expression was positively correlated with the postoperative T stage. Univariate and multivariable analysis demonstrated that COL3A1 expression was an independent poor prognostic factor for overall survival in the whole cohort. Silencing COL3A1 inhibited, while overexpressing COL3A1 promoted, the proliferation, migration, and invasion of ESCC cells. Furthermore, down-regulation of COL3A1 expression also suppressed the growth of ESCC in subcutaneous xenograft mouse models and inhibited ESCC metastasis in lung metastasis mouse models. In addition, we proved that the tumor-promoting effect of COL3A1 on ESCC cells was related to the activation of NF-κB signaling pathway. These findings indicate that COL3A1 confers a poor prognosis and malignant phenotype by activating the NF-κB pathway in ESCC, potentially representing a novel biomarker and/or providing a new curative target for ESCC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Increased ochratoxin A (OTA) or citrinin (CIT) concentrations in food correlate with increased prevalence of tubule-interstitial nephropathy. We tested the hypothesis that co-exposure of human proximal tubule-derived epithelial cells (HK-2) to OTA and CIT promotes synergistic events indicative for inflammation, epithelial-to-mesenchymal-transition (EMT) or fibrosis. We measured markers of inflammation, EMT and fibrosis and investigated the role of MAP-kinases. Only concurrent but not individual exposure to OTA and CIT at nanomolar concentrations led to (i) an increase of TNF protein and mRNA, (ii) a decrease of COX-2 protein and mRNA, (iii) a decrease of E-cadherin protein and (iv) an increase of vimentin and α-SMA protein. Cell shape shifted from a cobblestone- to a spindle-like phenotype indicating EMT. Extra- and intracellular collagen III protein content was increased. Concomitant mRNA expression changes were observed for TNF, COX-2, E-cadherin and α-SMA indicating transcriptional regulation. This was not the case for vimentin and collagen III mRNA indicating posttranscriptional regulation. Inhibition of ERK 1/2 and JNK 1/2 reduced the effect on TNF but not on α-SMA mRNA indicating an involvement of these kinases. Phosphorylation of ERK1/2 was increased by CIT, OTA alone and the mycotoxin combination. In contrast, the phosphorylation of JNK1/2 was unchanged. In conclusion, nanomolar OTA and CIT act synergistically favouring nephropathic processes., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Osthole, a natural coumarin and bioactive compound isolated from the fruit of Cnidium monnieri (L.) Cusson, was reported to prevent isoprenaline-induced myocardial fibrosis in mice by inhibiting the transforming growth factor-β1 (TGF-β1) expression, but the underlying mechanism is still unclear. The aim of this study is to illuminate whether the mechanism of osthole inhibiting collagen I and III expressions is associated with Smad signaling pathway in mouse cardiac fibroblasts (CFs) treated with TGF-β1., Methods: The mouse CFs stimulated with TGF-β1 were cultured and treated with osthole 1.25-5μg/ml for 24h. The expressions of α-SMA, collagen I, collagen III, TGF-β receptor I (TβRI), Smad2/3, phospho-Smad2/3 (P-Smad2/3), Smad4 and Smad7 were detected by real-time PCR method and western blot method, respectively., Results: After treatment with TGF-β1 and osthole in CFs, the levels of α-SMA expression and collagen I and III were reduced by osthole treatment. Accordingly, the ratio of collagen I/III had a similar changing trend. Besides, the levels of TβRI, Smad2/3, P-Smad2/3 and Smad4 expressions were decreased, while the level of Smad7 expression was increased after treatment with osthole., Conclusion: The present results demonstrated that osthole could inhibit the collagen I and III expressions and their ratio in CFs treated with TGF-β1 via Smad signaling pathway, which might be one of its anti-fibrotic action mechanisms., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Sphingosine 1-phosphate (S1P) is involved in multiple pathological processes, including fibrogenesis. S1P participates in mouse liver fibrogenesis via a paracrine manner. Herein, we investigated the involvement of S1P in human liver fibrosis. Human fibrotic samples were obtained from livers of patients undergoing liver transplantation. Expression of sphingosine kinase (SphK1), collagen (Col) α1(I), Col α1(III), α-smooth muscle actin, and p-Smad2/3 was characterized by immunofluorescence, real-time RT-PCR, high-content analysis, or Western blot analysis in the fibrotic liver, human bone marrow-derived mesenchymal stem cells, and human hepatogenic profibrotic cells. The effect of SphK1 was assessed using siSphK1 or SphK-specific inhibitor. SphK1, which was expressed in human fibrotic liver myofibroblasts, could be detected in human bone marrow-derived mesenchymal stem cells or human hepatogenic profibrotic cells activated by transforming growth factor β1 (TGF-β1). TGF-β1 evoked the activation of SphK1, increased intracellular S1P, and up-regulated expression of SphK1, Col α1(I), and Col α1(III) in a TGF-β receptor-dependent manner. TGF-β1 induced expression of Col α1(I) and Col α1(III) via SphK1, which was mediated by intracellular S1P, independent of S1P receptors. TGF-β1 evoked nuclear translocation of p-Smad2 and p-Smad3 in TGF-β receptor-dependent, but SphK1-independent, manner. In conclusion, intracellular S1P plays a crucial role in the TGF-β1-induced expression of Col α1(I) and Col α1(III), which is required for human fibrosis development. S1P exerts its effects in S1P receptor-independent manner.

Objective: The management of arterial pathology in individuals with vascular Ehlers-Danlos syndrome (vEDS) remains a challenge. Here we describe the correlation between COL3A1 gene mutation type and the clinical phenotype in individuals with vEDS., Methods: Individuals with confirmed molecular diagnoses of vEDS were enrolled in a multi-institutional natural history study. Data collected included demographics, clinical and family histories, arterial pathology (aneurysm, dissection, and rupture), operative details, and autopsy reports. Individuals were classified into two cohorts by the type of COL3A1 mutations and their effect on the amount of normal collagen produced: those with mutations that lead to minimal (MIN) production (10%-15%) of normal type III collagen and those with haploinsufficiency (HI) mutations that lead to production of 50% of the normal type III collagen., Results: A cohort of 68 individuals (72%) from 56 families had arterial pathology (44% male) with 13% HI. The HI group was older at the time of their first vascular event (mean, 42 [range, 26-58] years vs 33 [range, 8-62] years; P = .016) and had a higher incidence of aortic pathology than the MIN group (56% vs 21%; P = .025). Visceral arterial pathology was seen in 43 arteries in 23 individuals in the MIN group vs only one artery in five individuals in the HI group. Emergency surgical procedures were more likely to be undertaken when vEDS diagnosis was not known (81% vs 41%; P = .005), and 81% of these procedures were open surgical repair compared with 19% endovascular repairs (P = .019). Open and endovascular repairs were equally used in the elective setting. Postoperative complications were highest when the diagnosis of vEDS was not known (62% vs 14%; P < .001) and when procedures were undertaken in an emergency setting (5% vs 55% P < .001). Mortality due to arterial complications was 0% in the HI cohort and 21% in the MIN cohort (P = .132)., Conclusions: Arterial pathology in vEDS individuals is related to the underlying COL3A1 mutation type. The arterial pathology in individuals with HI mutations occurs at later ages with a higher incidence of aortic disease compared with other COL3A1 mutation types. Molecular diagnosis is recommended because diagnosis confirmation, appropriate surveillance, and prophylactic interventions in an elective setting improve surgical outcomes., (Copyright © 2014 Society for Vascular Surgery. All rights reserved.)

Introduction: In this study, we aimed to explore the expressions of α-smooth muscle actin, collagen type I, collagen type III, and osteocalcin in the periodontal ligament (PDL) under orthodontic loading, and to investigate the effect of myofibroblasts on collagen synthesis and osteocalcin production., Methods: The teeth in the right maxillae of the rats were orthodontically loaded while the contralateral teeth remained unloaded as controls. The total 30 rats were divided into 5 groups, with each group corresponding to a treatment duration (0, 3, 5, 7, or 14 days, respectively). The expressions of α-smooth muscle actin, collagen type I, collagen type III, and osteocalcin in the tension area of the PDL over time were analyzed by immunochemistry staining. For the in-vitro study, the expressions of α-smooth muscle actin, collagen type I, collagen type III, and osteocalcin in the myofibroblasts and human osteoblast-like cells (MG63) coculture and PDL cells-MG63 coculture systems were examined by Western blot and real-time polymerase chain reaction., Results: Enhanced expression of α-smooth muscle actin, collagen type I, collagen type III, and osteocalcin in the tension area of the PDL under orthodontic loading were observed in vivo, and increased expressions of α-smooth muscle actin, collagen type I, collagen type III, and osteocalcin in the myofibroblasts-MG63 coculture system were observed compared with the controls., Conclusions: Expressions of α-smooth muscle actin, collagen type I, collagen type III, and osteocalcin are up-regulated in the PDL under orthodontic tensile loading. Myofibroblasts have a more positive effect on collagen synthesis and osteocalcin expression than do PDL cells., (Copyright © 2014 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

Palmar fibromatosis is a benign fibroproliferative tumor of unknown etiology, with a high rate of recurrence after excision. The offending cells of palmar fibromatosis are myofibroblasts and the cellular origin of other myofibroblasts has previously been reported to be the bone marrow. However, further clarification of the relationship between bone marrow precursors and palmar fibromatosis is required. Stem cells (SCs) are known to exist in various tissues, but whether SCs can be isolated from fibromatosis tissue is still unclear. The purpose of this study was to isolate and identify stem cells from human palmar fibromatosis, and to evaluate the differences in the differentiation and fibrogenic capacities of bone marrow stem cells (BMSCs) and fibromatosis-derived stem cells (FSCs). We found that FSCs had better fibrogenic differentiation potential than BMSCs, whereas BMSCs had better adipogenic and chondrogenic differentiation capacities. Treatment with transforming growth factor-β1 increased the expression of α-smooth muscle actin, and types III and I collagen significantly more in FSCs than in BMSCs. An in vivo study further confirmed the results of fibrogenesis and suggested that FSCs can recapitulate the fibromatosis nodule. In summary, their myofibroblastic differentiation both in vivo and in vitro makes FSCs a potential cell source for future applications in murine models of fibromatosis or fibrogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fibrin is a frequently used biomaterial in surgery and tissue engineering. While it has been shown that fibrin supports cellular proliferation and biosynthesis, there is a scarcity of studies focusing on the effects of fibrin concentration. The objective of this study is to assess the effect of fibrin concentrations around the physiological concentration of 3mg/ml on the behavior of ligament fibroblasts. Fibroblasts were obtained from the anterior cruciate ligaments of four pigs and seeded throughout fibrin gels of either 1, 3, or 6 mg/ml fibrin. The gels were collected at 2, 6, and 10 days for measurement of DNA and collagen content. We found that both DNA and collagen content increased significantly over time in gels made with all concentrations of fibrin. However, the increases were significantly lower in gels made with the higher concentrations of fibrin (3 and 6 mg/ml). Microscopic assessment of FITC-labeled gels showed a decrease in pore size at high fibrin concentrations, which might be a reason for the observed effect on bioactivity. To enhance cell behavior and thus clinical results fibrin applications should build on physiologic or sub-physiologic concentrations, and those with higher concentrations, such as currently available sealants, should be used cautiously., (Copyright © 2009 Elsevier B.V. All rights reserved.)

In the present study, we tested the hypothesis of the indirect (via the sympathetic nervous system (SNS)) and direct (via AT1 receptors) contributions of Angiotensin II (Ang II) on the synthesis of collagen types I and III in the left ventricle (LV) in vivo. Sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA and protein synthesis of collagen types I and III were examined by Q-RT-PCR and immunoblotting in the LV. Collagen types I and III mRNA were decreased respectively by 53% and 22% after sympathectomy and only collagen type I mRNA was increased by 52% after AT1 receptor blockade. mRNA was not changed for collagen type I but was decreased by 25% for collagen type III after double treatment. Only collagen protein type III was decreased after sympathectomy by 12%, but collagen proteins were increased respectively for types I and III by 145% and 52% after AT1 receptor blockade and by 45% and 60% after double treatment. Deducted interpretations from our experimental approach suggest that Ang II stimulates indirectly (via SNS) and inhibits directly (via AT1 receptors) the collagen type I at transcriptional and protein levels. For collagen type III, it stimulates indirectly the transcription and inhibited directly the protein level. Therefore, the Ang II regulates collagen synthesis differently through indirect and direct pathways.

Objective: Angiotensin-(1-7) [ANG-(1-7)] has been reported to attenuate neointimal formation after vascular injury and stent implantation in rats, but the mechanism remains mostly unresolved. Interestingly, the levels of circulating transforming growth factor-beta1 (TGF-beta1) after myocardial infarction were suppressed by ANG-(1-7), which suggests a possible downstream target for the anti-remodeling action of ANG-(1-7). Our study focused on the effects of ANG-(1-7) on vascular remodeling, including neointimal formation and collagen synthesis, and determining whether or not these effects were dependent upon the TGF-beta signaling pathway., Methods: Thirty-two New Zealand white rabbits underwent sham surgery or angioplasty in abdominal aorta. The animals were divided into four groups, which were sham, control, ANG-(1-7), and ANG-(1-7)+A-779. Subsequently, an osmotic minipump was implanted to deliver saline, ANG-(1-7) (576 microg kg(-1)d(-1)) or ANG-(1-7)+A-779 (576 microg kg(-1)d(-1)) for 4 weeks., Results: The ANG-(1-7) group displayed a significant reduction in neointimal thickness (207.51+/-16.70 microm vs. 448.08+/-15.30 microm, P<0.001), neointimal area (0.266+/-0.009 mm(2) vs. 0.408+/-0.002 mm(2), P<0.001), and restenosis rate (28.13+/-2.74% vs. 40.13+/-2.74%, P<0.001) when compared to the control group. ANG-(1-7) also inhibited collagen synthesis by significantly decreasing the mRNA expression of Collagen I and Collagen III (vs., Control Group: 0.2190+/-0.0036 vs. 0.3852+/-0.0212, P<0.001 and 1.1328+/-0.0554 vs. 1.7378+/-0.1164, P<0.001, respectively). Furthermore, the expression of TGF-beta1 and phosphor-Smad2 (p-Smad2) were significantly suppressed by ANG-(1-7) (vs., Control Group: 1.21+/-0.07 vs. 1.54+/-0.08, P<0.001 and 0.31+/-0.01 vs. 0.43+/-0.02, P<0.001, respectively), but no effect on p38 phosphorylation was observed. [d-Ala(7)]-ANG-(1-7) (A-779), showed a tendency to attenuate the anti-remodeling effects of ANG-(1-7)., Conclusion: ANG-(1-7) decreases the amount of vascular remodeling, including a reduction in neointimal formation and collagen synthesis, after angioplasty in rabbits. The responsible mechanism may function through the possible down-regulation of TGF-beta1 levels and inhibition of the Smad2 pathway.

Transforming growth factor (TGF)-beta1 can cause fibrosis diseases by enhancing production of collagen. However, the intracellular signaling mechanism for TGF-beta1 stimulation of this process has not been fully elucidated. The present study focused on this mechanism and the cross-talk between the MAPK and Smad pathways. Extracellular signal-regulated kinase (ERK)2 ablation by a small interfering RNA led to marked inhibition of TGF-beta1-induced collagen synthesis and enhanced phosphorylation of the Smad2 linker site in NIH/3T3 fibroblast cells. However, ERK1 ablation had minimal effects. Ablation of either ERK2 or ERK1 had no effect on the phosphorylation of the Smad2 C-terminal site. Furthermore, a Smad2 mutant with reduced phosphorylation of the Smad2 linker site inhibited TGF-beta1-induced collagen synthesis. These results indicate that ERK2, rather than ERK1, plays a predominantly positive role in TGF-beta1-induced collagen synthesis, and that ERK2 enhances collagen synthesis, at least partially, through activation of the Smad2 linker site.

Background: Myocardial fibrosis is the major factor that regulates left ventricular (LV) diastolic function. Pioglitazone, an anti-diabetic drug, is reported to improve the LV diastolic function in diabetic patients, but its influence on myocardial fibrosis has not been clarified. We evaluated the effect of pioglitazone on LV diastolic function and myocardial fibrosis in type 2 diabetic (T2DM) patients., Methods and Results: Fifteen T2DM patients were enrolled in the ON group, and the parameters were examined before and after pioglitazone administration (15-30 mg/day) for 6 months. Twenty-four T2DM patients were assigned to the OFF group, and the parameters were examined before and 6 months after cessation of pioglitazone. We measured echocardiographic parameters such as early diastolic mitral annular velocity (E') and plasma concentration of aminoterminal propeptide of procollagen type III (PIIIP), a marker of myocardial fibrosis. In the ON group, pioglitazone significantly increased E' (6.04+/-1.70 cm/s vs. 6.51+/-1.64 cm/s, p<0.01) and decreased PIIIP (0.553+/-0.056 U/ml vs. 0.517+/-0.072 U/ml, p<0.05). There was a significant negative correlation between the change in PIIIP and the change in E' (r=-0.424, p=0.046). On the other hand, E' was significantly decreased (5.69+/-1.34 cm/s vs. 4.97+/-1.20 cm/s, p<0.01) in the OFF group. PIIIP was not significantly changed in the OFF group, but there was a significant negative correlation between the change in PIIIP and the change in E' (r=-0.374, p=0.035)., Conclusion: Six months of pioglitazone administration suppressed the synthesis of type III collagen, and this was associated with improved LV diastolic function in T2DM patients. Cessation of pioglitazone weakened the suppression of the synthesis of type III collagen, which in turn seemed to be associated with worse LV diastolic function.

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF - 1mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL's and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p<0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL.

Transforming growth factor-beta1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p<0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

The atrioventricular valves of 25 dogs of different breeds and age were examined grossly and microscopically following histochemical staining and immunohistochemical labelling for collagen types I, III and VI, and for fibronectin and laminin. Foci of cartilage were identified in the tricuspid septal leaflet within the fibrosa (n=21) or spongiosa (n=3). These were further characterized as either fibrocartilage, predominantly composed of collagens I and VI, or hyaline cartilage consisting of laminin and collagens III and VI. Eighteen of the dogs were of large breed and seven of small breed. Retrospective echocardiographic findings were available from five cases and in three of these a hyperechogenic structure was identified corresponding to the cartilage focus (0.1, 1.12 and 5.63 mm(2) in size). The clinical significance and mechanism of formation of these cartilaginous foci remain undetermined, although factors such as breed, size and concurrent chronic valvular disease may be significant.

Pulp capping is a treatment where a protective agent is applied to an exposed pulp to allow the maintenance of its vitality and function. The present study analyzed the immunohistochemical expression of fibronectin and type III collagen in human dental pulps submitted to direct pulp capping with calcium hydroxide [Ca(OH)2] or the Single Bond adhesive system (SBAS). The results demonstrated that both proteins were not expressed in the SBAS group, although in the group capped with Ca(OH)2 a diffuse labeling in the extracellular matrix was initially observed, followed by a late expression in the odontoblast-like layer and beneath the dentin bridge. It seems that application of adhesive systems in direct contact with healthy pulps will not lead to expression of proteins that are believed to be essential for pulpal repair. Moreover, Ca(OH)2 showed good biocompatibility properties with the dental pulp tissue, inducing the expression of reparative molecules, and therefore remains the material of choice for the treatment of accidental pulp exposures.

Burn wound excision is considered necessary to prepare skin for grafting, and the success of graft "take" is thought to be dependent on the vascular supply to the wound. We previously showed that photocrosslinkable chitosan hydrogel containing DMEM/F12 medium (medium-Az-CH-LA) is a biocompatible and biodegradable biomaterial that promotes re-epithelialization and neovascularization. The current study was designed to determine the effect of medium-Az-CH-LA on deep dermal burn. Sixteen male Wistar rats were randomly divided into two groups that were treated with medium-Az-CH-LA (n=5) or a collagen sponge (n=5). Under anesthesia, the dorsal fur was shaved and the skin was exposed to water at 95 degrees C for 10s. After 2h, damaged tissue was removed from the fascia and dressed with medium-Az-CH-LA or a collagen sponge. Specimens were obtained after 2, 4, 6, 8, 12, 16 and 32 days for histological analysis. There was no significant difference in the time required for wound closure between the two groups, but the thickness of the granulation tissue in the medium-Az-CH-LA-treated group was greater than that in the collagen sponge-treated group. Moreover, degradation and neovascularization occurred earlier in the group treated with medium-Az-CH-LA compared with the collagen sponge-treated group. These findings suggest that early degradative and angiogenic activities of medium-Az-CH-LA may be beneficial for granulation tissue formation in deep dermal burn wounds.

The purpose of present study is to investigate the effects of two different thawing temperatures on normal human fibroblast which dealt with -20 degrees C, hoping to provide a clue for further study in reducing excessive collagen formation after cryotherapy on skin diseases in vitro, as well as in differentiation disorders. In order to elucidate its action mechanism, a programmable freezing device was developed to apply freezing temperatures on cell cultures. The effects of two different thawing temperatures on frozen fibroblast proliferation, viability, collagen synthesis and alpha smooth muscle actin (alpha-SMA) expressing were investigated. We found that compared with 37 degrees C, thawing with 20 degrees C yielded same motility. But there are significant differences in terms of the alpha-SMA expression (P<0.05) of fibroblast and collagen I, III synthesis (P<0.01) between two groups after 72h. The results suggest that comparing with slow thawing; rapid thawing cannot only keep the same cell's damage, but also can modify collagen synthesis and differentiation of fibroblasts. It may be more suitable for the cryosurgical treatment of keloids and benign skin diseases.

Background: Keloids represent a dysregulated response to cutaneous wounding that result in an excessive deposition of extracellular matrix (ECM), especially types I and III collagen. In keloid scars, the ratio of 'type I to type III collagen' varies compared to normal skin. Transforming growth factor beta (TGF-beta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblasts. However, the underlying mechanisms are poorly understood. RNA interference (RNAi) is an evolutionally conserved mechanism for repressing targeted gene expression. In mammalian cells, RNAi is mediated by small interfering RNA (siRNA). In this paper, we examined the function of Sma and Drosophila mothers against decapentaplegic homolog 3 (Smad3), recently characterized as intracellular effector of TGF-beta signalling, in keloid fibroblasts using siRNA., Methods: Dermal fibroblasts obtained from one female patient aged 21 years were maintained in Dulbecco's modified Eagle's medium. Cells (<6 passages) were treated with or without Smad3 siRNA and the expression levels of related genes were examined by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot. Statistical analysis was performed using one-way ANOVA (Dunnett correction) and the Excel 7.0 software, with significance set at p<0.05., Results: The knockdown of Smad3 expression in mRNA and protein levels was confirmed using RT-PCR and Western Blot. Compared to blank, the mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad3 by siRNA (p<0.05). The results indicate that Smad3 plays an important role in the TGF-beta induced fibrosis in keloid. Downregulation of Smad3 expression in keloid fibroblasts can significantly decrease procollagen gene expression. SiRNA targeting Smad3 was an efficient reagent to reduce ECM deposition and attenuate process of fibrosis. It could be a new promising therapeutic approach to improve skin wound healing and inhibit progression of fibrotic conditions by interrupting the TGF-beta signal pathway.

Changes in the collagenous matrix may contribute to the pathogenesis and progression of human aortic valve stenosis (AS). To evaluate the significance of collagen I and III in the pathogenesis of AS, we studied their synthesis in diseased valves. Type I and type III collagen mRNA expression and the immunohistochemical localization of the collagen antigens were studied from 36 AS and 2 normal aortic valves. The concentrations of propeptides and telopeptide structure of type I (PINP, PICP, and ICTP) and those of III collagens (PIIINP and IIINTP) were measured by radioimmunoassays in soluble tissue extracts and trypsin-solubilized calcified and non-calcified matrices of 11 AS and 24 healthy aortic valves of different ages. The synthesis of type I collagen, localized in the myofibroblasts adjacent to calcified nodules, was two- to three-fold in the AS samples compared to the controls. The proportion of collagen in the total protein fraction was 90% in the healthy valves, 50% in the non-calcified matrix, and 10% in the calcified matrix of AS valves. In the calcified valves, the ICTP content was six-fold compared to the age-matched controls and two-fold compared to the young control group. In the controls, the amount of ICTP in type I collagen decreased with age (r=-0.908, p<0.001) and was replaced by other cross-linked C-telopeptide structure. The concentration of type III collagen decreased during aging (r=-0.753, p<0.001). The decrease in total collagen content, despite the increase in type I collagen synthesis indicates an increase in collagen turnover in AS. The calcification of the aortic valves is accompanied by increased amount of ICTP in type I collagen.

Introduction: Large trials have shown that angiotensin converting enzyme inhibitor (ACE-I) therapy reduces the risk of myocardial infarction and stroke. Acute vascular events are thought to be initiated by plaque rupture. Animal models of atherosclerosis show an increase in extra cellular matrix when given ACE-I therapy. ACE-I therapy could influence collagen synthesis, one of the major constituents of the atherosclerotic cap., Methods: A nested case-control study was performed within the Huntingdon Aneurysm Screening Project. Subjects were assessed for arterial disease, drug history and smoking. Blood samples were taken for a measure of collagen synthesis, the amino-terminal propeptide of type III procollagen (PIIINP), lipid levels, iron metabolism and cotinine levels., Results: Information was available for 420 subjects. Thirty-five were taking ACE-I therapy and 385 were not. Mean serum PIIINP level was 3.5 microg/l (sd 1.3 microg/l, range: 1.7-16.5 microg/l. There was a marked increase in mean collagen turnover between subjects taking ACE-I therapy compared to those not. Mean PIIINP level for cases and controls was 4.26 microg/l (95% CI: 3.73-4.79 microg/l) versus 3.61 microg/l (95% CI: 3.48-3.75 microg/l). No differences were found for patients taking other antihypertensive drugs. After adjusting for age, weight, height, lipid levels and ferritin, PIIINP levels remained significantly higher in cases than controls: 4.14 microg/l (95% CI: 3.72-4.57 microg/l) versus 3.62 microg/l (95% CI: 3.49-3.75 microg/l) (P-value 0.02)., Discussion: These results suggest that ACE-I therapy up-regulates collagen synthesis, and could improve plaque stabilisation. This may provide an explanation for the decrease in acute vascular events observed in patients on ACE-I therapy.

The purpose of this study was to examine whether mechanical tensile forces affect estrogen regulation of collagen synthesis of anterior cruciate ligament fibroblasts at the mRNA level. Estrogen was studied at three physiologic levels, 10(-11), 10(-10), and 10(-9)M. The results revealed that estrogen alone stimulated Type I and III collagen synthesis at the mRNA level, and application of mechanical force decreased the expression of collagen Type I and III genes at all tested estrogen levels. These findings suggest that estrogen may directly regulate ligament structure and function by alteration of Type I and III collagen synthesis. This regulation is dependent on mechanical loading.

Objective: Ovarian surface epithelium (OSE), the precursor of the epithelial ovarian carcinomas, has limited growth potential in culture. Epidermal growth factor+hydrocortisone (EGF+HC) enhances its growth but induces epitheliomesenchymal transition (EMT). This study was undertaken to define the effects of EGF+HC and their reversibility, to optimize growth-promoting media, and to relate OSE phenotypes in vitro to physiologic states in vivo., Methods: OSE was cultured in media 199/MDCB105 or EBM (Clonetics) with 2% or 10% fetal bovine serum with or without 10 ng/mL EGF, 1.0 microg/mL HC, and 1.0 microg/mL bovine brain extract. Growth rates and growth potentials (population doublings [PD] to senescence) were defined, and growth patterns and expression of keratin and collagen types III and IV were compared with the ovarian cancer cell lines OVCAR3 and SKOV3., Results: EGF+HC increased growth potentials from 12-14 PD to 40-42 PD and reduced PD time from 53 hours to 20 hours. Without EGF+HC, OSE cells remained uniformly epithelial. EGF+HC induced EMT (mesenchymal shapes, reduced keratin, and production of collagenous extracellular matrix), but the EMT response varied greatly among OSE from different women. EMT was reversed over 1-2 weeks by subculture into EGF+HC-free medium in passage 1, but inconsistently thereafter. EGF+HC had no effect on the differentiation of ovarian carcinoma lines., Conclusion: The phenotype of intact OSE in vivo is most closely reproduced in media without EGF+HC. EGF+HC enhances growth but initiates EMT, which likely mimics a repair response. Variations in EGF+HC-induced phenotypes point to the existence of OSE subpopulations with differing responsiveness to growth factors or steroids, which may relate to their susceptibility to malignant transformation.

Background/aims: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors called statins, have besides their cholesterol-lowering function, therapeutic value in conditions such as neo-angiogenesis and atherosclerosis. We investigated the effect of two statins on the proliferation rate and protein steady state levels of hepatic stellate cells (HSC)., Methods: Cellular DNA synthesis under the influence of statins and/or platelet derived growth factor (PDGF) and mevalonate was evaluated by measuring BrdU incorporation. Synthesis of collagens type I, III, IV and fibronectin was quantified by ELISA. Additionally, we examined the influence of simvastatin on isoprenylation of Ras and RhoA proteins., Results: Lovastatin and simvastatin induced a dose-dependent inhibition of the proliferation rate of HSC. Subsequent addition of PDGF and/or mevalonate, after long-term exposure of simvastatin to HSC, did not reverse simvastatins' antiproliferative effect. Lovastatin and simvastatin reduced the protein steady state level of collagens type I (-40%), III (-45%) and IV (-27%). Membrane bound Ras steady state levels decreased under the influence of simvastatin. Membrane bound RhoA remained unaltered, whereas, cytosolic RhoA protein level was strongly reduced., Conclusions: Our data showed that lovastatin and simvastatin inhibited HSC proliferation and collagen steady state levels by mechanisms independent of their lipid reducing activities.

Introduction: The aim of this study was to analyse the distribution pattern of extracellular matrix proteins in the irradiated and non-irradiated rat submandibular salivary gland in order to provide a more detailed profile of the radiation injury following radiotherapy of the head and neck., Material and Methods: External X-ray exposure, restricted to the left skull base and neck region, was performed in 60 female Wistar rats, fractionated to daily applications of 2 Gy, up to total dosages of 20, 40 or 60 Gy. Both submandibular glands were excised after supravital anaesthesia 6 months or 1 year after completion of the irradiation. Spatial and temporal patterns of extracellular matrix proteins were investigated histologically and immunohistochemically., Results: The polyclonal anti-human antisera used, identified the same antigens in rat tissue as in human tissues. The alterations in staining patterns and staining intensities between irradiated and non-irradiated salivary glands showed statistically significant differences. Different structures in irradiated glands reacted with different intensities, e.g. nerve tissue and the basement membranes of excretory ducts were intensely laminin-positive, fibronectin was predominantly found around the excretory ducts with transition to the interstitial tissues., Conclusion: Irradiation leads to statistically significant differences in the amount and composition of the extracellular matrix in salivary glands. The amount of extracellular matrix proteins in irradiated glands is dose-dependent. The higher the dosage the more extracellular matrix can be expected. Consecutively, total dosage is associated with greater loss of acini. Scatter effects of irradiation have also to be recognized. Immunohistochemical studies on salivary glands have to consider the pretreatment status, in particular those studies that investigate degenerative changes., (Copyright 2002 European Association for Cranio-Maxillofacial Surgery. Published by Elsevier Science Ltd.)