mRNA Expression Levels Of Collagen Research Articles

Effect of electroacupuncture on ventricular structure and function in rats with myocardial ischemia-reperfusion injury.

To observe the effect of electroacupuncture (EA) on changes of ventricular structure and function in rats with myocardial ischemia-reperfusion injury (MIRI), so as to explore its potential mechanisms underlying improvement of ventricular remodeling after MIRI. Forty male SD rats were randomly divided into 4 groups：sham operation group, model group, EA group and medication (sacubactril valsartan, LCZ696) group, with 10 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery and reperfusion. EA (2 Hz/100 Hz, 2 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once every other day for 21 d. Rats of the medication group received gavage of LCZ696 (60 mg·kg-1·d-1). After the intervention, echocardiography was used to detect the ejection fraction (EF) and fractional shortening (FS) of the left ventricle, and the contents of serum tumor necrosis factor-α(TNF-α), vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1) were assayed by enzyme-linked immunosorbent assay. The pathological changes of myocardial tissue were observed after HE staining. The Masson staining was used to evaluate the myocardial collagen deposition and myocardial fibrosis. The mRNA expression levels of collagen Ⅰ and Ⅲ and connective tissue growth factor (CTGF) in the myocardial tissue were detected by quantitative real-time PCR, and the expression levels of IL-1β and IL-18 were detected by Western blot. In contrast to the sham operation group, the EF and FS levels of the left ventricle were ob-viously decreased (P<0.001), while the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, the expression levels of IL-1β and IL-18 were significantly increased (P<0.001, P<0.000 1, P<0.05, P<0.01) in the model group. Compared with the model group, the EF and FS levels were remarkably increased (P<0.01), whereas the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, and the expression levels of IL-1β and IL-18 were significantly down-regulated (P<0.001, P<0.01, P<0.05) in both the medication and EA groups. No significant differences were found between the EA and medication groups in all the indexes mentioned above. EA can improve the left-ventricular fibrosis and function, delay or reverse ventricular remodeling in MIRI rats, which may be related to its functions in down-regulating myocardial inflammatory response and mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF.

Mouth breathing induces condylar remodelling and chondrocyte apoptosis via both the extrinsic and mitochondrial pathways in male adolescent rats

The prevalence of mouth breathing is high in children and adolescents. It causes various changes to the respiratory tract and, consequently, craniofacial growth deformities. However, the underlying mechanisms contributing to these effects are obscure. Herein, we aimed to study the effects of mouth breathing on chondrocyte proliferation and death in the condylar cartilage and morphological changes in the mandible and condyle. Additionally, we aimed to elucidate the mechanisms underlying chondrocyte apoptosis and investigate any variations in the related pathways. Subchondral bone resorption and decreased condylar cartilage thickness were observed in mouth-breathing rats; further, mRNA expression levels of Collagen II, Aggrecan, and Sox 9 were lower in the mouth breathing group, while those of matrix metalloproteinase 9 increased. TdT-mediated dUTP nick end labelling staining and immunohistochemistry analyses showed that apoptosis occurred in the proliferative and hypertrophic layers of cartilage in the mouth breathing group. TNF, BAX, cytochrome c, and cleaved-caspase-3 were highly expressed in the condylar cartilage of the mouth-breathing rats. These results suggest that mouth breathing leads to subchondral bone resorption, cartilage layer thinning, and cartilage matrix destruction, inducing chondrocyte apoptosis via both the extrinsic and mitochondrial apoptosis pathways.

Inhibiting S100A8/A9 attenuates airway obstruction in a mouse model of heterotopic tracheal transplantation

Although bronchiolitis obliterans syndrome (BOS) is a major cause of death after lung transplantation, an effective drug therapy for BOS has not yet developed. Here, we assessed the effectiveness of a neutralizing anti-S100 calcium binding protein (S100) A8/A9 antibody against BOS. A murine model of heterotopic tracheal transplantation was used. Mice were intraperitoneally administered control IgG or the S100A8/A9 antibody on day 0 and twice per week until they were sacrificed. Tissue sections were used to evaluate the obstruction ratio, epithelium-preservation ratio, α-smooth muscle actin (SMA)-positive myofibroblast infiltration, and luminal cell death. Quantitative reverse transcriptase-polymerase chain reaction analysis was performed to analyze the mRNA-expression levels of collagen, inflammatory cytokines, and chemokines on days 7, 14, and 21. The anti-S100A8/A9 antibody significantly improved the obstruction ratio and epithelium-preservation ratio, with less α-SMA-positive myofibroblast infiltration compared to the control group. Antibody treatment reduced the type-III collagen: type-I collagen gene-expression ratio. The antibody also significantly suppressed the number of dead cells in the graft lumen. The expression levels of tumor growth factor β1 and C–C motif chemokine 2 on day 21, but not those of interleukin-1β, interleukin-6, and tumor necrosis factor α, were significantly suppressed by S100A8/A9 antibody treatment. These findings suggest that S100A8/A9 may be a potential therapeutic target for BOS after lung transplantation.

A Simplified Murine Model to Imitate Flexor Tendon Adhesion Formation without Suture.

Peritendinous adhesion (PA) around tendons are daunting challenges for hand surgeons. Tenotomy with various sutures are considered classical tendon repair models (TRM) of tendon adhesion as well as tendon healing. However, potential biomimetic therapies such as anti-adhesion barriers and artificial tendon sheaths to avoid recurrence of PA are sometimes tested in these models without considering tendon healing. Thus, our aim is to create a simplified model without sutures in this study by using three 6 mm longitudinal and parallel incisions called the longitudinal incision model (LCM) in the murine flexor tendon. We found that the adhesion score of LCM has no significant difference to that in TRM. The range of motion (ROM) reveals similar adhesion formation in both TRM and LCM groups. Moreover, mRNA expression levels of collagen I and III in LCM shows no significant difference to that in TRM. The breaking force and stiffness of LCM were significantly higher than that of TRM. Therefore, LCM can imitate flexor tendon adhesion formation without sutures compared to TRM, without significant side effects on biomechanics with an easy operation.

Melittin and diclofenac synergistically promote wound healing in a pathway involving TGF-β1

A dysregulation of the wound healing process can lead to the development of various intractable ulcers or excessive scar formation. Therefore it is essential to identify novel pharmacological strategies to promote wound healing and restore the mechanical integrity of injured tissue. The goal of the present study was to formulate a nano-complex containing melittin (MEL) and diclofenac (DCL) with the aim to evaluate their synergism and preclinical efficacy in an in vivo model of acute wound. After its preparation and characterization, the therapeutic potential of the combined nano-complexes was evaluated. MEL-DCL nano-complexes exhibited better regenerated epithelium, keratinization, epidermal proliferation, and granulation tissue formation, which in turn showed better wound healing activity compared to MEL, DCL, or positive control. The nano-complexes also showed significantly enhanced antioxidant activity. Treatment of wounded skin with MEL-DCL nano-complexes showed significant reduction of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) pro-inflammatory markers that was paralleled by a substantial increase in mRNA expression levels of collagen, type I, alpha 1 (Col1A1) and collagen, type IV, alpha 1 (Col4A1), and hydroxyproline content as compared to individual drugs. Additionally, MEL-DCL nano-complexes were able to significantly increase hypoxia-inducible factor 1-alpha (HIF-1α) and transforming growth factor beta 1 (TGF-β1) proteins expression compared to single drugs or negative control group. SB431542, a selective inhibitor of type-1 TGF-β receptor, significantly prevented in our in vitro assay the wound healing process induced by the MEL-DCL nano-complexes, suggesting a key role of TGF-β1 in the wound closure. In conclusion, the nano-complex of MEL-DCL represents a novel pharmacological tool that can be topically applied to improve wound healing.

Inhibiting miR‑1 attenuates pulmonary arterial hypertension in rats.

MicroRNAs (miRs) are reported to serve key roles in pulmonary arterial hypertension (PAH). miR-1 has been found in cardiovascular diseases. The present study aimed to determine whether the knockdown of miR-1 could inhibit right ventricle (RV) remodeling and thereby control PAH in model rats. PAH model rats were established by exposing rats to hypoxia, while cardiac fibroblasts (CFs) obtained from PAH model rats were treated with hypoxia to establish an in vitro model, and RV remodeling was evaluated by Masson staining and the levels of collagen I, collagen III, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) evaluated by western blotting or reverse transcription-quantitative PCR. The results revealed that the expression levels of miR-1 were upregulated in the RV of PAH model rats induced with hypoxia and in the CFs treated with hypoxia. The mean pulmonary arterial pressure, RV systolic pressure, RV/(left ventricle + interventricular septum) and RV/tibia length were increased in PAH rats; however, the increases in all parameters were subsequently reversed by transfection with a miR-1 antagomiR in PAH model rats. The transfection with the miR-1 antagomiR inhibited the development of RV fibrosis and downregulated the mRNA expression levels of collagen I, collagen III, α-SMA and CTGF in the RV tissue of PAH model rats. The upregulation of collagen I, collagen III, α-SMA and CTGF expression levels in hypoxia-treated CFs was also subsequently reversed by miR-1 antagomiR transfection. The expression levels of collagen I, collagen III, α-SMA and CTGF were also upregulated in the CFs obtained from PAH model rats, and these increases were attenuated by miR-1 antagomiR transfection. The expression levels of phosphorylated (p)-PI3K and p-AKT were also upregulated in hypoxia-treated CFs, and these increases were also inhibited by transfection with miR-1 antagomiR. In conclusion, these results indicated that inhibiting miR-1 may attenuate RV hypertrophy and fibrosis in PAH model rats, a mechanism that may involve the PI3K/AKT signaling pathway.

Differences between the root and horn cells of the human medial meniscus from the osteoarthritic knee in cellular characteristics and responses to mechanical stress

BackgroundMany histological, mechanical, and clinical studies have been performed on the medial meniscus posterior root attachment, as it often tears in patients with osteoarthritic knee. Medial meniscal root repair is recommended in clinical situations; however, to date, no studies have examined the differences between meniscal root and horn cells. The aim of this study was, therefore, to investigate the morphology, reaction to cyclic tensile strain, and gene expression levels of medial meniscal root and horn cells. MethodsMeniscal samples were obtained from the medial knee compartments of 10 patients with osteoarthritis who underwent total knee arthroplasty. Root and horn cells were cultured in Dulbecco's modified Eagle's medium without enzymes. The morphology, distribution, and proliferation of medial meniscal root and horn cells, as well as the gene and protein expression levels of Sry-type HMG box 9 and type II collagen, were determined after cyclic tensile strain treatment. ResultsHorn cells had a triangular morphology, whereas root cells were fibroblast-like. The number of horn cells positive for Sry-type HMG box 9 and type II collagen was considerably higher than that of root cells. Although root and horn cells showed similar levels of proliferation after 48, 72, or 96 h of culture, more horn cells than root cells were lost following a 2-h treatment with 5% and 10% cyclic tensile. Sry-type HMG box 9 and α1(II) collagen mRNA expression levels were significantly enhanced in both cells after 2- and 4-h cyclic tensile strain (5%) treatment. ConclusionsMedial meniscal root and horn cells have distinct morphologies, reactions to mechanical stress, and cellular phenotypes. Our results suggest that physiological tensile strain is important to activate extracellular matrix production in horn cells.

The 3D-Printed PLGA Scaffolds Loaded with Bone Marrow-Derived Mesenchymal Stem Cells Augment the Healing of Rotator Cuff Repair in the Rabbits

The healing of tendon–bone in the rotator cuff is featured by the formation of the scar tissues in the interface after repair. This study aimed to determine if the 3D-printed poly lactic-co-glycolic acid (PLGA) scaffolds loaded with bone marrow-derived mesenchymal stem cells (BMSCs) could augment the rotator cuff repair in the rabbits. PLGA scaffolds were generated by the 3D-printed technology; Cell Counting Kit-8 assay evaluated the proliferation of BMSCs; the mRNA and protein expression levels were assessed by quantitative real-time polymerase chain reaction and western blot, respectively; immunohistology evaluated the rotator cuff repair; biomechanical characteristics of the repaired tissues were also assessed. 3D-printed PLGA scaffolds showed good biocompatibility without affecting the proliferative ability of BMSCs. BMSCs–PLGA scaffolds implantation enhanced the cell infiltration into the tendon-bone injunction at 4 weeks after implantation and improved the histology score in the tendon tissues after implantation. The mRNA expression levels of collagen I, III, tenascin, and biglycan were significantly higher in the scaffolds + BMSCs group at 4 weeks post-implantation than that in the scaffolds group. At 8 and 12 weeks after implantation, the biglycan mRNA expression level in the BMSCs–PLGA scaffolds group was significantly lower than that in the scaffolds group. BMSCs–PLGA scaffolds implantation enhanced collagen formation and increased collagen dimeter in the tendon–bone interface. The biomechanical analysis showed that BMSCs–PLGA scaffolds implantation improved the biomechanical properties of the regenerated tendon. The combination of 3D-printed PLGA scaffolds with BMSCs can augment the tendon–bone healing in the rabbit rotator cuff repair model.

Effect of aging on the tendon structure and tendon-associated gene expression in mouse foot flexor tendon.

To evaluate the biological changes in tendons during the aging process, the present study examined the effect of aging on the tendon structure, distribution of collagen types I and III, and expression of tendon-associated genes, using flexor tendons in a mouse model. Histological assessment of the tendon structure and distribution of collagen types I and III were performed, and the expression of tendon-associated genes was evaluated in flexor digitorium longus tendons of young (8 weeks) and aged (78 weeks) female C57BL/6 mice. The results indicated that the Soslowsky score, based on the analysis of cellularity, fibroblastic changes, and collagen fiber orientation and disruption, was significantly increased, or worsened, in the tendons of the aged group compared with those in the young group. Furthermore, in the aged group, the distribution of type I collagen was decreased and the distribution of type III collagen was relatively increased compared with the young group. Finally, the mRNA expression levels of collagen (type I and type III) and tenogenic markers (Mohawk homeobox, tenomodulin and scleraxis BHLH transcription factor) were significantly decreased in the aged group compared with the young group. The present observations demonstrated that the structure of the tendons, distribution of types I and III collagen and the expression of tendon-associated genes were modulated by aging in the flexor tendon, and that these changes may contribute to the degeneration of tendons in tendinopathy.

TGF-β3 improves bone mesenchymal stem cells toward chondrogenic differentiation under hypoxia environment

Objective: To investigate the impact of TGF-β3 on the chondrogenesis of bone marrow mesenchymal stem cells (BM-MSCs) under hypoxia environment. Methods: BM-MSCs were obtained from SD rat tibias and femora and cultured with whole bone marrow adherent method. Cell surface antigens were analyzed by flow cytometry and the multiple-directional differentiation capabilities were detected with special differentiation agents to affirm the reality of BM-MSCs. Under normoxia or hypoxia condition, BM-MSCs were induced with TGF-β3 or not. Then, alcian blue and immunofluorescence staining were performed to evaluate the expression level of aggrecan, collagen Ⅱ. qRT-PCR analysis were performed to analyze the expression of aggrecan, collagen Ⅱ and collagen Ⅹ. qRT-PCR and Western blot analysis was performed to detect the mRNA and protein level of HIF-1α, collagenⅡ and β-catenin. Results: BM-MSCs were fibroblast-like shape and had ablities of osteogeic, adipogenic and chondrogenic differentiation, with the expression of CD(29, )CD(44) and CD(90) but not CD(45). Alcian blue and immunofluorescence staining showed that BM-MSCs strongly expressed the aggrecan and collagen Ⅱ with the presence of TGF-β3 under hypoxia condition. qRT-PCR analysis showed the mRNA expression levels of collagen Ⅱ, aggrecan and collagen Ⅹ were up-regulated at 2.46, 2.20 and 1.80 folds, comparing with control group (all P<0.05). Western blot analysis showed that the protein levels of HIF-1α, collagenⅡ in BM-MSCs were up-regulated with the presence of TGF-β3 under hypoxia condition, but β-catenin level was down-regulated. Conclusion: TGF-β3 promotes the chondrogenic differentiation ability of BM-MSCs under hypoxia condition, which may be relative with the inhibition of Wnt/β-catenin signaling pathway.

Anti-fibrotic effects of polygonum plebeium r.br. in CCl4-induced hepatic damage and fibrosis in rats

Introduction: Asian countries have the highest burden of liver diseases. Polygonum plebeium (P. plebeium), a.k.a. the common knotweed, is a species of plant in the knotwood family that can act as a blood purifier and has been widely used in Pakistan to cure liver disorders like jaundice and hepatitis. The plant is also used in the treatment of pneumonia, bowel complaints, diarrhea, dysentery, eczema and ring worms. Tannins, flavonoids, saponins and alkaloids are the major components of P. plebeium. Since its use in folk medicine in Pakistan, there has been little scientific evidence or information on it. Therefore, this study was aimed at investigating the anti-fibrotic effects of P. plebeium in carbon tetrachloride (CCl4)-induced hepatic toxicity and fibrosis.&#x0D; Methods: The extracts of whole plant of P. plebeium were prepared and administered by oral gavage in rats. Liver fibrosis was induced by intraperitoneal (i.p.) administration of CCl4. To evaluate the hepatoprotective activity of P. plebeium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (γGT) levels were quantified. Histological evaluation of liver tissue revealed tissue necrosis and extracellular matrix deposition. Real-time PCR was done to evaluate mRNA expression of genes related to liver fibrosis.&#x0D; Results: The groups treated with P. plebeium extract showed an ablation in liver damage; the elevated enzyme levels of ALT, AST and γGT were decreased. Treatment with P. plebeium extract treatment restored the CCl4-induced tissue fibrosis by significantly suppressing alpha-smooth muscle actin (α-SMA), tumor growth factor beta (TGF-β) and collagen mRNA expression levels. Histology of liver sections also showed that the CCl4-induced fibrosis was improved in the treatment groups.&#x0D; Conclusion: Polygonum plebeium has therapeutic potential and can be used for preventing fibrosis in inflammatory liver disease.

P‑selectin increases angiotensin II‑induced cardiac inflammation and fibrosis via platelet activation.

Platelet activation is important in hypertension-induced cardiac inflammation and fibrosis. P-selectin expression significantly (P<0.05) increases when platelets are activated during hypertension. Although P-selectin recruits leukocytes to sites of inflammation, the role of P-selectin in cardiac inflammation and fibrosis remains to be elucidated. The present study aimed to investigate whether platelet-derived P-selectin promotes hypertensive cardiac inflammation and fibrosis. P-selectin knockout (P-sel KO) mice and wild-type (WT) C57BL/6 littermates were infused with angiotensin II (Ang II) at 1,500 ng/kg/min for 7 days and then cross-transplanted with platelets originating from either WT or P-sel KO mice. P-selectin expression was increased in the myocardium and plasma of hypertensive mice, and the P-sel KO mice exhibited significantly (P<0.05) reduced cardiac fibrosis. The fibrotic areas were markedly smaller in the hearts of P-sel KO mice compared with WT mice, as assessed by Masson's trichrome staining. In addition, α-smooth muscle actin and transforming growth factor β1 (TGF-β1) expression levels were decreased in the P-sel KO mice, as assessed by immunohistochemistry. Following platelet transplantation into P-sel KO mice, the number of Mac-2 (galectin-3)- and TGF-β1-positive cells was increased in mice that received WT platelets compared with those that received P-sel KO platelets, and the mRNA expression levels of collagen I and TGF-β1 were also increased. The results from the present study suggest that activated platelets secrete P-selectin to promote cardiac inflammation and fibrosis in Ang II-induced hypertension.

The effect of capacitively coupled (CC) electrical stimulation on human disc nucleus pulposus cells and the relationship between CC and BMP-7.

An in vitro study using human intervertebral disc nucleus pulposus cells to evaluate the effects of CC stimulation on disc-matrix macromolecule production. Nucleus pulposus cells were cultured in alginate beads and treated with CC stimulation. The effect of BMP on CC stimulation of the cells was evaluated by applying a BMP blocker (noggin) or by applying additional BMP-7 to the culture. The mRNA levels of the disc extracellular matrix genes (collagen I, II, aggrecan) and BMPs were measured by real-time PCR. The protein levels of aggrecan, collagen II, and BMPs were determined by ELISAs and Western blots. Sulfated glycosaminoglycan (sGAG) content was assayed using the DMMB method. (1) CC stimulation upregulates the production of the disc-matrix macromolecular components: sGAG, aggrecan and collagen II; (2) CC stimulation increases the ratio of mRNA expression levels of collagen II to collagen I; (3) CC stimulation induces the expression of endogenous BMP-4 and BMP-7; (4) inhibition of BMP activity (using noggin) reduces CC-mediated upregulation of aggrecan and collagen II; (5) CC and BMP-7 act in synergy to increase the upregulation of disc-matrix macromolecules. CC stimulation upregulates the production of the intervertebral disc-matrix macromolecules aggrecan, collagen II, and sGAG by a mechanism involving BMPs. CC stimulation acts in synergy with BMP-7 to increase the upregulation of these disc-matrix macromolecules.

Asiatic acid inhibits left ventricular remodeling and improves cardiac function in a rat model of myocardial infarction.

Left ventricular remodeling results in cardiac dysfunction and accounts for the majority of the morbidity and mortality following myocardial infarction (MI). The aim of the present study was to investigate the effect of asiatic acid (AA) on cardiac function and left ventricular remodeling in a rat model of MI and explore the underlying mechanisms. Rats were subjected to coronary artery ligation to model MI and orally treated with AA. After 4 weeks, cardiac function was assessed by echocardiography. Cardiomyocyte cross-sectional area was recorded, and the expression levels of a number of inflammatory cytokines were detected using ELISA. The degree of interstitial fibrosis was determined by evaluating the mRNA expression levels of collagen II and III. Western blot analysis was performed to detect the expression levels of total and phosphorylated p38 MAPK and ERK1/2, to investigate whether they are involved in the mechanism underlying the effect of AA on the heart. Rats subjected to MI displayed significantly impaired cardiac function compared with those subjected to a sham procedure, while this change was reversed by treatment with AA. Furthermore, AA markedly inhibited cardiac hypertrophy, reduced the mRNA expression levels of inflammatory cytokines and decreased interstitial fibrosis in the infarct border zone of MI model rats compared with those in vehicle-treated MI model rats. Furthermore, the phosphorylation of p38 MAPK and ERK1/2 was blocked by AA in the MI rats but not in the sham rats. In summary, AA treatment preserved cardiac function and inhibited left ventricular remodeling, potentially by blocking the phosphorylation of p38 MAPK and ERK1/2 in the infarct border zone of the ischemic myocardium, indicating that AA may be a novel candidate for development as a therapy for MI.

Silica promotes the transdifferentiation of rat circulating fibrocytes in vitro.

To investigate the effects of silica on circulating fibrocytes (cFbs), the present study established a primary culture model of rat alveolar macrophages and cFbs in vitro. Macrophages were treated with free silica, and their supernatant was used to stimulate cFbs. The mRNA expression levels of collagen I, collagen III and α-smooth muscle actin (SMA) in cFbs were analyzed by reverse transcription-quantitative polymerase chain reaction. The intracellular and extracellular protein expression levels of collagen I, collagen III and α-SMA were detected by ELISA and immunofluorescence staining. The results indicated that in the cell model, the free silica effectively increased the protein and mRNA expression levels of collagen-I, collagen-III and α-SMA. The free silica significantly promoted the transdifferentiation of cFbs into myofibroblasts in a dose-and time-dependent manner.

Role of microRNA-29b in the ochratoxin A-induced enhanced collagen formation in human kidney cells.

Ochratoxin A (OTA) is an ubiquitous mycotoxin suspected to cause fibrotic kidney diseases. The involvement of mircoRNAs in these processes is unknown. Here, we investigated the role of the anti-fibrotic miR-29b in OTA-induced alterations of cellular collagen homeostasis. OTA exposure of human embryonic kidney cells (HEK293) cells led to an increase of collagen I, III and IV protein amounts without changes in collagen mRNA expression levels, indicating post-transcriptionally mediated mechanisms potentially involving microRNAs and 3'UTRs of collagen mRNAs. This was confirmed by enhanced luciferase activity of a collagen1A1-3'UTR reporter plasmid after OTA exposure. OTA also enhanced the luciferase activity of a reporter plasmid containing the seed region of miR-29b showing that OTA diminishes miR-29b action. Additionally, OTA induced an altered intracellular distribution of miR-29b leading to decreased cytoplasmic abundance of miR-29b. Abundantly added miR-29b (miR-29b clamp) completely prevented OTA-induced collagen formation. In summary, we show that OTA has the potential to initiate or support the development of fibrotic kidney diseases by involving post-transcriptional regulation mechanisms comprising miR-29b. OTA reduces the impact of miR-29b and thus enhances collagen protein expression. These findings allow a new perspective on how the exposure to nanomolar OTA concentrations can lead to fibrotic tissue alterations.

Substance P Modulates Colitis-Asscociated Fibrosis

Substance P (SP) and the neurokinin-1 receptor (NK-1R) are involved in the development of colitis and mucosal healing after colonic inflammation. We studied whether SP modulates colonic fibrosis by using a chronic model of trinitrobenzenesulfonic acid (TNBS)-induced colitis in wild-type (WT) and NK-1R-deficient (NK-1R KD) mice. We found increased mRNA expression levels of collagen, vimentin, and the fibrogenic factors transforming growth factor β1 and insulin-like growth factor 1 in the chronically inflamed colons of WT mice treated with repeated intracolonic TNBS administrations. Fibrosis in TNBS-treated mice was also evident immunohistochemically by collagen deposition in the colon. Treatment of TNBS-exposed WT mice with the NK-1R antagonist CJ-12255 reduced colonic inflammation, colonic fibrosis, fibroblast accumulation, and expression levels of the fibrogenic factors. NK-1R knockout mice chronically exposed to TNBS had similar colonic inflammation compared with WT, but reduced colonic fibrosis, fibroblast accumulation, and expression levels of fibrogenic factors. Immunohistochemical staining also showed co-localization of NK-1R with fibroblasts in inflamed colons of mice and in colonic mucosa of patients with Crohn's disease. Exposure of human colonic CCD-18Co fibroblasts to SP (10 nmol/L) increased cell migration. SP stimulated collagen synthesis in CCD-18Co fibroblasts in the presence of transforming growth factor β1 and insulin-like growth factor 1, and this effect was reduced by Akt inhibition. Thus, SP, via NK-1R, promotes intestinal fibrogenesis after chronic colitis by stimulating fibrotic responses in fibroblasts.

Influence of recombinant transforming growth factor-beta3 on collagen synthesis and deposition: experiment with rat cell model of liver fibrosis

To investigate the influence of recombinant transforming growth factor-beta3 (TGF-beta3) on collagen synthesis and deposition. Plasmids pcDNA3.1 (+)-TGF-beta3 and pcDNA3.1 (+)-TGF-beta1 were constructed. Rat hepatic stellate cells (HSCs) of the strain HSC-T6 were cultured as cell model of fibrosis and divided into 4 groups: blank control group, pcDNA3.1-enhanced green fluorescent protein (EGFP)-transfected group (negative control group), pcDNA3.1 (+)-TGF-beta1 transfected group, and pcDNA3.1 (+)-TGF-beta3 transfected group. A positive cell clone stably and highly expressing TGF-beta1 was established after being screened by G418 medium. pcDNA3.1 (+)-TGF-beta3 was transfected into the positive clone. Real-time PCR was used to detect the mRNA expression of TGF-beta3. Western blotting was used to detect the protein expression of TGF-beta1, collagen I, matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metallo-proteinase (TIMP)-1. There was no significant difference in the TGF-beta1 mRNA expression between the TGF-beta1 positive clone group and the TGF-beta3 transfected group. The mRNA and protein expression levels of TGF-beta1, collagen I, MMP-2, and TIMP-1 of the TGF-beta1 positive clone group were all significantly higher than those of the blank control, negative control groups (all P < 0.05), and the MMP-9 mRNA expression of the TGF-beta1 positive clone group was significantly lower than those of the blank control and negative control groups (all P < 0.05); and the mRNA and protein expression levels of MMP-9 of the TGF-beta1 positive clone group were significantly lower than those of the blank control and negative control groups (all P < 0.05). The TGF-beta1 and MMP-2 mRNA expression levels of the TGF-beta3 transfected group were not significantly different from those of the positive clone group (all P > 0.05), the mRNA expression levels of collagen I and TIMP-1 of the TGF-beta3 transfected group were significantly lower than those of the positive clone group (both P < 0.05), and the mRNA expression level of MMP-9 of the TGF-beta3 transfected group was significantly higher than that of the positive clone group (P < 0.05). The protein expression levels of TGF-beta1, collagen I, and TIMP-1 of the TGF-beta3 transfected group were all significantly lower than those of the positive clone group (P < 0.05), the protein expression level of MMP-9 of the TGF-beta3 transfected group was significantly higher than that of the positive clone group (P < 0.05), and the protein expression level of MMP-2 of the TGF-beta3 transfected group was not significantly different from that of the positive clone group. Recombinant TGF-beta3 eukaryotic expression vector reduces the synthesis of collagen and inhibits the collagen deposition by adjusting the expression of matrix metalloproteinases and their inhibitors.

Biological characterisation of vascular grafts cultured in a bioreactor

In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.

Renoprotective effect of long-term combined treatment with adrenomedullin and omapatrilat in hypertensive rats

Previous studies demonstrated that adrenomedullin (AM) is metabolized by neutral endopeptidases and that the renal effect of AM is augmented by the inhibition of neutral endopeptidases. We have recently shown that the long-term administration of AM has renoprotective effects. This study assessed the chronic renoprotective effects of AM combined with a vasopeptidase inhibitor in hypertensive rats and attempted to elucidate the mechanism involved. We studied the following four groups: control Dahl salt-resistant (DR) rats, untreated Dahl salt-sensitive (DS) rats, omapatrilat (35 mg/kg per day)-treated DS rats; and human AM (500 ng/h) plus omapatrilat-treated DS rats. After 7 weeks' treatment, blood pressure, renal function, neurohumoral factors, gene expression levels, and histological findings were examined. DS rats were characterized by increased blood pressure, decreased renal function, abnormal histological findings, and increased gene expression of collagen I and III, transforming growth factor beta (TGF-beta), and NADPH oxidase subunits (p40phox, p47phox, and gp91phox) in the renal cortex compared with DR rats. Compared with DS rats, omapatrilat significantly decreased systolic blood pressure (-26 mmHg), improved renal function, histological findings, and messenger RNA expression levels of collagen I, collagen III, and TGF-beta. Combined treatment with omapatrilat and AM further improved renal function, histological findings, and mRNA expression levels of collagen I, collagen III, and TGF-beta, without a further reduction in blood pressure. Only combined treatment decreased mRNA levels of p40phox, p47phox, and gp91phox. There were no differences in plasma AM or atrial natriuretic peptide levels among three DS groups. Our results suggest that combined treatment with omapatrilat and AM provides additional renoprotective effects independent of blood pressure-lowering activity partly via inhibition of gene expressions of oxidative stress and extracellular matrix.