TGF-β1 Protein Expression Research Articles

The glucagon-like peptide-1 (GLP-1) analog exenatide ameliorates intrauterine adhesions in mice

ObjectiveThe purpose of the experiments in this study was to explore the effect of exenatide on intrauterine adhesions (IUAs) and to elucidate its mechanism to provide new ideas for the clinical treatment of IUAs. MethodsIn this study, an animal model of IUAs was established by double stimulation using mechanical curettage and inflammation. After modeling, the treatment group was injected subcutaneously with three doses of exenatide for two weeks. The model group was injected with sterile ultrapure water, and the sham operation group was treated the same as the normal group, except for the observation of abdominal wound changes. Two weeks later, all mice were sacrificed by cervical dysfunction. The obtained mouse uterine tissue was used for subsequent experimental detection, using HE and Masson staining for histomorphological and pathological analysis; qRT-PCR for the detection of TGF-β1, α-SMA, and MMP-9 gene expression in uterine tissue; and western blotting analysis of TGF-β1, α-SMA, and collagen 1 protein expression to verify whether exenatide has a therapeutic effect on IUAs in mice. ResultsIn the high-dose exenatide treatment group, the endometrial glands significantly increased in size, and the deposition area of collagen fibers in the endometrial tissue was significantly reduced. We observed that the mRNA expression of TGF-β1 and α-SMA in the endometrial tissue of IUAs mice in this group was significantly reduced, while the expression of MMP-9 was significantly increased. In addition, we found that the protein expression of TGF-β1, α-SMA, and collagen 1 remarkably decreased after treatment with exenatide. ConclusionExenatide may reduce the deposition of collagen fibers in the uterus of IUAs mice and promote the proliferation of endometrial glands in mice.

Downregulation of CR6-interacting factor 1 suppresses keloid fibroblast growth via the TGF-\u03b2/Smad signaling pathway

Keloids are a type of aberrant skin scarring characterized by excessive accumulation of collagen and extracellular matrix (ECM), arising from uncontrolled wound healing responses. While typically non-pathogenic, keloids are occasionally regarded as a form of benign tumor. CR6-interacting factor 1 (CRIF1) is a well-known CR6/GADD45-interacting protein, that has both nuclear and mitochondrial functions, and also exerts regulatory effects on cell growth and apoptosis. In this study, cell proliferation, cell migration, collagen production and TGF-β signaling was compared between normal fibroblasts (NFs) and keloid fibroblasts (KFs). Subsequently, the effects of CRIF1 deficiency were investigated in both NFs and KFs. Cell proliferation, cell migration, collagen production and protein expressions of TGF-β, phosphorylation of Smad2 and Smad3 were all found to be higher in KFs compared to NFs. CRIF1 deficiency in NFs and KFs inhibited cell proliferation, migration, and collagen production. In addition, phosphorylation of Smad2 and Smad3, which are transcription factors of collagen, was decreased. In contrast, mRNA expression levels of Smad7 and SMURF2, two important inhibitory proteins of Smad2/3, were increased, suggesting that CRIF1 may regulate collagen production. CRIF1 deficiency decreases the proliferation and migration of KFs, thereby inhibiting their overgrowth via the transforming growth factor-β (TGF-β)/Smad pathway. CRIF1 may therefore represent a potential therapeutic target in keloid pathogenesis.

Expression of miR-207 in renal tissue of renal fibrosis rats and its correlation analysis with protein expression of TGF-β1 and Smad3.

This study was designed to analyze the expression of miR-207 in renal tissue of renal fibrosis rats and its correlation with the protein expression of TGF-β1 and Smad3. Rat models with renal fibrosis were established via unilateral ureteral obstruction (UUO). Then, the expression levels of miR-207, TGF-β1 and Smad3 in renal tissue of rats were intervened by over-expression vector miR-207 mimic, miR-207 inhibitor and TGF-β/Smad3 signal SIS3 free base, and the effect and mechanism of action of miR-207 on renal fibrosis were analyzed. In UUO models established in this study, the expression levels of fibrosis related factors TGF-β1, Smad3, Smad2, α-SMA, BMP-7, MMP7 and MMP9 were elevated, and staining results showed that evident fibrosis occurred in renal tissue of rats. Moreover, we also found that the miR-207 expression increased in UUO model rats. After inhibiting miR-207 expression, their degree of renal fibrosis also reduced significantly, and the expression levels of TGF-β1, Smad3, Smad2, α-SMA, BMP-7, MMP7 and MMP9 were inhibited. Besides, miR-207 had a positive correlation with TGF-β1/Smad3 expression. We designed a group of rats, and found that while miR-207 expression was up-regulated, TGF-β1/Smad3 signals were inhibited, and compared with those with up-regulation of miR-207 expression, the severity of renal fibrosis reduced significantly, and the expression of other fibrosis indicators Smad2, α-SMA, BMP-7, MMP7 and MMP9 also reduced dramatically. The miR-207 expression in renal tissue of rats with renal fibrosis increased, which was positively correlated with TGF-β1/Smad3, and miR-207 could promote the progression of renal fibrosis through TGF-β1/Smad3 signals.

Effects of PPARγ agonist pioglitazone on cardiac fibrosis in diabetic mice by regulating PTEN/AKT/FAK pathway.

The aim of this study was to explore the role of pioglitazone (PIO), a peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, in cardiac fibrosis of diabetic mice. A total of 60 adult male C57/B6 mice were divided into 3 groups using a random number table, namely, control group (Sham group, n=20), diabetic cardiomyopathy group (DCM group, n=20), DCM + PIO group (n=20). Streptozocin (STZ) was injected into mice at a dose of 125 mg/Kg to induce the model of diabetes in vivo. After successful induction, mice in DCM + PIO group were intragastrically given PIO at 10 mg/kg/d once a day for 6 weeks. Meanwhile, those in Sham group and DCM group were given the same volume of normal saline. After 6 weeks, ejection fraction % (EF%), fraction shortening % (FS%) and heart rate of mice in each group were examined via echocardiography. Picrosirius red (PSR) staining assay was conducted to detect collagen deposition in myocardial tissues of mice in each group. The protein expression level of PPARγ in mouse myocardial tissues in each group was measured through Western blotting and immunohistochemical staining assays. Hematoxylin-eosin (H&E) staining assay was carried out to evaluate the myocardial hypertrophy of mice in each group. The protein expression level of transforming growth factor-β (TGF-β) in mouse myocardial tissues in each group was measured through immunohistochemical staining assay. In addition, Western blotting was employed to detect the expression of proteins related to the phosphate and tension homology deleted on chromsome ten (PTEN)/protein kinase B (AKT)/focal adhesion kinase (FAK) signaling pathway in myocardial tissues of mice in each group. The messenger ribonucleic acid (mRNA) and protein expression levels of PPARγ in mouse myocardial tissues were significantly lower in DCM group than those in Sham group (p<0.05). PPARγ agonist PIO could significantly increase the protein expression of PPARγ in myocardial tissues of DCM mice. The results of cardiac Doppler ultrasound revealed that PIO significantly upregulated EF% and FS% in DCM mice (p<0.05). Besides, PIO remarkably reduced collagen deposition and TGF-β protein expression in myocardial tissues in DCM mice (p<0.05). H&E staining results showed that PIO notably attenuated myocardial hypertrophy in DCM mice (p<0.05). Furthermore, it was discovered that PIO markedly elevated PTEN protein in myocardial tissues of DCM mice and inhibited the phosphorylation of AKT and FAK proteins (p<0.05). The protective effect of PIO against cardiac fibrosis in diabetic mice may be related to its regulation on the PTEN/AKT/FAK signaling pathway. Our findings suggest that PIO is expected to become a targeted drug for the treatment of DCM in clinical practice.

All-trans retinoic acid regulating angiopoietins-1 and alleviating extracellular matrix accumulation in interstitial fibrosis rats

All-trans retinoic acid (ATRA) is one of essentially active metabolite of vitamin A, and plays an important role in diverse physiological processes, such as cellular growth and function. Renal interstitial fibrosis (RIF) is a common pathological characteristic of chronic renal disease causing end-stage renal disease currently lacking effective treatment. Low level of Angiopoietins-1 (Angpt-1) is associated with extracellular matrix accumulation and fibrosis diseases. This study was performed to assess the association of ATRA with Angpt-1 in RIF disease. Rats were divided into three groups: group of sham (SHO group), group of unilateral ureteral obstruction group (UUO group), UUO mice administrated daily at the dose of ATRA (ATRA group). Masson-staining was used to detect the histologic lesion. Immunohistochemistry and Western-blot were applied to determine the targeted proteins. RIF score was significantly increased in UUO rats when compared with that of SHO group, and the fibrosis score was notably reduced in ATRA group. Transforming growth factor-β1 (TGF-β1), collagen IV (Col-IV) and fibronectin (FN) expressions in UUO group were significantly up-regulated, whereas Angpt-1 expression was significantly down-regulated compared with the SHO group. ATRA treatment reduced TGF-β1, Col-IV and FN expressions and improved Angpt-1 expression compared with the UUO group. The protein expression of Angpt-1 in kidney tissue of UUO group was negatively correlated with RIF index and protein expressions of Col-IV, FN and TGF-β1. In conclusion, low expression of Angpt-1 was associated with the RIF disease and ATRA treatment can increase the Angpt-1 and alleviate the RIF lesion in UUO rats.

Effects of Eucommia ulmoides extract against renal injury caused by long-term high purine diets in rats.

Diets of overloaded purine-rich foods for a long time are one of the important reasons to cause renal lesions. Eucommia ulmoides is one of the traditional Chinese medicine herbs, which has been used to recover functions of the kidney. However, its mechanism remains unclear. The aim of this study was to explore the effects and protective mechanism of Eucommia ulmoides extract on renal injury caused by long-term high purine diets in rats. SD rats underwent an intragastric adenine (200 mg kg-1 d-1) administration for 9 weeks and were treated for 15 weeks. The results demonstrated that Eucommia ulmoides extract significantly reduced serum Cre and BUN levels in rats. H&E and Masson's trichrome stains showed notable lowering of the infiltration of inflammatory cells, the formation of fibrous tissues and collagen fibers, and improvement in the pathological morphology of kidneys. It also suppressed the protein and mRNA expressions of TGF-β1 and α-SMA and enhanced E-cadherin expression. Meanwhile, Eucommia ulmoides extract prominently inhibited the mRNA expression of Col I, Col III, Col IV, TIMP-1, and TIMP-2 and promoted expressions of MMP-1, MMP-2 and MMP-9. Through our study, it is the first time to prove that Eucommia ulmoides extract could ameliorate renal interstitial fibrosis and may involve in the regulation of the extracellular matrix (ECM) degradation enzyme (MMPs/TIMPs) system, promotion of the expression of E-cadherin, and suppression of expressions of TGF-β1 and α-SMA. The results provide a significant implication for the utilization of Eunomia Ulmoides extract as functional foods to enhance renal functions and improve renal injury caused by high purine diets.

Dextran Sulfate Effects EMT of Human Gastric Cancer Cells by Reducing HIF-1α/ TGF-β.

The peritoneal implant metastasis is one of the main pathway and main cause for high mortality for gastric cancer metastasis. Researchs show that epithelial-mesenchymal transition (EMT) playing essential role in modulating gastric cancer metastasis, and the expression of hypoxia inducible factor-1α (HIF-1α) can promote EMT in tumor cells. This research aims to explore the influence and mechanism of Dextran Sulfate (DS) affecting EMT of human gastric cancer. In the present study, we found that DS can enter into the cytoplasm and function in it. Inhibition of HIF-1α or DS significantly inhibit the migration and invasion of human gastric cancer cells, and decrease the mRNA and protein expressions of HIF-1α, matrix metalloproteinase-2 (MMP-2), transforming growth factor-β (TGF-β), Twist and N-cadherin (N-cad), rise E-cadherin (E-cad) expression, DS with HIF-1α knockdown has a stronger effect. In vivo studies indicated that compared with using DS or HIF-1α knockdown alone, DS with HIF-1α knockdown can better suppress the volume and number of metastatic tumors, and reduce the mRNA and protein expressions of HIF-1α, MMP-2, TGF-β, Twist and N-cad in metastatic tumor tissues of nude mice. We further demonstrated that the expression of HIF-1α, MMP-2, TGF-β , Twist and N-cad were higher in well and poorly differentiated gastric cancer than paracancerous tissue, and poorly differentiated gastric cancer were even higher, while E-cad expression was opposite. Taken together, this study shows that DS can interfere the expression of HIF-1α, thereby inhibiting TGF-β-mediated EMT of gastric cancer cells, and demonstrated a promising application of DS in gastric cancer therapy.

Effect of intra-articular injection of adipose stem cells on traumatic osteoarthritis cartilage defects

Traumatic osteoarthritis with cartilage defects can lead to mobility problems. Mitotic activity in cartilage is extremely low, and once damaged, repairing can be difficult. The commonly used autologous or allogeneic cartilage transplantation techniques also have certain limitations. In recent years, directed induction of osteoblastic differentiation using adipocytes has been shown to be effective in repairing cartilage defects. However, it is often induced in vitro and is prone to incomplete or over-differentiation. In addition, because of the large differences in the in vivo and in vitro microenvironment, exploring the influence of these differences in the in vivo microenvironment on the directional differentiation of adipose-derived stem cells (ADSCs) and their effect on cartilage repair is necessary. In this study, a cartilage defect model in rabbits with traumatic osteoarthritis of the left knee was established, and different interventions were conducted in different groups. We determined the effect of directly injecting ADSCs into the joints on repairing cartilage defects in rabbits with traumatic osteoarthritis and analyzed the differences in repair time of newly developed cartilage defects and old cartilage frontal defects. The results indicated that the placement of a stent and injection of ADSCs improved the knee joint activity, increased the expression of BMP and TGF-β protein, and reduced the expression of inflammatory factors, including IL-1β, IL-6, IL-17, and TNF-α. No difference was found between the new cartilage defect and the old one. By directly observing the cartilage defect, intervention with ADSCs + scaffold increased the connection between the cartilage defect and the normal tissue and improved the cartilage repair effect. These results indicated that directly injecting ADSCs into the joints is an effective approach for repairing cartilage defects in traumatic osteoarthritis, and it was not affected by the age of the defect.

Inhibitory effects of alkaline extract from the pericarp of Citrus reticulata Blanco on collagen behavior in bleomycin-induced pulmonary fibrosis

Ethnopharmacological relevancePeel of Citrus reticulata, a Chinese herbal drug with functions of regulating Qi and expelling phlegm, has been used for the treatment of lung related diseases in Chinese medicine for a long time. Its detailed effects on collagen in anti-idiopathic pulmonary fibrosis (IPF) is still unclear. Aim of the studyTo explore the effects of citrus alkaline extract (CAE) on collagen synthesis, crosslinking and deposition in pulmonary fibrosis and understand the possible signal pathways involved in the activity. Materials and methodsCAE was prepared from C. reticulata. Bleomycin-induced pulmonary fibrosis mouse model was applied. Pulmonary fibrosis of lung was estimated with histopathology analysis, and collagen deposition was evaluated with immunohistochemistry. Collagen crosslinking related biomarkers and enzymes were analyzed with chemical methods, immunohistochemical and western blot analyses. ResultsCAE oral administration lowered hydroxyproline content, inhibited the collagen deposition including expressions of collagen I and III, and relieved bleomycin-induced pulmonary fibrosis in mice model. The productions of a collagen crosslink pyridinoline and crosslinking related enzymes including lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1) in lung were suppressed by CAE treatment. Furthermore, the protein expressions of TGF-β1 and Smad3 levels in lungs were also downregulated by CAE. ConclusionsThis study demonstrated that CAE inhibited collagen synthesis, crosslinking and deposition, and ameliorated bleomycin-induced pulmonary fibrosis. Preliminary mechanism study revealed that CAE exerted its bioactivity at least via downregulation of TGF-β1/Smad3 pathway. Our findings provided a great potential in fighting IPF based on CAE.

Chitosan and gelatin biopolymer supplemented with mesenchymal stem cells (Velgraft®) enhanced wound healing in goats (Capra hircus): Involvement of VEGF, TGF and CD31

AimCell-based therapy has emerged as promising strategy for chronic and impaired wounds treatment. Current research is focused on developing biomaterial systems that act as a niche for mesenchymal stem cells (MSCs) to promote wound healing through paracrine molecular cascading. This study was aimed to evaluate the wound healing potential of Velgraft, a ready-to-use biodegradable artificial skin substitute, on excision wound in goats. Materials and methodsTwelve male goats were randomized divided in to three groups of four animals each. After infliction of surgical wound, Velgraft and Soframycin were applied on wounds of the animals of Groups II and III while Group I (sham operated) served as control. Wound diameters were measured at pre-defined time-points for determination of progressive wound healing up to 28 days. Skin sections were stained using Hematoxylin and eosin (H&E) for examining the histoarchitectural changes, Masson trichome staining for ascertaining collagen synthesis and immunohistochemistry for expression of CD31, VEGF and TGF-β1 proteins to determine post-treatment angiogenesis in the inflicted wounds. ResultsVelgraft application appreciably enhanced wound closure by day 21 which was confirmed through restoration of the normal skin architecture as evident based on histopathological examination and characterized by complete regeneration of epidermal layers, collagen fibers, blood capillaries and hair follicular formation. Stimulation of angiogenesis markers was also observed at different time-points post-Velgraft application; which is suggestive of the improved angiogenesis and vasculogenesis. ConclusionVelgraft facilitates wound healing by augmenting early wound closure, enhancing collagen synthesis and deposition, trichosis development and promoting revascularization and epidermal layers restoration.

Natural flavonoid pectolinarigenin alleviated kidney fibrosis via inhibiting the activation of TGFβ/SMAD3 and JAK2/STAT3 signaling

Renal fibrosis is a final common manifestation of CKD resulting in progressive loss of kidney function. The activation of SMAD3 and STAT3 played central roles in the pathogenesis of renal fibrosis, which has been recognized as potential targets for antifibrotic therapy. As we known, the potential of natural products as the candidates for drug discovery has been well recognized. Here, we identified that pectolinarigenin (PEC), as a natural flavonoid and a reported STAT3 inhibitor, dose-dependently suppressed TGFβ/SMADs activity in HEK293 cells by luciferase reporter assay. In TGFβ1-stimulated NRK-49F fibroblast, PEC blocked the phosphorylation of SMAD3 and STAT3, and downregulated the major fibrotic gene and protein expression of TGFβ, α-SMA, COL-1, and FN. Notably, oral administration of PEC at a dose of 25 mg/kg/d for 7 days or 14 days effectively ameliorated kidney injury and tubulointerstitial fibrosis after unilateral ureteral obstruction (UUO) surgery in mice. Mechanically, PEC treatment inhibited the phosphorylated activation of SMAD3 and STAT3, which further reduced the protein expression of TGFβ, α-SMA, COL-1, and FN in the obstructed kidneys of UUO mice. In summary, our results suggested that pectolinarigenin alleviated tubulointerstitial fibrosis by inhibiting the activation of SMAD3 and STAT3 signaling.

Healing Effects of Curcumin Nanoparticles in Deep Tissue Injury Mouse Model.

Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in Deep Tissue Injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than the other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation. In contrast, control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylated-STAT3. Our results showed that CPNPs enhanced wound healing in DTI models through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

Inflammatory Mechanism of Total Flavonoids of Chrysanthemum and Medicated Serum on Castrated Dry Eye Animal and Cell Models

ObjectiveTo observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration. Methods(1) 150 male Japanese rabbits were randomly divided into five groups, with 30 rabbits in each group: normal control group (group A), sham group (group B), model group (group C), androgen control group (group D) and total flavonoids of chrysanthemum treatment group (group E). The androgen deficiency dry-eye model was established by bilateral castration in groups C, D and E. Normal saline was administered to groups A, B and C by gavage; androgen (testosterone propionate) was injected into muscle in group D; and group E was given total flavonoids of chrysanthemum by gavage. All white rabbits were tested the Schirmer I test (SIT) and tear break-up time (BUT). After euthanasia, tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue. The expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) was detected in the lacrimal gland tissue by immunohistochemistry. Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA. (2) Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels. The blank control group was set up without androgen culture, the control group with androgen culture, and the total flavonoids of chrysanthemum group without androgen. The MTT method was used to determine the optimal intervention dosage of drug-containing plasma. Western blot and QPCR were used to detect the expression of AR mRNA, NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells, and the androgen-like effect of total flavonoids of chrysanthemum was observed. Results(1) Immunohistochemistry showed that groups A, B, D and E had significantly lower expression of IL-1β and TNF-α than group C (P < 0.05); among these, group E had slightly higher expression than group D (P > 0.05). RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A, B, D and E was significantly higher than in group C (P < 0.05), and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B (P < 0.05). (2) Using the MTT method, the final concentration of interfering cells was calculated to be 13.2%. The expression of AR protein, NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced, and there were significant differences relative to the blank group (P < 0.01). The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group (P < 0.01). ConclusionsThe total flavonoids of chrysanthemum can inhibit IL-1β and TNF-α expression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1, thereby inhibiting the inflammatory response. The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA, upregulating expression of NF-κB, further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells, inhibiting inflammation by regulating related proteins, and ultimately alleviating the symptoms of dry eye.

Qualifying Osteogenic Potency Assay Metrics for Human Multipotent Stromal Cells: TGF-β2 a Telling Eligible Biomarker.

Potency assays are critical for regenerative medicine, addressing the known challenge of functional heterogeneity among human multipotent stromal cells (hMSC). Necessary laboratory cell expansion allows analysis before implantation in the patient. Levels of induction of five signature gene biomarkers, ALPL, COL1A2, DCN, ELN and RUNX2, constituted a previously reported proof-of-principle osteogenic potency assay. We tested assay modification to enhance reproducibility using six consistent bone marrow derived hBM-MSC and explored applicability to three adipose tissue derived hAT-MSC. Using a potent proprietary osteogenic induction factor, the GUSB/YWAHZ reference gene pair provided real time PCR consistency. The novel assay conditions supported the concept that genes encoding extracellular matrix proteins one week after osteogenic induction were informative. Nonetheless, relatively low induction of COL1A2 and ELN encouraged search for additional biomarkers. TGFB2 mRNA induction, important for osteogenic commitment, was readily quantifiable in both hBM-MSC and hAT-MSC. Combined with DCN, TGFB2 mRNA induction data provided discriminatory power for resolving donor-specific heterogeneity. Histomorphometric decorin and TGF-β2 protein expression patterns in eight-week heterotopic bone implants also discriminated the two non-bone-forming hMSC. We highlight progress towards prompt osteogenic potency assays, needed by current clinical trials to accelerate improved intervention with enhanced stem cell therapy for serious bone fractures.

Hesperidin inhibits L-NAME-induced vascular and renal alterations in rats by suppressing the renin-angiotensin system, transforming growth factor-β1, and oxidative stress.

The protective effect of hesperidin on vascular and renal alterations and possible underlying mechanisms involved in Nω -nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats were investigated in this study. Male Sprague-Dawley rats were administered L-NAME (40mg/kg/day), L-NAME plus hesperidin (30mg/kg/day), and L-NAME plus captopril (2.5mg/kg/day) for 5weeks. Hesperidin and captopril significantly prevented L-NAME-induced hypertension, vascular and renal dysfunction, intrarenal artery remodelling, glomerular extracellular matrix accumulation, and renal fibrosis. The preventive treatment with hesperidin and captopril also significantly decreased serum angiotensin-converting enzyme activity and plasma transforming growth factor-β1 (TGF-β1) levels and downregulated angiotensin II receptor type I and TGF-β1 protein expression in the kidneys. In addition, decreased malondialdehyde levels and increased superoxide dismutase activity in the plasma and kidney were observed after co-treatment with hesperidin or captopril. These findings suggest that hesperidin inhibits L-NAME-induced vascular and renal alterations in rats. The possible mechanism may be related to the suppression of the activation of the renin-angiotensin system and expression of TGF-β1, and reduction of oxidative stress.

Rectification of cavernosal fibrosis and veno‐occlusive dysfunction by administration of suberoylanilide hydroxamic acid in a rat model of cavernosal nerve injury: Comparison with a PDE5 inhibitor

Cavernosal fibrosis, which is induced by cavernosal nerve (CN) injury and progresses with time, is the main cause of cavernosal veno-occlusive dysfunction (CVOD) after radical prostatectomy. To determine whether daily oral administration of suberoylanilide hydroxamic acid (SAHA; vorinostat) for 5-weeks from the immediate post-injury period after CN injury would rectify CVOD by suppressing cavernosal fibrosis and normalizing HDAC pathway in a rat model of CN crush injury (CNCI) and to compare the results with those obtained using chronic administration of PDE5-inhibitors (a positive control). Fifty-six 12-week-old rats were randomized into the four groups: sham surgery (S), CNCI (I), and CNCI treated with daily administration of 25.0mg/kg SAHA (V) or 20.0mg/kg udenafil (P). Group-V and Group-P received the respective treatment for 5-weeks from the following day after CNCI. At 5weeks after surgery, dynamic infusion cavernosometry (DIC), histological staining, and Western blot analysis were performed. Group-I had a significantly decreased papaverine response, higher maintenance rate or drop rate, lower smooth muscle (SM)/collagen ratio, decreased SM content, and increased protein expression of HDAC2, HDAC3, TGF-β1, and collagen-1, compared with Group-S. The three DIC parameters in Group-V and Group-P significantly improved compared to those in Group-I. Except for the maintenance rate, the improvement in papaverine response and drop rate in Group-V was not significantly different from that in Group-P. Group-V and Group-P showed the rectification of SM/collagen ratio and protein expression of TGF-β1 or collagen-1. SM content was improved in Group-P, but not in Group-V. Group-V showed the normalization of protein expression of both HDAC2 and HDAC3, whereas protein expression of only HDAC2 was partially restored in Group-P. Treatment strategies targeting the HDAC pathway might be helpful to alleviate CVOD induced by CN injury. According to our data, chronic administration of SAHA improves post-injury CVOD by suppressing cavernosal fibrosis via rectifying the HDAC/TGF-β1 pathway in nerve-injured rats, comparable to that with PDE5 inhibitors.

Abstract 13025: Methotrexate Carried in Lipid Core Nanoparticles Prevented the Dilation and Dissection of the Aortic Arch in Mice With Marfan Syndrome by Increasing the Bioavailability of Intracellular Adenosine

Introduction: Mutations in the fibrillin-1 gene result in aneurysms and rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). Inflammation is associated with dissection and rupture of the aorta. We showed that methotrexate (MTX) carried in lipid core nanoparticles (LDE) had powerful anti-inflammatory effects on atherosclerotic lesions and myocardial infarction. Hypothesis: To investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in MFS. Methods: mgΔloxPneo MFS murine model and wild-type mice were allocated in 3 groups of treatment: LDE only; MTX in commercial formulation; LDE-MTX. The treatment occurred weekly at a dose of 1mg/kg ip, between the 3rd and 6th month of life, period in which both dilation and dissection of the aortic arch are observed. After 12 weeks, animals were submitted to analysis of the aortic arch. Results: Compared to LDE and MTX groups, LDE-MTX treatment showed smaller lumen area of ascending and descending aorta and aortic arch segments in MFS mice. LDE-MTX also decreased collagen volume fraction and the amount of aortic dissections, but did not affect the thickness of the vessel wall and the number of elastic fiber ruptures. Protein expression of the inflammatory markers CD68 (macrophages), CD3 (lymphocytes) and tumor necrosis factor alpha, of the apoptotic marker caspase 3 and of type 1 collagen were lower in LDE-MTX group when compared to LDE and MTX. Moreover, treatment with LDE-MTX reduced TGF-β, ERK and SMAD3 protein expression. Of note, CD68 and CD3 protein expression were positively correlated with the lumen area of the aortic arch (r2=0.36; p&lt;0.001), indicating the importance of inflammation for the aortic dilation. The increase in bioavailability of intracellular adenosine was suggested by higher expression of A2a adenosine receptor and lower levels of adenosine deaminase in the aortic arch. Conclusions: Possibly by increasing the bioavailability of intracellular adenosine, LDE-MTX treatment had the ability to reduce the processes of inflammation, apoptosis and fibrosis that are consequent to fibrillin-1 mutation. By these means, LDE-MTX may conceivably prevent the development of the dilation and dissection in the aortic arch, the hallmarks of MFS.

Attenuation of Abnormal Scarring Using Spherical Nucleic Acids Targeting Transforming Growth Factor Beta 1.

Abnormal scarring is a consequence of dysregulation in the wound healing process, with limited options for effective and noninvasive therapies. Given the ability of spherical nucleic acids (SNAs) to penetrate skin and regulate gene expression within, we investigated whether gold-core SNAs (AuSNAs) and liposome-core SNAs (LSNAs) bearing antisense oligonucleotides targeting transforming growth factor beta 1 (TGF-β1) can function as a topical therapy for scarring. Importantly, both SNA constructs appreciably downregulated TGF-β1 protein expression in primary hypertrophic and keloid scar fibroblasts in vitro. In vivo, topically applied AuSNAs and LSNAs downregulated TGF-β1 protein expression levels and improved scar histology as determined by the scar elevation index. These data underscore the potential of SNAs as a localized, self-manageable treatment for skin-related diseases and disorders that are driven by increased gene expression.

Alcohol induces TGF\u03b21 via downregulation of miR-1946a in murine lung fibroblast

Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFβ1 3′ untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFβ1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFβ1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFβ1 expression and TGFβ1 3′UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFβ1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFβ1 gene expression and TGFβ1 3′UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFβ1 gene and protein expression as well as alcohol-induced TGFβ1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFβ1 expression through direct interaction with TGFβ1 3′UTR. These findings identify a novel mechanism by which alcohol induces TGFβ1 in the lung.

Role of TGF-β1-mediated epithelial-mesenchymal transition in the pathogenesis of tympanosclerosis.

The present study aimed to explore the role of TGF-β1-mediated epithelial-mesenchymal transition (EMT) in the pathogenesis of tympanosclerosis. Sprague Dawley rats were injected with inactivated Streptococcus pneumoniae suspension to establish a rat model of tympanosclerosis. The rats were sacrificed 8 weeks after the model was established. H&E and von Kossa staining was used to observe the morphological changes of middle ear mucosa. Western blotting was used to detect the expression of TGF-β1 and EMT-associated proteins in the mucosa samples. Middle ear mucosal epithelial cells of rats were collected to establish a primary culture. The cultured cells were stimulated with TGF-β1 and the expression of EMT-associated proteins was detected by western blotting and immunofluorescence. In addition, the cells were treated with TGF-β receptor type I/II inhibitor and the expression level of EMT-associated proteins was detected by western blotting. Sclerotic lesions appeared on 72.4% of tympanic membranes, and marked inflammation, inflammatory cell infiltration and fibrosis were found in the middle ear mucosa of rat models of tympanosclerosis. In middle ear mucosa of rats with tympanosclerosis, the expression of mesenchymal cell markers increased and that of epithelial cell markers decreased compared with the control group. TGF-β1 stimulated the activation of the EMT pathway in middle ear mucosal epithelial cells, resulting in an increased expression of fibronectin and N-cadherin. In addition, a decreased expression level of EMT-associated proteins was observed when TGF-β1 was inhibited. In conclusion, the present study indicated that TGF-β1-mediated EMT may play an important role in the pathogenesis of tympanosclerosis.