Abstract 96: Simvastatin Prevents TGF-β1-induced SMAD2/3 Phosphorylation Involved in Ventricular Fibrosis: Role of Protein Phosphatases

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Farhan Rizvi ◽  
Ramail Siddiqui ◽  
Alessandra DeFranco ◽  
Alisher Holmuhamedov ◽  
Hao Xu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cardiac Fibrosis ◽  
Collagen Type ◽  
Protein Phosphatases ◽  
Collagen Type I ◽  
Type I ◽  
Smad Protein ◽  
The Media ◽  
Ventricular Fibrosis ◽  
Tgf Β1

Background: Ventricular fibrosis leads to progressive cardiac dysfunction and heart failure (HF). Statins are reported to reduce cardiac fibrosis through the cholesterol-independent pathway, but mechanisms remain elusive. We hypothesize simvastatin reduced TGF-β1-induced ventricular fibrosis through activation of SMAD protein phosphatase Mg 2+ /Mn 2+ -1A (PPM1A), -2A (PP2A). Methods: In the absence and presence of TGF-β1 (5ng) with or without simvastatin (1μM), the rate of fibroblast proliferation (doubling time), myofibroblast differentiation (ICC), α-SMA mRNA (RT-PCR) and protein expression (Western blot) and the release of collagen synthesis markers, pro-collagen type I C-terminal peptide (PICP) and pro-collagen type III N-terminal peptide (PIIINP), in the media (ELISA) were determined along with protein interaction between SMAD2/3 and PPM1A or PP2A (Co-IP) and SMAD2/3 phosphorylation (Western blot). Results: Simvastatin reduced the effect of TGF-β1 on hVF proliferation by 47% (50000 to 26500), p<0.01; myofibroblast differentiated population from 48% (avg 48/100) to 11% (avg 11/100), p<0.01; expression of α-SMA mRNA by 76%, p<0.01; and protein by 60%, p<0.05. Simvastatin also decreased release of PICP by 66%, p<0.01, and PIIINP by 83%, p<0.01, into the media. Time-dependent increases in SMAD2/3 phosphorylation were reduced by simvastatin through activation of protein phosphatases PPM1A and PP2A by interacting with SMAD2/3. Conclusion: Involvement of PPM1A and PP2A in the anti-fibrotic effect of simvastatin reveals novel signaling mediators that may be selectively targeted for prevention of myocardial injury-induced ventricular fibrosis and HF.

scholarly journals Mesenchymal Stem Cells Ameliorate Interstitial Fibrosis in Adenine-induced Nephropathy Based on Renal Proteomics

2020 ◽  
Author(s):  
Huajun Tang ◽  
Peiyue Zhang ◽  
Lianlin Zeng ◽  
Yu Zhao ◽  
Libo Xie ◽  
...  
Keyword(s):  
Western Blot ◽  
Therapeutic Target ◽  
Bioinformatics Analysis ◽  
Interstitial Fibrosis ◽  
Kidney Diseases ◽  
Histopathological Change ◽  
Collagen Type I ◽  
Tubulointerstitial Fibrosis ◽  
Type I ◽  
Tgf Β1

Abstract Background: Tubulointerstitial fibrosis (TIF) is one of the main pathological features of various progressive renal damages and chronic kidney diseases. Mesenchymal stromal cells (MSCs) have been verified with significant improvement in the therapy of fibrosis diseases, but the mechanism is still unclear. We attempted to explore the new mechanism and therapeutic target of MSCs against renal fibrosis based on renal proteomics.Methods: TIF model was induced by adenine gavage. Bone marrow derived MSCs was injected by tail vein after modeling. Fibrosis biomarkers or extracellular matrix proteins and histopathological change were assessed by Masson staining, Sirius red staining, immunohistochemistry, and western blot. Renal proteomics was analyzed using iTRAQ-based mass spectrometry.Results: MSCs treatment clearly decreased the expression of α-SMA, collagen type I, II, III, TGF-β1, p-Smad2/3, IL-6, IL-1β, and TNFα compared with model rats, while p38 MAPK increased. 6,213 proteins were identified, but only 40 proteins exhibited significant differences (30 upregulated, 10 downregulated) compared MSCs group with the model group. Bioinformatics analysis revealed that these proteins play important roles in the proliferation, inflammatory and immune responses, apoptosis, phagosome, etc. According to literatures and bioinformatics analysis, the most markedly downregulated protein, galectin3, was further assessed by quantitative PCR and western blot in renal tissues. Galectin3 levels were downregulated in adenine-induced renal tissues and TGF-β1 induced tubular epithelial cells and interstitial fibroblasts in consistent with iTRAQ after MSCs treatment.Conclusion: The founds suggest that galectin3 maybe involves in the antifibrotic mechanisms of MSCs therapy for tubulointerstitial fibrosis as well as a possible therapeutic target.

Abstract 48: Age-associated Imbalance of Vasorin/TGF-β1 Signaling in VSMC Facilitates Collagen Production

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Mingyi Wang ◽  
Gianfranco Pintus ◽  
Roberta Giordo ◽  
Jing Zhang ◽  
Liqun Jiang ◽  
...  
Keyword(s):  
Arterial Wall ◽  
Collagen Type ◽  
Collagen Type I ◽  
In Vivo Studies ◽  
Physical Interaction ◽  
Type I ◽  
Ang Ii ◽  
Collagen Production ◽  
At1 Antagonist ◽  
Tgf Β1

Collagen deposition, a hallmark of arterial aging that resembles post-injury arterial restenosis, is perpetrated by angiotensin II (Ang II) signaling in arterial wall. Collagen aggregation at sites of arterial injury is regulated by the coordinated signaling of pro-fibrotic TGF-β1 and anti-fibrotic vasorin within VSMCs. The Ang II/TGF-β1/vasorin signaling relationship within VSMCs with aging, however, remains unknown. In vivo studies in old vs. young FXBN rats show that aortic transcription and translation of vasorin markedly decrease with aging. In vitro studies in VSMCs isolated from old vs. young aortae. Ang II-associated reduction of vasorin protein abundance in young VSMCs and age-associated changes in vasorin protein levels are reversed by the AT1 antagonist, Losartan (Los) (Figure). Dual immunolabeling and co-immunoprecipitation demonstrate that the co-incidence and physical interaction of vasorin and TGF-β1 within VSMCs are significantly decreased with aging. Importantly, exposure of young VSMCs to Ang II that increases p-SMAD2/3 and collagen type I production, mimicking old cells, and this effect is abolished or substantially mitigated by Los treatment, overexpression of ectopic vasorin, or exogenous recombinant human-vasorin protein. In contrast, exposure of old VSMCs to Los decreases p-SMAD2/3 and collagen type I production.Thus, an imbalance of the Ang II/TGF-β1/vasorin signaling cascade, a feature of the aged arterial wall, enhances the collagen production by VSMCs. Maintaining this signaling balance is a novel measure to retard adverse extracellular matrix remodeling, a determinant of arterial stiffening with aging. (MW and GP co-first authors)

Losartan attenuates paraquat-induced pulmonary fibrosis in rats

2014 ◽  
Vol 34 (5) ◽  
pp. 497-505 ◽  
Author(s):  
F Guo ◽  
YB Sun ◽  
L Su ◽  
S Li ◽  
ZF Liu ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Lung Injury ◽  
Mrna Expression ◽  
Collagen Type ◽  
Collagen Type I ◽  
Control Group ◽  
Type I ◽  
Expression Levels ◽  
Relative Expression ◽  
Tgf Β1

Paraquat (PQ) is one of the most widely used herbicides in the world and can cause pulmonary fibrosis in the cases with intoxication. Losartan, an angiotensin II type 1 receptor antagonist, has beneficial effects on the treatment of fibrosis. The aim of this study was to examine the effect of losartan on pulmonary fibrosis in PQ-intoxicated rats. Adult male Sprague Dawley rats ( n = 32, 180–220 g) were randomly assigned to four groups: (i) control group; (ii) PQ group; (iii) PQ + losartan 7d group; and (iv) PQ + losartan 14d group. Losartan treatment (intragastrically (i.g.), 10 mg/kg) was performed for 7 and 14 days after a single i.g. dose of 40 mg/kg PQ. All rats were killed on the 16th day, and hematoxylin–eosin and Masson’s trichrome staining were used to examine lung injury and fibrosis. The levels of hydroxyproline and transforming growth factor β1 (TGF-β1), matrix metallopeptidase 9 (Mmp9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) messenger RNA (mRNA) expression and relative expression levels of collagen type I and III were also detected. PQ caused a significant increase in hydroxyproline content, mRNA expression of TGF-β1, Mmp9, and TIMP-1, and relative expression levels of collagen type I and III (  p < 0.05), while losartan significantly decreased the amount of hydroxyproline and downregulated TGF-β1, Mmp9, and TIMP-1 mRNA and collagen type I and III expressions (  p < 0.05). Histological examination of PQ-treated rats showed lung injury and widespread inflammatory cell infiltration in the alveolar space and pulmonary fibrosis, while losartan could markedly reduce such damage and prevent pulmonary fibrosis. The results of this study indicated that losartan could reduce lung damage and prevent pulmonary fibrosis induced by PQ.

scholarly journals Antifibrotic cardioprotection of berberine via downregulating myocardial IGF-1 receptor-regulated MMP-2/MMP-9 expression in diabetic rats

2018 ◽  
Vol 315 (4) ◽  
pp. H802-H813 ◽  
Author(s):  
Guohua Li ◽  
Wenjuan Xing ◽  
Min Zhang ◽  
Fenghao Geng ◽  
Hongyan Yang ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
High Glucose ◽  
Cardiac Fibrosis ◽  
Collagen Type ◽  
Cardiac Fibroblasts ◽  
Collagen Type I ◽  
Diabetic Rats ◽  
Type I ◽  
Antifibrotic Effect

Diabetic cardiac fibrosis increases ventricular stiffness and facilitates the occurrence of diastolic dysfunction. Our previous studies have shown that berberine, a natural alkaloid, attenuates cardiac ischemia-reperfusion injury in diabetic rats. The aim of present study was to investigate the effects of long-term berberine treatment on cardiac remodeling in diabetic rats and the underlying mechanisms. Diabetic rats induced by low-dose streptozotocin injection combined with 8 wk of high-fat diet displayed significant cardiac matrix collagen deposition and dysfunction, whereas berberine administration (200 mg·kg−1·day−1, gavage 4 wk) significantly ameliorated cardiac fibrosis and dysfunction and reduced cardiac IGF-1 receptor (IGF-1R) expression in diabetic rats. Interestingly, IGF-1R expression was upregulated in cardiac fibroblasts isolated from diabetic hearts or cultured in high-glucose conditions (30 mM). High glucose treatment or IGF-1R overexpression increased matrix metalloproteinase (MMP)-2/MMP-9 expression, α-smooth muscle actin (α-SMA), and collagen type I expression in cardiac fibroblasts. In contrast, berberine treatment significantly inhibited IGF-1R expression and exerted an antifibrotic effect in high glucose-cultured cardiac fibroblasts, as manifested by decreased MMP-2/MMP-9, α-SMA, and collagen type I expression, whereas IGF-1R siRNA plus berberine treatment did not further enhance this antifibrotic effect compared with berberine treatment alone. Taken together, long-term berberine treatment ameliorates cardiac fibrosis and dysfunction by downregulating IGF-1R expression in cardiac fibroblasts and subsequently reducing MMP-2/MMP-9, α-SMA, and collagen type I expression in diabetic hearts. The findings suggest the therapeutic potential of berberine for diabetic cardiomyopathy associated with cardiac fibrosis. NEW & NOTEWORTHY Berberine downregulated IGF-1 receptor expression and matrix metalloproteinase-2/matrix metalloproteinase-9 levels in cardiac fibroblasts and thus inhibited fibroblast differentiation and collagen overproduction in diabetic hearts, suggesting a novel mechanism for antifibrotic cardioprotection of berberine in type 2 diabetes.

Paeoniflorin inhibits TGF-β1-mediated collagen production bySchistosoma japonicumsoluble egg antigenin vitro

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1611-1621 ◽  
Author(s):  
D. CHU ◽  
Q. LUO ◽  
C. LI ◽  
Y. GAO ◽  
L. YU ◽  
...  
Keyword(s):  
Hepatic Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Peritoneal Macrophages ◽  
Type I ◽  
Smad3 Expression ◽  
Egg Antigen ◽  
Tgf Β1

SUMMARYThe main pathological characteristics of hepatic fibrosis in schistosomiasis are the proliferation of hepatic stellate cells (HSCs) and the deposition of collagen type I (Col I) and collagen type III (Col III). Transforming growth factor beta-1 (TGF-β1) plays an important role in hepatic fibrosis. Paeoniflorin (PAE) has been reported to have immunoregulatory effects; however, the mechanism of its anti-hepatic fibrosis inS. japonicumhas not been elucidated. In the present study, we found that mouse peritoneal macrophages (PMφs) stimulated by soluble egg antigen (SEA) ofS. japonicumcould secrete TGF-β1, and the TGF-β1 in the peritoneal macrophage-conditioned medium (PMCM) could induce proliferation of HSCs and secretion of Col I and III. We selected PMCM at 1:2 dilution as the optimum PMCM (OPMCM). Then we treated HSCs pre-incubated with OPMCM with PAE, and found that the inhibition of HSC proliferation or Col I and III production were closely correlated with the concentration of PAE. Further investigation found that PAE significantly decreased the Smad3 transcription and phosphorylation in HSCs stimulated by OPMCM. In conclusion, SEA plays a key role in hepatic fibrosis by inducing TGF-β1 from PMφs. PAE can exert anti-fibrogenic effects by inhibiting HSCs proliferation and down-regulating Smad3 expression and phosphorylation through TGF-β1 signalling.

scholarly journals Tenogenic differentiation of tonsil-derived mesenchymal stem cells

2017 ◽  
Vol 2 (3) ◽  
pp. 2473011417S0001
Author(s):  
Sangho Chun ◽  
Kyoung min Lee ◽  
Seung Yeol Lee
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Collagen Type ◽  
Collagen Type I ◽  
Immunofluorescence Staining ◽  
Type I ◽  
Stem Cell Markers ◽  
Pcr Assay ◽  
Tenogenic Differentiation

Category: Basic Sciences/Biologics Introduction/Purpose: Human palatine tonsil-derived mesenchymal stem cells (T-MSCs) are known to be a new source of progenitor cells. Using waste tissue after tonsillectomy as a cell provider can be the biggest benefit of T-MSCs, compared with other stem cells. The purpose of this study was to investigate tenogenic differentiation of T-MSCs and to access the differential effects of TGF-ß3 on the tenogenesis of T-MSCs. Methods: Human tonsil was obtained after tonsillectomy.Using a cytometric analysis, we were able to find that the T-MSCs had typical mesenchymal stem cell markers: positive for CD73, CD90 and CD105, and negative for CD14, CD34 and CD45. Using a Transforming growth factor beta 3 (TGF-ß3), the expressions of tenocyte-specific genes and proteins, such as Collagen type I (COL1), Tenomodulin (TNMD), and Scleraxis (SCX), were measured by a quantitative polymerase chain reaction (PCR) assay, immunofluorescence staining, immunohistochemistry and Western blot analysis. Results: Quantitative PCR assay showed that TGF-ß3 significantly increased the expressions of tenocyte lineage marker genes, including Collagen type I (COL1), Tenomodulin (TNMD), and Scleraxis (SCX), at a 3-day treatment, compared with control. However, these increases were not found at long-term exposures (7 or 10 days), except that TNMD expression was maintained at 50 ng/ml at 7-day exposure to TGF-ß3 (Fig). Like genes, the protein expression levels of COL1, TNMD, and SCX were also induced in TGF-ß3-treated T-MSCsin 3-day treatments. Moreover, the protein levels were maintained for 10 days, as evidenced by immunofluorescence staining, immunohistochemistry and Western blot analyses. Conclusion: This study demonstrated that T-MSCs in tenogenic stimulation with TGF-ß3 have high tenogenic differentiation potential.

microRNA-122 down-regulation may play a role in severe myocardial fibrosis in human aortic stenosis through TGF-β1 up-regulation

2013 ◽  
Vol 126 (7) ◽  
pp. 497-506 ◽  
Author(s):  
Javier Beaumont ◽  
Begoña López ◽  
Nerea Hermida ◽  
Blanche Schroen ◽  
Gorka San José ◽  
...  
Keyword(s):  
Aortic Stenosis ◽  
Myocardial Fibrosis ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Down Regulation ◽  
Enzymatic Systems ◽  
Tgf Β1 ◽  
Stimulation Of

We have found an association of miR-122 down-regulation with myocardial fibrosis in AS patients, probably through TGF-β1 up-regulation and stimulation of the enzymatic systems involved in extracellular collagen type I synthesis and deposition.

Stimulation of peroxisome-proliferator-activated receptor α (PPAR α) attenuates cardiac fibrosis and endothelin-1 production in pressure-overloaded rat hearts

2002 ◽  
Vol 103 (s2002) ◽  
pp. 284S-288S ◽  
Author(s):  
Takehiro OGATA ◽  
Takashi MIYAUCHI ◽  
Satoshi SAKAI ◽  
Yoko IRUKAYAMA-TOMOBE ◽  
Katsutoshi GOTO ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Myocardial Fibrosis ◽  
Cardiac Fibrosis ◽  
Collagen Type ◽  
Collagen Type I ◽  
Peroxisome Proliferator ◽  
Type I ◽  
Aortic Banding ◽  
Peroxisome Proliferator Activated Receptor ◽  
Rat Hearts

Endothelin-1 (ET-1) production is increased in hypertrophied hearts accompanied with fibrosis. ET-1 is a potent mitogen of fibroblasts and ET receptor antagonists are reported to inhibit the proliferation of fibroblasts and cardiac fibrosis. Peroxisome-proliferator-activated receptor α (PPARα), one of the nuclear hormone receptors, suppresses activator protein-1 (AP-1), one of the nuclear transcription factors. Activation of PPARα is reported to inhibit thrombin-induced ET-1 production by repressing the AP-1 signalling pathway in vascular endothelial cells. We investigated effects of the PPARα activator fenofibrate (80mg/kg per day, per os) on mRNA levels of ET-1, collagen type I and type III and histological features of myocardial fibrosis in hypertrophied rat hearts due to pressure-overload by abdominal aortic banding (AB). The treatment with fenofibrate or vehicle was started 7 days before the AB operation. Four days after the AB operation, fenofibrate treatment significantly reduced ET-1 mRNA expression compared with vehicle treatment in AB rat hearts. Collagen type I and type III mRNA expression, and interstitial and perivascular fibrosis were attenuated in the fenofibrate-treated AB rat group. Since the ET-1 gene has AP-1 response elements in the 5´-flanking region, it is suggested that myocardial fibrosis is effectively inhibited by fenofibrate through suppression of AP-1-mediated ET-1 gene augmentation in the pressure-overloaded heart caused by aortic banding in rats.

Mechanisms of skin toxicity of paclitaxel: An in vitro preclinical assessment.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15511-e15511
Author(s):  
Jose Alejandro Perez-Fidalgo ◽  
Cristina Estornut Navarro ◽  
Celia Sanz Garcia ◽  
Martin Perez-Leal ◽  
Jesus Poveda Ferriols ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
Collagen Type ◽  
Skin Toxicity ◽  
Collagen Type I ◽  
Tube Formation ◽  
Preclinical Model ◽  
Type I ◽  
Endothelial Tube Formation ◽  
Angiogenic Markers

e15511 Background: Paclitaxel skin toxicity is a frequent side effect extensively evaluated in the clinical setting. However little is known about the preclinical mechanisms that lead to this toxicity. The endpoint of this study was to analyse the cutaneous mechanisms that drive paclitaxel toxicity in a preclinical model. Methods: Primary human keratinocytes were co-cultured with human dermal fibroblast in collagen gel under air-liquid interface conditions to generate a multilayered 3D epidermis. Paclitaxel was added to 3D epidermis at 0.3 µM, 3 µM and 30 µM and total RNA and protein was extracted after 24h of incubation. Markers of cell senescence (p21 and p53), anti-apoptotic mediators (Bcl-2), skin elasticity (tropoelastin ELN, collagen type I and fibronectin), hidratation (aquaporin 3 AQP3), oxidative stress (NOX4), antioxidant (SOD1, Nrf2), and angiogenic markers (VEGFR, eNOS) were evaluated by RT-PCR and western blot. NfKB phosphorylation was measured by western blot and inflammatory citokines IL1α, IL-6 and IL-8 were measured in cultured supernatants by ELISA. Human primary melanocytes were cultured and stimulated with paclitaxel to measure melanogenesis through the expression of of TYR, TYRP1 and DCT genes. The effect of paclitaxel on skin endothelial cell angiogenesis was measured by endothelial tube formation. Results: In human 3D keratinocytesPaclitaxel inhibited the expression of Bcl-2, and increased the expression of p53 and p21. The angiogenic markers VEGF and eNOS were decreased. The expression of oxidative stress marker NOX4 was increased and the anti-oxidant mediators Nrf2 and SOD1 were decreased. Markers of elasticity, collagen type I, fibronectin and FN1 were decreased as occurs with AQP3 hidratation marker. Paclitaxel increased the phosphorylation of NfkB and elevated the secretion of IL1α, IL-6 and IL-8 cytokines. In skin endothelial cells, paclitaxel reduced the endothelial tube formation. In melanocytes, paclitaxel increased skin pigmentation represented by the increase of RNA expression of TYR, TYRP1 and DCT genes. Conclusions: This preclinical 3D model showed that paclitaxel impacts on the expression of proteins related with angiogenics, elasticity and senescence in human kerantinocytes. Moreover higher doses of paclitaxel increased inflammation parameters and confirmed phototoxic and anti-angiogenenic effects. This preclinical model could be a valuable tool to assess skin toxicity of new antineoplastic agents.

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