TGF-β1 + EGF-Initiated Invasive Potential in Transformed Human Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1–Dependent Collagen Remodeling Axis: Role for PAI-1

Abstract Abstract 1601 Objective: The dysfunction of human diabetic CD34+ endothelial progenitor cells limits their utility in autologous cell therapy for vascular complications. Previously, we showed that transient inhibition of transforming growth factor-beta 1 (TGF-β1) enhances vascular reparative function of human CD34+ cells isolated from diabetics (Bhatwadekar et al, 2010). Expression of PAI-1, the major gene product of TGF-β1 activation, is increased by high glucose and insulin exposure in endothelial cells and PAI-1 has been shown to be increased in the serum of diabetics. We asked whether the beneficial effects of TGF-β1 blockade on CD34+ cells function were mediated by inhibition of PAI-1 and whether blocking of PAI-1 could correct diabetes associated dysfunction of these cells. Research Design and Methods: Plasma determinations of PAI-1 and TGF-β1 (both measured by ELISA) were compared in type 2 (n=17) and type 1 (n=7) diabetic patients. CD34+ cells from these individuals were isolated and analyzed for cell survival (in the presence and absence of growth factors), cell proliferation, cell cycle analysis and migration. The effect of TGF-β1 phosphorodiamidate morpholino oligomers (PMO) treatment on PAI-1 level was determined in CD34+ cells. In CD34+ cells, PAI-1 was blocked using either lentivirus expressing PAI-1 shRNA or PAI-1 siRNA. In vivo homing ability of PAI-1 inhibited CD34+ cells was assessed using an ocular model of ischemia/reperfusion (I/R) Injury. Results: Plasma PAI-1 level was increased in type 2 diabetic patients compared to type 1 (p<0.05) and directly correlated with TGF-β1 plasma levels (r= 0.44). TGF-β1 PMO treatment resulted in a reduction of PAI-1 mRNA expression (p=0.0018 in diabetic, p=0.05 in non-diabetic). PAI-1 blockade promoted EPC proliferation in vitro and bypassed the inhibitory effect of TGF-β1 on cell survival (p<0.001) even in the absence of growth factors. PAI-1 blockade enhanced the migration of these cells in response to SDF-1α in (p<0.01) compared to cells treated with scrambled siRNA and improved the in vivo re-endothelialization by CD34+ cells in the I/R model. Conclusions: Our results suggest that the cytostatic activity of TGF-β1 in CD34+ cells is mediated largely through PAI-1. Blocking PAI-1 corrects multiple defects in CD34+ cells from type 2 diabetic patients. This approach may offer a promising therapeutic strategy for restoring vascular reparative function in diabetic cells and facilitate their use in autologous cell therapy. Disclosures: No relevant conflicts of interest to declare.

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Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts

Journal of Endocrinology ◽

10.1677/joe-07-0408 ◽

2007 ◽

Vol 196 (2) ◽

pp. 235-249 ◽

Cited By ~ 36

Author(s):

Jorge N Artaza ◽

Rajan Singh ◽

Monica G Ferrini ◽

Melissa Braga ◽

James Tsao ◽

...

Keyword(s):

Transforming Growth Factor ◽

Plasminogen Activator Inhibitor ◽

Mouse Cell ◽

Transforming Growth Factor Β1 ◽

Negative Regulator ◽

Public Health Burden ◽

Muscle Fibrosis ◽

Differentiated Cells ◽

Tgf Β1 ◽

Pai 1

Tissue fibrosis, the excessive deposition of collagen/extracellular matrix combined with the reduction of the cell compartment, defines fibroproliferative diseases, a major cause of death and a public health burden. Key cellular processes in fibrosis include the generation of myofibroblasts from progenitor cells, and the activation or switch of already differentiated cells to a fibrotic synthetic phenotype. Myostatin, a negative regulator of skeletal muscle mass, is postulated to be involved in muscle fibrosis. We have examined whether myostatin affects the differentiation of a multipotent mesenchymal mouse cell line into myofibroblasts, and/or modulates the fibrotic phenotype and Smad expression of the cell population. In addition, we investigated the role of follistatin in this process. Incubation of cells with recombinant myostatin protein did not affect the proportion of myofibroblasts in the culture, but significantly upregulated the expression of fibrotic markers such as collagen and the key profibrotic factors transforming growth factor-β1 (TGF-β1) and plasminogen activator inhibitor (PAI-1), as well as Smad3 and 4, and the pSmad2/3. An antifibrotic process evidenced by the upregulation of follistatin, Smad7, and matrix metalloproteinase 8 accompanied these changes. Follistatin inhibited TGF-β1 induction by myostatin. Transfection with a cDNA expressing myostatin upregulated PAI-1, whereas an shRNA against myostatin blocked this effect. In conclusion, myostatin induced a fibrotic phenotype without significantly affecting differentiation into myofibroblasts. The concurrent endogenous antifibrotic reaction confirms the view that phenotypic switches in multipotent and differentiated cells may affect the progress or reversion of fibrosis, and that myostatin pharmacological inactivation may be a novel therapeutic target against fibrosis.

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Effect of TGF-β1 on Re-Epithelialization of Human Keratinocytes In Vitro: An Organotypic Model

Journal of Investigative Dermatology ◽

10.1111/1523-1747.ep12396847 ◽

1994 ◽

Vol 103 (4) ◽

pp. 554-559 ◽

Cited By ~ 56

Author(s):

Jonathan A Garlick ◽

Lorne B Taichman

Keyword(s):

Human Keratinocytes ◽

Tgf Β1

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IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-β1 resulting in epithelial to mesenchymal transition

Cell Communication and Signaling ◽

10.1186/1478-811x-9-10 ◽

2011 ◽

Vol 9 (1) ◽

pp. 10 ◽

Cited By ~ 55

Author(s):

Logan A Walsh ◽

Sashko Damjanovski

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Epithelial To Mesenchymal Transition ◽

Invasive Potential ◽

Mesenchymal Transition ◽

Tgf Β1 ◽

Mcf 7

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Catalpol Protects Against Pulmonary Fibrosis Through Inhibiting TGF-β1/Smad3 and Wnt/β-Catenin Signaling Pathways

Frontiers in Pharmacology ◽

10.3389/fphar.2020.594139 ◽

2021 ◽

Vol 11 ◽

Author(s):

Fan Yang ◽

Zhen-feng Hou ◽

Hao-yue Zhu ◽

Xiao-xuan Chen ◽

Wan-yang Li ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathways ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Gene Expression Omnibus ◽

Matrix Remodeling ◽

Mesenchymal Transition ◽

Collagen Remodeling ◽

Gsk 3Β ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by fibroblast proliferation and extracellular matrix remodeling; however, the molecular mechanisms underlying its occurrence and development are not yet fully understood. Despite it having a variety of beneficial pharmacological activities, the effects of catalpol (CAT), which is extracted from Rehmannia glutinosa, in IPF are not known. In this study, the differentially expressed genes, proteins, and pathways of IPF in the Gene Expression Omnibus database were analyzed, and CAT was molecularly docked with the corresponding key proteins to screen its pharmacological targets, which were then verified using an animal model. The results show that collagen metabolism imbalance, inflammatory response, and epithelial-mesenchymal transition (EMT) are the core processes in IPF, and the TGF-β1/Smad3 and Wnt/β-catenin pathways are the key signaling pathways for the development of pulmonary fibrosis. Our results also suggest that CAT binds to TGF-βR1, Smad3, Wnt3a, and GSK-3β through hydrogen bonds, van der Waals bonds, and other interactions to downregulate the expression and phosphorylation of Smad3, Wnt3a, GSK-3β, and β-catenin, inhibit the expression of cytokines, and reduce the degree of oxidative stress in lung tissue. Furthermore, CAT can inhibit the EMT process and collagen remodeling by downregulating fibrotic biomarkers and promoting the expression of epithelial cadherin. This study elucidates several key processes and signaling pathways involved in the development of IPF, and suggests the potential value of CAT in the treatment of IPF.

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