Abstract 294: Differential Expression of Hedgehog/Notch and Transforming Growth Factor-ß in Human Abdominal Aortic Aneurysms

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Adam Doyle ◽  
Eileen M Redmond ◽  
John P Cullen ◽  
Paul A Cahill ◽  
David Morrow
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Notch Signaling ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Aortic Aneurysms ◽  
Causative Role ◽  
Differentiation Gene ◽  
Tgf Β1

Objective: The molecular mechanisms leading to the development of Adnominal Aortic Aneurysms (AAA) remain poorly understood. The aim of this study was to determine the expression of Sonic Hedgehog (SHh), Transforming Growth Factor Beta (TGF-β) and Notch signaling components in aneurysmal and non-aneurysmal aorta in vivo and demonstrate SHh control of Notch, TGF- β1 and SMC differentiation in vitro. Methods: Paired tissue samples were obtained from aneurysmal and non-aneurysmal (control) segments of the aortic wall of at least 8 patients with suitable anatomy undergoing open repair of infrarenal AAA. Protein and mRNA expression levels were determined by western blot analysis and quantitative Real-time PCR. Results: Aneurysm development resulted in a significant reduction in vascular smooth muscle (vSMC) differentiation gene protein and mRNA levels for α-actin and SMC22α, respectively. In parallel, significant reductions in Hh and Notch signaling component expression was observed in aneurysmal tissue when compared to control, concurrent with increased TGF-β1 expression. In vitro, Hh signaling inhibition with cyclopamine (40μmol/L) treatment for 24 h in human aortic smooth muscle cells (HASMC), resulted in decreased Hh/Notch signaling component and vSMC differentiation gene expression. Moreover, cyclopamine significantly increased TGF-β1 mRNA expression. Conclusion: These results suggest that SHh/Notch and TGFβ signaling are differentially regulated in aneurysmal tissue, compared with non-aneurysmal tissue. Changes in these signaling pathways and the resulting changes in vSMC content may play a causative role in the development of AAA.

Abstract 297: Cyclic GMP Reduces Vascular Smooth Muscle Migration through Inhibition of TGF-ß1/Smad Signaling

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Jackson R Vuncannon ◽  
Joshua D Stone ◽  
Danielle N Martin ◽  
Chintamani N Joshi ◽  
Shaquria P Adderley ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Cyclic Gmp ◽  
Transforming Growth Factor ◽  
Soluble Guanylate Cyclase ◽  
Sandwich Elisa ◽  
Cell Lysates ◽  
Tgf Β1

Abnormal vascular smooth muscle (VSM) growth remains an elemental foundation of vasculoproliferative disorders including atherosclerosis and restenosis. Many second messenger, cytokine, and growth factor signals mediate control of VSM growth, and among these is transforming growth factor (TGF)-β1, a pluripotent cytokine with wide-ranging yet often opposite effects in VSM. Cyclic nucleotide signaling also exerts powerful growth control of VSM, and our previous work has helped establish a biological link between cyclic GMP and TGF-β1 in injured carotid arteries. The current study characterized the influence of cyclic GMP on TGF-β1 and its receptor-activated Smad3 in rat primary VSM cells. The heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY41) significantly increased cyclic GMP and site-specific phosphorylation of vasodilator-activated serum phosphoprotein (VASP) in manner indicative of active protein kinase G (PKG) and PKA signaling. Recombinant TGF-β1 (10 ng/ml) significantly stimulated phospho-Smad3 (Ser 423/425 ) and decreased inhibitory Smad7 in VSM cell homogenates, and using flow cytometry significantly increased cells in G 2 /M and expression of cyclins D and E and Cdk2 and Cdk4 while decreasing expression of inhibitory p21 and p27 after 24 hours compared to vehicle controls. TGF-β1 also significantly increased cell numbers compared to controls after 48 hours, thus confirming growth promoting capacities of TGF-β1 in VSM. In cell lysates double-sandwich ELISA revealed that BAY41 significantly reduces total and active TGF-β1, and Western analyses showed it significantly decreases total and phospho-Smad3 Ser423/425 expression and reduces MMP-2 and MMP-9 expression and activity (via column zymography) in both cell lysates and conditioned media after 1 and 48 hours. BAY41 also significantly reduced serum- and PDGF-stimulated cell migration between 6 and 18 hours using an in vitro scrape injury and a transwell assay. In comparison, inclusive effects of BAY41 were replicated by its prototype YC-1 and by the heme-independent sGC activator BAY 60-2770. These data clearly support growth protective capacities of cGMP in VSM and propose it operates through attenuation of TGF-β1/Smad3 signaling.

Transforming growth factor-β1 signaling contributes to development of smooth muscle cells from embryonic stem cells

2004 ◽  
Vol 287 (6) ◽  
pp. C1560-C1568 ◽  
Author(s):  
Sanjay Sinha ◽  
Mark H. Hoofnagle ◽  
Paul A. Kingston ◽  
Mary E. McCanna ◽  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Model System ◽  
Embryoid Bodies ◽  
Regulatory Pathways ◽  
Tgf Β1

Knockout of transforming growth factor (TGF)-β1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-β1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle α-actin (SMαA), SM22α, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-β with an adenovirus expressing a soluble truncated TGF-β type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-β1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-β-Smad signaling for an early vs. late SMC marker gene in that SMαA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-β1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.

scholarly journals Pyrrole-Imidazole Polyamide Targeting Transforming Growth Factor β1 Ameliorates Encapsulating Peritoneal Sclerosis

2012 ◽  
Vol 32 (4) ◽  
pp. 462-472 ◽  
Author(s):  
Kazuo Serie ◽  
Noboru Fukuda ◽  
Shigeki Nakai ◽  
Hiroyuki Matsuda ◽  
Takashi Maruyama ◽  
...  
Keyword(s):  
Growth Factor ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Transforming Growth Factor ◽  
Fluorescein Isothiocyanate ◽  
Transforming Growth Factor Β1 ◽  
Vegf Mrna ◽  
Tgf Β1

ObjectiveEncapsulating peritoneal sclerosis (EPS) is a devastating fibrotic complication in patients treated with peritoneal dialysis (PD). Transforming growth factor β1 (TGF-β1) is a pivotal factor in the induction of EPS.MethodsTo develop pyrrole-imidazole (PI) polyamide, a novel gene silencer, targeted to the TGF-β1 promoter (Polyamide) for EPS, we examined the effects of Polyamide on messenger RNA (mRNA) expression of TGF-β 1, vascular endothelial growth factor (VEGF), and extracellular matrix (ECM) in mesothelial cells in vitro, and on the thickness of injured peritoneum evaluated by histology and high- resolution regional elasticity mapping in rats in vivo.ResultsPolyamide significantly lowered mRNA expression of TGF-β 1 and ECM in vitro. Polyamide labeled with fluorescein isothiocyanate was taken up into the injured peritoneum and was strongly localized in the nuclei of most cells. Polyamide 1 mg was injected intraperitoneally 1 or 3 times in rats receiving a daily intraperitoneal injection of chlorhexidine gluconate and ethanol (CHX) for 14 days. Polyamide significantly suppressed peritoneal thickening and the abundance of TGF-β 1 and fibronectin mRNA, but did not affect expression of VEGF mRNA in the injured peritoneum. Elasticity distribution mapping showed that average elasticity was significantly lower in Polyamide-treated rats than in rats treated solely with CHX.ConclusionsPolyamide suppressed the stiffness, ECM formation, and thickening of the injured peritoneum that occurs during EPS pathogenesis. These data suggest that PI polyamide targeted to the TGF-β 1 promoter will be a specific and feasible therapeutic strategy for patients with EPS.

Abstract 96: Aberrations in the Sonic Hedgehog/Notch Signaling Pathway in Abdominal Aortic Aneurysms

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Adam J Doyle ◽  
David L Gillespie ◽  
Eileen M Redmond ◽  
Peter Knight ◽  
Neil G Kumar ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Expression ◽  
Notch Signaling ◽  
Mrna Expression ◽  
Transforming Growth Factor Beta ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Aortic Aneurysms ◽  
Notch 1 ◽  
Tissue Samples

Objectives: The molecular mechanisms leading to the development of Adnominal Aortic Aneurysms (AAA) remain poorly understood. Vascular Smooth Muscle Cells (VSMCs) are fundamental to maintaining a healthy arterial wall and changes in VSMC phenotype may be pivotal to aneurysm development. We have recently determined a role for a Hedgehog (Hh)/Notch signaling cascade in regulating adult VSMC phenotype. The aim of this study was to investigate Hh/Notch signaling components in aneurysmal and non-aneurysmal aorta. As crosstalk between Notch and Transforming Growth Factor Beta (TGFβ) has been postulated we also investigated expression of this growth factor. Methods: Tissue samples were obtained from aneurysmal and non-aneurysmal segments of the aortic wall of at least 5 patients with suitable anatomy undergoing open repair of infrarenal AAA. All samples analyzed were paired from the same patient with aneurysmal and non-aneurysmal specimens. Protein and mRNA expression levels were determined by western blot analysis and quantitative Real-time PCR respectively. Results: SHh, Notch 1 and 3 IC protein expression was decreased by at least 50% in aneurysmal tissue when compared to non-aneurysmal tissue. In addition, SHh mRNA expression was also decreased by 65%, while there was a decrease in Dll4, Notch 1 and Notch 3 by 66%, 57% and 54% respectively. In contrast, aneurysmal tissue had significantly increased expression of TGFβ and MMP9. TGFB protein and mRNA expression was significantly increased by 5.45±2.13 and 2.5±.2 fold respectively in aneurysmal tissue when compared to non-aneurysmal tissue. Furthermore, MMP9 mRNA expression was significantly increased by 4.7±1.6 fold. In parallel experiments, SMC alpha actin protein expression was significantly decreased by 90% in aneurysmal tissue when compared to non-aneurysmal tissue. Conclusion: These results suggest that SHH/Notch and TGFβ signaling is altered in aneurysmal tissue, compared with non-aneurysmal tissue. Changes in these signaling pathways and resulting changes in VSMC phenotype may play a role in the development of AAA.

scholarly journals Differences in Transforming Growth Factor‐β1/BMP7 Signaling and Venous Fibrosis Contribute to Female Sex Differences in Arteriovenous Fistulas

2020 ◽  
Vol 9 (16) ◽  
Author(s):  
Chuanqi Cai ◽  
Sreenivasulu Kilari ◽  
Avishek K. Singh ◽  
Chenglei Zhao ◽  
Michael L. Simeon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Vascular Remodeling ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Tgf Β1

Background Women have decreased hemodialysis arteriovenous fistula (AVF) maturation and patency rates. We determined the mechanisms responsible for the sex‐specific differences in AVF maturation and stenosis formation by performing whole transcriptome RNA sequencing with differential gene expression and pathway analysis, histopathological changes, and in vitro cell culture experiments from male and female smooth muscle cells. Methods and Results Mice with chronic kidney disease and AVF were used. Outflow veins were evaluated for gene expression, histomorphometric analysis, Doppler ultrasound, immunohistologic analysis, and fibrosis. Primary vascular smooth muscle cells were collected from female and male aorta vessels. In female AVFs, RNA sequencing with real‐time polymerase chain reaction analysis demonstrated a significant decrease in the average gene expression of BMP7 (bone morphogenetic protein 7) and downstream IL17Rb (interleukin 17 receptor b) , with increased transforming growth factor‐β1 ( Tgf‐β1) and transforming growth factor‐β receptor 1 ( Tgfβ‐r1) . There was decreased peak velocity, negative vascular remodeling with higher venous fibrosis and an increase in synthetic vascular smooth muscle cell phenotype, decrease in proliferation, and increase in apoptosis in female outflow veins at day 28. In vitro primary vascular smooth muscle cell experiments performed under hypoxic conditions demonstrated, in female compared with male cells, that there was increased gene expression of Tgf‐β1 , Tgfβ‐r1 , and Col1 with increased migration. Conclusions In female AVFs, there is decreased gene expression of BMP7 and IL17Rb with increased Tgf‐β1 and Tgfβ‐r1 , and the cellular and vascular differences result in venous fibrosis with negative vascular remodeling.

scholarly journals Transforming Growth Factor β1 (TGF-β1) Promotes Endothelial Cell Survival during In Vitro Angiogenesis via an Autocrine Mechanism Implicating TGF-α Signaling

2001 ◽  
Vol 21 (21) ◽  
pp. 7218-7230 ◽  
Author(s):  
Francesc Viñals ◽  
Jacques Pouysségur
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Cell Survival ◽  
Network Formation ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
In Vitro Angiogenesis ◽  
Tgf Β1

ABSTRACT Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor β1 (TGF-β1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-β1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-β1-induced angiogenesis mainly by compromising cell survival. We established that TGF-β1 stimulated the expression of TGF-α mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-β1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-β1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-α alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-β1. We therefore propose that TGF-β1 promotes angiogenesis at least in part via the autocrine secretion of TGF-α, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.

Abstract 2681: Mutations in Smooth Muscle-Specific Contractile Proteins Cause Familial Thoracic Aortic Aneurysms and Dissections and Lead to Increased IGF-1 Expression

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Christina L Papke ◽  
Hariyadarshi Pannu ◽  
Dong-Chuan Guo ◽  
Nili Avidan ◽  
Van Tran-Fadulu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Contractile Proteins ◽  
Dermal Fibroblasts ◽  
Aortic Aneurysms ◽  
Trophic Factors ◽  
Aortic Tissue ◽  
Thoracic Aortic Aneurysms ◽  
Protein Levels ◽  
Tgf Β1

Aortic aneurysms and dissections are the most common disorders affecting the aorta, and are a major cause of morbidity and mortality in the United States. Familial thoracic aortic aneurysms and dissections (FTAAD) are inherited in an autosomal dominant manner with variable expression and decreased penetrance. The disorder is genetically heterogeneous with four loci and three genes identified. Mutations in either TGFBR2 , encoding the transforming growth factor β (TGF-β) type II receptor, or MYH11 , encoding the smooth muscle cell (SMC)-specific β-myosin heavy chain, were previously found to cause FTAAD. Recently, positional cloning identified smooth muscle α-actin ( ACTA2 ) mutations as a novel cause in 10% of FTAAD. Mutations in ACTC and MYH7 cause hypertrophic cardiomyopathy (HCM), characterized by myocyte disarray and upregulation of mitotic and trophic factors. Histologic examination of aortic tissue from patients with ACTA2 (n = 6) and MYH11 (n = 2) mutations revealed SMC disarray in the aortic media similar to that seen in HCM. Furthermore, we hypothesized that mutations in ACTA2 and MYH11 cause a similar increase of mitotic and trophic factors in SMCs. The expression of two factors known to be increased in HCM, TGF-β1 and insulin-like growth factor 1 (IGF-1), were analyzed in patients’ aortic SMCs and dermal myofibroblasts. No changes in TGF-β1 were found; however, both mRNA, as measured by Q-PCR (p<0.05), and protein levels, as assessed by immunostaining, of IGF-1 were markedly increased in MYH11 and two ACTA2 mutant SMCs and aortic tissue compared with control SMCs and tissue. Differentiation of dermal fibroblasts into myofibroblasts was accomplished using TGF-β1 treatment; myofibroblast differentiation was confirmed by assessing α-actin mRNA and protein levels in untreated vs. TGF-β1-treated fibroblasts. Upon differentiation, patients’ myofibroblasts (n = 3) demonstrated increased IGF-1 expression compared with controls (p<0.05), similar to the increased IGF-1 expression by SMCs. In conclusion, IGF-1 secretion is increased in response to defects in SMC contractile proteins in SMCs and myofibroblasts. Future studies will clarify the role of IGF-1 in FTAAD and identify the pathways leading to increased IGF-1 expression.

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