Phenotypic differentiation of adventitial fibroblasts into myofibroblasts is an essential feature of vascular remodeling. The present study was undertaken to test the hypothesis that reactive oxygen species (ROS) are involved in rat adventitial fibroblast differentiation to myofibroblast. Activation of alpha-smooth muscle actin (alpha-SMA) was used as a marker of myofibroblast. Angiotensin II increased intracellular ROS in adventitial fibroblasts that was completely inhibited by the free radical scavenger NAC, the NAD(P)H oxidase inhibitor DPI, and transfection of antisense gp91phox oligonucleotides. Myofibroblast differentiation was prevented by inhibition of ROS generation with DPI, NAC, and antisense gp91phox as shown by decreased expression of alpha-SMA. Angiotensin II rapidly induced phosphorylation of p38 MAPK and JNK, both of which were inhibited by DPI, NAC, antisense gp91phox, and the selective AT1 receptor antagonist, losartan. Inhibiting p38MAPK with SB202190 or JNK with SP600125 also reduced angiotensin II-induced alpha-SMA expression. These findings demonstrate that angiotensin II induces adventitial fibroblast differentiation to myofibroblast via a pathway that involves NADPH oxidase generation of ROS and activation of p38MAPK and JNK pathways.
"In fibroblasts of adventitia from vascular cells, ROS generated by NAD(P)H oxidase, activated the MAPK and finally the fibroblasts differentiated to MFs . To check the importance of MAPK during the transition process of HDF, HDF were exposed to rTGFβ1 in the presence and absence of non-toxic concentration of U0126, SP600125 and SB202190 (10 μM). "
[Show abstract][Hide abstract] ABSTRACT: Tumor-stroma interaction is a prerequisite for tumor progression in skin cancer. Hereby, a critical step in stromal function is the transition of tumor-associated fibroblasts to myofibroblasts by growth factors, for example transforming growth factor beta (TGFβ). In this study, the question was addressed of whether fibroblast associated NAD(P)H oxidase, known to be activated by TGFβ1, is involved in the fibroblast-to-myofibroblast switch. The up regulation of alpha smooth muscle actin (αSMA), a biomarker for myofibroblasts, is mediated by a TGFβ1-dependent increase in the intracellular level of reactive oxygen species (ROS). The source of these ROS was identified to be NAD(P)H oxidase, showing two activity peaks over time with an early and late activity peak after treatment with the growth factor. The late (second) activity peak of NAD(P)H oxidase was identified to be responsible for the downstream signaling resulting in reactive oxygen species triggered activation of the stress kinase p38 and expression of αSMA. These data suggest that inhibition of NAD(P)H oxidase activity prevents the fibroblast-to-myofibroblast switch and may be important for chemoprevention.
"We previously showed that Ang II activates MAPKs in adventitial fibroblasts . Therefore, we examined whether the MAPK activation was involved in Ang II–induced OPN expression in adventitial fibroblasts. "
[Show abstract][Hide abstract] ABSTRACT: Osteopontin is known to play important roles in various diseases including vascular disorders. However, little is known about its expression and function in vascular adventitial fibroblasts. Adventitial fibroblasts have been shown to play a key role in pathological vascular remodeling associating with various vascular disorders. In this study, we measured activation of Osteopontin and its biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results showed that angiotensin II and aldosterone increased Osteopontin expression in adventitial fibroblasts in a time- and concentration-dependent manner. MAPKs and AP-1 pathways were involved in Osteopontin upregulation. In addition, Adventitial fibroblast migration stimulated by Angiotensin II and aldosterone required OPN expression. Perivascular delivery of antisense oligonucleotide for Osteopontin suppressed neointimal formation post-injury. We concluded that upregulation of Osteopontin expression in adventitial fibroblasts might be important in the pathogenesis of vascular remodeling after arterial injury.
PLoS ONE 10/2011; 6(9):e23558. DOI:10.1371/journal.pone.0023558 · 3.23 Impact Factor
"Annexin A1 has been shown to modulate ERK but not p38 or JNK activity in lipopolysaccharride-induced responses20 and regulate cell proliferation by disruption of cell morphology and inhibition of cyclin D1 expression through sustained activation of the ERK1/2 MAPK pathway21. Our previous studies showed that ERK1/2 was activated during phenotypic differentiation of AFs into MFs22 and migration induced by Ang II23. Moreover, annexin A1 is a substrate for protein kinase C (PKC) and protein tyrosine kinases and has multiple phosphorylation sites as well as calcium and phospholipid binding properties. "
[Show abstract][Hide abstract] ABSTRACT: To identify proteins that could potentially be involved in adventitial remodeling in vascular adventitial fibroblasts (AFs) from spontaneously hypertensive rats (SHR).
AFs were isolated from thoracic aortas of 4-, 8-, 16-, and 24-week-old male SHR and Wistar-Kyoto (WKY) rats and cultured to passage 4. Proteomic differential expression profiles between SHR-AFs and WKY-AFs were investigated using 2-D electrophoresis (2-DE), whereas gel image analysis was processed using Image Master 2D Platinum. Protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Expression levels of annexin A1 in AFs and aortas from SHR and WKY rats were detected with Western blotting and immunofluorescence techniques.
In 4-, 8-, 16-, and 24-week-old SHR-AFs, 49, 59, 54, and 69 protein spots were found to have significant differences from the age-matched WKY-AFs. Fourteen spots with the same changes in patterns were analyzed in 4-, 8-, 16-, and 24-week-old SHR-AFs with mass spectrometry. Except for cytoskeleton proteins such as tubulin beta 5, it was found that annexin A1, translation elongation factor Tu, endoplasmic reticulum protein 29 and calcium-binding protein 1 were expressed in vascular AFs and their levels changed significantly in SHR-AFs compared with those in WKY-AFs. A decrease in annexin A1 in SHR-AFs was confirmed with Western blotting and immunofluorescence staining at the cell and tissue levels.
The application of proteomic techniques revealed a number of novel proteins involved in adventitial remodeling of AFs from SHR, which provide new mechanisms responsible for the occurrence and development of hypertension and potential targets for influencing vascular remodeling in hypertension.
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