Migration Of Adventitial Fibroblasts Research Articles

Smad2/3 signaling involved in urotensin II-induced phenotypic differentiation, collagen synthesis and migration of rat aortic adventitial fibroblasts

Objective. To investigate whether Smad2/3 signaling is involved in urotensin II (UII) induced activation of aortic adventitial fibroblasts. Materials and Methods. Growth-arrested adventitial fibroblasts were stimulated with UII in the presence or absence of urotensin II receptor (UT) antagonist SB710411 or transfected with Smad2/3 small inhibitory RNA (siRNA). UII stimulated Smad2/3 phosphorylation, α-smooth muscle actin (α-SMA), and collagen I expression and migration of adventitial fibroblasts were evaluated by western blot analysis, real-time reverse transcription polymerase chain reaction, immunofluorescence, ELISA, and transwell migration assay, respectively. Results. In cultured adventitial fibroblasts, UII time- and dose-dependently stimulated Smad2/3 protein phosphorylation, with maximal effect at 10-8 mol/l (increased by 147.2%, P<0.001). UII stimulated Smad2/3 upregulation and nuclear translocation. SB710411 significantly inhibited these effects. In addition, UII potently induced α-SMA and procollagen 1 protein or mRNA expression (P<0.01), which were completely blocked by Smad2 (decreased by 75.1%, 54.2% in protein, and by 73.3% and 38.2% in mRNA, respectively, P<0.01) or Smad3 siRNA (decreased by 80.3% and 47.0% in protein, and by 72.3% and 47.7% in mRNA, respectively, P<0.01). Meanwhile, Smad2 or smad3 siRNA significantly inhibited the UII-induced collagen 1 secretion and cell migration. Conclusions. UII may stimulate adventitial-fibroblast phenotype conversion, migration, and collagen I synthesis via phosphorylated-Smad2/3 signal transduction pathways.

Nuclear receptor subfamily 1 group D member 1 suppresses the proliferation, migration of adventitial fibroblasts, and vascular intimal hyperplasia via mammalian target of rapamycin complex 1/β-catenin pathway

ABSTRACT Background In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level. Objective Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia. Methods and Results We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of β-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of β-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of β-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo, we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery. Conclusion These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/β-catenin-dependent manner.

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation. Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function. Phosphorylation of p38, c-Jun N-terminal kinase (JNK), andextracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, andextracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression. These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation.

Homocysteine promotes migration of adventitial fibroblasts via angiotensin II type 1 receptor activation to aggravate atherosclerosis.

This study clarified the effect of homocysteine on adventitial fibroblasts (AFs) and its relationship with angiotensin II type 1 receptor (AT1R). Hyperhomocysteinemia aggravated the plaque area and increased the expression of IL-6, MCP-1, and macrophage infiltration in the plaque and adventitia of the aorta, whereas telmisartan improved this effect. Hyperhomocysteinemia induced the occurrence of the AFs marker protein ER-TR7 in the plaque and entire layer of the aorta, whereas telmisartan improved these effects, indicating that homocysteine induced AFs migration and that AT1R mediated this process. The migration experiments of AFs also reached the same conclusion. Homocysteine increased the phosphorylation levels of PKC and ERK1/2 in the AFs and HEK293A cells transfected with the AT1R plasmid, whereas telmisartan inhibited this effect, indicating that homocysteine activated AT1R intracellular signaling pathway. Homocysteine also increased the AFs At1R expression. Conclusion, homocysteine promoted adventitial inflammation, induced AFs migration, and aggravated atherosclerosis by activating AT1R.

A8374 Kruppel like factor 15 attenuates activation of adventitial fibroblasts-mediated angiotensin II-induced vascular remodeling

Objectives: Vascular remodeling is characterized by hypertrophy, vascular fibrosis and inflammation, but the precise mechanism underlying adventitial fibroblast-mediated vascular remodeling is not fully understood. We aim to determine whether transcription factor KLF15 (Krüppel-like factor 15) negatively regulate adventitial fibroblast activation contributing to vascular fibrosis and inflammation after Ang II (Angiotensin II) infusion. Methods: Male 8-week-old C57BL/6 mice were randomly divided into AdCTL group, Ang II + AdCTL group, Ang II + AdKLF15 group, Ang II + AdKLF15-ΔTAD (deletion of transactivation domain) group and Ang II + Losartan potassium group. Vascular morphology changes, adventitial fibrosis and inflammation, and KLF15 expression were detected. Primary culture of adventitial fibroblasts were used for cell function and mechanism study. Results: KLF15 was mainly expressed in vascular adventitia and intima. Ang II infusion-induced decrease of KLF15 expression especially in adventitia. Adenovirus-mediated overexpression of AdKLF15, but not AdKLF15-ΔTAD, attenuated vascular fibrosis, inflammation and TGF-beta signaling activation. Similarly, administered with Losartan potassium restored the expression of KLF15 and ameliorated Ang II-induced vascular remodeling. In adventitial fibroblasts, Ang II induced decrease of KLF15 in a dose- and time-dependent manner. Moreover, Ang II induced inflammation and fibrosis response, as well as the enhanced proliferation and migration of adventitial fibroblasts was suppressed by overexpression of KLF15 but not KLF15-ΔTAD. Conversely, these effects were aggravated by siRNA knockdown of KLF15. Conclusion: KLF15-dependent adventitial fibroblasts activation plays an important role in Ang II-induced vascular remodeling, likely through transactivation domain of KLF15.

A6321 Complement in Hypertension and Target Organ Damage

Objectives: Immunity and inflammation play pivotal roles in the development of hypertension, including innate and adaptive immunity. Complement system provides a bridge between innate and adaptive immune cells. We aim to elucidate the mechanism of complement activation in hypertension and related target organ damage. Methods: Complement C3, C3aR, C5aR knock out (KO) mice were utilized for performing hypertension model, including deoxycorticosterone acetate (DOCA)/salt and angiotensin II (Ang II)-induced hypertension. Macrophage and T cells were separated for stimulating with complement factors. Results: We firstly found that perivascular adipose tissue (PVAT) expressed abundant C3, which stimulated adventitial fibroblast migration and phenotype transdifferentiation. Subsequently, we showed that C5a regulated M1/M2 macrophage polarization and inhibited adiponectin production in the PVAT, which contributed to vascular inflammation in hypertension. More important, we demonstrated that double C3aR and C5aR KO (DKO) prevented Ang II-induced blood pressure elevation. Mechanically, DKO activated the immunosuppressive functio of regulatory T cells (Tregs). Adoptive transfer of DKO Tregs attenuated Ang II-induced hypertension and target organ damage. Conclusion: Our results demonstrate that complement activation-mediated immune cell infiltration is involved in the development of hypertension and target organ damage. Blockade of complement activation may be an alternative novel approach for hypertension treatment.

Activation and Migration of Adventitial Fibroblasts Contributes to Vascular Remodeling.

The rat carotid artery balloon injury model was used to prove the activation and migration of adventitial fibroblasts. We found that at day 7 after injury, adventitial fibroblasts proliferated, transformed into myofibroblasts under transmission electron microscopy in the model group. Simultaneously, we proved that the adventitial cells migrated to the media and intima on seventh day after injury by directly labeled the adventitial cells by the in vivo gene transfer technique. Moreover, we captured the precise moment when the adventitial fibroblasts migrated from the adventitia to the media through the external elastic plate under transmission electron microscope. This study provides direct evidences that adventitial fibroblasts activate and migrate to the media and intima, then actively take part in revascularization. Anat Rec, 301:1216-1223, 2018. © 2018 Wiley Periodicals, Inc.

Endothelial Cells Inhibit the Angiotensin II Induced Phenotypic Modulation of Rat Vascular Adventitial Fibroblasts.

The phenotypic modulation of vascular adventitial fibroblasts plays an important role in vascular remodeling. Evidence have shown that endothelial cells and adventitial fibroblasts interact under certain conditions. In this study, we investigated the influence of endothelial cells on the phenotypic modulation of adventitial fibroblasts. Endothelial cells and adventitial fibroblasts from rat thoracic aorta were cultivated in a co-culture system and adventitial fibroblasts were induced with angiotensin II (Ang II). Collagen I and alpha smooth muscle actin (α-SMA) expression and migration of adventitial fibroblasts were analyzed. Ang II upregulated the expression of collagen I and α-SMA and the migration of adventitial fibroblasts. Adventitial fibroblasts-endothelial cells co-culturing attenuated the effects of Ang II. Homocysteine-treated endothelial cells, which are functionally impaired, were less inhibitory of the phenotypic modulation of adventitial fibroblasts. Supplementation of endothelial cells with L-arginine (L-Arg) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) enhanced the trends, while with L-NG-nitroarginine methyl ester (L-NAME) or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) the opposite effect was observed. Under the influence of Ang II, adventitial fibroblasts were prone to undergo phenotypic modulation, which was closely related to vascular remodeling. Our study showed that endothelial cells influenced fibroblast phenotypic transformation and such effect would be mediated through the nitric oxide (NO)/cGMP signaling pathway. J. Cell. Biochem. 118: 1921-1927, 2017. © 2017 Wiley Periodicals, Inc.

Resveratrol inhibits adventitial fibroblast proliferation and induces cell apoptosis through the SIRT1 pathway.

Atherosclerosis is one of the most important causes of cardiovascular disease and studies have showed that adventitial fibroblasts, which are considered to be the most common cell type of the vascular adventitia, are involved in the development of early atherosclerotic plaques. Resveratrol is a plant polyphenolic compound confirmed to have anti-atherosclerotic and cardioprotective effects. The aim of the present study was to investigate the effects of resveratrol on adventitial fibroblasts in vitro and to clarify the underlying mechanism. Adventitial fibroblasts were isolated from the thoracic aorta of 8-week-old SPF Sprague-Dawley rats. Following pre-treatment with different concentrations of resveratrol, cell viability, DNA synthesis ability, cell apoptosis and cell migration ability were assessed in vitro. Through transfection with small interfering (si)RNA targeting sirtuin 1 (SIRT1), the role of the SIRT1 pathway in these processes was evaluated. Western blot analysis was used to assess the protein expression of SIRT1. It was demonstrated that resveratrol inhibited the cell viability, DNA synthesis and migratory ability of the adventitial fibroblasts, and induced cell apoptosis in a concentration-dependent manner in vitro. These effects were partly through the SIRT1 pathways. siRNA targeting SIRT1 successfully reversed the antiproliferative, antimigratory and pro-apoptotic effects of resveratrol on adventitial fibroblasts. In conclusion, the data showed that resveratrol inhibited cell viability, DNA synthesis and cell migration, and induced cell apoptosis in the rat adventitial fibroblasts in vitro through the SIRT1 signaling pathway. As the activation and migration of adventitial fibroblasts contributes to the early development of atherosclerosis, this may be a mechanism underlying the anti-atherosclerotic effect of resveratrol.

STAT3-mediated MMP-2 expression is required for 15-HETE-induced vascular adventitial fibroblast migration

ObjectiveVascular adventitial fibroblasts (VAFs) migration was involved in neointima formation, and increased 15-HETE levels contributed to vascular remodeling. However, how 15-HETE-induced VAF migration was not clear. Methods and results15-HETE-stimulated VAF phenotypic changes and migration as measured by the wound healing assay required STAT3 phosphorylation. JNK1 and CREB inhibition blocked 15-HETE-induced STAT3 activation and VAF changes. 15-HETE-induced MMP-2 expression and secretion were analyzed by Western blot and ELISA, respectively. MMP-2 knockdown blocked VAF migration and phenotypic alterations. JNK1, STAT3 and CREB blockade suppressed 15-HETE-induced MMP-2 expression in VAFs. MMP-2 promoter activity was assessed by chromatin immunoprecipitation using anti-STAT3 antibodies, which demonstrated that STAT3 was essential for 15-HETE-induced MMP-2 expression. Rats that suffered from hypoxia injury with or without treatment were examined. Pulmonary artery remodeling was obviously observed, and even the media was broken. MMP-2-positive staining was observed in the adventitia and intima. MMP-2 Serum secretion was enhanced as detected by ELISA, and MMP-2 and α-SMA protein expressions were increased after inducing hypoxia in the rats, which was restored in rats that had been administrated with NDGA. ConclusionThese results reveal that STAT3-mediated MMP-2 expression is required for 15-HETE induced-VAF migration.

Phenotypic transformation and migration of adventitial cells following angioplasty.

The present study was designed to investigate the phenotypic transformation and migration of adventitial fibroblasts using 5-bromo-2'-deoxyuridine (BrdU) labeling following angioplasty and to explore the correlation between adventitial cells and post-angioplasty restenosis. A vascular restenosis model was established in 23 rats by injuring the common carotid artery with a wire. BrDU was used to label the fibroblasts followed by immunohistochemistry for α-actin. Blood vessels were observed under light microscopy and scanning electron microscopy followed by image analysis. The number of BrDU-positive fibroblasts in the intima, media and adventitia of the blood vessels was determined 3, 7, 41 and 28 days after injury. The results demonstrated that at different time points, the number of BrDU-positive cells was significantly different in the intima, media and adventia (P<0.05). Electron microscopy indicated that the fibroblasts were full of cytoplasm. In addition, many secretory granules were noted on the rough endoplasmic reticulum and a large amount of microfilament bundles were noted after angioplasty. The fibroblasts transformed into myofibroblasts. Seven and 14 days after injury, the myofibroblasts formed wide pseudopods stretching to the fenestrae of the external and internal elastic lamina, and cells had a tendency to migrate into the lumen. The fibroblasts in the adventitia underwent transformation after percutaneous transluminal angioplasty and secreted α-actin. In conclusion, the fibroblasts in the adventitia transformed into myofibroblasts, migrated into and proliferated in the intima and became a component of the newly generated intima. Adventitial cells are thus related to vascular restenosis.

Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts

Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide ( P < 0.01) but not sense or mismatch oligonucleotides ( P > 0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10 −8 mol/l at 3 h for mRNA expression or at 12 h for protein secretion (both P < 0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10 −6 mol/l), and Ca 2+ channel blocker nicardipine (10 −5 mol/l), protein kinase C (PKC) inhibitor H7 (10 −5 mol/l), calcineurin inhibitor cyclosporine A (10 −5 mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10 −5 mol/l) and Rho kinase inhibitor Y-27632 (10 −5 mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca 2+ channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways.

Expression and Function of Osteopontin in Vascular Adventitial Fibroblasts and Pathological Vascular Remodeling

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.

E0159 Osteopontin is involved in urotensin II induced migration of adventitial fibroblasts from rat aorta

BackgroundRecent studies suggest osteopontin (OPN) plays a critical role in the progression of vascular remodelling, and that Urotensin II (UII) is a potent vasoconstrictor and stimulator of cellular migration. The...

Adiponectin Inhibits Lipopolysaccharide-Induced Adventitial Fibroblast Migration and Transition to Myofibroblasts via AdipoR1-AMPK-iNOS Pathway

Adiponectin is an important antiatherogenic adipocytokine that inhibits inflammation, insulin resistance, and oxide stress. Inflammation in the vascular adventitia is a crucial factor in the pathogenesis of atherosclerosis. Adventitial fibroblasts (AFs) can proliferate, divide into myofibroblasts, and migrate to the intima to become a new component of atherosclerotic plaque under inflammation and atherosclerosis. We investigated whether adiponectin might prevent AFs from proliferating, migrating, and transforming into myofibroblasts. Cultured AFs were stimulated with lipopolysaccharide (LPS) in the presence or absence of adiponectin. Methyl thiazolyl tetrazolium assay and migration and scratch-wound assays demonstrated that adiponectin reduced the AF proliferation and migration induced by LPS, respectively, whereas treatment with AdipoR1 small interfering (si) RNA (siAdipoR1), AMP-activated protein kinase (AMPK) siRNA (siAMPK), and an AMPK inhibitor reversed the effect. Immunocytochemistry and Western blot revealed that adiponectin reduced the transition of AFs to myofibroblasts, and treatment with siAdipoR1, siAMPK, and the AMPK inhibitor increased the transition. RT-PCR, Western blotting, and nitric oxide (NO) assay showed that adiponectin reduces induced NO synthase (iNOS) and nitrotyrosine expression and NO and ONOO(-) production induced by LPS. Treatment with siAdipoR1, siAMPK, and the AMPK inhibitor significantly attenuated adiponectin-induced phosphorylation of AMPK and its downstream target acetyl-coenzyme A carboxylase and up-regulated iNOS mRNA and protein expression, which resulted in a marked increase of NO and ONOO(-) production. In apolipoprotein E-deficient mice, immunohistochemistry of treated vascular adventitia showed that both iNOS expression and ONOO(-) production could be reversed with an adenovirus-adiponectin vector. Taken together, these results suggest that adiponectin reduces LPS-induced NO production and nitrosative stress and prevents AFs from proliferating, transforming to myoflbroblasts, and migrating to the intima, thus worsening atherosclerosis, by inhibiting the AdipoR1-AMPK-iNOS pathway in AFs.

Negligible contribution of coronary adventitial fibroblasts to neointimal formation following balloon angioplasty in swine

Adventitial fibroblasts have previously been proposed to be a major constituent of the neointima following coronary balloon angioplasty. The present study utilized the bromodeoxyuridine (BrdU) pulse-chase technique to track adventitial fibroblast migration early after balloon injury in swine. BrdU (30 mg/kg), a marker of proliferating cells, was given intravenously 1 or 2 days after balloon angioplasty. For each time point, one animal was euthanized 24 h after injection to identify the location of the proliferating cells, while a second animal was euthanized 25 days after angioplasty to determine whether the proliferating cells migrated to form the neointima. Our results demonstrate that BrdU-positive cells were located primarily in the adventitia with all three time points 24 h after balloon angioplasty. Furthermore, when BrdU was injected on day 1 or 2 only 0.65 +/- 0.17% and 1.7 +/- 0.64%, respectively, of neointimal cells were BrdU positive on day 25. In conclusion, these results demonstrate a negligible contribution of coronary adventitial fibroblasts to neointima formation following coronary balloon angioplasty, supporting the concept that the neointima is primarily of smooth muscle cell origin.

The effect of ecdysterone on cerebral vasospasm following experimental subarachnoid hemorrhage in vitro and in vivo

Objectives: Cerebral vasospasm has been the dreaded complication of ruptured intracranial aneurysms. Worldwide effort has led to many promising experimental treatments but none was confirmed to be effective in clinical trials. Ecdysterone is an insect steroid hormone. Our previous study showed that ecdysterone might prevent cerebral vasospasm in vitro. Even after all these works, rare attempts have been made to test the effect of ecdysterone on vascular adventitial fibroblast (VAF) proliferation, a process known to play an important role in various pathogenic vascular conditions. Thus, we tested the hypothesis that ecdysterone could affect VAF characteristics and have an effect on SAH induced cerebral vasospasm.Methods: OxyHb of 100 μM was used in the in vitro study to mimic the clinical situation. The effect of OxyHb on the cell proliferation and migration of cultured aortic smooth muscle cells was investigated. In the in vivo study, 20 rabbits were equally divided into four groups: control group, SAH group, SAH/nimodipine group and SAH/ecdysterone group. Changes in neurological function and cerebral angiograms were observed after SAH.Results: OxyHb increased the proliferation of vascular adventitial fibroblasts at 24 hours. Ecdysterone co-treatment was apparently similar to the suppression of proliferation. Cell cycle analysis indicated that ecdysterone inhibited the progression of vascular adventitial fibroblasts from G1 to S. The results of the migration assay showed that 100 μM OxyHb obviously prompted vascular adventitial fibroblast migration and that ecdysterone would attenuate this effect. In the SAH/nimodipine and SAH/ecdysterone groups, neurological deficit, cerebral vasospasm and structural changes in basilar artery were alleviated with nimodipine or ecdysterone treatment.Conclusion: Ecdysterone could affect vascular adventitial fibroblast characteristics and attenuate vasospasm after SAH.

Urotensin II induces phenotypic differentiation, migration, and collagen synthesis of adventitial fibroblasts from rat aorta

Urotensin II is a new potent vasoconstrictor. Nevertheless, little is known about its effects on the activation of adventitial fibroblasts. To explore the effects of urotensin II on phenotypic differentiation, migration, and collagen I synthesis of rat aortic adventitial fibroblasts. Growth-arrested adventitial fibroblasts were incubated in serum-free medium with urotensin II and some inhibitors of signal transduction pathways. The alpha-smooth muscle-actin expression, collagen I synthesis and migration of adventitial fibroblasts induced by urotensin II were evaluated by western blot, enzyme-linked immunosorbant assay, and the transwell technique, respectively. Urotensin II induced the [alpha]-smooth muscle-actin expression in a dose-dependent and time-dependent manner, with maximal effect at a concentration of 10(-8) mol/l at 24 h (79.9%); it also caused a dose-dependent increase in collagen I synthesis, with maximal effect at a concentration of 10(-7) mol/l (42.6%). The Ca2+ channel blocker nicardipine (10(-5) mol/l), protein kinase C inhibitor H7 (10(-5) mol/l), Rho protein kinase inhibitor Y-27632 (10(-5) mol/l), calcineurin inhibitor cyclosporine A (10(-5) mol/l), and mitogen-activated protein kinase inhibitor PD98059 (10(-5) mol/l) inhibited urotensin II-induced increases in [alpha]-smooth muscle-actin expression and collagen synthesis. Meanwhile, urotensin II stimulated the migration of adventitial fibroblasts dose dependently, with maximal effect at a concentration of 10(-8) mol/l, which was 5.7-fold greater than that of the control. This effect could also be inhibited by PD98059, H7, cyclosporine A, and Y-27632 but not nicardipine. Urotensin II may stimulate adventitial fibroblasts phenotypic conversion, migration, and collagen I synthesis through the protein kinase C, mitogen-activated protein kinase, calcineurin, Rho kinase, and/or Ca2+ signal transduction pathways, contributing to the development of vascular remodeling through adventitial fibroblasts activation.

Similar pathological effects of sympathectomy and hypercholesterolemia on arterial smooth muscle cells and fibroblasts

In a previous study, we showed that after sympathectomy, the femoral (FA) but not the basilar (BA) artery from non-pathological rabbits manifests migration of adventitial fibroblasts (FBs) into the media and loss of medial smooth muscle cells (SMCs). The aim of the present study was to verify whether similar behaviour of arteries occurred in the pathological context of atherosclerosis. Thus, similar experiments were conducted on hypercholesterolemic rabbits, which were chemically sympathectomized with 6-hydroxydopamine (n=4) or treated with vehicle for control (n=5). Cross-sections of BA and FA were immunolabelled for five markers of phenotypic modulation of vascular SMCs and FBs: vimentin, desmin, alpha-smooth muscle actin, beta-isoform of actin, and h-caldesmon and examined using a confocal microscope. Also, 3D images were constructed and morphometric analysis performed using image analysis software. Both intact and sympathectomized BA and FA developed atherosclerotic plaques, but the thickening of the intima was more advanced in sympathectomized animals, as judged by increased plaque frequency and by the phenotypic modulation of SMCs in the intima. Our results show that in the media of FAs hypercholesterolemia induces changes similar to those observed in sympathectomized rabbits in non-pathological conditions, i.e., migration of adventitial FBs to the media and loss of medial SMCs. These latter changes, which can be ascribed to pathological events, were accentuated after sympathectomy in the hypercholesterolemic rabbits. The present study reveals that pathological events, including migration and phenotypic modulation of vascular FBs and loss of SMCs, may be under the influence of sympathetic nerves.

Expression and function of vascular endothelial growth factor receptors (Flt-1 and Flk-1) in vascular adventitial fibroblasts

Vascular endothelial growth factor receptors (VEGFRs) are previously considered to exist exclusively in endothelial cells. However, little is known if the receptors are expressed in other non-endothelial cells. In this study, we measured activation of two VEGFRs, Flk-1 and Flt-1, and their biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results indicated that Flt-1, but not Flk-1, existed in adventitial fibroblasts. Angiotensin II increased Flt-1 protein expression in a time- and concentration-dependent manner. Adventitial fibroblast migration stimulated by vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) required Flt-1 expression. The Flt-1-induced adventitial fibroblast migration was blocked by anti-Flt-1 neutralizing antibody and soluble VEGFR1 protein (sFlt-1). However, Flt-1 activation did not enhance cell proliferation. In addition, Flt-1 expression was significantly increased in the neointima and adventitia in injured rat carotid arteries. We concluded that functional expression of Flt-1 in adventitial fibroblasts might be an important mediator in the pathogenesis of vascular remodeling after arterial injury.