Activation Of Adventitial Fibroblasts Research Articles

Hydrogen Sulfide Diminishes Activation of Adventitial Fibroblasts Through the Inhibition of Mitochondrial Fission.

:Activation of adventitial fibroblasts (AFs) on vascular injury contributes to vascular remodeling. Hydrogen sulfide (H2S), a gaseous signal molecule, modulates various cardiovascular functions. The aim of this study was to explore whether exogenous H2S ameliorates transforming growth factor-β1 (TGF-β1)–induced activation of AFs and, if so, to determine the underlying molecular mechanisms. Immunofluorescent staining and western blot were used to determine the expression of collagen I and α-smooth muscle actin. The proliferation and migration of AFs were performed by using cell counting Kit-8 and transwell assay, respectively. The mitochondrial morphology was assessed by using MitoTracker Red staining. The activation of signaling pathway was evaluated by western blot. The mitochondrial reactive oxygen species and mitochondrial membrane potential were determined by MitoSOX and JC-1 (5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethylbenzimidazolyl carbocyanine iodide) staining. Our study demonstrated exogenous H2S treatment dramatically suppressed TGF-β1–induced AF proliferation, migration, and phenotypic transition by blockage of dynamin-related protein 1 (Drp1)–mediated mitochondrial fission and regulated mitochondrial reactive oxygen species generation. Moreover, exogenous H2S reversed TGF-β1–induced mitochondrial fission and AF activation by modulating Rho-associated protein kinase 1–dependent phosphorylation of Drp1. In conclusion, our results suggested that exogenous H2S attenuates TGF-β1–induced AF activation through suppression of Drp1-mediated mitochondrial fission in a Rho-associated protein kinase 1–dependent fashion.

RNA-binding protein SFPQ cooperates with HDAC1 to suppress CD40 transcription in pulmonary adventitial fibroblasts.

Pulmonary artery adventitial fibroblasts, the most abundant cellular constituent of adventitia, are often the first to be activated and reprogrammed to then influence the tone and structure of the vessel wall in pulmonary arterial hypertension (PAH). Our previous study found that interruption of CD40 ligand (CD40L)-CD40 signaling improves the efficacy of transplanted endothelial progenitor cells in monocrotaline induced-PAH. However, whether CD40L-CD40 signaling is involved in the activation of adventitial fibroblasts in PAH and whether Drosophila behavior human splicing (DBHS) protein family members have any roles during adventitial fibroblasts activation are completely unclear. Here, we show that soluble CD40L (sCD40L) stimulation progressively increases pro-inflammatory activity, proliferation, and migration of pulmonary adventitial fibroblasts. Besides, sCD40L stimulation decreases splicing factor proline- and glutamine-rich protein (SFPQ) protein (one member of DBHS protein family) expression, while SFPQ overexpression suppresses sCD40L stimulation-induced proliferation and migration of pulmonary adventitial fibroblasts by repressing CD40 transcription. Moreover, ChIP assays found that sCD40L stimulation promotes histone H3 tri-methylation at lysine 4 (H3K4me3), H3K36me3, and H3K27 acetylation (H3K27ac) modifications on CD40 promoter region in pulmonary adventitial fibroblasts, while SFPQ overexpression decreases H3K36me3 modification and increases H3K36ac on CD40 promoter region by interacting with histone deacetylase-1 (HDAC1) to inhibit CD40 transcription. This in-depth study shows that CD40L-CD40 signaling promotes activation of pulmonary adventitial fibroblasts by increasing proliferation, migration, and pro-inflammatory activity of adventitial fibroblasts, and SFPQ could inhibit CD40 transcription though switching H3K36me3 to H3K36ac modifications on its promoter by interacting with HDAC1. This study, first, uncovers the roles of SFPQ on CD40L-CD40 signaling-mediated activation of pulmonary adventitial fibroblasts.

Abstract 628: Diabetes-Mediated Vascular Calcification is RAGE-Dependent

Type II diabetes mellitus (DM) is characterized by chronic hyperglycemia, and medial vascular calcification is a common cardiovascular complication of DM. This leads to aortic stiffening, which can leave patients at an increased risk for heart attack or stroke. Advanced Glycation End-Products (AGEs)/Receptor for AGEs (RAGE) signaling cascade has been implicated as a potentiator of diabetes-mediated vascular calcification, but it is not well understood. AGE/RAGE signaling influences both cellular and systemic responses to increase bone matrix proteins in hyperglycemic and calcification conditions and has also been shown to increase oxidative stress by promoting diabetes-mediated vascular calcification. This causes a phenotypic switch of vascular smooth muscle cells (VSMCs) to osteoblast-like cells and the hypothesized activation of adventitial fibroblasts (AFBs) to a myofibroblast phenotype. The purpose of this research is to understand AGE/RAGE mediated vascular calcification as a complication of diabetes. Calcification was induced for 7 days in primary mouse VSMCs and AFBs of non-diabetic, diabetic, non-diabetic RAGE knockout (RKO), and diabetic RKO, and then treated with AGEs to activate RAGE. Alizarin Red S staining was utilized to visualize calcification. Intracellular calcium levels were quantified and normalized to cell number (DAPI). Pronounced calcification was observed in non-diabetic VSMCs and the loss of RAGE resulted in decreased calcification in the non-diabetic RKO VSMCs. AFBs were exposed to the same experimental conditions as the VSMCs and calcification was increased in the diabetic AFBs while calcification was significantly decreased in the diabetic RKO AFBs. These data demonstrated that diabetes-mediated vascular calcification was RAGE-dependent in both cell types. Literature has cited the VSMC as the primary mediator for vascular calcification, but we have shown that the AFBs in the outer layer of the aorta have the ability to calcify and this is mediated by RAGE signaling, which elucidates a role for RAGE in diabetes-mediated vascular calcification. Understanding the role of AGE/RAGE signaling in diabetes-mediated vascular calcification will allow for possible targets for pharmacological intervention.

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.

Sonic hedgehog-dependent activation of adventitial fibroblasts promotes neointima formation.

Adventitial cells have been suggested to contribute to neointima formation, but the functional relevance and the responsible signalling pathways are largely unknown. Sonic hedgehog (Shh) is a regulator of vasculogenesis and promotes angiogenesis in the adult. Here we show that proliferation of vascular smooth muscle cells (SMC) after wire-induced injury in C57BL/6 mice is preceded by proliferation of adventitial fibroblasts. Simultaneously, the expression of Shh and its downstream signalling protein smoothened (SMO) were robustly increased within injured arteries. In vitro, combined stimulation with Shh and platelet-derived growth factor (PDGF)-BB strongly induced proliferation and migration of human adventitial fibroblasts. The supernatant of these activated fibroblasts contained high levels of interleukin-6 and -8 and strongly induced proliferation and migration of SMC. Inhibition of SMO selectively prevented fibroblast proliferation, cytokine release, and paracrine SMC activation. Mechanistically, we found that PDGF-BB activates protein kinase A in fibroblasts and thereby induces trafficking of SMO to the plasma membrane, where it can be activated by Shh. In vivo, SMO-inhibition significantly prevented the proliferation of adventitial fibroblasts and neointima formation following wire-induced injury. The initial activation of adventitial fibroblasts is essential for the subsequent proliferation of SMC and neointima formation. We identified SMO-dependent Shh signalling as a specific process for the activation of adventitial fibroblasts.

Soluble Epoxide Hydrolase Inhibition Protected against Angiotensin II-induced Adventitial Remodeling

Epoxyeicosatrienoic acids (EETs), the metabolites of cytochrome P450 epoxygenases derived from arachidonic acid, exert important biological activities in maintaining cardiovascular homeostasis. Soluble epoxide hydrolase (sEH) hydrolyzes EETs to less biologically active dihydroxyeicosatrienoic acids. However, the effects of sEH inhibition on adventitial remodeling remain inconclusive. In this study, the adventitial remodeling model was established by continuous Ang II infusion for 2 weeks in C57BL/6 J mice, before which sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered by gavage. Adventitial remodeling was evaluated by histological analysis, western blot, immunofluorescent staining, calcium imaging, CCK-8 and transwell assay. Results showed that Ang II infusion significantly induced vessel wall thickening, collagen deposition, and overexpression of α-SMA and PCNA in aortic adventitia, respectively. Interestingly, these injuries were attenuated by TPPU administration. Additionally, TPPU pretreatment overtly prevented Ang II-induced primary adventitial fibroblasts activation, characterized by differentiation, proliferation, migration, and collagen synthesis via Ca2+-calcineurin/NFATc3 signaling pathway in vitro. In summary, our results suggest that inhibition of sEH could be considered as a novel therapeutic strategy to treat adventitial remodeling related disorders.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes.

MiR-29a-3p attenuates hypoxic pulmonary hypertension by inhibiting pulmonary adventitial fibroblast activation.

Activation of pulmonary adventitial fibroblasts plays a key role in the pulmonary vascular remodeling in hypoxic pulmonary hypertension. Previous studies showed that miRNAs participated in the regulation of fibroblast activation. This study explored the role of miR-29 in the activation of pulmonary adventitial fibroblasts and the therapeutic potential in hypoxic pulmonary hypertension. We found that hypoxia-induced pulmonary adventitial fibroblasts activation was accompanied with a drastic decrease of miR-29a-3p expression. Knockdown of hypoxia-inducible factor-1 α or Smad3 reversed the hypoxia-induced decrease of miR-29-3p in cultured pulmonary adventitial fibroblasts. In vitro, miR-29a-3p mimic inhibited the hypoxia-induced proliferation, migration, and secretion of pulmonary adventitial fibroblasts, suppressed the hypoxia-induced expression of α-smooth muscle actin and extracellular matrix collagen in pulmonary adventitial fibroblasts; however, miR-29a-3p inhibitor mimicked the effect of hypoxia on the activation of pulmonary adventitial fibroblasts. Further studies revealed that preventative or therapeutic administration of miR-29a-3p significantly decreased pulmonary artery pressure and right ventricle hypertrophy index and ameliorated pulmonary vascular remodeling in hypoxic pulmonary hypertension rats. These findings suggest that miR-29a-3p regulates the activation and phenotype of pulmonary adventitial fibroblasts in hypoxia and has preventative and therapeutic potential in hypoxic pulmonary hypertension.

Serotonin drives the activation of pulmonary artery adventitial fibroblasts and TGF-β1/Smad3-mediated fibrotic responses through 5-HT(2A) receptors.

Pulmonary arterial remodeling is characterized by excessive proliferation, migration, and pro-differentiation and fibrotic activation of adventitial fibroblasts in pulmonary arterial hypertension (PAH) process. Several lines of evidence indicate that serotonin (5-HT) plays a central role in the pathogenesis of pulmonary arterial remodeling. In the present study, we investigated whether 5-HT is directly involved in the functional regulation of pulmonary artery adventitial fibroblasts (PAFs). Incubation of cultured rat PAFs with 5-HT caused a dose-dependent stimulation of cell proliferation, migration activity, and a time-dependent increase of α-SMA expression, a marker of fibroblast differentiation into myofibroblasts, and adventitia fibrosis, evaluating connective tissue growth factor (CTGF) and extracellular matrix (ECM) mRNAs and proteins. These effects were attenuated by the 5-HT2A receptor antagonist, ketanserin and mimicked by the 5-HT2A receptor agonist DOI. 5-HT-induced fibroblasts phenotypic alterations and ECM accumulation were dependent on stimulation of transforming growth factor (TGF)-β1 as demonstrated using a neutralizing antibody. 5-HT also caused Smad3 phosphorylation and ketanserin diminished 5-HT-induced Smad3 activation. These results demonstrated that 5-HT can directly activate PAFs through 5-HT2A receptor and promote fibroblasts phenotypic alterations and adventitia fibrosis depending on the signaling of the TGF-β1/Smad3 pathway.

MicroRNA-124 Controls the Proliferative, Migratory, and Inflammatory Phenotype of Pulmonary Vascular Fibroblasts

Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly proliferative, migratory, and inflammatory phenotype of hypertensive pulmonary adventitial fibroblasts. Thus, therapies directed at restoring miR-124 function, including histone deacetylase inhibitors, should be investigated.

Hyperhomocysteinemia Exaggerates Adventitial Inflammation and Angiotensin II−Induced Abdominal Aortic Aneurysm in Mice

A number of epidemiological studies have suggested an association of hyperhomocysteinemia (HHcy) and abdominal aortic aneurysm (AAA), but discrepancies exist. In addition, we lack direct evidence supporting a causal role. We determined the association and contribution of HHcy to AAA formation. We first performed a meta-analysis of studies involving 1489 subjects and found a strong association of HHcy and AAA (odds ratio, 7.39). Next, we used angiotensin II-infused male apolipoprotein E-deficient mice and tested whether HHcy contributes to AAA pathogenesis. Homocysteine (Hcy) supplement (1.8 g/L) in drinking water resulted in mild HHcy. Intriguingly, HHcy greatly increased the incidence of angiotensin II-induced AAA and aortic dissection in apolipoprotein E-deficient mice (vehicle versus Hcy: 50% versus 100%; P<0.05). Histology indicated HHcy markedly exaggerated aortic adventitial inflammation. Increased levels of proinflammatory interleukin-6 and monocyte chemoattractant protein-1 were preferentially colocalized within adventitial fibroblasts in HHcy plus angiotensin II mice, which suggested the importance of adventitial fibroblasts activation in Hcy-aggravated AAA. Hcy sequentially stimulated adventitial fibroblasts transformation into myofibroblasts, secretion of interleukin-6 and monocyte chemoattractant protein-1, and consequent recruitment of monocytes/macrophages to adventitial fibroblasts, which was abolished by the NADPH oxidase inhibitor diphenyliodonium. NADPH oxidase 4, but not other homologs of NADPH oxidase, was significantly upregulated by Hcy in adventitial fibroblasts, whereas NADPH oxidase 4 small interfering RNA silencing diminished Hcy-induced adventitial fibroblasts activation. Finally, folic acid supplement (0.071 μg/g per day) markedly reduced HHcy-aggravated angiotensin II-induced AAA formation in apolipoprotein E-deficient mice. HHcy may aggravate AAA formation at least partially via activating adventitial fibroblast NADPH oxidase 4.

Activation of Adventitial Fibroblasts in the Early Stage of the Aortic Transplant Vasculopathy in Rat

Transplant vasculopathy (TV) is the most significant obstacle to long-term success of organ transplantation. Increasing attention has been paid to the role of adventitia in vascular diseases. We evaluated the role of adventitial fibroblasts in the development of TV. Thoracic aortas from Sprague-Dawley (SD) rats transplanted into the abdominal aortas of Wistar rats worked as allografts, and isografts (SD to SD) were control. Grafts were removed on days 3, 7, and 14 for histologic, morphometric, and immunohistochemical detection of vimentin, alpha-smooth muscle actin, Ki-67, CD3, transforming growth factor-beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-7 (MMP-7), and quantitative real-time reverse transcriptase polymerase chain reaction for TGF-beta1, MCP-1, MMP-7, tumor necrosis factor-alpha, and interleukin-1beta. In the allografts, neointima thickness and neointima/media thickness ratios were slightly increased at 7 days and significantly increased at 14 days. Immunostaining of vimentin and alpha-smooth muscle actin showed adventitial fibroblasts activation and differentiation into myofibroblasts. Ki-67-positive nuclei were observed in the adventitia 3 days after allografting and subsequently in the neointima. No more than 4% CD3-positive cells were found in adventitia in all the groups. Compared with isografts, TGF-beta1, MMP-7, and MCP-1 were expressed in the adventitia before neointima formation and were significantly increased in allografts at all time points. Tumor necrosis factor-alpha and interleukin-1beta were also significantly increased in adventitia in allografts. These results demonstrated that adventitial fibroblasts are activated and can produce cytokines and chemokines before the neointimal hyperplasia. They may exert a potential effect on the development of neointimal hyperplasia in TV.

Coordinate activation of cardiac and pulmonary fibroblasts by hypoxia‐induced pulmonary hypertension in a transgenic mouse model

Activation of adventitial fibroblasts and myofibroblasts is an important determinant of pulmonary vascular remodeling in hypoxia‐induced pulmonary hypertension (PH). PH leads to right ventricular hypertrophy and failure. We hypothesized that hypoxia activates cardiac fibroblasts in concert with pulmonary fibroblasts. Transgenic mice expressing fibroblast phenotype‐sensitive promoter:reporter constructs (Collagen I promoter:Enhanced Green Fluorescent Protein, Coll I:EGFP; fibroblast phenotype; Smooth Muscle alpha‐Actin promoter:Red Fluorescent Protein, SMA:RFP, myofibroblast phenotype) were exposed to hypobaric hypoxia (6 weeks, 10% pO2). Fibroblast activation was monitored by reporter fluorescence in frozen tissue sections and by FACS analysis of enzymatically dispersed cells. Hypoxic exposure significantly elevated cardiac expression of SMA:RFP with a modest enhancement of CollI:EGFP. Increased RFP expression was greatest in right ventricle. Increased expression of RFP and EGFP were also observed in perivascular and parenchymal regions of lung. Distinct lung cell populations expressing fluorescent reporter proteins were observed by FACS analysis (EGFP+ &gt; RFP+EGFP+ &gt; RFP+). Coordinate activation of cardiac and pulmonary fibroblasts in response to hypoxia offers opportunities for novel fibroblast‐targeted therapies in PH. Supported by NIH HL014985.

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.

Activation of adventitial fibroblasts contributes to the early development of atherosclerosis: A novel hypothesis that complements the “Response-to-Injury Hypothesis” and the “Inflammation Hypothesis”

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This article introduces the hypothesis that the activation of the adventitia, specifically the fibroblasts, contributes to the formation of intimal atherosclerotic lesions. The evidence for this hypothesis includes: (a) the early proliferative changes seen in fibroblasts found in the adventitia; (b) the increase and the alteration of extracellular matrix deposition in the adventitia; (c) fibroblast differentiation into myofibroblasts and migration into the intima; and (d) fibroblast synthesis and release of cytokines that have potent effects on neighboring smooth muscle and endothelial cells prior to intimal lesion formation. In conclusion, the activation of adventitial fibroblasts is a key regulator of vascular function and structure from the "outside-in" and contributes to the development of atherosclerotic lesions. The outer location of the adventitia makes it a suitable location for drug delivery and gene therapy aimed at preventing and treating atherosclerosis.

Hypoxic Activation of Adventitial Fibroblasts : Role in Vascular Remodeling

Substantial experimental evidence supports the idea that the fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions. Fibroblasts have the ability to rapidly respond to hypoxic stress and to modulate their function to adapt rapidly to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions. Proliferative responses to hypoxia depend on the activation of Galpha(i) and Gq kinase family members, and on the subsequent stimulation of protein kinase C and mitogen-activated protein kinase family members. Extracellular nucleotides (eg, adenosine triphosphate [ATP]) are likely to be increased in the hypoxic adventitial compartment and can act as autocrine/paracrine modifiers of the hypoxia-induced proliferative response. The proliferative effects of ATP appear to be mediated largely through G-protein-coupled P2Y receptors in fetal and neonatal fibroblasts. Hypoxia, acting through Galpha(iota)-coupled pathways, also can directly up-regulate alpha-smooth muscle actin expression in fibroblast subpopulations, suggesting that hypoxia may play a direct role in mediating the "transdifferentiation" of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, similar to the heterogeneity described for vascular smooth muscle cells, numerous fibroblast subtypes exist in the vessel wall, and that each may respond in unique ways to hypoxia and other stimuli and thus serve special functions in response to injury. In fact, adventitia may be considered to be compartments in which cells with "stem-cell-like" characteristics reside. Future work is needed to determine more precisely the role of the fibroblast in the wide variety of vascular complications observed in many humans diseases, and in the genes and gene products that confer unique properties to this important vascular cell.

Role of matrix metalloproteinases and their tissue inhibitors in the regulation of coronary cell migration.

The migration of vascular cells is regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Because the activation of adventitial fibroblasts has been implicated in coronary repair, we have examined regional differences in cell outgrowth and the synthesis of MMPs/TIMPs in different layers of porcine coronary arteries. Coronary medial explants demonstrated significantly slower cell outgrowth than coronary adventitia in culture (P<0.001). These observations were paralleled by the predominant expression of TIMP-1 and -2 in the media (14-fold and 37-fold higher than in adventitia, respectively, P<0.001), whereas higher gelatinolytic activities (MMP-2 and -9) were released from adventitial explants. Smooth muscle cell outgrowth from the media was regulated by endogenous TIMPs, since TIMP inhibition (recombinant MMP-2 or neutralizing anti-TIMP antibodies) facilitated cell outgrowth (P<0.001). In contrast, the addition of recombinant TIMP-1 or -2 decreased adventitial cell outgrowth. In the coculture experiments, the presence of coronary media retarded adventitial cell outgrowth, whereas medial damage abrogated these effects, allowing for fibroblast migration (P<0.001). In conclusion, this study demonstrated differential migratory properties and distinct MMP/TIMP synthesis by coronary fibroblasts and smooth muscle cells. Endogenous TIMPs in the media may play an important role in maintaining coronary arterial wall homeostasis, whereas high levels of matrix-degrading activities confer the "invasive" characteristics of adventitial fibroblasts.