Protein Kinase C-Delta Mediates Adventitial Cell Migration Through Regulation of Monocyte Chemoattractant Protein-1 Expression in a Rat Angioplasty Model

Division of Vascular Surgery, University of Wisconsin Madison, 53705, USA.
Arteriosclerosis Thrombosis and Vascular Biology (Impact Factor: 6). 02/2012; 32(4):943-54. DOI: 10.1161/ATVBAHA.111.244921
Source: PubMed


The adventitia is increasingly recognized as an important player during the development of intimal hyperplasia. However, the mechanism of adventitial cell recruitment to the subintimal space remains largely undefined. We have shown previously that gene transfer of protein kinase C-delta (PKCδ) increases apoptosis of smooth muscle cells following balloon injury. In the current study, we investigated a potential role of PKCδ in regulating the recruitment of adventitial cells.
Conditioned media from PKCδ-overexpressing smooth muscle cells stimulated migration and CCR2 expression of adventitial fibroblasts through a MCP-1 dependent mechanism. Following balloon injury of rat carotid arteries, overexpression of PKCδ in smooth muscle cells significantly increased MCP-1 and CCR2 expression and the number of adventitia-originated cells detected in the neointima. Administration of an anti-MCP-1 antibody markedly diminished the recruitment of adventitial cells. Combined PKCδ overexpression and anti-MCP-1 inhibited intimal hyperplasia more effectively than either approach alone.
Our data suggest that PKCδ regulates recruitment of adventitial cells to the neointima via a mechanism involving upregulation of the MCP-1/CCR2 signaling axis in injured arteries. Blockage of MCP-1 while enhancing apoptosis may serve as a potential therapeutic strategy to attenuate intimal hyperplasia.

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    • "One possibility is that these de-differentiated cells translocate from another site. For example, there is substantial evidence that at least some cells that contribute to the neointima are myofibroblasts derived from the arterial adventitia [59]. There are a series of studies showing in the murine model, that neo-intimal cells are, in part, derived from the bone marrow [7], [60], [61], [62]. "
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    ABSTRACT: Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-β and its downstream signaling protein Smad3∶1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-β/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-β or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p<0.05) in TGF-β/Smad3 stimulated SMCs. We then performed GO term enrichment analysis using the DAVID bioinformatics database and found that TGF-β/Smad3 activated the expression of multiple genes related to either development or cell differentiation, several of which have been shown to be associated with multipotent stem or progenitor cells. Quantitative real-time PCR confirmed up-regulation of several developmental genes including FGF1, NGF, and Wnt11 (by 2.5, 6 and 7 fold, respectively) as well as stem/progenitor cell associated genes CD34 and CXCR4 (by 10 and 45 fold, respectively). In addition, up-regulation of these factors at protein levels were also confirmed by Western blotting, or by immunocytochemistry (performed for CXCR4 and NGF). Finally, TGF-β/Smad3 down regulated transcription of SMC contractile genes as well as protein production of smooth muscle alpha actin, calponin, and smooth muscle myosin heavy chain. These combined results suggest that TGF-β/Smad3 stimulation drives SMCs to a phenotypically altered state of de-differentiation through the up-regulation of developmental related genes.
    PLoS ONE 04/2014; 9(4):e93995. DOI:10.1371/journal.pone.0093995 · 3.23 Impact Factor
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    • "Myofibroblasts in the adventitia significantly contribute to neointimal hyperplasia. There is extensive data demonstrating that adventitial myofibroblasts migrate to the subintima and contribute to the formation of plaque [35], [36]. Thus inhibition of adventitial myofibroblast proliferation may enhance the ability of drugs such as rapamycin to protect against IH. "
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    ABSTRACT: Open vascular reconstructions frequently fail due to the development of recurrent disease or intimal hyperplasia (IH). This paper reports a novel drug delivery method using a rapamycin-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs)/pluronic gel system that can be applied periadventitially around the carotid artery immediately following the open surgery. In vitro studies revealed that rapamycin dispersed in pluronic gel was rapidly released over 3 days whereas release of rapamycin from rapamycin-loaded PLGA NPs embedded in pluronic gel was more gradual over 4 weeks. In cultured rat vascular smooth muscle cells (SMCs), rapamycin-loaded NPs produced durable (14 days versus 3 days for free rapamycin) inhibition of phosphorylation of S6 kinase (S6K1), a downstream target in the mTOR pathway. In a rat balloon injury model, periadventitial delivery of rapamycin-loaded NPs produced inhibition of phospho-S6K1 14 days after balloon injury. Immunostaining revealed that rapamycin-loaded NPs reduced SMC proliferation at both 14 and 28 days whereas rapamycin alone suppressed proliferation at day 14 only. Moreover, rapamycin-loaded NPs sustainably suppressed IH for at least 28 days following treatment, whereas rapamycin alone produced suppression on day 14 with rebound of IH by day 28. Since rapamycin, PLGA, and pluronic gel have all been approved by the FDA for other human therapies, this drug delivery method could potentially be translated into human use quickly to prevent failure of open vascular reconstructions.
    PLoS ONE 02/2014; 9(2):e89227. DOI:10.1371/journal.pone.0089227 · 3.23 Impact Factor
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    ABSTRACT: Adventitia acts as an active participant in vascular inflammation but the precise mechanism underlying adventitia-mediated vascular inflammation is not fully understood. In this study, we sought to determine whether vascular endothelial growth factor (VEGF) regulates osteopontin (OPN) expression through Flt-1 in adventitial fibroblasts (AFs) to mediate vascular inflammation and neointima formation. In primary cultured AFs, VEGF increased intracellular and secreted OPN expression in a time- and dose-dependent manner, which was effectively suppressed by a specific anti-Flt-1 hexapeptide. Interestingly, VEGF treatment of AFs enhanced the capability of AF-conditioned medium to stimulate macrophages chemotaxis, and this effect was attenuated after blockade of OPN from AF-conditioned medium. Furthermore, perivascular delivery of anti-Flt-1 peptide preferentially concentrated in the adventitia resulted in a decrease of neointima formation after balloon injury in carotid arteries. The inhibition of neointima formation was preceded by significant reduction of VEGF and OPN expression with concurrent macrophage infiltration into adventitia after injury. Activation of extracellular signal-regulated kinase 1/2 pathway was involved in OPN upregulation and macrophage chemotaxis. These results demonstrate that VEGF/Flt-1 signaling plays a significant role in vascular inflammation and neointima formation by regulating OPN expression in AFs and provide insight into Flt-1 as a potential therapeutic target for vascular diseases.
    Arteriosclerosis Thrombosis and Vascular Biology 07/2012; 32(9):2250-8. DOI:10.1161/ATVBAHA.112.255216 · 6.00 Impact Factor
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