Ribosomal Protein L17, RpL17, is an Inhibitor of Vascular Smooth Muscle Growth and Carotid Intima Formation
ABSTRACT BACKGROUND: Carotid intima-media thickening is associated with increased cardiovascular risk in humans. We discovered that intima formation and cell proliferation in response to carotid injury is greater in SJL/J (SJL) compared to C3HeB/FeJ (C3H/F) mice. The purpose of this study was to identify candidate genes contributing to intima formation. METHODS AND RESULTS: We performed microarray and bioinformatic analyses of carotid arteries from C3H/F and SJL mice. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the ribosome pathway was significantly up-regulated in C3H/F compared to SJL. Expression of a ribosomal protein, RpL17, was >40-fold higher in C3H/F carotids compared to SJL. Aortic vascular smooth muscle cells (VSMC) from C3H/F grew slower compared to SJL. To determine the role of RpL17 in VSMC growth regulation we analyzed the relationship between RpL17 expression and cell cycle progression. Cultured VSMC from mouse, rat, and human showed that RpL17 expression inversely correlated with growth as shown by decreased cells in S phase and increased cells in G(0)/G(1). To prove that RpL17 acted as a growth inhibitor in vivo we used pluronic gel delivery of RpL17 siRNA to C3H/F carotid arteries. This resulted in an 8-fold increase in the number of proliferating cells. Furthermore, following partial carotid ligation in SJL mice, RpL17 expression in the intima and media decreased while the number of proliferating cells increased. CONCLUSIONS: RpL17 acts as a VSMC growth inhibitor (akin to a tumor suppressor) and represents a potential therapeutic target to limit carotid intima-media thickening.
- [Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVE: We demonstrated that inflammatory cells and intima-media thickening are increased in carotids exposed to low-blood flow in the SJL/J (SJL) strain compared with other mouse strains. We hypothesized that the extent of inflammation associated with intima-media thickening is a genetically regulated trait.Approach and Results-We performed a whole genome approach to measure leukocyte infiltration in the carotid intima as a quantitative trait in a genetic cross between C3HeB/FeJ (C3H/F) and SJL mice. Immunostaining for CD45+ (a pan-specific leukocyte marker) was performed on carotids from C3H/F, SJL, F1, and N2 progeny to measure leukocyte infiltration. We identified a nearly significant quantitative trait locus for CD45+ on chromosome (chr) 11 (17 cM, LOD=2.3; significance was considered at threshold P=0.05). Interval mapping showed that the CD45+ locus on chr 11 accounted for 8% of the variation in the C3H/FxSJL backcross. Importantly, the CD45+ locus colocalized with the intima-modifier 2 (Im2) locus, which controls 17% of intima variation. We created 2 Im2 congenic lines of mice (C3H/F.SJL.11.1 and C3H/F.SJL.11.2) to better understand the regulation of intima-media thickening by the chr 11 locus. The C3H/F.SJL.11.1 congenic mouse showed ≈30% of the SJL trait, confirming that CD45+ cell infiltration contributed to the intima trait. CONCLUSIONS: We discovered a novel locus on chr 11 that controls leukocyte infiltration in the carotid. Importantly, this locus overlaps with our previously published Im2 locus on chr 11. Our study reveals a potential mechanistic relationship between leukocyte infiltration and intima-media thickening in response to decreased blood flow.Arteriosclerosis Thrombosis and Vascular Biology 02/2013; 33(5). DOI:10.1161/ATVBAHA.112.301129
- [Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVE: To determine the role of patched receptor (Ptc)-1 in mediating pulsatile flow-induced changes in vascular smooth muscle cell growth and vascular remodeling. APPROACH AND RESULTS: In vitro, HCASMC were exposed to normal or pathological low pulsatile flow conditions for 24 hours using a perfused transcapillary flow system. Low pulsatile flow increased vascular smooth muscle cell proliferation when compared with normal flow conditions. Inhibition of Ptc-1 by cyclopamine attenuated low flow-induced increases in Notch expression, whereas concomitantly decreasing HCASMC growth to that similar of normal flow conditions. In vivo, ligation injury-induced low flow increased vascular smooth muscle cell growth and vascular remodeling, although increasing Ptc-1/Notch expression. Perivascular delivery of Ptc-1 small interfering RNA by pluronic gel inhibited the pathological low flow-induced increases in Ptc-1/Notch expression and the subsequent increase in vascular remodeling. In addition, this pathological low flow-induced remodeling was returned to normal flow control levels after ptc-1 gene knockdown. CONCLUSIONS: These results suggest that pathological low flow stimulates smooth muscle cell growth in vitro and vascular remodeling in vivo via Ptc-1 regulation of Notch signaling.Arteriosclerosis Thrombosis and Vascular Biology 06/2013; 33(8). DOI:10.1161/ATVBAHA.113.301843
- [Show abstract] [Hide abstract]
ABSTRACT: Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, is expressed in vascular smooth muscle cells (VSMCs) of blood vessels and functions to repress VSMC migration and vascular remodeling. The goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration. Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 associated with the actin cytoskeleton. In response to chemoattractant stimulation, Csrp2 (mouse CRP2 gene symbol)-deficient (Csrp2(-/-)) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2(-/-) VSMCs following chemoattractant stimulation. Mammalian 2-hybrid and coimmunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibers in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs while CRP2 was restricted to FAs and stress fibers. Furthermore, we demonstrated that p130Cas expression and phosphorylation promote neointima formation following arterial injury. These studies demonstrate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.Cardiovascular Research 08/2013; DOI:10.1093/cvr/cvt207