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.
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- "In this study, we adopted a perivascular delivery system of gene specific siRNAs utilizing a pluronic gel  . Following pluronic gel delivery of Notch 1 siRNA, which targets the desired siRNA to an area below the bifurcation of the ligated carotid artery, we achieved a significant inhibition of whole vessel and medial Notch1 mRNA and protein expression, concomitant with decreased SMC medial growth and injury-induced remodeling. "
ABSTRACT: To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling. Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown. These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.PLoS ONE 01/2014; 9(1):e84122. DOI:10.1371/journal.pone.0084122 · 3.23 Impact Factor
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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 · 5.53 Impact Factor
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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 · 5.53 Impact Factor