Dania Mateu

The origin of post-injury neointimal cells in the rat balloon injury model

AIMS The origin of post-injury neointimal cells is still a matter of debate. This study aims to determine the anatomic source of neointimal cells in one of the most important animal models for the study of vascular stenosis in response to injury, the rat balloon injury model. METHODS AND RESULTS Chimeric rats were generated by rescuing lethally irradiated animals with green fluorescent protein (GFP)(+) bone marrow (BM) cells from transgenic rats. Neointimal formation was induced in the right iliac artery of these animals using a balloon angioplasty catheter. Injured and non-injured contra-lateral arteries were harvested at 7, 14, and 30 days post-surgery. BM-derived monocytes/macrophages (CD68(+) GFP(+)) were abundant in the media and adventitia of injured vessels harvested at 7 days as determined by immunofluorescence and confocal microscopy. The number of GFP(+) cells declined in the vascular wall with time. Post-injury neointimal cells were mostly GFP(-)/smooth muscle actin (SMA)(+), which indicated that those cells originated in the recipient. Only a few neointimal cells seemed to come from circulating progenitors (GFP(+) SMA(+), 2.34% +/- 1.61). The vascular origin of cells in the neointima was further confirmed by transplanting injured GFP arteries into wild-type recipients. In these grafts, 94.23 +/- 0.44% of medial and 92.95 +/- 19.34% of neointimal cells were GFP(+) SMA(+). Finally, we tested the capacity of vascular smooth muscle cells (VSMC) to migrate through the vascular wall using a novel in vivo assay. As expected, VSMC migrated and populated the neointima only in response to injury. CONCLUSION Our results suggest that neointimal cells in the rat balloon injury model mostly derive from pre-existing vascular cells and that only a small population of those cells come from BM-derived progenitors.

Structural Determinants for Phosphatidic Acid Regulation of Phospholipase C-β1

Signaling from G protein-coupled receptors to phospholipase C-β (PLC-β) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-β1 through a mechanism that requires the PLC-β1 C-terminal domain. PA also modulates Gαq stimulation of PLC-β1. These data suggest that PA may have a key role in the regulation of PLC-β1 signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-β1. We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-β1 proteins and with fragments of the Gαq binding domain. The results identify a region that includes the αA helix and flexible loop of the Gαq-binding domain as necessary for PA regulation. A mutant PLC-β1 with multiple alanine/glycine replacements for residues 944LIKEHTTKYNEIQN957 was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-β1 concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-β1 mutant. These data are consistent with a model for PA regulation of PLC-β1 that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Gαq-binding domain. PA regulation of PLC-β1 requires unique residues that are not required for Gαq stimulation or GTPase-activating protein activity.

Novel role of Egr-1 in nicotine-related neointimal formation

AIMS The aim of this study was to investigate the mechanisms by which nicotine increases vascular smooth muscle cell (VSMC) proliferation and post-injury neointimal formation. METHODS AND RESULTS Vascular injury was inflicted in the right iliac artery of nicotine-treated and control rats. Nicotine increased post-injury VSMC proliferation (Ki67(+) cells) and neointimal formation (neointima/media ratio, 0.42 ± 0.23 vs. 0.14 ± 0.07, P= 0.02). To determine the mechanisms by which nicotine exacerbates VSMC proliferation, cultured cells were exposed to nicotine, and signalling pathways leading to cell proliferation were studied. Nicotine activated extracellular signal-regulated kinase (ERK) 1/2 in a dose- and time-dependent manner. The blockade of this signalling axis abolished nicotine-mediated proliferation. Functional nicotinic acetylcholine receptors and Ca(2+) influx were necessary for ERK1/2 activation and nicotine-induced mitogenesis in VSMCs. Downstream to ERK1/2, nicotine induced the phosphorylation of Ets-like gene 1 in a timely co-ordinated manner with the up-regulation of the atherogenic transcription factor, early growth response 1 (Egr-1). The treatment of balloon-injured arteries with a lentivirus vector carrying a short hairpin RNA against Egr-1 abolished the deleterious effect of nicotine on vascular remodelling. CONCLUSION Nicotine acts through its receptors in VSMC to activate the ERK-Egr-1 signaling cascade that induces cell proliferation and exacerbates post-injury neointimal development.

Aging increases p16INK4a expression in vascular smooth-muscle cells

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMCs response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.

Aging increases p16 INK4a expression in vascular smooth-muscle cells.

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMCs response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.

An internal ribosome entry site mediates the initiation of soluble guanylyl cyclase β2 mRNA translation

The soluble guanylyl cyclases (sGC), the receptor for nitric oxide, are heterodimers consisting of an α‐ and β‐subunit. This study aimed to investigate the translational mechanism of the sGC β2‐subunit. Two mRNA species for sGC β2 were isolated from human kidney. These transcripts had dissimilar 5′‐untranslated regions (5′‐UTRs). The most abundant sGC β2 mRNA showed numerous upstream open reading frames (ORFs) and stable secondary structures that inhibited in vivo and in vitro translation. To evaluate whether these 5′‐UTRs harbored an internal ribosome entry site (IRES) that allows translation by an alternative mechanism, we inserted these regions between the two luciferase genes of a bicistronic vector. Transfection of those genetic constructs into HeLa cells demonstrated that both sGC β2 leaders had IRES activity in a cell‐type dependent manner. Finally, the secondary structural model of the sGC β2 5′‐UTR predicts a Y‐type pseudoknot that characterizes the IRES of cellular mRNAs. In conclusion, our findings suggest that sGC β2 5′‐UTRs have IRES activity that may permit sGC β2 expression under conditions that are not optimal for scanning‐dependent translation.

Aging increases p16INK4aexpression in vascular smooth-muscle cells

Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMCs response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.