Yuntao Wei

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.

Origin of Neointimal Cells in Arteriovenous Fistulae: Bone Marrow, Artery, or the Vein Itself?

To elucidate the source of neointimal cells, experimental fistulas were created in Lewis wild‐type (WT) and transgenic rats that constitutively expressed the green fluorescent protein (GFP) in all tissues. Arteriovenous fistula (AVFs) were created by anastomosing the left renal vein to the abdominal aorta. The contribution of bone marrow (BM)‐derived cells to the AVF neointima was examined in lethally irradiated WT rats that had been rescued with GFP BM cells. Neointimal cells in these chimeric rats were mostly GFP negative indicating the non‐BM origin of those cells. Then, the contribution of arterial cells to the AVF neointima was assessed in a fistula made with a GFP aorta that had been implanted orthotopically into a WT rat. Most of the neointimal cells were also GFP negative demonstrating that AVF neointimal cells are not derived from the feeding artery. Finally to study local resident cells contribution to the formation of neointimal lesions, a composite fistula was created by interposing a GFP vein between the renal vein and the aorta in a WT recipient rat. GFP neointimal cells were only found in the transplanted vein. This study suggests that neointimal cells originate from the local resident cells in the venous limb of the fistula.

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.

Macrophage-derived IL-18 and increased fibrinogen deposition are age-related inflammatory signatures of vascular remodeling.

Aging has been associated with pathological vascular remodeling and increased neointimal hyperplasia. The understanding of how aging exacerbates this process is fundamental to prevent cardiovascular complications in the elderly. This study proposes a mechanism by which aging sustains leukocyte adhesion, vascular inflammation, and increased neointimal thickness after injury. The effect of aging on vascular remodeling was assessed in the rat balloon injury model using microarray analysis, immunohistochemistry, and LINCOplex assays. The injured arteries in aging rats developed thicker neointimas than those in younger animals, and this significantly correlated with a higher number of tissue macrophages and increased vascular IL-18. Indeed, IL-18 was 23-fold more abundant in the injured vasculature of aged animals compared with young rats, while circulating levels were similar in both groups of animals. The depletion of macrophages in aged rats with clodronate liposomes ameliorated vascular accumulation of IL-18 and significantly decreased neointimal formation. IL-18 was found to inhibit apoptosis of vascular smooth muscle cells (VSMC) and macrophages, thus favoring both the formation and inflammation of the neointima. In addition, injured arteries of aged rats accumulated 18-fold more fibrinogen-γ than those of young animals. Incubation of rat peritoneal macrophages with immobilized IL-18 increased leukocyte adhesion to fibrinogen and suggested a proinflammatory positive feedback loop among macrophages, VSMC, and the deposition of fibrinogen during neointimal hyperplasia. In conclusion, our data reveal that concentration changes in vascular cytokine and fibrinogen following injury in aging rats contribute to local inflammation and postinjury neointima formation.

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.

Vascular Regeneration in Ischemic Hindlimb by Adeno‐Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor

Background Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno‐associated virus (AAV) and lentivirus have been tested in preclinical models. Methods and Results We present preclinical results of ischemia (hypoxia)‐regulated conditionally silenced (CS) AAV–human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV‐CS‐hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra‐arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV‐CS‐hVEGF. AAV‐CS‐hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch,SDF1, Angiopoietin, and Ephrin‐B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. Conclusions Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

A novel mouse model of in-stent restenosis

BACKGROUND AND AIMS In-stent restenosis (ISR) is the major complication that occurs after percutaneous coronary interventions to facilitate coronary revascularization. Herein we described a simple and cost-effective model, which reproduces important features of ISR in the mouse. METHODS AND RESULTS Microvascular bare metal stents were successfully implanted in the abdominal aorta of atherosclerotic ApoE-null mice. Patency of implanted stents was interrogated using ultrasound biomicroscopy. Aortas were harvested at different time points after implantation and processed for histopathological analysis. Thrombus formation was histologically detected after 1 day. Leukocyte adherence and infiltration were evident after 7 days and decreased thereafter. Neointimal formation, neointimal thickness and luminal stenosis simultaneously increased up to 28 days after stent implantation. Using multichannel fluorescence molecular tomography (FMT) for spatiotemporal resolution of MMP activities, we observed that MMP activity in the stented aorta of Apo-E null mice was 2-fold higher than that of wild-type mice. Finally, we compared neointimal formation in response to stenting in two genetically different mouse strains. In-stent neointimas in FVB/NJ mice were 2-fold thicker than in C57BL/6J mice (p=0.002). CONCLUSION We have developed a model that can take advantage of the multiple genetic resources available for the mouse to study the mechanisms of in-stent restenosis.

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.

CD4(+) lymphocytes improve venous blood flow in experimental arteriovenous fistulae.

BACKGROUND The role of immune cells in arteriovenous fistulae (AVF) maturation is poorly understood and has received, until quite recently, little attention. This study examines the function of T lymphocytes in AVF vascular remodeling. METHODS Experimental fistulae were created in athymic rnu nude rats lacking mature T lymphocytes and euthymic control animals by anastomosing the left superior epigastric vein to the nearby femoral artery. Blood flow rates, wall morphology, and histologic changes were assessed in AVF 21 days after creation. The effect of CD4(+) lymphocytes on AVF maturation in athymic animals was analyzed by adoptive transfer of cells after fistula creation. RESULTS The absence of T lymphocytes compromised blood flow in experimental fistulae. Histopathologic inspection of AVF from athymic rats revealed that T-cell immunodeficiency negatively affected venous vascular remodeling, as evidenced by a reduced lumen, a thick muscular layer, and a low number of inflammatory cells compared with control animals. Adoptive transfer of CD4(+) lymphocytes from euthymic rats into athymic animals after fistula creation improved blood flow and reduced intima-media thickness. CONCLUSION These results point at the protective role of CD4(+) lymphocytes in the remodeling of the AVF vascular wall.

PC202 Critical Role of Endothelial Notch1 Signaling in Atherosclerosis

establishment of a regulated inflammatory response during wounding. Macrophage function is dictated by metabolism, which alters gene expression. The mitochondrial deacylase enzyme, Sirtuin 3 (SIRT3), was recently shown to suppress inflammatory signaling and decrease reactive oxygen species. Therefore, we investigated the role of SIRT3 in inflammation in peripheral wound macrophages. Methods: C57BL/6 mice maintained on a normal or high-fat diet for 12 weeks underwent 4-mm hind limb wounding, and macrophages (CD3-CD19-NK1.1-CD11b cells) were isolated from wounds on days 1, 3, 5, and 7. SIRT3 expression was quantified by quantitative polymerase chain reaction. SIRT3 mice and their littermate controls underwent 4-mm hind limb wounds, and wound closure was compared daily between the two groups for 1 week. Macrophages were isolated from mouse wounds on day 3 using magnetic bead sorting. Quantitative polymerase chain reaction was used to quantify expression of inflammatory cytokines. Protein data were obtained using flow cytometry. Results: In this murine model of wound healing, CD11b cells (macrophages) isolated from wounds demonstrated a significant threefold increase in SIRT3 gene expression during the intermediate stage (day 3-5) of wound healing. Additionally, when we examined woundmacrophages from our diet-induced obese (DIO) model of diabetes, we found that they did not upregulate SIRT3. To directly examine the role of SIRT3 in wound healing, we wounded our SIRT3 and littermate controls. We found that the SIRT3 mice demonstrated significantly delayed wound healing compared to controls. Macrophages isolated from wounds of SIRT3 mice on day 3 revealed significantly higher expression of inflammatory cytokines (tumor necrosis factor-a, interleukin 1b, nitric oxide synthase 2) compared with controls (P < .01). Conclusions: SIRT3 is important in macrophages for normal wound healing and is decreased in macrophages from wounds of diabetic mice. These results suggest an important role for SIRT3 in regulating inflammation in wound healing.