Pathological consequences from the infusion of unstable lipid emulsion admixtures in guinea pigs.

The pathophysiologic effects of infusing unstable lipid emulsions are unclear, but these were shown to cause reticuloendothelial system (RES) dysfunction in animals and humans. We investigated the effects of unstable lipid emulsions in RES organs defined by two levels of the percent fat >5 microm (percentage of fat, PFAT>5 microm) in a guinea pig model. Two identical injectable lipid emulsions with differing (stable versus unstable) PFAT >5 microm levels, were infused for over 24h into two groups of animals (n=5/group). The PFAT>5 microm concentration was measured before and at the end of the infusion to ascertain the dose range of enlarged fat globules in each group. Animals were euthanized and specimens from the upper, middle and lower lung, and a single liver sample were examined histologically and for micromolar concentrations of malondialdehyde (MDA) per gram (micromol(-1)g) of wet tissue. The PFAT>5 microm concentrations pre-infusion were 0.004+/-0.001 and 2.418+/-0.273 for the stable and unstable injectable lipid emulsions respectively. At 24 h, the PFAT>5 microm level increased in both the groups (stable: 0.161+/-0.008; unstable: 7.861+/-0.291). MDA concentrations were significantly higher in the lungs of animals receiving the unstable (47.2+/-26.2 micromol(-1)g) versus stable (32.4+/-11.2 micromol(-1)g) injectable lipid emulsions (P=0.033), but was not different for the liver specimens (stable: 16.9+/-7.6 micromol(-1)g versus unstable: 17.7+/-2.2 micromol(-1)g, P=0.944). These preliminary data suggest that infusion of unstable injectable lipid emulsions has pathological consequences showing greater evidence of oxidative stress in the lungs.

Angiotensin receptor blockade with candesartan attenuates atherosclerosis, plaque disruption, and macrophage accumulation within the plaque in a rabbit model.

BACKGROUND: Little is known about whether direct angiotensin receptor blockade can reduce atherosclerosis and plaque disruption. This study evaluated the effect of angiotensin receptor blockade on both the development of atherosclerosis and the disruption of plaque in a modified Constantinides animal model. METHODS AND RESULTS: Twenty-eight New Zealand White rabbits underwent aortic balloon injury followed by a 1% cholesterol diet for 8 weeks. Thirteen rabbits received candesartan at 0.5 mg x kg(-1) x d(-1) beginning 2 days before aortic balloon injury and continued for the total 8 weeks of the cholesterol diet. The rabbits were then pharmacologically triggered and humanely killed, and their aortas were analyzed. The degree of atherosclerosis was determined by intima-media ratio of the infrarenal portion of the aorta. The frequency of intra-aortic thrombosis, a measure of plaque disruption, and the percentages of macrophage area and collagen-staining area of the plaque were determined. Candesartan-treated rabbits had less atherosclerosis (intima-media infrarenal aorta ratio of 1.18+/-0.08 versus 1.57+/-0.08 [mean+/-SEM] for the placebo group, P<0.001); fewer thrombi (3 of 13 versus 11 of 15; P<0.05); lower percentage area of macrophages to total plaque (18.8+/-2.7% versus 27+/-2.5%, P<0.05); and higher collagen to total plaque area (45+/-3% versus 35+/-2%, P<0.01). CONCLUSIONS: These results demonstrate that angiotensin receptor blockade attenuates the degree of atherosclerosis and reduces both plaque disruption and macrophage accumulation while increasing collagen deposition in the aortas of this animal model.

Temporal gene expression following prosthetic arterial grafting.

BACKGROUND: Following prosthetic arterial grafting, cytokines and growth factors released within the perianastomotic tissues stimulate smooth muscle cell proliferation and matrix production. While much in vitro work has characterized this response, little understanding exists regarding the sequential up- and down-regulation of genes following prosthetic arterial grafting. This study evaluates temporal gene expression at the distal anastomosis of prosthetic arterial grafts using microarray analysis. METHODS: Expanded polytetrafluoroethylene (ePTFE) carotid interposition grafts (n = 12) were surgically implanted into mongrel dogs. Distal anastomotic segments were harvested at 7, 14, 30, or 60 days. Contralateral carotid artery served as control. Total RNA was isolated from the anastomotic tissue and paired controls. Samples were probed with oligonucleotide microarrays consisting of approximately 10000 human genes to analyze differential gene expression at each time point. RESULTS: Forty-nine genes were found to be up-regulated and 37 genes were found to be down-regulated at various time points. Six genes were found to be consistently up-regulated at all time intervals, including collagen type 1 alpha-1 and alpha-2, 80K-L protein (MARCKS), and osteopontin. Six genes were found to be consistently down-regulated, including smoothelin and tropomyosin 2. RT-PCR and immunohistochemistry confirmed the microarray data. CONCLUSIONS: This study uses microarray analysis to identify genes that were temporally up- and down-regulated after prosthetic arterial grafting. Genes with similar patterns of expression have been identified, providing insights into related cellular pathways that may result in the formation of anastomotic intimal hyperplasia.

Temporal genomics of vein bypass grafting through oligonucleotide microarray analysis.

OBJECTIVE: Autologous vein is the conduit of choice for small artery reconstruction. Despite excellent patency, these conduits undergo remodeling over time. The purpose of this study was to identify temporal gene expression in vein grafts versus control veins through microarray analysis. METHOD: Cephalic vein grafts (n = 12) were used to bypass femoral arteries in canines. Vein grafts were harvested after 1, 7, 14, and 30 days. Normal contralateral cephalic vein served as control. Total RNA was isolated; its quantity and quality were confirmed with spectrophotometry and gel electrophoresis. Affymetrix U133A GeneChips, comprising approximately 15,000 genes, were used to analyze differential gene expression at each time point. Statistical analysis was performed with Affymetrix and dChip software to identify consistently upregulated and downregulated genes. Real-time, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to validate microarray data. RESULTS: Statistical analysis revealed that 49 genes were consistently upregulated and 31 genes were consistently downregulated in all three animals at various time points. qRT-PCR to quantitatively assess messenger RNA expression was performed on specific genes to validate the microarray data. Immunohistochemistry to qualitatively assess protein expression was used for further validation. Hierarchical clustering with dChip identified additional genes with similar temporal or functional expression patterns. CONCLUSIONS: This is the first study to use microarray analysis with confirmatory qRT-PCR to identify altered genes after vein bypass grafting. Oligonucleotide microarrays and hierarchical clustering are powerful tools to generate hypotheses as the basis for additional research on gene expression in vein graft remodeling. Ultimately, identification of a temporal sequence of differential gene expression may provide insights not preferred into the molecular mechanisms of vein graft remodeling, but also into the pathways leading to intimal hyperplasia.

Poly(ADP-Ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity.

BACKGROUND: We have previously shown that endothelial function is impaired not only in diabetes but also in subjects at risk of developing type 2 diabetes. We hypothesized that changes in the expression or activity of the endothelial isoform of nitric oxide synthase (eNOS), the receptor for advanced glycation end products (RAGE), and poly(ADP-ribose) polymerase (PARP) are related to this impairment. METHODS AND RESULTS: We included a control group of 21 healthy subjects, a group of 22 healthy individuals with parental history of type 2 diabetes, a group of 23 subjects with impaired glucose tolerance, and a group of 21 type 2 diabetic patients. Two 2-mm forearm skin biopsies were taken from each participant and used for measurements. The percentage of PARP-positive endothelial nuclei was higher in the group with parental history of type 2 diabetes and diabetic patients compared with the controls (P<0.001). Immunoreactivity for nitrotyrosine (a marker of reactive nitrogen species) was higher in the diabetic group compared with all other groups (P<0.01). No differences in the expression of eNOS and RAGE were found among all 4 groups. The polymorphism of the eNOS gene was also studied and was not found to influence eNOS expression or microvascular functional measurements. CONCLUSIONS: PARP activation is present in healthy subjects at risk of developing diabetes as well as in established type 2 diabetic patients, and it is associated with impairments in the vascular reactivity in the skin microcirculation.

Attenuated retinoblastoma gene product and associated E2F/retinoblastoma imbalance in anastomotic intimal hyperplasia.

OBJECTIVE: The retinoblastoma gene product is a cell cycle control protein that when inhibited allows cell proliferation to progress by releasing E2F. Retinoblastoma manipulation has been attempted to prevent intimal hyperplasia (IH) in injured native vessels by arresting vascular smooth muscle cell proliferation. However, no studies have identified the role, if any, of retinoblastoma in anastomotic IH formation after prosthetic arterial grafting. The goal of this study was to describe the relation of retinoblastoma and E2F to anastomotic IH with analyzing retinoblastoma/E2F levels, retinoblastoma phosphorylation, and transcription of retinoblastoma and E2F in prosthetic arterial grafting. METHODS: Six-mm-diameter expanded polytetrafluoroethylene carotid interposition grafts (n = 12) were implanted in 25-kg mongrel dogs. The intervening arterial segments were harvested as controls. The distal anastomoses were harvested at 14 and 30 days after implantation for immunoblot, messenger RNA (mRNA), and immunohistochemistry analyses. Tissue homogenate was separated with sodium dodecylsulfate-polyacrylamide gel electrophoresis and probed with antibody to total retinoblastoma, phosphorylated retinoblastoma at serine 795, serine 780, and serine 807/811, and E2F-1. Bands at each time point were quantitated and compared with control artery (n = 12). Each lane was standardized with reprobing with antibody to beta-tubulin. Immunohistochemistry was performed with antibody to retinoblastoma. Retinoblastoma and E2F mRNA expression levels in anastomotic IH and control artery were analyzed with an oligonucleotide microarray. RESULTS: Total retinoblastoma, from immunoblot analysis, was decreased at the 14-day and 30-day distal anastomoses by 35.7% and 33.6%, respectively, compared with control (P <.01). Furthermore, retinoblastoma at these time points was unphosphorylated at phosphorylation sites serine 795, serine 780, and serine 807/811. E2F-1 levels at 14 days and 30 days were unchanged compared with control. Positive staining for retinoblastoma was seen in endothelial and vascular smooth muscle cell from control, 14-day, and 30-day tissue. A qualitative decrease appeared to be seen in retinoblastoma in the neointima at 14 and 30 days compared with the native wall. No differential expression of retinoblastoma and E2F mRNA was seen in anastomotic IH compared with control. CONCLUSION: This study showed that total retinoblastoma levels are decreased and E2F-1 levels remain unchanged in anastomotic IH. Attenuated retinoblastoma is a novel concept to anastomotic IH after prosthetic arterial grafting. Retinoblastoma/E2F imbalance may not be the result of transcriptional regulation and may increase unbound E2F to promote cell proliferation. Hypophosphorylation of remaining retinoblastoma may minimize uncontrolled proliferation by preventing further increases in unbound E2F. Therefore, retinoblastoma/E2F imbalance may lead to the early but limited increase in cell proliferation seen after prosthetic arterial grafting and appears to contribute to the development and progression of anastomotic IH.

Vascular smooth muscle cells derived from atherosclerotic human arteries exhibit greater adhesion, migration, and proliferation than venous cells.

BACKGROUND: Phenotypic variation of vascular smooth muscle cells (VSMC) may result in altered biological behavior and responses. Within the vessel wall, arterial VSMC have a greater propensity to form atherosclerotic lesions as compared to venous VSMC. In this study the rates of proliferation, adhesion, and migration were compared between VSMC of atherosclerotic arterial and venous origin. MATERIALS AND METHODS: Human VSMC cultures were isolated from 18 infragenicular arteries at the time of below knee amputation and from 20 saphenous veins during lower extremity revascularization surgery. Cell cultures were isolated from the media of each specimen and maintained in distinct cell lines for all assays. Cells from passages 2 and 3 were assayed for their proliferative capacity using total DNA fluorescence photometry and for adhesion and migration using a modified Boyden chamber. RESULTS: Patient age and the incidence of atherosclerotic risk factors did not vary significantly between the arterial and the venous patient groups. VSMC of atherosclerotic arterial origin demonstrated greater proliferation (arterial, 162 +/- 59 absorption units, vs. venous, 106 +/- 56 absorption units, P < 0.001), adhesion (arterial, 74.1 +/- 22.6 cells/microscopic field, vs. venous, 41.3 +/- 12.8 cells/microscopic field, P < 0.001) and migration (arterial, 427 +/- 185 cells/microscopic field, vs venous, 119 +/- 101 cells/microscopic field, P < 0.001) than VSMC of venous origin. CONCLUSION: Human atherosclerotic arterial VSMC exhibit significantly increased rates of proliferation, adhesion, and migration as compared to human venous VSMC. These observations of VSMC in culture are consistent with the clinical predilection for the hyperplasic responses that result in the development of atherosclerosis in the arterial wall. Possible intrinsic differences in VSMC phenotype should be considered in designing methods to limit atherosclerosis.

A biologically active VEGF construct in vitro: implications for bioengineering-improved prosthetic vascular grafts.

Prosthetic arterial grafts are unable to develop an intact endothelial lining after implantation, predisposing them to fail. Strategies have been sought to enhance endothelialization using growth factors and cytokines. This study assessed the biologic activity of vascular endothelial growth factor (VEGF) covalently linked to bovine serum albumin (BSA). Native and modified VEGF were assayed for endothelial cell migration and proliferation. Migration assays were performed comparing the effects of 2% fetal bovine serum (FBS), 50 ng/mL, 100 ng/mL, and 200 ng/mL of native VEGF and VEGF-BSA. Proliferation assays were performed by using Alamar Blue comparing cellular growth in 1% FBS, 10% FBS, 100 ng/mL unbound VEGF, and 100 ng/mL VEGF-BSA. VEGF is a potent chemotactic agent for endothelial cells in both unbound and bound states. Native VEGF solutions (50 ng/mL, 100 ng/mL, and 200 ng/mL) stimulated 23.9 cells/high power field (HPF), 35.3 cells/HPF, and 49.1 cells/HPF (p < 0.005). VEGF-BSA solutions stimulated 25.9 cells/HPF, 39.1 cells/HPF, and 69.0 cells/HPF (p < 0.001). VEGF-BSA and native VEGF supported similar increased cellular proliferation compared with 1% FBS media (p < 0.002). Modified VEGF retains its chemotactic and proliferative properties in vitro. These findings suggest that bare prosthetic surfaces lined with VEGF bound to a "basecoat" albumin may support endothelial cell proliferation and migration and thereby offer new strategies to improve graft patency.