Improve endothelialization of metallic cardiovascular stent via femtosecond laser induced micro/nanostructure dependent cells proliferation and drug delivery control.

Regarding restenosis occurrence risk after metallic stent deployment in artery, stents with vascular smooth muscle cells antiproliferative agents sustained released from poly(lactic-co-glycolic acid) (PLGA) coating and endothelial cells proliferation favored surface textures were both attempted for endothelialization enhancement. In order to explore the interaction between the surface texture and performance of drug loaded PLGA coating, femtosecond laser surface treatment was used to change the surface characteristics of 316L stainless steel. Two different surface patterns in form of stripe (FSL100) and isolated island-like structure (FSL800) were firstly generated by femtosecond laser processing with 100 and 800 mW energy, then Rapamycin loaded PLGA coating was further deposited to polished and femtosecond laser processed 316L surfaces via a dip-coating method. The subsequent drug loading capacity and release profile studies confirmed the roles of surface texture. Morphological transition characteristics of the PLGA coating on the FLS100 sample indicate that the coating has integrity during degradation compared to the polished one. Finally, rapamycin eluting FLS100 stent was deployed to iliac arteries of New Zealand White rabbits with vascular plaques to demonstrate its endothelialization potential and resistance to restenosis.

Hyaluronic acid oligosaccharide-modified collagen nanofibers as vascular tissue-engineered scaffold for promoting endothelial cell proliferation.

Hyaluronic acid oligosaccharides (oHAs) have shown promising results in promoting vascular endothelial cell (EC) proliferation and endothelialization. To engineered develop tissue scaffold for promoting EC proliferation and vessel endothelialization, different sizes of oHAs were prepared and grafted onto collagen to improve the biological properties of the synthesized materials, especially in angiogenesis. Firstly, oHAs were successfully prepared and conjugated with collagen to construct glycosylated collagens by reductive amination. The glycosylated collagens were then electrospun into various nanofibrous structures and their morphology and hemocompatibility, as well as EC responses on these nanofibrous scaffolds, were studied to evaluate the potential for vascular tissue engineering. The results showed that the nanofibrous scaffolds grafted by oHAs promoted EC proliferation whereas high molecular weight HA inhibited proliferation. The scaffolds had no detectable degree of hemolysis and coagulation, suggesting their promise as engineered vascular tissue scaffolds.

Fumonisin B1 Induces Oxidative Stress and Breaks Barrier Functions in Pig Iliac Endothelium Cells.

Fumonisins (Fums) are types of mycotoxin that widely contaminante feed material crops, and can trigger potential biological toxicities to humans and various animals. However, the toxicity of Fums on porcine blood vessels has not been fully explored. Fumonisin B1 (FB1) is the main component of Fums. Therefore, the aim of this study was to explore the effects of FB1 on the oxidative stress and tight junctions of the pig iliac endothelial cells (PIECs) in vitro. The results showed that FB1 reduced the viability of PIECs, increased the contents of lipid peroxidation product malondialdehyde (MDA), decreased the activities of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and thioredoxin reductase (TrxR), and decreased the level of glutathione (GSH). In addition, the barrier functions were destroyed, along with the down-regulations on Claudin 1, Occludin and ZO-1 and the increase of paracellular permeability. Thus, this research indicates that FB1 facilitates oxidative stress and breaks barrier functions to damage pig iliac endothelium cells.

Circulating pig-specific DNA as a novel biomarker for monitoring xenograft rejection.

Monitoring for immune rejection is crucial for long-term survival of pig xenografts. Circulating DNA is a promising non-invasive biomarker for either organ injury or response to therapy. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection. Potential targets of cpsDNA were selected by in silico analysis, and species specificity of selected primers was confirmed by PCR. Subsequently, cpsDNA as a biomarker was evaluated using a complement-dependent cytotoxicity (CDC) assay in vitro. Then, early diagnosis and response to rapamycin were assessed by an in vivo imaging model of pig-to-mouse cell transplantation. Finally, cpsDNA was monitored in a pig-to-monkey artery patch transplantation model. The results showed that (a) a method of cpsDNA quantitation was established for application in mouse and nonhuman primate models; (b) cpsDNA reflected CDC in vitro; (c) cpsDNA in vivo mirrored xenograft rejection, and correlated with xenograft loss in pig-to-mouse cell transplantation; (d) cpsDNA was significantly reduced when rapamycin was administered; and (e) dynamic cpsDNA was detectable in pig-to-monkey artery patch transplantation. In conclusion, measurement of cpsDNA could prove to be a less invasive, but more specific and sensitive low-cost biomarker enabling monitoring of xenograft rejection and the response to immunosuppressive therapy.

Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

During hypoxia, a cellular adaptive response activates hypoxia-inducible factors (HIFs; HIF-1 and HIF-2) that respond to low tissue-oxygen levels and induce the expression of a number of genes that promote angiogenesis, energy metabolism, and cell survival. HIF-1 and HIF-2 regulate endothelial cell (EC) adaptation by activating gene-signaling cascades that promote endothelial migration, growth, and differentiation. An HIF-1 to HIF-2 transition or switch governs this process from acute to prolonged hypoxia. In the present study, we evaluated the mechanisms governing the HIF switch in 10 different primary human ECs from different vascular beds during the early stages of hypoxia. The studies demonstrate that the switch from HIF-1 to HIF-2 constitutes a universal mechanism of cellular adaptation to hypoxic stress and that HIF1A and HIF2A mRNA stability differences contribute to HIF switch. Furthermore, using 4 genome-wide mRNA expression arrays of HUVECs during normoxia and after 2, 8, and 16 h of hypoxia, we show using bioinformatics analyses that, although a number of genes appeared to be regulated exclusively by HIF-1 or HIF-2, the largest number of genes appeared to be regulated by both.-Bartoszewski, R., Moszynska, A., Serocki, M., Cabaj, A., Polten, A., Ochocka, R., Dell'Italia, L., Bartoszewska, S., Króliczewski, J., Dabrowski, M., Collawn, J. F. Primary endothelial cell-specific regulation of hypoxia-inducible factor (HIF)-1 and HIF-2 and their target gene expression profiles during hypoxia.

Analysis of non-Newtonian effects within an aorta-iliac bifurcation region.

The geometry of the arteries at or near arterial bifurcation influences the blood flow field, which is an important factor affecting arteriogenesis. The blood can act sometimes as a non-Newtonian fluid. However, many studies have argued that for large and medium arteries, the blood flow can be considered to be Newtonian. In this work a comprehensive investigation of non-Newtonian effects on the blood fluid dynamic behavior in an aorta-iliac bifurcation is presented. The aorta-iliac geometry is reconstructed with references to the values reported in Shah et al. (1978); the 3D geometrical model consists of three filleted cylinders of different diameters. Governing equations with the appropriate boundary conditions are solved with a finite-element code. Different rheological models are used for the blood flow through the lumen and detailed comparisons are presented for the aorta-iliac bifurcation. Results are presented in terms of the velocity profiles in the bifurcation zone and Wall Shear Stress (WSS) for different sides of the bifurcation both for male and female geometries, showing that the Newtonian fluid assumption can be made without any particular loss in terms of accuracy with respect to the other more complex rheological models.

Endothelial cell high-enrichment from endovascular biopsy sample by laser capture microdissection and fluorescence activated cell sorting.

BACKGROUND AND PURPOSE: Endovascular sampling and characterization from patients can provide very useful information about the pathogenesis of different vascular diseases, but it has been limited by the lack of an effective method of endothelial cell (EC) enrichment. We optimized the EC yield and enrichment from conventional guide wires by laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) technique, and addressed the feasibility of using these enriched ECs for downstream gene expression detection. METHODS: Iliac artery endovascular samples from 10 patients undergoing routine catheter angiography were collected using conventional 0.038 in. J-shape guide wires. Each of these samples was equally divided into two parts, which were respectively used for EC enrichment by immunocytochemistry-coupled LCM or multiple color FACS. After RNA extraction and reverse transcription, the amplified cDNA was used for quantitative polymerase chain reaction (qPCR). RESULTS: Fixed ECs, with positive CD31 or vWF fluorescent signal and endothelial like nucleus, were successfully separated by LCM and live single ECs were sorted on FACS by a seven color staining panel. EC yields by LCM and FACS were 51 ± 22 and 149 ± 56 respectively (P < 0.001). The minimum number of fixed ECs from ICC-coupled LCM for acceptable qPCR results of endothelial marker genes was 30, while acceptable qPCR results as enriched by FACS were attainable from a single live EC. CONCLUSION: Both LCM and FACS can be used to enrich ECs from conventional guide wires and the enriched ECs can be used for downstream gene expression detection. FACS generated a higher EC yield and the sorted live ECs may be used for single cell gene expression detection.

Endovascular biopsy: evaluating the feasibility of harvesting endothelial cells using detachable coils.

The absence of safe and reliable methods to harvest vascular tissue in situ limits the discovery of the underlying genetic and pathophysiological mechanisms of many vascular disorders such as aneurysms. We investigated the feasibility and comparable efficacy of endothelial cell collection using a spectrum of endovascular coils. Nine detachable coils ranging in k coefficient (0.15-0.24), diameter (4.0 mm-16.0 mm), and length (8.0 cm-47.0 cm) were tested in pigs. All coils were deployed and retrieved within the iliac artery of pigs (three coils/pig). Collected coils were evaluated under light microscopy. The total and endothelial cells collected by each coil were quantified. The nucleated cells were identified by Wright-Giemsa and DAPI stains. Endothelial and smooth muscle cells were identified by CD31 and α-smooth muscle actin antibody staining. Coils were deployed and retrieved without technical difficulty. Light microscopy demonstrated sheets of cellular material concentrated within the coil winds. All coils collected cellular material while five of nine (55.6%) coils retrieved endothelial cells. Coils collected mean endothelial cell counts of 89.0±101.6. Regression analysis demonstrated a positive correlation between increasing coil diameter and endothelial cell counts (R(2)=0.52, p = 0.029). Conventional detachable coils can be used to harvest endothelial cells. The number of endothelial cells collected by a coil positively correlated with its diameter. Given the widespread use of coils and their well-described safety profile their potential as an endovascular biopsy device would expand the availability of tissue for cellular and molecular analysis.

Automatic lumen segmentation in IVOCT images using binary morphological reconstruction.

BACKGROUND: Atherosclerosis causes millions of deaths, annually yielding billions in expenses round the world. Intravascular Optical Coherence Tomography (IVOCT) is a medical imaging modality, which displays high resolution images of coronary cross-section. Nonetheless, quantitative information can only be obtained with segmentation; consequently, more adequate diagnostics, therapies and interventions can be provided. Since it is a relatively new modality, many different segmentation methods, available in the literature for other modalities, could be successfully applied to IVOCT images, improving accuracies and uses. METHOD: An automatic lumen segmentation approach, based on Wavelet Transform and Mathematical Morphology, is presented. The methodology is divided into three main parts. First, the preprocessing stage attenuates and enhances undesirable and important information, respectively. Second, in the feature extraction block, wavelet is associated with an adapted version of Otsu threshold; hence, tissue information is discriminated and binarized. Finally, binary morphological reconstruction improves the binary information and constructs the binary lumen object. RESULTS: The evaluation was carried out by segmenting 290 challenging images from human and pig coronaries, and rabbit iliac arteries; the outcomes were compared with the gold standards made by experts. The resultant accuracy was obtained: True Positive (%) = 99.29 ± 2.96, False Positive (%) = 3.69 ± 2.88, False Negative (%) = 0.71 ± 2.96, Max False Positive Distance (mm) = 0.1 ± 0.07, Max False Negative Distance (mm) = 0.06 ± 0.1. CONCLUSIONS: In conclusion, by segmenting a number of IVOCT images with various features, the proposed technique showed to be robust and more accurate than published studies; in addition, the method is completely automatic, providing a new tool for IVOCT segmentation.

Assessment of the antimicrobial activity of acellular vascular grafts.

Several studies have reported biological vascular grafts to be more resistant to microbial infection than synthetic counterparts in vivo. Indeed, small intestinal submucosa (SIS) materials have previously been reported to be antimicrobial. The aim of this study was to assess the antimicrobial activity and the ability to resist biofilm formation of a novel acellular vascular graft and compare it to commercially available alternatives using a range of organisms: MRSA, MSSA, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Candida albicans. This was achieved using a modified disk diffusion assay and extraction of the materials into solution followed by minimum inhibitory concentration assays. To assess resistance to biofilm formation a novel biofilm assay was developed which compared the total colony forming units (CFU) recovered from each material and identification of the percentage of CFU which were loosely attached, residing within the biofilm or attached to the biomaterial. The results indicated a lack of antimicrobial activity for all materials tested, including SIS. The biological materials were more resistant to the formation of a biofilm compared to Dacron.

Dynamic regulation of vascular myosin light chain (MYL9) with injury and aging.

BACKGROUND: Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels. METHODOLOGY/PRINCIPAL FINDINGS: We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBI's GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers. CONCLUSIONS/SIGNIFICANCE: The consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty.

Hydrogen peroxide regulates glucose-regulated protein 78 expression via a cyclooxygenase-2 dependent mechanism.

This study was designed to investigate the effect of hydrogen peroxide on the expression of endoplasmic reticulum stress marker glucose-regulated protein 78 (GRP78) in endothelial cells and reveals the possible role of cyclooxygenase in this effect. The porcine endothelial cell line was cultured in 1640 medium. Western blot and immunocytochemistry were used to detect the expression of GRP78. The caspase-12 activity was analyzed with the immune fluorescence method. The results showed that after the endothelial cells were incubated with 250 µM of hydrogen peroxide for 12 h, apoptosis increased, which was antagonized by the cyclooxygenase-2 inhibitor nimesulide or the nonselective cyclooxygenase inhibitor aspirin, but not by the cyclooxygenase-1 inhibitor piroxicam. The expression of GRP78 was induced in endothelial cells after exposure to hydrogen peroxide for 12 h. The overexpression of GRP78 was inhibited by nimesulide and aspirin, but not by piroxicam. There are no significant differences in caspase-12 activity among all groups. The present study provides evidence that hydrogen peroxide induced GRP78 overexpression in endothelial cells by a mechanism involving cyclooxygenase-2-dependent pathway.

Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries.

Atherosclerotic plaques tend to form in the major arteries at certain predictable locations. As these arteries vary in atherosusceptibility, interarterial differences in endothelial cell biology are of considerable interest. To explore the origin of differences observed between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of harvested porcine coronary (CECs) and iliac artery endothelial cells (IECs) grown in static culture out to passage 4. Fewer differences were observed between the transcriptional profiles of CECs and IECs in culture compared with in vivo, suggesting that most differences observed in vivo were due to distinct environmental cues in the two arteries. One-class significance of microarrays revealed that most in vivo interarterial differences disappeared in culture, as fold differences after passaging were not significant for 85% of genes identified as differentially expressed in vivo at 5% false discovery rate. However, the three homeobox genes, HOXA9, HOXA10, and HOXD3, remained underexpressed in coronary endothelium for all passages by at least nine-, eight-, and twofold, respectively. Continued differential expression, despite removal from the in vivo environment, suggests that primarily heritable or epigenetic mechanism(s) influences transcription of these three genes. Quantitative real-time polymerase chain reaction confirmed expression ratios for seven genes associated with atherogenesis and over- or underexpressed by threefold in CECs relative to IECs. The present study provides evidence that both local environment and vascular bed origin modulate gene expression in arterial endothelium. The transcriptional differences observed here may provide new insights into pathways responsible for coronary artery susceptibility.

[Effect of tangtong recipe on the proliferation of cultured iliac arterial smooth muscle cells of diabetic rats in vitro].

OBJECTIVE: To explore the inhibition of Tangtong Recipe (TTR) on proliferation of vascular smooth muscle cells (VSMCs) in rat model of diabetes mellitus using seropharmacological and modern molecular biological technologies. METHODS: The drug-serum of TTR was prepared by feeding pure-breed New Zealand rabbits at various doses (18 mL/kg, 13 mL/kg, and 8 mL/kg) of TTR, and used to intervene the VSMCs get from the iliac artery of diabetic model rats cultured by tissue block plantation. The impact of drug-serum on VSMCs proliferation was estimated through measuring tritiated thymine deoxy-ribonucleotide (3H-TDR) incorporation using liquid-scintillation detector, and the cell cycle was detected using flow cytometry. RESULTS: The 3H-TDR incorporation in cells after intervention with various doses of TTR drug-serum were reduced significantly, in a concentration-dependent manner, to the level lower than that in the normal serum control group (P < 0.01). And the percentage of S and G2/M phase cells in the drug-serum treated groups also were markedly lower than that in the normal control group (P < 0.01). CONCLUSION: Drug-serum of TTR could inhibit the proliferation of VSMC of diabetic rat in vitro in a concentration-dependent manner.

Binding of anti-HLA class I antibody to endothelial cells produce an inflammatory cytokine secretory pattern.

Current methods are inadequate for the diagnosis of early chronic allograft rejection. The goal of this study was to determine whether ligation of anti-HLA antibodies to endothelial cells is associated with a distinctive cytokine secretory pattern. Human iliac artery endothelial cells (HIAEC) cultured in vitro were incubated with w6/32, an anti-HLA class I mAb. Culture supernatants collected daily for up to 4 days were tested for secretion of 13 cytokines using a multiplexed fluorescent microsphere immunoassay. Culture of HIAEC with medium containing mAb w6/32 supported the growth of HIAEC during the 4-day study period. Levels of the pro-inflammatory cytokines IL-1beta, IL-6, IL-8, and TNF-alpha became significantly increased in supernatants of HIAEC incubated with the mAb w6/32. We conclude that ligation of anti-HLA class I antibodies to HLA class I antigens in endothelial cells initiates an acute inflammatory process and detecting an inflammatory cytokine secretory pattern might be useful to diagnose sub-clinical chronic allograft rejection.

[Chemoattractive effects of chemokine-like factor 1 on human arterial smooth muscle cells].

OBJECTIVE: To investigate the chemotactic effect of chemokine-like factor 1 (CKLF1) on human arterial smooth muscle cells (ASMCs). METHODS: The recombinant eukaryotic expression vectors pEGFP-N1-CKLF1 (test group) and pEGFP-N1 (control group) were transiently transfected into 293T cells. The supernatants were harvested 72 h after transfection for bioactivity study. The chemotactic effect of CKLF1 on ASMCs were assayed by cellular chemotactic experiments. RESULTS: ASMCs number migrated from the test and control groups diluted by none and 10-fold supernatants had statistical significance (114+/-4 vs 41+/-4, P<0.05; 74+/-4 vs 34+/-3, P<0.01), but have no statistical significance (28+/-4 vs 25+/-5, P>0.05; 26+/-5 vs 23+/-5, P>0.05) between the two groups diluted by 100-fold and 1 000-fold supernatants. When ASMCs were treated at different concentrations of 0 and 2 microg/L of pertussis toxin (PTX), the cell number migrated from the test and control groups diluted by 10-fold supernatants, they had statistical significance (74+/-4 vs 34+/-3, P<0.01; 45+/-3 vs 34+/-3, P<0.01). And when ASMCs were treated at 10 microg/L of PTX, the cell number migrated from both groups diluted by 10-fold supernatants, they had no statistical significance (37+/-4 vs 34+/-3, P>0.05). CONCLUSION: CKLF1 has significant chemotactic effects on ASMCs and such a CKLF1-induced chemotaxis could be inhibited by PTX at concentration of 10 microg/L.

Porcine endothelial cells and iliac arteries transduced with AdenoIL-4 are intrinsically protected, through Akt activation, against immediate injury caused by human complement.

Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-alpha. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.

Nebivolol reduces nitroxidative stress and restores nitric oxide bioavailability in endothelium of black Americans.

BACKGROUND: Alterations in endothelial function may contribute to increased susceptibility of black Americans to cardiovascular disease. The ability to pharmacologically reverse endothelial dysfunction in blacks was tested with nebivolol, a beta1-selective agent with vasodilating and antioxidant properties. METHODS AND RESULTS: The effects of nebivolol on endothelial nitric oxide (NO), superoxide (O2-), and peroxynitrite concentration (ONOO-) release were studied in human umbilical vein endothelial cells and iliac artery endothelial cells isolated from age-matched black and white donors. Kinetics and concentrations of NO/O2-/ONOO- were measured simultaneously with nanosensors from single cells and shown to have significant interracial differences. The rate of NO release was &5 times slower in blacks than in whites (94 versus 505 nmol . L(-1).s(-1)), whereas the rates of release were faster by &2 times for O2- and &4 times for ONOO- (22.1 versus 9.4 nmol.L(-1).s(-1) for O2- and 810 versus 209 nmol.L(-1).s(-1) for ONOO-). Pretreatment with 1.0 to 5.0 micromol/L nebivolol restored NO bioavailability in endothelial cells from black donors with concurrent reductions in O2- and ONOO- release, similar to levels in the endothelium of whites. The effects of nebivolol were dose-dependent and not observed with atenolol; similar effects were observed with apocynin, an NAD(P)H oxidase inhibitor. CONCLUSIONS: Reduced endothelial NO bioavailability in American blacks is mainly due to excessive O2- and ONOO- generation by NAD(P)H and uncoupled endothelial NO synthase. Nebivolol decreased O2- and ONOO- concentrations and restored NO bioavailability in blacks to the level recorded in cells from whites, independently of beta1-selective blockade.

Tissue engineering of recellularized small-diameter vascular grafts.

A tissue-engineered small-diameter arterial graft would be of benefit to patients requiring vascular reconstructive procedures. Our objective was to produce a tissue-engineered vascular graft with a high patency rate that could withstand arterial pressures. Rat arteries were acellularized with a series of detergent solutions, recellularized by incubation with a primary culture of endothelial cells, and implanted as interposition grafts in the common femoral artery. Acellular grafts that had not been recellularized were implanted in a separate group of control animals. No systemic anticoagulants were administered. Grafts were explanted at 4 weeks for definitive patency evaluation and histologic examination; 89% of the recellularized grafts and 29% of the control grafts remained patent. Elastin staining demonstrated the preservation of elastic fibers within the media of the acellular grafts before implantation. Immunohistochemical staining of explanted grafts demonstrated a complete layer of endothelial cells on the lumenal surface in grafts that remained patent. Smooth muscle cells were observed to have repopulated the vessel walls. The mechanical properties of the matrix were comparable to native vessels. Such a strategy may present an alternative to autologous harvest of small vessels for use in vascular bypass procedures.

Nitric oxide mediates the effect of fluvastatin on intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression on human endothelial cells.

Leukocyte and platelet adhesion to endothelial cells, an early step in the pathogenesis of atherosclerosis, is mediated through adhesion molecules. It has been shown that statins decrease adhesion molecule expression. We examined the hypothesis that fluvastatin decreased intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression through a nitric oxide-mediated pathway. Human iliac artery endothelial cells were exposed to fluvastatin in the presence or absence of 2 mM N-monomethyl-L-arginine (L-NMMA). Flow cytometry analysis was used to measure ICAM-1 and PECAM-1 expression. In a separate experiment, confluent cell cultures were exposed in a serum-free medium to fluvastatin 20 microM, and the supernatant was collected for nitrate/nitrite determination after 6 and 48 hr of incubation. Protein was isolated and processed for immunoblotting with monoclonal antibodies specific for endothelial nitric oxide synthase (eNOS), Ser(1177)-phosphorylated eNOS, and AMP kinase. Relative band intensity was assessed with densitometry. Results are presented as the mean +/- standard deviation (SD), and p < 0.05 was considered significant. ICAM-1 and PECAM-1 were expressed constitutively. Human iliac artery endothelial cells (HIAECS) treated with 5 microM fluvastatin did not exhibit reduced expression of PECAM-1 or ICAM-1. Incubation with 10 microM fluvastatin reduced basal expression of both ICAM-1 and PECAM-1. Fluorescence intensity (FI) for these substance was as follows: 3638 +/- 1671, p = 0.01 and PECAM-1 vs. control FI 276 +/- 52 vs. 522 +/- 78, p = 0.02. In the presence of 2 mM L-NMMA, fluvastatin failed to decrease the expression of ICAM-1 (fluvastatin 10 microM + L-NMMA: FI was 3042 +/- 1378 vs. 3638 +/- 1671 for the control p = 0.01) or PECAM-1 (fluvastatin 10 microM + L-NMMA: FI was 415 +/- 188 vs. 522 +/- 78 for the control, p = 0.1). Incubation with 20 microM fluvastatin similarly reduced ICAM-1 expression (FI was 2014 +/- 1595 vs. 3638 +/- 1671 for the control, p = 0.02) and PECAM-1 expression (FI was 196 +/- 109 vs. 522 +/- 78 for the control, p = 0.02). This reduction was prevented in the presence of 2 mM L-NMMA. L-NMMA in a concentration of 2 mM had no significant effect on adhesion molecule expression (p > 0.05 for all comparisons of the control FI versus 2 mM L-NMMA mean FI). After a 48 hr incubation with 20 microM fluvastatin there was a 219 +/- 35% increase in the cell eNOS protein content (p = 0.01) and a 170 +/- 26% increase in the cell AMPK protein content (p = 0.02). Ser(1177)-phosphorylated eNOS protein levels were increased by 41 +/- 8% (p = 0.03). The nitric oxide concentration in the medium of the HIAEC treated with 20 microM fluvastatin for 48 hr was significantly higher than that in the control (p = 0.0004), pointing to increased production during the incubation period. Fluvastatin thus decreases basal expression of ICAM-1 and PECAM-1. Competitive inhibition of eNOS with L-NMMA abolishes the effect of fluvastatin on ICAM-1 and PECAM-1 expression. The statin up-regulates eNOS and AMP kinase, one of the enzymes that activates eNOS via phosphorylation at Ser(1177). We have shown that after a 48-hr exposure to fluvastatin there is an increased amount of the phosphorylated enzyme in the endothelial cells.