Effect of low shear stress on permeability and occludin expression in porcine artery endothelial cells.

INTRODUCTION: Although both fluid shear stress and mass transport of atherogenic substances into the vascular wall are known to be important factors in atherogenesis, there has been little research on the effect of shear stress on vascular permeability. Therefore, the effects of shear stress on the permeability of arteries and the expression of the endothelial cell tight junction molecule occludin, an important regulator of vascular permeability, were investigated. METHODS: Porcine carotid arteries were perfusion cultured ex vivo with low (1.5 dyne/cm(2)) or physiologic (15 dyne/cm(2)) shear stress and 100 mmHg pressure for 24 hours. Subsequently, 20 nm gold particles in solution were infused into the lumen of vessels at 100 mmHg for 30 minutes. Frozen sections were then cut and stained for gold particles. Image analysis was used to determine the density of the particles in the vessel walls. The expression of endothelial cell occludin mRNA and protein were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. RESULTS: Permeability results showed a 2.8-fold increase in the apparent permeability of vessels cultured with low versus physiologic shear stress. RT-PCR and Western blotting results showed significant decreases in occludin mRNA and protein expression at 12 and 24 hours in vessels cultured with low versus physiologic shear stress. CONCLUSIONS: These results demonstrate that low shear stress increases vascular permeability in porcine carotid arteries, possibly owing to decreased occludin expression. These results may have implications in the preferential formation of atherosclerotic vascular disease adjacent to branches and bifurcations where low mean shear stresses may occur.

HIV protease inhibitor ritonavir decreases endothelium-dependent vasorelaxation and increases superoxide in porcine arteries.

OBJECTIVE: Although HIV Protease inhibitors significantly reduce the viral load, they are associated with increased risk of cardiovascular disease. The aim of this study was to investigate the effects of HIV protease inhibitor ritonavir on vascular endothelial cell function. METHODS: Porcine carotid arteries were perfusion-cultured for 24 h as controls or with 15 microM of ritonavir. Vessels were precontracted with norepinephrine followed by endothelium-dependent vasorelaxation with acetylcholine. Rings of vessels were cultured as controls or with ritonavir for 24 h and basal and NADPH-stimulated superoxide levels were determined using lucigenin-enhanced chemiluminescence. Superoxide levels in situ were also examined using dihydroethidium (DHE) staining, and nitrotyrosine levels were examined using a nitrotyrosine antibody. RESULTS: Endothelium-dependent vasorelaxation was significantly reduced in ritonavir-treated vessels compared to controls. There were significant increases in basal and NADPH-stimulated superoxide production in vessel rings treated with ritonavir compared to control vessels. Dihydroethidium staining and nitrotyrosine staining were also elevated in endothelial cells of ritonavir-treated vessels, indicating increased superoxide production and increased oxidative stress, respectively, in ritonavir-treated vessels compared to controls. CONCLUSIONS: These data demonstrate that HIV protease inhibitor ritonavir causes a significant reduction in endothelium-dependent vasorelaxation in cultured porcine carotid arteries. Increased oxidative stress may be a possible mechanism of HIV protease inhibitor ritonavir-induced endothelial dysfunction.

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft.

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.

Estrogen blocks homocysteine-induced endothelial dysfunction in porcine coronary arteries(1,2).

BACKGROUND: The objective of this study was to examine the effect of estrogen combined with homocysteine on vasomotor function and endothelial integrity in intact porcine coronary arteries. MATERIALS AND METHODS: Pig coronary artery rings were incubated with estrogen, homocysteine, or estrogen and homocysteine for 24 h. Myographic analysis was performed with thromboxane A2 analog U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Endothelial nitric oxide synthase (eNOS) levels were determined by immunohistochemistry. Levels of superoxide anion were assessed by lucigenin-enhanced chemiluminescence analysis. RESULTS: Endothelium-dependent vasorelaxation (bradykinin) for the homocysteine alone group was 62% compared with control (P < 0.05), and endothelium-dependent vasorelaxation for the estrogen alone group was 85% compared with control (P > 0.05). Endothelium-dependent vasorelaxation for the estrogen-homocysteine combined group was 79% compared with 89% for control (P > 0.05). There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between all three groups and control. In addition, the eNOS immunoreactivity was declined in the homocysteine group and had no major change in the estrogen or estrogen plus homocysteine-treated group as compared with controls. The superoxide free radical measurement showed a marked increase in the homocysteine group, no major change from controls in the estrogen group, and a much-lessened effect in the combination of estrogen and homocysteine. CONCLUSIONS: These data demonstrate that combining estrogen with homocysteine significantly blocks the effect of homocysteine on impairing endothelium-dependent vasorelaxation as well as on decreasing eNOS expression and increasing oxidative stress in porcine coronary arteries. This study suggests that estrogen may play a role in preventing homocysteine-mediated endothelial dysfunction and may be of benefit in the hyperhomocysteinemic patient.

L-arginine improves endothelial vasoreactivity and reduces thrombogenicity after thrombolysis in experimental deep venous thrombosis.

PURPOSE: Nitric oxide (NO) is important in regulation of platelet aggregation, endothelial function, and intravascular thrombosis. The purposes of this study were to assess the effect of thrombolysis on endothelial function in a porcine model of deep venous thrombosis (DVT) and to evaluate the effect of NO precursor l-arginine on endothelial function after thrombolytic therapy. METHODS: DVT was created in bilateral iliac veins by deploying a self-expanding stent-graft that incorporated an intraluminal stenosis, from a groin approach. Five pigs underwent sham operation. After 7 days of DVT, animals were randomized to three groups: saline pulse-spray (saline group, n = 5), thrombolytic pulse-spray with tissue plasminogen activator (alteplase, 8 mg; t-PA group, n = 5), and thrombolytic pulse-spray plus intravenous l-arginine (20 mmol/L; arginine group, n = 5). At 2 weeks iliac vein patency was evaluated at venography and intravascular ultrasound scanning. NO level was determined with a chemiluminescent assay of the nitrite and nitrate metabolites (NO(x)). Thrombogenicity was evaluated with radiolabeled platelet and fibrin deposition. Veins were harvested and evaluated with light microscopy and scanning electron microscopy. Endothelial function was evaluated with organ chamber analysis. RESULTS: All iliac veins remained patent at 2 weeks. The luminal areas in the sham, saline, t-PA, and arginine groups were 53 +/- 23 mm(2), 14 +/- 11 mm(2), 34 +/- 19 mm(2), and 42 +/- 21 mm(2), respectively. No difference in endothelial cell structure was observed between the three treatment groups at light microscopy or scanning electron microscopy. Although no difference in fibrin deposition was noted among the three treatment groups, decreased platelet deposition occurred in the arginine group compared with the saline or t-PA groups (P <.05). The arginine group showed greater endothelial-dependent relaxation compared with the t-PA or saline groups (73% +/- 23% vs 49% +/- 18% and 32% +/- 21%; P <.05). Local NO(x) level in the arginine group was correspondingly higher compared with the saline or t-PA groups (1.8 +/- 0.3 micromol/L vs 0.3 +/- 0.05 micromol/L and 0.2 +/- 0.04 micromol/L; P <.05). CONCLUSIONS: NO precursor l-arginine supplementation enhances NO production at sites of venous thrombosis. Moreover, l-arginine preserves endothelial vasoreactivity and reduces platelet deposition after thrombolysis in iliac DVT. These data suggest that l-arginine may preserve endothelial function after thrombolysis and may reduce the likelihood of postthrombotic syndrome.

Red wine prevents homocysteine-induced endothelial dysfunction in porcine coronary arteries.

BACKGROUND: Hyperhomocysteinemia is an independent risk factor of coronary artery disease. Clinical studies have indicated that moderate red wine consumption is associated with a reduction of incidence of coronary artery disease. In this study, we determined the effect of red wine on homocysteine- induced endothelial dysfunction in porcine coronary arteries. MATERIALS AND METHODS: Porcine coronary arteries were dissected from 6 pig hearts and cut into 5-mm ring segments, which were assigned into 4 groups (9 rings/group): blank control, homocysteine treated (50 muM), red wine treated (0.08% alcohol), and homocysteine plus red wine treated. The rings were cultured in cell culture medium with or without treatment for 24 h. Myograph analysis was performed with U46619 (10(-7) M) for contraction and cumulative bradykinin (10(-9) to 10(-5) M) for endothelium-dependent relaxation. The endothelial nitric oxide synthase (eNOS) levels were analyzed by RT-PCR, Western blot, and immunohistochemistry. RESULTS: In response to 10(-5) M bradykinin, porcine coronary artery rings treated with homocysteine (50 muM) showed a significant reduction of endothelium-dependent vasorelaxation by 43% as compared to controls (P < 0.05). However, rings treated with red wine (0.08% alcohol) plus homocysteine showed no significant difference as compared to controls. Endothelium-dependent vasorelaxation was not different between control and red wine treated groups. Furthermore, eNOS mRNA density levels were significantly reduced by 36% in homocysteine treated group as compared to controls (P < 0.05). eNOS protein levels were also substantially reduced in the homocysteine-treated group. However, red wine treatment reversed the effect of homocysteine-induced eNOS downregulation. CONCLUSIONS: Homocysteine significantly impaired endothelial functions including endothelium-dependent vasorelaxation and eNOS mRNA and protein levels in porcine coronary arteries; and red wine effectively prevented homocysteine-induced endothelial dysfunction. This study suggests that protecting coronary endothelial cells from homocysteine damage may be an important mechanism of red wine for preventing coronary artery disease.

Hiv Tat protein causes endothelial dysfunction in porcine coronary arteries.

PURPOSE: Human immune deficiency virus (HIV) infection is often associated with chronic diseases, including atherosclerosis. However, the molecular mechanisms are largely unknown. We examined the effect of Tat protein, an HIV regulatory protein, on endothelial function in porcine coronary arteries. METHODS: Porcine coronary arteries were dissected from nine pig hearts and cut into 5-mm ring segments, which were incubated as controls or with Tat protein (10(-7), 10(-9), 10(-11) mol/L) or Tat protein plus anti-Tat antibody, for 24 hours. Myography was performed with thromboxane A(2) analog U46619 (10 (-7) mol/L) for contraction and with graded doses of bradykinin (10(-8), 10(-7), and 10(-6) mol/L) or sodium nitroprusside (10(-5) mol/L) for relaxation. Endothelial nitric oxide synthase (eNOS) messenger RNA was determined with reverse transcriptase polymerase chain reaction (RT-PCR), and protein levels were determined with Western blot analysis. Immunoreactivity of eNOS of treated rings was also detected. RESULTS: Endothelium-dependent vasorelaxation (10-7 mol/L of bradykinin) was significantly reduced (46.41%) in pig coronary artery rings treated with 10(-7) mol/L of Tat protein, as compared with control arteries (P <.05). Arteries treated with Tat protein plus anti-Tat antibody relaxed similarly as control arteries. There were no differences in smooth muscle contractility (U46619) or endothelium-independent vasorelaxation (sodium nitroprusside) between control and Tat protein-treated groups. RT-PCR for eNOS mRNA showed reduction in eNOS levels for Tat-treated coronary artery rings by 73%, as compared with control vessels (P <.05). Tat protein-treated vessels demonstrated substantially less eNOS protein band intensity and immunoreactivity compared with control vessels. CONCLUSIONS: Tat protein significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA and protein expression in endothelial cells of porcine coronary arteries. This study suggests that Tat protein-mediated endothelial dysfunction may be important in coronary heart disease in HIV-infected patients.

HIV protease inhibitor ritonavir induces cytotoxicity of human endothelial cells.

OBJECTIVE: Although HIV protease inhibitors have been successfully used against HIV infection, many metabolic side effects and premature cardiovascular diseases are often associated with this therapy. The mechanisms of these complications are not clear. In this study, we investigated the effect of the HIV protease inhibitor ritonavir on human endothelial cell cultures. METHODS AND RESULTS: By using nonradioactive cell proliferation and cytotoxicity assays, human endothelial cells treated with ritonavir showed a significant decrease in cell viability and an increase in cytotoxicity in a time- and dose-dependent fashion. Mitochondrial DNA was also substantially damaged with ritonavir treatment by long polymerase chain reaction analysis. In contrast, ritonavir had a very limited effect on endothelial apoptosis, as assessed by analyses of DNA fragmentation and cellular caspase-3 activity. CONCLUSIONS: These data demonstrate, for the first time, that the HIV protease inhibitor ritonavir at concentrations near clinical plasma levels is able to directly cause endothelial mitochondrial DNA damage and cell death mainly through necrosis pathways but not through apoptosis. This study suggests that HIV protease inhibitor-mediated endothelial injury may contribute to its cardiovascular complications.

Late complication of aortoiliac stent placement- atheroembolization of the lower extremities.

BACKGROUND: Atheroembolization following aortoiliac stent placement is uncommon. The purpose of this study was to examine the management and risk factors of lower extremity atheroembolization following aortoiliac stent placement for occlusive disease. MATERIALS AND METHODS: From March 1993 to February 2001, the hospital records of all patients who developed thromboembolic events following aortoiliac stent placement were reviewed. Risk factor analysis was performed by comparing with the control group, which consisted of 493 patients treated with aortoiliac stents during the study period who did not develop atheroembolic complications. Patients with cardiac etiologies or aortic aneurysms as the source of embolization as well as those who developed acute embolization following stent deployment (<30 days) were excluded. RESULTS: Atheroembolization occurred in eight patients (12 iliac artery stents and 1 aortic stent) at intervals ranging from 9 to 43 months (mean 22 months) following aortoiliac stent placement. Arteriography in all patients implicated the stented artery as the source of atheroembolism. Five corrective operations (two aorto-bifemoral bypasses, one ileofemoral bypass, and two aortoiliac endarterectomies) along with two concomitant femoropopliteal thrombectomies were performed successfully in five patients. The remaining three patients were treated with either thrombolysis and/or additional stent placement, which resulted in either iliac occlusion or recurrent embolic symptoms (P < 0.05). All 3 patients subsequently underwent bypass procedures (one ileofemoral and two femorofemoral bypasses). There was no perioperative mortality. During a mean follow-up of 16 months (range 3 to 45 months), two patients required minor amputations, whereas one required major leg amputation. No further episodes of atheroembolism occurred in the involved limbs following surgical bypass procedures. Risk factor analysis failed to identify potential variables that correlated with atheroembolism following aortoiliac stent placement. CONCLUSION: Patients with atheromatous embolization following aortoiliac stent placement should be evaluated aggressively. The treatment of choice is surgical correction or bypass with exclusion of the offending embolic source. Although intra-arterial stent placement in the atheroembolic stented iliac artery is feasible, it may provide a less durable result.

Percutaneous bedside femorofemoral bypass grafting for acute limb ischemia caused by intra-aortic balloon pump.

Acute limb ischemia is a common, recognized complication of intra-aortic balloon pump (IABP) placement in patients with failing myocardium, and an operative femorofemoral bypass graft is often necessary in IABP-dependent patients as a means of maintaining the lower-limb perfusion. In this report, we present a minimally invasive endovascular technique for creating a percutaneous temporary femorofemoral bypass graft at bedside in patients with IABP-induced limb ischemia. This temporary bypass grafting technique may obviate a potential graft infection or wound complications associated with a formal femorofemoral bypass graft.

Nicotine and cotinine up-regulate vascular endothelial growth factor expression in endothelial cells.

Cigarette smoking is an important risk factor for both vascular disease and various forms of cancer. Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen that is normally expressed only in low levels in normal arteries but may be involved in the progression of both vascular disease and cancer. Some clinical evidence suggests that cigarette smoking may increase plasma VEGF levels, but there is a lack of basic science studies investigating this possibility. We show here, using an intact porcine common carotid artery perfusion culture model, that nicotine and cotinine, the major product of nicotine metabolism, cause a significant increase in endothelial cell VEGF expression. VEGF mRNA levels were compared between groups using reverse transcriptase-polymerase chain reaction, whereas protein level changes were demonstrated with Western blotting and immunohistochemistry. Our results showed significant increases in endothelial cell VEGF mRNA and protein levels because of nicotine and cotinine at concentrations representative of plasma concentrations seen in habitual smokers. VEGF immunostaining also paralleled these results. These findings may give a clue as to the mechanisms by which nicotine and cotinine from cigarette smoking increase vascular disease progression and tumor growth and metastasis.

Shear stress regulates occludin and VEGF expression in porcine arterial endothelial cells.

BACKGROUND: One of the initiating factors of atherosclerosis is the accumulation of low-density lipoprotein in the intima. Despite the correlation between low shear stress and vascular lesion formation, there is little research on the effects of shear stress on the molecular regulators of endothelial cell permeability. In this study, the effects of shear stress on the expression of occludin and vascular endothelial growth factor (VEGF), two important regulators of endothelial permeability, were investigated. METHODS: Porcine carotid arteries were cultured in perfusion culture systems for 24 h with 100 mm Hg pressure and low or physiologic shear stress. Subsequently, vessel sections were taken for histology and endothelial cells were isolated for RNA and protein extraction. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine occludin and VEGF mRNA levels. Western blotting and immunohistochemistry were performed to examine occludin and VEGF protein levels. RESULTS: RT-PCR showed that endothelial cells from vessels cultured with low shear stress had an 11% decrease in occludin/GAPDH band density ratio (P < 0.05) and a 16% increase in VEGF/beta-actin band density ratio (P < 0.05) relative to the physiologic shear stress group. Western blot showed a 50% decrease in occludin protein expression (P < 0.01) and a 95% increase in VEGF protein expression in endothelial cells from vessels cultured with low shear stress relative to the physiologic shear stress group. Immunoreactivity of occludin and VEGF in vessels also reflected these changes. CONCLUSIONS: These results demonstrate that low shear stress both decreases endothelial cell occludin mRNA and protein expression and increases endothelial cell VEGF mRNA and protein expression. These changes may suggest a possible molecular mechanism for increased endothelial permeability due to low shear stress.

Homocysteine decreases endothelium-dependent vasorelaxation in porcine arteries.

BACKGROUND: Although hyperhomocysteinemia has long been recognized as an independent risk factor for vascular disease, the mechanisms of the pathogenesis of homocysteine are largely unknown. The objective of this study was to examine the effect of homocysteine on vasomotor function and endothelial integrity in intact porcine arteries. METHODS: Pig coronary artery rings were incubated with homocysteine (10, 50, or 100 microM) for 24 h. Myograph analysis was performed with thromboxane A2 analogue U46619 for contraction and bradykinin or sodium nitroprusside for relaxation. Pig carotid arteries were perfusion-cultured in control and 50 and 100 microM homocysteine treatment groups. The diameter change was analyzed in response to norepinephrine and acetylcholine, respectively. Endothelial morphology and nitric oxide synthase (eNOS) levels were determined by histology analysis. RESULTS: Endothelial-dependent vasorelaxation (bradykinin) was significantly reduced by 52, 87, and 97% in the pig coronary artery rings treated with 10, 50, and 100 microM homocysteine, respectively, compared to controls (P < 0.05). There were no differences in endothelium-independent vasorelaxation (sodium nitroprusside) or in smooth muscle contractility (U46619) between control and homocysteine-treated groups (P > 0.05). Acetylcholine-induced vasorelaxation was also significantly reduced by 44 and 98% in the pig carotid arteries treated with 50 and 100 microM homocysteine, respectively, compared to controls (P < 0.05). Variable degrees of endothelial cell injury, such as morphology change and detachment, were observed, and eNOS immunoreactivity was markedly reduced in both pig coronary and carotid arteries that were treated with high doses of homocysteine. CONCLUSION: These data demonstrated that homocysteine significantly decreased endothelium-dependent vasorelaxation and eNOS immunoreactivity as well as induced marked endothelial injury in both porcine coronary and carotid arteries. This study suggests that homocysteine-mediated endothelial dysfunction and injury may play important roles in vascular lesion formation in the hyperhomocysteinemic patient.