In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury

OBJECTIVE: Nitric oxide regulates vascular tone, inhibits platelet aggregation, and inhibits leukocyte adhesion, all of which are important modulators of ischemia-reperfusion injury. This study aimed to determine the effects of endothelial constitutive nitric oxide synthase gene transfer on ischemia-reperfusion injury in a rat lung transplant model. METHODS: In group I, donor animals were injected intravenously with 5 x 10(9) pfu of adenovirus-encoding endothelial constitutive nitric oxide synthase. Groups II and III served as controls, whereby donor animals were injected with either 5 x 10(9) pfu of adenovirus encoding beta-galactosidase or saline solution, respectively. Twenty-four hours after injection, left lungs were harvested and preserved for 18 hours at 4 degrees C, then implanted into isogeneic recipients, which were put to death 24 hours later. Recombinant endothelial constitutive nitric oxide synthase gene expression was evaluated by Western blotting and immunohistochemistry. Lung grafts were assessed by measuring arterial oxygenation, myeloperoxidase activity, and wet/dry weight ratios. RESULTS: Western blotting confirmed the overexpression of endothelial constitutive nitric oxide synthase in lungs so transfected compared with controls. Twenty-four hours after reperfusion, mean arterial oxygenation was significantly improved in group I compared with group II and III controls (189.4 +/- 47.1 mm Hg vs 71.7 +/- 8.9 mm Hg and 67.8 +/- 12.2 mm Hg, P =.02, P =.01, respectively). Myeloperoxidase activity, a reflection of tissue neutrophil sequestration, was also significantly reduced in group I compared with groups II and III (0.136 +/- 0.038 DeltaOD/mg/min vs 0. 587 +/- 0.077 and 0.489 +/- 0.126 DeltaOD/mg/min, P =.001, P =.01, respectively). CONCLUSION: Adenovirus-mediated gene transfer with endothelial constitutive nitric oxide synthase ameliorates ischemia-reperfusion injury as manifested by significantly improved oxygenation and decreased neutrophil sequestration in transplanted lung isografts. Endothelial constitutive nitric oxide synthase gene transfer may reduce acute lung dysfunction after lung transplantation.     

Monocyte adhesion to balloon-injured arteries: the influence of endothelial cell seeding

OBJECTIVE: Deendothelialization of injuries of the artery disrupts normal vascular homeostasis, affecting both the structural integrity of the blood vessel wall, as well as the interaction of the arterial surface with blood components such as platelets, leukocytes, and circulating proteins. Leukocyte and, in particular, monocyte recruitment to damaged vessels has been implicated in the pathogenesis of intimal hyperplasia. We hypothesize that reendothelialization is an important modulator of monocyte adhesion to healing arterial surfaces. METHODS: New Zealand white rabbits (n = 20) were subjected to bilateral iliofemoral artery balloon injury. Cultured, autologous venous endothelial cells (ECs) were immediately seeded onto one vessel, whereas the contralateral artery received medium alone, to accelerate endothelial relining. Vessels were harvested (5-9 days after injury) for analysis of permeability (Evans Blue dye exclusion), endothelial coverage (anti-CD31 immunohistochemistry), monocyte adhesion (ex vivo binding of 51Na2CrO4-labeled monocytic THP-1 cells), and monocyte recruitment (RAM-11 immunohistochemistry). RESULTS: Improved EC coverage was evidenced by positive staining for CD31 in the seeded vessels. Vessel wall permeability was markedly reduced in EC-seeded arteries (29% +/- 10% vs 99% +/- 0% surface Evans blue staining, P <.005), consistent with restoration of a functional endothelial barrier. EC seeding significantly reduced ex vivo THP-1 binding to vessels explanted at a mean of 8 days after injury (45,170 +/- 8939 vs 85,994 +/- 16,500 cells/cm2, P <.05). However, RAM-11 staining revealed no significant difference in overall macrophage accumulation between seeded and control vessels 1 week after injury (111 +/- 22 vs 95 +/- 14 cells/section, P =.36). CONCLUSIONS: Immediate seeding of a balloon-injured rabbit artery with cultured ECs results in accelerated restoration of the endothelial lining. At 1 week, barrier function is improved, and the seeded vessel surface is less adhesive to activated monocytes ex vivo, as compared with injured controls. Nonetheless, EC-seeded and nonseeded arteries demonstrate similar total macrophage accumulation over 1 week. These data suggest that after mechanical arterial injury, endothelial coverage may be one important variable influencing leukocyte adhesion.     

Effects of hypoxia-reoxygenation on microvascular endothelial function in the rat hippocampal slice.

BACKGROUND: Cerebral ischemia and hypoxia may cause injury to both neuronal and vascular tissue. The direct effects of hypoxia on endothelial function in intraparenchymal cerebral arterioles are unknown. Using a modification of the rat brain slice preparation, allowing continuous imaging of these previously inaccessible vessels, microvessel dilation was evaluated before and after a brief hypoxic episode. METHODS: Rat brain slices were superfused with oxygenated artificial cerebrospinal fluid. Hippocampal arterioles were visualized using computerized videomicroscopy, and their diameters (range, 12-27 microm) were measured using image analysis. After preconstriction with prostaglandin F2alpha and controlled pH and carbon dioxide tension, graded concentrations of either acetylcholine (endothelium-dependent vasodilation) or sodium nitroprusside (endothelium-independent vasodilation) were given before and after a 10-min period of hypoxia. RESULTS: Sodium nitroprusside (100 microM) caused similar dilation before and after hypoxia (mean +/- SEM: 9.6 +/- 0.6% vs. 13.0 +/- 0.9%). Acetylcholine (100 microM) caused significantly less dilation (P < 0.05) after hypoxia (mean +/- SEM: 9.3 +/- 1.8% vs. 3.6 +/- 1.2%). The decreased acetylcholine-induced dilation after hypoxia was not reversed by pretreatment with L-arginine (1 mM), the precursor of nitric oxide (mean +/- SEM: 8.8 +/- 1.3% vs. 4.4 +/- 0.7%). CONCLUSIONS: Even brief periods of hypoxia may cause endothelial dysfunction in intraparenchymal cerebral arterioles. This does not seem to be related to a deficiency of the nitric oxide substrate, L-arginine. Endothelial dysfunction and impaired endothelium-dependent dilation of microvessels may decrease oxygen delivery and increase neuronal injury during cerebral hypoxia-reoxygenation.     

Inhibition of endothelial cell migration by cigarette smoke condensate

Cigarette smoking is among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanism, however, that links cigarette smoking to an increased incidence of atherosclerosis is poorly understood. Endothelial cell (EC) integrity is critical in preventing vascular lesion formation, and after a loss of EC integrity reendothelialization must be rapid and complete. We therefore investigated whether cigarette smoke affected the ECs ability to migrate or altered the intracellular signals generated during migration. The DMSO-soluble fraction of cigarette smoke condensate (CSC), derived from the standard research cigarette, was tested on cultured ECs (HUVEC) derived from human umbilical vein. The addition of CSC caused a dose-dependent decrease in the ability of EC to migrate as measured over a 24-h time period. Nicotine and cadmium sulfate, two constituents of cigarette smoke, individually or in combination, had no effect on migration. Examination of the tyrosine phosphorylation state of various intracellular proteins by Western blot analysis showed that CSC caused the hyperphosphorylation of a 130-kDa protein. In addition, other intracellular proteins showed changes in their phosphorylation states after CSC addition. These results support the hypothesis that CSC is detrimental to normal EC function in maintaining vascular integrity and suggest that smokers are more likely to develop complications of vascular disease due to delayed or incomplete reendothelialization as a consequence of decreased EC migration.     

Accelerated enlargement of experimental abdominal aortic aneurysms in a mouse model of chronic cigarette smoke exposure

BACKGROUND: Cigarette smoking and pulmonary emphysema are strongly associated with abdominal aortic aneurysms (AAAs), but the biologic mechanisms linking these conditions are undefined. STUDY DESIGN: To determine if exposure to cigarette smoke influences formation and growth of experimental AAAs, 129/SvEv mice were acclimated to daily cigarette smoke exposure for 2 weeks followed by transient elastase perfusion of the abdominal aorta to induce aneurysmal degeneration. Smoking was continued for intervals of either 2 or 12 weeks (8 mice per group). Nonsmoking 129/SvEv controls (n = 29) underwent elastase perfusion and followup evaluation at the same time intervals. In all animals, abdominal aortic diameter (AD) was measured to determine interval increases in AD (Delta AD), with AAAs defined as a Delta AD > 100%. RESULTS: Preperfusion and immediate postperfusion ADs were not significantly different between experimental groups. Aneurysmal dilatation was present 2 weeks after elastase perfusion in both smoking mice and nonsmoking controls, with no significant difference in final AD (mean +/- SEM: smoking, 1.23 +/- 0.11 mm versus nonsmoking, 1.22 +/- 0.05 mm). There were also no differences in the overall extent of aortic dilatation (Delta AD smoking, 136 +/- 24% versus nonsmoking, 138 +/- 10%), or the incidence of AAAs (smoking, 75% versus nonsmoking, 79%). Although all animals had developed AAAs by 12 weeks after elastase perfusion, the overall extent of aortic dilatation was 50% greater in smoking mice compared with nonsmoking controls (Delta AD smoking, 204 +/- 23% versus nonsmoking, 135 +/- 17%; p < 0.05). CONCLUSIONS: Short-term exposure to cigarette smoke did not alter initial development of experimental AAAs, but chronic smoke exposure was associated with a substantial increase in the late progression of aneurysmal dilatation. This novel combination of in vivo experimental models offers a new approach to investigate mechanisms by which cigarette smoking promotes aneurysmal degeneration.     

Low molecular weight fucoidan prevents neointimal hyperplasia after aortic allografting

BACKGROUND: Fucoidan, a new low molecular weight sulfated polysaccharide (LMWF), has previously been shown to mobilize bone marrow-derived progenitors cells via stimulation of stromal derived factor (SDF)-1 release. Mobilized progenitor cells have been suggested to repair intimal lesions after immune-mediated endothelial injury and thus prevent intimal proliferation. The aim of this study was to evaluate the effect of LMWF treatment in a rat aortic allograft model of transplant arteriosclerosis (TA). METHODS: Aortic grafts were performed in Brown Norway (BN, donor) and Lewis (Lew, recipient) rats. The recipient rats were treated with LMWF (5 mg/kg/day) and sacrificed at 30 days. To determine the role of SDF-1 in mediating the effects of LMWF, a specific inhibitor of the SDF-1 receptor CXCR4, AMD 3100 (20 microg/kg/day), was used. The grafted segments were evaluated by morphometric (histochemical) analyses. RESULTS: Untreated aortic allografts exhibited severe intimal proliferation, indicative of TA. In contrast, LMWF treatment significantly prevented allograft intimal proliferation as compared with controls (5.7+/-3 vs. 66.2+/-6 microm, P<0.01) and permitted a normalization of the intima/media ratio (0.1+/-0.1 vs. 1.7+/-0.3, P<0.01). Further, LMWF treatment stimulated allograft reendothelialization, as evidenced by strong intimal endothelial nitric oxide synthase antibody and CD31 signals. Unexpectedly, AMD treatment failed to prevent the protective effect of LMWF on intimal thickening and AMD treatment alone was found to reduced intimal proliferation in allografts. CONCLUSIONS: We found that LMWF treatment reduced intimal thickness and induced the presence of an endothelial cell lining in the vascular graft at 30 days. Our findings may suggest a novel therapeutic strategy in the prevention of TA.     

Measurement of tibial endothelial cell function after cigarette smoking, cessation of smoking and hyperbaric oxygen therapy

SUMMARY: Cigarette smoking is hazardous to a range of human tissues. For instance, cigarette smoke inhalation has been proven to delay bone healing. This study analysed the effects of cigarette smoking on tibial vascular endothelium and blood flow using the bone-chamber model. The effects of smoking cessation and hyperbaric oxygen (HBO) on the damage caused by smoking were also compared. 54 adult New Zealand rabbits were divided into three groups. Group 1: control, Group 2: 1 week smoking, and Group 3: 6 weeks' smoking. This study on rabbits confirmed that both short-term and long-term cigarette smoking is dangerous to the bony vascular endothelium of the tibia. The vasodilatation caused by nitric oxide production was significantly attenuated in Group 2 and 3's tibia. Long-term smoking damaged the vascular endothelium more severely than short-term smoking (P<.01). Cessation of smoking effectively reduces the adverse effects of smoking when the cessation time equals the smoking time. HBO also effectively reduces the adverse effects of smoking.     

Cigarette smoking and kidney involvement

Cigarette smoking has adverse effects on health causing ischemic heart disease, stroke, chronic obstructive lung disease and cancers of the respiratory and upper digestive tract, pancreas, kidney and urinary tract. Smoking causes an acute increase in mean arterial pressure and heart rate mediated by catecholamines and beta-adrenergic mechanisms. Chronic cigarette smoking reduces renal plasma flow, probably increasing synthesis of the vasoconstrictor endothelin and reducing generation of the vasodilatory endothelial nitric oxide. There is clinical evidence that cigarette smoking has important adverse effects on renal outcome in primary hypertension, diabetic nephropathy, primary glomerular diseases, systemic diseases involving the kidney and in patients on chronic hemodialysis, or after renal transplantation.     

L-arginine improves endothelial and myocardial function after brain death

BACKGROUND: Recently, we showed that brain death (BD) leads to a severe impairment of endothelial function. METHODS: To test the hypothesis, that nitric oxide supply improves endothelial function, we infused L-arginine (40 mg/kg) in 6 dogs after BD induction (subdural balloon). Six vehicle-treated BD animals served as controls. Coronary blood flow (CBF), preload recruitable stroke work (PRSW), and plasma L-arginine and nitrite/nitrate levels were measured before and 6 hr after BD induction. In addition, endothelium-dependent vasodilatation after intracoronary application of acetylcholine (ACH) and endothelium-independent vasodilation after sodium nitroprusside (SNP) were assessed. RESULTS: Six hours after BD, CBF decreased significantly in the control group (38.2+/-3.5 vs. 26.8+/-3.1 ml/min, P<0.05), whereas the decrease was less pronounced in the L-arginine group (41.8+/-6.9 vs. 36.0+/-1.2 ml/min, P<0.05 vs. control). Before BD, ACH led to a similar vasodilative response in both groups (81+/-6 vs. 75+/-7%). After BD, a paradox vasoconstriction occurred after ACH in the control group, while the vasodilative response did not change in the L-Arginine group (36+/-6 vs. 69+/-7%, P<0.05). The response to SNP did not differ between the groups and over the time. After BD PRSW decreased in both groups, however, it was still significantly higher in the L-arginine group (56+/-7 vs. 71+/-7 kerg, P<0.05). L-arginine (711+/-144 vs. 234+/-54 microM P<0.05) and nitrite/nitrate (39+/-3 vs. 27+/-3 microM P<0.05) levels were significantly higher in the L-arginine group. CONCLUSION: L-arginine treatment prevents endothelial dysfunction and improves myocardial performance after BD via enhancement of endogenous nitric oxide synthesis.     

Pulmonary vascular endothelial growth factor and nitric oxide interaction during total cardiopulmonary bypass in neonatal pigs

OBJECTIVE: Lung injury after cardiopulmonary bypass includes pulmonary hypertension and lung edema. Both complications are related to endothelial pulmonary vascular dysfunction, leukocyte sequestration, and increased capillary permeability. This study was done in an attempt to better define the endothelial dysfunction and the cause of edema. METHODS: Twenty-five neonatal piglets were subjected to total cardiopulmonary bypass for 90 minutes without crossclamping of the aorta. After weaning from cardiopulmonary bypass, they were allowed to survive 2 hours, at which time they were killed. Preoperative and postoperative hemodynamic studies, lung (n = 16) and muscular (n = 5) vascular endothelial growth factor contents, and exhaled nitric oxide (n = 8) were recorded. Immediately after the animals were killed, pulmonary arterial rings were obtained from 12 piglets and mounted in organ chamber for assessment of endothelial function with receptor-dependent (acetylcholine) or non-receptor-dependent (calcium ionophore A23187) studies and compared with control pulmonary arterial rings. The left lungs of 13 piglets were mounted in isolated perfused lung chambers for filtration coefficient assessment and comparison with control preparations. RESULTS: After cardiopulmonary bypass, pulmonary vascular resistance increased from 953.7 +/- 302.6 dyne x s x cm(-5) to 1973.6 +/- 925.4 dyne x s x cm(-5) (P =.03). This was associated with an increase in lung vascular endothelial growth factor content from 91.07 +/- 5.314 pg/100 mg tissue to 151.6 +/- 11.4 pg/100 mg tissue (P <.0001), an increase in muscle vascular endothelial growth factor from 76.02 +/- 11.53 pg/100 mg tissue to 81.58 +/- 7.7 pg/100 mg tissue (P not significant), and a decrease in exhaled nitric oxide from 6 +/- 1.7 ppb to 3.12 +/- 1.4 ppb (P =.003). The filtration coefficient was statistically significantly higher after cardiopulmonary bypass than in control preparations (0.259 +/- 0.02 vs 0.525 +/- 0.07, P <.0001). Variations in lung vascular endothelial growth factor accumulation were statistically significantly higher than in muscular vascular endothelial growth factor accumulation (60.5 +/- 9.1 vs 5.5 +/- 5.9, P =.0008). In addition, a statistically significant correlation was found between postbypass lung vascular endothelial growth factor and lung filtration coefficient (P =.0058), as well as between change in lung vascular endothelial growth factor and change in lung filtration coefficient (P =.03). Pulmonary vascular endothelial receptor-dependent (acetylcholine) function was statistically significantly blunted after bypass relative to control values (15.44% +/- 4.8% vs 55.5% +/- 5.96% of maximal relaxation, P =.0001), whereas non-receptor-dependent endothelial function was unaffected by cardiopulmonary bypass (110.77% +/- 8.9% vs 120.63% +/- 15.46% of maximal relaxation, P not significant). CONCLUSIONS: These findings suggest that lung ischemia that occurs during cardiopulmonary bypass affects the signal transduction from membrane receptors to intracellular calcium mobilization and nitric oxide synthase activation. Lung edema after bypass is probably due in part to lung accumulation of vascular endothelial growth factor, a finding that was not found in systemic muscular nonischemic territories.     

Ultrastructural effects of cigarette smoke on rat testis

The purpose of this study was to investigate the ultrastructural changes taking place in the testicular tissue of adult male Wistar rats following exposure to cigarette smoke. Twenty rats were exposed to smoke in a smoking machine for 2 h daily for 60 days; 10 control rats were placed in the machine for the same amount of time but were exposed to room air. After ether anesthesia and dissection of the animals, the testes were removed and fixed in 2.5% glutaraldehyde. The sections were examined under a transmission electron microscope. Evident degeneration and dissociation of the spermatogenetic cells were common findings. Basal lamina of the tubuli seminiferi contorti was thickened and significantly irregular. Cigarette smoke inhalation damaged the seminiferous epithelium. The primary spermatocytes as well as Sertoli cells appeared to be targets for the smoke. The results indicated that cigarette smoke inhalation could cause specific lesions in the development of spermatozoon, and it might be either directly or indirectly toxic to spermatogenesis.     

Luminal thrombus disrupts nitric oxide-dependent endothelial physiology

BACKGROUND: The goals of this study were: (1) to develop a large animal model to study endothelial function, and (2) to determine if arterial thrombosis induces endothelial dysfunction in vivo. METHODS: Surgical exposure of the porcine iliac and femoral arteries was performed. Normal porcine arteries were compared with arteries subjected to 90 min of arterial thrombosis. External iliac artery (EIA) luminal diameters were measured using M- and B-mode duplex ultrasound. Endothelium-dependent relaxation (EDR) and endothelium-independent relaxation (EIR) were measured using acetylcholine (ACh) and sodium nitroprusside (NTP), respectively. Endothelial integrity was determined by factor VIII immunohistochemistry (F8) and scanning electron microscopy (SEM). Nitric oxide levels were determined using a chemiluminescence assay of nitrite/nitrate metabolites (NO(x)). Continuous variables were analyzed using the two-tailed Student t test. RESULTS: Control artery EDR was 80 +/- 7.1% (+/- SE), while arteries exposed to luminal thrombus for 90 min had an EDR of 55.2 +/- 5.7% (ACh = 15 microg/min, n = 11, P = 0.0231). EIR was preserved in normal and thrombosis groups with uniform response to NTP (4.92 +/- 0.1 cm vs 5.07 +/- 0.42 cm, P = 0.76). F8 staining identified endothelium in all groups. SEM analysis revealed an intact monolayer of endothelium after thrombosis. Local NO(x) levels were 17.3% lower after 90 min of thrombosis (49.3 microM vs 40.8 microM, n = 16, P < 0.001). CONCLUSIONS: Luminal thrombus induces arterial dysfunction acutely without causing endothelial cell loss. EIR remains unaffected, indicating normal smooth muscle cell function. NO(x) levels suggest that nitric oxide levels are decreased acutely after thrombosis. The development of this porcine large animal model allows the in vivo study of vasospasm and alternative thrombolytic regimens.     

Autologous endothelial progenitor cells transplantation promoting endothelial recovery in mice

Transplantation of endothelial progenitor cells (EPCs) restores endothelial function. The present study was designed to determine the effect of autologous EPCs transplantation on the regeneration of endothelium in mice. Mice splenectomy was performed 14 days before carotid artery injury, and mononuclear cells were isolated and cultured in endothelial growth media for 7 days. EPCs were confirmed by immunostaining (CD31, endothelial nitric oxide synthase (eNOS) and double positive for 1,1'dioctadecyl-3,3,3',3-tetramethylindocarbocyanine (DiI)-low-density lipoprotein and ulex europaeus agglutinin (UEA)). Cell counts and fluorescence-activated cell sorting for stem cell marker were performed. 1 x 10(6) 4-,6-Diamidino-2-phenylindole- labeled EPCs or saline were injected through tail vein after wire injury. Two weeks after transplantation, cell tracking and immunohistochemical staining showed homing and incorporation of labeled EPCs in injury artery. Administration of EPCs enhanced reendothelialization (P < 0.05) after 1 week and inhibition of neointima formation at 3 weeks compared with that of saline (P < 0.05, n = 6). These data demonstrate that delivery of autologous EPCs is associated with accelerated reendothelialization and reduced neointimal formation. Thus, delivery of autologous EPCs represents an important vasculoprotective approach to attenuate the response to acute vascular injury.     

Cardiac surgery increases the activity of matrix metalloproteinases and nitric oxide synthase in human hearts

OBJECTIVES: Heart function is variably impaired after cardiopulmonary bypass. We hypothesized that, similar to other myocardial injury states, cardiopulmonary bypass leads to enhanced activity of nitric oxide synthase and matrix metalloproteinases. METHODS: We obtained right atrial biopsy specimens and plasma samples at the onset and termination of cardiopulmonary bypass in 10 patients. Biopsy specimens were analyzed for nitric oxide synthase activity by using a citrulline assay, whereas plasma and tissue were analyzed for matrix metalloproteinase-9 and matrix metalloproteinase-2 activity by using zymography. Tissue inhibitor of metalloproteinase-4 was analyzed by means of Western blotting. The cellular expression of inducible nitric oxide, endothelial nitric oxide synthase, matrix metalloproteinase-2, and matrix metalloproteinase-9 was determined in right atrial biopsy samples from 3 additional patients by using the appropriate conjugated antibodies. RESULTS: Nitric oxide synthase activity increased from the beginning to the end of bypass (4.46 +/- 1.07 vs 16.77 +/- 4.86 pmol citrulline/mg of protein per minute, respectively; P =.018). Pro-matrix metalloproteinase-9 activity increased in hearts (199 +/- 41 vs 660 +/- 177 density units/mg protein; P =.008) and plasma (14.1 +/- 4.6 vs 52.2 +/- 5.9 density units/mg protein; P =.008). Pro-matrix metalloproteinase-2 activity increased in the heart (201 +/- 23 vs 310 +/- 35 density units/mg protein, P <.05) but not in plasma. Tissue inhibitor of metalloproteinase-4 expression in the heart decreased (1574 +/- 280 vs 864 +/- 153 density units, P =.014). CONCLUSIONS: Cardiopulmonary bypass activates enzymes mediating acute inflammation and organ injury (ie, nitric oxide synthase, matrix metalloproteinase-9, and matrix metalloproteinase-2). Decreased tissue inhibitor of metalloproteinase-4 expression allows relatively unopposed increases in matrix metalloproteinase tissue activity. We postulate that these changes play a role in the pathogenesis of heart dysfunction after bypass surgery.     

Immunophilin ligands promote penile neurogenesis and erection recovery after cavernous nerve injury

PURPOSE: We investigated the long-term effect of immunophilin ligands on erection physiology and cavernous tissue histology using rat models of unilateral and bilateral cavernous nerve (CN) injury. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were administered the immunophilin ligand FK506 (5 mg/kg subcutaneously daily for 5 days), the nonimmunosuppressant FK506 derivative GPI1046 (3 to 3-pyridyl)-1-propyl(2 seconds)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate) (40 mg/kg subcutaneously daily for 5 or 28 days) or saline immediately upon induction of 2 paradigms of cavernous nerve injury, namely focal transection of the CN unilaterally, and a combination of focal transection of the CN unilaterally and excision of a 5 mm segment of contralateral CN (BIL treated). At 28 days following CN injury electrical stimulation of the transected CN and penile erection monitoring were performed. Whole penes were removed and evaluated immunohistochemically for the nitric oxide generator neuronal nitric oxide synthase, the neuronal marker synaptophysin, the endothelial marker CD31 and the smooth muscle marker alpha-actin. RESULTS: In unilaterally treated groups erection recovery was significantly greater in FK506 treated than in saline treated animals ((83.5% +/- 9.2% vs 24.3% +/- 8.1%, p <0.001) and similarly greater in GPI1046 treated animals than in the respective saline treated controls for this group (57.9% +/- 12.6% vs 23.8% +/- 7.0%, p <0.05). In BIL treated groups erection recovery for FK506 and GPI1046 treated rats exceeded saline treated values by approximately 70% (p <0.05) and 40% (p = 0.14), respectively. After 28 days of continuous treatment in BIL treated groups erection recovery for GPI1046 treated rats exceeded saline treated values by 140% (p <0.05). Neuronal and endothelial staining was preserved after immunophilin ligand treatment. CONCLUSIONS: Immunophilin ligands exert neurotrophic effects on the penile innervation that preserve cavernous tissue structure and promote erectile function recovery in rats after extensive CN injury.     

Novel cardioprotective effects of tetrahydrobiopterin after anoxia and reoxygenation: Identifying cellular targets for pharmacologic manipulation

OBJECTIVES: Contemporary cardioprotective strategies to prevent perioperative ischemia-reperfusion injury have focused on the l-arginine nitric oxide pathway. Tetrahydrobiopterin is an absolute cofactor required for the enzyme nitric oxide synthase and is thus a critical determinant of nitric oxide production. We hypothesized that ischemia-reperfusion results in diminished levels of tetrahydrobiopterin, which might represent a key cellular defect underlying endothelial and myocyte dysfunction after ischemia-reperfusion. To this aim, we examined the effects of tetrahydrobiopterin supplementation in (1) an in vivo experimental model of global ischemia-reperfusion and (2) an in vitro human ventricular heart cell model of simulated ischemia-reperfusion. Measures of endothelial function, oxidant production, cell survival, and cardiac function were used to assess outcome. METHODS: In study 1 Wistar rats were divided into one of 2 groups (n = 10 per group). One group received tetrahydrobiopterin (25 mg x kg(-1) x d(-1) for 7 days), and the other group served as the control group. Hearts were subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion, and left ventricular developed pressure, left ventricular systolic pressure, and left ventricular end-diastolic pressure were determined by using the modified Langendorff technique. In study 2 we quantitated myocardial malondialdehyde, a marker of lipid peroxidation, in ventricular tissues from both groups of animals using butanol phase extraction and spectrophotometric analysis. In study 3 coronary vascular responses were determined in vascular segments of the left coronary artery in both groups of animals after ischemia-reperfusion. Endothelium-dependent and endothelium-independent vasodilatation to acetylcholine and sodium nitroprusside, respectively, were compared between groups. In study 4, using a human ventricular heart cell model of simulated ischemia-reperfusion, we studied the effects of tetrahydrobiopterin (20 micromol/L) on cellular injury (as assessed by means of trypan blue uptake). RESULTS: After ischemia-reperfusion, myocardial dysfunction was evidenced by a decrease in left ventricular developed pressure and an increase in left ventricular end-diastolic pressure (P =.01 compared with baseline). Hearts from tetrahydrobiopterin-treated rats exhibited protection against ischemia-reperfusion injury (left ventricular developed pressure: 74 +/- 4 vs control 42 +/- 8 mm Hg, P =.01; left ventricular end-diastolic pressure: 12 +/- 3 vs 34 +/- 7 mm Hg, P =.01). Furthermore, tetrahydrobiopterin treatment attenuated the rise in malondialdehyde levels after ischemia-reperfusion (P =.01). After reperfusion, coronary endothelial function to acetylcholine was attenuated (P =.003 vs sham-treated mice), whereas responses to sodium nitroprusside remained unchanged. Tetrahydrobiopterin-treated rats exhibited an improvement in acetylcholine-mediated vasorelaxation (P =.01 vs ischemia-reperfusion group). Cellular injury, as assessed by means of trypan blue uptake, was higher in human ventricular heart cells subjected to simulated ischemia-reperfusion; this effect was prevented with tetrahydrobiopterin treatment (P =.001). CONCLUSIONS: Supplemental tetrahydrobiopterin provides a novel cardioprotective effect on left ventricular function, endothelial-vascular reactivity, oxidative damage, and cardiomyocyte injury after ischemia-reperfusion injury and might represent an important cellular target for future operative myocardial protection strategies.     

Cigarette smoke condensate aggravates renal injury in the renal ablation model

BACKGROUND: Cigarette smoking increases the risk of progression of diabetic and non-diabetic renal diseases. The mechanisms underlying the adverse effects of smoking are largely unknown. We examined the subtotally nephrectomized rat (i) to investigate whether components of cigarette smoke dissolved in acetone (cigarette smoke condensate) aggravate structural renal damage and (ii) to establish whether this provides an animal model that can be used to investigate potential pathomechanisms of cigarette smoke-induced renal damage. Since nicotine activates the sympathetic nerve system in humans, we investigated whether interference with this system modulates the effects of cigarette smoke condensate on the damaged kidney. METHODS: One group of Sprague-Dawley rats was subtotally nephrectomized (SNX). Acetone (SNX + solvent) or cigarette smoke condensate (SNX + cigarette) was applied daily to the oral mucosa. Another group of Sprague-Dawley rats was sham-operated and received the same treatments (sham + solvent, sham + cigarette). To investigate whether increased activity of the sympathetic nerve system is involved, the remnant kidney was denervated by microsurgical technique in one SNX + cigarette group. The control group for this intervention was a solvent-treated SNX group with denervated remnant kidney. Blood pressure (BP) was measured weekly by tail plethysmography. The experiment was terminated after 12 weeks. Structural renal damage was assessed by morphometric techniques (indices of glomerulosclerosis, tubulointerstitial and vascular damage) and urinary albumin and endothelin-1 excretion were measured. RESULTS: Indices of structural renal damage were increased in all SNX-groups. Treatment with cigarette smoke condensate further increased the indices of glomerulosclerosis and tubulointerstitial damage in SNX, but not sham-operated rats. This increase was completely prevented by renal denervation. No differences in systemic blood pressure were observed in the different SNX groups. Urinary albumin excretion went in parallel with the indices of glomerulosclerosis and tubulointerstitial damage and urinary endothelin-1 excretion was significantly increased in SNX + cigarette animals. CONCLUSION: These findings document that acetone soluble components in cigarette smoke aggravate glomerulosclerosis and tubulointerstitial damage in the renal ablation model. Renal injury induced by cigarette smoke condensate in this model is reversed by renal denervation. We conclude that cigarette smoke-induced renal damage is due, at least in part, to activation of the sympathetic nerve system.     

Effect of endothelial and adventitial injury on somatostatin receptor expression

BACKGROUND: The somatostatin analog, angiopeptin, inhibits intimal hyperplasia formation; although the specific somatostatin receptor (SSTR) subtypes transducing this effect are unknown. The purpose of this study was to determine the expression of SSTR subtypes in rat iliac arteries after balloon catheter endothelial injury and perivascular dissection. METHODS: Male rats received balloon endothelial injury to their left common and external iliac arteries with or without circumferential arterial dissection. The right arteries served as controls. At 1 and 2 months after intimal injury, animals were killed and their iliac arteries harvested and studied for SSTR expression by using immunocytochemical and molecular techniques. Quantitative polymerase chain reaction was used to determine the level of SSTR expression. RESULTS: Normal rat iliac arteries expressed only SSTR2 and 3. After balloon endothelial injury, there was significant upregulation of SSTR2 messenger RNA at 1 and 2 months after injury as compared with controls (1 month, 1.8 +/- 0.3 vs 0.4 +/- 0.1 zmol, P < .001; 2 months, 2.7 +/- 0.5 vs 1.1 +/- 0.2 zmol, P < .001). The addition of adventitial dissection to endothelial injury also showed a significant increase in SSTR2 expression (1 month, 2.4 +/- 0.4 vs 0.8 +/- 0.2, P < .05; 2 months, 1.3 +/- 0.3 vs 0.7 +/- 0.3, P < .05), but not significantly greater than that seen after balloon endothelial injury alone. Immunocyto-chemical studies also demonstrated an increase in SSTR2 immunoreactivity on the luminal surface of the endothelial cells in the balloon catheter-injured arteries. CONCLUSIONS: These findings show that SSTR2 is the primary SSTR that is upregulated after injury and likely mediates the effects of somatostatin analogs on intimal hyperplasia.     

Effects of tetrahydrobiopterin on endothelial dysfunction in rats with ischemic acute renal failure

The role of nitric oxide (NO) in ischemic renal injury is still controversial. NO release was measured in rat kidneys subjected to ischemia and reperfusion to determine whether (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), a cofactor of NO synthase (NOS), reduces ischemic injury. Twenty-four hours after bilateral renal arterial clamp for 45 min, acetylcholine-induced vasorelaxation and NO release were reduced and renal excretory function was impaired in Wistar rats. Administration of BH4 (20 mg/kg, by mouth) before clamping resulted in a marked improvement of those parameters (10(-8) M acetylcholine, delta renal perfusion pressure: sham-operated control -45 +/- 5, ischemia -30 +/- 2, ischemia + BH4 -43 +/- 4%; delta NO: control +30 +/- 6, ischemia + 10 +/- 2, ischemia + BH4 +23 +/- 4 fmol/min per g kidney; serum creatinine: control 23 +/- 2, ischemia 150 +/- 27, ischemia + BH4 48 +/- 6 microM; mean +/- SEM). Most of renal NOS activity was calcium-dependent, and its activity decreased in the ischemic kidney. However, it was restored by BH4 (control 5.0 +/- 0.9, ischemia 2.2 +/- 0.4, ischemia + BH4 4.3 +/- 1.2 pmol/min per mg protein). Immunoblot after low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the dimeric form of endothelial NOS decreased in the ischemic kidney and that it was restored by BH4. These results suggest that the decreased activity of endothelium-derived NO may worsen the ischemic tissue injury, in which depletion of BH4 may be involved.     

Smoking and mouse MCL healing

Cigarette smoking has been shown to delay soft tissue healing and complicate musculoskeletal recovery. Its deleterious effects have been proven in bone and soft tissue, although to a much more limited fashion in ligament or tendon. We exposed 120 experimental mice to two cigarettes per day for 2 months and then induced a blunt medial collateral ligament (MCL) injury. Mice were assigned to three groups: the first group exposed to two cigarettes per day after injury, the second group exposed to one cigarette per day after injury, and the third group not exposed after injury. A fourth, no-smoking group served as control. The cut and contralateral intact ligaments were biomechanically tested to failure at 7 and 28 days. The ligaments of mice exposed to cigarette smoke were weaker (p = 0.02) and less stiff (p = 0.0004) at 28 days (3.2 N, 3.5 N/mm) compared with those of mice exposed to cigarette smoke for 7 days (3.9 N, 4.7 N/mm). The ligaments of mice exposed to cigarette smoke were weaker (p = 0.02) and less stiff (p = 0.01) at 28 days compared with control mice at 28 days. Between 7 and 28 days, smoking had a deleterious effect on healing manifested as weaker and less stiff ligaments. However, our findings did not support a dose-dependent effect of cigarette exposure on the tensile mechanical properties of ligaments.