Acute arterial thrombosis causes endothelial dysfunction: a new paradigm for thrombolytic therapy

PURPOSE: The goals of this study were to delineate the time course of endothelial dysfunction after arterial thrombosis, to determine the cause of endothelial dysfunction in this setting, and to determine whether modulating standard thrombolytic therapy would ameliorate the thrombosis-mediated endothelial dysfunction. METHODS: Male adult rats underwent infrarenal aortic occlusion by means of clip ligature to induce arterial thrombosis. After 30 minutes, 1, 2, and 3 hours, ring segments from the infrarenal aorta were harvested and placed into physiologic buffer baths. With the use of a force transducer, both endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR) were measured. Endothelial function and presence were determined by means of factor VIII immunohistochemical staining. Endothelial morphology was evaluated with scanning electron microscopy (SEM). Nitric oxide (NO) levels were determined with a chemiluminescent assay of its nitrite/nitrate metabolites (NO(x)). Standard thrombolytic therapy with urokinase (UK) was infused into thrombosed aortic ring segments and compared with UK supplemented with both low-dose L -arginine (2 mmol) and high-dose L -arginine (20 mmol). RESULTS: Arterial thrombosis decreases EDR. The nadir of EDR occurs 1 hour after thrombosis (mean +/- SE, 13% +/- 6.4% vs 94% +/- 2.6% for controls, P <.005), with persistent lowering of EDR as long as 3 hours after thrombosis. EIR is preserved, and vasoconstriction with norepinephrine or potassium buffer is unaltered. Both endothelial function and presence (n = 6 per group) were documented by means of factor VIII immunohistochemistry. An intact monolayer of endothelium at all time intervals after thrombosis was revealed by means of SEM analysis. No differences between control and thrombosed specimens were revealed by means of the grading of SEM images. Local NO(x) levels were lower after 1 hour of thrombosis, with an increase higher than baseline values at 3 hours. The addition of low-dose L -arginine resulted in a minor increase in EDR. However, high-dose L -arginine resulted in a significant increase in EDR versus controls receiving UK alone (64% +/- 6.3% vs 38% +/- 4.4%, P <.05). Correspondingly, local NO(x) levels were 20-fold higher after the high-dose L -arginine supplementation when compared with UK thrombolysis alone (2.8 +/- 0.52 micromol/L vs 0.133 +/- 0.02 micromol/L, n = 6 samples/group, P <.005). CONCLUSION: Acute arterial thrombosis causes endothelial dysfunction, without causing endothelial cell loss. Endothelial function reaches a nadir after 1 hour of thrombosis. EIR and vasoconstriction remain unaffected, indicating normal smooth muscle cell function. NO(x) levels suggest that NO levels are decreased acutely after thrombosis. Supplementing standard thrombolytic therapy with the NO precursor, l-arginine, ameliorates the endothelial dysfunction seen after acute thrombosis by increasing local NO production.     

The effects of thrombus, thrombectomy and thrombolysis on endothelial function.

OBJECTIVE: this study was undertaken to examine and compare the effects of thrombus, thrombectomy, and thrombolysis on endothelial function as measured by endothelium-dependent vasorelaxation (EDR). METHODS: adult, male New Zealand white rabbits underwent ligation of the left common iliac to femoral artery to induce thrombosis and were then randomly assigned to one of five groups, n=6 in each. Group A consisted of ligation and thrombosis for 4 h. Group B underwent similar ligation for 4 h, but without intraluminal thrombus present. Following 4 h of ligation and thrombosis, Group C underwent thrombectomy while group D was treated with urokinase (UK), 4000 U/min for 30 min. Group E underwent UK infusion alone. The right external iliac artery served as control vessel in each group. All arteries were removed and endothelial function was determined by measuring EDR. RESULTS: the presence of thrombus reduced EDR by 50% (group A) compared to control. Vessels with interrupted flow, but not exposed to thrombus, retained normal EDR (group B). Thrombectomy decreased EDR significantly (group C) compared to thrombolysis (group D) and control. UK did not significantly alter EDR (groups D, E). CONCLUSIONS: exposure of endothelium to thrombus significantly decreases EDR. EDR was not affected by interruption of blood flow in the absence of thrombus. Thrombectomy appeared to cause a further additive insult to the endothelium. In contrast, thrombolysis with UK preserved residual endothelial function. These data suggest that it is important to differentiate the effects of thrombus on endothelium from effects due to thrombectomy or thrombolysis when evaluating treatment modalities for arterial thrombosis.     

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.     

Arginase activity is increased by thrombin: a mechanism for endothelial dysfunction in arterial thrombosis

BACKGROUND: Earlier observations implicate arterial thrombosis causing endothelial dysfunction by decreasing nitric oxide (NO) levels. NO levels are restored by regional L-arginine supplementation in animal models. The purpose of this study was to investigate the roles of thrombus components in NO generation. STUDY DESIGN: Human umbilical vein endothelial cells were harvested and cultured. The thrombus components thrombin, thrombin receptor agonist peptide (TRAP), and fibrin were added to a media of confluent human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) activity was assayed by measuring conversion of L-arginine to L-citrulline. Endothelial NOS mRNA levels were quantitated using real-time polymerase chain reaction. Cellular membrane transport of L-arginine through the y+ channel was assayed with (14)C-labeled L-arginine. Arginase activity was determined as the conversion of (14)C L-arginine to (14)C urea and trapped as Na(2)(14)CO(3) for scintillation counting. Arginase protein amounts were assessed using Western blotting. RESULTS: Endothelial cells exposed to thrombin for 4 hours led to increased arginase activity. Thrombin (10 U/mL) caused a 1.6-fold increase compared with that in controls (320+/-29 microM urea/min versus 194+/-10 microM urea/min, p=0.03), and thrombin (30 U/mL) increased arginase activity 2.1-fold (398+/-27 microM urea/min, p < 0.001, versus controls); thrombin at 1 U/mL and fibrin had no effect. TRAP (50 microM) had an effect similar to that of thrombin 10 U/mL (316+/-21 microM urea/min, p < 0.01, versus controls). Protein amounts of arginase corresponded with activity levels. Neither eNOS nor inducible nitric oxide synthase (iNOS) activities were affected by exposure to thrombin and TRAP for 4 hours. Similarly, quantification of eNOS, iNOS, and endothelin-1 mRNA did not change, although CL-100, a known thrombin-inducible gene, was upregulated. Finally, transport of L-arginine into endothelial cells was unaffected by thrombin, TRAP, and fibrin exposure. CONCLUSIONS: Endothelial cells exposed to thrombin have increased arginase enzymatic activity, and the remainder of NO generation capability is unaffected. L-arginine supplementation or arginase blockade may counteract endothelial dysfunction in the setting of acute arterial thrombosis.     

Thrombus-induced endothelial dysfunction: Hemoglobin and fibrin decrease nitric oxide bioactivity without altering eNOS

BACKGROUND: Arterial thrombosis is associated with endothelial dysfunction. The purpose of this study was to determine which components of thrombus induce endothelial dysfunction and to determine their effect on endothelial nitric oxide synthase (eNOS) activity and expression. MATERIALS AND METHODS: Human aortic endothelial cells were grown to confluence. Group 1 consisted of control cells exposed to media. Group 2 was exposed to cellular components of thrombus, including erythrocytes (RBCs) (1.5, 3, and 6 x 10(4) cells/ml) and platelets (0.5, 1, and 2 x 10(5) platelets/ml). Group 3 was exposed to extracellular thrombus components, including hemoglobin (1.25, 2.5, and 5.0 g/dl), thrombin (0.4, 4.0, and 10.0 units/ml), and fibrin. The exposure time was 20 h. Nitric oxide (NO) levels were measured following exposure. Global cellular integrity was evaluated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) conversion. eNOS mRNA expression was measured using semiquantitative PCR and eNOS protein activity evaluated via a l-citrulline conversion assay. Studies were done in replicates of six and expressed as percentages of controls. RESULTS: NO levels decreased following exposure to hemoglobin (1.25 g/dl = 59 +/- 5%, 2.5 g/dl = 38 +/- 3%, 5 g/dl = 37 +/- 6%, P < 0.001). MTS metabolism was likewise suppressed (1.25 g/dl = 57 +/- 2%, 2.5 g/dl = 55 +/- 3%, 5 g/dl = 44 +/- 6%, P < 0.001). Fibrin diminished NO levels (37 +/- 5%, P < 0.01) and MTS metabolism (49 +/- 5%, P < 0.01). RBCs and platelets had no effect on NO production or MTS metabolism (P > 0.05). Thrombin's effect was concentration dependent, inhibiting nitric oxide production at low doses while stimulating it at high doses (P < 0.01). All thrombin doses enhanced MTS metabolism (P < 0.05). eNOS activity was not altered by fibrin, thrombin, or hemoglobin exposure (P > 0.05). Moreover, eNOS mRNA expression was unaffected (P > 0.05). CONCLUSIONS: Intact cellular components of thrombus do not cause endothelial dysfunction in vitro. However, free hemoglobin and fibrin diminish NO bioactivity, likely via scavenging. Thrombin affects NO levels in a concentration-dependent manner. Neither eNOS expression nor eNOS activity is diminished, indicating that thrombus does not cause permanent changes in NO generation capability.     

Normal endothelial function and decreased vasoreactivity of experimental arterial grafts

During autogenous vascular grafting endothelial cell damage inevitably occurs. In this study we examined the functional and morphologic integrity of experimental arterial grafts. The right external iliac artery was grafted into the right carotid artery of 12 male New Zealand white rabbits. The left external iliac artery was used as the control artery. Four weeks after surgery, isometric tension studies were performed on rings of control artery and arterial graft. Control arteries and arterial grafts precontracted with norepinephrine demonstrated endothelium-dependent relaxation of 28.3% +/- 5.6% and 16.1% +/- 1.7%, respectively, in response to acetylcholine (5 x 10(-6) mol/L). Both control arteries and arterial grafts contracted in response to norepinephrine, histamine, and serotonin. The median effective dose (ED50) of norepinephrine was 4.1 +/- 1.3 x 10(-7) mol/L for control arteries and 62.1 +/- 17.9 x 10(-7) mol/L for arterial grafts (p less than 0.005). Similarly, arterial grafts were less sensitive to histamine; the ED50 of histamine for control arteries was 4.6 +/- 1.3 x 10(-7) mol/L and 51.4 +/- 13.0 x -7 mol/L for arterial grafts (p less than 0.005). In contrast, reactivity to serotonin was unaltered. The ED50 was 4.1 +/- 1.3 x 10(-7) mol/L for control arteries and 4.5 +/- 1.3 x 10(-7) mol/L for arterial grafts. Scanning and transmission electron microscopy revealed a largely intact endothelial surface. These data are markedly different from our previous findings with vein grafts in which serotonin supersensitivity was associated with an absence of endothelium-mediated relaxation to acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atorvastatin improves endothelial function in renal-transplant recipients.

BACKGROUND: Hyperlipidaemia and endothelial dysfunction are common features in cyclosporin A (CsA)-treated renal transplant recipients. Endothelial dysfunction may contribute to the risk of premature atherosclerosis and cardiovascular death in these patients. A beneficial effect of statin therapy beyond cholesterol lowering may be an improvement of endothelial function. The present study was designed to assess the effect of atorvastatin on serum lipids and endothelial function in CsA treated renal transplant recipients. METHODS: This pilot study was an open trial of 4 weeks atorvastatin (10 mg per day) treatment in renal transplant recipients (n=22). All patients received a CsA- and prednisolone-based immunosuppressive regimen. Endothelial function was assessed in the forearm skin microvasculature by acetylcholine stimulation and laser Doppler flowmetry, before and after atorvastatin treatment. Serum lipids, plasma endothelin-1 (ET-1), nitric oxide (NO), and von Willebrand factor (vWF) were also measured. RESULTS: Both total and LDL cholesterol were significantly reduced by 26.8 +/- 8.4 and 41.5 +/- 11.0% respectively, after 4 weeks of treatment. Endothelial function was significantly improved during atorvastatin treatment, area under the flux versus time curve (AUC)(ACh) was 538 +/- 362 AU x min before and 682 +/- 276 AU x min after treatment (P=0.042). Plasma NO levels also showed a borderline significant increase from 49 +/- 30 to 57 +/- 37 micromol/l during the treatment period (P=0.051), though plasma ET-1 (0.37+/-0.08 vs 0.37+/-0.12 fmol/ml) and vW (196+/-57 vs 197+/-37%) were unchanged. CONCLUSION: Atorvastatin lowered serum cholesterol significantly and improved endothelial function in renal transplant recipients after 4 weeks of treatment. Plasma NO levels were increased during atorvastatin treatment, indicating a possible endothelial protective effect through an "endothelial-NO pathway".     

Experimental treatment of thrombotic vascular occlusion

The role of laser energy in the treatment of thrombotic vascular occlusion was evaluated in two sets of experiments. First, 10 polytetrafluoroethylene grafts were used to replace segments of the superficial femoral arteries in dogs and were thrombosed by distal ligation. Occlusion was maintained for one hour, or for 7, 14, 21, and 28 days in each of two grafts. Patency was restored in all 10 grafts without perforation or anastomotic disruption using a 2 mm hot tip probe powered by an Argon laser. However, increased organization of thrombus related to the duration of occlusion lead to decreased laser channel diameters, and 75% of the 28 day thrombus remained in the graft after recanalization. The second experiments tested the added benefit of thrombolytic infusion following laser recanalization. Bilateral external iliac artery thrombosis was induced in dogs by operative vessel isolation, de-endothelialization, and thrombin injection. At 7 days the efficacy of laser-assisted thrombolysis (LAT) versus enzymatic thrombolysis (ET) alone was compared. Eight vessels underwent ET by urokinase (4000 I.U./min.); 14 vessels were laser recanalized prior to thrombolytic infusion. LAT was performed from a carotid artery approach in 8 vessels (antegrade) and from a femoral artery in 6 vessels (retrograde). In contrast to studies using the hot tip alone, both ET and LAT accomplished complete thrombus removal. However, LAT lead to significant iliac arterial flow in 9 +/- 8 min. (antegrade) and 25 +/- 8 min. (retrograde) while ET required 109 +/- 47 min (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of folinic acid on forearm blood flow in patients with end-stage renal disease.

BACKGROUND: Abnormalities of endothelial function are likely to contribute to the accelerated atherosclerotic risk in subjects with end-stage renal disease (ESRD). While folates can improve endothelial function, their role in ESRD has not been fully studied. The objective was to determine the acute and 12 week-effect of folinic acid on endothelium-dependent vasodilation in subjects with ESRD. METHODS: Forearm blood flow (FBF) was assessed by strain gauge plethysmography at baseline and after 12 weeks in 34 ESRD patients (57 +/- 14 years). Vascular function was assessed with acetylcholine (ACh), and sodium nitroprusside (SNP). Patients were randomized to receive folinic acid (50 mg i.v. once weekly) or a matching placebo. A subset of 25 subjects also received folinic acid (500 microg/min intra-arterially) or placebo to determine the acute effect on ACh and SNP mediated dilation at the time of the baseline vascular study. RESULTS: Folinic acid acutely improved the maximum change in ACh mediated FBF (10.0 +/- 2.4 to 12.8 +/- 2.2 ml/min/100 ml, P = 0.017), but did not change SNP responses. Chronic active therapy did not change ACh or SNP-mediated increases in FBF. Folinic acid resulted in a non-significant decrease in homocysteine (21 +/- 6 vs 28 +/- 18 micromol/l, P = 0.16) and diastolic blood pressure was significantly reduced (P = 0.05). CONCLUSIONS: The present study demonstrated that folinic acid acutely improved endothelium-dependent vasodilatation in patients with ESRD suggesting a direct vascular effect. Chronic treatment with folinic acid did not show benefit in endothelial function, but did lower diastolic blood pressure. Further work is required to determine the optimal regime to protect vascular health in subjects with ESRD.     

The effects of propofol on neural and endothelial control of in situ rat mesenteric vascular smooth muscle transmembrane potentials

We indirectly assessed the in vivo effect of propofol on sympathetic neural and endothelial control of vascular smooth muscle (VSM) tone in Sprague-Dawley rats by measurement of in situ responses of VSM transmembrane potential (E(m)) in intact, small mesenteric arteries and veins superfused with physiologic salt solution. Measurements were made before, during, and after propofol infusion (10 and 30 mg x kg(-1) x h(-1)) in sympathetically innervated and locally denervated vessels. Propofol's effect on E(m) response to superfusion with acetylcholine (ACh), in physiologic salt solution also containing NG-nitro-L-arginine-methyl-ester and indomethacin, was determined in innervated vessels. At 30 mg x kg(-1) x h(-1), propofol caused greater arterial VSM hyperpolarization in innervated compared with denervated vessels (4.8 +/- 2.0 mV versus 2.8 +/- 1.5 mV, respectively). ACh hyperpolarized arterial, but not venous, VSM (e.g., 11.7 +/- 2.4 mV at 10(-4) M). ACh-induced hyperpolarization was eliminated by 30 mg x kg(-1) x h(-1) propofol. Assuming a close inverse correlation between magnitude of VSM E(m) and contractile force, these results suggest that propofol attenuates both sympathetic neural and nonneural regulation of VSM tone. They also suggest that propofol and ACh may act competitively in the second messenger cascade regulating VSM K+ channel activity in mesenteric resistance arteries. IMPLICATIONS: Vascular smooth muscle (VSM) contractile force responses to the IV anesthetic, propofol, were indirectly assessed by VSM membrane potential changes to clarify the mechanisms underlying attenuation of peripheral vascular control of arterial blood pressure. Results indicate that propofol-induced VSM membrane hyperpolarization and coupled reduction of VSM contractile force underlie such attenuation.     

Endothelial dysfunction and reduced nitric oxide in resistance arteries in autosomal-dominant polycystic kidney disease

BACKGROUND: Patients with autosomal-dominant polycystic kidney disease (ADPKD) have defective endothelium-dependent relaxation (EDR). We investigated the relationship between endothelial dysfunction and nitric oxide generation in hypertension and chronic renal insufficiency (CRI) in ADPKD. METHODS: We contrasted acetylcholine (ACh)-induced EDR, 3-morphollinosydnonimine (SIN-1)-induced endothelium-independent relaxation (EIDR) and constitutive nitric oxide synthase (cNOS) activity in subcutaneous resistance vessels and plasma levels and excretion of NO2-/NO3- (NOX) in normal, control (N = 10) patients with ADPKD or essential hypertension. RESULTS: EDR was decreased significantly in normotensive ADPKD (N = 9), but more severely in hypertensive ADPKD (N = 6), or those with CRI (N = 5) and in essential hypertension (N = 9). The increases in EDR with l-arginine and decreases with LG-nitro-l-arginine methyl ester (L-NAME) were lost in all groups of patients with ADPKD and in essential hypertension except for a modest effect of L-NAME in normotensive ADPKD. EIDR was unimpaired throughout. Vascular cNOS activity and renal NOX excretion were reduced profoundly in patients with all categories of ADPKD and especially in those with hypertension. CONCLUSION: EDR in resistance vessels from patients with ADPKD is impaired even in the absence of hypertension or CRI, but becomes more marked as hypertension develops. Patients with ADPKD have defective nitric oxide generation from diminished cNOS activity. Endothelial dysfunction and impaired cNOS activity in ADPKD may predispose to hypertension whose occurrence is accompanied by a further sharp deterioration in EDR.     

Evaluation of thrombolysis in a porcine model of chronic deep venous thrombosis: an endovascular model

PURPOSE: The advancement of catheter-based interventions for vascular recanalization has underscored the need for an experimental animal model of vascular thrombosis that can be used for the evaluation of interventional therapies. In this model, a porcine model of deep venous thrombosis with a novel endovascular technique was described, and the efficacy of thrombolytic therapy with urokinase was evaluated. METHODS: An endovascular device that consisted of a tapered polytetrafluoroethylene graft attached within a self-expanding nitinol stent was delivered to bilateral common iliac veins in 20 pigs. Venous thrombosis occurred as a result of flow stasis created by the intrastent stenosis. Catheter-directed pulse-spray thrombolysis with urokinase (250,000 units) and heparin (5000 IU) was performed on one limb while the contralateral limb received control saline solution. Thrombolysis was performed in 1 hour (n = 4), 8 hours (n = 4), 3 days (n = 4), 7 days (n = 4), and 14 days (n = 4) after the stent-graft deployment. Venography and intravascular ultrasound were used to evaluate the efficacy of thrombolysis. Light microscopy was used for histologic analysis of the thrombus. RESULTS: Complete thrombolysis was achieved in groups with deep vein thrombosis that were younger than 1 day. Angioplasty of the tapered stent-grafts in the completely thrombolysed iliac vein was successful in restoring venous flow. The efficacy of thrombolysis in 3-day, 7-day, and 14-day groups was 86% +/- 7%, 73% +/- 13%, and 42% +/- 23%, respectively. The thrombolytic efficacy was enhanced to 92% +/- 16% and 86% +/- 18% (P <.05) in 3-day and 7-day groups, respectively, when doses of the pulse-spray thrombolysis were doubled. Increased dosages of the thrombolytic agent, however, did not significantly enhance the thrombus dissolution in the 14-day group. CONCLUSION: The thrombolytic efficacy of urokinase correlated with the chronicity of deep venous thrombosis in our model. An increased dose of urokinase may be used to enhance the efficacy of thrombolysis in a 1-week-old thrombus.     

Lipopolysaccharide impairs endothelial nitric oxide synthesis in rat renal arteries

BACKGROUND: Impaired endothelium-dependent vasodilation may contribute to hypoperfusion and failure of abdominal organs, including the kidneys during endotoxin or septic shock. In this study, the short-term (2 h) effects of bacterial lipopolysaccharide (LPS) on endothelium-dependent vasodilation in rat renal and superior mesenteric arteries were documented. METHODS: Rat renal and mesenteric arteries were dissected and exposed in vitro to LPS for two hours. The effects of LPS on vascular reactivity were determined and compared with time-matched controls. Endothelial nitric oxide (NO) release was determined using an NO microsensor in adjacent vessel segments. RESULTS: LPS impaired maximal acetylcholine (ACh)-induced endothelium-dependent vasodilation in renal arteries (62.5 +/- 8.8% vs. 34.4 +/- 7.5% in controls and LPS-exposed arteries), but not in mesenteric arteries. LPS did not alter the sensitivity of renal arteries to exogenous NO. ACh-dependent vasodilation was abolished after blocking NO synthesis with 10-4 mol/L L-NA in control and LPS-incubated renal arteries. When compared with controls, NO release induced by ACh and the receptor-independent calcium ionophore A23187 was significantly decreased (P < 0.05) in LPS-exposed renal segments and was fully abolished in endothelium-denuded segments, indicating that LPS attenuated receptor-dependent as well as receptor-independent endothelial NO release. In contrast, ACh- and A23187-induced NO release was normal in LPS-exposed mesenteric arteries. CONCLUSIONS: These results indicate that LPS-induced selective impairment of ACh-induced endothelium-dependent relaxation in rat renal arteries is caused by decreased endothelial NO release. This may contribute to the propensity for acute renal failure during septic shock.     

Maintenance of physiological coronary endothelial function after 3.3 h of hypothermic oxygen persufflation preservation and orthotopic transplantation of non-heart-beating donor hearts.

OBJECTIVE: The use of non-heart-beating donors (NHBD) might increase the number of grafts available for transplantation. Experiments on heart transplantation from NHBDs demonstrated the necessity for oxygenation during preservation to allow sufficient myocardial recovery. It has been shown that, after 16 min normothermic ischemia followed by 3.3-h hypothermic preservation, excellent myocardial and cardiovascular recovery is attained, if coronary oxygen persufflation (COP) is included in the preservation protocol. Here tests are presented on the recovery of coronary endothelium derived relaxation (EDR) of NHBD hearts after preservation including COP. METHODS: After 16 min normothermic ischemia, pig hearts were stored for 3.3 h at 0-1 degrees C in modified HTK plus COP (mBHTK+COP, n=6) or in two control groups without COP: (1) with mBHTK (n=6); and (2) with HTK (n=4). Following orthotopic transplantation and 3 h of reperfusion with full blood, coronary EDR was tested in vitro using Substance P (SP) under indomethacin for prostaglandin blockage. Additional tests were performed adding L-NIL to block the NO-production by iNOS or L-NNA to block total NO production. RESULTS: The EDR in percent of precontraction was 78 +/- 7% after mBHTK+COP and 77 +/- 20% (mBHTK) or 72 +/- 7% (HTK) in the controls without significant differences between the groups. Physiologic values of normal coronaries were 75 +/- 9%. L-NIL for blockage of NO-production by iNOS resulted in unchanged relaxations. After blockage of total NO production by L-NNA, the SP-induced dilation was significantly reduced to 58 +/- 8% (mBHTK+COP) and to 48 +/- 8% (mBHTK) or 55 +/- 13% (HTK) in the controls. CONCLUSIONS: Even after 16 min of warm ischemia followed by 3.3 h of preservation with gaseous oxygen persufflation, orthotopic transplantation, and reperfusion the endothelium derived coronary dilatation was unchanged from physiologic values and similar to the controls without COP. Blockage of NO production by L-NNA resulted in equal values of EDR with or without COP, while blockage of NO production by iNOS did not influence the EDR reaction. Thus COP preservation, which has been shown to allow excellent recovery of preserved NHBD hearts, caused no damage to the coronary EDR mechanisms.     

Thermal laser arterial injury and prostacyclin administration in dogs: thrombotic and hyperplastic consequences.

Angioplasty is considered as an alternative to surgical reconstruction of arteriosclerotic vessels especially since lasers and atherectomy devices have become clinically available. However, the resulting arterial injury may lead to acute thrombotic occlusion and chronic restenosis because of hyperplastic vascular repair. The purpose of this experimental study was to evaluate the consequences of thermal laser arterial injury on platelet deposition and myointimal hyperplasia in dog femoral arteries. An intraarterial, short-term prostacyclin (PGI2) infusion was given to evaluate the antithrombotic and antiproliferative effects of this drug. Severe arterial necrosis, partly carbonized and vacuolized, extending to the adventitia was induced by a transluminal heated laser probe motion. The platelet deposition after one hour was 33.62 +/- 6.56 (x 10(6)/cm2.) (mean +/- SEM) without prostacyclin, after 40 ng/kg/min prostacyclin (PGI2) 24.70 +/- 5.45 and after 400 ng/kg/min 9.3 +/- 2.26 (p less than 0.005 no PGI2 vs 400 ng/kg/min PGI2). Myointimal hyperplasia was present eight weeks after thermal laser vascular injury independent of the initially administered prostacyclin. In conclusion, acutely thrombotic and chronically hyperplastic femoral arteries were found following transluminal thermal arterial injury in dogs. Prostacyclin administration could be clinically beneficial in reducing acute vascular thrombosis following thermal angioplasty. Short-term use of this substance, however, may not prevent a hyperplastic response to angioplasty.     

Atherosclerosis-induced chronic arterial insufficiency causes clitoral cavernosal fibrosis in the rabbit

In our previous studies we found that aging-associated fibrosis of clitoral cavernosal tissue correlated with the prevalence of cardiovascular disease in elderly women. The aim of this study was to determine specifically, arterial insufficiency-related structural changes of clitoral cavernosal tissue in a rabbit model. New Zealand white female rabbits were divided into clitoral cavernosal ischemia (CCI, n = 5) and control (n = 5) groups. The CCI group underwent balloon endothelial injury of the iliac arteries and received 0.5% cholesterol diet. The control group received a regular diet. After 16 weeks, arteriography was performed then the animals were sacrificed. The iliac arteries and the entire clitoris were removed. Cross-sections of the iliac arteries and clitoris were processed for histologic evaluation The percentage of smooth muscle and connective tissue in trichrome stained sections of clitoral cavernosal tissue was determined by computer-assisted histomorphometry. Arteriography revealed diffused occlusive disease in the common iliac, internal iliac and pudendal arteries in the CCI group. Histology showed that arterial occlusive disease spreads from the site of balloon injury to the smaller branches involving the clitoral cavernosal arteries. Diffuse fibrosis was observed in the clitoral cross-sections of the CCI group. The percentage of clitoral cavernosal smooth muscle (mean +/- standard error) in the CCI group (53% +/- 0.9%) was significantly decreased compared with the control group (62% +/- 0.8%) (P = 0.0001). Chronic clitoral cavernosal ischemia causes significant fibrosis and loss of smooth muscle in the clitoral cavernosal tissue. These findings suggest that chronic clitoral cavernosal arterial insufficiency may play a role in the pathophysiology of female sexual arousal disorders.     

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.     

The effect of increasing clamping forces on endothelial and arterial wall damage: an experimental study in the sheep.

PURPOSE: This study aimed to relate the level of physical force applied to the arterial wall by atraumatic clamps to the degree of endothelial and wall damage. METHODS: Sixteen sheep carotid and femoral arteries were each demarcated into four segments 1 cm apart (total 64 segments). Each segment was clamped for 15 min with a standard angled DeBakey vascular clamp. Four levels of force were generated by closing the clamp at three, four, five and six notches of closure. The extent of endothelial injury was assessed by using a dedicated computer assisted image acquisition program to measure the area stained by Evan's blue dye. The extent of damage to the layers of the arterial wall was analyzed and compared by scanning electron microscopy and light microscopy. RESULTS: For femoral arteries, the area of endothelial injury was considerably less for three notch (3.76 +/- 0.28 newtons) and four notch (5.68 +/- 0.29 newtons) closure compared with that for five notch (6.19 +/- 0.31 newtons) and six notch (6.61 +/- 0.16 Newtons) closure (p = 0.01). For carotid arteries, three notch (5.68 +/- 0.28 newtons) closure caused less damage than did four notch (7.98 +/- 0.29 newtons), five notch (9.17 +/- 0.40 newtons) and six notch (9.57 +/- 0.64 newtons) closure (P = 0.02). Scanning electron microscopy confirmed the extent and depth of arterial injury corresponded directly to the forces generated by the vascular clamps. CONCLUSIONS: The closing forces generated by arterial clamps correlated positively with the extent of artery wall injury. Vascular clamps should be applied at the minimum level of force that will arrest blood flow.     

Acute administration of L-arginine does not improve arterial endothelial function in chronic renal failure.

BACKGROUND: Reduced activity of the nitric oxide (NO) pathway has been implicated in the endothelial dysfunction that occurs in patients with renal failure. NO is generated from L-arginine by NO synthase, and certain uremic toxins including asymmetrical dimethyl-L-arginine (ADMA), inhibit NO synthase and might contribute to endothelial dysfunction. We hypothesized that exogenous L-arginine might improve endothelial function in patients with renal failure by overcoming the effects of uremic toxins. METHODS: Endothelial function of the forearm resistance vasculature was assessed using plethysmography to measure the dilator response to intra-arterial acetylcholine (25 to 100 nmol/min). Endothelial function of radial and brachial arteries was assessed using vascular ultrasound to measure the dilator response to flow during reactive hyperemia (flow-mediated dilation; FMD). Studies were performed before and after administration of L-arginine by intra-arterial infusion (50 micromol/min) in 8 pre-dialysis patients or by intravenous infusion (10 g) in 18 hemodialysis patients. RESULTS: Local L-arginine did not improve the dilator response of forearm resistance vessels (AUC 23.1 +/- 6.4 pre, 23.1 +/- 5.1 post; P = 0.9) or FMD of the radial artery (6.5 +/- 1.2% pre, 6.3 +/- 0.8% post; P = 0.8). Systemic L-arginine did not improve FMD of the brachial artery (4.1 +/- 1.1% pre, 3.0 +/- 1.1% post; P = 0.07). These data demonstrate that acute local or systemic administration of L-arginine did not improve endothelial function in resistance or conduit arteries of patients with chronic renal failure. CONCLUSION: The results suggest that competitive inhibition of nitric oxide synthase (NOS) by circulating inhibitors is not the principal explanation for impaired endothelial dilator function in chronic renal failure.