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.     

Impaired renal function is associated with markers of endothelial dysfunction and increased inflammatory activity.

BACKGROUND: Patients with end-stage renal disease (ESRD) as well as those with mild renal insufficiency are at increased risk for the development of cardiovascular disease, which cannot be attributed entirely to traditional risk factors. Endothelial dysfunction and chronic inflammatory activity, two important phenomena in atherogenesis, can be found in ESRD. At present, it is unclear whether endothelial dysfunction and chronic inflammatory activity are related to renal function in the pre-dialysis stage. METHODS: In a cross-sectional, single-centre study, four groups of 20 subjects with renal function ranging from a normal, calculated creatinine clearance (>90 ml/min) to a pre-dialysis situation (<31 ml/min) were investigated. We measured markers of endothelial function [von Willebrand factor (vWf), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and E-selectin (ES)], and markers of inflammatory activity [secretory phospholipase A(2) (sPLA(2)) and C-reactive protein (CRP)]. Using these markers, composite endothelial function and inflammatory activity scores were constructed. RESULTS: Creatinine clearance correlated with the endothelial function score (r=-0.43, P<0.001), the inflammatory activity score (r=-0.53, P<0.05), vWf (r=-0.54, P<0.001), sVCAM-1 (r=-0.50, P<0.001), sPLA(2) (r=-0.28, P<0.05), homocysteine (r=-0.61, P<0.001), age (r=-0.54, P<0.001) and blood pressure (r=-0.44, P<0.001). In multivariate analyses, creatinine clearance was an independent determinant of the endothelial function score (beta=-0.34, P=0.006), plasma vWf (beta=-0.37, P=0.022) and sICAM-1 (beta=-0.33, P=0.012). The relationship of creatinine clearance with sVCAM-1 and endothelial function score was not significant when plasma homocysteine was added to the model. Creatinine clearance was also a determinant of the inflammatory activity score (beta=-0.31, P=0.025) and sPLA(2) (beta=-0.32, P=0.024), although this was no longer significant after correction for systolic blood pressure. CONCLUSIONS: Renal dysfunction is associated with markers of endothelial dysfunction and inflammatory activity. Plasma homocysteine may be an intermediate factor in the relationship between endothelial dysfunction and renal function, while blood pressure may modulate the association between inflammatory activity and renal function.     

Local gene delivery: arterial thrombosis model for endothelial cell-targeted thrombolytic gene therapy research

The authors describe a rabbit arterial thrombosis model that employs vessel division followed by microsurgical anastomosis and transluminal sutures, to reliably induce thrombus formation in the common femoral artery (CFA). Using two objective measures, "evacuation/refill" evaluation of patency and computer-aided histomorphometric analysis of the thrombus area, thrombus formation was confirmed and characterized in the model at both short- and long-term observation time-points. In addition, a gene delivery method was developed in the CFA that employs an adenoviral vector solution injected through the inferior epigastric artery (IEA). Using this method, a marker transgene (beta-galactosidase) was delivered to endothelial cells locally and without trauma. By subsequently performing beta-galactosidase staining, effective endothelial transfection was demonstrated simultaneously with endothelial viability, with preserved endothelial synthetic function in the immediate environment of the occluding thrombus. The results suggest that these two techniques can be used together in one model, to effectively introduce a foreign therapeutic transgene into endothelial cells and to evaluate the effect of the expressed protein product in a consistent in vivo thrombosis system. This combined model may be used as one of several assays of efficacy in future endothelial cell-targeted thrombolytic/antithrombotic vascular gene therapy research.     

Serum Arginase II level can be a novel indicator for erectile dysfunction in patients with vasculogenic erectile dysfunction: a comparative study

Purpose

The purpose of the study was to compare serum level of Arginase II in patients with vasculogenic erectile dysfunction (ED) versus healthy controls and to assess if its level is affected by severity of ED.

Methods

This is a prospective study that compared Arginase II in 40 patients with ED versus 40 healthy controls. Patients were excluded if they had any pelvic trauma or pelvic surgery, hormonal disorders, Peyronie’s disease, smoking, drug addiction or systemic illnesses. ED was evaluated by the validated Arabic version of the abbreviated five-item form of the international index of erectile function (IIEF-5). Serum arginase II level was assayed using ELIZA. Mann–Whitney, Kruskal–Wallis and Chi-square tests and Spearman correlation were used as appropriate and confirmed by logistic regression model.

Results

22 (55%) patients had DM. 15 (37.5%), 7 (17%), 6 (15%) and 12 (30%) patients suffered from severe, moderate, mild to moderate and mild ED, respectively. The level of serum Arginase II was significantly higher in patients than controls (p?<?0.001) and confirmed by multivariate logistic regression analysis. It also correlated significantly with age (r²?=?0.22; p?<?0.001) and IIEF-5 score (r²?=?0.8; p?<?0.001). Serum Arginase II increased significantly with more severe ED (p?<?0.001). Arginase II was also significantly higher in diabetic patients (p?<?0.001).

Conclusion

Serum level of Arginase II is significantly higher in patients with vasculogenic ED compared to healthy controls. It correlates significantly with age and IIEF-5 and was significantly affected by the severity of ED.

     

Platelet activation is increased in peripheral arterial disease

OBJECTIVE: Platelet activation was assessed in patients with peripheral arterial disease compared with healthy control subjects. METHODS: This prospective comparative study included 100 subjects: 40 consecutive patients with intermittent claudication, 20 consecutive patients with critical ischemia and tissue loss, and 40 healthy control subjects. Whole blood flow cytometric analysis was performed to determine resting and stimulated platelet P-selectin expression and resting and stimulated platelet fibrinogen binding. Results are presented as platelet percentage and also as mean fluorescence intensity. RESULTS: P-selectin expression was significantly increased in patients with intermittent claudication (median, 0.85%; range, 0.31%-4.77%; P =.023) and critical ischemia (median, 1.11%; range, 0.2%-3.26%; P =.028) compared with control subjects (median, 0.59%; range, 0.16%-4.58%). The percentage of platelets binding fibrinogen was also significantly higher in patients with intermittent claudication (median, 2.89%; range, 1.08%-9.59%; P <.001) compared with control subjects (median, 1.57%; range, 0.17%-10.7%). There was no significant difference in percentage of platelet fibrinogen binding between control subjects and patients with critical ischemia. Fibrinogen binding by stimulated platelets was significantly diminished in patients with critical limb ischemia compared with control subjects (67.2% vs 77.9%; P =.006). CONCLUSIONS: Platelet activation is increased in patients with peripheral arterial disease, suggesting an underlying prothrombotic state. Platelets from patients with critical limb ischemia are less responsive to in vitro stimulation.     

Circulating monocyte oxidative activity is increased in patients with type 2 diabetes and erectile dysfunction

PURPOSE: We investigated the relationship between oxidative stress and diabetic erectile dysfunction. MATERIALS AND METHODS: A total of 23 patients with a mean +/- SD age of 56.7 +/- 5.6 years, a history of type 2 diabetes for 10.0 +/- 8.3 years and erectile dysfunction, as tested by the International Index of Erectile Function questionnaire, but without vascular and neurological complications, and 15 age matched patients with diabetes without erectile dysfunction were recruited. Circulating monocyte oxidative activity by cytofluorometry, and endothelin-1, intercellular adhesion molecule-1, plasminogen activator inhibitor-1 by enzyme linked immunosorbent assay were evaluated in all patients in the study. RESULTS: Monocyte free radical production, and total and low density lipoprotein cholesterol were higher in patients with than in those without erectile dysfunction (p <0.03, <0.02 and <0.05, respectively). In all patients the International Index of Erectile Function score inversely correlated with low density lipoprotein (p <0.05), while in patients with erectile dysfunction it negatively correlated with age (p <0.03), body mass index (p <0.02), endothelin-1 (p <0.02) and intercellular adhesion molecule-1 (p <0.05). Endothelin-1, intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 were not different in patients with diabetes with and without erectile dysfunction. CONCLUSIONS: In men with type 2 diabetes who have erectile dysfunction but are asymptomatic for cardiovascular disease oxidative activation of monocytes is increased and it is related to other risk factors of endothelial dysfunction.     

Profile of endothelial dysfunction in arterial hypertension. Vascular ultrasound assessment

Assessment of endothelial dysfunction in arterial hypertension using brachial ultrasound is a common method. Because of prognostic value of flow mediated dilation, vascular ultrasound is considered the standard noninvasive method. MATERIAL AND METHOD: In our study we assessed 92 hypertensive patients (very carefully selected) in a prospective and long term follow-up manner. We used the technical indications of actual guidelines. RESULTS: We confirmed the importance of a complete and complex assessment of endothelial dysfunction, using different parameters. Our study demonstrated that endothelial dysfunction exists in hypertensives, has a large interindividual variability and can be improved using adequate treatment. We also proposed a new parameter--the velocity ratio, and tested the value of normalized diameter ratio, as a nonconventional variable.     

Late arterial thrombosis in a replanted thumb: a case report

A case of arterial occlusion occurred nine years after replantation of a thumb. Factors implicated were smoking and repeated blunt trauma to the first web space. The digit was successfully revascularized after excision of a segment of thrombosed arterial vein graft.     

Vasopeptidase inhibition: a new treatment approach for endothelial dysfunction

Angiotensin-converting enzyme (ACE) inhibition is a well-established principle in the treatment of endothelial dysfunction. Numerous preclinical and clinical studies have clearly demonstrated the beneficial effects of inhibiting the renin-angiotensin-aldosterone system (RAS) in states of impaired endothelial function. The successful use of ACE inhibitors encouraged attempts to inhibit other key enzymes in the regulation of vascular tone, such as the neutral endopeptidase (NEP). Similar to ACE, NEP is an endothelial cell surface metalloproteinase that is involved in the degradation of several regulatory peptides, including the natriuretic peptides, and, thus, NEP inhibition augments vasodilatation and natriuresis through increased levels of atrial natriuretic peptide (ANP). By inhibiting the RAS and potentiating the natriuretic peptide system at the same time, combined NEP/ACE inhibitors, the so-called vasopeptidase inhibitors, reduce vasoconstriction and enhance vasodilatation, thereby decreasing peripheral vascular resistance and blood pressure. Within the vessel wall this may lead to a reduction of vasoconstrictor and proliferative mediators, such as angiotensin II and endothelin-1, and may increase local levels of bradykinin as well as natriuretic peptides. Even though first results of both preclinical and clinical studies indicate that combined inhibition of ACE and NEP by vasopeptidase inhibitors represents a promising strategy in the treatment of hypertension and heart failure, angioedema occurs more frequently on vasopeptidase inhibition as compared to ACE inhibition. To establish vasopeptidase inhibition as a novel option in the treatment of cardiovascular disease, further validation of efficacy and safety of this promising therapeutic principle is mandatory.     

Circulating endothelial progenitor cells and endothelial microparticles in patients with arterial erectile dysfunction and metabolic syndrome

Blood endothelial progenitor cells (EPC) and microparticles (EMP) have been proposed as markers of endothelial dysfunction. The aim of this study was to evaluate both EPCs and EMPs in patients with arterial erectile dysfunction (ED) and metabolic syndrome (MetS). To accomplish this, 100 patients (ages 45-60 years) with ED and MetS (Adult Treatment Panel III [ATP III] 1999 criteria) and 17 healthy men (ages 44-57 years) were selected. EPC (CD45(neg)/CD34(pos)/CD144(pos)) and EMP (CD45(neg)/CD144(pos)/Annexin V(pos)) blood concentrations were evaluated by flow cytometry, before and after administration of tadalafil (20 mg) on demand for 3 months. Before treatment, EPCs and EMPs were significantly higher in patients compared with healthy men. EPCs increased significantly after tadalafil administration, whereas EMPs did not differ significantly. EPCs correlated positively or negatively with body mass index and with some cavernous artery indices, both before and after tadalafil administration. EMPs showed only positive correlations with body mass index and some cavernous artery indices, both before and after tadalafil administration. Patients with arterial ED and MetS have higher EPCs and EMPs compared with healthy men; hence, these cells may be regarded as markers of cavernous artery dysfunction. Tadalafil administration increased EPCs but not EMPs, suggesting that this compound may play a role in the endothelial repair response.     

Urokinase treatment preserves endothelial and smooth muscle function in experimental acute arterial thrombosis

Purpose: Pharmacologic lysis or balloon thrombectomy are options to treat acute arterial thrombosis; however, little is known about their effects on functional changes in the arterial wall. The aim of this study was to determine function of the endothelium and smooth muscle in canine arteries revascularized after acute thrombosis with balloon thrombectomy or lytic therapy.Methods: Acute thrombosis was obtained by bilateral proximal and distal ligation of 8-cm segments of the femoral arteries in dogs. After 24 hours, the ties were removed and the arteries randomized to treatment groups: group 1, balloon thrombectomy (#4 Fogarty balloon catheter at 60 grams linear shear × 1 pass, n = 7); group 2, untreated, tie removal only (n = 6); group 3, regional intra-arterial urokinase infusion (4000 U/min × 90 min, n = 6); group 4, regional intra-arterial carrier infusion (0.43 ml/min × 90 min, n = 6); group 5, unoperated normal vessels (n = 5). After treatment, the arteries were removed and endothelial and smooth muscle responses examined in organ chambers. Endothelial loss was graded with light microscopy of vessel rings from each animal by an observer blinded to the treatment group. Findings were confirmed with scanning electron microscopy.Results: Treatment with urokinase did not alter endothelium-dependent relaxations or smooth muscle contractions compared with carrier infusion or untreated alone. Balloon catheter thrombectomy significantly reduced endothelium-dependent relaxations compared with all other groups in response to acetylcholine, bradykinin, and thrombin (p < 0.001). Contractions of smooth muscle in response to potassium chloride (60 mol/L) and phenylephrine (1 × 10^-6 mol/L) were also reduced (p < 0.05). Rings from balloon thrombectomized arteries contracted in response to calcium ionophore A23187 (p < 0.001); these contractions were endothelium dependent and not reduced by indomethacin or blockade of endothelin A and B receptors. No significant differences in percentage of endothelial coverage between groups were assessed by light and electron microscopy.Conclusion: Thrombolysis with urokinase caused no or minimal abnormalities in endothelial and smooth muscle function. Endothelium present after balloon thrombectomy produces contractile factors. Although the duration and recovery of these abnormalities in function are unknown, these findings support preferential use of urokinase over balloon thrombectomy when possible in acute arterial thrombosis or embolism. (J Vasc Surg 1996;23:851-9.)     

Notch通路在高浓度葡萄糖腹膜透析液所致大鼠腹膜纤维化模型中的活化

目的 观察Notch通路在高浓度葡萄糖透析液所致大鼠腹膜纤维化模型中的变化并探讨其可能机制。 方法 给予SD雄性大鼠每日腹腔注射高浓度葡萄糖腹膜透析液，于实验后2周和4周杀检。取壁层腹膜组织行光镜检查；免疫印迹检测转化生长因子β1 （TGF-β1）、 E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA) 和I型胶原蛋白（Col I）的表达；RT-PCR检测Notch通路的下游靶基因Hes-1的表达；免疫印迹和RT-PCR检测Notch配体Jagged-1和Notch通路的负性调节因子Numb的表达。 结果 HE染色显示模型组腹膜明显增厚，间皮细胞减少；Masson染色显示壁层腹膜中可见明显的胶原沉积。与健康对照组相比，模型组的TGF-β1、α-SMA 和 Col I的表达增加而E-cadherin的表达下降（均P < 0.01）。4周模型组与对照组相比Jagged-1表达明显增加（P < 0.05），同时Hes-1的表达亦明显增加（P < 0.01），而Notch通路的负性调节因子Numb的表达下降（P < 0.01）。 结论 在高浓度葡萄糖腹膜透析液所致的大鼠腹膜纤维化模型中有Notch通路的活化，而该通路的活化可能与Notch通路的负性调节因子Numb表达的下调有关。高表达Notch通路的负性调节因子，如Numb，可能是治疗腹膜透析患者腹膜纤维化的新途径。     

Adjuvant radiotherapy is associated with increased sexual dysfunction in male patients undergoing resection for rectal cancer: a predictive model

OBJECTIVES: The objectives of this study were to evaluate the effect of radiotherapy (RT) on sexual function in patients undergoing oncologic resection for rectal cancer, and to develop a mathematical model for quantifying the risk of sexual dysfunction through time for this group of patients. METHODS: Data were prospectively collected on patients undergoing proctosigmoidectomy (group 1: n = 101) or adjuvant radiotherapy (40-50 Gy) and resection (group 2: n = 100) for rectal cancer at a tertiary referral center between December 1998 and July 2004. Study end points were recorded at 7 time intervals (preoperatively, 4 months, 8 months, 1 year, 2 years, 3 years, and 4 years after surgery) and included: 1) ability to have an erection, 2) maintain an erection, 3) attain orgasm, 4) dry orgasm, and 5) whether they were sexually active. Multilevel logistic regression analysis for repeated measures was used to identify factors associated with the sexual dysfunction. A predictive model was developed and internally validated by comparing observed and model-predicted outcomes. RESULTS: Radiotherapy had an adverse effect on the ability to get an erection, maintain an erection, attain orgasm, and being sexually active in comparison with patients undergoing surgery alone (7.4%, 12.6%, 16.2%, and 13.7% reduction 8 months after surgery respectively; P < 0.05). The effect of sexual dysfunction deteriorated with age (odds ratio for erectile function, 0.40 per 10-year increase in age; 95% confidence interval, 0.29-0.49; P < 0.001). A significant variability in sexual function was present among the 7 time points with a maximal deterioration occurring at 8 months after surgery with subsequent slow but not complete recovery (P < 0.001). The predictive model showed adequate discrimination on 4 of the 5 domains of sexual dysfunction (area under the receiver operating characteristic curve >0.70). CONCLUSIONS: Radiotherapy has an adverse effect on sexual function, the effect being maximal at 8 months after surgery. The risk of sexual dysfunction can be quantified preoperatively using the proposed index and can assist patients in making better informed choices on the type of treatment they receive.     

Local heparin is superior to systemic heparin in preventing arterial thrombosis

Thrombosis poses a significant problem in microvascular surgery, despite antithrombotic therapy. The purpose of the present study was to investigate whether a topical application of unfractionated heparin is equally efficient as a systemic bolus in avoiding thrombosis. A rat femoral artery model was used. Three different doses of systemic heparin (50, 100, and 200 U/kg) and one dose of locally administered heparin (100 U/ml) were evaluated and compared to a control group receiving isotonic saline. A thrombogenic injury, simulating poor microsurgical technique, was applied to the artery. The thrombus area was visualized by transillumination, and recorded for 60 min on video, for subsequent measurement. In addition, the level of activated partial thromboplastin time (APTT) and of anti-activated clotting factor X (aXa) was determined. Local heparin significantly reduced thrombus size as compared to isotonic saline and systemic heparin (50 and 100 U/kg). High-dose systemic heparin (200 U/kg) was equally potent, but local heparin had significantly less influence on the hemostatic parameters.