Consequences of hyperhomocyst(e)inemia on vascular function in atherosclerotic monkeys

Moderate elevation of plasma homocyst(e)ine is associated with increased risk for atherosclerotic vascular disease. In a previous study, we observed impaired vascular function in nonatherosclerotic monkeys with moderate hyperhomocyst(e)inemia. In this study, we tested the hypothesis that dietary intervention to lower plasma homocyst(e)ine corrects vascular dysfunction in atherosclerotic monkeys. Cynomolgus monkeys were fed an atherogenic diet that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. After 17 months, the atherogenic diet was supplemented with B vitamins (5 mg folic acid, 400 micrograms vitamin B-12, and 20 mg vitamin B-6 daily) for 6 months. Total plasma homocyst(e)ine decreased from 12.8 +/- 2.8 to 3.5 +/- 0.3 mumol/L (n = 9; mean +/- SE; P < .01) after vitamins were added to the diet, but plasma cholesterol remained elevated (522 +/- 63 versus 514 +/- 41 mg/dL; P > .05). In response to intra-arterial infusion of collagen, blood flow to the leg decreased by 30 +/- 3% and 38 +/- 5%, respectively, before and after vitamin supplementation (P > .05). In vivo responses of resistance vessels to endothelium-dependent vasodilators (acetylcholine or ADP) were impaired at baseline and did not improve after vitamin supplementation. In carotid artery studied ex vivo, relaxation to low doses of acetylcholine improved after vitamin supplementation, but maximal relaxation remained impaired. Ex vivo thrombomodulin anticoagulant activity was threefold higher in monkeys fed the atherogenic diet (with or without B vitamins) than in normal monkeys (P < .05). We conclude that normalization of plasma homocyst(e)ine is insufficient to restore normal vascular function in atherosclerotic monkeys with persistent hypercholesterolemia and that atherosclerosis, with or without hyperhomocyst(e)inemia, is associated with elevated thrombomodulin activity.     

Hemodynamic sequelae of regression of experimental atherosclerosis

Regression of experimental atherosclerosis is characterized by decreased intimal thickness and luminal enlargement, but intimal fibrosis becomes more dense. We tested the hypothesis that fibrosis of arteries during regression might limit vasodilator capacity and restrict hemodynamic improvement despite luminal improvement. We studied limb, coronary, and cerebral hemodynamics in 11 normal cynomolgus monkeys, 10 monkeys given an atherogenic diet for 20 mo and 8 monkeys given a regression diet for an additional 18 mo. The atherogenic diet induced lesions of moderate severity (50-60% stenosis); owing to characteristic vessel growth during the atherogenic period, luminal size did not decrease correspondingly. Regression monkeys showed typical changes of regression with luminal enlargement but increased fibrosis. The iliac artery was perfused at constant blood flow and maximal vasodilatation was produced with papaverine. Blood flow was measured with microspheres during maximal vasodilatation in the coronary bed (adenosine) and cerebral bed (hypercapnia). In normal monkeys, minimal vascular resistances were 1.95 +/- 0.19 mm Hg/ml/min X 100 g (mean +/- SE) (limb), 0.13 +/- 0.01 (coronary), and 0.44 +/- 0.02 (cerebral). In atherosclerotic monkeys minimal resistance increased (P less than 0.05) 108, 62, and 166% in the limb, coronary, and cerebral beds, respectively. In regression monkeys, minimal resistance increased from values found in atherosclerotic animals in the limb (+22%), decreased inconsistently in the coronary bed (-19%), and decreased significantly in the cerebral bed (-44%, P less than 0.05). Thus morphologic regression was accompanied by significant hemodynamic improvement during maximal dilatation only in cerebral vessels. We conclude that increases in luminal size during regression of atherosclerotic lesions may not be associated with increases in vasodilator capacity, as intimal fibrosis may limit physiologically important hemodynamic improvement.     

Ultralow concentrations of proenkephalin and [met5]-enkephalin differentially affect IgM and IgG production by B cells

Adenovirus-mediated gene transfer is a promising method for studies of vascular biology and potentially for gene therapy. Intravascular approaches for gene transfer to blood vessels in vivo generally require interruption of blood flow and have several limitations. We have used two alternative approaches for gene transfer to blood vessels in vivo using perivascular application of vectors. First, replication-deficient adenovirus expressing nuclear-targeted bacterial beta-galactosidase was injected into cerebrospinal fluid via the cisterna magna of rats. Leptomeningeal cells over the major arteries were efficiently transfected, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. When viral suspension was injected with the rat in a lateral position, the reporter gene was expressed extensively on the ipsilateral surface of the brain. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, positioning of the head may 'target' specific regions of the brain. Second, vascular gene delivery was accomplished by perivascular injection of virus in peripheral vessels. Injection of the adenoviral vector within the periarterial sheath of monkeys resulted in gene transfer to the vessel wall that was substantial in magnitude although limited to cells in the adventitia. Approximately 20% of adventitial cells expressed the transgene, with no gene transfer to cells in the intima or media. These approaches may provide alternative approaches for gene transfer to blood vessels, and may be useful for studies of vascular biology and perhaps vascular gene therapy.     

Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.     

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. METHODS: Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. RESULTS: Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. CONCLUSIONS: These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.     

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.     

Enhanced monocyte tissue factor response after experimental balloon angioplasty in hypercholesterolemic rabbit: inhibition with dietary L-arginine

BACKGROUND: There is evidence that tissue factor (TF) is a major contributor to the thrombogenicity of a ruptured atherosclerotic plaque. Nitric oxide (NO) has antiatherogenic and antithrombotic properties. We investigated whether L-arginine (L-arg), the endogenous precursor of NO, might affect the ability of monocytes to produce TF. METHODS AND RESULTS: We studied TF expression in 18 rabbits with atherosclerosis induced by bilateral iliac damage and 10 weeks of a 2% cholesterol diet. Six weeks after the initiation of the diet, an angioplasty was performed. After angioplasty, the surviving rabbits (n=15) were randomized to receive L-arg (2.25%) supplementation in drinking water (L-arg group, n=8) or no treatment (untreated group, n=7). TF expression was evaluated in mononuclear cells from arterial blood in the presence and absence of endotoxin stimulation. Monocyte TF expression, as assessed with an amidolytic assay, did not differ significantly before or after the induction of atherosclerotic lesions (87+/-15 versus 70+/-12 mU of TF/1000 monocytes, P=NS). Endotoxin-stimulated TF activity increased significantly 4 weeks after angioplasty (138+/-22 versus 70+/-12 mU of TF/1000 monocytes, P=0.02). This increase was blunted by L-arg (43+/-16 mU of TF/1000 monocytes, P=0.01). CONCLUSIONS: This study demonstrates that angioplasty-induced plaque rupture is associated with a marked increase in monocyte TF response that is blunted by the oral administration of L-arg. This suggests that the documented antithrombotic properties of NO may be related in part to an inhibitory effect on monocyte TF response.     

Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis

BACKGROUND: NO regulates vascular tone and structure, platelets, and monocytes. NO is synthesized by endothelial NO synthase (eNOS). Endothelial dysfunction occurs in atherosclerosis. METHODS AND RESULTS: With a porphyrinic microsensor, NO release was measured in atherosclerotic human carotid arteries and normal mammary arteries obtained during surgery. eNOS protein expression was analyzed by immunohistochemistry. In normal arteries, the initial rate of NO release after stimulation with calcium ionophore A23187 (10 micromol/L) was 0.42+/-0.05 (micromol/L)/s (n=10). In contrast, the initial rate of NO release was markedly reduced in atherosclerotic segments, to 0.08+/-0.04 (micromol/L)/s (n=10, P<0.0001). NO peak concentration in normal arteries was 0.9+/-0.09 micromol/L (n=10) and in atherosclerotic segments, 0.1+/-0.03 micromol/L (n=10, P<0.0001). Reduced NO release in atherosclerotic segments was accompanied by marked reduction of immunoreactive eNOS in luminal endothelial cells, although specific endothelial cell markers (CD31) were present (n=13). Endothelial cells of vasa vasorum of atherosclerotic segments, however, remained positive for eNOS, as was the endothelium of normal arteries. CONCLUSIONS: In clinically relevant human atherosclerosis, eNOS protein expression and NO release are markedly reduced. This may be involved in the progression of atherosclerosis.     

Oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerotic rabbits

The goal of the present study was to determine whether oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerosis. Baroreceptor activity was measured from the carotid sinus nerve during pressure ramps in isolated carotid sinuses of anesthetized rabbits. Rabbits fed a 0.5% to 1.0% cholesterol diet for 7.9 +/- 0.4 months (mean +/- SE; range, 5.5 to 10) developed atherosclerotic lesions in the carotid sinuses. Maximum baroreceptor activity measured at 140 mm Hg and the slope of the pressure-activity curve were reduced in atherosclerotic (n = 15) compared with normal (n = 13) rabbits (425 +/- 34 versus 721 +/- 30 spikes per second and 6.2 +/- 0.6 versus 10.8 +/- 0.8 spikes per second per mm Hg, respectively, P < .05). The level of activity was inversely related to plasma cholesterol concentration (r = .86, P < .001) and total cholesterol load (plasma concentration x duration of diet, r = .92). Mean arterial pressure was normal in both groups. Exposure of the carotid sinus to the free-radical scavengers superoxide dismutase (SOD) and catalase significantly increased maximum baroreceptor activity by 25 +/- 4% in atherosclerotic rabbits (n = 6) but caused only small and irreversible changes in activity in normal rabbits (n = 8). Catalase alone but not SOD also increased baroreceptor activity in atherosclerotic rabbits (n = 7). Exposure of the carotid sinus of normal rabbits to exogenous free radicals generated from the reaction between xanthine and xanthine oxidase inhibited baroreceptor activity in a dose-dependent and reversible manner (n = 8, P < .05). The inhibition of activity was attenuated by SOD and catalase but was not attenuated by the inhibitor of hydroxyl radical formation, deferoxamine. Neither restoration of baroreceptor activity in atherosclerotic rabbits by catalase nor inhibition of activity by xanthine/xanthine oxidase could be explained by changes in the carotid pressure-diameter relation or prostacyclin formation. These results indicate that oxidant stress inhibits baroreceptor activity and that endogenous oxyradicals produced in atherosclerotic carotid sinuses contribute to baroreceptor dysfunction.     

Cystic adventitial degeneration of the popliteal artery. Rare cause of intermittent claudication in middle-aged adults

We report on a 36-year old male patient with clinically prolonged intermittent claudication of the right leg. An operative specimen measuring 8.0 cm of the A. poplitea was sent for examination, which was sheathed by an aneurysmal cyst having a maximal diameter of 1.5 cm. Examination under the light microscope showed an incomplete septated ganglion-like cystic formation with flat connective tissue-like cells or even larger histiocytoid cell elements as inner lining. Immunohistochemically, the cells on the inner lining of the cyst showed to be strongly positive to macrophage marker PG-M1, KP1 and Ki-Mp1. The endothelium of the vessels of the wall of the cyst showed a marked expression of cytokeratin 18. Immunohistochemically as well as in conventional histology, there are general parallels between cysts in cystic adventitial degeneration, ganglia and normal synovia as the two last mentioned tissues also show an expression of cytokeratin 18 at the endothelia of smaller vessels as well as an expression of macrophage-associated antigens on cells of the inner lining. Aetiopathologically, the cysts developing in the course of the disease are rather adventitial ganglia.     

Human and murine osteocalcin gene expression: conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo

Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6+/-3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2+/-0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2+/-7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5+/-0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly upregulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.     

Thy-1, a novel marker for angiogenesis upregulated by inflammatory cytokines

We identified the cell surface glycoprotein Thy-1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti-Thy-1 staining showed that Thy-1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy-1-Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy-1 staining. Thy-1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy-1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha upregulated Thy-1 mRNA by 8- and 14-fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor-BB had no effect on Thy-1 mRNA. Thus, Thy-1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy-1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis.     

Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene

The human cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins. The activity of CETP results in a reduction in HDL cholesterol levels, but CETP may also promote reverse cholesterol transport. Thus, the net impact of CETP expression on atherogenesis is uncertain. The influence of hypertriglyceridemia and CETP on the development of atherosclerotic lesions in the proximal aorta was assessed by feeding transgenic mice a high cholesterol diet for 16 wk. 13 out of 14 (93%) hypertriglyceridemic human apo CIII (HuCIII) transgenic (Tg) mice developed atherosclerotic lesions, compared to 18 out of 29 (62%) controls. In HuCIII/CETPTg, human apo AI/CIIITg and HuAI/CIII/CETPTg mice, 7 of 13 (54%), 5 of 10 (50%), and 5 of 13 (38%), respectively, developed lesions in the proximal aorta (P < .05 compared to HuCIIITg). The average number of aortic lesions per mouse in HuCIIITg and controls was 3.4 +/- 0.8 and 2.7 +/- 0.6, respectively in HuCIII/CETPTg, HuAI/CIIIg, and HuAI/CIII/CETPTg mice the number of lesions was significantly lower than in HuCIIITg and control mice: 0.9 +/- 0.4, 1.5 +/- 0.5, and 0.9 +/- 0.4, respectively. There were parallel reductions in mean lesion area. In a separate study, we found an increased susceptibility to dietary atherosclerosis in nonhypertriglyceridemic CETP transgenic mice compared to controls. We conclude that CETP expression inhibits the development of early atherosclerotic lesions but only in hypertriglyceridemic mice.     

Isolation of hMRE11B: failure to complement yeast mre11 defects due to species-specific protein interactions

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5'-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 microg) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 +/- 0.2 microg/mg total protein) was 10 times that in normal tissue (0.1 +/- 0.01 microg/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 +/- 0.3 microg/mg) was 15 times that in normal tissue (0.12 +/- 0.02 microg/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.     

Long-term efficacy of a self-retaining intraurethral catheter for the treatment of prostatic obstruction

The aim of the present study was to estimate insulin secretion, insulin sensitivity (SI), and glucose effectiveness at basal insulin (SG) in subjects with bulimia nervosa. Eight bulimic patients and eight age-, body mass index-, and sex-matched healthy control subjects without a family history of diabetes were studied. The subjects all had normal glucose tolerance. They underwent a modified frequently sampled intravenous glucose tolerance test; glucose (300 mg/kg body weight) was administered, and insulin (4 mU/kg body weight/min) was infused from 20 to 25 minutes after administration of glucose. SI and SG were estimated by Bergman's minimal model method. Basal insulin (27 +/- 3 v 45 +/- 3 pmol/L) was significantly lower in bulimic patients than in normal controls (P < .05), but basal glucose was similar between the two groups (4.5 +/- 0.1 v 4.9 +/- 0.1 mmol/L, P > .05). The glucose disappearance rate (KG) and acute insulin response to glucose estimated by the intravenous glucose tolerance test (AIR(glucose)) were similar between the two groups (KG, 1.35 +/- 0.29 v 2.20 +/- 0.21 min(-1), P > .05; AIR(glucose), 2,920 +/- 547 v 2,368 +/- 367 pmol/L x min, P > .05). No significant difference was observed in SI between the two groups (1.34 +/- 0.18 v 1.25 +/- 0.20 x 10(-4) x min(-1) x pmol/L(-1), P > .05). On the other hand, glucose effectiveness at basal (SG) and zero (GEZI) insulin was significantly diminished in comparison to normal controls (SG, 0.011 +/- 0.002 v 0.024 +/- 0.002 min(-1), P < .01; GEZI, 0.008 +/- 0.002 v 0.017 +/- 0.003 min(-1), P < .01). Thus, bulimic patients with normal glucose tolerance without a family history of diabetes were characterized by normal insulin secretion, normal SI, and reduced SG and GEZI.     

Hyporeactivity of rat diaphragmatic arterioles after exposure to hypoxia in vivo. Role of the endothelium

The effect of prior in vivo hypoxia on the in vitro responses to changes in transmural pressure, alpha-adrenoceptor activation, and depolarization with KCl were evaluated in first-order diaphragmatic arterioles. Rats (n = 14 per group) were exposed to normoxia (controls) or to hypoxia (inspired O2 concentration = 10%) for 12 or 48 h. The arteriolar pressure-diameter relationships were recorded over a pressure range from 10 to 200 mm Hg. In separate groups of arterioles (n = 12 per group), the diaphragmatic arteriolar responses to phenylephrine (10(-8) to 10(-5 M) or KCl (10 to 100 mM) were determined after exposure to either room air or hypoxia for 48 h. In half of the arterioles studied, the endothelium was removed. After 12 h of hypoxia, the pressure-diameter relationship was normal in endothelialized arterioles but was shifted upward in de-endothelialized vessels (p < 0.05). After 48 h of hypoxia, the constrictor response to increasing transmural pressure was severely suppressed in all arterioles. The intraluminal diameters during activation with phenylephrine and KCl were larger in arterioles from rats exposed to hypoxia (103 +/- 8 and 81 +/- 7 microns, respectively) than in control arterioles (41 +/- 5 and 54 +/- 6 microns, respectively; p < 0.05 for differences). During maximum phenylephrine- and KCl-induced constriction in de-endothelialized arterioles, diameters averaged 125 +/- 8 and 105 +/- 8 microns, respectively, for arterioles from hypoxic rats and 32 +/- 6 and 40 +/- 5 microns, respectively, for arterioles from control vessels. Exposure to hypoxia results in impairment of diaphragmatic arteriolar smooth muscle reactivity and reversal of the normal inhibitory influence of the endothelium on diaphragmatic arteriolar tone.     

Effect of 8-iso prostaglandin E2 on aqueous humor dynamics in monkeys

OBJECTIVE: To evaluate the effects of 8-iso prostaglandin E2 (8-iso PGE2; prosta-5,13-dien-1-oic acid,11,15-dihydroxy-9-oxo-,[5Z,8beta-11X,13E,15 S]-) on the intraocular pressure (IOP), outflow facility, and aqueous humor flow rates in normal monkeys and monkeys with glaucoma. METHODS: The IOP was measured before and as long as 6 hours after the topical application of 8-iso PGE2 to 1 eye of 6 normal monkeys and to the glaucomatous eye of 8 monkeys with unilateral laser-induced glaucoma. The pupil diameter was measured at the same times as the IOP measurements in the normal monkeys. Tonographic outflow facility and fluorophotometric flow rates of aqueous humor were measured in 6 normal monkeys before and after drug treatment. RESULTS: In normal monkeys, a single dose of 0.1% 8-iso PGE2 reduced (P<.01) the IOP for 4 hours in the treated eyes with a maximum (mean +/- SEM) reduction of 3.2 +/- 0.2 mm Hg, compared with the contralateral control eyes. The pupil size was smaller (P<.01) in the treated eyes by as much as 1.0 +/- 0.2 mm for 4 hours. In 8 glaucomatous monkey eyes, the application of 0.05% and 0.1% 8-iso PGE2 reduced the IOP (P<.01) for as long as 2 and 5 hours, respectively. The maximum reduction in the IOP was 4.6 +/- 0.8 mm Hg (0.05%) and 6.0 +/- 0.8 mm Hg (0.1%) compared with baseline measurements. The magnitude and duration of the ocular hypotensive effect were enhanced with twice-a-day administration for 5 consecutive days. Outflow facility in normal monkey eyes was increased (P<.05) by 48% in the treated eyes, and aqueous humor flow was unchanged (P>.10), compared with vehicle-treated contralateral control eyes. Mild eyelid edema, conjunctival edema, hyperemia, and discharge appeared in some eyes treated with the 0.1% drug concentration. CONCLUSIONS: The use of 8-iso PGE2 reduces the IOP in both normal and glaucomatous monkey eyes. An increase in outflow facility appears to account for most of the IOP reduction in normal monkeys. Clinical Relevance: The application of 8-iso PGE2 may have potential for the treatment of glaucoma as an outflow facility-increasing drug.     

Correction of hypertriglyceridemia and impaired fat tolerance in lipoprotein lipase-deficient mice by adenovirus-mediated expression of human lipoprotein lipase

Humans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5 x 10(9) plaque-forming units (pfu) of an E1/E3-deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial beta-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n = 57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834 +/- 133 vs 313 +/- 89 mU/mL, P < .01), and correlated with human-specific LPL activity (522 +/- 219 mU/mL) and mass (9214 +/- 782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17 +/- 0.07 vs 1.90 +/- 0.89 mmol/L, P < .01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.     

Adherent leukocytes to the aortic wall promotes atherosclerosis in the Watanabe heritable hyperlipidemic rabbits

It is well known that leukocytes adherence to the endothelium of arterial wall occurs in diet-induced hypercholesterolemic animals. We examined the relationship between leukocytes adherence and atherosclerosis in the thoratic aorta of the Watanabe heritable hyperlipidemic rabbits (WHHL rabbits). Five of 2 or 3 months old WHHL rabbits were sacrificed and after perfusion fixation with 2.5% gulutaraldehyde, thoratic aorta was taken out carefully and divided into the 4 portions: Portion 1; cranial side of aortic arch, Portion 2; caudal side of aortic arch, Portion 3; upper side of thoratic aorta except aortic arch, Portion 4; lower side of thoratic aorta except aortic arch. Three to 6 samples from each portion except branching sites were examined using electron microscopy, and the counts of adherent leukocytes (LC) in each portion were calculated. Seven of 6 to 12 months old WHHL rabbits were sacrificed and the internal side of thoratic aorta was cut opened from the ventral side and the atherosclerotic lesions were copied. From these copies, the % area of atherosclerotic plaques (%AT) in each 4 portions as described was calculated using microcomputer. LC in Portion 1 to 4 was 265 +/- 62, 234 +/- 46, 53 +/- 8 and 41 +/- 13/mm2 respectively. LC in Portion 1 or 2 was significantly larger than that in Portion 3 or 4 (p < 0.05). The endothelium to which leukocytes adhered was intact. %AT in Portion 1 to 4 was 68 +/- 8, 63 +/- 8, 40 +/- 8 and 34 +/- 8% respectively. %AT in Portion 1 or 2 was significantly larger than that in Portion 3 or 4 (p < 0.05). It is concluded that leukocytes adherence to the intact endothelium of the arterial wall was one of the geneses and promoters of atherosclerosis in WHHL rabbits.