Overexpression of PAI-1 prevents the development of abdominal aortic aneurysm in mice.

Vessel wall inflammation and matrix destruction are critical to abdominal aortic aneurysm (AAA) formation and rupture. We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in experimental AAA and is essential for AAA formation and expansion. In this study, we examined the effects of overexpression of a natural inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), on the development of angiotensin (Ang) II-induced AAA in ApoE-deficient (ApoE(-/-)) mice. Mice were treated with recombinant adenovirus containing either the human PAI-1 gene (Ad5.CMV.PAI-1) or the luciferase gene (Ad5.CMV.Luc) delivered either locally by intra-adventitial injection or systemically by tail vein injection. Our results show that local delivery of the PAI-1 gene completely prevented AAA formation (0 vs 55.6% in Ad5.CMV.Luc controls, P<0.05). In contrast, systemic delivery of the PAI-1 gene did not affect AAA incidence (78 vs 90% in Ad5.CMV.Luc controls, P=0.125). Local delivery of the PAI-1 gene 2 weeks after Ang II infusion prevented further expansion of small aneurysms, but had no significant effect on the progression of larger aneurysms. These data suggest that local PAI-1 gene transfer could be used to stabilize small AAA and reduce the rate of expansion and risk of rupture.

Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption.

The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice. 125I-17beta-estradiol binding in aortic tissue showed significantly more high affinity cytosolic- nuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by NG-nitro-arginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endothelium-dependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.

Role of endothelial nitric oxide in bone marrow-derived progenitor cell mobilization.

Mobilization and recruitment of bone marrow-derived progenitor cells (BMDPCs) play an important role in postischemic tissue repair. Patients with coronary artery disease (CAD) or peripheral vascular disease (PVD) exhibit endothelial dysfunction, and as a result are likely to have a reduced number of progenitor cells mobilized in their peripheral circulation following ischemic injury. Identification of eNOS independent pathways for BMDPC mobilization may have important therapeutic value in this patient population. To identify such mechanisms we investigated the effect of granulocyte-colony stimulating factor (GCSF) and stem cell factor (SCF) in eNOS-KO mice with and without surgical hind-limb ischemia. Our results suggest that BMDPC mobilization can be achieved via activation of NO-independent pathways.

Clot stability as a determinant of effective factor VIII replacement in hemophilia A.

BACKGROUND: Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. OBJECTIVE: To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties. PATIENTS/METHODS: In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. RESULTS: FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. CONCLUSIONS: Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.

Identification and function probing of an antithrombin IIIß conformation-specific antibody.

UNLABELLED: ESSENTIALS: Antithrombin III (AT)ß binds heparin with higher affinity than ATα. A conformation-specific antibody against ATß, TPP2009, was made to investigate ATß in hemostasis. TPP2009 bound specifically to heparin-ATß and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATß in hemostasis. BACKGROUND: Antithrombin III (AT)ß is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. OBJECTIVES: To characterize a conformation-specific antibody against ATß and begin to investigate the role of ATß in maintaining hemostasis. METHODS: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATß (ATß*H) by the use of phage display. RESULTS: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATß*H and glycosaminoglycan-associated ATß on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATß and heparin. In purified systems with ATß and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. CONCLUSIONS: TPP2009 specifically targets a unique conformational epitope on ATß*H and blocks ATß-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATß in hemostasis.

Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse.

Most previous studies of atherosclerosis in hyperlipidemic mouse models have focused their investigations on lesions within the aorta or aortic sinus in young animals. None of these studies has demonstrated clinically significant advanced lesions. We previously mapped the distribution of lesions throughout the arterial tree of apolipoprotein E knockout (apoE(-/-)) mice between the ages of 24 and 60 weeks. We found that the innominate artery, a small vessel connecting the aortic arch to the right subclavian and right carotid artery, exhibits a highly consistent rate of lesion progression and develops a narrowed vessel characterized by atrophic media and perivascular inflammation. The present study reports the characteristics of advanced lesions in the innominate artery of apoE(-/-) mice aged 42 to 60 weeks. In animals aged 42 to 54 weeks, there is a very high frequency of intraplaque hemorrhage and a fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap. By 60 weeks of age, the lesions are characterized by the presence of collagen-rich fibrofatty nodules often flanked by lateral xanthomas. The processes underlying these changes in the innominate artery of older apoE(-/-) mice could well be a model for the critical processes leading to the breakdown and healing of the human atherosclerotic plaque.

Gender difference in endothelial dysfunction in the aorta of spontaneously hypertensive rats.

We investigated endothelium-dependent responses of thoracic aorta isolated from age-matched male and female spontaneously hypertensive rats (SHR) to explore gender differences in endothelial dysfunction that may contribute to the sexual dimorphism observed in the development of hypertension in this strain. Endothelium-dependent relaxation in response to acetylcholine (10(-9) to 10(-4) mol/L) was significantly greater in female rats than in male rats, although impaired responses were seen in both sexes compared with normotensive controls. Inhibition of cyclooxygenase by indomethacin (10(-5) mol/L) improved endothelium-dependent relaxation, but it did not abolish the gender difference. Relaxations in response to sodium nitroprusside were identical in denuded aortic rings from male and female SHR. Acetylcholine at higher concentrations (10(-6) to 10(-4) mol/L) induced endothelium-dependent contraction in intact, quiescent aortic rings from male SHR but not in those from female SHR. After incubation with NG-nitro-L-arginine (10(-4) mol/L), contraction in response to acetylcholine became apparent in rings from female SHR, but it was still significantly less pronounced than in similarly treated rings from male SHR. Endothelium-dependent contraction was prevented by indomethacin in both sexes, suggesting that a cyclooxygenase product such as endoperoxide may be mediating this effect. Because responses evoked by the thromboxane/endoperoxide receptor agonist U46619 (10(-10) to 10(-6) mol/L) were not greater in rings from male SHR than those from female SHR, increased smooth muscle responsiveness or higher thromboxane/endoperoxide receptor density in the males could not account for the differences in endothelium-dependent contraction. These results suggest that sex steroid hormones may control endothelium-dependent vascular reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bibrotoxin, a novel member of the endothelin/sarafotoxin peptide family, from the venom of the burrowing asp Atractaspis bibroni.

A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing asp Atractaspis bibroni by reversed-phase FPLC. The amino acid sequence of BTX differs from SRTX-b in the substitution Ala4 instead of Lys4, which suggests that it represents the peptide isoform of Atractaspis bibroni corresponding to SRTX-b. BTX competed for [125I]ET-1 binding to human ETB-type receptor with a Ki of 3.2 x 10(-9) M compared to 4.2 x 10(-9) M for SRTX-b. In rat thorax aorta BTX induced vasoconstrictions with a threshold concentration of 3 x 10(-8) M compared to 1 x 10(-9) for ET-1.

Accelerated atherosclerosis and premature calcified cartilaginous metaplasia in the aorta of diabetic male Apo E knockout mice can be prevented by chronic treatment with 17 beta-estradiol.

Epidemiological data indicate that estrogens significantly reduce the risk of morbidity and mortality due to cardiovascular diseases in postmenopausal women. Although numerous animal studies demonstrated inhibition of early atheromatous lesion formation by estrogen treatment in several species, information about the potential benefits of estrogens on complex, advanced atherosclerotic lesions is still lacking. The present study was designed to test whether chronic treatment with 17 beta-estradiol affects hyperglycemia-induced premature advanced lesion formation in 40-week-old male apolipoprotein E-deficient (Apo E-KO) mice. In order to accelerate advanced lesion formation, we treated male Apo E-KO mice with streptozotocin (STZ) at the age of 6 weeks. Two weeks later the STZ-treated mice received a slow release pellet containing either 17 beta-estradiol or placebo. STZ treatment caused sustained hyperglycemia without changes in serum total cholesterol or triglyceride levels compared to citrate control mice. STZ-treated Apo E-KO mice developed significantly more lesions in some (but not all) parts of the aorta and its main branches, and caused premature calcified cartilaginous metaplasia in the lesions of the proximal aorta. Chronic treatment with 17 beta-estradiol lead to a significant decrease in blood glucose and triglyceride levels, reduced the lesion area in all vascular segments studied and prevented cartilaginous metaplasia in STZ-treated Apo E-KO mice. The results of this study show that STZ treatment leads to significant acceleration of atherosclerotic lesion formation and premature occurrence of calcified cartilaginous areas in Apo E-KO mice, which could be effectively prevented by chronic estrogen treatment.

Effect of 17beta-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta.

1. Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17beta-oestradiol on cytokine-induced nitric oxide production by the inducible nitric oxide synthase (iNOS). 2. Treatment of the isolated aortic rings with interleukin-1beta (IL-1beta, 20 micro ml(-1)) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM-10 microM)-evoked contraction. 3. Cycloheximide (1 microM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL-1beta on the isolated aortic rings. 17Beta-oestradiol (1 nM-10 microM) and the partial oestrogen receptor agonist 4-OH-tamoxifen (1 nM-10 microM) produced concentration-dependent inhibition of IL-1beta-induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 microM). 4. Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL-1beta-induced and 17beta-oestradiol-treated rings. Western blot analysis of rat aorta homogenates revealed that 17beta-oestradiol treatment resulted in a reduction in IL-1beta-induced iNOS protein level. 5. Incubation with tumour necrosis factor alpha (TNF alpha, 1 ng ml(-1)) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL-1beta. The effects of TNF alpha were also inhibited by co-incubation of the rings with 17beta-oestradiol and 4-OH-tamoxifen (1 microM). 6. The anti-transforming growth factor-beta1 (TGF-beta1) antibody, which inhibited TGF-beta1-induced suppression of nitrite production from IL-1beta-treated vascular rings, did not affect the inhibitory action of 17beta-oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF-beta1. 7. These results show that the ovarian sex steroid, 17beta-oestradiol is a modulator of cytokine-induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

Mechanisms of pressure-induced myogenic activation of cerebral and renal arteries: role of the endothelium.

Control of blood flow is mediated by a variety of metabolic, neurogenic, myogenic and other mechanisms. The basic response of the myogenic mechanisms is a mechanical stimulus that activates vascular muscle via a sensor within the vessel wall. We studied the way in which increases in transmural pressure within the physiological range can activate cerebral and renal vascular muscle by depolarizing the muscle cell membrane. These membrane electrical effects depend on the external calcium concentration [( Ca2+]o) and on the presence of an intact vascular endothelium. Bioassay experiments have shown that an elevation in pressure releases a vasoactive product(s) from endothelial cells, which mediates the pressure-dependent membrane depolarization in these vessels. These responses may affect the autoregulation of blood flow in that vessels become smaller as pressures increase between 60 and 160 mmHg.

Effective treatment of vascular endothelial growth factor refractory hindlimb ischemia by a mutant endothelial nitric oxide synthase gene.

Gene delivery of angiogenic growth factors is a promising approach for the treatment of ischemic cardiovascular diseases. However, success of this new therapeutic principle is hindered by the lack of critical understanding as to how disease pathology affects the efficiency of gene delivery and/or the downstream signaling pathways of angiogenesis. Critical limb ischemia occurs in patients with advanced atherosclerosis often exhibiting deficiency in endothelial nitric oxide production. Similar to these patients, segmental femoral artery resection progresses into severe ischemic necrosis in mice deficient in endothelial nitric oxide synthase (ecNOS-KO) as well as in balb/c mice. We used these models to evaluate the influence of severe ischemia on transfection efficiency and duration of transgene expression in the skeletal muscle following plasmid injection in combination with electroporation. Subsequently, we also explored the potential therapeutic effect of the phosphomimetic mutant of ecNOS gene (NOS1177D) using optimized delivery parameters, and found significant benefit both in ecNOS-KO and balb/c mice. Our results indicate that NOS1177D gene delivery to the ischemic skeletal muscle can be efficient to reverse critical limb ischemia in pathological settings, which are refractory to treatments with a single growth factor, such as vascular endothelial growth factor.

Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae.

Gender differences in the production/release of endothelium-derived nitric oxide (EDNO) was assessed by determining the ability of intact endothelium to suppress serotonin- (10(-7)-10(-5) M) and phenylephrine-induced (10(-9)-(10(-5) M) contractions in thoracic aortae isolated from male and female Wistar rats mounted in organ chambers for isometric tension recording or tested in bioassay experiments. The endothelium suppressed these contractions significantly more in aortae from female than from male rats. In the bioassay, the perfusate from intact female thoracic aortic segments produced a significantly greater relaxation of the detector rings than that from the aortae isolated from male rats. Acetylcholine (10(-9)-10(-5) M), used to investigate agonist-induced release of EDNO, evoked significantly greater endothelium-dependent relaxation in aortae from female rats. The unstimulated release of 6-ketoprostaglandin F1 alpha and thromboxane B2 from intact thoracic aortic rings from male and female rats was not significantly different. There was no difference in smooth muscle reactivity to sodium nitroprusside (10(-10)-10(-6) M) in rings without endothelium. These results indicate that EDNO production/release is higher in thoracic aortae isolated from female rats.

Bradykinin-induced, N omega-nitro-L-arginine-insensitive endothelium-dependent relaxation of porcine coronary arteries is not mediated by bioassayable relaxing substances.

The effect of the arginine analogue, N omega-nitro-L-arginine (L-NNA) was studied on bradykinin-induced relaxation in porcine coronary arteries. In the presence of indomethacin (3 x 10(-6) M) and captopril (10(-6) M), treatment with L-NNA (10(-4) M) had no effect on the bradykinin-induced (10(-10)-10(-7) M) relaxations in strips contracted with U-46619. In contrast to the findings in organ chamber experiments, bradykinin-induced release of endothelium-derived relaxing factor(s) (EDRFs) was abolished after 45 min of treatment of perfused porcine coronary artery segments with L-NNA (10(-4) M) in a superfusion bioassay system. These results show that, in addition to the release of nitric oxide, endothelium-dependent relaxation of porcine coronary arteries to bradykinin involves an alternative mechanism(s), which accounts for the relaxation in the presence of L-NNA. Since the release of a relaxing mediator could not be detected from L-NNA-treated porcine coronary artery segments under bioassay conditions, it is postulated that either no diffusible factor(s) is involved in the L-NNA-insensitive endothelium-dependent relaxation, or it is mediated by an extremely labile endothelium-derived substance(s).

Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen.

Experimental and clinical studies have provided ample evidence that estrogens exert a significant antiatherosclerotic effect and reduce morbidity and mortality due to cardiovascular diseases. The exact cellular mechanism of this vasculoprotective action of estrogen is not known, but recent work in our and other laboratories suggests that upregulation of endothelial nitric oxide (NO) production may significantly contribute to the mechanism. The vascular endothelium of female animals and humans produces more NO than that of males. Estrogen treatment significantly increases endothelial NO generation in ovariectomized animals and in postmenopausal women. Reduced endothelial NO production in the aorta of estrogen-receptor-deficient mice indicates that the nuclear estrogen receptor mediates the effect of estrogen. The most probable mechanism of estrogen-induced upregulation of endothelial NO production is the transcriptional stimulation of NOS III gene expression. However, the following alternative mechanisms may be involved as well: (1) inhibition of cytokine-induced downregulation of NOS III gene expression, (2) posttranslational modification of NOS III protein, (3) increased cofactor or L-arginine availability, (4) nongenomic activation of second messengers (e.g., Ca2+, cAMP) and tyrosine kinase, and (5) modulation of NO degrading systems (e.g., reactive oxygen radical generation and antioxidants). This paper reviews current data supporting these potential mechanisms.

Effect of cilazapril and indomethacin on endothelial dysfunction in the aortas of spontaneously hypertensive rats.

Chronic hypertension is associated with impaired endothelial function [i.e., reduced synthesis/release of endothelium-derived relaxing factor (EDRF) and increased synthesis/release of endothelium-derived contracting factor (EDCF)] in both animals and humans. Although it is not known whether endothelial dysfunction is the consequence and/or an important pathogenetic cause of hypertension, the goal of effective antihypertensive therapy should include restoration of normal endothelial function as well. Because angiotensin I-converting enzyme (ACE) inhibitors are effectively used in the treatment of hypertension, the aim of the present study was to test whether in vivo treatment of spontaneously hypertensive rats (SHRs) with the ACE inhibitor cilazapril improves endothelial function in the isolated thoracic aorta of SHRs. Treatment of SHRs with cilazapril (10 mg/kg/day orally for 2 weeks) resulted in a significant decrease in blood pressure and normalization of endothelium-dependent relaxation evoked by acetylcholine (ACh) and adenosine diphosphate (ADP). However, cilazapril treatment had no significant effect on endothelium-dependent contractions evoked by 5-hydroxytryptamine (5-HT; serotonin) and prostaglandin F2 alpha (PGF2 alpha). In contrast, in vitro treatment of isolated thoracic aortas with indomethacin (10(-5) M) normalized endothelium-dependent relaxations to ACh and ADP as well as inhibited endothelium-dependent contractions to 5-HT and PGF2 alpha. These results suggest that the ACE inhibitor cilazapril increases the synthesis/release of EDRFs whereas indomethacin prevents the synthesis/release of cyclo-oxygenase-derived EDCFs in the endothelium of rat aorta. The exact mechanism of action of ACE inhibitors on endothelial dysfunction remains to be determined.

17 beta-Estradiol attenuates endotoxin-induced excessive nitric oxide production in ovariectomized rats in vivo.

Experiments were designed to examine the effect of 17 beta-estradiol on lipopolysaccharide (LPS)-induced excessive nitric oxide production in vivo. Ovariectomized and sham-operated female rats were injected with LPS (5 mg/kg ip), and different groups of ovariectomized, LPS-injected animals were treated with either 17 beta-estradiol, dexamethasone, or aminoguanidine. Nitric oxide generation was estimated by measuring plasma nitrite levels 12 h after LPS injection. LPS treatment of the rats resulted in a four- to fivefold increase in circulating plasma nitrite level, significantly higher in the ovariectomized animals compared with the sham-operated animals. The LPS-induced plasma nitrite increase could be prevented by dexamethasone (3 mg/kg ip) injected 1 h before LPS treatment. 17 beta-Estradiol (3 micrograms/rat sc), administered 48 h before LPS treatment, significantly attenuated the LPS-induced elevation in plasma nitrite levels. This effect was comparable to that achieved by aminoguanidine (200 microM/kg), an inhibitor of inducible nitric oxide synthase, injected 1 h after LPS treatment. The present findings suggest that estrogens may be beneficial in pathological conditions associated with excessive nitric oxide generation.

Effect of 17beta-estradiol in hypercholesterolemic rabbits with severe endothelial dysfunction.

17beta-Estradiol prevents early vascular lesion development and may also affect advanced atherosclerosis. To test the antiatherosclerotic effect of estrogen under conditions that resemble more advanced human atherosclerosis with severe endothelial dysfunction, we have investigated the effect of 17beta-estradiol in hypercholesterolemic rabbits treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). Chronic L-NAME administration attenuated endothelial nitric oxide (EDNO)-mediated vascular responses leading to significantly accelerated atherosclerotic plaque development. 17beta-Estradiol treatment alone inhibited aortic lesion formation with concurrent increase in EDNO-mediated responses. The beneficial effect of estrogen persisted in the L-NAME-treated rabbits, suggesting that the antiatherogenic action of 17beta-estradiol involves NO-independent mechanisms as well. Serum cholesterol levels were not altered by any of the treatments. 17beta-Estradiol treatment significantly increased EDNO production under these conditions as well. The reduction in plaque size by 17beta-estradiol was always accompanied by increased EDNO production, suggesting a strong association between these two events. The results demonstrate that estrogen treatment may exert protection against atherosclerosis even in patients with severe endothelial dysfunction.

Endothelium-dependent modulation of endothelin-induced vasoconstriction and membrane depolarization in cat cerebral arteries.

Effects of endothelin, the newly discovered, endothelium-derived vasoactive peptide, were examined on isolated, pressurized cat arteries. Responses to increasing concentrations of porcine endothelin (10(-12) to 10(-8) M) were compared in endothelium intact and denuded vessel segments utilizing diameter and membrane potential measurements. Endothelin produced a concentration-dependent contraction in arteries with intact endothelium with a maximal response of 26 +/- 2.8% of the initial diameter in physiological saline solution at 100 mm Hg of intraluminal pressure. Removal of the endothelium sensitized the smooth muscle to endothelin and increased the maximal response to 48 +/- 2.3% of the initial diameter. After incubation of the intact vessels with 10(-5) M indomethacin, the endothelin response was potentiated to the maximal value of 66 +/- 1.26% of the initial diameter of the intact vessels, and to 61 +/- 2.6% of the denuded arteries. The arterial smooth muscle cells were depolarized by 6 +/- 0.45 mV after administration of endothelin to the vessels, and by 10 +/- 0.8 mV in the denuded vessels. This depolarization was not altered by indomethacin. Our data suggest that endothelin has a direct effect on the smooth muscle of cat cerebral vascular tissue, and the sensitivity of these vessels to endothelin may be modulated by the release of dilatory cyclooxygenase products derived from the intact endothelium.

Pressure releases a transferable endothelial contractile factor in cat cerebral arteries.

When exposed to an increasing transmural pressure, middle cerebral arteries of the cat exhibit reduction of internal diameter which is mediated by vascular muscle cell depolarization. This laboratory has recently demonstrated that this "pressure-induced" activation is dependent upon the presence of an intact endothelium. The present studies were undertaken to determine if this phenomenon is due to inhibition of tonically released endothelium-derived relaxing factors (EDRF) or release of a contractile substance. When cerebral arterial segments were pressurized to between 40 and 160 mm Hg there was 13.2% reduction in internal diameter accompanied by significant muscle cell depolarization from -53 +/- 2.7 to -22 +/- 1.4 mV. There was a significant positive correlation between the delta Em and step increases in transmural pressure. These excitatory responses were lost and vessels dilated to pressure when the endothelium was removed. Upon exposing the denuded vessel to a pressurized intact donor, the denuded vessel recovered its ability to contract and depolarize suggesting that a contractile substance might be released from the vascular endothelium upon pressurization. The EDRF antagonist oxyhemoglobin did not alter the excitatory response to pressure in these isolated cerebral arteries further suggesting that the reduction in diameter and muscle cell depolarization results from the release of a contractile substance from the vascular endothelium and not inhibition of EDRF.