Eberhard Bassenge

Flow-dependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibition.

Endothelial damage or removal abolishes the dilation of epicardial coronary arteries induced by increments in flow through these arteries in vitro. Therefore, we tested whether or not the release of a cyclooxygenase product from endothelial cells in vivo is the mechanism of this flow-dependent dilation. In eight conscious dogs, instrumented to register the external diameter of two epicardial branches--anterior descending and circumflex--of the left coronary artery, increments in coronary flow increased and reductions in coronary flow decreased the diameter of the left circumflex epicardial artery by 182 +/- 11 micron/100% change in flow. When mean coronary flow in one epicardial branch was kept constant by a distal, flow-limiting stenosis during the application of flow-augmenting stimuli (temporal coronary occlusion or 80-400 micrograms/kg adenosine i.v.), no dilation of this artery was observed. Cyclooxygenase inhibition (suppressing the bradykinin-induced elevation of plasma 6-keto-PGF1 alpha) by indomethacin (5 mg/kg) or by diclofenac (10 mg/kg) increased smooth muscle tone in both epicardial arteries, but did not modify the flow-diameter relation (181 +/- 10 and 179 +/- 9 microns/100% change in flow, respectively). It is concluded that a tonic, instantaneous influence of coronary flow on the smooth muscle tone of the epicardial coronary arteries exists in vivo. It is unlikely that prostacyclin or another prostanoid is a mediator of this endothelium-mediated influence of flow on smooth muscle tone.

Postprandial hypertriglyceridemia impairs endothelial function by enhanced oxidant stress

AIMS it appears that hypertriglyceridemia (HTG) is a risk factor of atherosclerosis as demonstrated by recent studies. In this study, we analyzed the effects of acute HTG on endothelial function and oxidative stress, which are important mechanisms in the pathogenesis of atherosclerosis. METHODS AND RESULTS in a high fat meal group (n = 11), serum triglycerides and PMA-activated leukocyte O(2)(-)* production were significantly (P < 0.005) increased from 146 +/- 69 mg/dl and 4.09 +/- 0.93 nmol/10(6) cells/min preprandially to 198 +/- 88 mg/dl and 5.49 +/- 1.19 nmol/10(6) cells/min, respectively, 2 h after eating a high-fat meal. The flow-mediated endothelium-dependent brachial artery dilation (FMD; high-resolution ultrasound) was decreased from 13.7 +/- 3.3% preprandially to 8.2 +/- 3.7%, 2 h after eating a high-fat meal (P < 0.005). However, following a low-fat meal (n = 9), there were no significant changes in triglycerides, leukocyte O(2)(-)* production and FMD. Changes of serum triglycerides were correlated negatively (r = -0.650, P < 0.005) with changes of FMD, but were correlated positively (r = 0.798, P < 0.001) with changes of leukocyte O(2)(-)* production, which - in turn - were correlated negatively (r = -0.784, P < 0.001) with changes of FMD in all study subjects (mean age: 56 years, n = 20). CONCLUSIONS this study suggests that acute HTG causes endothelial dysfunction via enhanced oxidant stress and this may pave the way for the development of atherosclerosis under chronic conditions.

Endothelial modulation of coronary tone.

T HE VASCULAR endothelium lining as a monolayer the entire circulatory system does not represent a passive diffusion barrier but displays the features of a distributed organ (1 to 1.5 kg) with a variety of biological functions. It serves as an antithrombogenic surface, and it is significantly involved in a well-regulated balance between coagulation and fibrinolysis. To this end its surface contains heparan sulfate and it is able to secrete antithrombin III, prostacyclin, and plasminogen activator. In combination with the negative surface charges of the endothelial glycocalyx, these compounds maintain the fluidity of the blood at the interface between the streaming blood and the vessel wall and prevent red cells from adhering. A number of compounds are synthesized by the endothelium, which may be involved in localized thrombus formation (Table 1). Other important functions comprise the metabolism of norepinephrine (NE), serotonin (5-HT), and adenine nucleotides and the conversion of angiotensin I and bradykinin (Bk) by angiotensin-converting enzyme and other peptidases (Table 2). (See Endothelial Metabolism and its Influence on Vascular Tone.) In spite of the fact that endothelial cells display such a variety of synthetic, secretory, and cleavage functions,’ it was not recognized until 1980 when Furchgott and Zawadzki* made pioneering observations as to how the endothelium exerts a significant role in the modulation of local vascular tone by the release of an endotheliumderived vascular relaxant factor (EDRF) (for recent reviews see references 3 through 7; see also Addendum). In this review we try to summarize recent findings regarding the regulatory function of this endothelium-derived relaxant factor under physiologic and pathophysiologic conditions. In addition, some biochemical properties of endothelial cells that may influence vascular homeostasis and vascular tone are briefly outlined. This role of the endothelium may be especially important in pathophysiologic conditions, when endothelial function is impaired (eg, under arteriosclerotic conditions and during hypercholesterolemia) and its local dilator potential is either reduced or absent. Under such conditions the net balance between dilatory signals from the endothelium, acting indirectly, and direct constrictor impulses affecting the vasculature is disturbed: failure of vasodilation may result in enhanced or excessive local vasoconstriction and spasm, and thus partial or complete ischemia. The decisive role of such an endotheliumdependent vasomotor balance becomes obvious when one considers that the majority of vasoactive agonists (such as [5-HT], histamine, adenosine diphosphate [ADP], adenosine triphosphate [ATP], acetylcholine (ACh), thrombin [Th], vasopressin) display an endothelium-dependent vasodilator activity in addition to a simultaneous direct constrictor effect on vascular smooth muscle. In this context it is surprising that various a-agonists or substances stimulating a-receptors (eg, NE and various ergot alkaloids like ergonovine, which are used to provoke spasm in Prinzmetal patients) exhibit a simultaneous endothelium-dependent dilator component. Consequently, the resultant action on the coronary arterial wall depends on the balance of these two opposing actions (Table 3). EDRF-mediated modulation of vascular tone has been primarily demonstrated in large conductance arteries like the coronaries’ (for review see reference 6), wherein vasomotion does not determine the rate of tissue perfusion unless critical stenoses are present. The role of EDRFmediated vasomotion in resistance vessels, which are responsible for control of tissue perfusion, has not yet been well established, because of difficulties in functionally inhibiting or destroying the endothelium in a defined manner without causing simultaneous damage to the adjacent tissue. The problems caused by increasing overlap with concurrent metabolically induced vasomotion in the microcirculation renders such a quantitative

Effects of native and oxidized low density lipoproteins on formation and inactivation of endothelium-derived relaxing factor

The influence of native (N-) and oxidized (Ox-) low density lipoproteins (LDLs) on endothelium-dependent vasomotion is still controversial. We investigated the short-term effects of N-LDL and Ox-LDL on the formation of endothelium-derived relaxing factor (EDRF) in native and cultured endothelial cells and on its inactivation after release from the cells. N-LDL was isolated from fresh human plasma via sequential ultracentrifugation and oxidized by incubation with Cu2+. EDRF released from cultured endothelial cells was inactivated by both N-LDL and Ox-LDL (1 mg/ml) as detected in a bioassay system. N-LDL reduced the EDRF-mediated vasodilations of the detector segments by 38.5 +/- 5.3%, and Ox-LDL, by 55.5 +/- 4.6%. The effects of lipoproteins on EDRF formation were studied in cultured endothelial cells preincubated with either N-LDL or Ox-LDL (1 mg/ml for 1 hour) and stimulated for EDRF release with bradykinin after washout of the lipoproteins. EDRF was assessed by measuring its stimulatory effect on the activity of a purified, soluble guanylate cyclase. Both N-LDL and Ox-LDL did not reduce the bradykinin-induced EDRF formation. Consistent with this finding, acetylcholine-induced, EDRF-mediated dilations of intact rabbit femoral artery segments were not impaired by luminal exposure to N-LDL or Ox-LDL (1 mg/ml for 1 hour). However, these relaxations were significantly reduced by preincubation of aortic ring preparations with the same concentrations of the same charges of N-LDL and Ox-LDL. In conclusion, neither N-LDL nor Ox-LDL acutely impairs the formation of EDRF but does inactivate EDRF after its release from endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of endothelium in the control of vascular tone

SummaryIn the last few years, experimental evidence has accumulated which suggests a substantial role for the endothelium in the control of vascular tone. Endothelium-dependent dilatations have been demonstrated in various arteries of numerous mammalian species including man. Among the stimuli which elicit endothelium-dependent dilatation are such varying stimuli as increases in blood flow and hypoxia, as well as endogenous (acetylcholine, ATP, ADP, bradykinin, substance P) and pharmacological agents (calcium ionophore A 23187, ergometrine, hydralazine, melittin). The functional importance of endothelium-dependent dilatation is emphasized by the fact that the direct vasoconstrictor effects of some of these substances (acetylcholine, histamine, norepinephrine, serotonin) on vascular smooth muscle is attenuated or even reversed by their simultaneuos stimulatory effect on endothelial cells, resulting in the release of a vasodilator signal. Bioassay experiments have shown that a humoral vasodilator agent with a biological half-life in the range of seconds is released from the endothelium (native or cultured) during stimulation with acetylcholine, ATP and calcium ionophore. Experimental data are presented, which suggest that EDRF may act by direct stimulation of guanylate cyclase, resulting in smooth muscle relaxation due to increased smooth muscle cyclic GMP levels. The chemical nature of this nonprostaglandin endothelium-derived relaxant factor (EDRF) is still not known.The possible physiological and pathophysiological significance of endothelium-dependent dilatation in situ is discussed. Special attention is paid in this context to the potential role of EDRF activity in coronary vasomotor control.

Endothelium-derived relaxant factor inhibits platelet activation

SummaryExperiments were designed to investigate whether platelet activation is modulated by endothelium-derived relaxant factor (EDRF) which has been shown to induce vascular smooth muscle relaxation by direct stimulation of soluble guanylate cyclase. EDRF was released from cultured bovine endothelial cells, grown on microcarrier beads, by stimulation with thimerosal in the presence of indomethacin. EDRF had no effect on the intracellular free calcium concentration (Cai2+, measured with the fluorescent indicator indo-1) of resting washed human platelets but significantly attenuated the thrombin-induced rise of Cai2+ from 896 ± 99 (SEM) to 509 ± 48 nmol/l. EDRF significantly increased platelet cyclic GMP levels from 0.25 ± 0.04 to 2.5 ± 0.4 pmol/108 platelets and reduced the thrombin-induced aggregation to 23 ± 3% of control. EDRF had no effect on Caiz+, cyclic GMP or aggregation after a 3 min storage interval, but superoxide dismutase (shown to increase stability of the labile factor) significantly augmented the EDRF effects on Cai2+. The antiaggregatory potency of EDRF was completely abolished in the presence of hemoglobin. The results characterize EDRF as a potent cyclic GMP-dependent antiaggregatory factor which may act synergistically in vivo with the cyclic AMP-dependent inhibitory effect of prostacyclin.

Endothelial and neuro-humoral control of coronary blood flow in health and disease

1 Mechanical Determinants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 1.1 Perfusion Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 1.2 Extravascular Coronary Compression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 1.3 Gregg and Gardenhose Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Oxidized low density lipoproteins potentiate vasoconstrictions to various agonists by direct interaction with vascular smooth muscle.

In hypercholesterolemia, low density lipoproteins (LDLs) may be oxidized by monocytes/macrophages in the arterial wall. Therefore, we investigated the effect of LDL and its oxidative derivatives (ox-LDL) on vascular tone in isolated perfused rabbit femoral arteries. Perfusion of endothelium-intact and endothelium-denuded segments with ox-LDL (80 micrograms protein/ml) caused no or only weak vasoconstrictions in the absence of contractile agonists. However, in the presence of ox-LDL, vasoconstrictions to threshold concentrations of norepinephrine, serotonin, phenylephrine, or potassium were significantly enhanced. This enhancement correlated with the degree of oxidation. When ox-LDL was administered at higher concentrations (greater than 200 micrograms protein/ml), it evoked moderate vasoconstrictions even in the absence of contractile agonists. Native LDL had no effect on vascular tone. Preincubation with verapamil, diltiazem, and nitrendipine inhibited vasoconstrictions evoked by ox-LDL, both in the presence and in the absence of a contractile agonist. The contractile responses to ox-LDL were significantly greater in endothelium-denuded segments than in endothelium-intact segments. At the above concentrations, ox-LDL had no influence on endothelium-derived relaxing factor-mediated vasodilations. These data indicate that ox-LDL enhances agonist-induced vasoconstrictions by a direct effect on the vascular smooth muscle. We therefore suggest that ox-LDL is an important factor that may increase the risk of inappropriate vasoconstriction in hypercholesterolemia, independent of its putative cytotoxic effect on the endothelium.

Nitric oxide synthesis in endothelial cytosol: Evidence for a calcium-dependent and a calcium-independent mechanism

SummaryRelease of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 μM free calcium; EC50 0.3 μM). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 μM) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation NG-nitro-L-arginine (1 mM) and by LY 83583 (1 μM), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells.

Prostacyclin and nitric oxide contribute to the vasodilator action of acetylcholine and bradykinin in the intact rabbit coronary bed

Summary: The relative contribution of nitric oxide (NO) and cyclo-oxygenase products in the dilator response to equieffective doses of acetylcholine (ACh) and bradykinin (Bk) was studied in the isolated, saline-perfused rabbit heart under constant flow conditions. ACh (1μ.M) and Bk (10 nAf) induced a similar vasodilation, with a maximum reduction in coronary perfusion pressure (CPP) of 27 ± 2%. The vasodilation induced by both agonists was associated with an enhanced release of 6-keto-PGF1α from the coronary bed, with the Bk-induced increase in 6-keto-PGF1α being threefold greater than that induced by ACh. The angiotensin converting enzyme (ACE) inhibitor ramiprilat (0.3μ.M) selectively enhanced both the 6-keto-PGF1α outflow and the dilator response to Bk. The B2-receptor antagonist Hoe 140 (0.1μ.M) blocked both Bk effects. The cyclo-oxygenase inhibitor diclofenac (1 μM) halved the dilator response to Bk, but did not affect the vasodilation to ACh. Both agonists induced the release of NO, as assessed by the increase in cyclic GMP content of platelets passing through the vascular bed. However, ACh induced a 2.5-fold greater increase in platelet cyclic GMP content, compared to Bk. Treatment of hearts with NG-nitro-L-arginine (L-NNA, 30 u.Af) halved the ACh-and Bk-induced maximum reduction in CPP. Combined infusion of L-NNA and diclofenac completely blocked the dilator response to Bk, and inhibited the vasodilation to ACh more efficiently than L-NNA alone. We conclude that both NO and PGI2 contribute to the coronary dilator response to Bk and ACh in the rabbit Langendoriff heart. Bradykinin induces a balanced release of these autacoids, which both contribute to and account for the vasodilation observed with this peptide. The relatively more important release of NO with ACh overwhelms the contribution of PGI2. However, the latter plays a significant role in the presence of an impaired L-arginine-NO pathway.