William M. Chilian

Tumor Necrosis Factor-α Induces Endothelial Dysfunction in the Prediabetic Metabolic Syndrome

Inflammation is a condition that underscores many cardiovascular pathologies including endothelial dysfunction, but no link is yet established between the vascular pathology of the metabolic syndrome with a particular inflammatory cytokine. We hypothesized that impairments in coronary endothelial function in the obese condition the prediabetic metabolic syndrome is caused by TNF-&agr; overexpression. To test this, we measured endothelium-dependent (acetylcholine) and -independent vasodilation (sodium nitroprusside) of isolated, pressurized coronary small arteries from lean control and Zucker obese fatty (ZOF, a model of prediabetic metabolic syndrome) rats. In ZOF rats, dilation to ACh was blunted compared with lean rats, but sodium nitroprusside–induced dilation was comparable. Superoxide (&OV0151;) generation was elevated in vessels from ZOF rats compared with lean rats, and administration of the &OV0151; scavenger TEMPOL, NAD(P)H oxidase inhibitor (apocynin), or anti–TNF-&agr; restored endothelium-dependent dilation in the ZOF rats. Real-time PCR and Western blotting revealed that mRNA and protein of TNF-&agr; were higher in ZOF rats than that in lean rats, whereas eNOS protein levels were reduced in the ZOF versus lean rats. Immunostaining showed that TNF-&agr; in ZOF rat heart is localized in endothelial cells and vascular smooth muscle cells. Expression of NAD(P)H subunits p22 and p40-phox were elevated in ZOF compared with lean animals. Administration of TNF-&agr; more than 3 days also induced expression of these NAD(P)H subunits and abrogated endothelium-dependent dilation. In conclusion, the results demonstrate the endothelial dysfunction occurring in the metabolic syndrome is the result of effects of the inflammatory cytokine TNF-&agr; and subsequent production of &OV0151;.

Longitudinal Gradients for Endothelium-Dependent and -Independent Vascular Responses in the Coronary Microcirculation

BACKGROUND Coronary microvessels (< 300 microns in diameter) have been demonstrated to be important in the regulation of local resistance and flow. Recent studies also suggest that these microvessels are more responsive to physiological and pharmacological stimuli than conduit vessels. However, little is known regarding the relative sensitivity of different microvascular segments in response to flow (shear stress) and agonists. The goal of this study was to test the hypothesis that a longitudinal gradient for shear stress- and agonist-induced dilation exists in the coronary microcirculation. METHODS AND RESULTS Experiments were performed in four different sizes of porcine subepicardial coronary arterial microvessels: small arterioles (40 +/- 1-micron ID with resting tone); intermediate arterioles (60 +/- 1 micron); large arterioles (106 +/- 4 micron); and small arteries (179 +/- 9 microns). Vessels were isolated and cannulated to allow luminal pressure and flow to be independently controlled. All vessels developed active tone (to approximately 65% to 75% of maximum diameter) at their control luminal pressures and showed graded dilations to stepwise increases in shear stress (0 to 10 dynes/cm2). For arterioles, the magnitude of the dilations increased as vessel size increased. The highest shear stress produced 21 +/- 3%, 32 +/- 2%, and 52 +/- 5% increases in diameter in small, intermediate, and large arterioles, respectively. Small arteries dilated only 22 +/- 6%. The endothelium-dependent vasodilator substance P (SP) produced dose-dependent dilation of all vessels with a threshold at 10(-16) mol/L. Arterioles were maximally dilated at 10(-9) mol/L SP. However, this dose produced only 80% dilation in small arteries. The ED50 for SP was shifted to the right by two orders of magnitude in small arteries compared with the arterioles. Adenosine preferentially dilated small arterioles, and the dose-response curves shifted to the right for larger vessels. The thresholds for adenosine-induced dilation were 10(-12), 10(-11), and 10(-9) mol/L for small, intermediate, and large arterioles, respectively. The endothelium-independent vasodilator nitroprusside produced identical dose-dependent dilations in all vessel segments. CONCLUSIONS The results indicate that the pig coronary circulation exhibits a heterogeneity in physiological and pharmacological responses along the microvascular network. Small arterioles are more sensitive to adenosine, but large arterioles are more responsive to shear-stress stimulation. We speculate that site-specific preferential responses may play a crucial role in coordinating overall vascular function in the coronary microvascular network.

Ischemia-Induced Coronary Collateral Growth Is Dependent on Vascular Endothelial Growth Factor and Nitric Oxide

Background—We hypothesized that ischemia-induced expression of vascular endothelial growth factor (VEGF) and the production of NO stimulate coronary collateral growth. Methods and Results—To test this hypothesis, we measured coronary collateral blood flow and VEGF expression in myocardial interstitial fluid in a canine model of repetitive myocardial ischemia under control conditions and during antagonism of NO synthase. Collateralization was induced by multiple (1/h; 8/d), brief (2 minutes) occlusions of the left anterior descending coronary artery for 21 days. In controls, collateral blood flow (microspheres) progressively increased to 89±9 mL · min−1 · 100 g−1 on day 21, which was equivalent to perfusion in the normal zone. Reactive hyperemic responses (a measure of the severity of ischemia) decreased as collateral blood flow increased. In NG-nitro-l-arginine methyl ester (L-NAME)– and L-NAME+nifedipine–treated dogs, to block the production of NO and control hypertension, respectively, collateral blood flow did not increase and reactive hyperemia was robust throughout the occlusion protocol (P <0.01 versus control). VEGF expression (Western analyses of VEGF164 in myocardial interstitial fluid) in controls peaked at day 3 of the repetitive occlusions but waned thereafter. In sham-operated dogs (instrumentation but no occlusions), expression of VEGF was low during the entire protocol. In contrast, VEGF expression was elevated throughout the 21 days of repetitive occlusions after L-NAME. Reverse transcriptase–polymerase chain reaction analyses revealed that the predominant splice variant expressed was VEGF164. Conclusions—NO is an important regulator of coronary collateral growth, and the expression of VEGF is induced by ischemia. Furthermore, the induction of coronary collateralization by VEGF appears to require the production of NO.

Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation. Restoration of endothelium-dependent responses by L-arginine.

The goals of this study were 1) to quantitate the effects of atherosclerosis on physiological and pharmacological endothelium-dependent vasoactive responses in coronary arterioles downstream from arterial lesions and 2) to determine if administration of L-arginine, the precursor for endothelium-derived was induced in pigs, and vasomotor responses of isolated, cannulated coronary arterioles (30-70 microns in diameter) were assessed by measuring diameter changes in vitro. To assess pharmacological alterations of endothelium-dependent responses, dose-response curves were constructed to ADP, serotonin, and histamine. To assess physiological alterations in endothelial function, different flow rates were established across the vessel. Arteriolar diameters were measured in vessels from normal and atherosclerotic pigs under control conditions, after administration of L-arginine, and after endothelial denudation. In arterioles from normal pigs, administration of serotonin, histamine, or ADP produced dose-dependent vasodilation, which was abolished by endothelial denudation. In arterioles from atherosclerotic pigs, administration of histamine, serotonin, and ADP produced dilation at only the highest doses (10(-6)-10(-7) M), and the extent of dilation was only 20-30% of that observed in arterioles from normal pigs. Initiation of flow also produced vasodilation in arterioles from normal pigs that was completely abolished after endothelial denudation. In arterioles from atherosclerotic pigs, flow-induced responses were absent. These abnormal physiological and pharmacological responses (i.e., blunted vasodilation to pharmacological stimulation and to flow) were restored after administration of L-arginine for 40 minutes. The vascular responses after administration of L-arginine were not different from those observed under control conditions in arterioles from normal pigs. In addition, L-arginine did not restore vasodilation to the endothelium-dependent agonists in denuded segments. From these data in arterioles downstream from atherosclerotic lesions, we conclude that 1) the ED50 and maximal responses of endothelium-dependent vasodilation to ADP, histamine, and serotonin are attenuated; 2) the physiological response to flow, that is, flow-mediated endothelium-dependent vasodilation, is absent; and 3) the abnormality in arteriolar responsiveness during large vessel disease involves an impairment of the synthesis and/or release of endothelium-derived relaxing factor.

α-Adrenergic Coronary Vasoconstriction and Myocardial Ischemia in Humans

The use of quantitative coronary angiography, combined with Doppler and PET, has recently been directed at the study of alpha-adrenergic coronary vasomotion in humans. Confirming prior animal experiments, there is no evidence of alpha-adrenergic coronary constrictor tone at rest. Again confirming prior experiments, responses to alpha-adrenoceptor activation are augmented in the presence of coronary endothelial dysfunction and atherosclerosis, involving both alpha(1)- and alpha(2)-adrenoceptors in epicardial conduit arteries and microvessels. Such augmented alpha-adrenergic coronary constriction is observed during exercise and coronary interventions, and it is powerful enough to induce myocardial ischemia and limit myocardial function. Recent studies indicate a genetic determination of alpha(2)-adrenergic coronary constriction.

Coronary arteriolar myogenic response is independent of endothelium.

The purpose of this study was to investigate if myogenic responses of isolated coronary arterioles were dependent on an intact, functional endothelium. Arterioles were located in situ by intracoronary perfusion with india ink-gelatin solution and then dissected and cannulated at both ends with glass micropipettes. Intraluminal pressure was initially set at 60 cm H2O; then the pressure was altered in steps of 20 cm H2O over a range of 20-140 cm H2O. Arterioles developed spontaneous tone and exhibited a significant myogenic response in physiological saline solution (36 degrees -37 degrees C). Arteriolar dilation and constriction were observed at lower (20-60 cm H2O) and higher (60-140 cm H2O) pressures, respectively. The presence of a functional and automatically intact endothelium was confirmed by relaxation to the endothelium-dependent vasodilator bradykinin and by transmission electron microscopy, respectively. After mechanical denudation of the endothelium with a specially designed abrasive micropipette, spontaneous tone and myogenic responses were preserved. Denudation of the endothelium was verified functionally (no response to bradykinin) and with transmission electron microscopy. Moreover, the mechanical denudation technique did not deleteriously affect smooth muscle because vasoconstrictor and vasodilator responses to nonendothelial-dependent drugs were the same before and after denudation. In summary, the present study demonstrates that pressure-dependent responses occur in isolated coronary arterioles and that this response is not dependent on the endothelium. Therefore, pressure-induced changes in coronary arteriolar tone are a true myogenic response in that they originate from smooth muscle.

Coronary Microcirculation in Health and Disease Summary of an NHLBI Workshop

This article summarizes a 2-day workshop on the coronary microcirculation held in Bethesda, Md, in September 1994 and sponsored by the National Heart, Lung, and Blood Institute of the National Institutes of Health. The workshop explored a variety of topics pertaining to coronary microvascular physiology and pathophysiology. The latest methodologies that are being used to investigate the coronary microvasculature, including endoscopic microscopy of the intramural coronary microvasculature and micro-x-ray computerized tomography, were discussed. The most recent advances in the regulation of the coronary microcirculation-for example, myogenic and flow-dependent responses, KATP channels, and regional heterogeneity-were reported. The workshop touched on the relation of the microcirculation to clinically important conditions and offered recommendations for future research in this important area. Comparisons are made to recent advances in the peripheral circulation and current gaps in our knowledge concerning the coronary microcirculation. In recent years, research on the coronary microcirculation has made substantial advances, in part as a result of investigations in the peripheral microcirculation but also because of the application of unique methodologies. This research is providing new ways to investigate abnormalities of myocardial perfusion, an area of inquiry that until recently has been limited to examination of coronary pressure-flow relationships.

Role of Nitric Oxide in the Coronary Microvascular Responses to Adenosine and Increased Metabolic Demand

BACKGROUND The purpose of this study was to test the hypothesis that endothelium-derived nitric oxide (NO) participates in coronary microvascular responses to adenosine and pacing-induced increases in metabolic demand by maintaining an optimal distribution of coronary resistance. METHODS AND RESULTS Coronary microvascular diameters were measured by stroboscopic epi-illumination and intravital microscopy in open-chest dogs (n = 20). Epicardial coronary blood velocity (CBV) was measured by Doppler flowmetry. Responses to adenosine (1 and 10 micrograms.kg-1.min-1 IC) and left atrial pacing (180 beats per minute) were recorded before and after inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME, 30 micrograms.kg-1.min-1 IC). At baseline, adenosine dilated arterioles (< 100 microns) (11 +/- 4% and 25 +/- 3% diameter changes, P < .05) more than small arteries (> 100 microns) (-4 +/- 6% and 7 +/- 3%, P < .05 for the higher dose) and increased CBV (43 +/- 31% and 118 +/- 25%, P < .05). Left atrial pacing dilated arterioles (12 +/- 2%, P < .05) and small arteries (8 +/- 3%, P < .05) and also increased CBV (68 +/- 9%, P < .05). L-NAME abolished CBV increases caused by acetylcholine (10 and 100 ng.kg-1.min-1 IC; 53 +/- 33% and 168 +/- 82% versus -12 +/- 15% and -1 +/- 14%, P < .05) but not papaverine. Small arteries were constricted by L-NAME (-8 +/- 2%, P < .05), arterioles were dilated (10 +/- 4%, P < .05), and CBV was unchanged. After L-NAME, adenosine failed to dilate arterioles further (3 +/- 3% and 2 +/- 2%; P < .05 versus prior responses), and CBV changes were attenuated (14 +/- 16% and 8 +/- 13%; P < .05 versus prior responses). Pacing also failed to dilate arterioles (-4 +/- 2%, P < .05 versus prior response), resulting in an attenuated CBV change (34 +/- 13%, P < .05 versus prior response). The possibility that adenosine stimulates NO release in canine coronary arterioles was investigated in isolated arterioles (diameters, 81 +/- 4 microns; n = 8). Adenosine caused dose-dependent dilation to maximal diameter, which was unaffected by inhibition of NO synthesis by L-NAME. CONCLUSIONS Inhibition of NO synthesis attenuates coronary dilation during adenosine infusions and during pacing-induced increases in metabolic demand. Inhibition of NO synthesis may shift the major site of coronary resistance into small arteries through autoregulatory adjustments in arterioles. These data therefore suggest that NO, by dilating predominantly small coronary arteries, promotes metabolic coronary dilation by preserving the tone and vasodilator reserve of arterioles.

Endothelium-dependent relaxation competes with alpha 1- and alpha 2-adrenergic constriction in the canine epicardial coronary microcirculation.

Background. The purpose of this study was to determine whether endothelium‐dependent relaxation competes with &agr;1‐ and &agr;2‐adrenergic coronary microvascular constriction in the beating heart in vivo. Methods and Results. Coronary microvascular diameters were measured using stroboscopic epiillumination and intravital microscopy during fluorescein microangiography in open‐chested dogs (n=20). Both &agr;1‐ and &agr;2‐adrenergic receptors were selectively activated by intracoronary infusions of norepinephrine (0.05 and 0.2 &mgr;g · kg‐1 · min‐1) in the presence of the &agr;2‐adrenergic antagonist rauwolscine (0.2 mg/kg) or the &agr;1‐adrenergic antagonist prazosin (0.75 mg/kg) during &bgr;‐adrenergic blockade (1 mg/kg propranolol). Microvascular diameters during selective &agr;‐adrenergic receptor activation were measured under baseline conditions and after inhibition of endogenous nitric oxide synthesis by an analogue of l‐arginine, either NG‐nitro‐L‐arginine (L‐NA, 30 mg/kg) or NG‐nitro‐l‐arginine methyl ester (L‐NAME, 30 mg/kg). Under baseline conditions, &agr;1‐adrenergic activation constricted small arteries (vessels with diameters between 100 and 300 &mgr;m) (4±1% and 5±1% decrease in diameter for the low and high doses of norepinephrine, respectively, both p<0.05) but did not change the diameter of arterioles (vessels with diameters <100 &mgr;m). In contrast, &agr;2‐adrenergic activation by the lower but not the higher dose of norepinephrine induced constriction of arterioles (6±2% and 3±4% decrease in diameter, p<0.05 and NS, respectively) but not small arteries. Inhibition of nitric oxide synthase activity by either L‐NA or L‐NAME produced constriction of small coronary arteries (9±2% decrease in diameter, p<0.01) and arterioles (6±1% decrease in diameter, p<0.05). The dilatation of small arteries and arterioles by acetylcholine (0.05 &mgr;g‐1 · kg‐1 · min‐1 intracoronary infusion; 10±1% increase in diameter under baseline conditions, p<0.05) was abolished by either analogue. Both &agr;1‐ and &agr;2‐adrenergic coronary microvascular constriction were markedly potentiated after L‐NA or L‐NAME. &agr;1‐Adrenergic constriction was unmasked in arterioles (7±3% and 10±4% decrease in diameter, p<0.05), although it was not significantly increased in small arteries. Conversely, &agr;2‐adrenergic constriction was unmasked in small arteries (8±1% and 6±2% decrease in diameter, both p<0.05) and potentiated in arterioles (12±1% and 8±4% decrease in diameter, both p<0.05). After L‐NA or L‐NAME, microvessels retained the ability to dilate to sodium nitroprusside (0.1 &mgr;g · kg‐1 · min‐1 intracoronary infusion; 10±2% increase in diameter, p<0.05). &agr;‐Adrenergic constriction was not accentuated by increased tone alone, since it was either attenuated or converted to dilatation during a similar degree of preconstriction by the endothelium‐independent vasoconstrictor angiotensin II (p<0.05 for both &agr;1‐ and &agr;2‐adrenergic activation). Conclusions. These data confirm that &agr;‐adrenergic receptors are widespread in the coronary microcirculation, with the baseline functional responses to &agr;1‐adrenergic activation predominating in small arteries and those to &agr;2‐adrenergic activation predominating in arterioles. Furthermore, coronary microvascular constriction caused by both &agr;1‐ and &agr;2‐adrenergic receptor activation is significantly modulated by endothelium‐dependent relaxation, being markedly potentiated by inhibition of nitric oxide synthase activity. The data imply that &agr;‐adrenergic activation will assume considerable importance as a determinant of coronary microvascular resistance in pathophysiological situations associated with coronary endothelial impairment. (Circulation 1993;87:1264‐1274)

Integrin Signaling Transduces Shear Stress-Dependent Vasodilation of Coronary Arterioles

A direct relationship exists between shear stress and endothelium-dependent NO-mediated vasodilation of blood vessels. The transduction of shear stress to the biochemical signals resulting in the production of NO is, however, unknown. We tested the hypothesis that integrin binding to Arg-Gly-Asp(RGD) peptide sequences in extracellular matrix proteins is a critical step in initiation of the signaling sequence whereby shear stress activates endothelial tyrosine kinase(s) and induces vasodilation of isolated arterioles. Isolated coronary arterioles were exposed to increasing shear stress under control conditions and in the presence of a synthetic peptide, GRGDNP, to competitively inhibit integrin binding to extracellular matrix proteins containing RGD peptide sequences. Intraluminal GRGDNP (0.1, 0.5, and 1.0 mmol/L) inhibited shear stress-induced vasodilation in a concentration-dependent manner. Application of GRGDNP had no effect on endothelium-dependent relaxation to substance P (10(-12) to 10(-8) mol/L). An inactive structural analogue, GRGESP, did not alter shear stress-induced vasodilation. To further elucidate the integrin involved in shear stress-induced vasodilation, we administered a blocking antibody to the integrin beta 3 chain (F11) and observed significant attenuation of the vasodilation. Shear stress was also associated with an increase in tyrosine kinase activity, as assessed by anti-phosphotyrosine binding. Application of GRGDNP significantly decreased anti-phosphotyrosine binding during shear stress, suggesting a link between tyrosine kinase activation and integrin signaling during this vasodilatory response. Taken together, these results indicate that integrin-matrix interactions, possibly at focal adhesions, are of cardinal importance in the signaling pathway of shear stress-induced vasodilation.