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Am J Physiol Heart Circ Physiol 275: H1411-H1418, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 4, H1411-H1418, October 1998

Effects of coronary artery disease on expression and microvascular response to VEGF

Caroline Métais1, Jianyi Li1, Jian Li2, Michael Simons2, and Frank W. Sellke1

1 Division of Cardiothoracic Surgery, Department of Surgery, and 2 Cardiovascular Division, Department of Medicine of Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effects of coronary artery disease (CAD) on human coronary microvascular responses to vascular endothelial growth factor (VEGF) and the alterations of the myocardial expressions of VEGF and its flk-1 and flt-1 receptors were examined in 48 patients. Microvascular studies were performed in vitro with video microscopy. The expressions of VEGF and its receptors were examined using Northern analysis of total mRNA, and the expressions of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were examined by RT-PCR. VEGF and hepatocyte growth factor (HGF) caused potent relaxations of microvessels. These responses were reduced in the presence of NG-nitro-L-arginine and the tyrosine kinase inhibitor genistein or in microvessels from patients with CAD. Relaxations to substance P and sodium nitroprusside were similar in both groups. The substance P response was abolished in the presence of NG-nitro-L-arginine. The expression of VEGF and its receptors and the expression of cNOS and iNOS were not altered in patients with CAD. In conclusion, VEGF and HGF elicit the release of nitric oxide through activation of tyrosine kinase receptors. CAD is associated with reduced vascular responses to both VEGF and HGF; this is not likely due to a reduced expression of VEGF or flt-1 or flk-1 receptors and not due to a generalized endothelium dysfunction despite the presence of mild hypercholesterolemia in these patients with CAD. These findings may have important implications regarding the efficacy of endogenous and exogenous VEGF in patients with risk factor for CAD.

growth factor; coronary microcirculation; human heart; endothelium; inducible nitric oxide synthase; vascular endothelial growth factor

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PATIENTS WITH OBSTRUCTIVE coronary artery disease (CAD) often present with anginal symptoms due to inadequate blood flow to the myocardium, necessitating the need for antianginal medication, catheter-based coronary intervention such as angioplasty, or in severe cases, coronary artery bypass surgery. However, there is significant variation in the propensity of patients to develop collateral vessels. Whereas some patients have inadequate perfusion to the recipient myocardium with relatively moderate CAD, other patients develop new collateral vessels sufficient to provide normal myocardial perfusion even in cases of severe CAD.

Hypercholesterolemia, hypertension, and diabetes mellitus are important risk factors for the development of obstructive coronary atherosclerosis. Impaired endothelium-dependent vasorelaxation of the large coronary arteries is often evident even before the formation of atherosclerotic lesions (29, 48). In addition, these and other risk factors for the development of atherosclerosis are associated with reduced endothelium-dependent vasodilation of the coronary microcirculation in animals and human patients (18, 41) in the absence of overt atherosclerotic disease. Impaired endothelium-dependent relaxation is not only a cause of altered vasomotor regulation in conditions associated with coronary occlusive disease but perhaps more importantly is an indicator of a general impairment of endothelial cell function.

Vascular endothelial growth factor (VEGF) is a 46-kDa heparin-binding glycoprotein and a specific mitogen for vascular endothelial cells in angiogenesis (12, 13). VEGF induces increased microvascular permeability (33, 44, 45) and monocyte migration through endothelial layers (10), which are important in early and advanced atherogenesis. In addition, VEGF has been shown to increase intracellular calcium and affect vascular tone through a constitutive nitric oxide synthase (cNOS) resulting in endothelium-dependent relaxation in coronary arteries (24). Because VEGF receptors are located only on endothelium and patients with CAD often have an angiogenic response inadequate to prevent coronary insufficiency and symptoms of myocardial ischemia, it is uncertain whether patients with occlusive CAD have the potential for normal angiogenesis induced by the release of VEGF. This may have implications regarding the efficacy of both endogenous VEGF and exogenous VEGF delivered during trials of therapeutic angiogenesis.

The expression of VEGF mRNA is increased in cardiac myocytes (2, 25) and vascular smooth muscle cells (5, 47) by various insults, including vascular injury (27), acute hypoxia (5), acute ischemia (1, 23, 26), and chronic myocardial ischemia (43), but little is known regarding the expression of VEGF mRNA in the human heart and the alteration of vascular reactivity in patients with CAD.

The purpose of this study is to examine the effects of CAD on gene expression of VEGF and its receptors flk-1 and flt-1, cNOS, and inducible nitric oxide synthase (iNOS) in the human heart. In addition, the microvascular responses to VEGF and other physiologically important vasoactive substances were examined to confirm a functional correlate to the possible alterations in expressions.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Patient population. We studied 48 patients. Twenty-nine patients with CAD with at least two coronary arteries with >60% stenoses underwent coronary artery bypass grafting. Nineteen patients without CAD (No CAD) and with angiographically normal coronary arteries (no stenosis >20%) underwent solitary atrial septal defect repair or mitral or aortic valve repair or replacement. The clinical characteristics of the patients are indicated in Table 1. The atrial appendage was taken at the beginning of surgery at the time of atrial cannulation before initiation of cardiopulmonary bypass. The atrial appendage was immediately frozen in liquid nitrogen for molecular biology studies. Other atrial tissue segments were placed in a cold (5-10°C) Krebs buffer solution of the following composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, and 11.1 glucose, in preparation for vascular reactivity studies. The study was approved by the clinical research committee of Beth Israel Deaconess Medical Center.

                              
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  		Table 1.   Clinical characteristics of patients

In vitro atrial microvascular studies. Atrial microvessels (57-183 µm in internal diameter) were dissected using a ×10-60 microscope (Olympus Optical, Tokyo, Japan). Microvessels were placed in a Plexiglas microvessel chamber, cannulated with dual glass micropipettes measuring 40-80 µm in diameter, and secured with 10-0 nylon monofilament suture (Ethicon, Somerville, NJ). Oxygenated (95% O2-5% CO2) Krebs buffer solution warmed to 37°C was continuously circulated through the microvessel chamber. The vessels were pressurized to 40 mmHg in a no-flow state using a burette manometer filled with a Krebs buffer solution. With an inverted microscope (×40-200, Olympus CK2, Olympus Optical) connected to a video camera, the vessel image was projected onto a black and white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, VT) was used to measure internal lumen diameter. Vessels were allowed to bathe in the organ chamber for at least 30 min before an intervention.

Microvessel study protocols. Relaxation responses of microvessels were examined after development of spontaneous tone with or without supplemental precontraction with the thromboxane A2 analog U-46619. Baseline diameter was defined as the internal diameter within minutes of cannulation and placement in the bath when the diameter tended to be at a maximum and spontaneous contraction has not yet occurred. At the completion of an experiment, papaverine (10-4 M) was applied to confirm that the initial diameter reading was similar to the maximally dilated diameter. If the spontaneous contraction was <30% of the initial baseline diameter, incremental concentrations of U-46619 (10-8 to 10-6 M) were applied so that the final precontraction was 30-60% of the initial baseline diameter. Vascular responses to VEGF (10-16 to 10-11 M), hepatocyte growth factor (HGF) (10-16 to 10-11 M), sodium nitroprusside (SNP) (10-9 to 10-4 M), ADP (10-9 to 10-4 M), and substance P (10-15 to 10-6 M) were examined. Selected experiments were performed in the presence of 10-4 M NG-nitro-L-arginine (L-NNA), 10-5 M genistein, or 10-6 M indomethacin. Blocking drugs were applied at least 20 min before we performed a dose-response intervention. All drugs were applied extraluminally. Measurements were made and recorded 2-3 min after the drug administration when the response had stabilized. In measuring the response to VEGF, it took ~5-8 min for the response to be stabilized. Once VEGF or substance P was applied to a vessel, the vessel was discarded to avoid tachyphylaxis. One to four interventions were performed on each vessel. The order of drug administration was random. The vessels were washed three times with a Krebs buffer solution and allowed to equilibrate in a drug-free Krebs buffer solution for 15-30 min between interventions.

mRNA analysis of VEGF expression. Approximately 1 g of atrial appendage (n = 4 in each group) was snap frozen in liquid nitrogen and then homogenized in 4 M guanidium isothiocyanate followed by centrifugation through 5.7 M cesium chloride at 200,000 g for 16 h. The RNA pellet was dissolved in water and precipitated in ethanol. For Northern blots, 10 mg of total RNA were fractionated on 1.3% formaldehyde-agarose gel and transferred to a GeneScreen Plus (DuPont) membrane. The VEGF, its receptors flt-1 and flk-1, and 18S cDNA probes were labeled with [32P]deoxycytidine triphosphate (New England Nuclear) by use of a random-priming labeling kit (Boehringer, Indianapolis, IN) and purified of unincorporated nucleotides with the use of G-50 Quick Spin Columns (Boehringer). The typical specific activity of the probes used in the experiments was 1-2 × 109 counts/min (cpm)/mg. The blots were hybridized at 68°C for 3 h in QuickHyb solutions (Stratagene, La Jolla, CA). After the hybridization, blots were washed twice in 2× standard saline citrate, 0.1% sodium dodecyl sulfate for 15 min at room temperature, and then twice in 0.1% sodium dodecyl sulfate for 15 min at 60°C. Autoradiography was carried out with Kodak XAR film at 80°C for 16-20 h. For quantitative analysis of expression, the blots were exposed on PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed using Image-Quant software (Molecular Dynamics).

RT-PCR analysis of cNOS and iNOS expression. For cNOS and iNOS mRNA studies, the RT-PCR was performed because the intensity of signals for cNOS and iNOS was not sufficient for quantitative analysis by Northern hybridization. Primers were designed based on the published cNOS and iNOS sequence. The primers of the sense 5'-CAGTGTCCAACATGCTGCTGGAAATTG-3', corresponding to bases 1050-1076, and the antisense 5'-TAAAGGTCTTCTTCCTGGTGATGCC-3', corresponding to bases 1511-1535, were used to amplify a 486-base pair fragment of cNOS. For iNOS, the primer of sense 5'-GCCTCGCTCTGGAAAGA-3', corresponding to bases 1425-1441, and the antisense 5'-TCCATGCAGACAACCTT-3', corresponding to bases 1908-1924, were used to amplify a 500-base pair fragment of iNOS.

Equal amount of total RNA was used for RT-PCR. For quantification, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified from the same amount of RNA to correct for variation of different samples. The PCR products were subjected to electrophoresis on 1.5% agarose gel then scanned and quantitated using Image-Quant software (Molecular Dynamics).

Drugs. Human recombinant VEGF-165 and human recombinant HGF were obtained from Genentech (South San Francisco, CA). Substance P was obtained from RBI (Natick, MA). L-NNA, indomethacin, genistein, ADP, and SNP were obtained from Sigma (St. Louis, MO). VEGF and HGF were dissolved in phosphate-buffered saline with 0.1% bovine serum albumin to make stock solutions that were stored at -80°C. The other drugs were dissolved in ultrapure distilled water. All solutions were prepared daily.

Data analysis. The relaxation responses were expressed as the percent relaxation of the spontaneous and/or U-46619-induced vascular contraction (means ± SE). Comparisons of dose-response curves were performed by a two-way analysis of variance for repeated measures, followed by Scheffé's multiple range test when indicated. Student's t-test was used to compare changes in gene expressions and hematologic and hemodynamic data. Chi square analysis was used to examine descriptive variables. All P values <0.05 were considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The clinical characteristics of the two patient groups (CAD and No CAD) are shown in Table 1. Patients in the CAD group were more often male and smokers and were older and more often diabetic compared with the patients in the No CAD group. Of the 29 patients with CAD, 21 patients were hypertensive (blood pressure > 140/90 mmHg), but none had echocardiographic evidence of left ventricular hypertrophy. Nineteen patients in the CAD group had a history of hypercholesterolemia that was treated with pharmacological therapy. Total serum cholesterol concentration was not significantly different between the two groups, but serum low-density lipoprotein was higher and high-density lipoprotein was lower in the CAD group versus the respective levels in patients of the No CAD group. A mean of 3.4 ± 0.2 bypass grafts was placed at the time of surgery. Concomitant aortic or mitral valve surgery was performed in six patients in the CAD group at the time of coronary artery bypass grafting. Of 19 patients in the No CAD group, 3 had a history of hypercholesterolemia that required pharmacological treatment. Left ventricular function was similar in both groups.

Vessels characteristics. Atrial microvessels ranged from 57 to 183 µm in internal diameter, averaging 109 ± 4 µm in the CAD group and 111 ± 5 µm in the No CAD group. Percent precontraction after spontaneous constriction or after application of the thromboxane A2 analog U-46619 (1 nM to 1 µM) was 43 ± 3% in the No CAD group and 43 ± 3% in the CAD group. Although the application of L-NNA, genestein, or indomethacin did not have an acute effect on vessel diameter, it appears that all of these blockers increased the development of spontaneous tone within the No CAD group (Table 2).

                              
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  		Table 2.   Microvessel data

In vitro response to VEGF of HGF. In the No CAD group, VEGF caused a potent dilation of atrial microvessels (EC50 = 10-13) (Fig. 1). After treatment of vessels from the No CAD group with either 10-4 M L-NNA (P < 0.01) or 10-5 M genistein (P < 0.01), the VEGF-induced relaxation was markedly reduced, suggesting that the major portion of VEGF-induced relaxation is through the release of nitric oxide (NO) via a tyrosine kinase receptor-mediated mechanism. The relaxation elicited by VEGF was markedly decreased in atrial microvessels from the CAD group (P < 0.05) versus the response of vessels from the No CAD group.


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  		Fig. 1.   Plot of in vitro response to vascular endothelium growth factor (VEGF) of atrial microvessels from patients with coronary artery disease (CAD, n = 8) and patients without coronary artery disease (No CAD, n = 6). Selected experiments were performed on vessels in No CAD group in presence of nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA, n = 7) or tyrosine kinase inhibitor genistein (n = 6). Responses are percent relaxation of spontaneous and/or U-46619-induced contraction. Data are expressed as means ± SE. * P < 0.05 vs. No CAD.

Similarly to the response profile of vessels to VEGF, HGF induced a significant relaxation of vessels in the No CAD group (EC50 = 10-13) (Fig. 2). This relaxation was significantly reduced in the presence of 10-4 M L-NNA (P < 0.01) or 10-5 M genistein (P < 0.01). The relaxation response to HGF in the CAD group was also decreased (P < 0.05) versus the response of vessels in the No CAD group.


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  		Fig. 2.   Plot of in vitro response to hepatocyte growth factor (HGF) of atrial microvessels from patients with CAD (n = 8) and patients without CAD (n = 6). Selected experiments were performed on vessels in No CAD group in presence of NOS inhibitor L-NNA (n = 6) or tyrosine kinase inhibitor genistein (n = 6). Responses are percent relaxation of spontaneous and/or U-46619-induced contraction. Data are expressed as means ± SE. * P < 0.05 vs. No CAD.

In vitro response to substance P and ADP. The response of microvessels to substance P was similar in patients without CAD and those with CAD. The response of atrial microvessels from the No CAD group to substance P was nearly abolished in the presence of the NOS inhibitor L-NNA (Fig. 3). Responses of microvessels to ADP were not altered in the presence of L-NNA, and they were not altered in vessels from patients with CAD. However, the response was significantly impaired in the presence of the cyclooxygenase inhibitor indomethacin (P < 0.05) (Fig. 4).


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  		Fig. 3.   Plot of in vitro response to substance P of atrial microvessels from patients with CAD (n = 9) and patients without CAD (n = 9). Selected experiments were performed on vessels in No CAD group in presence of NOS inhibitor L-NNA (n = 6) or tyrosine kinase inhibitor genistein (n = 6). Responses are percent relaxation of spontaneous and/or U-46619-induced contraction. Data are expressed as means ± SE. * P < 0.05 vs. No CAD.


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  		Fig. 4.   Plot of in vitro response to ADP of atrial microvessels from patients with CAD (n = 8) and patients without CAD (n = 6). Selected experiments were performed on vessels in No CAD group in presence of NOS inhibitor L-NNA (n = 6) or cyclooxygenase inhibitor indomethacin (n = 6). Responses are percent relaxation of spontaneous and/or U-46619-induced contraction. Data are expressed as means ± SE. * P < 0.05 vs. No CAD.

In vitro response to SNP. The relaxation of atrial microvessels to SNP, which operates through an endothelium-independent cGMP-mediated mechanism, was similar in both groups. This suggests that CAD causes no alteration of the smooth muscle's ability to relax through the cGMP pathway (Fig. 5).


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  		Fig. 5.   Plot of in vitro response to sodium nitroprusside (SNP) of atrial microvessels from patients with CAD (n = 8) and patients without CAD (n = 6). Responses are percent relaxation of spontaneous and/or U-46619-induced contraction. Data are expressed as means ± SE.

Gene expression of VEGF, cNOS, and iNOS. To examine whether the decreased sensitivity of microvessels from patients with CAD compared with those from patients without CAD is due to altered growth factor receptor expression or altered expression of one of the isoforms of NOS, the expression of VEGF and its receptors was examined using Northern analysis, and the expressions of cNOS and iNOS were analyzed by RT-PCR. Expression of VEGF mRNA and its flt-1 and flk-1 receptors was similar in both groups (Fig. 6). Similarly, gene expression of cNOS and iNOS was not different. The density ratio of cNOS to GAPDH was 1.54 ± 0.26 in the No CAD group and 1.28 ± 0.25 in the CAD group. The density ratio of iNOS to GAPDH was 1.30 ± 0.08 in the No CAD group and 1.34 ± 0.0.05 in the CAD group. This suggests that altered cNOS or iNOS expression is not involved in the reduced microvascular relaxation to VEGF (Fig. 7).


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  		Fig. 6.   Northern blot mRNA level expression of VEGF and its receptors flt-1 and flk-1 in atrial tissues in CAD group (n = 4) and in No CAD group (n = 4) expressed as percentage of 18S ribosomal RNA.


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  		Fig. 7.   Inducible nitric oxide synthase (iNOS), constitutive nitric oxide synthase (cNOS), and GAPDH gene expression in human atrial tissue from patients with CAD and without CAD. One microgram of atrial tissue was subjected to RT reaction, and reaction mixtures were amplified by PCR and then analyzed by agarose gel electrophoresis. Positions and sizes of DNA markers in base pairs (bp) are shown at right.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The main findings of this study are that 1) VEGF is a potent coronary vasodilator in the human coronary microcirculation, 2) VEGF and HGF both mediate their vascular effects predominantly by the release of endothelium-derived NO via tyrosine kinase receptors, 3) CAD is associated with a reduction in vascular response to these angiogenic growth factors, 4) this reduced response is neither due to a reduced expression of the tyrosine kinase receptors nor due to an altered expression of either iNOS or eNOS, and finally, 5) the reduced response to VEGF and HGF appears to be either due to a functional alteration of the tyrosine kinase receptor or due to another alteration in the transduction cascade, leading to the generation of NO as a consequence of risk factors for CAD and not because of a generalized defect in endothelial function.

VEGF is a glycoprotein that is mitogenic for endothelial cells in vitro and in vivo. VEGF protein and receptor expression is increased in ischemic and hypoxic myocytes (1, 26, 43) and in response to cytokine stimulation (5). VEGF can induce marked vasorelaxation in animals (24, 43) and increase vascular permeability (30). VEGF binds to high-affinity tyrosine kinase receptors encoded by flt-1 and flk-1 genes (16, 46) on vascular endothelial cells, which leads to the release of NO by the constitutive Ca2+ calmodulin-dependent NOS (24). It has been shown in vivo that there is a reduced basal activity of NOS and NO in epicardial arteries and in the coronary microcirculation in patients with atherosclerosis compared with that in normal control subjects (38). However, the mechanism appears to be multifactorial, complex, and more likely related to increased oxidative stress leading to reduced efficacy of NO or increased degradation of NO rather than an absolute decrease in NO concentration (22). The defect may lie in the availability of substrate for NO production within the endothelial cell (14, 17, 28), in the intracellular signal transduction pathway that carries the message from the membrane receptors to the enzyme that synthesizes NO (20), in the NOS enzyme itself, or even in the breakdown of NO by superoxide anions in the interstitium (36).

Whereas altered gene expression of either inducible or constitutive isoforms of NOS as a contributing cause of endothelial dysfunction was ruled out in the present study, the protein content or absolute activity of these enzymes was not examined. Thus it is possible that cNOS or iNOS protein level was changed due to alterations in mRNA or protein half-life. Alternatively, the ability of eNOS and iNOS to generate NO may have been altered as well. However, studies with substance P argue against these possibilities. Substance P is a neuropeptide that stimulates the release of vasoactive factors from the endothelial cell by binding to a tachykinin receptor (40). Substance P-induced atrial microvessel dilation is primarily due to the release of NO, since its effects are markedly inhibited in the presence of L-NNA. Surprisingly, in this study the response of atrial microvessels to substance P was not reduced in vessels from patients with CAD compared with responses of vessels from patients without CAD. This suggests that the reduced vascular effects of VEGF and HGF are related to changes to the respective tyrosine kinase receptors rather than reduced sensitivity to NOS secondary to endothelial dysfunction. Thus it is possible that the blunted vascular responses to VEGF and HGF are due to a specific alteration of the tyrosine kinase receptors, to an abnormality of the signal transduction pathway, or to defects of the final processes that lead to the production and release of NO. These may include a decreased synthesis or increased breakdown by superoxide anions, which account for the endothelial dysfunction in patients with CAD. Because markedly increased levels of NO have been shown to decrease the activity of cNOS, it was hypothesized that increased expression of iNOS in the atrium of patients with CAD could contribute to the decreased endothelium-dependent relaxation elicited by VEGF. However, neither iNOS expression nor that of cNOS was altered in the atrial tissue of patients with CAD, making this mechanism unlikely.

Previous studies have concurred with the findings of the present study that substance P stimulates the release of endothelium-derived NO (3, 9). In addition, substance P may evoke the release of endothelium-derived hyperpolarizing factor and histamine in some species (4, 19). No constrictor action or direct smooth muscle dilator effects of substance P have been reported (21). In vivo, human coronary arteries relax in response to substance P, and this is partially attenuated in strips of atherosclerotic epicardial coronary arteries from the ventricular myocardium (3, 8, 9). In vivo, substance P causes forearm microvascular vasodilation in humans and both epicardial and microvascular coronary vasodilation (6, 31, 37). Few patients with atherosclerosis have been studied with substance P, and the results have been inconclusive (15) but generally demonstrate a reduced relaxation response. Endothelial dysfunction was demonstrated in asymptomatic children and young adults with risk factors for atherosclerosis such as hypercholesterolemia and cigarette smoking (7). It has been shown (39) that hypertension and hypercholesterolemia, even in the presence of angiographically normal coronary arteries, are associated with depression of coronary epicardial and microvascular dilation in response to substance P. In our study, there was no difference in response to substance P in atrial microvessels between the two groups. It is possible that the presence of risk factors of coronary artery disease present in the No CAD group could have affected the relaxation response to substance P in a similar way that it has altered the relaxation response of the vessels in the CAD group. Alternatively, it is possible, although unlikely, that atrial arterioles are not as severely affected by hypercholesterolemia and other risk factors for CAD as are large epicardial coronary arteries. Third, since the degree of hypercholesterolemia was relatively mild in our patients with CAD because of a tendency of close medical surveillance in these patients, this degree of elevated low-density lipoprotein and reduced level of high-density lipoprotein may not be sufficient to cause impaired vascular relaxation to endothelium-dependent agonists such as substance P. Finally, the present study did not examine the possible altered mechanisms of vasorelaxation elicited by substance P in patients with CAD. Studies by Najibi and co-workers (34, 35) have demonstrated that the endothelium-dependent agonist acetylcholine may elicit similar vasorelaxation in vessels from hypercholesterolemic and normal rabbits but that the relative contributions of NO released from the endothelium and that of activation of potassium channels may be altered. However, other studies from the same group (11) have demonstrated impaired endothelium-dependent relaxation to substance P in a hypercholesterolemic porcine model, and the studies in the patients mentioned above demonstrated impaired relaxations to substance P in hypercholesterolemic patients. Thus, although the present study suggests that the impaired vascular response to VEGF and HGF is due to decreased stimulated release of NO specific to these growth factors, additional studies will be required to rule out possible contributions of potassium channels and other mechanisms in maintaining vascular relaxation to substance P and other endothelium-dependent agonist before this can be concluded with certainly.

Limitations and implications. There are several limitations of the present study. First, because of the nature of the study, microvessels were obtained from patients who had multiple medical problems in addition to CAD rather than from patients or animals with one disease state such as hypercholesterolemia or hypertension. However, this is a potential criticism of nearly all studies involving patients with CAD. Second, the effects of multiple factors placing patients at risk for CAD likely contributed to development of the alterations in vascular reactivity in this study. Thus the exact process or mechanism leading to an altered response to VEGF cannot be discerned from the present set of experiments. Furthermore, patients were taking medication for the treatment of other ailments that could adversely affect vascular responses. However, the response of vessels to substance P and ADP, which elicit relaxation predominantly through the release of NO and prostaglandins, respectively, were not altered in the CAD group compared with that in vessels from patients without significant obstructive CAD. Therefore, differences in medications taken by patients in the two groups probably did not contribute significantly to the altered pattern of responses to VEGF or HGF. Finally, it remains to be determined whether the reduced atrial microvascular relaxation responses to VEGF and HGF in patients with CAD have an impact on the angiogenic and other actions of these growth factors in ventricular myocardium. Whereas VEGF stimulates proliferation of postcapillary endothelial cells through the production of NO and cGMP accumulation (32) in some models, other mechanisms may be operating in patients and the VEGF-induced release of NO may be inconsequential. It is possible that the decreased release of NO in response to VEGF and other angiogenic growth factors is countered by other pathways in the angiogenic process.

The finding that VEGF and another growth factor, HGF, elicit less relaxation of coronary microvessels from patients with CAD than that in vessels from patients without CAD may have implications regarding not only the efficacy of endogenous VEGF released during inflammation and ischemia but also regarding the efficacy of exogenous VEGF delivered during trials of therapeutic angiogenesis. Patients who are candidates for therapeutic angiogenesis are generally not candidates for coronary artery bypass surgery or catheter-based techniques of revascularization. In addition, these patients nearly always have globally severe, diffuse CAD and multiple risk factors for the development of atherosclerosis. Therapeutic angiogenesis can be performed using adenoviral and other viral vectors containing DNA-encoding angiogenic factors or by the direct administration of growth factor proteins such as VEGF. With either method, a relatively lack of efficacy may have a negative impact on the potential for therapeutic benefit for this new method of treatment for patients with inoperable CAD. A recent preliminary human trial suggested that whereas some patients benefit from growth factor-induced angiogenesis, some patients do not develop new vascularity to the ischemic territory (42). The issues raised above will need to be addressed by further investigation in vivo in patients and in vitro using isolated tissues.

    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants RO1 HL-46716, RO1 HL-53793, and P50 HL-56993.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: F. W. Sellke, Division of Cardiothoracic Surgery, Beth Israel-Deaconess Medical Center, East Campus, Dana 905, 330 Brookline Ave., Boston, MA 02215.

Received 10 April 1998; accepted in final form 29 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Banai, S., D. Shweiki, A. Pinson, M. Chandra, G. Lazarovici, and E. Keshet. Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis. Cardiovasc. Res. 28: 1176-1179, 1994[Abstract/Free Full Text].

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Am J Physiol Heart Circ Physiol 275(4):H1411-H1418
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