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(Circulation Research. 1999;85:192-198.)
© 1999 American Heart Association, Inc.

Original Contribution

Bradycardia-Induced Coronary Angiogenesis Is Dependent on Vascular Endothelial Growth Factor

Wei Zheng, Margaret D. Brown, Tommy A. Brock, Robert J. Bjercke, Robert J. Tomanek

From the Department of Anatomy and Cell Biology and The Cardiovascular Center, (W.Z., R.J.T.) University of Iowa, Iowa City, Iowa; The School of Sport and Exercise Science (M.D.B.), University of Birmingham, United Kingdom; and Department of Pharmacology (T.A.B., R.J.B.), Texas Biotechnology Corporation, Houston, Tex.

Correspondence to Robert J. Tomanek, PhD, Department of Anatomy and Cell Biology, Bowen Science Building, University of Iowa, Iowa City, IA 52242. E-mail robert-tomanek{at}uiowa.edu

	Abstract

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Abstract—A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm³) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased >2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.

Key Words: angiogenesis • bradycardia • alinidine • vascular endothelial growth factor • basic fibroblast growth factor

	Introduction

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Because the formation of new blood vessels by spouting (angiogenesis) may occur in the fully differentiated heart under some conditions,¹ attention has been focused on neovascularization and collateral vessel growth in ischemic heart disease. A recent review² indicates that a number of growth factors are candidates for therapeutic augmentation of myocardial perfusion. These include vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factors (aFGF and bFGF, also known as FGF-1 and FGF-2), transforming growth factor-ß (TGF-ß), insulin-like growth factor, and scatter factor/hepatocyte growth factor. Coronary angiogenesis also may occur in nonischemic models, eg, exercise, some models of hypertrophy (reviewed in Reference 1¹ ), and chronic bradycardia.^{3 4 5}

Bradycardia appears to be a particularly good model of coronary angiogenesis, because the magnitude of capillary growth is impressive^{4 5} and the model is physiological rather than pathological. The growth factor(s) that provide an angiogenic stimulus have not been identified, although after 5 weeks of pacing, capillary density and a low molecular endothelial cell stimulating factor were found to be correlated.⁶ TGF-ß was also increased in these long-term–paced hearts. The experiments described in this study were initiated to characterize the time course of angiogenesis in chronic bradycardia and to determine whether VEGF or bFGF plays a role in this growth process. The bradycardic drug alinidine was used to avoid invasive protocols.⁶ We hypothesized that angiogenesis in this model is regulated by bFGF and/or VEGF. This hypothesis was based on a variety of studies implicating these growth factors in coronary angiogenesis in normal and pathological conditions. Both growth factors are known to be direct mitogens for endothelial cells, and both have been shown to promote several events in the angiogenic cascade.^{2 7 8 9}

VEGF, a secreted, direct-acting endothelial cell mitogen, expressed in many different cell types, ie, macrophage, fibroblast,¹⁰ smooth muscle cell,¹¹ and endothelial cell,¹² stimulates angiogenesis and vascular permeability.¹³ VEGF binding to receptor tyrosine kinase Flk-1 and Flt-1¹⁴ expressed in endothelial cells is required for normal vascularization.¹⁵ Disruption of VEGF, Flk-1, or Flt-1 genes results in embryonic lethality due to failure of vascular development.^{16 17} Myocardial ischemia induced by coronary artery occlusion has been shown to increase VEGF expression.¹⁸ Moreover, intracoronary injection of VEGF was found to enhance the development of small coronary arteries and to improve flow to the ischemic myocardium.¹⁹

bFGF, a known mitogen for most nonterminally differentiated cells of both embryonic mesodermal and neuroectodermal origin,^{2 20} has been shown to stimulate most of the individual components of new capillary growth.^{21 22} Our previous studies indicated that cell migration from embryonic heart explants on collagen gels is enhanced when bFGF is added to culture medium.²³ Exogenous bFGF has been shown to increase the arteriolar and capillary numbers on the surface of infarcted rat hearts and enhance collateral flow in the ischemic canine heart.^{24 25} Most recently, we found that myocardial bFGF mRNA is upregulated after thyroxine administration.⁹

	Materials and Methods

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Bradycardic Model
All experiments used male Sprague-Dawley rats, which were maintained at 17°C to 24°C with a 12-hour light cycle and were provided rat chow and water ad libitum. All protocols were approved by the Animal Care and Use Committee of the University of Iowa and conform to Public Health Service guidelines. Most of the experiments used rats with initial body weights of 200 to 240 g. The experiments using VEGF-neutralizing antibodies are based on rats with initial body weights of 125 to 150 g.

A long-term bradycardia was achieved in rats by IP injection of 3 mg/kg alinidine twice daily for 1 to 3 weeks. Under anesthesia (ketamine, 100 mg/kg, IM), heart rate was recorded with implanted subcutaneous ECG electrodes after 10, 20, 30, 40, 50, 60, 90, and 120 minutes of injection of alinidine, and 18 hours after the last drug administration in rats chronically treated with the bradycardic agent for 1, 2, and 3 weeks. Control rats received 0.9% saline injections.

Measurement of Hemodynamics
Measurements of cardiac output and arterial pressure were made once heart rate had stabilized at its lowest point (20 to 40 minutes after injection of alinidine). In control animals, measurements were made at a comparable time after saline injection. Rats were anesthetized by sodium pentobarbital (30 mg/kg IP), supplemented as necessary via an intravenous cannula. Arterial pressure was measured via a brachial artery catheter and a Bell and Howell transducer. To estimate cardiac output, the left ventricle was cannulated for injection of radiolabeled microspheres, with reference withdrawal from a brachial artery. Microspheres labeled with ⁴⁶Sc, ⁵⁷Co, or ¹¹³Sn were used to measure coronary flow. Withdrawal of reference blood at 0.5 mL/min commenced 15 seconds before injection of 0.5 to 0.9 mL of saline containing 200 000 to 500 000 15-µm microspheres and continued for 30 seconds after flushing the microspheres with 1 mL of 1% BSA. After these measurements were taken, the rat was killed by anesthetic overdose, and tissues were removed for radioactivity counting in preweighed tubes in a Hewlett-Packard gamma counter. Right and left kidneys were sampled to assess adequacy of microsphere mixing. Lung was sampled to assess the degree of any arteriovenous shunting. Results were discarded if they failed to meet the criteria of a <10% difference between kidney flows, which would indicate inadequate mixing, and/or >5% total counts in the lung, which would indicate significant nontrapping of microspheres during their first circulation. Total isotope activity in the volume injected was calculated from the counts of a known volume and weight of a retained sample of microspheres. Cardiac output was computed from radioactivity levels in withdrawn blood relative to the sample microsphere counts.

Assessment of Capillary Growth
After 1, 2, or 3 weeks of alinidine or saline treatment, the rats were weighed and anesthetized with ketamine (100 mg/kg IM). After exposing the heart and great vessels via a thoracotomy, sodium heparin (1000 units) and 1.5 mL 2% procaine were injected into the lumen of the left ventricle to prevent blood clots and to arrest the heart in diastole, respectively. The heart was excised, mounted on a Langendorff-type apparatus, flushed with Lock's solution, and perfused with 2.5% glutaraldehyde at 120 mm Hg pressure. After perfusion fixation, the heart was cleared of fat, and weights of the whole heart and right and left ventricles were determined. Samples of the left ventricle were fixed in the glutaraldehyde overnight at 4°C, processed, and embedded in JB-4 plastic. All specimens were dissected so that sections representing cross-sectional fields of myocytes and capillaries could be prepared. Subsequently, 2-µm sections were stained with hematoxylin and eosin.

Capillary growth was assessed by length density measurements obtained by image analysis of light microscopic sections as previously described.²⁶ Length density (L_V) was calculated from capillary long (a) and short (b) axes and numerical density (N_A) according to the following relationship: L_V(mm/mm³)=(a/b)N_A.

Administration of VEGF Neutralizing Antibodies
Capillary volume percentage was calculated from lumen cross-sectional areas of capillary profiles divided by the total field area times 100.

To determine whether VEGF is necessary for angiogenesis in this model, an additional protocol was conducted using 3 groups of rats. We administered large doses of monoclonal VEGF-neutralizing antibodies (Texas Biotechnology Corp) during a 2-week period to rats being treated with alinidine. The neutralizing antibody was administered according to the protocol of Couffinhal et al.²⁷ On day 1, 0.3 mg/100 g IP was injected, and then on days 3, 6, 9, and 12, 0.6 mg/100 g was injected. Hearts were harvested from these rats on day 14. The group receiving alinidine and VEGF-neutralizing antibodies was compared with a group receiving alinidine alone and with a control group that was given daily injections of saline.

Northern Blot Analysis
For the temporal expression of VEGF and bFGF mRNA, the left ventricle, inclusive of the septum, was frozen in liquid nitrogen immediately after excision. These samples were also used for protein analysis. Total RNA was extracted by modification of the method using the RNA isolation reagent, RNA STAT-60 (Tel-Test Inc). The RNA pellet was dissolved in diethyl pyrocarbonate–treated water. For Northern blots, 10 µg of total RNA was separated on 1.2% formaldehyde-agarose gel, transferred to nylon filter (Hoefer Scientific Instruments) in 25 mmol/L sodium phosphate buffer by capillary blotting overnight, and cross-linked by ultraviolet irradiation. Prehybridization of the filters was performed for 1 to 2 hours at 55°C in 50% formamide, 5x SSC, 4x Denhardt's solution, 1% SDS, 10% dextran sulfate, and 150 µg/mL heat-denatured, sheared salmon sperm DNA. Hybridization was performed for 16 to 20 hours by adding VEGF, bFGF, or 18S RNA probes labeled with [-³²P]dUTP to the same solution. After hybridization, the filters were washed twice in SSC, 0.2% SDS for 10 minutes at room temperature and then twice in 0.1x SSC, 1.0% SDS for 60 minutes at 55°C. Autoradiography was carried out with Fuji RX film at –70°C for 16 to 20 hours. Plasmid pGEM-VEGF (kindly provided by Dr. Kenneth Thomas, Merck Research Laboratories) was digested with EcoRI, and plasmid pGEM-bFGF was digested with HindIII. VEGF RNA probe, transcribed by SP6 polymerase, and bFGF RNA probe, transcribed by T7 polymerase, were labeled with [-³²P]dUTP. The typical specific activity of the probes used in the experiments was 3x10⁶ cpm/mL hybridization solution. Probe for 18S RNA was used as a control of RNA loading.

Western Blot Analysis
The samples for protein analysis were harvested as described above and homogenized in ice-cold PBS containing protease inhibitors. Proteins (50 µg) were run out on 15% SDS-PAGE, subsequently transferred to nitrocellulose membranes (Schleicher & Schuell) by electrotransfer, and blocked with 5% nonfat milk for 1 hour at room temperature. The blots were incubated with VEGF rabbit polyclonal IgG diluted 1:500 and bFGF rabbit polyclonal IgG diluted 1:500 in 1% milk and 0.05% TBS-Tween 20. The antigen-antibody complexes were visualized using anti-rabbit IgG–horseradish peroxidase diluted 1:5000 and the enhanced chemiluminescence detection system (Amersham). All antibodies were purchased from Santa Cruz Biotechnology.

Statistical Analysis
The data were analyzed using ANOVA and the Student t test followed by a Bonferroni adjustment for multiple comparisons. P0.05 was selected to denote statistical significance. Data are presented as mean±SE throughout the figures.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acute and Chronic Effects of Alinidine on Heart Rate
Heart rate responses to a single injection of alinidine were monitored for 2 hours in anesthetized rats (Figure 1). Heart rate decreased by 32% for 20 to 40 minutes after injection and then gradually rose but remained depressed by 20% to 25% for up to 2 hours. We then assessed the chronic effect of twice-daily injections of alinidine by recording heart rate before daily injection (Figure 2). Heart rate was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment. Left ventricular weight, body weight, and their ratio were not affected by alinidine-induced bradycardia (Table 1). Therefore, long-term bradycardia in rats, induced with alinidine, did not alter ventricular mass.

View larger version (11K): [in this window] [in a new window]   Figure 1. Changes in acute heart rate in rats (n=6) at various times after alinidine treatment. The changes are expressed as percentage of pretreatment values. Heart rate decreased by 32% for 20 to 40 minutes after alinidine treatment and then gradually rose but remained depressed by 20% to 25% up to 2 hours.

View larger version (33K): [in this window] [in a new window]   Figure 2. Reduction of heart rate in rats treated chronically with alinidine. Measurements were made 2 to 3 hours after the last alinidine injection. The decrease in heart rate is 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Five rats were used for each time point.

View this table: [in this window] [in a new window]   Table 1. Left Ventricular Weights, Body Weights, and Their Ratios

Heart Performance
Data in Table 2 show the effects of acute intraperitoneal injection of either alinidine or saline on hemodynamics. There were no significant differences between the 2 groups in arterial pressure; cardiac output was maintained in the face of the decreased heart rate by a significant increase of 34% in stroke volume index.

View this table: [in this window] [in a new window]   Table 2. Effects of Acute Injection of Alinidine on Hemodynamics

Capillary Growth
Figure 3 shows that left ventricular capillary length density (mm/mm³) increased gradually, with significant growth (19%) noted after 2 weeks of alinidine administration, and a 23% increase documented after 3 weeks of treatment. These data indicate that long-term bradycardia resulted in a marked increase in capillary supply in the absence of changes in left ventricular weight. Capillary volume percentage increased by 30% and 34% after 2 and 3 weeks of treatment (control, 8.00±0.75; alinidine, 11.13±0.47; [mean±SE]). Capillary diameters were similar in the treated and control groups.

View larger version (34K): [in this window] [in a new window]   Figure 3. Assessment of left ventricular capillary length density (CLD) (mm/mm3) after 1, 2, and 3 weeks of alinidine treatment. The increase of CLD is 19% (*P<0.01 vs control) and 23% (**P<0.01 vs control) after 2 and 3 weeks of treatment, respectively. Numbers of rats are indicated in parentheses.

Expression of VEGF and bFGF mRNA
Expression of left ventricular VEGF mRNA (Figure 4) and bFGF mRNA (Figure 5) at various time intervals after alinidine treatment were quantified by Northern blot analysis. In all cases, the same blot was hybridized initially with the probe for VEGF and bFGF and subsequently with that for 18S RNA. VEGF and bFGF specific counts in each lane were then adjusted for the 18S counts. Representative Northern blots are illustrated in Figures 4A and 5A, and a quantitative analysis is shown in Figures 4B and 5B. The latter was obtained by scanning the blots and then normalizing the data by calculating VEGF mRNA/18S RNA ratio. VEGF mRNA increased significantly 1 week after alinidine treatment, reaching a 2.1-fold increase over the control level (P<0.01 versus control). The level remained elevated (P=0.015) for 2 weeks, declining to 1.3-fold after 3 weeks. Hybridization with bFGF RNA probe showed mRNA bands of 4 kb, but no difference in bFGF mRNA expression was found between the control and alinidine-treated groups in this study. Thus, our data show that VEGF, but not bFGF, is involved in capillary growth due to chronic bradycardia.

View larger version (32K): [in this window] [in a new window]   Figure 4. Northern blot analysis showing time course of VEGF mRNA expression in hearts of rats treated with alinidine for 1, 2, and 3 weeks (A1W, A2W, and A3W), along with their controls (C1W, C2W, and C3W). A, Representative Northern blot. B, VEGF mRNA quantified by scanning and normalizing by calculating VEGF mRNA/18S RNA ratio. VEGF mRNA increased significantly 1 week after alinidine injection, reaching a 2.1-fold increase over control level (**P<0.01 vs C1W). The level remained elevated at 2 weeks (*P=0.015 vs C2W), declining to 1.3-fold over control (C3W) at 3 weeks. Data in panel B are means of 3 experiments.

View larger version (29K): [in this window] [in a new window]   Figure 5. Northern blot analysis showing time course of bFGF mRNA expression after treatment of alinidine. A, Representative Northern blot. B, Quantitative analysis of mRNA. No significant change of bFGF mRNA expression occurred in hearts of rats treated for 1, 2, and 3 weeks (A1W, A2W, and A3W). Data in panel B are means of 3 experiments.

VEGF Protein
Western Blots for VEGF, which are shown in Figure 6, indicate that VEGF protein level rose gradually in the alinidine-treated rats during the treatment period. VEGF protein increased by 15% at 2 weeks and by 75% at 3 weeks.

View larger version (19K): [in this window] [in a new window]   Figure 6. Time course of VEGF protein expression (Western blot analysis) in hearts of rats treated with alinidine for 1, 2, and 3 weeks (A1W, A2W, and A3W). A, Immunoreactivity for VEGF is located at 27 kDa. B, Quantitative analysis of proteins confirmed that VEGF was higher in the alinidine-treated rats than in the controls after 2 weeks of treatment (*P<0.05) and was increased further at 3 weeks (**P<0.01). Data in panel B are means of 3 experiments.

Bradycardia-Induced Angiogenesis Is VEGF Dependent
To test the hypothesis that the coronary angiogenesis characteristic of bradycardia is VEGF dependent, we administered VEGF-neutralizing antibodies to a group of rats undergoing alinidine treatment. The neutralizing antibodies were administered 5 times during a 2-week period. As seen in Figure 7, the increase in capillary length density that occurs with alinidine treatment was completely blocked in rats that received the VEGF-neutralizing antibodies. The mean for the alinidine and anti-VEGF group was 38% lower than that for the group that received alinidine alone. Because capillary length density in the alinidine and anti-VEGF group was significantly lower than that in the controls, we noted that the neutralizing antibodies compromised the angiogenesis that occurs during normal growth. The rats grew considerably during the 2-week treatment period; ie, their weights increased by 40 to 50 g. This growth is normally associated with a nearly proportional capillary angiogenesis.

View larger version (29K): [in this window] [in a new window]   Figure 7. Capillary length density (CLD) in rats treated with alinidine for 2 weeks. The VEGF monoclonal neutralizing antibody prevented the alinidine-associated increase in CLD (**P<0.001). The values of the alinidine+anti-VEGF group are 38% lower than those of the alinidine group and 26% lower than those of the nontreated controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The most important finding from this study is that myocardial angiogenesis in response to chronic bradycardia is dependent on VEGF. We found that VEGF is upregulated during chronic bradycardia and that administration of VEGF-neutralizing antibodies during 2 weeks of alinidine-induced bradycardia prevents the angiogenesis associated with bradycardia and also compromises the angiogenesis associated with normal growth during this period. Our data also show that bFGF mRNA was unchanged in rats with chronic bradycardia and, therefore, is not likely a factor in angiogenesis in this model, unless it plays a permissive role in conjunction with VEGF. These data support the hypothesis that bradycardia, which facilitates an increase in end-diastolic filling, enhances stretch on the myocardium and serves as a stimulus for VEGF and angiogenesis.

Bradycardia-Induced Coronary Angiogenesis
We used a model of chronic bradycardia, because previous work has shown a strong angiogenic response of left ventricular capillaries to either electrical pacing³ or the bradycardic drug alinidine.⁷ Bradycardic pacing in rabbits was found to increase capillary density in relation to the duration of pacing.⁴ In rabbits with aortic valve–lesioned hearts, chronic bradycardic pacing resulted in a 43% increase in left ventricular capillary density.⁶ In a subsequent study, alinidine was used to affect bradycardia in rats,⁷ and capillary angiogenesis was documented by an increase in capillary/myocyte ratio after 5 weeks of treatment.

Alinidine reduces heart rate by decreasing sinus and ventricular rate without altering blood pressure.²⁸ The drug appears to function in minimizing the effects of ischemia, as indicated by data that document attenuation of increased heart rate and the onset of ischemia in dogs subjected to coronary artery occlusion and reperfusion.²⁹ Moreover, it also has been shown to prevent hypoperfusion of the endomyocardium during low perfusion of the isolated rat heart.³⁰

Myocardial angiogenesis in the heart has been consistently documented in models of (1) chronic increases in coronary perfusion, eg, via vasodilators or administration of thyroxine, and (2) in mechanically or pharmacologically induced bradycardia.^{1 7} Chronic increases in myocardial perfusion and chronic bradycardia both favor enhancement of mechanical factors. Increased flow provides for increased wall tension and stretch of the capillary wall. In bradycardia, the prolongation of diastole facilitates (1) a longer period in which capillary diameters are maximal and (2) enhanced diastolic filling, which stretches the myocytes and capillaries. We submit that such mechanical factors provide the primary stimulus for myocardial angiogenesis during bradycardia.

Role of VEGF in Angiogenesis in the Heart
VEGF not only is an endothelial cell mitogen, which mediates its effect by binding to tyrosine kinase receptors and activating PKC and PLC,³¹ but it also stimulates other important angiogenic events, eg, cell migration,³² tube formation,²³ maintenance and repair of luminal endothelium, and a local endogenous regulation of endothelial cell integrity.³³ These events play major roles in the multiple steps required for angiogenesis. Data from our laboratory implicate VEGF in coronary angiogenesis in both prenatal and early postnatal development, which are time periods of marked myocardial neovascularization.⁴⁰ Upregulation of the growth factor has also been reported in the adult heart during ischemia or hypoxia.^{18 34 35 36} Li et al¹⁴ observed an initial rapid rise in mRNA expression for VEGF and its receptors throughout the heart 1 hour after myocardial infarction; after 6 hours, the increase was limited to the edge of the myocardial infarction zone, where angiogenesis was occurring.

VEGF Is a Link Between Bradycardia and Coronary Angiogenesis
Initially, heart rate was depressed after an alinidine injection for at least 2 hours (Figure 1). By the second week of treatment, we were able to document a chronic bradycardia, which became more marked after 2 and 3 weeks. Accordingly, heart rate was chronically depressed after 1 week of treatment (Figure 2).

In contrast to our recent work that documented an upregulation of bFGF during the first 2 days of thyroxine administration concomitant with capillary proliferation,⁹ we found no change in bFGF mRNA after 1, 2, or 3 weeks of alinidine administration. Therefore, even though bFGF is a potent angiogenic factor, it does not appear to be triggered during bradycardia-induced myocardial neovascularization. Hudlicka et al⁷ also did not observe an increase in bFGF mRNA in rabbits in which bradycardia was induced by electrical pacing. In contrast, mRNA for TGF-ß was increased in paced hearts. The latter finding suggests an important link to the current data, indicating an increase in VEGF mRNA in hearts with chronic bradycardia, given that TGF-ß can upregulate VEGF.³⁷ TGF-ß appears to facilitate myocardial VEGF upregulation by stretch, as indicated by the work of Li et al.³⁸ When they elevated end-diastolic pressure to 35 mm Hg in an isolated perfused Langendorff preparation for 30 minutes, they found nearly a 6-fold increase in VEGF mRNA level not only in the chamber subjected to stretch (left ventricle) but also in the unstretched right ventricle, thus raising the possibility of a soluble factor mediating stretch-induced induction of VEGF expression. This increase was negated by administering anti-TGF-ß neutralizing antibodies. Their data, thus, indicate a TGF-ß–mediated upregulation of VEGF mRNA in a nonischemic, nonhypoxic model of stretch. Unpublished data from our laboratory (W. Zheng, R.J. Tomanek, 1999) document that cyclic stretch of isolated cardiac myocytes causes a marked increase in VEGF mRNA. This finding is consistent with previous studies that have documented that stretch of the ventricle³⁸ or isolated cardiac myocytes³⁹ causes a marked increase in VEGF mRNA. Moreover, our preliminary data indicate that the conditioned media from stretched cardiac myocytes enhances in vitro DNA synthesis, migration, and tube formation of coronary microvascular endothelial cells.

Conclusions
Our data indicate that the marked capillary angiogenesis in response to bradycardia induced by alinidine is associated with upregulation of VEGF mRNA and followed by enhanced VEGF protein. We were able to completely block the angiogenic response with VEGF-neutralizing antibodies. These data suggest that mechanisms associated with bradycardia provide a signal for the enhancement of this angiogenic polypeptide, which is responsible for the myocardial angiogenesis observed. In contrast, bFGF expression is unchanged during this period of angiogenesis, which suggests that it does not play a direct role.

	Acknowledgments

 
This work was supported by funds from NIH Grant R01 HL-48961. We acknowledge Boehringer Ingelheim Ltd for their kind gift of alinidine.

Received March 23, 1999; accepted April 23, 1999.

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