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Hypoxic Preconditioning Triggers Myocardial Angiogenesis: a Novel Approach to Enhance Contractile Functional Reserve in Rat with Myocardial Infarction
J Mol Cell Cardiol 34, 335–348 (2002) doi:10.1006/jmcc.2001.1516, available online at http://www.idealibrary.com on
Hypoxic Preconditioning Triggers Myocardial Angiogenesis: a Novel Approach to Enhance Contractile Functional Reserve in Rat with Myocardial Infarction Hiroaki Sasaki1, Shoji Fukuda1, Hajime Otani2, Li Zhu1, Genbu Yamaura1, Richard M. Engelman3, Dipak K. Das1 and Nilanjana Maulik1 1
Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030-1110, 2 Department of Thoracic and Cardiovascular Surgery, Kansai Medical Center, Osaka, Japan and 3 Baystate Medical Center, Springfield MA, USA (Received 9 November 2001, accepted 11 December 2001)
H. S, S. F, H. O, L. Z, G. Y, R. M. E, D. K. D N. M. Hypoxic Preconditioning Triggers Myocardial Angiogenesis: a Novel Approach to Enhance Contractile Functional Reserve in Rat with Myocardial Infarction. Journal of Molecular and Cellular Cardiology (2002) 34, 335–348. A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of “angiogens”, mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore we sought to examine a novel method of stimulating myocardial angiogenesis through hypoxic preconditioning at both capillary (using anti-CD31) and arteriolar (using anti- smooth muscle actin) levels and also investigate whether such treatments could preserve left ventricular contractile functional reserve and regional blood flow by increasing vascular endothelial growth factor (VEGF). Male Sprague-Dawley rats were randomly divided into four groups: normoxia+sham surgery (CS), normoxia+permanent left anterior descending coronary artery (LAD) occlusion (CMI), hypoxic preconditioning+sham surgery (HS) and hypoxic preconditioning+permanent LAD occlusion (HMI). Rats in the preconditioned groups were subjected to systemic hypoxemic hypoxic exposure (10±0.4% O2) for 4 h followed by a 24 h period of normoxic reoxygenation prior to undergoing LAD occlusion. Rats in the normoxia group were time matched with the preconditioned group and maintained under normoxic conditions for a 28 h period prior to LAD occlusion. Western blot analysis was performed to measure VEGF expression and TUNEL staining with endothelial cell-specific antibody, anti-VWF, was used to examine endothelial apoptosis. One, two and three weeks after the LAD occlusion, baseline left ventricular pressures were monitored and recorded. Pharmacological stress tests with dobutamine infusion in progressively increasing doses revealed significantly elevated contractile reserve at each dose point in the HMI group compared to the CMI group. The HMI group displayed statistically significant increases in capillary as well as arteriolar density after 1, 2 and 3 weeks post-operation. Blood flow was also significantly elevated in the HMI groups when compared to the CMI group. The extent of endothelial cell apoptosis was found to be inversely proportional to VEGF expression. It was concluded that hypoxic preconditioning stimulates myocardial angiogenesis to an extent sufficient to exert significant cardioprotection in a rat model of myocardial infarction progressing to heart failure as evidenced by increased capillary/arteriolar density and enhanced ventricular contractile functional reserve. 2002 Elsevier Science Ltd. All rights reserved.
K W: Myocardial infarction; Hypoxic preconditioning; Oxidative stress; Angiogenesis; VEGF; Apoptosis. 0022–2828/02/030335+14 $35.00/0
2002 Elsevier Science Ltd. All rights reserved.
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Introduction Various coronary interventions have made a remarkable contribution to the treatment of coronary artery disease. The ultimate goal of these interventions is to improve arterial blood supply through the formation of coronary collateral vessels (angiogenesis) to potentially ischemic myocardium. Factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) which stimulate collateral growth, are expected to exert a protective effect against myocardial infarction. Indeed, VEGF is a major regulator of angiogenesis and vasculogenesis.1 A strong temporal and spatial correlation exists between VEGF expression and angiogenesis in both animals and humans.2,3 The biological functions of VEGF, triggered by external stimuli, are initiated through the activation of intracellular signal transduction cascades involving specific kinases.4 VEGF behaves as a classical stressinduced gene in this respect. Recent studies documented that hypoxia induces VEGF gene by a 5′promoter sequence which binds to the hypoxiainducible factor-1 (HIF-1),5 which is also induced and stabilized by hypoxia.6 Among various triggers of angiogenesis, tissue hypoxia has been identified as being an important stimulus for the induction of new vessel growth, especially at the capillary level.7 In the myocardium, hypoxia occurs during ischemic insult, usually followed by reoxygenation and revascularization. Preconditioning induced by cyclic episodes of brief periods of ischemia and reperfusion or hypoxia and reoxygenation has been found to possess profound cardioprotective ability in lowering myocardial infarction by reducing both cell necrosis and apoptosis and in improving post-ischemic contractile function.8,9 The cardioprotective abilities of preconditioning last up to 4–6 h and reappear after 24 h (2nd window of preconditioning), lasting for another 48 h.10 To the best of our knowledge, no study has ever been performed to examine the prolonged effects of preconditioning on myocardial angiogenesis. The aim of this study was, therefore, to determine the effects of hypoxic preconditioning on myocardial angiogenesis including blood flow and cardiac performance in the myocardial infarction model. The results documented that hypoxic preconditioning improved myocardial performance and angiogenesis at the level of capillary and arteriolar density after 1 week of LAD
occlusion, the effect lasting even after 3 weeks. Such angiogenic effects of preconditioning were found to be due to its ability to augment VEGF protein expression which presumably played a crucial role in reducing endothelial cell apoptosis, a proven determinant for congestive heart failure.
Materials and Methods All animals used in this study received humane care in compliance with the principles of laboratory animal care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals published by NIH.
In vivo hypoxia/reoxygenation Rats weighing 275–300 g were randomly divided into four groups as outlined in Figure 1. Based on previous experiments performed in our laboratory, we have determined that a 4 h duration of systemic hypoxemic hypoxia followed by 24 h of reoxygenation induces a very strong induction of angiogenic factors.11 We therefore used this duration of hypoxic exposure to effect hypoxic preconditioning in our preconditioned animal groups. Rats in the hypoxic preconditioned group (HMI) were subjected to a hypoxic challenge, using a gas mixture (10% O2/90% N2),12 of 4 h duration in an anesthesia chamber. An in-flow aperture was used for the gas mixture and an out-flow aperture connected to a gas absorption cannister (A.M. Bickford Inc., Wales Center, NY, USA). A KE-25 galvanic cell O2 sensor (Kent Scientific, Litchfield, CT, USA) was used to continuously monitor the %O2 concentration, the signal being amplified through a TRN-005 amplifier (Kent Scientific). The ambient O2 concentration was gradually lowered from 20.9% down to 10% over the course of 30 min. Once stabilization was obtained, the in-flow rate was adjusted in order to maintain the O2 concentration at 10±0.4% for a 4 h duration. These rats were then maintained in a normoxic environment for 24 h until preoperative preparation. Rats in the control group (CMI) were similarly maintained in a normoxic environment in a time-matched manner (29 h) up until preoperative preparation. Two to three animals were excluded from each permanent
Please address all correspondence to: Nilanjana Maulik, PhD, Molecular Cardiology Laboratory, Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, Farmington, CT 06030-1110, USA. Fax: (860) 679-4606; Tel: (860) 6792857; E-mail: [email protected]
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Figure 1 Outline of experimental protocol. Rats randomly assigned to various experimental groups (n=12) were subjected to LAD ligation following different exposure protocols. CMI group: normoxia+LAD occlusion. HMI group: hypoxia/reoxygenation+LAD occlusion. Rats which were randomly assigned to the next two groups served as the respective sham-operated controls for groups CMI and HMI, respectively. CS group: normoxia+sham surgery; HS group: hypoxia/reoxygenation+sham surgery.
occlusion group either due to post-operative mortality or morbidity within 24 h, or based on infarct size being less than 50%.
Survival surgery and LAD occlusion After the 24 h reoxygenation period/time-matched normoxic period, rats were anesthetized with Ketamine HCl (100 mg/kg i.p.) and Xylazine (10 mg/ kg i.p.).13,14 Cefazolin (25 mg/kg i.p.) was administered by i.v. injection as preoperative antibiotic cover. The animals were placed in the supine position and body temperature was maintained at 37°C by means of a water circulating thermal heating pad. A midline neck incision was made and the trachea was identified. Tracheostomy was performed by making a vertical incision approximately 2 mm long in the trachea. A polyethylene tube (2 mm O.D.)15 was introduced through the opening and positive pressure ventilation with room air was commenced (stroke volume 12 ml/kg,15–17 tidal volume 2.5 ml18 and respiratory rate 70/min19 using a small animal respirator (model 683, Harvard Apparatus, Holliston, MA, USA). The tube was secured in place by means of stay sutures. An intercostal thoracotomy in the left fourth intercostal space was
performed.13,14,16,19 The heart was exposed and the pericardium incised. The left coronary artery was identified between the left atrial appendage and the pulmonary artery outflow tract. A 6-0 suture was passed beneath the vessel 1 to 2 mm from its origin. Care was taken not to enter the ventricular cavity. In the sham-operated groups (CS and HS), the suture was left in place up until chest closure but was not ligated. After completion of all protocols, the chest wall was closed in three layers using 40 silk sutures. A catheter fashioned from PE-190 tubing attached to a 12 ml syringe with a stopcock was used as a chest tube to evacuate air from the pleural space as the last stitch was tightened. Postoperative pain relief was achieved by the administration of buprenorphine (0.05–2.5 mg/kg s.c.).
Non-survival surgery and baseline cardiac function One, two and three weeks after the operative intervention, the rats were anesthetized and ventilated as described above. A small incision was made to the right of the midline in the neck. The right internal jugular vein was identified and a PE 50 catheter was introduced into the vein. The proximal end of the catheter was connected to a low pressure
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transducer. The inserted catheter tip was then advanced until it reached the right atrium and the central venous pressure (CVP) signal was obtained. The same procedure was used to place a catheter in the right carotid artery where the catheter was used to monitor aortic blood pressure. The inserted tip of this catheter was advanced down until it reached the left ventricular lumen and the left ventricular pressure (LVP) signal was obtained. All pressure signals were monitored, analysed and recorded in real time using the Digimed data acquisition and analysis system (Micromed, Louisville, KY, USA). Heart rate (HR), developed pressure, and dP/dtmax were all calculated from the continuously obtained LVP signal.
Cardiac stress testing with dobutamine After baseline left ventricular functional parameters were recorded, rats were subjected to pharmacological cardiac stress testing with dobutamine infusion in progressively incremental doses to reveal the extent of left ventricular contractile functional reserve. Dobutamine was infused i.v. through the venous catheter by means of a microinjection pump (Harvard Apparatus, Holliston, MA, USA) to achieve doses of 1, 2, 3 and 5 g/kg/min. Based on previous studies in our laboratory, we have found that administration up to a dose of 5 g/kg/ min is necessary to observe the plateau of the doseresponse curve. Administration was maintained at each dose for a duration of 2 min. dP/dtmax and other left ventricular functional parameters were recorded for each dose 90 s after the start of each dose.
Tissue retrieval and processing After measurement of all functional parameters, the animals were systemically heparinized to prevent intravascular coagulation. The animals were killed with 1 ml of 30 m KCl injected i.v. Hearts were excised, the ventricular lumen was flushed clean with Ringer’s lactate solution and the ventricles were either frozen in liquid nitrogen for later use in biochemical studies (Western Blot analysis) or were sectioned approximately 5–6 mm from the apex into approximately 2 mm thick transverse sections. The sections were then fixed in 10% buffered formalin and embedded in paraffin using standard procedures to perform immunohistochemical analysis.
Measurement of capillary and arteriolar density The standard deparaffinization protocol was used. Endothelial cells were labeled using mouse monoclonal anti-CD31/PECAM-1 (1:100, Pharmingen, San Diego, CA, USA) followed by a biotinylated horse anti-mouse secondary antibody (1:200 dilution). The reaction product (brown) was visualized with DAB substrate using the Vector ABC Vectastain Elite Kit (Vectorlabs, Burminghame, CA, USA) and was counterstained with methyl green (Vectorlabs). On separate slides, vascular smooth muscle cells were labeled using mouse monoclonal anti-smooth muscle actin (1:50, Biogenex, San Ramon, CA, USA) followed by a biotinylated ratadsorbed horse anti-mouse secondary antibody (1: 200 dilution). The reaction product (violet) was visualized with VIP substrate using the Vector ABC Vectastain Elite Kit (Vectorlabs). Images were captured and stored in digital tiff file format for later image analysis. Counts of capillary density and arteriolar density per mm2 were obtained after superimposing a calibrated morphometric grid on each digital image using Adobe Photoshop Software.
Western blot analysis for VEGF To quantify the abundance of the angiogenic factor VEGF, we performed Western blot analysis using a specific primary antibody.11
TUNEL assay for apoptotic endothelial cells At the end of the experiments, the heart was perfusion-fixed with 10 ml of 10% formaldehyde in 0.1 mol/l phosphate buffer (pH 7.2) at room temperature. Transverse ventricular slices were embedded in paraffin, cut into 4 micrometer sections and deparaffinized with a graded series of xylene and ethanol solutions. The sections were then assayed for in situ terminal transferase labeling (TUNEL) using Apop Tag Plus (Oncor Inc., Gaithersburg, MD, USA). Negative control slides were processed with the TdT enzyme excluded. The slides were first stained with peroxidase-conjugated sheep polyclonal anti-digoxigenin antibodies and diaminobenzidine and with methyl green as a counterstain. For the detection of endothelial cell apoptosis, the serial sections were first stained by TUNEL with fluorescein isothiocyanate (FITC) and then incubated with polyclonal rabbit anti-von Willebrand factor antibodies (Dako Japan, Tokyo,
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Japan) followed by incubation with tetrarhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Dako Japan). FITC and TRITC fluorescence were viewed with a confocal laser microscopy (Fluo View, Olympus Co., in Tokyo, Japan). The number of TUNEL-positive endothelial cells was counted on 60 high power fields (magnification ×600) from the endocardium through the epicardium of the left ventricular free wall.
Measurement of percent circumferential left ventricular infarct size Infarct size and area at risk were measured after myocardial infarction (n=6).20 One ml of 5% Evan’s blue dye was infused into the heart through the apex to mark the risk zone as unstained (not blue) tissue. Frozen hearts were then cut into 2 mm thick slices parallel to the atrioventricular groove. Sections were thawed and incubated in a 1% tetrazolium chloride (TTC) phosphate buffered solution (pH 7.4) at 37°C for 15 min and fixed in 10% formalin to increase the contrast of the Evan’s blue and TTC staining. Tissue sections were compressed to a uniform 2 mm thickness by placing them between two glass plates separated by a 2 mm spacer. The infarct size was determined between the point of ligation and the apex. The area at risk was determined with Evan’s blue dye staining, whereas the non-infarcted (stained red) and infarcted (unstained) areas were determined after incubation with 1% triphenyltetrazolium chloride. With the use of NIH imaging, the volumes were calculated. Infarct size was reported as a percent of the area at risk (volume of infarcted tissue divided by volume of area at risk) ×100.
Measurement of blood flow by neutron microsphere technique We used neutron activation technique for the assay of stable isotopically labeled gold (Au) microsphere for measuring regional rat myocardial perfusion.21 Neutron activation is a very accurate procedure in which trace quantities of isotope of interest in a sample are activated and the radiation emitted by this method is measured with a high-resolution detection technique. Stable gold labeled microspheres (15×10,15 15 m) were injected in an in vivo rat model of myocardial infarction through the left atrium under anesthesia. Simultaneously, a reference blood sample was drawn from a femoral arterial catheter using a withdrawal pump (model
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PHD 2000 programmable, Harvard Apparatus, Millis, MA, USA) to measure absolute myocardial blood flow. The withdrawal rate was set at 1 ml/min and collected over a 2 min interval; a total of 2 ml reference blood was collected. At the end of the experiments the animals were killed and hearts (risk area or border zone of the left ventricle) from the various groups were collected and weighed. The tissue and the blood samples were dried in a 70°C oven overnight. All tissue and blood samples were sent for further analysis to BioPAL, (Worcester, MA, USA), where they were exposed to a field of neutrons. The samples were stored for 48 h to allow short-lived activation products to decay. After this period, spectrophotometric analysis was performed. The samples were counted for 2 min and during this period samples were corrected for tracer decay. The absolute blood flow was determined in ml/min/ gram tissue. The microsphere concentration of each segment (in disintegrations per min (dpm)/g) was normalized to the microsphere concentration evaluated in the 2 min reference blood collection (in dpm.min/ml).
Statistical analysis Results are expressed as mean±standard error of the mean (...). Differences between groups were tested for statistical significance by two-way analysis of variance (ANOVA) and Student’s t-test, with the Bonferroni adjustment for multiple comparisons. The dose response curves to dobutamine of the various groups were tested for significant differences by two way repeated measure ANOVA. Differences were considered significant at P<0.05.
Results Effects of hypoxic preconditioning on capillary and arteriolar density For the measurement of capillary density, eight non-overlapping random fields were selected from the endocardial regions of non-infarcted (border zone) zones of the left ventricles and examined at 400× magnification. The HMI group displayed a statistically significant increase in capillary density after 1 week of operation when compared to the control sham-operated group (CS) [1862±67 counts/mm2 in HMI v 1600±16 counts/mm2 in control baseline (BL)]. There was mild-to-moderate increase in capillary density in the HMI group
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Figure 2 Left ventricular endocardial capillary density. Tissue sections were processed for CD31 staining. Eight nonoverlapping random fields were selected from endocardial regions of the left ventricle from each heart (16 fields per region per heart, 64 fields per region per group, magnification ×400 were used, n=6). Images were captured and stored in digital tiff file format for image analysis. Where ∗ P<0.01, compared to sham operation; † P<0.01, compared to CMI; § P<0.05, compared to CS.
compared to the HS animals 2 days post-operatively (1811±56 v 1862±37 counts/mm2). However, the capillary density increased significantly 4 days post-operatively when compared to the CMI and HMI groups. The increased capillary density was maintained even 1, 2 and 3 weeks post-operation when compared to sham-operated groups (Fig. 2). The most interesting observation was the increased capillary density (CD) in the hypoxic sham (HS) group compared to the control sham group (CS). CD was higher in the hypoxic sham group after 2 days (1814±56 counts/mm2) compared to the control baseline group (CS) (1642±43 counts/ mm2). Therefore, hypoxic preconditioning itself demonstrated increase in CD compared to the nonhypoxic group of control (Fig. 2). Differences between sham groups were only demonstrable in capillary density. For the measurements of arteriolar density, eight non-overlapping random fields were selected from the endocardial regions of non-infarcted zones of
the left ventricles and examined at 2000× magnification. Arteriolar counts (purple color) for each field in the same region were averaged to yield the value of the particular density measurement. The arteriolar density was significantly elevated in the HMI group after 1 week (2.23±0.12 HMI v 1.53±0.02 counts/mm2 in CMI) (Fig. 3). Again, the increased arteriolar density was maintained even 2 and 3 weeks post-operatively in the HMI group compared to the CMI group.
Effect of hypoxic preconditioning on dobutamine stress test: left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and heart rate (HR), maximal dP/dt dP/dtmax However, statistically significant differences were noticed in LVSP and LVEDP between the group’s representative of the sham/baseline v all the other permanently occluded groups (CMI and HMI) after
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Figure 3 Left ventricular endocardial arteriolar density. Left ventricular tissue sections were labeled using monoclonal anti-smooth muscle actin and eight non-overlapping random fields were selected from the endocardial region of the left ventricle. Where ∗ P<0.01, compared to sham operation; † P<0.01, compared to CMI; § P<0.05, compared to CS.
1, 2 and 3 weeks (Fig. 4, only 3 weeks’ data shown). There was no difference in baseline LV function between the groups. No statistical (two-way ANOVA analysis) difference was found for heart rate between the groups even after 3 weeks of LAD occlusion (data not shown). Pharmacological cardiac stress testing with dobutamine infusion in incremental doses revealed differences in the extent of cardiac contractile reserve between groups (CMI and HMI). This was evident from differences in the extent of change in dP/dtmax values displayed by various groups during the course of such stress testing after 1 week of intervention. The differences remained statistically significant even after 2 and 3 weeks (Fig. 5, only 1 and 3 weeks’ results are shown). The hypoxic preconditioned group displayed significantly elevated contractile reserve at each dose point of evaluation compared to the CMI group, but the extent of such reserve was lower than that displayed by sham operated groups or by control rats (all of which exhibited similar and statistically indifferent values of dP/dtmax throughout the course of stress testing). This enhanced preservation of contractile reserve in the hypoxic preconditioned group was apparent at the 1 g/kg/ min dose (6419±488 mmHg/s in the HMI group
v 5594±197 mmHg/s in the CMI group after 3 weeks) and this trend persisted at all subsequent doses up to 5 g/kg/min.
Measurement of infarct size Heart sections obtained from hypoxic preconditioned animals (HMI) after 24 h demonstrated significantly lowered infarct size, as expected (30.56±3.7% of the area at risk) compared to the infarct size in non-preconditioned animals (CMI) (51.69±6.01% of the area at risk) (Fig. 6).
Measurement of left ventricular blood flow Left ventricular blood flow was measured in rat myocardium 1 and 3 weeks post-operatively by neutron microsphere technique. It was found that in the HMI group, the blood flow measured in the risk area was significantly higher compared to the CMI group. Therefore, hypoxic preconditioning was not only able to enhance the capillary density and arteriolar density but at the same time it was also able to increase the blood flow to make it functional.
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Figure 5 Dose response curves of dP/dtmax during dobutamine stress test. Changes are shown in dP/dtmax in mm of mercury per second (mmHg/s) from baseline with increasing dobutamine boluses (0–5 g/kg/min) in hearts 7 days and 3 weeks after MI. The dose response curves for dobutamine for the various doses were tested for significant differences by repeated measure by ANOVA. Differences were considered significant at P<0.05. Where ∗ P<0.01 compared to sham, † P<0.01 compared to CMI.
Figure 4 Dose response curves of LVSP and LVEDP during dobutamine stress test. Changes are shown in the systolic and end-diastolic pressure (mmHg) from baseline with increasing dobutamine boluses (0–5 g/kg/min) in hearts 3 weeks after MI. The dose response curve for dobutamine for the various doses was tested for significant differences by repeated measure by ANOVA. Differences were considered significant at P<0.05. Where ∗ P<0.01 compared to sham operation.
Therefore in the CMI group the flow rate was 2.22±1.31 whereas in the HMI group it was 3.83±1.41 after 1 week. After 3 weeks, the blood flow rate in the HMI group was increased further to 4.0±0.22 v 1.8±0.20 in the CMI group (Fig. 7).
Effect of hypoxic preconditioning on VEGF expression Protein expression profile of VEGF was found to be significantly elevated after 2 (33%) and 3 (63.3%) weeks of LAD occlusion in the HMI group compared to CMI and/or baseline control or sham group. However, in the CMI group, after 3 weeks of LAD occlusion the VEGF expression level was increased moderately (20%) compared to the baseline control
Figure 6 Infarct size of the hearts, expressed as a percentage of the area at risk in rat myocardium subjected to permanent LAD occlusion for 24 h. Results are expressed as means±... of six hearts/group. ∗ P<0.05 compared to CMI.
(Fig. 8). Hypoxic preconditioning induced VEGF in the hypoxic group even before MI (HS) (50% compared to the non-hypoxic control).11 The protein level was measured by densitometry scanning and normalized with -actin.
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Effect of hypoxic preconditioning on endothelial cell apoptosis
Figure 7 Regional blood flow with neutron microsphere injection. Regional blood flow was estimated after 1 week and after 3 weeks of LAD occlusion. Differences were considered significant at P<0.05. Where ∗ P<0.01 compared to CMI. Ε CMI, Γ HMI.
The number of apoptotic endothelial cells was found to be inversely proportional to the extent of VEGF expression in our model. A higher proportion of endothelial cells was observed to be apoptotic in the CMI group of myocardium when compared to the HMI group after 2 days (30±0.47% in CMI v 14±2.3% in HMI); 4 days (18±0.47% in CMI v 7.8%±1.7% in HMI); 1 week (9±0.47% in CMI group v 3±1.2% in HMI), 2 weeks (10±0.5% in CMI v 2±0.7 in HMI) and 3 weeks (9±0.4% in CMI v 1.2±0.4% in HMI) after LAD occlusion. Thus, in both the CMI and HMI groups, the extent of apoptosis was found to be extremely significant in the early stage after LAD occlusion (2 and 4 days and 1 week). However, the number of apoptotic cells was reduced in the CMI group after 1, 2 and
Figure 8 Representative Western blots showing the effects of systemic hypoxia and LAD occlusion on the expression of VEGF in rat myocardium in vivo after 2, 4, 7 and 21 days. VEGF proteins were expressed as 40 kDa. Similar results were obtained in six independent experiments performed in triplicate. Densitometric scanning of blots was used to determine levels of proteins relative to baseline control (sham). Where ∗ P<0.01 compared to baseline.
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3 weeks, but remained significantly higher when compared to the HMI group (Fig. 9).
Discussion The results of our study demonstrate for the first time that hypoxic preconditioning induced by whole body hypoxia/reoxygenation induces myocardial angiogenesis in a rat model of myocardial infarction as evidenced by increased capillary/arteriolar density and blood flow. Our findings also documented increased VEGF expression along with decreased endothelial apoptosis during hypoxic adaptation. Several recent studies have shown that hypoxic preconditioning, like ischemic preconditioning, can attenuate post-ischemic ventricular dysfunction caused by stunning.22,23 Although the exact mechanism of preconditioning is under considerable debate, there is a general agreement that myocardial preservation is achieved through a biphasic intrinsic mechanism. The early effect is most likely to occur through the modulation of intracellular signaling mechanisms, and the late effect is believed to be mediated by gene expression and protein synthesis.24 Cellular injury occurs both during hypoxia and after reoxygenation. It was observed that brief hypoxia followed by reoxygenation causes more damage at the cellular level than does more prolonged hypoxia alone. A major component of hypoxiainduced death was due to apoptosis, as observed by DNA laddering.25 Recently we have shown for the first time a comprehensive evaluation of the early effects (4 h hypoxia followed by 24 h reoxygenation) of in vivo systemic hypoxemic hypoxia on the protein expression and distribution profiles of the VEGF system and the Ang-Tie system in adult rat myocardium tissue. In this report we have demonstrated that hypoxia-reoxygenation increases the expression of both VEGF and Flk-1/ KDR in the myocardium.11 Hypoxic preconditioning induced VEGF and increased capillary density even before MI (HS). The relative time course of protein expression in response to hypoxic preconditioning, as indicated from our previous experiments,11 seems to suggest the involvement of the VEGF system as well as the Ang-Tie system in early angiogenesis.
In vivo, the early angiogenic response to systemic hypoxemic hypoxia in adult rat myocardium appears11 to be mediated not only through the induction of VEGF and its receptors Flk-1 and Flt-1, but also through the concurrent induction of Ang1, Ang-2 ,Tie-1 and Tie-2 (Table 1). The increased presence of Ang-2 may actually be pro-angiogenic in nature by effecting dissolution of surrounding matrix, thereby setting up a suitable environment in which endothelial cell migration and capillary sprouting can occur. Examination of non-MI left ventricle (border zone tissue) by anti-CD31 revealed a significant increase in the capillary density in the hypoxic preconditioned group (1800±46 v 1650±30 counts/mm2) compared to the control non-hypoxic group, confirming that modulation of angiogenic factors and their receptors by hypoxia/ reoxygenation was able to stimulate capillary proliferation even in the non-MI animals. Differences between sham groups were only demonstrable in capillary density, as shown in the Result’s section. VEGF-induced angiogenesis has been found to be associated with enhanced cell survival in human umbilical vein endothelial cells in vitro.26 This effect is thought to be due to a reduction in apoptotic endothelial cell death. Increased expression of the anti-apoptotic protein Bcl-2 and reduced expression of the pro-apoptotic protein caspase3 are possible biochemical mediators of such effects of VEGF.27 The observed effect of VEGF is mediated via the PI3 Kinase/Akt pathway, for which activation of the VEGF receptor Flk-1/KDR has been shown to be essential.26 Studies have also shown that hypoxia induces NFB activation in HUVEC cells, which leads to cell survival after exposure to TNF-alpha. In addition, we have demonstrated that in vivo, oxidative stress induced by systemic inhalational hypoxemic hypoxia can increase the DNA binding activity of NFB along with the increased expression pattern of VEGF in rat myocardium.28 According to our previous observations baseline VEGF expression was mainly in the ventricular myocardium with strong localization in the region of the coronary arterial wall where vascular smooth muscle appeared to stain positive. Hearts obtained from rats subjected to hypoxia displayed an increase in intensity of staining for VEGF in the 1 h hypoxia group and this enhanced level of immunoreactivity
Figure 9 TUNEL assay for apoptotic endothelial cells was performed on confocal laser microscopy as described in the Methods section. Representative photomicrographs show immunohistochemical staining of extended DNA. Where A= CS; B=HS; C=CMI, 1 week; D= HMI, 1 week; E=CMI, 2 weeks; F=HMI, 2 weeks; G=CMI, 3 weeks; H=HMI, 3 weeks. Note that apoptotic endothelial cells are lying on the luminal surface of the coronary vessels. Magnification ×1200.
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Table 1 Angiogenic protein expression profile in the non-MI hypoxic preconditioned myocardium compared to the normoxic control (% normoxic control), where H=hypoxia, R=24 h reoxygenation Angiogenic factors and their receptors VEGF Flk-1 Flt-1 Angiopoietin-1 Angiopoietin-2 Tie-1 Tie-2
1 h H/R (%)
2 h H/R (%)
4 h H/R (%)
50.8 41.5 325.0 23.8 31.3 533.0 93.9
51.9 22.0 201.0 30.0 14.8 586.7 120.5
50.0 12.8 149.9 50.0 same as normoxic control same as normoxic control 53.4
persisted in the 2 h and 4 h groups, where surrounding myocardium also displayed positive staining.11 In another study, myocytes from a sham group of animals showed detectable amounts of VEGF mRNA in both the ventricles. However, after 6 h of coronary ligation, decreased expression of VEGF mRNA was observed in the infarcted area of the anterior left ventricle. Six weeks post-ligation, mRNA levels in myocytes were not clearly distinguishable from the control hearts.29 In our experimental set up, increased VEGF in HMI (33% after 1 week and 64% after 3 weeks of hypoxic preconditioning) probably potentiates a survival signal to reduce the extent of apoptosis associated with myocardial infarction when compared to the non-preconditioned CMI group of animals. This phenomenon perhaps accelerated the capillary and arteriolar density and increased regional myocardial blood flow in the hypoxic preconditioned myocardium after coronary occlusion. As expected, the extent of infarction after coronary occlusion was reduced significantly after 24 h and the amount of viable myocardium was found to be more abundant in the HMI group compared to the CMI group. Possible mechanisms to salvage infarcted myocardium in the HMI group compared to the CMI group may be due to increased VEGF expression which increases flow through the artery, opening of latent collateral vessels by its vasodilating effect, induction of vascular growth and direct cellular protection against ischemia, as we have observed by reduced endothelial apoptosis. All these parameters no doubt significantly protect the cardiac function in the HMI group compared to the CMI group of animals. Thus, our results suggest that a relationship exists between programmed cell death (endothelial) and blood flow, and such a relationship is dependent on the level of survival factor VEGF. Existing evidence indicates that hypoxia results in the expression of a number of growth factors that influence vascular endothelial
cell viability.30 Furthermore, it was suggested that in the abnormal regression of retinal capillaries (due to hypoxia), VEGF acts as a survival factor and can prevent apoptosis associated with regression.31,32 Thus VEGF isoforms are considered the prime candidates for circulating survival stimuli. Pharmacological cardiac stress testing with dobutamine proved successful in effectively revealing the ability of hypoxic preconditioning to exert a pronounced and significant effect in maintaining LV contractile reserve even after 3 weeks. Hypoxic preconditioning was also able to reduce the extent of myocardial infarction significantly in the permanent LAD occluded group and also demonstrated a significantly enhanced level of contractile function and increased blood flow. These results, therefore, also demonstrate that hypoxic preconditioning was not only able to increase capillary and arteriolar density but it also helped them to remain more functional than the corresponding CMI group. This is the first study to report long-term improvement (3 weeks) in post-myocardial infarction and cardiac function induced by prior hypoxic preconditioning. The observed enhancement in preservation of cardiac reserve elicited during dobutamine challenge may be attributed at least partially to the stimulation of myocardial angiogenesis in the infarcted myocardium and the reduction of endothelial apoptotic cell death with a concomitant increase in the level of the most important survival factor, VEGF.
Acknowledgement This study was supported by National Institutes of Health Grant HL 56803, HL 22559.
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endothelial growth factor and basic fibroblast growth factor in children with cyanotic congenital heart disease. J Thorac Cardiovasc Surg 2000; 119: 534– 539. M B, S LK, P KH, R W, U A. Glioblastoma growth inhibited in vivo by a dominant-negative FLK-1 mutant. Nature (Lond.) 1994; 367: 576–579. A AP, M M, B MT, D’A DJ, F J, Y TK, Y KT. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994; 118: 445–450. G K, K S, L W, G L, R S, G P, L PY, H RP. VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling. Expt Cell Res 1999; 247: 495–504. F N, D-S T. The biology of vascular endothelial growth factor. Endocrine Revs 1997; 18: 4–25. H LE, A Z, L DM, B HF. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem 1996; 271: 32253– 32259. S DT, A AP, F N, Y KT, Y TK, A R, F J, D’A PA. Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen. Mol Med 1995; 1: 182–193. E DT, W M, E RM, R JA, K E, K VE, M N, D DK. Hypoxic preconditioning preserves antioxidant reserve in the working rat heart. Cardiovas Res 1995; 29: 133–140. M N, K VR, T VA, D DK. Redistribution of phosphatidylethanolamine and phosphatidylserine precedes reperfusion-induced apoptosis. Am J Physiol 1998; 274: H242–H248. K RA, B R, M E, R L, B E. Medical and cellular implications of stunning, hibernation, and preconditioning. Circulation 1998; 97: 1848–1867. R PS, E-H T, S H, Z L, M N. Early effects of hypoxia/reoxygenation on VEGF, Ang-1, Ang-2 and their receptors in the rat myocardium: implications for myocardial angiogenesis. Mol Cell Biochem 2000; 213: 145– 153. G E, G D. NFB induction during in vivo hypoxia in dorsocaudal brain stem of rat: effect of MK 801 and L-NAME. J Appl Physiol 1998; 85: 372–376. D RL, H S, M HR, C R, C KR, R J J. Insulin-like growth factor1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure. J Clin Invest 1995; 95: 619–627. O S, B V, O S, M L, H G, R HA, R J J. In vivo assessment of left ventricular remodelling after myocardial infarction by digital videocontrast angiography in the rat. Cardiovasc Res 1994; 28: 349–357. X Z, G M, B S, K T. The capillary
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implications for retinopathy of prematurity. Nat Med 1995; 1: 1024–1028. 32. S D, I A, S C, K E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359: 843–845.
Hypoxic Preconditioning Triggers Myocardial Angiogenesis: a Novel Approach to Enhance Contractile Functional Reserve in Rat with Myocardial Infarction Hiroaki Sasaki1, Shoji Fukuda1, Hajime Otani2, Li Zhu1, Genbu Yamaura1, Richard M. Engelman3, Dipak K. Das1 and Nilanjana Maulik1 1
Department of Surgery, University of Connecticut Health Center, Farmington, Connecticut 06030-1110, 2 Department of Thoracic and Cardiovascular Surgery, Kansai Medical Center, Osaka, Japan and 3 Baystate Medical Center, Springfield MA, USA (Received 9 November 2001, accepted 11 December 2001)
H. S, S. F, H. O, L. Z, G. Y, R. M. E, D. K. D N. M. Hypoxic Preconditioning Triggers Myocardial Angiogenesis: a Novel Approach to Enhance Contractile Functional Reserve in Rat with Myocardial Infarction. Journal of Molecular and Cellular Cardiology (2002) 34, 335–348. A modern experimental strategy for treating myocardial ischemia is to induce neovascularization of the heart by the use of “angiogens”, mediators that induce the formation of blood vessels, or angiogenesis. Studies demonstrated that coronary collateral vessels protect ischemic myocardium after coronary obstruction; therefore we sought to examine a novel method of stimulating myocardial angiogenesis through hypoxic preconditioning at both capillary (using anti-CD31) and arteriolar (using anti- smooth muscle actin) levels and also investigate whether such treatments could preserve left ventricular contractile functional reserve and regional blood flow by increasing vascular endothelial growth factor (VEGF). Male Sprague-Dawley rats were randomly divided into four groups: normoxia+sham surgery (CS), normoxia+permanent left anterior descending coronary artery (LAD) occlusion (CMI), hypoxic preconditioning+sham surgery (HS) and hypoxic preconditioning+permanent LAD occlusion (HMI). Rats in the preconditioned groups were subjected to systemic hypoxemic hypoxic exposure (10±0.4% O2) for 4 h followed by a 24 h period of normoxic reoxygenation prior to undergoing LAD occlusion. Rats in the normoxia group were time matched with the preconditioned group and maintained under normoxic conditions for a 28 h period prior to LAD occlusion. Western blot analysis was performed to measure VEGF expression and TUNEL staining with endothelial cell-specific antibody, anti-VWF, was used to examine endothelial apoptosis. One, two and three weeks after the LAD occlusion, baseline left ventricular pressures were monitored and recorded. Pharmacological stress tests with dobutamine infusion in progressively increasing doses revealed significantly elevated contractile reserve at each dose point in the HMI group compared to the CMI group. The HMI group displayed statistically significant increases in capillary as well as arteriolar density after 1, 2 and 3 weeks post-operation. Blood flow was also significantly elevated in the HMI groups when compared to the CMI group. The extent of endothelial cell apoptosis was found to be inversely proportional to VEGF expression. It was concluded that hypoxic preconditioning stimulates myocardial angiogenesis to an extent sufficient to exert significant cardioprotection in a rat model of myocardial infarction progressing to heart failure as evidenced by increased capillary/arteriolar density and enhanced ventricular contractile functional reserve. 2002 Elsevier Science Ltd. All rights reserved.
K W: Myocardial infarction; Hypoxic preconditioning; Oxidative stress; Angiogenesis; VEGF; Apoptosis. 0022–2828/02/030335+14 $35.00/0
2002 Elsevier Science Ltd. All rights reserved.
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Introduction Various coronary interventions have made a remarkable contribution to the treatment of coronary artery disease. The ultimate goal of these interventions is to improve arterial blood supply through the formation of coronary collateral vessels (angiogenesis) to potentially ischemic myocardium. Factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) which stimulate collateral growth, are expected to exert a protective effect against myocardial infarction. Indeed, VEGF is a major regulator of angiogenesis and vasculogenesis.1 A strong temporal and spatial correlation exists between VEGF expression and angiogenesis in both animals and humans.2,3 The biological functions of VEGF, triggered by external stimuli, are initiated through the activation of intracellular signal transduction cascades involving specific kinases.4 VEGF behaves as a classical stressinduced gene in this respect. Recent studies documented that hypoxia induces VEGF gene by a 5′promoter sequence which binds to the hypoxiainducible factor-1 (HIF-1),5 which is also induced and stabilized by hypoxia.6 Among various triggers of angiogenesis, tissue hypoxia has been identified as being an important stimulus for the induction of new vessel growth, especially at the capillary level.7 In the myocardium, hypoxia occurs during ischemic insult, usually followed by reoxygenation and revascularization. Preconditioning induced by cyclic episodes of brief periods of ischemia and reperfusion or hypoxia and reoxygenation has been found to possess profound cardioprotective ability in lowering myocardial infarction by reducing both cell necrosis and apoptosis and in improving post-ischemic contractile function.8,9 The cardioprotective abilities of preconditioning last up to 4–6 h and reappear after 24 h (2nd window of preconditioning), lasting for another 48 h.10 To the best of our knowledge, no study has ever been performed to examine the prolonged effects of preconditioning on myocardial angiogenesis. The aim of this study was, therefore, to determine the effects of hypoxic preconditioning on myocardial angiogenesis including blood flow and cardiac performance in the myocardial infarction model. The results documented that hypoxic preconditioning improved myocardial performance and angiogenesis at the level of capillary and arteriolar density after 1 week of LAD
occlusion, the effect lasting even after 3 weeks. Such angiogenic effects of preconditioning were found to be due to its ability to augment VEGF protein expression which presumably played a crucial role in reducing endothelial cell apoptosis, a proven determinant for congestive heart failure.
Materials and Methods All animals used in this study received humane care in compliance with the principles of laboratory animal care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals published by NIH.
In vivo hypoxia/reoxygenation Rats weighing 275–300 g were randomly divided into four groups as outlined in Figure 1. Based on previous experiments performed in our laboratory, we have determined that a 4 h duration of systemic hypoxemic hypoxia followed by 24 h of reoxygenation induces a very strong induction of angiogenic factors.11 We therefore used this duration of hypoxic exposure to effect hypoxic preconditioning in our preconditioned animal groups. Rats in the hypoxic preconditioned group (HMI) were subjected to a hypoxic challenge, using a gas mixture (10% O2/90% N2),12 of 4 h duration in an anesthesia chamber. An in-flow aperture was used for the gas mixture and an out-flow aperture connected to a gas absorption cannister (A.M. Bickford Inc., Wales Center, NY, USA). A KE-25 galvanic cell O2 sensor (Kent Scientific, Litchfield, CT, USA) was used to continuously monitor the %O2 concentration, the signal being amplified through a TRN-005 amplifier (Kent Scientific). The ambient O2 concentration was gradually lowered from 20.9% down to 10% over the course of 30 min. Once stabilization was obtained, the in-flow rate was adjusted in order to maintain the O2 concentration at 10±0.4% for a 4 h duration. These rats were then maintained in a normoxic environment for 24 h until preoperative preparation. Rats in the control group (CMI) were similarly maintained in a normoxic environment in a time-matched manner (29 h) up until preoperative preparation. Two to three animals were excluded from each permanent
Please address all correspondence to: Nilanjana Maulik, PhD, Molecular Cardiology Laboratory, Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, Farmington, CT 06030-1110, USA. Fax: (860) 679-4606; Tel: (860) 6792857; E-mail: [email protected]
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Figure 1 Outline of experimental protocol. Rats randomly assigned to various experimental groups (n=12) were subjected to LAD ligation following different exposure protocols. CMI group: normoxia+LAD occlusion. HMI group: hypoxia/reoxygenation+LAD occlusion. Rats which were randomly assigned to the next two groups served as the respective sham-operated controls for groups CMI and HMI, respectively. CS group: normoxia+sham surgery; HS group: hypoxia/reoxygenation+sham surgery.
occlusion group either due to post-operative mortality or morbidity within 24 h, or based on infarct size being less than 50%.
Survival surgery and LAD occlusion After the 24 h reoxygenation period/time-matched normoxic period, rats were anesthetized with Ketamine HCl (100 mg/kg i.p.) and Xylazine (10 mg/ kg i.p.).13,14 Cefazolin (25 mg/kg i.p.) was administered by i.v. injection as preoperative antibiotic cover. The animals were placed in the supine position and body temperature was maintained at 37°C by means of a water circulating thermal heating pad. A midline neck incision was made and the trachea was identified. Tracheostomy was performed by making a vertical incision approximately 2 mm long in the trachea. A polyethylene tube (2 mm O.D.)15 was introduced through the opening and positive pressure ventilation with room air was commenced (stroke volume 12 ml/kg,15–17 tidal volume 2.5 ml18 and respiratory rate 70/min19 using a small animal respirator (model 683, Harvard Apparatus, Holliston, MA, USA). The tube was secured in place by means of stay sutures. An intercostal thoracotomy in the left fourth intercostal space was
performed.13,14,16,19 The heart was exposed and the pericardium incised. The left coronary artery was identified between the left atrial appendage and the pulmonary artery outflow tract. A 6-0 suture was passed beneath the vessel 1 to 2 mm from its origin. Care was taken not to enter the ventricular cavity. In the sham-operated groups (CS and HS), the suture was left in place up until chest closure but was not ligated. After completion of all protocols, the chest wall was closed in three layers using 40 silk sutures. A catheter fashioned from PE-190 tubing attached to a 12 ml syringe with a stopcock was used as a chest tube to evacuate air from the pleural space as the last stitch was tightened. Postoperative pain relief was achieved by the administration of buprenorphine (0.05–2.5 mg/kg s.c.).
Non-survival surgery and baseline cardiac function One, two and three weeks after the operative intervention, the rats were anesthetized and ventilated as described above. A small incision was made to the right of the midline in the neck. The right internal jugular vein was identified and a PE 50 catheter was introduced into the vein. The proximal end of the catheter was connected to a low pressure
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transducer. The inserted catheter tip was then advanced until it reached the right atrium and the central venous pressure (CVP) signal was obtained. The same procedure was used to place a catheter in the right carotid artery where the catheter was used to monitor aortic blood pressure. The inserted tip of this catheter was advanced down until it reached the left ventricular lumen and the left ventricular pressure (LVP) signal was obtained. All pressure signals were monitored, analysed and recorded in real time using the Digimed data acquisition and analysis system (Micromed, Louisville, KY, USA). Heart rate (HR), developed pressure, and dP/dtmax were all calculated from the continuously obtained LVP signal.
Cardiac stress testing with dobutamine After baseline left ventricular functional parameters were recorded, rats were subjected to pharmacological cardiac stress testing with dobutamine infusion in progressively incremental doses to reveal the extent of left ventricular contractile functional reserve. Dobutamine was infused i.v. through the venous catheter by means of a microinjection pump (Harvard Apparatus, Holliston, MA, USA) to achieve doses of 1, 2, 3 and 5 g/kg/min. Based on previous studies in our laboratory, we have found that administration up to a dose of 5 g/kg/ min is necessary to observe the plateau of the doseresponse curve. Administration was maintained at each dose for a duration of 2 min. dP/dtmax and other left ventricular functional parameters were recorded for each dose 90 s after the start of each dose.
Tissue retrieval and processing After measurement of all functional parameters, the animals were systemically heparinized to prevent intravascular coagulation. The animals were killed with 1 ml of 30 m KCl injected i.v. Hearts were excised, the ventricular lumen was flushed clean with Ringer’s lactate solution and the ventricles were either frozen in liquid nitrogen for later use in biochemical studies (Western Blot analysis) or were sectioned approximately 5–6 mm from the apex into approximately 2 mm thick transverse sections. The sections were then fixed in 10% buffered formalin and embedded in paraffin using standard procedures to perform immunohistochemical analysis.
Measurement of capillary and arteriolar density The standard deparaffinization protocol was used. Endothelial cells were labeled using mouse monoclonal anti-CD31/PECAM-1 (1:100, Pharmingen, San Diego, CA, USA) followed by a biotinylated horse anti-mouse secondary antibody (1:200 dilution). The reaction product (brown) was visualized with DAB substrate using the Vector ABC Vectastain Elite Kit (Vectorlabs, Burminghame, CA, USA) and was counterstained with methyl green (Vectorlabs). On separate slides, vascular smooth muscle cells were labeled using mouse monoclonal anti-smooth muscle actin (1:50, Biogenex, San Ramon, CA, USA) followed by a biotinylated ratadsorbed horse anti-mouse secondary antibody (1: 200 dilution). The reaction product (violet) was visualized with VIP substrate using the Vector ABC Vectastain Elite Kit (Vectorlabs). Images were captured and stored in digital tiff file format for later image analysis. Counts of capillary density and arteriolar density per mm2 were obtained after superimposing a calibrated morphometric grid on each digital image using Adobe Photoshop Software.
Western blot analysis for VEGF To quantify the abundance of the angiogenic factor VEGF, we performed Western blot analysis using a specific primary antibody.11
TUNEL assay for apoptotic endothelial cells At the end of the experiments, the heart was perfusion-fixed with 10 ml of 10% formaldehyde in 0.1 mol/l phosphate buffer (pH 7.2) at room temperature. Transverse ventricular slices were embedded in paraffin, cut into 4 micrometer sections and deparaffinized with a graded series of xylene and ethanol solutions. The sections were then assayed for in situ terminal transferase labeling (TUNEL) using Apop Tag Plus (Oncor Inc., Gaithersburg, MD, USA). Negative control slides were processed with the TdT enzyme excluded. The slides were first stained with peroxidase-conjugated sheep polyclonal anti-digoxigenin antibodies and diaminobenzidine and with methyl green as a counterstain. For the detection of endothelial cell apoptosis, the serial sections were first stained by TUNEL with fluorescein isothiocyanate (FITC) and then incubated with polyclonal rabbit anti-von Willebrand factor antibodies (Dako Japan, Tokyo,
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Japan) followed by incubation with tetrarhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Dako Japan). FITC and TRITC fluorescence were viewed with a confocal laser microscopy (Fluo View, Olympus Co., in Tokyo, Japan). The number of TUNEL-positive endothelial cells was counted on 60 high power fields (magnification ×600) from the endocardium through the epicardium of the left ventricular free wall.
Measurement of percent circumferential left ventricular infarct size Infarct size and area at risk were measured after myocardial infarction (n=6).20 One ml of 5% Evan’s blue dye was infused into the heart through the apex to mark the risk zone as unstained (not blue) tissue. Frozen hearts were then cut into 2 mm thick slices parallel to the atrioventricular groove. Sections were thawed and incubated in a 1% tetrazolium chloride (TTC) phosphate buffered solution (pH 7.4) at 37°C for 15 min and fixed in 10% formalin to increase the contrast of the Evan’s blue and TTC staining. Tissue sections were compressed to a uniform 2 mm thickness by placing them between two glass plates separated by a 2 mm spacer. The infarct size was determined between the point of ligation and the apex. The area at risk was determined with Evan’s blue dye staining, whereas the non-infarcted (stained red) and infarcted (unstained) areas were determined after incubation with 1% triphenyltetrazolium chloride. With the use of NIH imaging, the volumes were calculated. Infarct size was reported as a percent of the area at risk (volume of infarcted tissue divided by volume of area at risk) ×100.
Measurement of blood flow by neutron microsphere technique We used neutron activation technique for the assay of stable isotopically labeled gold (Au) microsphere for measuring regional rat myocardial perfusion.21 Neutron activation is a very accurate procedure in which trace quantities of isotope of interest in a sample are activated and the radiation emitted by this method is measured with a high-resolution detection technique. Stable gold labeled microspheres (15×10,15 15 m) were injected in an in vivo rat model of myocardial infarction through the left atrium under anesthesia. Simultaneously, a reference blood sample was drawn from a femoral arterial catheter using a withdrawal pump (model
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PHD 2000 programmable, Harvard Apparatus, Millis, MA, USA) to measure absolute myocardial blood flow. The withdrawal rate was set at 1 ml/min and collected over a 2 min interval; a total of 2 ml reference blood was collected. At the end of the experiments the animals were killed and hearts (risk area or border zone of the left ventricle) from the various groups were collected and weighed. The tissue and the blood samples were dried in a 70°C oven overnight. All tissue and blood samples were sent for further analysis to BioPAL, (Worcester, MA, USA), where they were exposed to a field of neutrons. The samples were stored for 48 h to allow short-lived activation products to decay. After this period, spectrophotometric analysis was performed. The samples were counted for 2 min and during this period samples were corrected for tracer decay. The absolute blood flow was determined in ml/min/ gram tissue. The microsphere concentration of each segment (in disintegrations per min (dpm)/g) was normalized to the microsphere concentration evaluated in the 2 min reference blood collection (in dpm.min/ml).
Statistical analysis Results are expressed as mean±standard error of the mean (...). Differences between groups were tested for statistical significance by two-way analysis of variance (ANOVA) and Student’s t-test, with the Bonferroni adjustment for multiple comparisons. The dose response curves to dobutamine of the various groups were tested for significant differences by two way repeated measure ANOVA. Differences were considered significant at P<0.05.
Results Effects of hypoxic preconditioning on capillary and arteriolar density For the measurement of capillary density, eight non-overlapping random fields were selected from the endocardial regions of non-infarcted (border zone) zones of the left ventricles and examined at 400× magnification. The HMI group displayed a statistically significant increase in capillary density after 1 week of operation when compared to the control sham-operated group (CS) [1862±67 counts/mm2 in HMI v 1600±16 counts/mm2 in control baseline (BL)]. There was mild-to-moderate increase in capillary density in the HMI group
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Figure 2 Left ventricular endocardial capillary density. Tissue sections were processed for CD31 staining. Eight nonoverlapping random fields were selected from endocardial regions of the left ventricle from each heart (16 fields per region per heart, 64 fields per region per group, magnification ×400 were used, n=6). Images were captured and stored in digital tiff file format for image analysis. Where ∗ P<0.01, compared to sham operation; † P<0.01, compared to CMI; § P<0.05, compared to CS.
compared to the HS animals 2 days post-operatively (1811±56 v 1862±37 counts/mm2). However, the capillary density increased significantly 4 days post-operatively when compared to the CMI and HMI groups. The increased capillary density was maintained even 1, 2 and 3 weeks post-operation when compared to sham-operated groups (Fig. 2). The most interesting observation was the increased capillary density (CD) in the hypoxic sham (HS) group compared to the control sham group (CS). CD was higher in the hypoxic sham group after 2 days (1814±56 counts/mm2) compared to the control baseline group (CS) (1642±43 counts/ mm2). Therefore, hypoxic preconditioning itself demonstrated increase in CD compared to the nonhypoxic group of control (Fig. 2). Differences between sham groups were only demonstrable in capillary density. For the measurements of arteriolar density, eight non-overlapping random fields were selected from the endocardial regions of non-infarcted zones of
the left ventricles and examined at 2000× magnification. Arteriolar counts (purple color) for each field in the same region were averaged to yield the value of the particular density measurement. The arteriolar density was significantly elevated in the HMI group after 1 week (2.23±0.12 HMI v 1.53±0.02 counts/mm2 in CMI) (Fig. 3). Again, the increased arteriolar density was maintained even 2 and 3 weeks post-operatively in the HMI group compared to the CMI group.
Effect of hypoxic preconditioning on dobutamine stress test: left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and heart rate (HR), maximal dP/dt dP/dtmax However, statistically significant differences were noticed in LVSP and LVEDP between the group’s representative of the sham/baseline v all the other permanently occluded groups (CMI and HMI) after
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Figure 3 Left ventricular endocardial arteriolar density. Left ventricular tissue sections were labeled using monoclonal anti-smooth muscle actin and eight non-overlapping random fields were selected from the endocardial region of the left ventricle. Where ∗ P<0.01, compared to sham operation; † P<0.01, compared to CMI; § P<0.05, compared to CS.
1, 2 and 3 weeks (Fig. 4, only 3 weeks’ data shown). There was no difference in baseline LV function between the groups. No statistical (two-way ANOVA analysis) difference was found for heart rate between the groups even after 3 weeks of LAD occlusion (data not shown). Pharmacological cardiac stress testing with dobutamine infusion in incremental doses revealed differences in the extent of cardiac contractile reserve between groups (CMI and HMI). This was evident from differences in the extent of change in dP/dtmax values displayed by various groups during the course of such stress testing after 1 week of intervention. The differences remained statistically significant even after 2 and 3 weeks (Fig. 5, only 1 and 3 weeks’ results are shown). The hypoxic preconditioned group displayed significantly elevated contractile reserve at each dose point of evaluation compared to the CMI group, but the extent of such reserve was lower than that displayed by sham operated groups or by control rats (all of which exhibited similar and statistically indifferent values of dP/dtmax throughout the course of stress testing). This enhanced preservation of contractile reserve in the hypoxic preconditioned group was apparent at the 1 g/kg/ min dose (6419±488 mmHg/s in the HMI group
v 5594±197 mmHg/s in the CMI group after 3 weeks) and this trend persisted at all subsequent doses up to 5 g/kg/min.
Measurement of infarct size Heart sections obtained from hypoxic preconditioned animals (HMI) after 24 h demonstrated significantly lowered infarct size, as expected (30.56±3.7% of the area at risk) compared to the infarct size in non-preconditioned animals (CMI) (51.69±6.01% of the area at risk) (Fig. 6).
Measurement of left ventricular blood flow Left ventricular blood flow was measured in rat myocardium 1 and 3 weeks post-operatively by neutron microsphere technique. It was found that in the HMI group, the blood flow measured in the risk area was significantly higher compared to the CMI group. Therefore, hypoxic preconditioning was not only able to enhance the capillary density and arteriolar density but at the same time it was also able to increase the blood flow to make it functional.
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Figure 5 Dose response curves of dP/dtmax during dobutamine stress test. Changes are shown in dP/dtmax in mm of mercury per second (mmHg/s) from baseline with increasing dobutamine boluses (0–5 g/kg/min) in hearts 7 days and 3 weeks after MI. The dose response curves for dobutamine for the various doses were tested for significant differences by repeated measure by ANOVA. Differences were considered significant at P<0.05. Where ∗ P<0.01 compared to sham, † P<0.01 compared to CMI.
Figure 4 Dose response curves of LVSP and LVEDP during dobutamine stress test. Changes are shown in the systolic and end-diastolic pressure (mmHg) from baseline with increasing dobutamine boluses (0–5 g/kg/min) in hearts 3 weeks after MI. The dose response curve for dobutamine for the various doses was tested for significant differences by repeated measure by ANOVA. Differences were considered significant at P<0.05. Where ∗ P<0.01 compared to sham operation.
Therefore in the CMI group the flow rate was 2.22±1.31 whereas in the HMI group it was 3.83±1.41 after 1 week. After 3 weeks, the blood flow rate in the HMI group was increased further to 4.0±0.22 v 1.8±0.20 in the CMI group (Fig. 7).
Effect of hypoxic preconditioning on VEGF expression Protein expression profile of VEGF was found to be significantly elevated after 2 (33%) and 3 (63.3%) weeks of LAD occlusion in the HMI group compared to CMI and/or baseline control or sham group. However, in the CMI group, after 3 weeks of LAD occlusion the VEGF expression level was increased moderately (20%) compared to the baseline control
Figure 6 Infarct size of the hearts, expressed as a percentage of the area at risk in rat myocardium subjected to permanent LAD occlusion for 24 h. Results are expressed as means±... of six hearts/group. ∗ P<0.05 compared to CMI.
(Fig. 8). Hypoxic preconditioning induced VEGF in the hypoxic group even before MI (HS) (50% compared to the non-hypoxic control).11 The protein level was measured by densitometry scanning and normalized with -actin.
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Effect of hypoxic preconditioning on endothelial cell apoptosis
Figure 7 Regional blood flow with neutron microsphere injection. Regional blood flow was estimated after 1 week and after 3 weeks of LAD occlusion. Differences were considered significant at P<0.05. Where ∗ P<0.01 compared to CMI. Ε CMI, Γ HMI.
The number of apoptotic endothelial cells was found to be inversely proportional to the extent of VEGF expression in our model. A higher proportion of endothelial cells was observed to be apoptotic in the CMI group of myocardium when compared to the HMI group after 2 days (30±0.47% in CMI v 14±2.3% in HMI); 4 days (18±0.47% in CMI v 7.8%±1.7% in HMI); 1 week (9±0.47% in CMI group v 3±1.2% in HMI), 2 weeks (10±0.5% in CMI v 2±0.7 in HMI) and 3 weeks (9±0.4% in CMI v 1.2±0.4% in HMI) after LAD occlusion. Thus, in both the CMI and HMI groups, the extent of apoptosis was found to be extremely significant in the early stage after LAD occlusion (2 and 4 days and 1 week). However, the number of apoptotic cells was reduced in the CMI group after 1, 2 and
Figure 8 Representative Western blots showing the effects of systemic hypoxia and LAD occlusion on the expression of VEGF in rat myocardium in vivo after 2, 4, 7 and 21 days. VEGF proteins were expressed as 40 kDa. Similar results were obtained in six independent experiments performed in triplicate. Densitometric scanning of blots was used to determine levels of proteins relative to baseline control (sham). Where ∗ P<0.01 compared to baseline.
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3 weeks, but remained significantly higher when compared to the HMI group (Fig. 9).
Discussion The results of our study demonstrate for the first time that hypoxic preconditioning induced by whole body hypoxia/reoxygenation induces myocardial angiogenesis in a rat model of myocardial infarction as evidenced by increased capillary/arteriolar density and blood flow. Our findings also documented increased VEGF expression along with decreased endothelial apoptosis during hypoxic adaptation. Several recent studies have shown that hypoxic preconditioning, like ischemic preconditioning, can attenuate post-ischemic ventricular dysfunction caused by stunning.22,23 Although the exact mechanism of preconditioning is under considerable debate, there is a general agreement that myocardial preservation is achieved through a biphasic intrinsic mechanism. The early effect is most likely to occur through the modulation of intracellular signaling mechanisms, and the late effect is believed to be mediated by gene expression and protein synthesis.24 Cellular injury occurs both during hypoxia and after reoxygenation. It was observed that brief hypoxia followed by reoxygenation causes more damage at the cellular level than does more prolonged hypoxia alone. A major component of hypoxiainduced death was due to apoptosis, as observed by DNA laddering.25 Recently we have shown for the first time a comprehensive evaluation of the early effects (4 h hypoxia followed by 24 h reoxygenation) of in vivo systemic hypoxemic hypoxia on the protein expression and distribution profiles of the VEGF system and the Ang-Tie system in adult rat myocardium tissue. In this report we have demonstrated that hypoxia-reoxygenation increases the expression of both VEGF and Flk-1/ KDR in the myocardium.11 Hypoxic preconditioning induced VEGF and increased capillary density even before MI (HS). The relative time course of protein expression in response to hypoxic preconditioning, as indicated from our previous experiments,11 seems to suggest the involvement of the VEGF system as well as the Ang-Tie system in early angiogenesis.
In vivo, the early angiogenic response to systemic hypoxemic hypoxia in adult rat myocardium appears11 to be mediated not only through the induction of VEGF and its receptors Flk-1 and Flt-1, but also through the concurrent induction of Ang1, Ang-2 ,Tie-1 and Tie-2 (Table 1). The increased presence of Ang-2 may actually be pro-angiogenic in nature by effecting dissolution of surrounding matrix, thereby setting up a suitable environment in which endothelial cell migration and capillary sprouting can occur. Examination of non-MI left ventricle (border zone tissue) by anti-CD31 revealed a significant increase in the capillary density in the hypoxic preconditioned group (1800±46 v 1650±30 counts/mm2) compared to the control non-hypoxic group, confirming that modulation of angiogenic factors and their receptors by hypoxia/ reoxygenation was able to stimulate capillary proliferation even in the non-MI animals. Differences between sham groups were only demonstrable in capillary density, as shown in the Result’s section. VEGF-induced angiogenesis has been found to be associated with enhanced cell survival in human umbilical vein endothelial cells in vitro.26 This effect is thought to be due to a reduction in apoptotic endothelial cell death. Increased expression of the anti-apoptotic protein Bcl-2 and reduced expression of the pro-apoptotic protein caspase3 are possible biochemical mediators of such effects of VEGF.27 The observed effect of VEGF is mediated via the PI3 Kinase/Akt pathway, for which activation of the VEGF receptor Flk-1/KDR has been shown to be essential.26 Studies have also shown that hypoxia induces NFB activation in HUVEC cells, which leads to cell survival after exposure to TNF-alpha. In addition, we have demonstrated that in vivo, oxidative stress induced by systemic inhalational hypoxemic hypoxia can increase the DNA binding activity of NFB along with the increased expression pattern of VEGF in rat myocardium.28 According to our previous observations baseline VEGF expression was mainly in the ventricular myocardium with strong localization in the region of the coronary arterial wall where vascular smooth muscle appeared to stain positive. Hearts obtained from rats subjected to hypoxia displayed an increase in intensity of staining for VEGF in the 1 h hypoxia group and this enhanced level of immunoreactivity
Figure 9 TUNEL assay for apoptotic endothelial cells was performed on confocal laser microscopy as described in the Methods section. Representative photomicrographs show immunohistochemical staining of extended DNA. Where A= CS; B=HS; C=CMI, 1 week; D= HMI, 1 week; E=CMI, 2 weeks; F=HMI, 2 weeks; G=CMI, 3 weeks; H=HMI, 3 weeks. Note that apoptotic endothelial cells are lying on the luminal surface of the coronary vessels. Magnification ×1200.
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Table 1 Angiogenic protein expression profile in the non-MI hypoxic preconditioned myocardium compared to the normoxic control (% normoxic control), where H=hypoxia, R=24 h reoxygenation Angiogenic factors and their receptors VEGF Flk-1 Flt-1 Angiopoietin-1 Angiopoietin-2 Tie-1 Tie-2
1 h H/R (%)
2 h H/R (%)
4 h H/R (%)
50.8 41.5 325.0 23.8 31.3 533.0 93.9
51.9 22.0 201.0 30.0 14.8 586.7 120.5
50.0 12.8 149.9 50.0 same as normoxic control same as normoxic control 53.4
persisted in the 2 h and 4 h groups, where surrounding myocardium also displayed positive staining.11 In another study, myocytes from a sham group of animals showed detectable amounts of VEGF mRNA in both the ventricles. However, after 6 h of coronary ligation, decreased expression of VEGF mRNA was observed in the infarcted area of the anterior left ventricle. Six weeks post-ligation, mRNA levels in myocytes were not clearly distinguishable from the control hearts.29 In our experimental set up, increased VEGF in HMI (33% after 1 week and 64% after 3 weeks of hypoxic preconditioning) probably potentiates a survival signal to reduce the extent of apoptosis associated with myocardial infarction when compared to the non-preconditioned CMI group of animals. This phenomenon perhaps accelerated the capillary and arteriolar density and increased regional myocardial blood flow in the hypoxic preconditioned myocardium after coronary occlusion. As expected, the extent of infarction after coronary occlusion was reduced significantly after 24 h and the amount of viable myocardium was found to be more abundant in the HMI group compared to the CMI group. Possible mechanisms to salvage infarcted myocardium in the HMI group compared to the CMI group may be due to increased VEGF expression which increases flow through the artery, opening of latent collateral vessels by its vasodilating effect, induction of vascular growth and direct cellular protection against ischemia, as we have observed by reduced endothelial apoptosis. All these parameters no doubt significantly protect the cardiac function in the HMI group compared to the CMI group of animals. Thus, our results suggest that a relationship exists between programmed cell death (endothelial) and blood flow, and such a relationship is dependent on the level of survival factor VEGF. Existing evidence indicates that hypoxia results in the expression of a number of growth factors that influence vascular endothelial
cell viability.30 Furthermore, it was suggested that in the abnormal regression of retinal capillaries (due to hypoxia), VEGF acts as a survival factor and can prevent apoptosis associated with regression.31,32 Thus VEGF isoforms are considered the prime candidates for circulating survival stimuli. Pharmacological cardiac stress testing with dobutamine proved successful in effectively revealing the ability of hypoxic preconditioning to exert a pronounced and significant effect in maintaining LV contractile reserve even after 3 weeks. Hypoxic preconditioning was also able to reduce the extent of myocardial infarction significantly in the permanent LAD occluded group and also demonstrated a significantly enhanced level of contractile function and increased blood flow. These results, therefore, also demonstrate that hypoxic preconditioning was not only able to increase capillary and arteriolar density but it also helped them to remain more functional than the corresponding CMI group. This is the first study to report long-term improvement (3 weeks) in post-myocardial infarction and cardiac function induced by prior hypoxic preconditioning. The observed enhancement in preservation of cardiac reserve elicited during dobutamine challenge may be attributed at least partially to the stimulation of myocardial angiogenesis in the infarcted myocardium and the reduction of endothelial apoptotic cell death with a concomitant increase in the level of the most important survival factor, VEGF.
Acknowledgement This study was supported by National Institutes of Health Grant HL 56803, HL 22559.
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