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Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue
Life Sciences 79 (2006) 1781 – 1788 www.elsevier.com/locate/lifescie
Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue Bin Gong a , Gregory K. Asimakis b , Zhenping Chen c , Thomas B. Albrecht c , Paul J. Boor d , Todd C. Pappas a , Brent Bell a , Massoud Motamedi a,⁎ a
c
Center of Biomedical Engineering, University of Texas Medical Branch, Galveston, TX 77555, USA b Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA d Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA Received 16 February 2006; accepted 2 June 2006
Abstract Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 °C for 15 min. The control (n = 23) and heated (n = 55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart. © 2006 Published by Elsevier Inc. Keywords: Hyperthermia; Vascular endothelial growth factor; Heat shock protein 70; Platelet endothelial cell adhesion molecular-1; Angiogenesis
Introduction Brief, sub-lethal stress such as ischemia, hypoxia, and wholebody hyperthermia (WBH) can induce sub-cellular changes that enhance the tolerance of the heart muscle to a subsequent lethal ischemic insult {Marber, 1993 1/id; Vanden et al., 2000 12/id; Yamashita, 1998 11/id; Joyeux–Faure, 2003 59 /id}. In recent years, several laboratories have also shown that sub-lethal ischemia or hypoxia induces angiogenesis in the heart. New vessels thus formed can limit the damage associated with coronary artery occlusion (Operschall et al., 2000; Schaper and Ito,
⁎ Corresponding author. Tel.: +1 409 772 6118; fax: +1 409 772 0751. E-mail address: [email protected] (M. Motamedi). 0024-3205/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.lfs.2006.06.025
1996b) by enhancing the supply of oxygen and nutrients as well as providing washout of metabolic end products. Recent developments in the field of molecular cardiology and vascular biology have significantly increased our understanding of the role of angiogenic factors in the development and growth of blood vessels. In particular, vascular endothelial growth factor (VEGF) has been shown to be one of the most potent angiogenic factors that is directly stimulating, via cellsurface receptors, to vascular endothelial cell proliferation and migration in both physiological and pathological processes (Carmeliet and Collen, 1999, 2000). In recent studies, the administration of VEGF constructs has been shown to stimulate neovascularization in ischemic hearts (Amant et al., 1999; Neufeld et al., 1999; Schwarz et al., 2000; Schaper and Ito, 1996a) and in ischemic limbs (Chawla et al., 1999). It would be of
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interest; however, to develop new means to promote angiogenesis in the heart. A number of studies suggest the possibility that differing stressors can up regulate VEGF, thus potentially promoting angiogenesis. Hyperthermia has been used previously as a means to stress cardiac tissue and promote myocardial protection against ischemia (Marber et al., 1993; Yamashita et al., 1998). Previous studies also demonstrated up-regulation of VEGF in transplantable murine tumors (Kanamori et al., 1999) and experimental Pseudomonas corneal ulceration (Nanbu et al., 2004) following hyperthermia. These studies collectively suggest the possibility that heat stress can provide dual protection in the heart by increasing tolerance to acute ischemia and by promoting angiogenesis in the long-term due to up-regulation of angiogenic factors such as VEGF. The purpose of the present study was to examine the ability of brief sub-lethal WBH to promote endogenous up-regulation of VEGF in cardiac tissue, to determine the time course of thermal modulation of VEGF and HSP, and to morphologically assess (Couffinhal et al., 1998) the potential for thermallyinduced neovascularization in the heart. Materials and methods Experimental animal Seventy-eight male Sprague–Dawley rats weighing 300– 400 g were divided into 10 groups. Experimental groups were exposed to WBH at 42 °C for 15 min (see below), which control groups underwent the same procedure at 37 °C. The control (C) and heated (H) groups were allowed to recover for 4 (C4: n = 11, H4: n = 11), 12 (C12: n = 3, H12: n = 11), 24 (C24: n = 3, H24: n = 11), 48 (C48: n = 3, H48: n = 11), and 72 h (C72: n = 3, H72: n = 11) prior to harvesting the whole heart for histologic assessment or, in separate animals, the dissected right or left ventricles only for molecular study. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 85-23, revised 1985).
After the recovery period, the animals were re-anesthetized with the same dosage of ketamine and xylazine as in the primary step. A median sternotomy was performed and hearts were rapidly excised and rinsed in 0.9% saline. Hearts from 6 rats in each group of C4, H4, H12, H24, H48, and H72 and 3 rats in each group of C12, C24, C48, and C72 were sectioned along the intraventricular septum. The right and left ventricles were snapped frozen in liquid nitrogen for use in Western Blot assays. Additional groups of hearts were harvested for histopathologic and immunohistochemical studies. Following immersion fixation in 10% formalin in phosphate-buffered saline solution at physiologic pH, these hearts were routinely dehydrated, embedded in paraffin, and sectioned for light microscopy. Tissue temperatures during WBH In separate experiments, temperatures were monitored in left and right ventricles in vivo in control (n = 2) and heat-treated rats (n = 3), and subjected to WBH at 42 °C for 15 min as above after operative placement of temperature probes in the left and right ventricles. After administration of anesthesia as above, the rats were intubated and mechanically ventilated with 1% isoflurane of inhaled air. Animals were placed on the heating blanket and monitored by a rectal thermometer throughout the experiment. A left thoracotomy through the fifth intercostal space was performed to expose the epicardial surface of the left and right ventricles. A 6–0 polypropylene suture to the posterior or anterior walls of left and right ventricles secured temperature probes, respectively. The thoracotomy was closed and air was evacuated from the thorax using a 20-gauge intravenous catheter connected to a 10-mL syringe. Animals were then allowed to stabilize for 15 min. Temperatures of the left ventricle, right ventricle, rectum, and heating pad were recorded with a data acquisition system (Fig. 1). The animals in the control group were wrapped in a blanket and maintained at 37 °C.
WBH protocol Rats were anesthetized with ketamine (50 mg/kg) and xylazine (20 mg/kg) given intraperitoneally, then wrapped in a heating blanket connected to a circulating water bath heated at 47.6 °C while keeping the animal's head out of the blanket to protect the brain. A rectal thermometer using a computer-based data acquisition system (Omega Engineering, Standford, CT) continuously monitored the body temperature. The core temperatures of the heat-treated animals were increased from the basal temperature 37 °C to 41.5 °C during a period of about 20 min. The core temperatures were then kept at 41.5–42.5 °C for 15 min during WBH, and were allowed to return to basal level by removing the heating blanket. Control groups were anesthetized and wrapped in the heating blanket, but the core temperature was maintained at a normal body temperature of 37 °C. Following heat treatment, the animals were returned to their cages and permitted access to food and water.
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Fig. 1. Temperature profiles during WBH. Temperatures of right ventricle (●), left ventricle ( ), rectum (line only), and the heating pad (☉) are shown during the course of a representative experiment. The heating pad was turned on at the 2-minute point. After the rectal temperature reached 41 °C, the heating pad temperature was adjusted downward in order to maintain the core (rectal) temperature between 41° and 42 °C.
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VEGF and HSP70 quantification Approximately 0.3 g of right or left ventricular tissue was homogenized in 1 mL cold buffer containing 50 mmol/L Tris– HCl, pH 7.4, 1.0 mmol/L dithiothreitol, and protease inhibitors. Samples were centrifuged and a protein concentration was determined before equal amounts of soluble protein (100 μg/lane) were resolved by electrophoresis on 4–12% NuPAGE Bis-Tris Novex gradient gels (Invitrogen, Carlsbad, CA). Western blot analysis was performed as described (Gowda et al., 1998a), incubating membranes with a 1:1000 dilution of each primary antibody for 90 min, respectively, followed by incubation with secondary antibody at 1:2000 for 45 min. All antibodies (antiVEGF mouse monoclonal, anti-Flk-1 mouse monoclonal, antiFlt-1 rabbit polyclonal, and anti-HSP70 mouse monoclonal) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The results were quantified using an AlphaImager™ 2000 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). Differences in optical densities were
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determined relative to the density of the band of interest for the appropriate control ventricles harvested at 4 h after mock treatment. A Western Blot for β-actin was used to verify equal loading and transfer. Morphology study Cross-sections of hearts taken at multiple levels were routinely examined in hemotoxylin and eosin stained sections. Immunohistochemical staining was used to assess cellular distribution and intensity of VEGF protein in cardiac tissue. Deparaffinized and rehydrated sections were incubated for 30 min with anti-VEGF, anti-HSP70 antibody (dilution, 1:30 and 1:400 respectively), and anti-PECAM-1 (Serotic, UK; dilution, 1:5) followed by staining by the labeled streptavidin– biotin method with a Dako LSAB2 kit (KO684). Dako DAB chromogen (S300) solution was used as chromogen (Dako, Carpinteria, CA, USA), and counter-staining was performed with methyl-green. Negative controls, consisting of samples in
Fig. 2. Effect of WBH on expression of VEGF. A. A western blot of VEGF in the left ventricle (LV) and right ventricle (RV) following WBH (H, 42 °C for 15 min) or normothermia (C, 37 °C for 15 min) for each recovery time. B. Densitometric analysis of the western blots of VEGF in the LV and RVof heated and control hearts. The results are expressed relative to the 4-hour control groups. The values for all results are expressed as the means (±SEM) for 4–6 rats (heated) or 3–6 (control). * p < 0.05 vs. respective control value. + p < 0.001 vs. LV at same time point.
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which primary antibody was omitted, were evaluated in parallel to myocardium of control rats and rats undergoing WBH. Fullthickness sections of left ventricular free wall allowed for evaluation of staining in all cellular components of endocardial, epicardial, and myocardial tissue. The density of vessels was examined micrographically with PECAM-1 stained sections. A total of 90 randomly selected fields (3 samples/animal × 30 fields/sample) adjacent to the epicardium were evaluated by a single observer with blinded preparations. The number of capillaries per field were counted using a 60× objective as described previously (Couffinhal et al., 1998) and the vessel density determined.
ing to the protocol provided by manufacture. The DeadEnd™ assay measures nuclear fragmentation quantitatively using a fluorescein-tagged reagent to bind to 3′-OH DNA ends (Promega Incorporated Technical Bulletin No. 235, DeadEnd™ Fluorometric TUNEL system. 2001). Formalin-fixed, paraffin-embedded tissue sections 4 μm thick were used for this experiment. The slides were analyzed with a fluorescence microscope using a standard filter set optimized for detection of fluorescein. Yellowgreen fluorescence was visualized at 520 nm and fluorescein (DAPI Vector Lab, Burlingame, CA) was observed at 460 nm.
Apoptosis assessment
Groups were compared by one-way analysis of variance (ANOVA). If ANOVA indicated statistical differences, multiple group comparisons were performed by the Student–Newman– Kuels procedure. Statistic significance was recognized at p < 0.05.
PARP cleavage and TUNEL staining in sections including both ventricles were used to assess apoptosis of the rat hearts. The antibody (SC-7150; Santa Cruz Biotechnology, Santa Cruz, CA) used to detect PARP is specific for the carboxy terminal end of PARP, and thus binds both PARP and its major cleavage product (85 kDa) associated with apoptosis. TUNEL (TdT-mediated dUTP nick-end labeling) assays were performed using a Lab-Tek chamber slide (Nunc, Naperville, IL) and the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI) accord-
Statistical analysis
Results WBH increased the abundance of VEGF but not VEGF receptors The density of the VEGF-immunoreactive band was increased significantly in heat-treated hearts as early as 4 h post-heating
Fig. 3. Effect of WBH on expression of HSP 70. A. Western blot of HSP70 in the left ventricle (LV) and right ventricle (RV) following WBH (H, 42 °C for 15 min) or normothermia (C, 37 °C for 15 min) for each recovery time. B. Densitometry analysis of the western blots of HSP70 in the LV and RVof heated and control hearts. The results are expressed relative to the 4-hour control groups. The values are expressed as the means (±SEM) for 4–6 rats (heated) or 3–6 (control). * p < 0.05 vs. respective control value. + p < 0.001 vs. LV at same time point.
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Fig. 4. Morphologic and immunohistochemical studies. A. Right ventricular myocardium from control rats at 4 h H and E. Note normal, uninterrupted epicardial surface of right ventricle. B. Immunohistochemical staining of epicardium from a control heart at 4 h for VEGF. C. Immunohistochemistry of endocardium of heattreated rats at 48 h. Note homogeneous, slight staining of interstitium and capillaries in both B and C. D. H and E staining of right ventricular epicardial surface in rats undergoing WBH with recovery for 48 h. Note multiple focal areas of extensive capillary proliferation (c: indicates capillary lumen) with no evidence of myocyte damage. E. Immunohistochemical detection of VEGF in the RV (epicardial surface shown) 48 h after WBH. Note the markedly increased presence of VEGF in the interstitial matrix and in proliferative capillaries (c: indicates capillary lumen). All illustrations 330×.
(Fig. 2). However, there were differences in the kinetics and magnitude of the increase in VEGF between the left and right heart (Fig. 2). In the left ventricle, the maximum increase occurred at 12 h, when a 4.1-fold increase was observed; while in the right heart, the maximum increase (5.1-fold) was obtained at
24 h (Fig. 2). Although the differences in the abundance of VEGF in the left and right heart at the time of maximal increase were not great (4.1- versus 5.1-fold), they were significant from control (p < 0.05). By 72-hour post-heating, the level of VEGF was declining to baseline levels in both the left and right heart.
Fig. 5. Increased PECAM-1staining after WBH. PECAM-1 staining in the right ventricles from control animal hearts (A) or 48 h after WHB (B). Note the little evidence of vascular staining in control hearts; however, the brown staining of the capillary lumen walls (arrows) in right ventricular epicardium verifies the presence of endothelial neovascular sprouts at 48 h following WBH (B). All illustrations 460×.
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Fig. 6. Western blot analysis of PARP in right ventricles (RV). PARP analysis was performed following WBH (42 °C for 15 min) or normothermia (37 °C for 15 min) in a representative animal from each experimental group after the recovery times indicated. Lane 1, positive control for PARP cleavage, consisting of human pulmonary fibroblasts with apoptosis induced by infection with human CMV (5 PFU/cell, 12 h post-infection). The PARP cleavage product is indicated by its apparent molecular mass (85 kDa) in the positive control.
Furthermore, Western Blot analysis revealed that WBH did not significantly alter the expression of VEGF receptors, Flk-1 and Flt1 in the left or right ventricle of the heated hearts (data not shown). WBH increased the abundance of HSP70 in rat hearts To assess the stress response to WBH, we evaluated the expression of HSP70 using the same protein extracts used in the investigation of VEGF abundance. Western Blot analysis indicated that WBH caused a significant increase in the level of HSP70 in both the left and right heart from 4 to 72 h postheating. Similar to the results for VEGF, there were differences in both the kinetics and magnitude of the increase between the left and right heart (Fig. 3). In the left ventricle, the maximum increase of HSP70 occurred at 12 h, when a 4.5-fold increase was observed; while in the right heart, the maximum increase (6.3-fold) was obtained at 24 h (Fig. 3). The increases observed in both the left and right heart were significant compared to control (p < 0.01) from 4 to 72 h and the difference between the maximum abundance of HSP70 in the left and right heart also were significant (p < 0.001). WBH induced morphological change In ventricular sections inmmunostained for VEGF, reactivity was focally increased in the endothelium of capillaries, small arteries, and in the interstitium following WBH (Fig. 4). The most prominent staining for VEGF was observed in the right ventricle tissue and was most intense near the epicardial surface at 48 h after heating (Fig. 4). However, no detectable difference was observed in the extent of VEGF staining in the endocardium of the right ventricles of heated heart versus the control heart (Fig. 4). At 24 h following heating, histopathologic examination and specific PECAM-1 staining did not reveal the presence of any new vessels in either the right or the left ventricles (data not
shown). At 48 h (Fig. 4D and E) and 72 h (data not shown) following WBH, focal areas of extensive neovascularization were observed in the right ventricle of the superficial epicardium with no evidence of myocyte damage and little or no inflammatory infiltration (Fig. 4D). The presence of neovascularization was confirmed by specific staining of endothelial cells of the microvessels in the right ventricles by PECAM-1 (Fig. 5). Morphometric analysis of the density of new vessels as determined by PECAM-1 staining demonstrated a maximum increase at 48 h post-treatment, when the number of vessels per microscopic field increased from 30.2 ± 3.0 for the unheated control to 62.8 ± 8.8 for heat-treated hearts (p < 0.01). A statistically significant increase in vessel density was also observed in the right ventricular tissue of heated animals (49.0 ± 2.9, p < 0.05) at 72 h. However, no significant vessel formation was observed in the left ventricle at any time after WBH (data not shown). WBH did not cause detectable necrosis or apoptosis in the cardiac tissue The above data indicates that WBH can induce up-regulation of VEGF and new vessel proliferation. However, the potential beneficial effects of neovascularization could be negated if accompanied by necrosis or apoptosis. In heat-treated hearts, no morphological evidence of necrosis was observed. Inflammatory cells were not observed and interstitial edema was minimal (Fig. 4). Heat-induced apoptosis was assessed using a fluorometric TUNEL assay for nuclear fragmentation (DeadEnd™, Promega) and PARP cleavage tests of two consequences of apoptosis (Garrity et al., 2003; Nicoletti and Stella, 2003). The characteristic fluorescence for these assays was not evident in the sections of hearts exposed to normothermia or hyperthermia (data not shown). Image analysis with the Universal Imaging Inc. Metamorph program also failed to reveal appreciable fluorescence at the emission wavelength of fluorescein. To confirm the lack of detection of apoptotic cells, we analyzed extracts of heated and control hearts for evidence of PARP cleavage using antibodies that react with both PARP and its major cleavage product (Nicoletti and Stella, 2003). Although the 85 kDa PARP cleavage product was evident in a positive control for apoptosis, no evidence of PARP cleavage was evident in either the heated or non-heated hearts (Fig. 6). Thus, WBH and up-regulation of VEGF and HSP70 were not associated with an appreciable induction of apoptosis. Discussion WBH and up-regulation of VEGF expression in the heart Selected stimulation and enhancement of angiogenesis is a promising strategy for lessening myocardial cellular injury due to anoxia or other injurious agents. A mismatch between energy demand and energy production can result in the release of factors that promote compensatory neovascularization (Gu et al., 1999; Ruef et al., 1997). Of these factors, VEGF is notable because it helps maintain normal vascularity under physiological conditions (Carmeliet et al., 1996; Gu et al., 1999), and it is a potent
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angiogenic factor under conditions of prolonged hypoxia (Ladoux and Frelin, 1993; Shweiki et al., 1992) or ischemia (Miraliakbari et al., 2000). Vascular smooth muscle cells secrete VEGF and its up-regulation by hypoxia occurs at the transcriptional and translational levels. Hypoxia induces VEGF transcription by causing the accumulation of hypoxia-inducible factor-1, which binds to a specific promoter region of the VEGF gene (Ladoux and Frelin, 1993). Moreover, VEGF levels are increased due to a hypoxia-inducible protein complex that binds to and inhibits degradation of VEGF mRNA (Levy et al., 1996). While ischemia or hypoxia are clear promoters of VEGF up-regulation and angiogenesis, the present study shows that a potentially safer stress, mild hyperthermia, can also up regulate VEGF in the heart with the potential for neovascularization and vessel proliferation. We found by immunohistochemistry that the myocardial VEGF was localized to the endothelium of the capillaries and small arteries. This increase in VEGF expression was greatest in right ventricle. This localization may reflect the method of heating being nearer to right ventricle, or some inherent difference between left ad right ventricles. This is consistent with studies showing that smooth muscle cells can produce VEGF and VEGF primarily targets endothelial cells, which have two VEGF cellsurface receptors, Flt-1 and Flk-1 (Carmeliet and Collen, 1999, 2000; Larrivee and Karsan, 2000; Veikkola and Alitalo, 1999). A previous study showed that although a sub-lethal ischemic stress can increase VEGF levels in the heart, VEGF receptors were down regulated 3 and 6 h post-stress (Miraliakbari et al., 2000), which could have an adverse effect on angiogenesis. However, we observed that heat stress did not significantly affect the expression of the Flt-1 or Flk-1 receptors from 4 to 72 h post-stress, indicating the effect of VEGF up-regulation is not minimized by downregulation of its receptors. The possible association of HSP70 with VEGF signature and angiogenesis Our study revealed a close association between up-regulation of VEGF and HSP70. As chaperones, heat shock proteins play important roles in cell cycle regulation and protection against the adverse effects of stress. Elevated HSPs are involved in the nuclear localization of regulatory proteins, since they transiently associate with key molecules of the cell cycle control system, such as Cdk4, Wee-1, pRb, p53, and p27/Kip1 in proliferating mammalian cells (Helmbrecht et al., 2000). Furthermore, some HSPs, including HSP70, are associated with kinases of the mitogen-activated signal cascade (Helmbrecht et al., 2000). Since both types of VEGF receptors possess intrinsic tyrosine kinase activity and Src kinases are involved in the control of angiogenesis stimulated by VEGF (Schlessinger, 2000), concomitant activation of HSP70 may contribute to overall signaling effects in stressed cells. Moreover, the activation of MAP kinases may lead to the transactivation of VEGF expression (Berra et al., 2000). Of particular concern is that hyperthermia could cause cellular injury that would negate any positive effects of thermallyinduced up-regulation of cardio-protective proteins. Our histological data indicated neither significant edema nor necrosis at
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the time of peak expression of VEGF in cardioventricular tissue. In this study, we found no significant cleavage of PARP and no fluorescent signal in TUNEL assay through the experimental time course indicating that apoptosis was not a significant factor in these changes. Furthermore, in the focal area with apparent angiogenesis observed in right ventricle, VEGF was markedly increased in vessels and interstitial cells in the absence of evident cell injury. Parallel increases in HSP70 were also documented. Collectively, these observations suggest that the induced HSP70 expression may contribute to the effectiveness of the VEGFinduced signaling cascade directly while protecting the endothelial cells from damage associated with increased permeability. Differential effects of WBH on the right and left ventricles It may be noteworthy that the maximum increases in the expression of both VEGF and HSP70 occurred in the left ventricle before they were observed in the right ventricle although the maximum levels obtained were consistently greater in the right ventricle. A possible explanation for this observation is that in our heating model, the right ventricle is positioned closer to the chest wall resulting in a greater heat stimulus because this region of the heart is heated faster and for a longer time since it is closer to the heating source. Temperature probes placed in the right and left ventricles support this view; however, the difference in temperature is less than 1 °C (Fig. 1). We previously showed that applying a heating probe to induce local hyperthermia in the left ventricle alone produced elevated levels of HSP70 in both the left and right ventricles, although hyperthermia was absent in the right ventricle (Gowda et al., 1998b). Collectively, this data suggest that there may be intrinsic differences between the right and left ventricles that result in greater up-regulation of VEGF and HSP70 in the right heart. This could have significant consequences on the relative abilities of the right and left ventricles to undergo neovascularization in response to a stress stimulus. The current study revealed the presence of vessel sprouts exclusively in the right ventricle. It is possible that the lack of observable heat-induced neovascularization in the left heart may be due to the insufficient up-regulation of VEGF. If so, a greater exposure to heat stress may be required to initiate angiogenesis in the left heart. Moreover, whether heat-induced angiogenesis would provide a significant collateral flow to have therapeutic effects is unknown. However, these are important issues to be addressed in future studies. In conclusion, the results of this study show that sub-lethal heat stress can up regulate the expression of VEGF and HSP70 accompanied by enhanced local proliferation of blood vessels without causing cardiac tissue injury. Moreover, the heat stress does not down-regulate VEGF receptors. Therefore, future studies may demonstrate further the potential application of sublethal heat stress as a safe and effective intervention for promotion of neovascularization. Acknowledgements This research was supported in part by a grant from the Advanced Technology Program, Texas Higher Education
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Coordinating Board, Award No. 004952-0318-1999 and a Grant-in-Aid from the American Heart Association, Texas Affiliate (G. Asimakis). References Amant, C., Berthou, L., Walsh, K., 1999. Angiogenesis and gene therapy in man: dream or reality? Drugs 58, 33–36 Spec No 1. Berra, E., Pages, G., Pouyssegur, J., 2000. MAP kinases and hypoxia in the control of VEGF expression. Cancer and Metastasis Reviews 19 (1–2), 139–145. Carmeliet, P., Collen, D., 1999. Role of vascular endothelial growth factor and vascular endothelial growth factor receptors in vascular development. Current Topics in Microbiology and Immunology 237, 133–158. Carmeliet, P., Collen, D., 2000. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Annals of the New York Academy of Sciences 902, 249–262. Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., Nagy, A., 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380 (6573), 435–439. Chawla, P.S., Keelan, M.H., Kipshidze, N., 1999. Angiogenesis for the treatment of vascular diseases. International Angiology 18 (3), 185–192. Couffinhal, T., Silver, M., Zheng, L.P., Kearney, M., Witzenbichler, B., Isner, J.M., 1998. Mouse model of angiogenesis. American Journal of Pathology 152 (6), 1667–1679. Garrity, M.M., Burgart, L.J., Riehle, D.L., Hill, E.M., Sebo, T.J., Witzig, T., 2003. Identifying and quantifying apoptosis: navigating technical pitfalls. Modern Pathology 16 (4), 389–394. Gowda, A., Yang, C., Asimakis, G.K., Rastegar, S., Motamedi, M., 1998a. Heat shock improves recovery and provides protection against global ischemia after hypothermic storage. Annals of Thoracic Surgery 66 (6), 1991–1997. Gowda, A., Yang, C.J., Asimakis, G.K., Ruef, J., Rastegar, S., Runge, M.S., Motamedi, M., 1998b. Cardioprotection by local heating: improved myocardial salvage after ischemia and reperfusion. Annals of Thoracic Surgery 65 (5), 1241–1247. Gu, J.W., Brady, A.L., Anand, V., Moore, M.C., Kelly, W.C., Adair, T.H., 1999. Adenosine upregulates VEGF expression in cultured myocardial vascular smooth muscle cells. Am.J.Physiol 277 (2 Pt 2), H595–H602. Helmbrecht, K., Zeise, E., Rensing, L., 2000. Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Proliferation 33 (6), 341–365. Kanamori, S., Nishimura, Y., Okuno, Y., Horii, N., Saga, T., Hiraoka, M., 1999. Induction of vascular endothelial growth factor (VEGF) by hyperthermia and/or an angiogenesis inhibitor. International Journal of Hyperthermia 15 (4), 267–278. Ladoux, A., Frelin, C., 1993. Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochemical and Biophysical Research Communications 195 (2), 1005–1010.
Larrivee, B., Karsan, A., 2000. Signaling pathways induced by vascular endothelial growth factor (review). International Journal of Molecular Medicine 5 (5), 447–456. Levy, A.P., Levy, N.S., Goldberg, M.A., 1996. Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel–Lindau protein. Journal of Biological Chemistry 271 (41), 25492–25497. Marber, M.S., Latchman, D.S., Walker, J.M., Yellon, D.M., 1993. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation 88 (3), 1264–1272. Miraliakbari, R., Francalancia, N.A., Lust, R.M., Gerardo, J.A., Ng, P.C., Sun, Y.S., Chitwood Jr., W.R., 2000. Differences in myocardial and peripheral VEGF and KDR levels after acute ischemia. Annals of Thoracic Surgery 69 (6), 1750–1753. Nanbu, P.N., Wakabayashi, T., Yamashita, R., Hayashi, H., Hisano, S., Oshika, T., 2004. Heat treatment enhances healing process of experimental pseudomonas corneal ulcer. Ophthalmic Research 36 (4), 218–225. Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z., 1999. Vascular endothelial growth factor (VEGF) and its receptors. FASEB Journal 13 (1), 9–22. Nicoletti, V.G., Stella, A.M., 2003. Role of PARP under stress conditions: cell death or protection? Neurochemical Research 28 (2), 187–194. Operschall, C., Falivene, L., Clozel, J.P., Roux, S., 2000. A new model of chronic cardiac ischemia in rabbits. Journal of Applied Physiology 88 (4), 1438–1445. Ruef, J., Hu, Z.Y., Yin, L.Y., Wu, Y., Hanson, S.R., Kelly, A.B., Harker, L.A., Rao, G.N., Runge, M.S., Patterson, C., 1997. Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis. Circulation Research 81 (1), 24–33. Schaper, W., Ito, W.D., 1996a. Molecular mechanisms of coronary collateral vessel growth. Circulation Research 79 (5), 911–919. Schaper, W., Ito, W.D., 1996b. Therapeutic targets in cardiovascular disorders. Current Opinion in Biotechnology 7 (6), 635–640. Schlessinger, J., 2000. New roles for Src kinases in control of cell survival and angiogenesis. Cell 100 (3), 293–296. Schwarz, E.R., Speakman, M.T., Patterson, M., Hale, S.S., Isner, J.M., Kedes, L.H., Kloner, R.A., 2000. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat-angiogenesis and angioma formation. Journal of the American College of Cardiology 35 (5), 1323–1330. Shweiki, D., Itin, A., Soffer, D., Keshet, E., 1992. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359 (6398), 843–845. Vanden, H.T., Becker, L.B., Shao, Z.H., Li, C.Q., Schumacker, P.T., 2000. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circulation Research 86 (5), 541–548. Veikkola, T., Alitalo, K., 1999. VEGFs, receptors and angiogenesis. Seminars in Cancer Biology 9 (3), 211–220. Yamashita, N., Hoshida, S., Taniguchi, N., Kuzuya, T., Hori, M., 1998. Wholebody hyperthermia provides biphasic cardioprotection against ischemia/ reperfusion injury in the rat. Circulation 98 (14), 1414–1421.
Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue Bin Gong a , Gregory K. Asimakis b , Zhenping Chen c , Thomas B. Albrecht c , Paul J. Boor d , Todd C. Pappas a , Brent Bell a , Massoud Motamedi a,⁎ a
c
Center of Biomedical Engineering, University of Texas Medical Branch, Galveston, TX 77555, USA b Department of Surgery, University of Texas Medical Branch, Galveston, TX 77555, USA Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA d Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA Received 16 February 2006; accepted 2 June 2006
Abstract Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 °C for 15 min. The control (n = 23) and heated (n = 55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart. © 2006 Published by Elsevier Inc. Keywords: Hyperthermia; Vascular endothelial growth factor; Heat shock protein 70; Platelet endothelial cell adhesion molecular-1; Angiogenesis
Introduction Brief, sub-lethal stress such as ischemia, hypoxia, and wholebody hyperthermia (WBH) can induce sub-cellular changes that enhance the tolerance of the heart muscle to a subsequent lethal ischemic insult {Marber, 1993 1/id; Vanden et al., 2000 12/id; Yamashita, 1998 11/id; Joyeux–Faure, 2003 59 /id}. In recent years, several laboratories have also shown that sub-lethal ischemia or hypoxia induces angiogenesis in the heart. New vessels thus formed can limit the damage associated with coronary artery occlusion (Operschall et al., 2000; Schaper and Ito,
⁎ Corresponding author. Tel.: +1 409 772 6118; fax: +1 409 772 0751. E-mail address: [email protected] (M. Motamedi). 0024-3205/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.lfs.2006.06.025
1996b) by enhancing the supply of oxygen and nutrients as well as providing washout of metabolic end products. Recent developments in the field of molecular cardiology and vascular biology have significantly increased our understanding of the role of angiogenic factors in the development and growth of blood vessels. In particular, vascular endothelial growth factor (VEGF) has been shown to be one of the most potent angiogenic factors that is directly stimulating, via cellsurface receptors, to vascular endothelial cell proliferation and migration in both physiological and pathological processes (Carmeliet and Collen, 1999, 2000). In recent studies, the administration of VEGF constructs has been shown to stimulate neovascularization in ischemic hearts (Amant et al., 1999; Neufeld et al., 1999; Schwarz et al., 2000; Schaper and Ito, 1996a) and in ischemic limbs (Chawla et al., 1999). It would be of
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interest; however, to develop new means to promote angiogenesis in the heart. A number of studies suggest the possibility that differing stressors can up regulate VEGF, thus potentially promoting angiogenesis. Hyperthermia has been used previously as a means to stress cardiac tissue and promote myocardial protection against ischemia (Marber et al., 1993; Yamashita et al., 1998). Previous studies also demonstrated up-regulation of VEGF in transplantable murine tumors (Kanamori et al., 1999) and experimental Pseudomonas corneal ulceration (Nanbu et al., 2004) following hyperthermia. These studies collectively suggest the possibility that heat stress can provide dual protection in the heart by increasing tolerance to acute ischemia and by promoting angiogenesis in the long-term due to up-regulation of angiogenic factors such as VEGF. The purpose of the present study was to examine the ability of brief sub-lethal WBH to promote endogenous up-regulation of VEGF in cardiac tissue, to determine the time course of thermal modulation of VEGF and HSP, and to morphologically assess (Couffinhal et al., 1998) the potential for thermallyinduced neovascularization in the heart. Materials and methods Experimental animal Seventy-eight male Sprague–Dawley rats weighing 300– 400 g were divided into 10 groups. Experimental groups were exposed to WBH at 42 °C for 15 min (see below), which control groups underwent the same procedure at 37 °C. The control (C) and heated (H) groups were allowed to recover for 4 (C4: n = 11, H4: n = 11), 12 (C12: n = 3, H12: n = 11), 24 (C24: n = 3, H24: n = 11), 48 (C48: n = 3, H48: n = 11), and 72 h (C72: n = 3, H72: n = 11) prior to harvesting the whole heart for histologic assessment or, in separate animals, the dissected right or left ventricles only for molecular study. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 85-23, revised 1985).
After the recovery period, the animals were re-anesthetized with the same dosage of ketamine and xylazine as in the primary step. A median sternotomy was performed and hearts were rapidly excised and rinsed in 0.9% saline. Hearts from 6 rats in each group of C4, H4, H12, H24, H48, and H72 and 3 rats in each group of C12, C24, C48, and C72 were sectioned along the intraventricular septum. The right and left ventricles were snapped frozen in liquid nitrogen for use in Western Blot assays. Additional groups of hearts were harvested for histopathologic and immunohistochemical studies. Following immersion fixation in 10% formalin in phosphate-buffered saline solution at physiologic pH, these hearts were routinely dehydrated, embedded in paraffin, and sectioned for light microscopy. Tissue temperatures during WBH In separate experiments, temperatures were monitored in left and right ventricles in vivo in control (n = 2) and heat-treated rats (n = 3), and subjected to WBH at 42 °C for 15 min as above after operative placement of temperature probes in the left and right ventricles. After administration of anesthesia as above, the rats were intubated and mechanically ventilated with 1% isoflurane of inhaled air. Animals were placed on the heating blanket and monitored by a rectal thermometer throughout the experiment. A left thoracotomy through the fifth intercostal space was performed to expose the epicardial surface of the left and right ventricles. A 6–0 polypropylene suture to the posterior or anterior walls of left and right ventricles secured temperature probes, respectively. The thoracotomy was closed and air was evacuated from the thorax using a 20-gauge intravenous catheter connected to a 10-mL syringe. Animals were then allowed to stabilize for 15 min. Temperatures of the left ventricle, right ventricle, rectum, and heating pad were recorded with a data acquisition system (Fig. 1). The animals in the control group were wrapped in a blanket and maintained at 37 °C.
WBH protocol Rats were anesthetized with ketamine (50 mg/kg) and xylazine (20 mg/kg) given intraperitoneally, then wrapped in a heating blanket connected to a circulating water bath heated at 47.6 °C while keeping the animal's head out of the blanket to protect the brain. A rectal thermometer using a computer-based data acquisition system (Omega Engineering, Standford, CT) continuously monitored the body temperature. The core temperatures of the heat-treated animals were increased from the basal temperature 37 °C to 41.5 °C during a period of about 20 min. The core temperatures were then kept at 41.5–42.5 °C for 15 min during WBH, and were allowed to return to basal level by removing the heating blanket. Control groups were anesthetized and wrapped in the heating blanket, but the core temperature was maintained at a normal body temperature of 37 °C. Following heat treatment, the animals were returned to their cages and permitted access to food and water.
▪
Fig. 1. Temperature profiles during WBH. Temperatures of right ventricle (●), left ventricle ( ), rectum (line only), and the heating pad (☉) are shown during the course of a representative experiment. The heating pad was turned on at the 2-minute point. After the rectal temperature reached 41 °C, the heating pad temperature was adjusted downward in order to maintain the core (rectal) temperature between 41° and 42 °C.
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VEGF and HSP70 quantification Approximately 0.3 g of right or left ventricular tissue was homogenized in 1 mL cold buffer containing 50 mmol/L Tris– HCl, pH 7.4, 1.0 mmol/L dithiothreitol, and protease inhibitors. Samples were centrifuged and a protein concentration was determined before equal amounts of soluble protein (100 μg/lane) were resolved by electrophoresis on 4–12% NuPAGE Bis-Tris Novex gradient gels (Invitrogen, Carlsbad, CA). Western blot analysis was performed as described (Gowda et al., 1998a), incubating membranes with a 1:1000 dilution of each primary antibody for 90 min, respectively, followed by incubation with secondary antibody at 1:2000 for 45 min. All antibodies (antiVEGF mouse monoclonal, anti-Flk-1 mouse monoclonal, antiFlt-1 rabbit polyclonal, and anti-HSP70 mouse monoclonal) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The results were quantified using an AlphaImager™ 2000 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). Differences in optical densities were
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determined relative to the density of the band of interest for the appropriate control ventricles harvested at 4 h after mock treatment. A Western Blot for β-actin was used to verify equal loading and transfer. Morphology study Cross-sections of hearts taken at multiple levels were routinely examined in hemotoxylin and eosin stained sections. Immunohistochemical staining was used to assess cellular distribution and intensity of VEGF protein in cardiac tissue. Deparaffinized and rehydrated sections were incubated for 30 min with anti-VEGF, anti-HSP70 antibody (dilution, 1:30 and 1:400 respectively), and anti-PECAM-1 (Serotic, UK; dilution, 1:5) followed by staining by the labeled streptavidin– biotin method with a Dako LSAB2 kit (KO684). Dako DAB chromogen (S300) solution was used as chromogen (Dako, Carpinteria, CA, USA), and counter-staining was performed with methyl-green. Negative controls, consisting of samples in
Fig. 2. Effect of WBH on expression of VEGF. A. A western blot of VEGF in the left ventricle (LV) and right ventricle (RV) following WBH (H, 42 °C for 15 min) or normothermia (C, 37 °C for 15 min) for each recovery time. B. Densitometric analysis of the western blots of VEGF in the LV and RVof heated and control hearts. The results are expressed relative to the 4-hour control groups. The values for all results are expressed as the means (±SEM) for 4–6 rats (heated) or 3–6 (control). * p < 0.05 vs. respective control value. + p < 0.001 vs. LV at same time point.
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which primary antibody was omitted, were evaluated in parallel to myocardium of control rats and rats undergoing WBH. Fullthickness sections of left ventricular free wall allowed for evaluation of staining in all cellular components of endocardial, epicardial, and myocardial tissue. The density of vessels was examined micrographically with PECAM-1 stained sections. A total of 90 randomly selected fields (3 samples/animal × 30 fields/sample) adjacent to the epicardium were evaluated by a single observer with blinded preparations. The number of capillaries per field were counted using a 60× objective as described previously (Couffinhal et al., 1998) and the vessel density determined.
ing to the protocol provided by manufacture. The DeadEnd™ assay measures nuclear fragmentation quantitatively using a fluorescein-tagged reagent to bind to 3′-OH DNA ends (Promega Incorporated Technical Bulletin No. 235, DeadEnd™ Fluorometric TUNEL system. 2001). Formalin-fixed, paraffin-embedded tissue sections 4 μm thick were used for this experiment. The slides were analyzed with a fluorescence microscope using a standard filter set optimized for detection of fluorescein. Yellowgreen fluorescence was visualized at 520 nm and fluorescein (DAPI Vector Lab, Burlingame, CA) was observed at 460 nm.
Apoptosis assessment
Groups were compared by one-way analysis of variance (ANOVA). If ANOVA indicated statistical differences, multiple group comparisons were performed by the Student–Newman– Kuels procedure. Statistic significance was recognized at p < 0.05.
PARP cleavage and TUNEL staining in sections including both ventricles were used to assess apoptosis of the rat hearts. The antibody (SC-7150; Santa Cruz Biotechnology, Santa Cruz, CA) used to detect PARP is specific for the carboxy terminal end of PARP, and thus binds both PARP and its major cleavage product (85 kDa) associated with apoptosis. TUNEL (TdT-mediated dUTP nick-end labeling) assays were performed using a Lab-Tek chamber slide (Nunc, Naperville, IL) and the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI) accord-
Statistical analysis
Results WBH increased the abundance of VEGF but not VEGF receptors The density of the VEGF-immunoreactive band was increased significantly in heat-treated hearts as early as 4 h post-heating
Fig. 3. Effect of WBH on expression of HSP 70. A. Western blot of HSP70 in the left ventricle (LV) and right ventricle (RV) following WBH (H, 42 °C for 15 min) or normothermia (C, 37 °C for 15 min) for each recovery time. B. Densitometry analysis of the western blots of HSP70 in the LV and RVof heated and control hearts. The results are expressed relative to the 4-hour control groups. The values are expressed as the means (±SEM) for 4–6 rats (heated) or 3–6 (control). * p < 0.05 vs. respective control value. + p < 0.001 vs. LV at same time point.
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Fig. 4. Morphologic and immunohistochemical studies. A. Right ventricular myocardium from control rats at 4 h H and E. Note normal, uninterrupted epicardial surface of right ventricle. B. Immunohistochemical staining of epicardium from a control heart at 4 h for VEGF. C. Immunohistochemistry of endocardium of heattreated rats at 48 h. Note homogeneous, slight staining of interstitium and capillaries in both B and C. D. H and E staining of right ventricular epicardial surface in rats undergoing WBH with recovery for 48 h. Note multiple focal areas of extensive capillary proliferation (c: indicates capillary lumen) with no evidence of myocyte damage. E. Immunohistochemical detection of VEGF in the RV (epicardial surface shown) 48 h after WBH. Note the markedly increased presence of VEGF in the interstitial matrix and in proliferative capillaries (c: indicates capillary lumen). All illustrations 330×.
(Fig. 2). However, there were differences in the kinetics and magnitude of the increase in VEGF between the left and right heart (Fig. 2). In the left ventricle, the maximum increase occurred at 12 h, when a 4.1-fold increase was observed; while in the right heart, the maximum increase (5.1-fold) was obtained at
24 h (Fig. 2). Although the differences in the abundance of VEGF in the left and right heart at the time of maximal increase were not great (4.1- versus 5.1-fold), they were significant from control (p < 0.05). By 72-hour post-heating, the level of VEGF was declining to baseline levels in both the left and right heart.
Fig. 5. Increased PECAM-1staining after WBH. PECAM-1 staining in the right ventricles from control animal hearts (A) or 48 h after WHB (B). Note the little evidence of vascular staining in control hearts; however, the brown staining of the capillary lumen walls (arrows) in right ventricular epicardium verifies the presence of endothelial neovascular sprouts at 48 h following WBH (B). All illustrations 460×.
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Fig. 6. Western blot analysis of PARP in right ventricles (RV). PARP analysis was performed following WBH (42 °C for 15 min) or normothermia (37 °C for 15 min) in a representative animal from each experimental group after the recovery times indicated. Lane 1, positive control for PARP cleavage, consisting of human pulmonary fibroblasts with apoptosis induced by infection with human CMV (5 PFU/cell, 12 h post-infection). The PARP cleavage product is indicated by its apparent molecular mass (85 kDa) in the positive control.
Furthermore, Western Blot analysis revealed that WBH did not significantly alter the expression of VEGF receptors, Flk-1 and Flt1 in the left or right ventricle of the heated hearts (data not shown). WBH increased the abundance of HSP70 in rat hearts To assess the stress response to WBH, we evaluated the expression of HSP70 using the same protein extracts used in the investigation of VEGF abundance. Western Blot analysis indicated that WBH caused a significant increase in the level of HSP70 in both the left and right heart from 4 to 72 h postheating. Similar to the results for VEGF, there were differences in both the kinetics and magnitude of the increase between the left and right heart (Fig. 3). In the left ventricle, the maximum increase of HSP70 occurred at 12 h, when a 4.5-fold increase was observed; while in the right heart, the maximum increase (6.3-fold) was obtained at 24 h (Fig. 3). The increases observed in both the left and right heart were significant compared to control (p < 0.01) from 4 to 72 h and the difference between the maximum abundance of HSP70 in the left and right heart also were significant (p < 0.001). WBH induced morphological change In ventricular sections inmmunostained for VEGF, reactivity was focally increased in the endothelium of capillaries, small arteries, and in the interstitium following WBH (Fig. 4). The most prominent staining for VEGF was observed in the right ventricle tissue and was most intense near the epicardial surface at 48 h after heating (Fig. 4). However, no detectable difference was observed in the extent of VEGF staining in the endocardium of the right ventricles of heated heart versus the control heart (Fig. 4). At 24 h following heating, histopathologic examination and specific PECAM-1 staining did not reveal the presence of any new vessels in either the right or the left ventricles (data not
shown). At 48 h (Fig. 4D and E) and 72 h (data not shown) following WBH, focal areas of extensive neovascularization were observed in the right ventricle of the superficial epicardium with no evidence of myocyte damage and little or no inflammatory infiltration (Fig. 4D). The presence of neovascularization was confirmed by specific staining of endothelial cells of the microvessels in the right ventricles by PECAM-1 (Fig. 5). Morphometric analysis of the density of new vessels as determined by PECAM-1 staining demonstrated a maximum increase at 48 h post-treatment, when the number of vessels per microscopic field increased from 30.2 ± 3.0 for the unheated control to 62.8 ± 8.8 for heat-treated hearts (p < 0.01). A statistically significant increase in vessel density was also observed in the right ventricular tissue of heated animals (49.0 ± 2.9, p < 0.05) at 72 h. However, no significant vessel formation was observed in the left ventricle at any time after WBH (data not shown). WBH did not cause detectable necrosis or apoptosis in the cardiac tissue The above data indicates that WBH can induce up-regulation of VEGF and new vessel proliferation. However, the potential beneficial effects of neovascularization could be negated if accompanied by necrosis or apoptosis. In heat-treated hearts, no morphological evidence of necrosis was observed. Inflammatory cells were not observed and interstitial edema was minimal (Fig. 4). Heat-induced apoptosis was assessed using a fluorometric TUNEL assay for nuclear fragmentation (DeadEnd™, Promega) and PARP cleavage tests of two consequences of apoptosis (Garrity et al., 2003; Nicoletti and Stella, 2003). The characteristic fluorescence for these assays was not evident in the sections of hearts exposed to normothermia or hyperthermia (data not shown). Image analysis with the Universal Imaging Inc. Metamorph program also failed to reveal appreciable fluorescence at the emission wavelength of fluorescein. To confirm the lack of detection of apoptotic cells, we analyzed extracts of heated and control hearts for evidence of PARP cleavage using antibodies that react with both PARP and its major cleavage product (Nicoletti and Stella, 2003). Although the 85 kDa PARP cleavage product was evident in a positive control for apoptosis, no evidence of PARP cleavage was evident in either the heated or non-heated hearts (Fig. 6). Thus, WBH and up-regulation of VEGF and HSP70 were not associated with an appreciable induction of apoptosis. Discussion WBH and up-regulation of VEGF expression in the heart Selected stimulation and enhancement of angiogenesis is a promising strategy for lessening myocardial cellular injury due to anoxia or other injurious agents. A mismatch between energy demand and energy production can result in the release of factors that promote compensatory neovascularization (Gu et al., 1999; Ruef et al., 1997). Of these factors, VEGF is notable because it helps maintain normal vascularity under physiological conditions (Carmeliet et al., 1996; Gu et al., 1999), and it is a potent
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angiogenic factor under conditions of prolonged hypoxia (Ladoux and Frelin, 1993; Shweiki et al., 1992) or ischemia (Miraliakbari et al., 2000). Vascular smooth muscle cells secrete VEGF and its up-regulation by hypoxia occurs at the transcriptional and translational levels. Hypoxia induces VEGF transcription by causing the accumulation of hypoxia-inducible factor-1, which binds to a specific promoter region of the VEGF gene (Ladoux and Frelin, 1993). Moreover, VEGF levels are increased due to a hypoxia-inducible protein complex that binds to and inhibits degradation of VEGF mRNA (Levy et al., 1996). While ischemia or hypoxia are clear promoters of VEGF up-regulation and angiogenesis, the present study shows that a potentially safer stress, mild hyperthermia, can also up regulate VEGF in the heart with the potential for neovascularization and vessel proliferation. We found by immunohistochemistry that the myocardial VEGF was localized to the endothelium of the capillaries and small arteries. This increase in VEGF expression was greatest in right ventricle. This localization may reflect the method of heating being nearer to right ventricle, or some inherent difference between left ad right ventricles. This is consistent with studies showing that smooth muscle cells can produce VEGF and VEGF primarily targets endothelial cells, which have two VEGF cellsurface receptors, Flt-1 and Flk-1 (Carmeliet and Collen, 1999, 2000; Larrivee and Karsan, 2000; Veikkola and Alitalo, 1999). A previous study showed that although a sub-lethal ischemic stress can increase VEGF levels in the heart, VEGF receptors were down regulated 3 and 6 h post-stress (Miraliakbari et al., 2000), which could have an adverse effect on angiogenesis. However, we observed that heat stress did not significantly affect the expression of the Flt-1 or Flk-1 receptors from 4 to 72 h post-stress, indicating the effect of VEGF up-regulation is not minimized by downregulation of its receptors. The possible association of HSP70 with VEGF signature and angiogenesis Our study revealed a close association between up-regulation of VEGF and HSP70. As chaperones, heat shock proteins play important roles in cell cycle regulation and protection against the adverse effects of stress. Elevated HSPs are involved in the nuclear localization of regulatory proteins, since they transiently associate with key molecules of the cell cycle control system, such as Cdk4, Wee-1, pRb, p53, and p27/Kip1 in proliferating mammalian cells (Helmbrecht et al., 2000). Furthermore, some HSPs, including HSP70, are associated with kinases of the mitogen-activated signal cascade (Helmbrecht et al., 2000). Since both types of VEGF receptors possess intrinsic tyrosine kinase activity and Src kinases are involved in the control of angiogenesis stimulated by VEGF (Schlessinger, 2000), concomitant activation of HSP70 may contribute to overall signaling effects in stressed cells. Moreover, the activation of MAP kinases may lead to the transactivation of VEGF expression (Berra et al., 2000). Of particular concern is that hyperthermia could cause cellular injury that would negate any positive effects of thermallyinduced up-regulation of cardio-protective proteins. Our histological data indicated neither significant edema nor necrosis at
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the time of peak expression of VEGF in cardioventricular tissue. In this study, we found no significant cleavage of PARP and no fluorescent signal in TUNEL assay through the experimental time course indicating that apoptosis was not a significant factor in these changes. Furthermore, in the focal area with apparent angiogenesis observed in right ventricle, VEGF was markedly increased in vessels and interstitial cells in the absence of evident cell injury. Parallel increases in HSP70 were also documented. Collectively, these observations suggest that the induced HSP70 expression may contribute to the effectiveness of the VEGFinduced signaling cascade directly while protecting the endothelial cells from damage associated with increased permeability. Differential effects of WBH on the right and left ventricles It may be noteworthy that the maximum increases in the expression of both VEGF and HSP70 occurred in the left ventricle before they were observed in the right ventricle although the maximum levels obtained were consistently greater in the right ventricle. A possible explanation for this observation is that in our heating model, the right ventricle is positioned closer to the chest wall resulting in a greater heat stimulus because this region of the heart is heated faster and for a longer time since it is closer to the heating source. Temperature probes placed in the right and left ventricles support this view; however, the difference in temperature is less than 1 °C (Fig. 1). We previously showed that applying a heating probe to induce local hyperthermia in the left ventricle alone produced elevated levels of HSP70 in both the left and right ventricles, although hyperthermia was absent in the right ventricle (Gowda et al., 1998b). Collectively, this data suggest that there may be intrinsic differences between the right and left ventricles that result in greater up-regulation of VEGF and HSP70 in the right heart. This could have significant consequences on the relative abilities of the right and left ventricles to undergo neovascularization in response to a stress stimulus. The current study revealed the presence of vessel sprouts exclusively in the right ventricle. It is possible that the lack of observable heat-induced neovascularization in the left heart may be due to the insufficient up-regulation of VEGF. If so, a greater exposure to heat stress may be required to initiate angiogenesis in the left heart. Moreover, whether heat-induced angiogenesis would provide a significant collateral flow to have therapeutic effects is unknown. However, these are important issues to be addressed in future studies. In conclusion, the results of this study show that sub-lethal heat stress can up regulate the expression of VEGF and HSP70 accompanied by enhanced local proliferation of blood vessels without causing cardiac tissue injury. Moreover, the heat stress does not down-regulate VEGF receptors. Therefore, future studies may demonstrate further the potential application of sublethal heat stress as a safe and effective intervention for promotion of neovascularization. Acknowledgements This research was supported in part by a grant from the Advanced Technology Program, Texas Higher Education
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Coordinating Board, Award No. 004952-0318-1999 and a Grant-in-Aid from the American Heart Association, Texas Affiliate (G. Asimakis). References Amant, C., Berthou, L., Walsh, K., 1999. Angiogenesis and gene therapy in man: dream or reality? Drugs 58, 33–36 Spec No 1. Berra, E., Pages, G., Pouyssegur, J., 2000. MAP kinases and hypoxia in the control of VEGF expression. Cancer and Metastasis Reviews 19 (1–2), 139–145. Carmeliet, P., Collen, D., 1999. Role of vascular endothelial growth factor and vascular endothelial growth factor receptors in vascular development. Current Topics in Microbiology and Immunology 237, 133–158. Carmeliet, P., Collen, D., 2000. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Annals of the New York Academy of Sciences 902, 249–262. Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., Nagy, A., 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380 (6573), 435–439. Chawla, P.S., Keelan, M.H., Kipshidze, N., 1999. Angiogenesis for the treatment of vascular diseases. International Angiology 18 (3), 185–192. Couffinhal, T., Silver, M., Zheng, L.P., Kearney, M., Witzenbichler, B., Isner, J.M., 1998. Mouse model of angiogenesis. American Journal of Pathology 152 (6), 1667–1679. Garrity, M.M., Burgart, L.J., Riehle, D.L., Hill, E.M., Sebo, T.J., Witzig, T., 2003. Identifying and quantifying apoptosis: navigating technical pitfalls. Modern Pathology 16 (4), 389–394. Gowda, A., Yang, C., Asimakis, G.K., Rastegar, S., Motamedi, M., 1998a. Heat shock improves recovery and provides protection against global ischemia after hypothermic storage. Annals of Thoracic Surgery 66 (6), 1991–1997. Gowda, A., Yang, C.J., Asimakis, G.K., Ruef, J., Rastegar, S., Runge, M.S., Motamedi, M., 1998b. Cardioprotection by local heating: improved myocardial salvage after ischemia and reperfusion. Annals of Thoracic Surgery 65 (5), 1241–1247. Gu, J.W., Brady, A.L., Anand, V., Moore, M.C., Kelly, W.C., Adair, T.H., 1999. Adenosine upregulates VEGF expression in cultured myocardial vascular smooth muscle cells. Am.J.Physiol 277 (2 Pt 2), H595–H602. Helmbrecht, K., Zeise, E., Rensing, L., 2000. Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Proliferation 33 (6), 341–365. Kanamori, S., Nishimura, Y., Okuno, Y., Horii, N., Saga, T., Hiraoka, M., 1999. Induction of vascular endothelial growth factor (VEGF) by hyperthermia and/or an angiogenesis inhibitor. International Journal of Hyperthermia 15 (4), 267–278. Ladoux, A., Frelin, C., 1993. Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochemical and Biophysical Research Communications 195 (2), 1005–1010.
Larrivee, B., Karsan, A., 2000. Signaling pathways induced by vascular endothelial growth factor (review). International Journal of Molecular Medicine 5 (5), 447–456. Levy, A.P., Levy, N.S., Goldberg, M.A., 1996. Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel–Lindau protein. Journal of Biological Chemistry 271 (41), 25492–25497. Marber, M.S., Latchman, D.S., Walker, J.M., Yellon, D.M., 1993. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation 88 (3), 1264–1272. Miraliakbari, R., Francalancia, N.A., Lust, R.M., Gerardo, J.A., Ng, P.C., Sun, Y.S., Chitwood Jr., W.R., 2000. Differences in myocardial and peripheral VEGF and KDR levels after acute ischemia. Annals of Thoracic Surgery 69 (6), 1750–1753. Nanbu, P.N., Wakabayashi, T., Yamashita, R., Hayashi, H., Hisano, S., Oshika, T., 2004. Heat treatment enhances healing process of experimental pseudomonas corneal ulcer. Ophthalmic Research 36 (4), 218–225. Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z., 1999. Vascular endothelial growth factor (VEGF) and its receptors. FASEB Journal 13 (1), 9–22. Nicoletti, V.G., Stella, A.M., 2003. Role of PARP under stress conditions: cell death or protection? Neurochemical Research 28 (2), 187–194. Operschall, C., Falivene, L., Clozel, J.P., Roux, S., 2000. A new model of chronic cardiac ischemia in rabbits. Journal of Applied Physiology 88 (4), 1438–1445. Ruef, J., Hu, Z.Y., Yin, L.Y., Wu, Y., Hanson, S.R., Kelly, A.B., Harker, L.A., Rao, G.N., Runge, M.S., Patterson, C., 1997. Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis. Circulation Research 81 (1), 24–33. Schaper, W., Ito, W.D., 1996a. Molecular mechanisms of coronary collateral vessel growth. Circulation Research 79 (5), 911–919. Schaper, W., Ito, W.D., 1996b. Therapeutic targets in cardiovascular disorders. Current Opinion in Biotechnology 7 (6), 635–640. Schlessinger, J., 2000. New roles for Src kinases in control of cell survival and angiogenesis. Cell 100 (3), 293–296. Schwarz, E.R., Speakman, M.T., Patterson, M., Hale, S.S., Isner, J.M., Kedes, L.H., Kloner, R.A., 2000. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat-angiogenesis and angioma formation. Journal of the American College of Cardiology 35 (5), 1323–1330. Shweiki, D., Itin, A., Soffer, D., Keshet, E., 1992. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359 (6398), 843–845. Vanden, H.T., Becker, L.B., Shao, Z.H., Li, C.Q., Schumacker, P.T., 2000. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circulation Research 86 (5), 541–548. Veikkola, T., Alitalo, K., 1999. VEGFs, receptors and angiogenesis. Seminars in Cancer Biology 9 (3), 211–220. Yamashita, N., Hoshida, S., Taniguchi, N., Kuzuya, T., Hori, M., 1998. Wholebody hyperthermia provides biphasic cardioprotection against ischemia/ reperfusion injury in the rat. Circulation 98 (14), 1414–1421.