Thermal Preconditioning Before Rat Arterial Balloon Injury

Limitation of Injury and Sustained Reduction of Intimal Thickening

From the Departments of Surgery (D.G.N., S.D.S., M.L.D., A.K.H., M.A.G.), Medicine (P.N.R., D.J.L., K.K., E.S.B.), and Pathology (J.E.T.), University of Pennsylvania Medical Center, Philadelphia, Pa.

Correspondence to Michael A. Golden, MD, Department of Surgery, Hospital of the University of Pennsylvania, 4 Silverstein Pavilion, 3400 Spruce St, Philadelphia, PA 19104. E-mail goldenm{at}mail.med.upenn.edu.

	Abstract

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Abstract—Heat shock proteins (HSPs) are a family of highly conserved proteins, essential to cell survival, that are induced during times of physiological stress. These proteins, when induced, can provide tolerance to subsequent injury. Several studies have documented that HSPs play an important role in the response of vascular cells to injury or stress. Whether the vasculature itself can be effectively preconditioned before arterial injury is unknown. Vascular HSP induction by whole-body hyperthermia (WBH) was evaluated with regard to its effects on the vascular response to balloon injury. WBH treatment of Sprague-Dawley rats (colonic temperatures of 41 to 42°C for 15 minutes) resulted in maximal arterial HSP expression within 8 to 12 hours. Rats (male, 300 g, n=59) were randomly assigned to undergo either WBH or no treatment 8 hours before standard carotid balloon injury. At 14 (n=26) and 90 (n=21) days after balloon injury, histomorphometric analysis revealed a significant limitation of intimal accumulation in preconditioned arteries as compared to controls (intimal/medial area ratios±SEM: 14 days, 0.57±0.07 versus 0.86±0.08, P=0.01; 90 days, 0.78±0.12 versus 1.19±0.14, P<0.05). The medial cell proliferation index at 4 days (n=12) was significantly reduced in the treated group as well (3.6±0.9% versus 7.2±1.3%, P<0.05). Conversely, the mean total cell number in the media of heated arteries was higher (393±20 versus 328±17, P<0.05). Vascular preconditioning with brief WBH induces a heat shock response in the arterial wall that is associated with a significant and sustained reduction in intimal accumulation. This effect appears to be due in part to preservation of medial cell integrity and limitation of the proliferative response. These results suggest that thermal preconditioning of vascular tissue may be an effective strategy to improve long-term results after revascularization procedures.

Key Words: heat shock proteins • arteries • restenosis • smooth muscle • balloon

	Introduction

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The term heat shock proteins (HSPs) refers to a family of proteins essential for normal cell viability and thermotolerance, reported by Tissières and colleagues¹ in 1974 after the observation of heat-inducible 70-kd proteins in Drosophila. This followed the first description of a "puffing pattern" induced by temperature shock in Drosophila by Ritossa² in 1962. Commonly referred to as "molecular chaperones," the HSPs bind ATP and are believed to participate in energy-dependent processes such as the disassembly of the clathrin coat of coated vessels, ATP-reversible binding of various abnormal proteins, and the translocation of proteins across membranes.³

These structurally related proteins are grouped according to their molecular weight, of which the HSP60, HSP90, and HSP70 families are the most abundant.⁴ The HSP70 family of proteins have been extensively studied, in part because the inducible form of HSP70 is the most closely related to the magnitude and duration of a thermal stress.⁵ Previous investigators have reported that the induction of HSP70 protein imparts at least some degree of thermotolerance and is not merely a marker of a generalized heat shock response. Microinjection of anti-HSP70 antibodies into rat cells has been shown to prevent nuclear accumulation of HSP70 and has resulted in increased lethality to heat treatment.⁶ Similarly, plasmid transfection of rat fibroblasts with human HSP70 conferred resistance to heat stress.⁷

A wide variety of studies have demonstrated that preconditioning of organs by induction of HSP expression can limit subsequent injuries. For example, pretreatment with WBH demonstrated a protective effect on myocardial tissue subjected to subsequent ischemia and reperfusion injury in rabbits.⁸ As further evidence of the protective role of HSPs, Plumier and coworkers⁹ recently demonstrated that a transgenic mouse expressing the human inducible form of HSP70 had increased protection of the myocardium from ischemia and reperfusion injury.

Johnson et al¹⁰ demonstrated, in atherosclerotic plaques, a decrease in HSP levels in vascular SMCs residing near areas of necrosis, suggesting a role of HSPs in SMC survival. Their study was prompted by earlier work demonstrating the induction of HSPs in areas of atherosclerotic plaque subjected to increased stress.¹¹ To date, however, the issue of whether the vasculature itself can be preconditioned in vivo to limit subsequent injury has not been addressed. This is particularly important because restenosis occurs after angioplasty in 35% to 45% of patients at 6 months^{12 13} and because coronary stenting, which has gained widespread acceptance as a method of limiting restenosis, is still associated with a proliferative intimal thickening response that may be even greater than that seen with conventional angioplasty.^{14 15}

For heat stress to induce a protective effect in a particular tissue, that tissue must be able to mount an effective heat shock response. Udelsman and colleagues¹⁶ have shown that the application of external stress can induce the production of HSP70 in rat aorta and that this ability decreases with increasing age of the animal. This same group also demonstrated that acute hypertension upregulates HSP production in aorta.¹⁷

Balloon vascular injury has been previously demonstrated, at least in rats, to lead to a loss of a portion of the vascular SMCs, presumably because of cell necrosis.¹⁸ The degree of the proliferative response to this injury appears to be directly proportional to the degree of injury.¹⁹ The mechanism of this graded response has been suggested to be due to release of bFGF by injured and dying cells.²⁰ These data suggest that limiting the initial injury to SMCs might limit the subsequent proliferative response. We therefore hypothesized that pretreatment of an animal with WBH, designed to generate a heat shock response in the arterial wall, might confer a protective effect on SMCs subjected to a properly timed subsequent balloon injury, resulting in a reduced neointimal response.

	Methods

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Animals
All experimentation was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. Male Sprague-Dawley rats weighing approximately 300 to 350 g (Harlan Sprague Dawley, Indianapolis, Ind) were housed in an environmentally controlled facility at a temperature of 72°F with a 12-hours-light/12-hours-dark cycle. All animals were allowed to acclimate for at least 48 hours before experimentation. Rats had access to rat chow and water ad libitum except in the immediate (8 hour) postoperative period.

Passive Hyperthermia
Rats were placed in a clear, ventilated Plexiglas® restraining device. A flexible thermometer was placed rectally, and serial measurements of colonic temperature were made every 5 minutes. A commercial heating blanket was wrapped around the restraining device with care to allow adequate ventilation and observation. This unit was then placed in an open plastic animal container 6 in under an 80-W incandescent lamp. Animals were heated in this manner until colonic temperature reached 41°C (average time, 35 minutes), at which time the light source and heating blanket were removed as needed to maintain the temperature between 41 and 42°C for 15 more minutes. The rats were then immediately released from the restraining device and allowed to cool. Rats returned to baseline temperature within 30 minutes after release and did not show any adverse effect from the heating. There were no mortalities related to heating when this protocol was used.

RNA Extraction and Northern Analysis
Harvested tissues were homogenized and the RNA extracted with RNAzol B. Total RNA (10 µg per lane), run in duplicate, was separated by electrophoresis on formaldehyde-agarose gels and transferred to nylon membranes. Hybridization was performed using a ³² P-labeled cDNA HSP70 probe as previously described.²¹ The cDNA probe was a gift from Dr Reuben Mestril²² and was labeled using a random priming technique with -[³²P] dCTP. Accuracy of loading and transfer was confirmed by hybridization with an 18S rRNA-specific cDNA probe. Autoradiographs of the blots were obtained and quantified by PhosphorImage analysis.

Western Analysis
Harvested tissues were pulverized and suspended in a cell lysis solution containing 1% SDS, 1 µM PMSF, and 10 µg/mL leupeptin. After centrifugation, a portion of the supernatant was used for protein determination. The remainder was diluted 1/1 in sample buffer containing 5% ß-mercaptoethanol and boiled for 5 minutes. The protein concentration of each sample was determined by protein assay. Equal amounts of total protein were loaded in duplicate onto a 10% polyacrylamide gel, electrophoresed, and transferred to nitrocellulose. The blot was then incubated at 4°C overnight in a 5% dried-milk reagent dissolved in 1x Tris-buffered saline with 0.1% Tween-20. The blot was then incubated with a murine monoclonal anti-HSP70 antibody diluted 1/2000 in Tris-buffered saline. A biotinylated sheep anti-mouse immunoglobulin was used as the secondary antibody. The blots were incubated with a streptavidin–alkaline phosphatase complex. The complex was visualized with nitroblue tetrazolium in dimethyl formamide in combination with 5-bromo-4-chloro-3-indolyl phosphate in dimethyl formamide as substrate for the alkaline phosphatase.

Carotid Balloon Injury Model
Anesthesia was induced, after a brief exposure to inhaled methoxyflurane, by intramuscular injection of ketamine (50 mg/kg), xylazine (5 mg/kg), and acepromazine maleate (1 mg/kg). Additional doses equivalent to one-half the initial doses of ketamine and xylazine were given when required. A midline incision was made in the anterior neck, and a controlled injury of the left common carotid artery was induced by three passes of a 2F Fogarty balloon catheter as previously described.¹⁸ Suture materials were a gift from United States Surgical Corporation.

Tissue Preparation
Rats underwent WBH as described above. The aorta and both carotid arteries were harvested, rinsed with PBS, and immediately frozen in foil in a 2-methylbutane/dry ice bath. These samples were used for analysis of HSP70 RNA or protein.

Harvest time points for study of mRNA in aortic tissue were immediately after and 1, 4, 8, 12, 16, 24, 48, 72, and 96 hours after heating. Time points for carotid tissue were immediately after and 1, 2, and 6 hours after heating. Four carotid arteries had to be pooled for each time point to obtain sufficient tissue for RNA analysis, whereas sufficient tissue was available in a single aorta. In addition, tissue from nonstressed rats, rats that were exposed to inhaled methoxyflurane for 2 minutes, and rats that were restrained but not heated were studied as controls.

Similarly, harvest time points for protein analysis in aortic tissue were immediately after and 4, 8, 12, 16, 24, 48, 72, and 96 hours after heating. Time points for carotid tissue were 6, 8, 12, 16, and 24 hours after heating. Two carotid arteries were pooled for each time point to obtain sufficient tissue for analysis of protein. Control vessels were harvested from unstressed animals.

At 14 and 90 days after balloon injury, injured (described above) and contralateral uninjured carotid arteries from heat-pretreated and nonheated controls were harvested for histomorphometric analysis in the following manner. Under anesthesia (as described above), 1 mL of a 5.0% solution of Evan's Blue was administered intravenously 20 minutes before killing. By staining deendothelialized vessels a deep blue, adequacy of balloon injury was confirmed in all animals studied. Laparotomy was then performed, with exposure of the abdominal aorta and cannulation with a 22-gauge catheter. The external jugular vein was then exposed and incised for exsanguination. The aorta was perfused retrogradely with lactated Ringer solution at 120 to 150 mm Hg of pressure until the effluent was clear, followed by 10% neutral buffered formalin (also at 120 to 150 mm Hg) for fixation. This was followed by at least 24 hours of immersion in the same fixative. Proximal, middle, and distal segments of the balloon-injured carotid arteries were embedded in paraffin and sectioned. Slides were stained with hematoxylin and eosin. The intimal area, medial area, and I/M ratio of each of the three segments were obtained by computerized planimetry (Summa Sketch II Plus with BioQuant System IV software). Mean values for each artery were calculated. An additional series of control and heat-pretreated rats were killed 4 days after balloon injury and were used to measure vascular SMC proliferation.

Immunohistochemistry
Segments of aorta and carotid artery were harvested from heated rats at various times after heating and from nonheated controls. These segments were then immersion fixed in 10% buffered formalin, paraffin embedded, and sectioned. Immunohistochemistry was performed using a test kit relying on a rat absorbed goat anti-mouse biotinylated secondary antibody and a horseradish-peroxidase-conjugated–streptavidin complex. A 1/250 dilution of mouse anti-HSP70 primary antibody was incubated at 4°C overnight, followed by a 1-hour incubation at room temperature. The final complex was visualized with 3'3'diaminobenzidine and hydrogen peroxide in PBS. Parallel staining was performed with nonimmune IgG1 isotype as the primary antibody. Samples were studied for localization of HSP70 containing cells and for timing of maximal HSP70 expression.

In Vivo Quantitation of Proliferation: BrdU Labeling
To study the mechanism of heat shock preconditioning on the vascular remodeling response, heated rats and nonheated controls were balloon injured as described above. Exsanguination under anesthesia and formalin fixation was performed as described above 4 or 14 days after arterial injury. At 19, 8, and 1 hour before killing, rats received an intramuscular dose of BrdU (50 mg/kg). BrdU, a thymidine analog, is taken up by proliferating cells and can be visualized by immunohistochemical techniques. Injured carotid arteries, contralateral negative controls, and small intestine segments (positive control) were paraffin embedded and sectioned, and a random section from each animal was incubated with antibody to BrdU at a concentration of 10 µg/mL. Intracellular BrdU was detected as previously described²³ using an avidin-peroxide complex and 3,3 diaminobenzidine substrate. Slides were counterstained with hematoxylin to enable counting of the total number of cells in each compartment.

Data Analysis
Results are reported as mean±SEM. Values from heat-pretreated and nonheated control animals were compared using a two-tailed Student's unpaired t test. Differences were considered significant at the level of P<0.05. All experiments were performed in a blinded manner such that the investigator performing the balloon injury did not know which animals were pretreated.

	Results

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Induction of HSP70 mRNA and Protein Expression
Our first objective was to study the time course of HSP induction in rat arterial tissue. Aortic samples were studied initially because there was sufficient tissue from a single aorta to extract the required amounts of RNA and protein for analysis. Time points selected for analysis in carotid tissue were based on the results in the aorta. Northern analysis was used to examine the relative level of expression of HSP70 mRNA in the aorta (Fig 1, A and B) and in the carotid arteries (Fig 1, C and D) at the indicated time points following WBH and in controls. This comparison illustrates an intense induction of HSP70 mRNA in both carotid artery and aortic tissue, present immediately after completion of the heating protocol (which averaged 45 minutes from start to finish) and at 1 and 2 hours after termination of the heating protocol. HSP70 mRNA levels rapidly decreased thereafter, returning to essentially baseline by 6 hours. After quantitation by PhosphorImaging analysis and normalization to 18S rRNA levels, we detected a maximum 100-fold increase in HSP70 mRNA levels in the aorta and a maximum 35-fold increase in HSP70 mRNA levels in the carotid artery relative to nonstressed controls. Restraint for 45 minutes alone elicited a smaller but detectable increase in HSP70 mRNA expression, a finding similar to that of other investigators.¹⁶ An additional faint band at 24 hours is noted in Fig 1, A; however, its significance is unclear.

View larger version (25K): [in this window] [in a new window]   Figure 1. Northern analysis of steady-state HSP70 mRNA levels after WBH. Total RNA was extracted from aortas (A) and carotid arteries (C) of male Sprague-Dawley rats that were not stressed (NS), exposed to a brief (2-minute) period of inhaled anesthesia (In), restrained only (R), or exposed to WBH. Rats were killed at the times indicated (hours) after the heating protocol. The Northern blots (10 µg per lane) were then sequentially probed with labeled cDNA probes for HSP70 and 18s rRNA. Quantitative analyses (B and D) of the Northern blots shown in A and C, respectively, were performed using a PhosphorImager. Values were normalized to the level of 18S rRNA in each lane and are expressed relative to the level of HSP70 mRNA in a nonstressed control animal (A) or an animal exposed to brief inhaled anesthesia only (C). This figure shows an early and transient upregulation of HSP70 mRNA in both aortic and carotid tissues.

To confirm that balloon injury itself did not induce large amounts of HSP70 mRNA, further studies of steady-state mRNA levels were performed at time points ranging from 0 to 96 hours after balloon injury. Normal, noninjured rat carotid artery had essentially undetectable levels by Northern analysis, and no induction of HSP70 mRNA was detected as a result of balloon injury alone in nonheated rats at any of the time points indicated from 1 hour to 2 weeks after injury (Fig 2).

View larger version (54K): [in this window] [in a new window]   Figure 2. Effect of balloon injury on HSP70 mRNA expression: Northern blot of steady-state HSP70 mRNA levels in unheated rat carotid tissue at indicated times after balloon injury. The Northern blot was performed as described in the legend of Fig 1. NI indicates not injured; and C, positive control (aorta 1 hour after heat stress). No significant induction of HSP70 mRNA in response to balloon injury alone was shown.

Western analysis was performed on protein extracted from the aorta and carotid arteries at the indicated time points after heating and from controls to determine the timing of maximal HSP70 protein expression (Fig 3). Levels of HSP70 protein was maximal at 8 to 12 hours after heating, with a steady decrease in protein levels thereafter. No significant HSP70 protein induction was noted in control arteries. We therefore elected to perform all balloon injury experiments on heat-pretreated rats 8 hours after the heating protocol to coincide with rapidly rising HSP70 protein levels to maximize the hypothesized protective effect.

View larger version (15K): [in this window] [in a new window]   Figure 3. Time course of HSP70 induction in aorta (A) and carotid arteries (B) after WBH: Western blot analysis. Protein was extracted from rat aorta and carotid arteries and separated using reducing 10% SDS-PAGE (10 µg per lane). HSP70 protein was identified using a monoclonal anti-rat HSP70 antibody and an enzyme-conjugated secondary antibody detection system. Peak protein induction was present 8 to 12 hours after WBH.

Immunohistochemistry: Confirmation of Timing of HSP70 Expression and Protein Localization
Immunohistochemistry was performed both to confirm results obtained by Western analysis as to the timing of the robust HSP70 protein response as well as to specifically localize protein expression within arterial wall (Fig 4). Maximal expression of HSP70 protein was detected in the endothelium and media of aortic tissue at 8 and 12 hours after heating, with a diminished response at 16 hours (Fig 4, A, C, and E). Low-level HSP70 expression was detectable in aortas of unheated rats as well (Fig 4, G). Maximal expression of HSP70 protein was detected in a similar time course and distribution in the carotid artery, with peak expression at 12 hours (Fig 4, D) after heating and a diminished response at 16 hours (Fig 4, F). The majority of HSP70 protein was localized in medial SMCs. No significant staining was detected in either nonstressed control carotid arteries stained with anti-HSP70 antibody (Fig 4, H) or heated carotid arteries stained with nonimmune IgG1 as an additional control (Fig 4, B).

View larger version (108K): [in this window] [in a new window]   Figure 4. Immunohistochemical localization of HSP70 protein. Aortas and carotid arteries from either unstressed rats or rats exposed to WBH were harvested after heating at the times indicated, fixed in formalin, sectioned, and stained with a monoclonal anti-HSP70 antibody1:250 (A and C through H) or an equal concentration of nonimmune isotype control IgG1(B). The antibodies were detected with an enzyme-conjugated detection system that yields a brown color in positive cells. Samples of aorta (A, C, E, and G) and carotid artery (B, D, F, and H) were harvested after heating for 8 hours (A), 12 hours (B, C, and D), and 16 hours (E and F), or with no preheating (G and H). The sample shown in B was treated with a nonimmune IgG primary antibody as an additional negative control. (Original magnification x400.)

Inhibition of the Increased I/M Ratio Response to Injury
Animals were harvested 14 days after balloon injury as described above. The intimal area, medial area, and I/M ratio was obtained from each animal (values are reported as mean±SEM). There was a trend toward lower absolute intimal area in the HS group (0.078±0.008 versus 0.103±0.009 mm², P= 0.06), while the medial area in the HS animals was only slightly, but significantly, higher than that of controls (0.139±0.004 versus 0.122±0.007 mm², P<0.05). Fig 5 depicts a representative section of control and preconditioned carotid arteries 14 days after balloon injury. The I/M ratio is used to analyze effects on intimal accumulation as it controls for variability due to animal size or perfusion artifact. The I/M ratio was significantly lower in the HS group (n=14) than in controls (n=12), (0.57±0.07 versus 0.86±0.08, P=0.01) (Fig 6). These data suggest that preconditioning may both limit intimal accumulation and preserve medial integrity, thus maintaining a more normal wall architecture.

View larger version (113K): [in this window] [in a new window]   Figure 5. Hematoxylin and eosin-stained micrograph of rat carotid artery sections 14 days after balloon injury. A shows uninjured carotid artery; B, control artery (injured but not heat- pretreated); and C, heat-pretreated artery 8 hours before balloon injury. (Original magnification x400.)

View larger version (30K): [in this window] [in a new window]   Figure 6. I/M ratio 14 and 90 days after balloon injury. Animals in the heated groups were exposed to WBH (41 to 42°C) for 15 minutes. Balloon injury of the left common carotid artery was performed 8 hours after heating. Rats were killed and perfusion fixed 14 and 90 days after balloon injury. Histomorphometry was performed on heated rats and nonstressed controls. I/M ratios were obtained and are expressed as mean±SEM.

To determine whether heat preconditioning had a sustained effect on the reduction of intimal accumulation, an HS group and a control group of rats were harvested 90 days after balloon injury. The mean I/M ratio of the heat-preconditioned rats (n=12) was significantly lower than that of the control animals (n=9, 0.78±0.12 versus 1.19±0.14, P<0.05), demonstrating a sustained effect (Fig 6). The mean absolute intimal area showed a trend toward being lower in the heat-preconditioned group (0.11±0.02 versus 0.15±0.02 mm², P=.09). There were no significant difference in the absolute mean medial area of the two groups (0.136±0.006 versus 0.127±0.005 mm², HS versus control, P=0.29). These data suggest a sustained reduction in intimal accumulation in the heat-preconditioned animals 3 months after balloon injury.

Inhibition of SMC Proliferation and Medial Preservation
To investigate the mechanism of heat shock preconditioning on the vascular remodeling response, we studied BrdU incorporation in carotid arteries 4 and 14 days after injury and in contralateral uninjured arteries and small intestine segments (as controls) for both heated and control animals. The proliferation index was determined by dividing the number of positively staining cells by the total number of cells. The mean medial proliferation index was significantly lower in the heated group (3.6±0.9%) than in the control group (7.2±1.3%) 4 days after balloon injury (P<0.05). Despite a significant inhibition of medial cell proliferation, the mean total medial cell count was significantly higher in the heat-preconditioned arteries compared to control arteries 4 days after balloon injury (393±20 versus 328±17, P< 0.05). Heating alone, without injury, did not result in any changes in the medial cell number (389±28 versus 356±15, heated, uninjured versus control, uninjured, P=0.38). There were no significant differences in the intimal or medial proliferative indices between heated animals and controls when studied 14 days after balloon injury. These data, which demonstrate less proliferation but more total medial cells, suggest that preconditioning may lower proliferation by limiting the loss initially, thereby lessening the subsequent proliferative stimulus.

	Discussion

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This study demonstrates for the first time that the vasculature itself can be preconditioned by appropriately timed thermal stress to limit both the amount of medial cell loss and the severity of the resultant proliferation and intimal accumulation induced by balloon injury. The protection afforded by preconditioning was durable in that the same degree of limitation of intimal accumulation seen at 2 weeks was also observed at 3 months, which represents a long-term result in this model.

Numerous investigators have elicited a heat shock response in various animals and tissues and have demonstrated a protective effect. Yellon and colleagues⁸ have demonstrated the protective role of WBH in decreasing the extent of injury following an ischemia-reperfusion event in rabbit myocardium. Saad et al²⁴ used WBH to precondition rats before an ischemia-reperfusion event in the liver. These investigators demonstrated a significant improvement in survival and liver function studies in the preconditioned animals. In the field of transplantation, Perdrizet and colleagues²⁵ have demonstrated a significant improvement in the ability to recover functional pancreatic islet cells from donors that underwent a heat stress. These same investigators have also reported improved function in porcine renal allografts taken from donors that experienced a heat shock before organ harvest.²⁶ However, no studies have addressed the use of thermal preconditioning in vivo to ameliorate the effects of vascular injury.

Previous investigators have demonstrated the ability of arterial tissue to respond to a heat stress by upregulating the production of HSP70 proteins.¹⁶ The addition of HSP72/73 proteins to cultures of arterial cells from normal and atherosclerotic cynomolgus macaques increased arterial cell survival to subsequent stress.²⁷ More recently, the same investigators suggested that insufficient HSP70 accumulates in SMCs located near necrotic areas of atherosclerotic lesions, possibly leading to loss of protection of these cells to plaque toxicity.¹⁰ Acute hypertension has also been shown to induce an HSP response in the rat aorta; this ability may be lost or lessened as the animal ages.¹⁷ Our results confirm and extend these observations in that we demonstrated that both aorta and carotid artery significantly upregulate HSP70 mRNA and protein expression after brief WBH. Of note is that balloon injury itself, in our study, did not elicit a significant upregulation of HSP70 expression, whereas brief WBH did. However, by 6 hours after heating, levels of HSP70 mRNA had returned to near baseline. In both aortic and carotid tissue, the levels of HSP70 protein, as demonstrated by Western analysis, was highest at 12 hours, with a rapid decline to near baseline levels after 24 hours. We selected the timing of balloon injury to coincide with a rapid increase in, and high levels of, HSP70 protein. It is unclear at the present time whether this is the optimal timing to confer the maximum degree of protection. It is also not certain whether a longer duration of HSP expression, a higher magnitude of expression, or both would convey more protection. Additional studies exploring these issues seem warranted.

WBH may not be the only way or the best way to initiate a vascular preconditioning response. Direct application of heat to the vessel wall has been accomplished via specialized angioplasty catheters,²⁸ but the temperatures (50 to 60°C) and timing (at the time of angioplasty) used make it unlikely to have conveyed a protective effect. Various genes have also been overexpressed via gene-transfer approaches in the vessel wall after angioplasty²⁹ in an attempt to limit intimal thickening, but few studies to date have attempted to precondition the vessel wall sufficiently before injury to enable optimal gene expression at the time of the angioplasty. Overexpression of HSPs in the vessel wall at an early enough time before vascular intervention could confer a similar or greater degree of protection, but this concept needs to be tested directly. It is also unclear from our studies whether overexpression of a single HSP would give as protective an effect as demonstrated here.

The precise mechanism of protection can only be inferred from the data at the present time. Intimal thickening after vascular injury in this model is a process initiated by deendothelialization, barotrauma, and cell death, followed by SMC proliferation, migration to the intima, and subsequent neointimal cell proliferation associated with extracellular matrix deposition. Studies by Lindner and Reidy²⁰ have suggested that administration of an antibody to bFGF around the time of balloon injury can limit the initial proliferative response in the rat carotid artery injury model. Additionally, they have demonstrated that SMC migration is significantly enhanced when deendothelialization of the rat carotid artery is accompanied by traumatic injury to the underlying smooth muscle or by the systemic injection of bFGF in animals that underwent gentle deendothelialization.³⁰ These data would suggest that release of bFGF from dead or dying medial SMCs may play an important role in augmenting the response to injury.³⁰ Similarly, Indolfi and colleagues¹⁹ have demonstrated that the degree of neointimal thickening and SMC proliferation is proportional to the severity of a balloon injury in the rat carotid artery. Increased balloon injury severity was also associated with a proportional increase in c-fos proto-oncogene expression in this study.

It has been reported that FGF-1 can be released by cells in response to heat stress.³¹ Additionally, Nabel and colleagues³² have demonstrated that direct gene transfer of FGF-1, delivered into the arterial wall, could induce substantial intimal hyperplasia. These findings could suggest a potential limitation to the use of thermal preconditioning of the vasculature. However, Shi et al³¹ note that FGF-2, well known to be a SMC mitogen, is not released by cells in response to heat stress. Furthermore, Nabel et al³² demonstrated enhanced reendothelial repair in response to FGF-1 induction. It could be postulated that heat stress induces the release of extracellular FGF-1, which may lead to both enhanced reendothelialization and intimal thickening, with the reendothelialization being a more efficient process, leading to an overall reduction in intimal thickening. Our 14-day data were generated exclusively from deendothelialized segments; therefore, early protection is probably not related to efficient reendothelialization. However, at the 3-month time point, most of the vessel had reendothelialized in both the heat-pretreated and control groups. It is possible that increased reendothelialization efficiency in the heat-pretreated group is responsible for the durability of the protective effect, but this would need to be studied more specifically. In our studies, heating alone did not result in intimal thickening in any of the uninjured arteries. The heat shock response involves the production of multiple proteins, one of which is HSP70. Further studies are warranted to determine the specific proteins involved in vascular protection.

Our findings of preserved medial SMC numbers despite a lower proliferative index suggest that WBH enhances the ability of medial SMCs to withstand the original insult, and this subsequently leads to a less intense neointimal response. This concept is significantly different from most other strategies to limit this response. Many approaches involve therapies to suppress the intensity of the proliferative response after the fact rather than limiting the extent of the injury.³³ Further studies focusing on early events and cell death following thermal preconditioning and balloon injury are needed to help clarify the specific mechanisms of protection. The current study suggests that preconditioning of vascular tissue may be an effective, novel strategy to improve long-term results after revascularization procedures.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor BrdU = 5-bromo-2-deoxyuridine FGF = fibroblast growth factor HS = heat stress HSP = heat shock protein I/M = intimal/medial area SMC = smooth muscle cell WBH = whole-body hyperthermia

	Acknowledgments

 
This work was supported in part by the National Institutes of Health (grant HL47839), the American Heart Association, and the Lifeline Foundation.

Received July 16, 1997; accepted October 3, 1997.

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