73 and impaired wound healing in diabetic and hypercortisolemic states

Heat-shock protein 72/73 and impaired wound healing in diabetic and hypercortisolemic states Milad S. Bitar, MSc, PhD, FCP, Thameem Farook, PhD, Bency John, BSc, and Issam M. Francis, MD, PhD, Safat, Kuwait

Background. Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Emerging evidence favors the involvement of glucocorticoids (GCs) in the pathogenesis of this diabetic complication. Recent data indicated that a heat-shock protein (HSP) with a molecular weight of about 70 kd is expressed in wound healing and it is under the control of the hypothalamic-pituitaryadrenal axis. In view of these findings, the current study was designed to examine the influence of diabetes and the hypercortisolemic state on the expression of HSP 72/73 during wound healing. Methods. Induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol (PVA) sponges were used as a wound healing model. Control and diabetic animals received, respectively, subcutaneous 30-day timed-release pellets of GC (200 mg) and RU 486 (25 mg). Corresponding animals received placebo pellets. Expression of HSP 72/73 within the PVA sponges was assayed with use of Western blotting and immunohistochemical techniques. Results. GCs caused a Cushing-like syndrome with weight loss and adrenal atrophy. A pronounced accumulation of constitutive HSP 72/73 was observed in the cytoplasm of various cell types including fibroblasts, macrophages, and endothelium of nondiabetic controls. The PVA sponge contents of HSP 72/73 were decreased as a function of diabetes. A similar phenomenon was seen in control animals receiving high doses of GCs. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the GC receptor block RU 486 (hypercortisolemia) ameliorated the diabetes-related decrease in PVA sponge contents of HSP 72/73. Conclusions. The current study provides evidence that both diabetes and the hypercortisolemic state are associated with a reduction in PVA sponge content of HSP 72/73. An amelioration of these changes was achieved by the institution of RU 486 therapy. Although our data may point to the possibility that the diabetes-related decrease in HSP 72/73 is mediated at least in part by GCs, a confirmation regarding this premise awaits further investigation. (Surgery 1999;125:594-601.) From the Departments of Pharmacology and Toxicology and Pathology, Faculty of Medicine, Kuwait University, Safat, Kuwait

WOUND HEALING IS A COMPLEX phenomenon that proceeds with inflammation and granulation tissue formation, followed by extracellular matrix deposition and remodeling.1 Clinical and experimental diabetes has been reported to alter most of these processes, including a reduction in angiogenesis, collagen biosynthesis, and inflammatory cell infiltration.2-7 A possible mediator of diabetes-related deficit in wound repair may include an elevation in Accepted for publication Feb 20, 1999. Supported by Kuwait University grant No. MR033. Reprint requests: Milad S. Bitar, MSc, PhD, FCP, Department of Pharmacology, Faculty of Medicine, PO Box 24923, 13110 Safat, Kuwait. Copyright © 1999 by Mosby, Inc. 0039-6060/99/$8.00 + 0

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the plasma level of glucocorticoids (GCs).8 These hormones may regulate the expression of multiple genes that are likely to influence cell ability to respond to the stress of wounding.9 Cells derived from various organs respond to metabolic stress by increasing the formation of a set of highly conserved proteins termed heat-shock or stress proteins (HSPs). A certain class of these HSPs with a molecular weight of about 70 kd appears to exhibit a cytoprotective role in that it makes a cell better able to survive an acute stress or injury.10-17 Indeed, a loss of heat stress tolerance has been demonstrated in fibroblasts and neurons after they were microinjected with an anti-HSP 70 antibody.18,19 The HSPs 70 are also involved in normal cellular functions including protein folding, assembly, and translocation as well as DNA replication and repair.20-22

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Fig 1. Body weights (BW) in control and diabetic animals receiving placebo or drug treatment. Diabetes (Diab) inductions, drug treatment, and wound initiation were conducted as described in Table. Values are expressed as means ± SEM of at least 8 animals. Asterisk, Significantly different from corresponding initial body weight at P < .05. Cont, Control; Ins, insulin.

In view of these HSP-related functions and the fact that most of them are recognized features of tissue repair dynamics, we initiated the current study with the aim of examining wound tissue expression of HSP 72/73 in normal, diabetic, and hypercortisolemic states. MATERIAL AND METHODS Animals. Adult male Sprague-Dawley rats (Kuwait University breeding colony) weighing 175 to 200 g were housed individually on a 12-hour light/dark cycle (light on from 6 AM to 6 PM). The ambient temperature was kept at 21°C and the rats had free access to standard laboratory food and tap water. Diabetes induction and insulin treatment. Diabetes was induced by an intravenous injection of streptozotocin (STZ, 55 mg/kg body weight) diluted in 0.05 mol/L sodium citrate, pH 4.5; control rats received buffer alone by the same route. Diabetic animals were randomly subdivided into 2 groups, with one group receiving no antidiabetic treatment and the other receiving a subcutaneous injection of crystalline zinc insulin/lente insulin (1:3 ratio), 5 to 8 U daily, starting 3 days after STZ injection and continuing throughout the experimental period. The dose of insulin was determined on the basis of the daily urine and weekly blood tests for glucose levels. Steroid and RU 486 therapy. A hypercorti-

solemic state was induced in nondiabetic control rats by the subcutaneous implantation of slowreleasing pellets containing a supraphysiologic dose of GCs. One group of the hypercortisolemic animals was subjected to treatment with the GC receptor blocker RU 486, and the other received placebo pellets by the same subcutaneous route. RU 486 was also given to a group of 5-day STZ diabetic rats. The pellets were calibrated to release GC (6.7 mg/d) or RU 486 (0.84 mg/d) for 30 days. A preliminary study in our laboratory has indicated that a supraphysiologic dose of GC reduced both thymus tissue weight and polyvinyl alcohol (PVA) sponge hydroxyproline content and increased hepatic activity of tryptophan dioxygenase, a hemecontaining enzyme that is inducible with ethanol and GCs.23,24 All the above GC-related actions were counteracted by RU 486 given for 30 days at a rate of 0.84 mg/day. Lower dosages (0.21, 0.42 mg/d) were also used but the degree of effectiveness was minimal. RU 486 (a selective GC antagonist) possesses a high binding affinity for the GC receptor with negligible agonistic activity, even at high doses.25-28 Indeed, it has been reported that RU 486 is the first known GC antagonist with a full capacity to negate the acute and chronic effects of GCs in vivo as well as in vitro.25-28 Wound healing model. Twenty days after placebo or insertion of the slow-releasing pellets, animals were subjected to wounding under pentobarbital

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Fig 2. Representative Western blot of PVA sponge content of constitutive HSP 72/73 as function of duration of diabetes. Diabetes induction, wound initiation, and drug treatment were conducted as described in the Table. Total cellular protein was isolated from PVA sponges derived from control and diabetic rats. HSP 72/73 protein levels were determined in individual samples (40 mg) by immunoblotting. Lane 1, Control; lane 2, 1-day diabetic; lane 3, 3-day diabetic; lane 4, 7-day diabetic; lane 5, 30-day diabetic.

anesthesia (50 mg/kg). After the backs were shaved and scrubbed with an organic iodine solution, a single 5-cm dorsal skin incision was made down to the level of the paniculus carnosus. Subcutaneous pockets were created at the poles and margins of the wound into which sterile moistened PVA sponges (25 to 40 mg) were inserted. Four sponges were used for each rat. Wounds were then closed with interrupted 4-0 stainless steel sutures. Wound tissue expression of HSP 72/73 as a function of the duration of diabetes was studied with the inclusion of groups of 1-, 3-, 7-, and 30-day diabetic rats. Seven or 14 days after wounding 6-hour fasted animals were killed by a lethal dose of pentobarbital and PVA sponges were dissected free of connective tissue, wrapped in aluminum foil, and stored at –70°C for subsequent biochemical and immunohistochemical testing (3 to 7 days). Protein extraction and immunoblotting. Total cellular protein was isolated from PVA sponges by homogenization in ice-cold buffer containing 50 mmol/L tris-hydroxymethyl amino methane (TRIS)–hydrochloric acid (pH.7.5), 3% sodium dodecyl sulfate (SDS), 0.2 mmol/L dithiothreitol, 10% glycerol, 0.5 mmol/L phenylmethylsulfonylflouride, and 0.5 mmol/L leupeptin at 4°C. The homogenate was centrifuged at 14,000g (5 minutes, 4°C), and the soluble protein concentration was determined with the bicinichoninic acid colorimetric protein quantitation kit (Pierce, Rockford, Ill) and the method of Lowry et al.29 Individual soluble protein samples (40 µg) in nonreducing SDS buffer were heated at 95°C for 5

minutes, followed by cooling and then separation on a 12% SDS polyacrylamide gel with the Xcell minicell system (Novex, Encinitas, Calif). After electrophoresis, the proteins were transferred to a 45 µmol/L nitrocellulose membrane (Bio-Rad, Richmond, Va) for 12 hours at 150 mA with the miniblot module (Novex). The molecular weight of the HSP 72/73 was estimated by comparison with the prestained molecular weight standards (Bio-Rad). Immunoblotting was performed by first treating the nitrocellulose membranes with 5% nonfat dry milk for 1 hour at room temperature. This was followed by incubation with the human HSP 72/73 monoclonal antibody (kindly provided by Dr W. Welch, University of California, San Francisco), diluted 1:1000. A peroxidase-labeled antimouse immunoglobulin G (1:1000) was used to form a protein antibody complex that was detected with a chemiluminescent reaction (ECL assay kit, Amersham, Arlington Heights, Ill) followed by exposure to x-ray film. Immunohistochemistry. PVA sponges were dissected free of connective tissues, fixed for 12 hours in a 10% solution of neutral buffered formalin, and then embedded in paraffin by standard techniques. The paraffin-embedded PVA sponges were cut at 5 µm and the sections were mounted on slides coated with 0.5% Elmer’s glue (PGC Scientific, Frederic, Md). Slide-mounted sections were deparaffinized in xylene and rehydrated in a graded solution of ethanol. All were treated with 3% hydrogen peroxide–sodium azide solution to eliminate endogenous peroxidase activity. Slides were

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Fig 3. PVA sponge contents of constitutive HSP 72/73 during acute and semichronic diabetes. STZ (55 mg/kg intravenously) was injected 23 days before or at 0, 4, and 6 days after wound initiation. Animals were killed 7 days after wounding and PVA sponges were removed and processed for immunoblotting. Values are means ± SEM of at least 8 animals. Asterisk, Significantly different from controls at P < .05.

then incubated in 5% nonfat dry milk in 0.01 mol/L phosphate-buffered saline solution for 30 minutes at room temperature to block nonspecific binding. Primary incubation was performed overnight with the human HSP 72/73 monoclonal antibody (kindly provided by W. J. Welch) at 1:500 dilution. After 3 washes with phosphate-buffered saline solution, the bound primary antibody was detected with use of the avidin-biotin technique (DAKO, Glostrub, Denmark). Diaminobenzidinetreated slides were counterstained with hematoxylin, after which they were dehydrated, cleared in xylene, and mounted with DPX (Winlab, Maidenhead, UK). Control slides were treated in the same way with the omission of the anti-HSP 72/73 antibody incubation step to evaluate nonspecific staining. Immunostaining intensity was graded from the lowest (+) to the highest (+++) intensity for each cell type in the section studied. Evaluation of the immunoreaction product was conducted blindly by a pathologist unaware of the different animal treatment. To determine the relative changes in the levels of HSP 72/73 immunoreactive product, slides from at least 3 animals per group probed with the HSP 72/73 monoclonal antibody were analyzed at ×40 magnification with the image cell analysis system (CAS 200, Becton Dickinson, Elmhurst, Ill). For each treatment group measurement of the total areas of granulation tissue and the HSP 72/73 immunostaining

were conducted on each of the randomly selected microscopic fields. Statistical analysis. Data are expressed as means ± SEM or SD. Differences between treatment groups were determined by analysis of variance followed by a multiple range test (Student–NewmanKeuls or paired t test). The level for statistical significance was set at P < .05. RESULTS Nondiabetic control animals demonstrated a gain in body weight of about 30 g/wk for a total duration of 4 to 5 weeks (Fig 1). In contrast, the rate of growth was markedly decreased in diabetic and control animals receiving high doses of GCs (Fig 1). Insulin- and RU 486–treated animals gained 20 g and 7 g per week, respectively (Fig 1). Fig 2 is a representative Western blot that shows the degree of expression of HSP 72/73 in 7-day postwounding PVA sponges of 1-, 3-, 7-, and 30-day diabetic animals. As can be seen, the band densities of diabetics compared with controls were reduced, but only in 30-day diabetic animals. Fig 3 demonstrates a summary of multiple individual Western blots of PVA sponge contents of HSP 72/73 as a function of the duration of diabetes. Optical density–based quantification revealed that the levels of HSP 72/73 in the 30-day diabetic animals were markedly decreased compared with the corresponding control values. In contrast, no changes in

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Fig 4. Effects of diabetes on PVA sponge contents of constitutive HSP 72/73 in 7- and 14-day wounds. Wound initiation and implantation of PVA sponges were performed 16 or 23 days after diabetes induction. At time of death (7 or 14 days after wounding) all animals had been diabetic for about 30 days. PVA sponges were removed and processed for immunoblotting. Values are means ± SEM of at least 8 animals. Asterisk, Significantly different from corresponding control values at P < .05.

Table. Glucose and constitutive HSP 72/73 contents in control and diabetic animals receiving placebo or drug treatment Glucose (mg/100 mL) Treatment Control Control-GC Diabetic Diabetic + insulin Diabetic + RU 486 Control-GC + RU 486

Plasma 135 ± 32 118 ± 23 467 ± 61 168 ± 62 414 ± 48 126 ± 20

Constitutive HSP 72/73

Wound fluid 85 ± 28 78 ± 22 410 ± 50* 163 ± 76 387 ± 55 81 ± 12

Optical density 1.48 ± 0.66 0.39 ± 0.12* 0.52 ± 0.36* 1.29 ± 0.46 1.13 ± 0.34 1.38 ± 0.43

Area percent 34 ± 12 11 ± 10* 17 ± 9* 27 ± 14 29 ± 18 37 ± 13

STZ was injected intravenously at a dose of 55 mg/kg. Five days after diabetes induction, slow-releasing pellets of hydrocortisone or RU 486 were inserted subcutaneously under pentobarbital anesthesia. Twenty-one days thereafter 5-cm skin incision was made with concomitant insertion of PVA sponges. Animals were killed 7 days after wounding and PVA sponges were removed and processed for immunohistochemistry and Western blotting as described in Material and methods. HSP 72/73 content was expressed in terms of optical density (Western blotting) and percent of stained area relative to total granulation tissue (immunohistochemistry). Values were expressed as means ± SD of at least 8 animals. *Indicates significantly different control and insulin or RU 486–treated diabetic rats.

the levels of this stress protein were detected during acute diabetes, as exemplified by the 1-, 3-, and 7-day STZ-treated rats. Plasma glucose levels in control and 1-, 3-, 7-, and 30-day diabetics were 143 ± 12, 433 ± 23, 462 ± 31, and 461 ± 43, respectively. It appears from these data that the degree of hyperglycemia is similar in acutely (1-, 3-, and 7-day diabetic) and semichronically (30-day diabetic) diabetic rats. Fig 4 shows the effects of diabetes on PVA sponge contents of HSP 72/73 in the 7- and 14-day wounds. At 7 and 14 days after wounding the PVA sponge contents of HSP 72/73 in the 30-day diabetic animals were reduced by 57% and 59%,

respectively compared with the corresponding control values. The Table summarizes the effects of diabetes and drug or placebo treatment on PVA sponge contents of HSP 72/73. Western blotting data revealed that the optical density of HSP 72/73 was decreased by 65% and 80%, respectively, in diabetic and GC-treated control animals. Amelioration of these deficits in HSP 72/73 levels during diabetes and the hypercortisolemic state was achieved by the therapeutic institution of insulin (diabetic) and GC receptor blocker RU 486 (diabetic, GC-control, Table). These Western blotting data appear to compare favorably with the immunohistochemical

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A

B

D

C

E Fig 5. Immunohistochemical localization of constitutive HSP 72/73 in sections of PVA sponges derived from control and diabetic animals receiving placebo or drug treatment. Diabetes induction, wound initiation, and drug treatment were conducted as described in the Table. Diabetic and GC-treated controls exhibited weak immunostaining (+), whereas moderate to strong staining was seen in control (+++), insulin-treated (++), or RU 486–treated (++) diabetic rats. (Original magnification ×40.) Curved, straight, and open arrows, respectively, indicate endothelium, macrophage, and fibroblastic localization of HSP 72/73. A, Control. B, GC-treated control. C, Vehicle-treated diabetic. D, Insulin-treated diabetic. E, RU 486–treated diabetic.

determination of HSP 72/73 within granulation tissue (Table). Fig 5 shows the immunohistochemical distribution of HSP 72/73 in sections of PVA sponges derived from control and diabetic animals receiving placebo or drug treatment. An intense HSP

72/73 immunostaining was localized to the cytoplasm of various cell types, including fibroblasts, macrophages, and endothelium (Fig 5, A). In contrast, a faint staining was apparent in PVA sponges of diabetic and GC-treated control animals (Fig 5, B and C). A significant increase in the intensity of

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the immunostaining was detected, however, after treatment of diabetic animals with insulin or RU 486 (Fig 5, D and E). DISCUSSION HSPs represent a family of proteins that are expressed constitutively in all cell types under normal conditions. These proteins perform a highly conserved “molecular chaperone” role in proper folding, glycosylation, assembly, and turnover of other proteins.20,22,30 Moreover, they also play a role in protecting cells against injuries and other types of stress.10-17 The current immunohistochemical and Western blotting data revealed the presence of a certain class of HSP 70 exemplified by HSP 72/73 in various cells of PVA sponge model of wound healing. Indeed, a dense immunostaining for HSP 72/73 was seen in fibroblasts, macrophages, and endothelium (Fig 5). Our findings are consistent with the recent view indicating that a certain class of HSPs with molecular weights of about 70 kd are expressed in wound tissues obtained from animal and human subjects.31,32 The emerging novelty of our data, however, is that the PVA sponge contents of HSP 72/73 are diminished as a function of STZ-induced diabetes (Table, Fig 3). This attenuation in HSP 72/73 expression appears to be selective because total soluble proteins in PVA sponges were not changed in response to diabetes. A similar phenomenon was seen in control animals receiving high doses of GCs (Table, Fig 5, B). Exogenously administered insulin prevented semichronic diabetes-related suppression of HSP 72/73 in a PVA sponge model of wound healing (Table). Plasma insulin levels were not determined; instead, we considered the insulin deficiency to be appropriately corrected when blood glucose levels had been normalized (Table). These data alone do not allow discrimination between hyperglycemiainduced versus hypoinsulinemia-induced suppression of HSP 72/73. Accordingly, we designed an experiment in which plasma and wound fluid concentrations of glucose in nondiabetic control animals were elevated to 425 and 380 mg/100 mL, respectively, by intraperitoneal injection of glucose at a concentration of 20 mmol/L. The data of these studies revealed that the level of HSP 72/73 in acute hyperglycemic animals was similar to saline solution–treated normoglycemic values. Thus the reduced level of HSP 72/73 in STZ-diabetic animals are more likely a result of the effects of the insulinopenic rather than the hyperglycemic state. An additional consideration was also given to the duration of hypoinsulinemia and hyperglycemia. In this connection, animals rendered insulinopenic and hyperglycemic for 1, 3, or 7

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days with STZ did not demonstrate any changes in the level of HSP 72/73. In contrast, in 30-day diabetic animals the level of this stress protein was significantly decreased compared with control values. Overall, the above data point to the possibility that the suppression of the PVA sponge content of HSP 72/73 during wounding is a manifestation of the semichronic degenerative changes of diabetes. The mechanism(s) by which diabetes suppresses the cell expression of HSP 72/73 during wound healing has not been fully defined in this study. However, strong evidence is emerging for the role of GCs in regulating the expression of stress-induced HSP 70.32,33 In this connection, high therapeutic doses of GCs have been shown to reduce adrenal and wound tissue levels of a certain class of HSP with molecular weights of about 70 kd.31,34 Our laboratory has documented a diabetes-related increase in plasma corticosterone levels at various times in the diurnal cycle.8 We have also shown that the levels of hippocampal GC-receptors (indicators of the efficiency of the corticosterone-induced negative feedback mechanism at the corticotropin-releasing hormone–corticotropin axis) are diminished as a function of diabetes.8 A similar finding was noticed in human diabetes.35,36 Collectively, these data indicate that the hypothalamic-pituitary-adrenal axis is overactive during diabetes. Accordingly, the current study examined the relationship between GCs and HSP 70 during wound healing in diabetes mellitus. A pharmacologic manipulation involving the GC receptor blocker RU 486 was used to ameliorate the associated hypercortisolemic state of diabetes. The success of GC receptor blockade with RU 486 was confirmed by its ability to first reduce the activity of hepatic tryptophan dioxygenase, an enzyme that is positively regulated by GCs, and second to counteract the GC-induced decrease in thymus weight, the PVA sponge content of hydroxyproline, and the skin tensile strength. The data derived from these studies revealed that both diabetes and the hypercortisolemic state induced by the exogenous administration of a supraphysiologic dose of GCs are associated with a reduction in the PVA sponge content of HSP 72/73. Such a decrease in the degree of expression of HSP 72/73 in these pathophysiologic states was counteracted by the use of RU 486. Overall, these findings may point to the possibility that the diabetes-related decrease in HSP 72/73 is mediated at least in part by GCs. This proposition is of interest in the light of our previous data and those of others showing that the plasma GC level is elevated during diabetes.8,35,36 Studies are in progress to confirm or refute the above hypothesis.

Surgery Volume 125, Number 6 We thank Ms Karen Grimes of Pharmacia Upjohn, Kalamazoo, Mich, for the generous gift of streptozotocin. REFERENCES 1. Clark RAF. Cutaneous tissue repair: basic biologic consideration, I. J Am Acad Dermatol 1985;13:701-25. 2. Rosenthal SP. Acceleration of primary wound healing by insulin. Arch Surg 1968;96:53-5. 3. Prokash MK, Sharma LK. Studies in wound healing in experimental diabetes. Int Surg 1974;59:25-8. 4. Goodson WH, Hunt TK. Wound healing and the diabetic patient. Surg Gynecol Obstet 1979;149:600-8. 5. Yue DK, McLennan S, Marsh M, Mai YW, Spaliviero J, Delbridge L, et al. Effects of experimental diabetes, uremia and malnutrition on wound healing. Diabetes 1987;36:2959. 6. Fahey TJ, Sadaty A, Jones WG, Barber A, Smoller B, Shires GT. Diabetes impairs the late inflammatory response to wound healing. J Surg Res 1991;50:308-18. 7. Goodson WH, Hunt TK. Studies of wound healing in experimental diabetes. J Surg Res 1977;22:221-7. 8. Bitar MS. Glucocorticoid dynamics and impaired wound healing in diabetes mellitus. Am J Pathol 1998;152:547-54. 9. Rokowski RJ, Sheehy J, Cutroneo KR. Glucocorticoid mediated selective reduction of functioning collagen messenger ribonucleic acid. Arch Biochem Biophys 1981;210:74-81. 10. Hightower LE. Heat shock, stress proteins, chaperones and proteotoxicity. Cell 1991;66:191-7. 11. Walsh DA, Klein NW, Hightower LE, Edwards MJ. Heat shock and thermotolerance during early rat embryo development. Teratology 1987;36:181-91. 12. Currie RW, Karmazyn M, Kloc M, Mailer K. Heat shock response is associated with enhanced post-ischemic ventricular recovery. Circ Res 1988;63:543-9. 13. Sanchez Y, Lindquist SL. HSP 104 required for induced thermotolerance. Science 1993;248:1112-4. 14. Yufu Y, Nishimura J, Ideguchi H, Nawata H. Enhanced synthesis of heat shock proteins and augmented thermotolerance after induction of differentiation in HL 60 human leukemia cells. FEBS Lett 1990;268:173-6. 15. Mivechi NF, Monson JM, Hahn GM. Expression of HSP-28 and three HSP-70 genes during the development and decay of thermotolerance in leukemia and nonleukemic human tumors. Cancer Res 1991;51:6608-14. 16. Solomon JM, Rossi JM, Golic K, McGarry T, Lindquist S. Changes in HSP-70 after thermotolerance and heat shock regulation in Drosophila. New Biol 1991;3:1106-20. 17. Lee YJ, Hou ZZ, Curetly L, Corry PM. Expression, synthesis and phosphorylation of HSP 28 family during development and decay of thermotolerance in CHO plateau-phase cells. J Cell Physiol 192;150:441-6. 18. Khan NA, Sotelo J. Heat shock stress is deletrious to CNS cultured neurons microinjected with anti-HSP 70 antibodies. Biol Cell 1989;65:199-202.

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19. Riabowol KT, Mizzen LA, Welch WJ. Heat shock is lethal to fibroblasts microinjected with antibodies against HSP-70. Science 1988;242:433-6. 20. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992;355:33-44. 21. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress protein and implications for medicine and disease. Physiol Rev 1992;72:1063-81. 22. Jindal S. Heat shock proteins: applications in health and disease. TIBTECH 1996;14:17-20. 23. Knox WF, Auerback VH. The hormonal control of tryptophan peroxidase in the rat. J Biol Chem 1955;214:307-13. 24. Gjerd H, Morland J, Olsen H. The antiglucocorticoid RU 486 inhibits the ethanol-induced increase of tryptrophan oxygenase. J Steroid Biochem 1985;23:1091-2. 25. Bertagan X, Bertagan C, Luton JP, Husson JM, Girard F. The new steroid analog RU 486 inhibits glucocorticoid action in man. J Clin Endocrinol Metab 1984;59:25-8. 26. Jung-Testas I, Baulieu EE. Inhibition of glucocorticoid action in cultured L-929 mouse fibroblasts by RU 486, a new antiglucocorticoid high affinity for the glucocorticoid receptor. Exp Cell 1983;147:177-82. 27. Moguilewsky M, Philibert D. RU 38486: potent antiglucocorticoid activities correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J Steroid Biochem 1984;20:271-6. 28. Chrousos GP, Laue L, Nieman LK, Kawai S, Udelsman RU, Brandon DD, et al. Glucocorticoids and glucocorticoid antagonists: lessons from RU 486. Kidney Int 1988;34(26 Suppl):S18-23. 29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75. 30. Beckmann RP, Mizzsen LA, Welch WJ. Interaction of HSP 70 with newly synthesized protein: implications for protein folding and assembly. Science 1990;248:850-4. 31. Gordon CB, Li DG, Stagg CA, Manson P, Udelsman R. Impaired wound healing in Cushing’s syndrome: the role of heat-shock proteins. Surgery 1994;116:1082-7. 32. Oberringer M, Baum HP, Jung V, Welter C, Frank J, Kuhlmann M, et al. Differential expression of heat shock protein 70 in well healing and human wound tissue. Biochem Biophys Res Commun 1995;214:1009-14. 33. Holbrook NJ. Stress-induced HSP 70 expressing in adrenal cortex: a glucocorticoid-sensitive, age-dependent response. Proc Natl Acad Sci U S A 1991;88:9873-7. 34. Udelsman R, Blake MJ, Stagg CA, Holbrook NJ. Endocrine control of stress-induced heat shock protein 70 expression in vivo. Surgery 1994;115:611-6. 35. Cameron OG, Thomas B, Tiongeo D, Greden J. Hypercortisolism in diabetes mellitus. Diabetes Care 1987;10:662-4. 36. Roy M, Collier B, Roy A. Hypothalamic-pituitary adrenal axis dysregulation among diabetic outpatients. Psychol Res 1990;31:31-7.