- Email: [email protected]
Correlation of lymphocyte heat shock protein 70 levels with neurologic deficits in elderly patients with cerebral infarction
Correlation of Lymphocyte Heat Shock Protein 70 Levels with Neurologic Deficits in Elderly Patients with Cerebral Infarction Xingfang Jin, MD, Chengfeng Xiao, MD, PhD, Robert M. Tanguay, PhD, Li Yang, MD, Feng Wang, MD, Ming Chen, MD, Xiaoye Fu, MD, Ru Wang, MD, Jie Deng, MD, Zhijun Deng, MD, Yongyong Zheng, MD, Qingyi Wei, MD, PhD, Tangchun Wu, MD, PhD PURPOSE: To assess the association between heat shock protein 70 (HSP70) levels and the severity of ischemic stroke in elderly patients. METHODS: We conducted a case-control study to investigate the changes in lymphocyte HSP70 levels by immunoblot in 65 elderly patients with mild (n ⫽ 22), intermediate (n ⫽ 21), or severe (n ⫽ 22) stroke, and in 34 healthy controls. We analyzed correlations between HSP70 levels and neurologic deficit scores on days 1, 15, and 30 after the onset of stroke. RESULTS: Mean (⫾ SD) HSP70 levels were higher in all stroke patients compared with controls (mild stroke: 709 ⫾ 194 units; intermediate: 585 ⫾ 165 units; severe: 421 ⫾ 124 units; controls: 86 ⫾ 34 units on day 1). Patients with mild stroke had higher levels at day 1 and 15 than did patients with severe stroke.
HSP70 levels decreased rapidly from days 1 to 30 in all patients, except in patients with severe stroke, in whom levels decreased slowly between days 15 and 30. There were significant negative correlations between HSP70 levels and neurologic deficit scores in patients on days 1 (r ⫽ ⫺0.53, P ⬍0.001) and 15 (r ⫽ ⫺0.54, P ⬍0.001), but a positive correlation on day 30 (r ⫽ 0.49, P ⬍0.001). CONCLUSION: These data suggest that HSP70 may be a marker for neuroprotection in the early stage of ischemic stroke and a marker for a crisis in the later stages of severe cerebral infarction. Further studies on the use of lymphocyte HSP70 levels in predicting clinical outcomes and underlying mechanisms in cerebral infarction are warranted. Am J Med. 2004; 117:406 – 411. ©2004 by Elsevier Inc.
H
(5–9), and protect against ischemic injury in the heart, brain, and kidney (10 –15). Heat shock proteins are important in the pathogenesis and prognosis of some diseases (16 –19). The heat shock protein 70 (HSP70) family is one of the most extensively studied subgroups of heat shock proteins, and the inducible HSP70 has been shown to exert cytoprotective effects in the heart (10 –14) and brain (20,21). Whether there is an association between HSP70 levels and the severity of ischemic stroke is not known. Here, we investigated the possible protective role of HSP70 in cerebral ischemia by measuring the dynamic changes in lymphocyte HSP70 levels in elderly patients with cerebral infarction and by correlating these levels with neurologic deficits during recovery from infarction.
eat shock proteins are conserved proteins induced by heat and numerous noxious stimuli, including xenobiotics, chemical stressors (heavy metals, free radicals), and pathologic stresses (ischemia, fever, and infection) (1,2). The ubiquitous nature of the heat shock response suggests that heat shock proteins are essential for cell survival. Indeed, these proteins function as molecular chaperones, facilitating synthesis, folding, assembly, and intracellular trafficking of proteins (1,3,4). They also protect against cell damage, a role that has been well documented for thermotolerance in cultured cells
From Institute of Occupational Medicine (XJ, CX, FW, QW, TW), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Neurological Medicine (XJ, LY, MC, XF, RW, JD, ZD, YZ), Yanan Hospital, Kunming, China; Laboratory of Cell and Developmental Genetics (RMT), Department of Medicine, Faculty of Medicine, Pav. C.E. Marchand, Université Laval, Québec, Canada; and CHUL Research Center (RMT), CHUQ, Québec, Canada. This work was supported in part by research funds from the National Key Basic Research and Development Program (2002CB512905) and from the National Natural Science Foundation of China (30128021 and 30371204) to Dr. Wu. Drs. Wu and Tanguay received financial support from the National Natural Science Foundation of China and the Canadian Institute of Health Research of Canada (CIHR) for a research exchange program and an operating CIHR grant. Requests for reprints should be addressed to Tangchun Wu, MD, PhD, Institute of Occupational Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, Hubei 430030, China, or [email protected]. Manuscript submitted August 27, 2003, and accepted in revised form March 25, 2004. 406
© 2004 by Elsevier Inc. All rights reserved.
METHODS Sample The study included 65 elderly patients with ischemic cerebral infarction and 34 apparently healthy control subjects. Patients aged 60 and 86 years (mean [⫾ SD] age, 74 ⫾ 6 years) had been admitted to Yanan Hospital, Kunming City, China, between January 2000 and December 2001. The diagnosis of cerebral infarction was based on Chinese national diagnostic criteria (22). Briefly, the diagnostic parameters were age, sex, history of hypertension and atherosclerosis, history of sudden onset of focal brain deficit, and signs and symptoms through neuro0002-9343/04/$–see front matter doi:10.1016/j.amjmed.2004.03.026
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
logic examination. Routine laboratory tests of blood and urine, brain computed tomography scan or magnetic resonance imaging, and electrocardiography were also included. Patients with conditions mimicking acute stroke syndromes, such as hypoglycemia, migraine, intracranial hemorrhages, and cerebral embolism, were excluded. Patients with transient ischemic attacks were not included. Patients were treated with regimens such as systemic thrombolytic agents, platelet anticoagulants, and medications to improve circulation and protect neurons. The controls lived in the same city. None had any complaints such as nausea, vomiting, diarrhea, weakness, headache, dizziness, visual disturbances, and confusion, as well as any active diseases such as hypertension, coronary heart disease, central nervous system or psychiatric diseases, cancer, and other acute and chronic diseases. Patients and controls were matched in living and working environments, and use of tobacco and alcohol was similar between the two groups. Informed consent was obtained from all participants in accordance with the ethics committees of Tongji Medical College and Yanan Hospital.
Evaluation of Cerebral Deficits To determine the level of brain injury in patients with stroke, neurologic deficits were evaluated quantitatively on days 1, 15, and 30 after the onset of stroke by neurologists. Evaluations were performed in joint conferences, and the level of brain injury was assessed according to the Chinese National Scoring Criteria for Neurological Deficits in Patients with Stroke (22). These criteria are similar to the National Institutes of Health stroke scale (23) and are a quantitative measure of stroke-related neurologic deficits (stroke scale), determined by the following neurologic examinations: level of consciousness, including questions and commands; gaze and visual fields; facial symmetry; language function; left and right arm motors; left and right hand motors; left and right leg motors; and walking capability. The highest score is 45 (most severe cerebral injury) and the lowest is 0 (normal function). Based on the stroke scores on day 1 after the onset of stroke, the 65 elderly patients were divided into three clinical subtypes: mild (n ⫽ 22 patients; score: ⱕ15), intermediate (n ⫽ 21; score: 16 to 30), and severe (n ⫽ 22; score: 31 to 45) cerebral infarctions (22). The infarction site and the number of sites were also determined using computed tomography and magnetic resonance imaging.
Blood Sampling Venous blood was collected after overnight fasting on days 1, 15, and 30 after the onset of stroke. Lymphocytes were isolated using a standard separation medium (Biochemical Reagent Co., Shanghai, China), washed twice with phosphate buffer solution, and counted. The cell viability was ⬎95%, and cell numbers were within normal range (4 to 10 ⫻ 103/L), for patients and controls.
The lymphocyte concentration was adjusted to 5000 per L in phosphate buffer solution; 200 L of lymphocytes was centrifuged, the buffer was removed rapidly, and the cells were stored at ⫺80°C until analyzed.
Determination of HSP70 Levels in Lymphocytes The HSP70 assay was performed with the frozen cells within 1 month of blood collection (18,24,25). Briefly, lymphocyte proteins were separated on sodium dodecyl sulfate (Sigma Chemical Company, St. Louis, Missouri)– polyacrylamide (Promega Corporation, Madison, Wisconsin) gels and transferred to nitrocellulose membranes (Pall Corporation, New York, New York). Membranes were saturated with blocking buffer (phosphate buffer solution containing 5% skim milk powder) in a plastic container for 1 hour at 37°C, washed with phosphate buffer solution 0.05% Tween 80, and incubated at 37°C for 1 hour with rabbit anti-human HSP70 antibodies (26) diluted 1:1000. After washing the membranes a second time, horseradish peroxidase–labeled goat anti-rabbit immunoglobulin G (1:1000) (Beijing Zhongshan Biotechnology Company, Beijing, China) was added and incubated for 1 hour at 37°C. After a final wash, HSP70 was detected using 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical Company) for 3 to 5 minutes and quantified by integrated optical density using an imaging densitometer (Shimadzu CS-930, Shimadzu, Japan).
Statistical Analysis Comparisons were made with the Student t test, analysis of variance (one-way or two-way), or univariate and multivariate linear regression models, as appropriate. Stratification analysis was performed for evaluating subgroup differences. The general linear model was used for the trend test for changes over time. Statistical inferences were based on a significance level of P ⬍0.05. All data analyses were two-sided and used SAS software, version 8.0 (SAS Institute, Inc., Cary, North Carolina).
RESULTS There was no difference in age, distribution of sex, smoking habits, or body mass index (⬎28 kg/m2) between patients and controls (Table 1). However, significantly more patients had total cholesterol levels ⬎224 mg/dL and hypertension as compared with controls (P ⬍0.001 for all comparisons). Further comparisons between each patient subgroup (by stroke severity) and controls suggested that the differences remained significant (P ⬍0.001), except for elevated total cholesterol level in the mild stroke group and for diabetes in the intermediate stroke group (P ⬍0.01). The proportions of these clinical conditions did not differ among the various infarction severity groups (P ⬎0.05 for all comparisons).
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
Volume 117 407
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
Table 1. Characteristics of Patients with Stroke and Healthy Controls Patients with Stroke Characteristic
Controls (n ⫽ 34)
Mild (n ⫽ 22)
Intermediate (n ⫽ 21)
P Value
Severe (n ⫽ 22)
All (N ⫽ 65)
All Patients vs. Controls
Between Group
Number (%) or Mean ⫾ SD Age (years) Male sex Smoking (pack-year) Body mass index ⬎28 kg/m2 Total cholesterol ⬎224 mg/dL Hypertension Diabetes
72 ⫾ 8 8 (24) 22 ⫾ 6 4 (12)
73 ⫾ 5 5 (23) 24 ⫾ 7 5 (23)
75 ⫾ 6 5 (24) 22 ⫾ 8 3 (14)
74 ⫾ 8 3 (14) 23 ⫾ 5 5 (23)
74 ⫾ 6 13 (20) 23 ⫾ 6 13 (20)
0.17 0.68 0.43 0.30
0.88 0.70 0.89 0.73
0
6 (27)*
7 (33)†
8 (36)†
21 (32)
⬍0.001
0.81
0 0
15 (68)† 7 (32)†
17 (81)† 6 (29)*
20 (91)† 10 (45)†
52 (80) 23 (35)
⬍0.001 ⬍0.001
0.17 0.47
* P ⬍0.01 for comparisons with controls. † P ⬍0.001 for comparisons with controls.
234 ⫾ 73 units; mild stroke: 157 ⫾ 49 units; intermediate stroke: 204 ⫾ 54 units), although these differences were not statistically significant (P ⬎0.05 for all comparisons).
Changes in Neurologic Deficit Scores during the First 30 Days after Stroke Onset The neurologic deficit scores of patients with mild stroke did not change in the first 15 days after the onset but decreased significantly between days 15 and 30 (P ⬍0.001) (Table 2). In the intermediate stroke group, scores decreased steadily between days 1 and 30 after the onset of stroke, but the decrease was only significant between days 1 and 15 (P ⬍0.05). Similarly, for patients with severe stroke, neurologic deficit scores decreased steadily between days 1 and 30, but only significantly between days 1 and 15 (P ⬍0.001); the decrease between days 15 and 30 was of borderline significance (P ⫽ 0.05).
HSP70 Levels by Site of Cerebral Infarction Mean HSP70 levels did not vary by stroke location or number of infarction sites: basal ganglia, 457 ⫾ 144 units; cerebral cortex in frontal and parietal lobe, 433 ⫾ 139 units; occipital lobe, 398 ⫾ 143 units; thalamus, 390 ⫾ 125 units; temporal lobe, 408 ⫾ 113 units; brainstem, 512 ⫾ 145 units (P ⬎0.05 for all comparisons).
Correlation between HSP70 Levels and Neurologic Deficit Scores during Recovery There were significant negative correlations between HSP70 level and neurologic deficit score on days 1 (r ⫽ ⫺0.53, P ⬍0.001) and 15 (r ⫽ ⫺0.54, P ⬍0.001) after the onset of stroke, and a significant positive correlation on day 30 (r ⫽ 0.49, P ⬍0.001).
Changes in HSP70 Levels after Stroke Mean (⫾ SD) HSP70 levels in patients with mild, intermediate, or severe stroke were higher (Figure) than in controls (86 ⫾ 34 units, P ⬍0.05 for all comparisons) from days 1 to 30 after the onset of stroke. There was a significant decreasing trend in HSP70 levels among stroke patients during this period (P for trend ⬍0.001 for all three groups), with the decrease appearing to be faster in patients with mild or intermediate stroke. Mean HSP70 levels were higher in patients with mild or intermediate stroke on days 1 and 15 than in those with severe stroke. This trend was reversed by day 30 (severe stroke:
DISCUSSION Ischemic stroke, one of the most common life-threatening neurologic diseases, is associated with serious long-term disability (27). Animal models suggest that HSP70 can protect the brain from various insults, including cerebral ischemia
Table 2. Neurologic Deficit Scores among Patients with Stroke, by Day after Onset of Stroke Neurologic Deficit Stroke (units) Type of Cerebral Infarction
Day 1
Day 15
Day 30
P Value Day 1 vs. 15
Day 1 vs. 30
Day 15 vs. 30
0.35 0.03 ⬍0.001
⬍0.001 0.002 ⬍0.001
⬍0.001 0.41 0.05
Number (%) or Mean ⫾ SD Mild (n ⫽ 22) Intermediate (n ⫽ 21) Severe (n ⫽ 22) 408
September 15, 2004
10 ⫾ 3 21 ⫾ 3 41 ⫾ 3
10 ⫾ 2 19 ⫾ 2 38 ⫾ 2
THE AMERICAN JOURNAL OF MEDICINE威
7⫾1 18 ⫾ 1 37 ⫾ 2 Volume 117
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
Figure. Changes in lymphocyte heat shock protein 70 (HSP70) levels among patients with mild, intermediate, or severe ischemic stroke from days 1 to 30 after disease onset. P values obtained from the test for trend were ⬍0.001 for patients with mild, intermediate, or severe stroke. IOD ⫽ integrated optical density units. Diamonds, circles, and triangles represent the mean values; error bars represent the SD.
(13,20,21,28 –30). Previous studies suggest that quantitative measures of stroke-related neurologic deficits correspond well with neurologic injuries (23,27). In the present study, we found that lymphocyte HSP70 levels correlated with neurologic deficit scores in patients with stroke, after onset and during 30-day recovery, which suggests that lymphocyte HSP70 levels may be an early marker for stroke severity within 1 day of onset and may serve as an indicator of prognosis in the elderly, particularly for those with milder stroke. Our results also suggest that HSP70 is associated with neuroprotective effects during the early stages (up to 15 days) of stroke and may be a marker for poor prognosis, particularly at the later stages (30 days after onset) of severe stroke. Several studies suggest that cells with overexpression of HSP70 are resistant to various stresses, including ischemia, and have better survival (10 –15). In mouse and rat models, a moderate pre– heat shock treatment or transgenic expression of the HSP70 gene provided protection from ischemic injury in the heart, brain, and kidneys. In mice, transgenic expression of human HSP70 resulted in a 24-hour improvement in hippocampal neuron survival, although the overall infarction size was not affected (13). Cerebral infarction after 6 hours of ischemia was less severe in transgenic mice overexpressing rat HSP70 than in wild type mice (28). In knockout mice, reduced expression of HSP70 increased neuron damage during acute focal cerebral ischemia (31). Furthermore, gene therapy with HSP70 improved neuron survival after focal cerebral ischemia in a rat model (20).
Thus, the synthesis of HSP70 is thought to be protective and an indicator of ongoing active repair or a compensatory mechanism related to neuron remodeling, facilitating the recovery of postischemic neurologic deficits (32). In contrast, little is known about the long-term effects of HSP70 on neuroprotection in humans. Our data show that there is a positive correlation between lymphocyte HSP70 levels and the neurologic deficit scores in patients with cerebral infarction on day 30 after disease onset. This phenomenon seems to be counterintuitive considering the beneficial functions of HSP70 as discussed above. Alternatively, this correlation may indicate that the stress response was still ongoing in patients with severe ischemia. Thus, lymphocyte HSP70 level may be a marker for detrimental effects in the late stages of cerebral infarction. While heat shock proteins are expressed at low levels under normal physiologic conditions, HSP70 levels increase under stressful or pathologic conditions, such as cerebral infarction (1,2,27–29). Indeed, high levels of HSP70 are indicative of injury, and persistently high levels during the late stages of severe stroke may be indicative of the continued presence of noxious stimuli even after 30 days. Of the three patient groups in our study, patients with mild cerebral infarction had the highest HSP70 levels on day 1 after stroke onset and the lowest levels on day 30. It is not known how the expression of HSP70 in lymphocytes is regulated in these patients. Heat shock proteins are generally expressed under complex regulatory controls operating at both the transcriptional and translational levels (33). Basal levels of HSP70 in leukocytes were found to be lower in athletes than in untrained persons, but the response of leukocytes to an in vitro heat shock was higher among athletes (34), suggesting that HSP70 levels are low under normal conditions but high under stressful conditions, consistent with a protective role that can be regulated. Basal levels of HSP70 in lymphocytes vary between persons and may contribute to interindividual variation in response to stresses (19,35). As HSP70 is polymorphic, variants may also affect individual disease susceptibility or recovery from pathologic stresses (36). In animal models, HSP70 expression differs in neurologic cells and tissues under normal and stress conditions (37–39). HSP70 expression is a useful marker for cellular injury and may help identify previously unrecognized areas of vulnerability in the nervous system after a neurotoxic stimulus (28). We found that lymphocyte HSP70 levels were not related to the location of cerebral infarction, consistent with findings from a study of 51 patients undergoing open heart surgery (40), in which HSP70 levels in the myocardium were not correlated with HSP70 levels in lymphocytes. Our data show that HSP70 levels in lymphocytes were correlated with both the severity of the stroke and improvement in neurologic deficit scores after stroke. Since we could not measure HSP70 levels in the brain, whether changes in lymphocytes can predict
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
Volume 117 409
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
changes in the brain remains unknown. Further investigations aimed at correlating HSP70 levels in lymphocytes on the first day of recovery with the level of functional recovery at 30 days after stroke onset, as measured by neurologic deficit scores in ischemic animal models and in humans, are warranted. It will also be important to take into account the role of HSP70 polymorphisms and inducibility, preferably in the target tissues, in the susceptibility to trauma and during recovery from stroke.
13.
14.
15.
16.
ACKNOWLEDGMENTS
17.
We are particularly grateful to the patients and their family members who volunteered to participate in this study and to the many members of the medical staff of Yanan Hospital for their help in the examination and sampling of subjects. We thank Professor R. W. Currie (Dalhousie University, Halifax, Canada) for revising the manuscript and for his suggestions.
18.
19.
20.
REFERENCES
21.
1. Lindquist S, Craig EA. The heat shock proteins. Ann Rev Genet. 1988;22:631– 677. 2. Progress and perspectives in the biology of heat shock proteins and molecular chaperones. In: Morimoto RI, Tissières A, Georgopoulos C, eds. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1994:1–30. 3. Hightower LE. Heat shock, stress protein, chaperones, and proteotoxicity. Cell. 1991;66:191–197. 4. Hartl F-U. Molecular chaperones in protein folding. Nature. 1996; 381:571–580. 5. Angelidis CE, Lazaridis I, Pagoulatos GN. Constitutive expression of heat shock protein 70 in mammalian cells confers thermotolerance. Eur J Biochem. 1991;199:35–39. 6. Li GC, Li LY, Liu K, et al. Thermal response of rat fibroblasts stably transfected with the human 70kDa heat shock protein encoding gene. Proc Natl Acad Sci USA. 1988;88:1681–1685. 7. Rollet E, Lavoie JN, Landry J, Tanguay RM. Expression of Drosophila 27kDa heat shock protein in rodent cell confers thermal resistance. Biochem Biophys Res Commun. 1992;185:116 –120. 8. Mehlen P, Preville X, Chareyron P, et al. Constitutive expression of human hsp27, Drosophila hsp27 and human alpha-B crystallin confers resistance to tumor necrosis factor alpha and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J Immunol. 1995;154:364 –374. 9. Parsell DA, Lindquist S. Heat shock proteins and stress tolerance. In: Morimoto RI, Tissières A, Georgopoulos C, eds. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1994:457– 494. 10. Currie RW, Tanguay RM, Kingma JG. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation. 1993;87:963–971. 11. Marber MS, Mestril R, Chi S, et al. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest. 1995;96: 1446 –1456. 12. Plumier C, Ross BM, Currie RW, et al. Transgenic mice expressing 410
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
22. 23.
24.
25.
26.
27. 28. 29.
30.
31.
32.
33.
34.
the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest. 1995;95:1854 –1860. Plumier C, Krueger AM, Currie RW, et al. Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones. 1997;2:162–167. Radford NB, Fina M, Benjamin IJ, et al. Cardioprotective effects of 70-kDa heat shock protein in transgenic mice. Proc Natl Acad Sci USA. 1996;93:2339 –2342. Beck FX, Neuhofer W, Müller E. Molecular chaperones in the kidney: distribution, putative roles, and regulation. Am J Physiol Renal Physiol. 2000;279:F203–F215. Welch WJ. Mammalian stress response: cell physiology, structure/ function of stress proteins and implications for medicine and disease. Physiol Rev. 1992;72:1063–1081. Minowada G, Welch WJ. Clinical implications of the stress response. J Clin Invest. 1995;95:3–12. Xiao C, Chen S, Li J, et al. Association of HSP70 and genotoxic damage in lymphocytes of workers exposed to coke-oven emission. Cell Stress Chaperones. 2002;7:396 – 402. Xiao C, Wu T, Ren A, et al. Basal and inducible levels of Hsp70 in patients with acute heat-induced illness induced during training. Cell Stress Chaperones. 2003;8:86 –92. Yenari MA, Fink SL, Sun GH, et al. Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol. 1998;44:584 –591. Rajdev S, Hara K, Kokubo Y, et al. Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Ann Neurol. 2000;47:782–791. Stroke. In: Huang R, Su Z, eds. Rehabilitation of Stroke. Beijing, China: People Health Press; 2001:90 –356. Brott TG, Adams HP, Olinger CP, et al. Measurements of acute cerebral infarction: a clinical examination scale. Stroke. 1989;20: 864 – 870. Wu T, Chen S, Sun Y, et al. Presence of antibody against the inducible Hsp71 in patients with acute heat-induced illness. Cell Stress Chaperones. 2001;6:113–120. Wu T, Ma J, Chen S, et al. Association of plasma antibodies against the inducible Hsp70 with hypertension and harsh working conditions. Cell Stress Chaperones. 2001;6:394 – 401. Tanguay RM, Wu Y, Khandjian EW. Tissue-specific expression of heat shock stress proteins of the mouse in the absence of stress. Dev Genet. 1993;14:112–118. Stapf C, Mohr JP. Ischemic stroke therapy. Annu Rev Med. 2002;53: 453– 475. Rajdev S, Sharp FR. Stress proteins as molecular markers of neurotoxicity. Toxicol Pathol. 2000;28:105–112. Lee JE, Yenari MA, Sun GH, et al. Differential neuroprotection from human heat shock protein 70 overexpression in vitro and in vivo models of ischemia and ischemia-like conditions. Exp Neurol. 2001;170:129 –139. Kelly S, Bieneman A, Horsburgh K, et al. Targeting expression of hsp70i to discrete neuronal populations using Lmo-1 promoter: assessment of the neuroprotective effects of hsp70i in vivo and in vitro. J Cereb Blood Flow Metab. 2001;21:972–981. Lee SH, Kim M, Yoon BW, et al. Targeted hsp70.1 disruption increases infarction volume after focal cerebral ischemia in mice. Stroke. 2001;32:2905–2912. Fredduzzi S, Mariucci G, Tantucci M, Ambrosini MV. Generalized induction of 72-kDa heat shock proteins after transient focal ischemia in rat brain. Exp Brain Res. 2001;136:19 –24. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 1998;12: 3788 –3796. Fehrenbach E, Niess AM, Schlotz E, et al. Transcriptional and trans-
Volume 117
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al lational regulation of heat shock proteins in leukocytes of endurance runners. J Appl Physiol. 2000;89:704 –710. 35. Boschoff T, Lombard F, Eiselen R, et al. Differential basal synthesis of Hsp70/Hsc70 contributes to interindividual variation in Hsp70/ Hsc70 inducibility. Cell Mol Life Sci. 2000;57:1317–1325. 36. Favatier F, Bornman L, Hightower LE, et al. Variation in hsp gene expression and Hsp polymorphism: do they contribute to differential disease susceptibility and stress tolerance? Cell Stress Chaperones. 1997;2:141–155. 37. Vass K, Welch WJ, Nowak TS Jr. Localization of 70-kDa stress pro-
tein induction in gerbil brain after ischemia. Acta Neuropathol. 1995;77:128 –135. 38. Gaspary H, Graham SH, Sagar SM, Sharp FR. HSP70 heat shock protein induction following global ischemia in the rat. Brain Res Mol Brain Res.1995;34:327–332. 39. Leppa S, Kajanne R, Arminen L, Sistonen L. Differential induction of Hsp70-encoding genes in human hematopoietic cells. J Biol Chem. 2001;276:31713–31719. 40. Demidov ON, Tyrenko VV, Svistov AS, et al. Heat shock proteins in cardiosurgery patients. Eur J Cardio Thorac Surg. 1999;16:444 – 449.
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
Volume 117 411
HSP70 levels decreased rapidly from days 1 to 30 in all patients, except in patients with severe stroke, in whom levels decreased slowly between days 15 and 30. There were significant negative correlations between HSP70 levels and neurologic deficit scores in patients on days 1 (r ⫽ ⫺0.53, P ⬍0.001) and 15 (r ⫽ ⫺0.54, P ⬍0.001), but a positive correlation on day 30 (r ⫽ 0.49, P ⬍0.001). CONCLUSION: These data suggest that HSP70 may be a marker for neuroprotection in the early stage of ischemic stroke and a marker for a crisis in the later stages of severe cerebral infarction. Further studies on the use of lymphocyte HSP70 levels in predicting clinical outcomes and underlying mechanisms in cerebral infarction are warranted. Am J Med. 2004; 117:406 – 411. ©2004 by Elsevier Inc.
H
(5–9), and protect against ischemic injury in the heart, brain, and kidney (10 –15). Heat shock proteins are important in the pathogenesis and prognosis of some diseases (16 –19). The heat shock protein 70 (HSP70) family is one of the most extensively studied subgroups of heat shock proteins, and the inducible HSP70 has been shown to exert cytoprotective effects in the heart (10 –14) and brain (20,21). Whether there is an association between HSP70 levels and the severity of ischemic stroke is not known. Here, we investigated the possible protective role of HSP70 in cerebral ischemia by measuring the dynamic changes in lymphocyte HSP70 levels in elderly patients with cerebral infarction and by correlating these levels with neurologic deficits during recovery from infarction.
eat shock proteins are conserved proteins induced by heat and numerous noxious stimuli, including xenobiotics, chemical stressors (heavy metals, free radicals), and pathologic stresses (ischemia, fever, and infection) (1,2). The ubiquitous nature of the heat shock response suggests that heat shock proteins are essential for cell survival. Indeed, these proteins function as molecular chaperones, facilitating synthesis, folding, assembly, and intracellular trafficking of proteins (1,3,4). They also protect against cell damage, a role that has been well documented for thermotolerance in cultured cells
From Institute of Occupational Medicine (XJ, CX, FW, QW, TW), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Neurological Medicine (XJ, LY, MC, XF, RW, JD, ZD, YZ), Yanan Hospital, Kunming, China; Laboratory of Cell and Developmental Genetics (RMT), Department of Medicine, Faculty of Medicine, Pav. C.E. Marchand, Université Laval, Québec, Canada; and CHUL Research Center (RMT), CHUQ, Québec, Canada. This work was supported in part by research funds from the National Key Basic Research and Development Program (2002CB512905) and from the National Natural Science Foundation of China (30128021 and 30371204) to Dr. Wu. Drs. Wu and Tanguay received financial support from the National Natural Science Foundation of China and the Canadian Institute of Health Research of Canada (CIHR) for a research exchange program and an operating CIHR grant. Requests for reprints should be addressed to Tangchun Wu, MD, PhD, Institute of Occupational Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, Hubei 430030, China, or [email protected]. Manuscript submitted August 27, 2003, and accepted in revised form March 25, 2004. 406
© 2004 by Elsevier Inc. All rights reserved.
METHODS Sample The study included 65 elderly patients with ischemic cerebral infarction and 34 apparently healthy control subjects. Patients aged 60 and 86 years (mean [⫾ SD] age, 74 ⫾ 6 years) had been admitted to Yanan Hospital, Kunming City, China, between January 2000 and December 2001. The diagnosis of cerebral infarction was based on Chinese national diagnostic criteria (22). Briefly, the diagnostic parameters were age, sex, history of hypertension and atherosclerosis, history of sudden onset of focal brain deficit, and signs and symptoms through neuro0002-9343/04/$–see front matter doi:10.1016/j.amjmed.2004.03.026
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
logic examination. Routine laboratory tests of blood and urine, brain computed tomography scan or magnetic resonance imaging, and electrocardiography were also included. Patients with conditions mimicking acute stroke syndromes, such as hypoglycemia, migraine, intracranial hemorrhages, and cerebral embolism, were excluded. Patients with transient ischemic attacks were not included. Patients were treated with regimens such as systemic thrombolytic agents, platelet anticoagulants, and medications to improve circulation and protect neurons. The controls lived in the same city. None had any complaints such as nausea, vomiting, diarrhea, weakness, headache, dizziness, visual disturbances, and confusion, as well as any active diseases such as hypertension, coronary heart disease, central nervous system or psychiatric diseases, cancer, and other acute and chronic diseases. Patients and controls were matched in living and working environments, and use of tobacco and alcohol was similar between the two groups. Informed consent was obtained from all participants in accordance with the ethics committees of Tongji Medical College and Yanan Hospital.
Evaluation of Cerebral Deficits To determine the level of brain injury in patients with stroke, neurologic deficits were evaluated quantitatively on days 1, 15, and 30 after the onset of stroke by neurologists. Evaluations were performed in joint conferences, and the level of brain injury was assessed according to the Chinese National Scoring Criteria for Neurological Deficits in Patients with Stroke (22). These criteria are similar to the National Institutes of Health stroke scale (23) and are a quantitative measure of stroke-related neurologic deficits (stroke scale), determined by the following neurologic examinations: level of consciousness, including questions and commands; gaze and visual fields; facial symmetry; language function; left and right arm motors; left and right hand motors; left and right leg motors; and walking capability. The highest score is 45 (most severe cerebral injury) and the lowest is 0 (normal function). Based on the stroke scores on day 1 after the onset of stroke, the 65 elderly patients were divided into three clinical subtypes: mild (n ⫽ 22 patients; score: ⱕ15), intermediate (n ⫽ 21; score: 16 to 30), and severe (n ⫽ 22; score: 31 to 45) cerebral infarctions (22). The infarction site and the number of sites were also determined using computed tomography and magnetic resonance imaging.
Blood Sampling Venous blood was collected after overnight fasting on days 1, 15, and 30 after the onset of stroke. Lymphocytes were isolated using a standard separation medium (Biochemical Reagent Co., Shanghai, China), washed twice with phosphate buffer solution, and counted. The cell viability was ⬎95%, and cell numbers were within normal range (4 to 10 ⫻ 103/L), for patients and controls.
The lymphocyte concentration was adjusted to 5000 per L in phosphate buffer solution; 200 L of lymphocytes was centrifuged, the buffer was removed rapidly, and the cells were stored at ⫺80°C until analyzed.
Determination of HSP70 Levels in Lymphocytes The HSP70 assay was performed with the frozen cells within 1 month of blood collection (18,24,25). Briefly, lymphocyte proteins were separated on sodium dodecyl sulfate (Sigma Chemical Company, St. Louis, Missouri)– polyacrylamide (Promega Corporation, Madison, Wisconsin) gels and transferred to nitrocellulose membranes (Pall Corporation, New York, New York). Membranes were saturated with blocking buffer (phosphate buffer solution containing 5% skim milk powder) in a plastic container for 1 hour at 37°C, washed with phosphate buffer solution 0.05% Tween 80, and incubated at 37°C for 1 hour with rabbit anti-human HSP70 antibodies (26) diluted 1:1000. After washing the membranes a second time, horseradish peroxidase–labeled goat anti-rabbit immunoglobulin G (1:1000) (Beijing Zhongshan Biotechnology Company, Beijing, China) was added and incubated for 1 hour at 37°C. After a final wash, HSP70 was detected using 3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical Company) for 3 to 5 minutes and quantified by integrated optical density using an imaging densitometer (Shimadzu CS-930, Shimadzu, Japan).
Statistical Analysis Comparisons were made with the Student t test, analysis of variance (one-way or two-way), or univariate and multivariate linear regression models, as appropriate. Stratification analysis was performed for evaluating subgroup differences. The general linear model was used for the trend test for changes over time. Statistical inferences were based on a significance level of P ⬍0.05. All data analyses were two-sided and used SAS software, version 8.0 (SAS Institute, Inc., Cary, North Carolina).
RESULTS There was no difference in age, distribution of sex, smoking habits, or body mass index (⬎28 kg/m2) between patients and controls (Table 1). However, significantly more patients had total cholesterol levels ⬎224 mg/dL and hypertension as compared with controls (P ⬍0.001 for all comparisons). Further comparisons between each patient subgroup (by stroke severity) and controls suggested that the differences remained significant (P ⬍0.001), except for elevated total cholesterol level in the mild stroke group and for diabetes in the intermediate stroke group (P ⬍0.01). The proportions of these clinical conditions did not differ among the various infarction severity groups (P ⬎0.05 for all comparisons).
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
Volume 117 407
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
Table 1. Characteristics of Patients with Stroke and Healthy Controls Patients with Stroke Characteristic
Controls (n ⫽ 34)
Mild (n ⫽ 22)
Intermediate (n ⫽ 21)
P Value
Severe (n ⫽ 22)
All (N ⫽ 65)
All Patients vs. Controls
Between Group
Number (%) or Mean ⫾ SD Age (years) Male sex Smoking (pack-year) Body mass index ⬎28 kg/m2 Total cholesterol ⬎224 mg/dL Hypertension Diabetes
72 ⫾ 8 8 (24) 22 ⫾ 6 4 (12)
73 ⫾ 5 5 (23) 24 ⫾ 7 5 (23)
75 ⫾ 6 5 (24) 22 ⫾ 8 3 (14)
74 ⫾ 8 3 (14) 23 ⫾ 5 5 (23)
74 ⫾ 6 13 (20) 23 ⫾ 6 13 (20)
0.17 0.68 0.43 0.30
0.88 0.70 0.89 0.73
0
6 (27)*
7 (33)†
8 (36)†
21 (32)
⬍0.001
0.81
0 0
15 (68)† 7 (32)†
17 (81)† 6 (29)*
20 (91)† 10 (45)†
52 (80) 23 (35)
⬍0.001 ⬍0.001
0.17 0.47
* P ⬍0.01 for comparisons with controls. † P ⬍0.001 for comparisons with controls.
234 ⫾ 73 units; mild stroke: 157 ⫾ 49 units; intermediate stroke: 204 ⫾ 54 units), although these differences were not statistically significant (P ⬎0.05 for all comparisons).
Changes in Neurologic Deficit Scores during the First 30 Days after Stroke Onset The neurologic deficit scores of patients with mild stroke did not change in the first 15 days after the onset but decreased significantly between days 15 and 30 (P ⬍0.001) (Table 2). In the intermediate stroke group, scores decreased steadily between days 1 and 30 after the onset of stroke, but the decrease was only significant between days 1 and 15 (P ⬍0.05). Similarly, for patients with severe stroke, neurologic deficit scores decreased steadily between days 1 and 30, but only significantly between days 1 and 15 (P ⬍0.001); the decrease between days 15 and 30 was of borderline significance (P ⫽ 0.05).
HSP70 Levels by Site of Cerebral Infarction Mean HSP70 levels did not vary by stroke location or number of infarction sites: basal ganglia, 457 ⫾ 144 units; cerebral cortex in frontal and parietal lobe, 433 ⫾ 139 units; occipital lobe, 398 ⫾ 143 units; thalamus, 390 ⫾ 125 units; temporal lobe, 408 ⫾ 113 units; brainstem, 512 ⫾ 145 units (P ⬎0.05 for all comparisons).
Correlation between HSP70 Levels and Neurologic Deficit Scores during Recovery There were significant negative correlations between HSP70 level and neurologic deficit score on days 1 (r ⫽ ⫺0.53, P ⬍0.001) and 15 (r ⫽ ⫺0.54, P ⬍0.001) after the onset of stroke, and a significant positive correlation on day 30 (r ⫽ 0.49, P ⬍0.001).
Changes in HSP70 Levels after Stroke Mean (⫾ SD) HSP70 levels in patients with mild, intermediate, or severe stroke were higher (Figure) than in controls (86 ⫾ 34 units, P ⬍0.05 for all comparisons) from days 1 to 30 after the onset of stroke. There was a significant decreasing trend in HSP70 levels among stroke patients during this period (P for trend ⬍0.001 for all three groups), with the decrease appearing to be faster in patients with mild or intermediate stroke. Mean HSP70 levels were higher in patients with mild or intermediate stroke on days 1 and 15 than in those with severe stroke. This trend was reversed by day 30 (severe stroke:
DISCUSSION Ischemic stroke, one of the most common life-threatening neurologic diseases, is associated with serious long-term disability (27). Animal models suggest that HSP70 can protect the brain from various insults, including cerebral ischemia
Table 2. Neurologic Deficit Scores among Patients with Stroke, by Day after Onset of Stroke Neurologic Deficit Stroke (units) Type of Cerebral Infarction
Day 1
Day 15
Day 30
P Value Day 1 vs. 15
Day 1 vs. 30
Day 15 vs. 30
0.35 0.03 ⬍0.001
⬍0.001 0.002 ⬍0.001
⬍0.001 0.41 0.05
Number (%) or Mean ⫾ SD Mild (n ⫽ 22) Intermediate (n ⫽ 21) Severe (n ⫽ 22) 408
September 15, 2004
10 ⫾ 3 21 ⫾ 3 41 ⫾ 3
10 ⫾ 2 19 ⫾ 2 38 ⫾ 2
THE AMERICAN JOURNAL OF MEDICINE威
7⫾1 18 ⫾ 1 37 ⫾ 2 Volume 117
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
Figure. Changes in lymphocyte heat shock protein 70 (HSP70) levels among patients with mild, intermediate, or severe ischemic stroke from days 1 to 30 after disease onset. P values obtained from the test for trend were ⬍0.001 for patients with mild, intermediate, or severe stroke. IOD ⫽ integrated optical density units. Diamonds, circles, and triangles represent the mean values; error bars represent the SD.
(13,20,21,28 –30). Previous studies suggest that quantitative measures of stroke-related neurologic deficits correspond well with neurologic injuries (23,27). In the present study, we found that lymphocyte HSP70 levels correlated with neurologic deficit scores in patients with stroke, after onset and during 30-day recovery, which suggests that lymphocyte HSP70 levels may be an early marker for stroke severity within 1 day of onset and may serve as an indicator of prognosis in the elderly, particularly for those with milder stroke. Our results also suggest that HSP70 is associated with neuroprotective effects during the early stages (up to 15 days) of stroke and may be a marker for poor prognosis, particularly at the later stages (30 days after onset) of severe stroke. Several studies suggest that cells with overexpression of HSP70 are resistant to various stresses, including ischemia, and have better survival (10 –15). In mouse and rat models, a moderate pre– heat shock treatment or transgenic expression of the HSP70 gene provided protection from ischemic injury in the heart, brain, and kidneys. In mice, transgenic expression of human HSP70 resulted in a 24-hour improvement in hippocampal neuron survival, although the overall infarction size was not affected (13). Cerebral infarction after 6 hours of ischemia was less severe in transgenic mice overexpressing rat HSP70 than in wild type mice (28). In knockout mice, reduced expression of HSP70 increased neuron damage during acute focal cerebral ischemia (31). Furthermore, gene therapy with HSP70 improved neuron survival after focal cerebral ischemia in a rat model (20).
Thus, the synthesis of HSP70 is thought to be protective and an indicator of ongoing active repair or a compensatory mechanism related to neuron remodeling, facilitating the recovery of postischemic neurologic deficits (32). In contrast, little is known about the long-term effects of HSP70 on neuroprotection in humans. Our data show that there is a positive correlation between lymphocyte HSP70 levels and the neurologic deficit scores in patients with cerebral infarction on day 30 after disease onset. This phenomenon seems to be counterintuitive considering the beneficial functions of HSP70 as discussed above. Alternatively, this correlation may indicate that the stress response was still ongoing in patients with severe ischemia. Thus, lymphocyte HSP70 level may be a marker for detrimental effects in the late stages of cerebral infarction. While heat shock proteins are expressed at low levels under normal physiologic conditions, HSP70 levels increase under stressful or pathologic conditions, such as cerebral infarction (1,2,27–29). Indeed, high levels of HSP70 are indicative of injury, and persistently high levels during the late stages of severe stroke may be indicative of the continued presence of noxious stimuli even after 30 days. Of the three patient groups in our study, patients with mild cerebral infarction had the highest HSP70 levels on day 1 after stroke onset and the lowest levels on day 30. It is not known how the expression of HSP70 in lymphocytes is regulated in these patients. Heat shock proteins are generally expressed under complex regulatory controls operating at both the transcriptional and translational levels (33). Basal levels of HSP70 in leukocytes were found to be lower in athletes than in untrained persons, but the response of leukocytes to an in vitro heat shock was higher among athletes (34), suggesting that HSP70 levels are low under normal conditions but high under stressful conditions, consistent with a protective role that can be regulated. Basal levels of HSP70 in lymphocytes vary between persons and may contribute to interindividual variation in response to stresses (19,35). As HSP70 is polymorphic, variants may also affect individual disease susceptibility or recovery from pathologic stresses (36). In animal models, HSP70 expression differs in neurologic cells and tissues under normal and stress conditions (37–39). HSP70 expression is a useful marker for cellular injury and may help identify previously unrecognized areas of vulnerability in the nervous system after a neurotoxic stimulus (28). We found that lymphocyte HSP70 levels were not related to the location of cerebral infarction, consistent with findings from a study of 51 patients undergoing open heart surgery (40), in which HSP70 levels in the myocardium were not correlated with HSP70 levels in lymphocytes. Our data show that HSP70 levels in lymphocytes were correlated with both the severity of the stroke and improvement in neurologic deficit scores after stroke. Since we could not measure HSP70 levels in the brain, whether changes in lymphocytes can predict
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
Volume 117 409
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al
changes in the brain remains unknown. Further investigations aimed at correlating HSP70 levels in lymphocytes on the first day of recovery with the level of functional recovery at 30 days after stroke onset, as measured by neurologic deficit scores in ischemic animal models and in humans, are warranted. It will also be important to take into account the role of HSP70 polymorphisms and inducibility, preferably in the target tissues, in the susceptibility to trauma and during recovery from stroke.
13.
14.
15.
16.
ACKNOWLEDGMENTS
17.
We are particularly grateful to the patients and their family members who volunteered to participate in this study and to the many members of the medical staff of Yanan Hospital for their help in the examination and sampling of subjects. We thank Professor R. W. Currie (Dalhousie University, Halifax, Canada) for revising the manuscript and for his suggestions.
18.
19.
20.
REFERENCES
21.
1. Lindquist S, Craig EA. The heat shock proteins. Ann Rev Genet. 1988;22:631– 677. 2. Progress and perspectives in the biology of heat shock proteins and molecular chaperones. In: Morimoto RI, Tissières A, Georgopoulos C, eds. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1994:1–30. 3. Hightower LE. Heat shock, stress protein, chaperones, and proteotoxicity. Cell. 1991;66:191–197. 4. Hartl F-U. Molecular chaperones in protein folding. Nature. 1996; 381:571–580. 5. Angelidis CE, Lazaridis I, Pagoulatos GN. Constitutive expression of heat shock protein 70 in mammalian cells confers thermotolerance. Eur J Biochem. 1991;199:35–39. 6. Li GC, Li LY, Liu K, et al. Thermal response of rat fibroblasts stably transfected with the human 70kDa heat shock protein encoding gene. Proc Natl Acad Sci USA. 1988;88:1681–1685. 7. Rollet E, Lavoie JN, Landry J, Tanguay RM. Expression of Drosophila 27kDa heat shock protein in rodent cell confers thermal resistance. Biochem Biophys Res Commun. 1992;185:116 –120. 8. Mehlen P, Preville X, Chareyron P, et al. Constitutive expression of human hsp27, Drosophila hsp27 and human alpha-B crystallin confers resistance to tumor necrosis factor alpha and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J Immunol. 1995;154:364 –374. 9. Parsell DA, Lindquist S. Heat shock proteins and stress tolerance. In: Morimoto RI, Tissières A, Georgopoulos C, eds. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1994:457– 494. 10. Currie RW, Tanguay RM, Kingma JG. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation. 1993;87:963–971. 11. Marber MS, Mestril R, Chi S, et al. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest. 1995;96: 1446 –1456. 12. Plumier C, Ross BM, Currie RW, et al. Transgenic mice expressing 410
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
22. 23.
24.
25.
26.
27. 28. 29.
30.
31.
32.
33.
34.
the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest. 1995;95:1854 –1860. Plumier C, Krueger AM, Currie RW, et al. Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones. 1997;2:162–167. Radford NB, Fina M, Benjamin IJ, et al. Cardioprotective effects of 70-kDa heat shock protein in transgenic mice. Proc Natl Acad Sci USA. 1996;93:2339 –2342. Beck FX, Neuhofer W, Müller E. Molecular chaperones in the kidney: distribution, putative roles, and regulation. Am J Physiol Renal Physiol. 2000;279:F203–F215. Welch WJ. Mammalian stress response: cell physiology, structure/ function of stress proteins and implications for medicine and disease. Physiol Rev. 1992;72:1063–1081. Minowada G, Welch WJ. Clinical implications of the stress response. J Clin Invest. 1995;95:3–12. Xiao C, Chen S, Li J, et al. Association of HSP70 and genotoxic damage in lymphocytes of workers exposed to coke-oven emission. Cell Stress Chaperones. 2002;7:396 – 402. Xiao C, Wu T, Ren A, et al. Basal and inducible levels of Hsp70 in patients with acute heat-induced illness induced during training. Cell Stress Chaperones. 2003;8:86 –92. Yenari MA, Fink SL, Sun GH, et al. Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol. 1998;44:584 –591. Rajdev S, Hara K, Kokubo Y, et al. Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Ann Neurol. 2000;47:782–791. Stroke. In: Huang R, Su Z, eds. Rehabilitation of Stroke. Beijing, China: People Health Press; 2001:90 –356. Brott TG, Adams HP, Olinger CP, et al. Measurements of acute cerebral infarction: a clinical examination scale. Stroke. 1989;20: 864 – 870. Wu T, Chen S, Sun Y, et al. Presence of antibody against the inducible Hsp71 in patients with acute heat-induced illness. Cell Stress Chaperones. 2001;6:113–120. Wu T, Ma J, Chen S, et al. Association of plasma antibodies against the inducible Hsp70 with hypertension and harsh working conditions. Cell Stress Chaperones. 2001;6:394 – 401. Tanguay RM, Wu Y, Khandjian EW. Tissue-specific expression of heat shock stress proteins of the mouse in the absence of stress. Dev Genet. 1993;14:112–118. Stapf C, Mohr JP. Ischemic stroke therapy. Annu Rev Med. 2002;53: 453– 475. Rajdev S, Sharp FR. Stress proteins as molecular markers of neurotoxicity. Toxicol Pathol. 2000;28:105–112. Lee JE, Yenari MA, Sun GH, et al. Differential neuroprotection from human heat shock protein 70 overexpression in vitro and in vivo models of ischemia and ischemia-like conditions. Exp Neurol. 2001;170:129 –139. Kelly S, Bieneman A, Horsburgh K, et al. Targeting expression of hsp70i to discrete neuronal populations using Lmo-1 promoter: assessment of the neuroprotective effects of hsp70i in vivo and in vitro. J Cereb Blood Flow Metab. 2001;21:972–981. Lee SH, Kim M, Yoon BW, et al. Targeted hsp70.1 disruption increases infarction volume after focal cerebral ischemia in mice. Stroke. 2001;32:2905–2912. Fredduzzi S, Mariucci G, Tantucci M, Ambrosini MV. Generalized induction of 72-kDa heat shock proteins after transient focal ischemia in rat brain. Exp Brain Res. 2001;136:19 –24. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 1998;12: 3788 –3796. Fehrenbach E, Niess AM, Schlotz E, et al. Transcriptional and trans-
Volume 117
HSP70 Levels and Ischemic Stroke in Elderly Patients/Jin et al lational regulation of heat shock proteins in leukocytes of endurance runners. J Appl Physiol. 2000;89:704 –710. 35. Boschoff T, Lombard F, Eiselen R, et al. Differential basal synthesis of Hsp70/Hsc70 contributes to interindividual variation in Hsp70/ Hsc70 inducibility. Cell Mol Life Sci. 2000;57:1317–1325. 36. Favatier F, Bornman L, Hightower LE, et al. Variation in hsp gene expression and Hsp polymorphism: do they contribute to differential disease susceptibility and stress tolerance? Cell Stress Chaperones. 1997;2:141–155. 37. Vass K, Welch WJ, Nowak TS Jr. Localization of 70-kDa stress pro-
tein induction in gerbil brain after ischemia. Acta Neuropathol. 1995;77:128 –135. 38. Gaspary H, Graham SH, Sagar SM, Sharp FR. HSP70 heat shock protein induction following global ischemia in the rat. Brain Res Mol Brain Res.1995;34:327–332. 39. Leppa S, Kajanne R, Arminen L, Sistonen L. Differential induction of Hsp70-encoding genes in human hematopoietic cells. J Biol Chem. 2001;276:31713–31719. 40. Demidov ON, Tyrenko VV, Svistov AS, et al. Heat shock proteins in cardiosurgery patients. Eur J Cardio Thorac Surg. 1999;16:444 – 449.
September 15, 2004
THE AMERICAN JOURNAL OF MEDICINE威
Volume 117 411