Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue

Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 degrees C for 15 min. The control (n=23) and heated (n=55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart.     

Heat stress protection against mesenteric I/R-induced alterations in intestinal mucosa in rats.

Prior induction of heat shock protein 70 (HSP70) protects against ischemia-reperfusion (I/R) mucosal injury, but the ability of HSP70 to affect I/R-induced alterations in epithelial cell function is unknown. Rats subjected to whole body hyperthermia (41.5-42 degrees C for 6 min) increased HSP70 and heat shock factor 1 mRNA expression, reaching a maximum 2 h after heat stress and declining thereafter. HSP70 production was maximally elevated at 4 h after heat stress and remained elevated until after 12 h. Heat stress alone had no effect on mucosal function except to enhance secretion in response to ACh. Heat stress provided complete morphological protection against I/R-induced mucosal injury but did not confer a similar protection against I/R-induced decreases in mucosal resistance, sodium-linked glucose absorption, or tachykinin-mediated chloride secretion. Heat stress, however, attenuated the I/R-induced suppression of ACh response, and this effect was dependent on enteric nerves. Thus induction of heat shock protein 70 is associated with the preservation of mucosal architecture and attenuation of some specific functional alterations induced by I/R.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

Preoperative treatment of rectal cancer with radiation, chemotherapy and hyperthermia: analysis of treatment efficacy and heat-shock response

Preoperative treatment of locally advanced rectal cancer with radiation, chemotherapy and hyperthermia is analyzed with regard to heat-shock response. In 23 patients with locally advanced rectal cancer (uT3/uT4), hyperthermia was administered in combination with radiotherapy and chemotherapy. In parallel, the effect of the treatment on levels of the heat-shock proteins HSP27 and inducible HSP70 in tumors and surrounding tissues was investigated by Western blotting. The patients' sera were also examined for autoantibodies against HSPs. HSP27 and inducible HSP70 were detected in most rectal tumors and surrounding tissues before and after treatment. HSP27 and inducible HSP70 levels had changed in 10 tumors after treatment. However, prior to treatment, there existed an unexpected diversity in HSP levels in the tumors and surrounding tissue. Hyperthermia doses in cumulative minutes for which 90% of the tumor is above the reference temperature (cum min T90 > or = 15 min) led to increased survival and response compared to that of a control group of patients treated without or with low-dose hyperthermia (cum min T90 < 15 min). However, there was no correlation to different expression of the HSPs. Hyperthermia as used in this setting does not lead to any sustained expression of HSPs in either the tumor or the surrounding tissue.     

Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of “heat-controlled necrosis” with heat shock protein expression

Heat shock proteins (HSPs) are highly conserved proteins whose syntheses are induced by a variety of stresses, including heat stress. Since the expression of HSPs, including HSP70, protects cells from heat-induced apoptosis, HSP expression has been considered to be a complicating factor in hyperthermia. On the other hand, recent reports have shown the importance of HSPs, such as HSP70, HSP90 and glucose-regulated protein 96 (gp96), in immune reactions. If HSP expression induced by hyperthermia is involved in tumor immunity, novel cancer immunotherapy based on this novel concept can be developed. In such a strategy, a tumor-specific hyperthermia system, which can heat the local tumor region to the intended temperature without damaging normal tissue, would be highly advantageous. To achieve tumor-specific hyperthermia, we have developed an intracellular hyperthermia system using magnetite nanoparticles. This novel hyperthermia system can induce necrotic cell death via HSP expression, which induces antitumor immunity. In the present article, cancer immunology and immunotherapy based on hyperthermia, and HSP expression are reviewed and discussed. This article forms part of the Symposium in Writing "Thermal stress-related modulation of tumor cell physiology and immune responses", edited by Elfriede Noessner.     

Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography-tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPbeta6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.     

Inducible and constitutive HSP70s confer synergistic resistance against metabolic challenges

Chaperonic proteins, including inducible HSP70 (HSP70i) and constitutive HSP70 (HSC70), have been implicated as essential players in the cellular adaptive protection. Ensuing studies demonstrated that overexpression of either protein individually protects against thermal and oxidative challenges. The present study aimed to determine whether a concurrent overexpression of both HSC70 and HSP70i confers a better metabolic protection than the expression of each protein alone. Using a rat heart-derived H9c2 cardiac myoblast cell line, we found that HSP70i was rapidly induced within 2–8 h following a mild thermal preconditioning (43 °C for 20 min) in both parental cells and an established H9/70c clonal sub-line overexpressing HSC70. The level of HSP70i protein in heat pretreated H9/70c clonal cells reached only 50% of that in heat pretreated H9c2 parental cells. Nevertheless, protection against lethal hyperthermia, menadione (an oxidant) and hydrogen peroxide (H₂O₂) exposure in the pretreated H9/70c clonal cells was significantly higher than the sum of protection afforded by the early induction of HSP70i in the pretreated parental cells and protection afforded by the pre-existing HSC70 in the H9/70c cells without preconditioning. Using dosimetric analysis, we also found that menadione resistance in the pretreated parental cells increased linearly with cellular HSP70i level (10–300 ng/mg total protein). However, the resistance in the pretreated H9/70c cells showed a biphasic relationship with cellular HSP70i level; when HSP70i concentration reached >250 ng/mg protein, survivability after menadione exposure was markedly enhanced. Similar results were observed in H9c2 cells genetically manipulated to overexpress both HSC70 and HSP70i. The survival benefit against lethal hyperthermia, oxidant treatment, and hypoxia/reoxygenation conferred by a concerted HSC70 and HSP70i overexpression was greater than the sum of benefits contributed by individual protein overexpression. Together, these findings suggest that HSC70 and HSP70i may complement each other in a synergistic manner to preserve cellular integrity during metabolic challenges.     

Role of heat shock protein 70 in hepatic ischemia-reperfusion injury in mice

It is well established that liver ischemia-reperfusion induces the expression of heat shock protein (HSP) 70. However, the biological function of HSP70 in this injury is unclear. In this study, we sought to determine the role of HSP70 in hepatic ischemia-reperfusion injury in mice. Male mice were subjected to 90 min of partial hepatic ischemia followed by up to 8 h of reperfusion. HSP70 was rapidly upregulated after reperfusion. To explore the function of HSP70, sodium arsenite (8 mg/kg iv) was injected before surgery. We found that this dose induced HSP70 expression within 6 h of treatment. Induction of HSP70 with arsenite resulted in a >50% reduction in liver injury as determined by serum transaminases and histology. In addition, arsenite similarly reduced liver neutrophil recruitment and liver nuclear factor-kappaB activation, and attenuated serum levels of tumor necrosis factor-alpha and macrophage inflammatory protein-2, but increased levels of interleukin (IL)-6. In HSP70 knockout mice, arsenite did not protect against liver injury but did reduce liver neutrophil accumulation. Arsenite-induced reductions in neutrophil accumulation in HSP70 knockout mice were found to be mediated by IL-6. To determine whether extracellular HSP70 contributed to the injury, recombinant HSP70 was injected before surgery. Intravenous injection of 10 microg of recombinant HSP70 had no effect on liver injury after ischemia-reperfusion. The data suggest that intracellular HSP70 is directly hepatoprotective during ischemia-reperfusion injury and that extracellular HSP70 is not a significant contributor to the injury response in this model. Targeted induction of HSP70 may represent a potential therapeutic option for postischemic liver injury.     

Oxidative and nitrosative mediators in hepatic injury caused by whole body hyperthermia in rats

The involvement of oxidative and nitrosative mediators in liver injury caused by heat stress remains unclear. This study aimed to elucidate the role of endothelial nitric oxide synthase (eNOS), and inducible NOS (iNOS)-derived NO and nitrotyrosine in the whole-body hyperthermia (WBH)-induced liver injury. Rats were anesthetized with intraperitoneal pentobarbital, and were exposed to a heating lamp for 60 min to raise the core temperature to 42.5 degrees C. The rats were maintained at the hyperthermic state for an additional 50 min. Blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, creatine phosphokinase, amylase, lipase, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines (tumor necrosis factoralpha, interleukin-1beta and interleukin-10) were measured before and 14 h after hyperthermia. Immunohistochemical staining was employed to detect the eNOS, iNOS and nitrotyrosine levels. Western blotting was used to examine the expression of heatshock protein 70 (HSP 70). Histopathological examination of the liver tissue was performed. WBH caused liver injury accompanied with significant increases in biochemical factors, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines. In addition, WBH enhanced the eNOS, iNOS, nitrotyrosine and HSP 70 levels. WBH caused hepatic injury. The pathogenetic mechanism is likely mediated through the NOS-derived NO, free radical, proinflammatory cytokines and nitrotyrosine. The enhanced expression of HSP 70 may play a protective role.     

Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts

Perturbation of oxidant/antioxidant cellular balance, induced by cellular metabolism and by exogenous sources, causes deleterious effects to proteins, lipids, and nucleic acids, leading to a condition named "oxidative stress" that is involved in several diseases, such as cancer, ischemia-reperfusion injury, and neurodegenerative disorders. Among the exogenous agents, both H(2)O(2) and hyperthermia have been implicated in oxidative stress promotion linked with the activation of apoptotic or necrotic mechanisms of cell death. The goal of this work was to better understand the involvement of some stress-related proteins in adaptive responses mounted by human fibroblasts versus the oxidative stress differently induced by 42 degrees C hyperthermia or H(2)O(2.) The research was developed, switching off inducible nitric oxide synthase (iNOS) expression through antisense oligonucleotide transfection by studying the possible coregulation in the expression of HSP32 (also named HO-1), HSP70, and iNOS and their involvement in the induction of DNA damage. Several biochemical parameters, such as cell viability (MTT assay), cell membrane integrity (lactate dehydrogenase release), reactive oxygen species formation, glutathione levels, immunocytochemistry analysis of iNOS, HSP70, and HO-1 levels, genomic DNA fragmentation (HALO/COMET assay), and transmembrane mitochondrial potential (deltaPsi) were examined. Cells were collected immediately at the end of the stress-inducing treatment. The results, confirming the pleiotropic function of i-NOS, indicate that: (i). HO-1/HSP32, HSP70, and iNOS are finely tuned in their expression to contribute all together, in human fibroblasts, in ameliorating the resistance to oxidative stress damage; (ii). ROS exposure, at least in hyperthermia, in human fibroblasts contributes to growth arrest more than to apoptosis activation; and (iii). mitochondrial dysfunction, in presence of iNOS inhibition seems to be clearly involved in apoptotic cell death of human fibroblasts after H(2)O(2) treatment, but not after hyperthermia.     

Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia using magnetite nanoparticles

In this study we demonstrated that heat shock protein (HSP) 70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. Our hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. We purified the HSP70-peptide complexes from the tumor after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity, and we found that in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. Our hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity. This phenomenon, which may be termed in situ vaccination, has important implications for the development of novel antitumor therapies.     

Extracellular matrix components affect the pattern of protein synthesis of endothelial cells responding to hyperthermia

Summary The biosynthetic profile of endothelial cells responding to hyperthermia is altered by extracellular matrix components. The extracellular matrix components influence the quantitative expression of members of the HSP70 family and HSP90. The expression of several HSP70 mRNA species, which are strictly stress inducible, are modulated by extracellular matrix components. Both laminin and collagen type IV decrease the amount of HSP70 protein and mRNA expressed by endothelial cells exposed to hyperthermia relative to control cultures attached to virgin plastic. In contrast, both laminin and collagen type IV increased the amount of HSP90 mRNA constitutively expressed by endothelial cells at 37° C. When endothelial cells were exposed to elevated temperatures, these two extracellular matrix proteins decrease the amount of HSP90 mRNA relative to control cultures attached to virgin plastic. Our observations are consistent with the proposal that the extracellular matrix components regulate gene expression and cell behavior in regard to thermotolerance.     

The use of transgenic mice to study cytoprotection by the stress proteins

Heat shock or stress proteins (HSPs) have been shown to be able to confer cytoprotection in a diversity of cell types and organisms. We were interested in assessing if HSPs, in particular HSP70, were protective against pathophysiological stresses such as myocardial ischemia. Our approach was to generate a transgenic mouse line that would constitutively express high levels of an inducible rat HSP70 isoform in the heart. The hearts of the transgenic mice were then used in an isolated perfused mouse heart model to assess whether increased expression of HSP70 alone was protective against ischemia-reperfusion injury. Our study showed that there was a significant improvement in contractile recovery, less cellular damage, and a reduction in infarct size in the hearts of transgenic mice as compared to non-transgenic mice following global ischemia in our isolated perfused mouse heart model. Additional studies have since shown that increased expression of HSP70 as well as other stress proteins in transgenic mice protects against different forms of pathological stresses. We present here the methods we used to generate HSP70 transgenic mice and assess their increased tolerance to ischemia-reperfusion injury.     

Antitumor effects of combined therapy of recombinant heat shock protein 70 and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma

Heat shock proteins (HSPs) are recognized as significant participants in cancer immunity. We previously reported that HSP70 expression following hyperthermia using magnetic nanoparticles induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by administration of recombinant HSP70 protein into the tumor in situ. Hyperthermia was conducted using our original magnetite cationic liposomes (MCLs), which have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs and recombinant mouse HSP70 (rmHSP70) were injected into melanoma nodules in C57BL/6 mice, which were subjected to AMF for 30 min. Temperature within the tumor reached 43°C and was maintained by controlling the magnetic field intensity. The combined treatment strongly inhibited tumor growth over a 30-day period and complete regression of tumors was observed in 20% (2/10) of mice. It was also found that systemic antitumor immunity was induced in the cured mice. This study suggests that novel combined therapy using exogenous HSP70 and hyperthermia has great potential in cancer treatment.     

Preinduction of HSP70 promotes hypoxic tolerance and facilitates acclimatization to acute hypobaric hypoxia in mouse brain

It has been shown that induction of HSP70 by administration of geranylgeranylacetone (GGA) leads to protection against ischemia/reperfusion injury. The present study was performed to determine the effect of GGA on the survival of mice and on brain damage under acute hypobaric hypoxia. The data showed that the mice injected with GGA survived significantly longer than control animals (survival time of 9.55 ± 3.12 min, n = 16 vs. controls at 4.28 ± 4.29 min, n = 15, P < 0.005). Accordingly, the cellular necrosis or degeneration of the hippocampus and the cortex induced by sublethal hypoxia for 6 h could be attenuated by preinjection with GGA, especially in the CA2 and CA3 regions of the hippocampus. In addition, the activity of nitric oxide synthase (NOS) of the hippocampus and the cortex was increased after exposure to sublethal hypoxia for 6 h but could be inhibited by the preinjection of GGA. Furthermore, the expression of HSP70 was significantly increased at 1 h after GGA injection. These results suggest that administration of GGA improved survival rate and prevented acute hypoxic damage to the brain and that the underlying mechanism involved induction of HSP70 and inhibition of NOS activity.     

Lipopolysaccharide pretreatment protects against ischemia/reperfusion injury via increase of HSP70 and inhibition of NF-κB

It has been reported that pretreatment of rats with lipopolysaccharide (LPS) increases myocardial functional recovery in ischemia/reperfusion (I/R) hearts. However, the mechanisms by which LPS induces cardioprotection against I/R injury have not been fully elucidated. In this study, we pretreated rats with LPS (1.0 mg/kg) 24 h before they were subjected to I/R injury, and then examined the roles of heat shock protein-70 (HSP70) and nucleus factor-κB (NF-κB) in LPS-induced cardioprotection. We observed that pretreatment with low-dose LPS resulted in significantly increased levels of HSP70 in the myocardium, which could dramatically inhibit NF-κB translocation and reduce degradation of inhibitory κB. Inhibition of NF-κB, in turn, attenuated release of inflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-1β, and IL-6) and reduced apoptosis of myocardium and infarct area following I/R injury. Moreover, HSP70 could ameliorate oxidative stress following I/R injury. To further investigate whether increase of HSP70 might be responsible for protection of the myocardium against I/R injury, we co-administered the HSP70 inhibitor, quercetin, with LPS before I/R injury. We found that LPS-induced cardioprotection was attenuated by co-administration with quercetin. Herein, we concluded that increased levels of HSP70 through LPS pretreatment led to inhibition of NF-κB activity in the myocardium after I/R injury. Our results indicated that LPS-induced cardioprotection was mediated partly through inhibition of NF-κB via increase of HSP70, and LPS pretreatment could provide a means of reducing myocardial I/R injury.     

Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury

The major heat shock protein, HSP70, is known to be involved in cytoprotection against environmental stresses mediated by their function as a "molecular chaperone". Monochloramine (NH(2)Cl) is a potent cytotoxic oxidant generated by neutrophil-derived hypochlorous acid and Helicobacter pylori urease-induced ammonia. In this study, to evaluate the cytoprotective effect of HSP70 against NH(2)Cl-induced gastric mucosal cell injury, rat gastric mucosal cells (RGM-1) were stably transfected with pBK-CMV containing the human HSP70 gene (7018-RGM-1) or pBK-CMV alone (pBK-CMV-12) as control cells. These cells were treated with various concentrations of NH(2)Cl. Cell Viability was determined by MTT assay and the direct plasma membrane damage was analyzed by lactate dehydrogenase (LDH) release assay. Apoptosis was determined by DNA fragmentation analysis. NH(2)Cl caused injury to pBK-CMV-12 cells in a concentration-dependent manner. NH(2)Cl-induced gastric cell injury was significantly diminished in HSP70 over-expressing cell line (7018-RGM-1) both necrosis and apoptosis compared to the control cell line (pBK-CMV-12) transfected with CMV vector alone. These result suggest that overexpression of HSP70 plays an important role in protecting gastric cells against NH(2)Cl-induced injury.     

Acquisition of thermotolerance in bay scallops, Argopecten irradians irradians, via differential induction of heat shock proteins

Many cells and organisms are rendered transiently resistant to lethal heat shock by short exposure to sublethal temperatures. This induced thermotolerance is thought to be related to increased amounts of heat shock proteins (HSPs) which, as molecular chaperones, protect cells from stress-induced damage. As part of a study on bivalve stress and thermotolerance, work was undertaken to examine the effects of sublethal heat shock on stress tolerance of juveniles of the northern bay scallop, Argopecten irradians irradians, in association with changes in the levels of cytoplasmic HSP70 and 40. Juvenile bay scallops heat-shocked at a sublethal temperature of 32 °C survived an otherwise lethal heat treatment at 35 °C for at least 7 days. As determined by ELISA, acquisition of induced thermotolerance closely paralleled HSP70 accumulation, whereas HSP40 accrual appeared less closely associated with thermotolerance. Quantification of scallop HSPs following lethal heat treatment, with or without conditioning, suggested a causal role for HSP70 in stress tolerance, with HSP40 contributing to a lesser, but significant extent. Overall, this study demonstrated that sublethal heat shock promotes survival of A. irradians irradians juveniles upon thermal stress and the results support the hypothesis that HSPs have a role in this induced thermotolerance. Exploitation of the induced thermotolerance response shows promise as a means to improve survival of bay scallops in commercial culture.