Extracellular Heat Shock Protein Research Articles

Lectin-Like Oxidized LDL Receptor 1 Mediates the Uptake of the C-Terminal Domain of Hsp70 (A Promising Immune Adjuvant Molecule) and Antigen Peptide Complexes

Aims: Extracellular heat shock protein 70 (Hsp70) is an adjuvant molecule that stimulates the immune system. The C-terminal domain of Hsp70 (C70), without the ATPase domain, is sufficient for antigen cross-presentation. However, the mechanism by which the receptor mediates the uptake of C70–peptide complex remains unclear. We therefore aimed to determine the process by which the receptor mediates the uptake of antigenic peptide-bound C70. Methodology: Hsp70 and C70 individually cloned into pET28a were expressed in Escherichia coli BL21 (DE3) and were purified on Ni-NTA agarose and MonoQ HR5/5. Hsp70 and C70 were labeled with Alexa 555 and Alexa 633, respectively, to detect cellular binding. HEK293 cells stably expressing lectin-like oxidized LDL receptor-1 (LOXOriginal Research Article International Journal of Biochemistry Research & Review, 4(4): 284-294, 2014 285 1) and KG-1 human dendritic-like cells were incubated with Alexa-labeled Hsp70 and C70 individually or with C70 and antigenic complexes and were observed using fluorescence microscopy. The affinity of LOX-1 toward Hsp70 and C70 was analyzed by chip assay using surface plasmon resonance, which immobilized LOX-1 ligand recognition domain. Results: HEK293 cells stably expressing LOX-1 and KG-1 cells accepted the C70– peptide and Hsp70–peptide complexes. Anti-LOX-1-neutralizing antibody inhibited the uptake of the C70–peptide complexes by KG-1 cells. The dissociation constant (KD) of C70 toward the LOX-1 extracellular domain, measured by surface plasmon resonance, was 4.02 × 10 −7 M and that of the C70–peptide complex was 6.6 × 10 −8 M. C70 increased the LOX-1 affinity by forming a complex with the antigen peptide. Conclusion: Our findings suggest that LOX-1 is the primary receptor for the C70–peptide and the Hsp70–peptide complexes. C70 is a promising adjuvant molecule that is internalized via LOX-1. In addition, it is convenient to prepare C70 using an E. coli expression system and C70 is more stable than full-length Hsp70.

Early response roles for prolactin cortisol and circulating and cellular levels of heat shock proteins 72 and 90α in severe sepsis and SIRS.

Objective. To evaluate the early heat shock protein (HSP) and hormonal stress response of intensive care unit (ICU) patients with severe sepsis/septic shock (SS) or systemic inflammatory response syndrome (SIRS) compared to healthy subjects (H). Methods. Patients with early (first 48 hrs) SS (n = 29) or SIRS (n = 29) admitted to a university ICU and 16 H were enrolled in the study. Serum prolactin, cortisol, and plasma ACTH were determined using immunoassay analyzers. ELISA was used to evaluate extracellular HSPs (eHSP90α, eHSP72) and interleukins. Mean fluorescence intensity (MFI) values for intracellular HSPs (iHSP72, iHSP90α) were measured using 4-colour flow-cytometry. Results. Prolactin, cortisol, and eHSP90α levels were significantly increased in SS patients compared to SIRS and H (P < 0.003). ACTH and eHSP72 were significantly higher in SS and SIRS compared to H (P < 0.005). SS monocytes expressed lower iHSP72 MFI levels compared to H (P = 0.03). Prolactin was related with SAPS III and APACHE II scores and cortisol with eHSP90α, IL-6, and lactate (P < 0.05). In SS and SIRS eHSP90α was related with eHSP72, IL-6, and IL-10. Conclusion. Prolactin, apart from cortisol, may have a role in the acute stress response in severe sepsis. In this early-onset inflammatory process, cortisol relates to eHSP90α, monocytes suppress iHSP72, and plasma eHSP72 increases.

Extracellular HSP70/HSP70-PCs Promote Epithelial-Mesenchymal Transition of Hepatocarcinoma Cells

BackgroundExtracellular heat shock protein 70 and peptide complexes (eHSP70/HSP70-PCs) regulate a variety of biological behaviors in tumor cells. Whether eHSP70/HSP70-PCs are involved in the epithelial-mesenchymal transition (EMT) of tumor cells remains unclear. AimsTo determine the effects of eHSP70/HSP70-PCs on EMT of hepatocarcinoma cells. MethodsThe expressions of E-cadherin, HSP70, α-smooth muscle actin protein (α-SMA) and p-p38 were detected immunohistochemically in liver cancer samples. Immunofluorescence, western blotting and real-time RT-PCR methods were used to analyze the effects of eHSP70/HSP70-PCs on the expressions of E-cadherin, α-SMA and p38/MAPK in vivo. ResultsHSP70, E-cadherin, α-SMA and p-p38 were elevated in hepatocellular carcinoma tissues. The expression of HSP70 was positively correlated with malignant differentiated liver carcinoma. The expressions of HSP70, α-SMA and p-p38 correlated with recurrence-free survival after resection. eHSP70/HSP70-PCs significantly promoted the expressions of α-SMA and p-p38 and reduced the expressions of E-cadherin in vivo. The effect was inhibited by SB203580. ConclusionThe expressions of HSP70, E-cadherin, α-SMA and p-p38 may represent indicators of malignant potential and could discriminate the malignant degree of liver cancer. eHSP70/HSP70-PCs play an important role in the EMT of hepatocellular carcinoma via the p38/MAPK pathway.

Extracellular Hsp72 concentration relates to a minimum endogenous criteria during acute exercise-heat exposure

Extracellular heat shock protein 72 (eHsp72) concentration increases during exercise-heat stress when conditions elicit physiological strain. Differences in severity of environmental and exercise stimuli have elicited varied response to stress. The present study aimed to quantify the extent of increased eHsp72 with increased exogenous heat stress, and determine related endogenous markers of strain in an exercise-heat model. Ten males cycled for 90 min at 50 % [Formula: see text] in three conditions (TEMP, 20 °C/63 % RH; HOT, 30.2 °C/51%RH; VHOT, 40.0 °C/37%RH). Plasma was analysed for eHsp72 pre, immediately post and 24-h post each trial utilising a commercially available ELISA. Increased eHsp72 concentration was observed post VHOT trial (+172.4 %) (p < 0.05), but not TEMP (-1.9 %) or HOT (+25.7 %) conditions. eHsp72 returned to baseline values within 24 h in all conditions. Changes were observed in rectal temperature (Trec), rate of Trec increase, area under the curve for Trec of 38.5 and 39.0 °C, duration Trec ≥38.5 and ≥39.0 °C, and change in muscle temperature, between VHOT, and TEMP and HOT, but not between TEMP and HOT. Each condition also elicited significantly increasing physiological strain, described by sweat rate, heart rate, physiological strain index, rating of perceived exertion and thermal sensation. Stepwise multiple regression reported rate of Trec increase and change in Trec to be predictors of increased eHsp72 concentration. Data suggests eHsp72 concentration increases once systemic temperature and sympathetic activity exceeds a minimum endogenous criteria elicited during VHOT conditions and is likely to be modulated by large, rapid changes in core temperature.

Correlation between Extracellular Heat Shock Protein 60 (exHSP60) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) in Non Diabetic Men

BACKGROUND: Adipose tissue expansion in obesity leads to abnormal adipocyte function, chronic low grade inflammation, primary reticulum stress, and mitochondrial stress. This induces mitochondrial unfolded protein response (UPRmt) that selectively upregulates mitochondrial chaperone protein. Heat shock Protein 60 (HSP60) is the primary chaperone in mitochondrial matrix. Inflammatory stress promotes HSP60 released from adipocytes and induces insulin resistance. In this study we attempted to investigate the correlation between exHSP60 and HOMA-IR in men with different ranges of waist circumference (WC).METHODS: This study was an observational cross sectional study carried out on 141 non diabetic men, aged 30-55 years old, who were divided into three groups based on WC; WC ≤90 cm, 90 cm &lt; WC ≤ 100 cm and WC &gt; 100 cm. Fasting plasma glucose, triglyceride, HDL-C, hsCRP, HSP60 serum and anti-HSP60 antibody, serum IL-1β serum, insulin, were examined. For statistical analysis, Kolmogorov-Smirnov and Spearman’s correlation analysis were conducted.RESULTS: There were a significant correlation between exHSP60 and HOMA-IR (r=0.281; p=0.041) in WC ≤ 90 cm group; and a negative significant correlation between exHSP60 and HOMA-IR (r=-0.508; p=0.007) in WC &gt; 100 cm group.CONCLUSION: This study showed that there was a dynamic correlation between exHSP60 and HOMA-IR in WC ≤ 90 cm group compared with WC &gt; 100 cm group. We also found inverse correlation patterns between exHSP60 and HOMA-IR, and between anti-HSP60 antibody and HOMA-IR in non diabetic subjects.KEYWORDS: obesity, insulin resistance, mitochondrial stress, exHSP60, hsCRP, HOMA-IR

Extracellular heat shock protein A9 is a novel interaction partner of podoplanin in oral squamous cell carcinoma cells

In previous studies, we have shown several lines of evidence that podoplanin (PDPN) plays an important role in cell adhesion via its association with extracellular components in neoplastic conditions, though there has been no trial to search for PDPN-interaction molecules in the extracellular milieu. To screen for those molecules, we performed proteomics-based analysis using liquid chromatography-tandem mass spectrometry followed by co-immunoprecipitation for PDPN in ZK-1, an oral squamous cell carcinoma (SCC) cell system whose cell membrane molecules were cross-linked with each other in their extracellular compartments, and we identified heat shock protein (HSP) A9 as one of the extracellular PDPN bound molecules. Effects of transient PDPN knockdown by siRNA in ZK-1 were also comparatively examined for cellular behaviors in terms of HSPA9 expression and secretion. Finally, HSPA9 expression modes were immunohistochemically visualized in oral SCC tissue specimens. HSPA9 was secreted from ZK-1 cells, and the expression and secretion levels of HSPA9 gene and protein were well coordinated with those of PDPN. Immunohistochemically, HSPA9 and PDPN were co-localized in ZK-1 cells and oral SCC foci, especially in the peripheral zone. In conclusion, the results indicate that HSPA9 secreted by oral SCC cells interacts with PDPN on their cell surface in an autocrine manner and regulates their growth and invasiveness.

Effects of Habitual Exercise on the eHsp72-Induced Release of Inflammatory Cytokines by Macrophages from Obese Zucker Rats

Regular exercise is a good non-pharmacological treatment of metabolic syndrome in that it improves obesity, diabetes, and inflammation. The 72 kDa extracellular heat shock protein (eHsp72) is released during exercise, thus stimulating the inflammatory responses. The aim of the present work was to evaluate the effect of regular exercise on the eHsp72-induced release of IL-1β, IL-6, and TNFα by macrophages from genetically obese Zucker rats (fa/fa) (ObZ), using lean Zucker (LZ) rats (Fa/fa) to provide reference values. ObZ presented a higher plasma concentration of eHsp72 than LZ, and exercise increased that concentration. In response to eHsp72, the macrophages from ObZ released less IL-1β and TNFα, but more IL-6, than macrophages from LZ. While eHsp72 stimulated the release of IL-1β, TNFα, and IL-6 in the macrophages from healthy LZ (with respect to the constitutive release), it inhibited the release of IL-1β and IL-6 in macrophages from ObZ. The habitual exercise improved the release of inflammatory cytokines by macrophages from ObZ in response to eHsp72 (it increased IL-1β and TNFα, and decreased IL-6), tending to values closer to those determined in healthy LZ. A deregulated macrophage inflammatory and stress response induced by eHsp72 underlies MS, and this is improved by habitual exercise.

Effects of chronic heat stress on plasma concentration of secreted heat shock protein 70 in growing feedlot cattle1

Sixty Angus steers (449.2±11.0 kg) with implanted body temperature (BT) transmitters were used in a 110-d study to determine the effect of chronic stress (housing, diet, and climate) on extracellular heat shock protein 70 (eHsp70) concentration in plasma. The steers were a subset of a larger study involving 164 steers. Before the start of the study (d -31), 63 steers were implanted with a BT transmitter between the internal abdominal muscle and the peritoneum at the right side flank. Steers were housed in 20 pens (10 with shade and 10 without). Within each pen, 3 steers had a transmitter, and BT was recorded at 30-min intervals throughout the study. On d 0, 30, 60, 90, and 110, steers were weighed, BCS assessed (1 to 9 scale in which 1=emaciated and 9=obese), and 10 mL of blood from the coccygeal vein was collected for determination of inducible heat shock protein 70 (Hsp70) concentration by ELISA. Climatic variables (ambient temperature, relative humidity, solar radiation, black globe temperature, and wind speed) were obtained every 30 min from an on-site weather station. The relationship between the climatic variables and Hsp70 concentration were examined. As we failed to detect an effect of shade, all data were pooled. Mean BT over the duration of the study was 39.6±0.10°C. Mean BT was lowest (38.7±0.10°C) on d 0 and highest on d 110 (40.2°C±0.10). The Hsp70 concentration was least on d 0 (2.33±0.47 ng/mL) and greatest on d 30 (8.08±0.78 ng/mL). The Hsp70 concentration decreased from d 30 but remained above the d-0 concentrations on d 60, 90, and 110. There was a strong relationship between Hsp70 concentration and ambient temperature (r2=0.86; P<0.0001) and Hsp70 concentration and photoperiod (r2=0.94; P<0.0001) and no relationship with BT (r2=0.06; P<0.0001). When assessed with both BCS and BT, the relationship was moderate (r2=0.48; P<0.001). The relationship between Hsp70 and change in BT (BTΔ) above 38.6°C was also moderate (r2=0.54; P<0.0001). The BT at a given time does not appear to be related to Hsp70 concentration. However, Hsp70 expression may be a useful indictor for BTΔ when BT>38.6°C. The Hsp70 concentration is a reliable indicator of chronic stress but is not a reliable indicator of a single stressor when animals are exposed to multiple chronic stressors.

New Paradigm for Intrinsic Function of Heat Shock Proteins as Endogenous Ligands in Inflammation and Innate Immunity

Recently, growing evidences that extracellular heat shock protein (HSP) functions as endogenous immunomodulator for innate and adaptive immune responses have been demonstrated. Because HSPs inherently act as chaperones within the cells, passive release such as cell necrosis and active release including secretion in the form of exosome have been suggested for HSP release into extracellular milieu. Such extracellular HSPs have been shown to be activators for innate immune responses through Toll-like receptors (TLRs). However, it has also been suggested that HSPs augmented the ability of associated innate ligands such as LPS to stimulate cytokine production and dendritic cell (DC) maturation. More interestingly, recent study demonstrated that innate immune responses elicited by both endogenous and exogenous danger signals were spatially and temporally regulated and this can be manipulated using Hsp90 or oxygen-regulated protein 150 (ORP150), thereby controlling the immune responses. We will discuss how spatiotemporal regulation of HSP-chaperoned molecules within antigen-presenting cells affects the antigen cross-presentation and innate immune responses. Precise analysis of HSP biology can lead us to establish outstanding HSPbased immunotherapy.

116. The functional role of exosome associated extracellular heat shock protein 72 following exposure to acute stress

One component of the adaptive stress response is that innate immunity is primed by circulating endogenous danger associated molecular patterns (DAMPs). Extracellular heat shock protein 72 (eHsp72) is a DAMP that is upregulated intracellularly after acute stress, but its mechanism of release is unknown. Previous in vitro studies have demonstrated the presence of Hsp72 on extracellular vesicles known as exosomes, which are biologically active vesicles secreted from all known cells in the body. We hypothesize that stress-evoked eHsp72 is released into the blood via an exosome pathway and that these exosomes are a critical component of stress-enhanced immunity. Male Fisher 344 rats (8/group) were subjected to tail shock stress or no stress, and exosome isolation from plasma was verified by size (EM and Nanosight) and common exosome markers (acetylcholinesterase, CD63, and Rab5b). We report that exposure to an acute stressor increases exosome expression of eHsp72, but not other stress-inducible proteins (IL-1β and IL-6). Additionally, exosomes from stressed, but not control, rats facilitates in vivo bactericidal inflammatory response ( p < 0.05) and an in vitro LPS-evoked inflammatory responses ( p < 0.05). These data suggest that exposure to stress can alter the proteomic composition of circulating exosomes, thereby enhancing the innate immune response.

Conference Scene: Taking the Heat out of Chaperokine Function

The field of heat-shock protein (HSP) research has been revitalized when extracellular HSPs were reported in the literature in the 1990s. Emerging findings suggest that HSPs can perform a wide variety of biological functions, more than their traditional role as molecular chaperones. With prominent speakers from various HSP fields, the 1(st) Heat Shock Protein - Multiple Roles in Inflammation, Vaccination and Homeostasis Conference covered topics on intracellular and extracellular HSPs along with the role of bacterial and self-HSPs. This conference, organized by Euroscicon, brought together more than 20 delegates from industry and academia for a lively discussion of the current state of HSP research.

Heat-Shock Proteins as Endogenous Ligands Building a Bridge Between Innate and Adaptive Immunity

There has been growing evidence that heat-shock protein (HSP) functions as an endogenous immunomodulator for innate and adaptive immune responses. Since HSPs inherently act as chaperones within cells, passive release (e.g., by cell necrosis) and active release (including release by secretion in the form of an exosome) have been suggested as mechanisms of HSP release into the extracellular milieu. Such extracellular HSPs have been shown to be activators of innate immune responses through Toll-like receptors. However, it has also been suggested that HSPs augment the ability of associated innate ligands such as lipopolysaccharides to stimulate cytokine production and dendritic cell maturation. More interestingly, a recent study has demonstrated that innate immune responses elicited by danger signals were regulated spatiotemporally and that can be manipulated by HSPs, thereby controlling immune responses. We will discuss how spatiotemporal regulation of HSP-chaperoned molecules within antigen-presenting cells affects adaptive immunity via antigen cross-presentation and innate immune responses. Precise analysis of HSP biology should lead to the establishment of effective HSP-based immunotherapy.

Abstract 544: Extracellular Heat Shock Protein 27 Inhibits Foam Cell Formation by Targeting Scavenger Receptor-A in the Vessel Wall

Background: We showed that injection of recombinant heat shock protein 27 (rHSP27) into ApoE -/- mice reduces atherosclerosis lesions and total serum cholesterol levels. Extracellular HSP27 also interacts with scavenger receptor (SR)-A and inhibits acLDL uptake in macrophages, but the role of SR-A in HSP27 atheroprotection remains unclear. Purpose: To elucidate the mechanism of HSP27 atheroprotection by exploring if the presence of SR-A is required to facilitate its salutary effects. Methods/Results: rHSP27 treatment of bone-marrow derived macrophages from ApoE -/- mice reduced acLDL uptake (38%, p&lt;0.05) using flow cytometry - effects not observed (p=0.171) in cells from ApoE -/- SR-A -/- mice. rHSP27 reduced acLDL binding (38%, p&lt;0.01) and uptake (34%, p&lt;0.01) in THP-1 macrophages associated with downregulation of SR-A transcript (46%, p&lt;0.01) and total protein levels (63%, p&lt;0.001). Flow cytometry and confocal microscopy revealed reduction in SR-A expression at the cell surface. To explore the signaling involved, NF-kB activation was assessed in THP-1 cells stably transfected with an NF-kB reporter. rHSP27 increased NF-kB activity (7 fold, p&lt;0.001) and induced translocation of the NF-kB p65 subunit from the cytosol to the nucleus - effects that were inhibited (p&lt;0.05) by the NF-kB antagonist, BAY. Addition of BAY inhibited the effect of rHSP27 on SR-A gene and protein expression and acLDL uptake (p&lt;0.05). To explore the importance of SR-A in HSP27 atheroprotection, rHSP25 (mouse homologue) or PBS was administered to ApoE -/- and ApoE -/- SR-A -/- mice for 3 weeks on a high fat diet. Treatment of ApoE -/- mice revealed reductions of en face aortic lesions (39%, p&lt;0.001), SR-A expression in the intima, and total serum cholesterol levels (32%, p&lt;0.01). In the absence of SR-A, HSP27 atheroprotection was abolished (p=0.79) despite similar decreases in serum cholesterol levels (25%, p&lt;0.05). Conclusions: Extracellular HSP27 inhibits foam cells by downregulating SR-A expression via an NF-κB-dependent mechanism. HSP27 atheroprotection requires the presence of SR-A expression in vivo. In the absence of SR-A, loss of atheroprotection occurs despite reductions in total serum cholesterol levels, suggesting SR-A as a novel target for HSP27 in the vessel wall.

Abstract 549: Extracellular Heat Shock Protein 27 Signals Through NF-κB to Favorably Modulate Macrophage Inflammation

Background: Previously we demonstrated that recombinant Heat Shock Protein 27 (rHSP27) reduces atherosclerotic lesion formation when administered to ApoE -/- mice. In addition, administration of rHSP27 to ApoE -/- mice with established atherosclerotic lesions halts lesion progression and is associated with lesion modifications that are consistent with resilience to plaque rupture. However, the mechanism(s) for these therapeutic effects remain elusive. Objective: To determine whether rHSP27 favorably modulates macrophage inflammation by focusing on NF-kB signaling. Methods/Results: Activation of the NF-kB signaling pathway by rHSP27 was observed in peritoneal macrophages from ApoE -/- mice. Treatment with rHSP27 for 30 minutes activated the translocation of the NF-kB p65 subunit from the cytosol to the nucleus as observed by immunolabeling. A dose-dependant increase in rHSP27 mediated NF-kB activation was observed in RAW 264.7 macrophages stably transfected with an NF-kB inducible reporter gene (15 fold; p&lt;0.05). The use of an N-terminal deletion mutant of rHSP27, rC1, at equimolar concentrations did not induce NF-kB activation, demonstrating specificity of the full-length protein. In addition, NF-kB inhibitors (BAY 11-7082 and MG-132) attenuated the induction of the NF-kB reporter gene by rHSP27, thereby implicating the involvement of IkBa phosphorylation and degradation by the proteasome. A consequence of rHSP27 signaling in macrophages was the up-regulation of the transcript for the haematopoietic growth factor/regulator, GM-CSF (300 fold; p&lt;0.05) and subsequently its secretion (400 fold, p&lt;0.05). In the presence of BAY, GM-CSF expression was inhibited suggesting the involvement of NF-kB. Conclusions: rHSP27 promotes the nuclear translocation and activation of NF-kB in macrophages which results in the up-regulation of GM-CSF, a haematopoietic growth factor/regulator that may be responsible for the observed therapeutic effects of rHSP27 in vivo. Current studies are testing rHSP27 therapy in ApoE -/- mice deficient in GM-CSF in order to evaluate the importance of macrophage modulation for the beneficial affects of rHSP27 in atherogenesis.

Abstract 348: Extracellular heat shock protein 90 (eHsp90) initiates EMT events and enhances cell motility in prostate cancer

Abstract Prostate Cancer (PCa) is the most commonly diagnosed malignancy in men, responsible for the second highest lethality. Although early cancer detection and treatment is often curative, subsequent metastatic spread of tumor cells renders the disease incurable. Activation of the epithelial to mesenchymal transition (EMT) genetic program is considered a key event contributing to metastatic potential and subsequent lethality. Although targeting EMT may be a promising approach for improving treatment outcome, the main, potentially druggable, targets driving this pathway in PCa remain poorly defined. Multiple reports indicate that extracellular Hsp90 (eHsp90) promotes cell motility, invasion, and metastasis in a number of cancer cell types. Intriguingly, eHsp90 has been preferentially detected in the serum or plasma from aggressive cancers. Its presence in the serum of metastatic PCa patients implicates a potential causative role in PCa. Therefore, our objective was to investigate whether eHsp90 may influence EMT events, thereby influencing the metastatic potential of PCa. We now report that paired sets of differentially aggressive PCa tumor cell lines exhibited higher expression of eHsp90. Functionally, exposure of PCa cells to eHsp90 elicted a significant increase in cell motility. Inversely, blockade of eHsp90 attenuated the activation of several pro-motility effectors, concomitant with the significant suppression of cell migration. These events were associated with a profound alteration of cellular morphology towards a mesenchymal phenotype. Changes in cell phenotype were coupled with a loss and redistribution of the EMT suppressive gatekeeper protein E-cadherin. eHsp90 was also found to impact on the localization of other junctional proteins known to maintain a cuboidal epithelial phenotype. Finally, eHsp90 was found to transcriptionally upregulate several of the established transcriptional factors known to initiate EMT events. Confirmation of the EMT inducing ability of eHsp90 was determined by focused quantitative real time EMT PCR arrays. Our results therefore highlight a novel role for eHsp90 as a central regulator of EMT events in PCa. Our findings further support the premise that therapeutic targeting of eHsp90 may have the potential to suppress EMT activation and diminish the dissemination and metastatic propensity of PCa tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 348. doi:1538-7445.AM2012-348

Heat Stress and Cardiovascular, Hormonal, and Heat Shock Proteins in Humans

Conditions such as osteoarthritis, obesity, and spinal cord injury limit the ability of patients to exercise, preventing them from experiencing many well-documented physiologic stressors. Recent evidence indicates that some of these stressors might derive from exercise-induced body temperature increases. To determine whether whole-body heat stress without exercise triggers cardiovascular, hormonal, and extracellular protein responses of exercise. Randomized controlled trial. University research laboratory. Twenty-five young, healthy adults (13 men, 12 women; age = 22.1 ± 2.4 years, height = 175.2 ± 11.6 cm, mass = 69.4 ± 14.8 kg, body mass index = 22.6 ± 4.0) volunteered. Participants sat in a heat stress chamber with heat (73°C) and without heat (26°C) stress for 30 minutes on separate days. We obtained blood samples from a subset of 13 participants (7 men, 6 women) before and after exposure to heat stress. Extracellular heat shock protein (HSP72) and catecholamine plasma concentration, heart rate, blood pressure, and heat perception. After 30 minutes of heat stress, body temperature measured via rectal sensor increased by 0.8°C. Heart rate increased linearly to 131.4 ± 22.4 beats per minute (F₆,₂₄ = 186, P < .001) and systolic and diastolic blood pressure decreased by 16 mm Hg (F₆,₂₄ = 10.1, P < .001) and 5 mm Hg (F₆,₂₄ = 5.4, P < .001), respectively. Norepinephrine (F₁,₁₂ = 12.1, P = .004) and prolactin (F₁,₁₂ = 30.2, P < .001) increased in the plasma (58% and 285%, respectively) (P < .05). The HSP72 (F₁,₁₂ = 44.7, P < .001) level increased with heat stress by 48.7% ± 53.9%. No cardiovascular or blood variables showed changes during the control trials (quiet sitting in the heat chamber with no heat stress), resulting in differences between heat and control trials. We found that whole-body heat stress triggers some of the physiologic responses observed with exercise. Future studies are necessary to investigate whether carefully prescribed heat stress constitutes a method to augment or supplement exercise.

Extracellular heat shock protein 72 protects schwann cells from hydrogen peroxide‐induced apoptosis

The cytoprotective role of extracellular heat shock protein (Hsp) 72 has been demonstrated in various cell types, including neuronal cells; however, few studies have investigated the actual role of Hsp72 in the survival of Schwann cells (SCs). In the present study, we investigated the effect of exogenous Hsp72 on Schwann cell apoptotic cell death induced by H₂O₂. We determined that extracellular exposure to Hsp72 reduced cell death in rat SCs in a dose-dependent manner, with the protection resulting from downregulation of apoptosis induced by H₂O₂ (as shown by TUNEL and annexin V flow cytometry analyses). Moreover, we observed that Hsp72 suppressed caspase-3 and -9 activation induced by H₂O₂. This was accompanied by upregulation of the antiapoptotic protein Bcl-2. These findings indicate that extracellular Hsp72 can afford neuroprotection to peripheral nerves via its ability to inhibit Schwann cell apoptosis and diminish oxidative stress-mediated injuries.

Stress-induced facilitation of host response to bacterial challenge in F344 rats is dependent on extracellular heat shock protein 72 and independent of alpha beta T cells

Activation of the in vivo stress response can facilitate antibacterial host defenses. One possible mechanism for this effect is stress-induced release of heat shock protein 72 (Hsp72) into the extracellular environment. Hsp72 is a ubiquitous cellular protein that is up-regulated in response to cellular stress, and modulates various aspects of immune function including macrophage inflammatory/bactericidal responses and T-cell function when found in the extracellular environment. The current study tested the hypothesis that in vivo extracellular Hsp72 (eHsp72) at the site of inflammation contributes to stress-induced restricted development of bacteria, and facilitated recovery from bacteria-induced inflammation, and that this effect is independent of alpha beta (αβ) T cells. Male F344 rats were exposed to either inescapable electrical tail-shocks or no stress, and subcutaneously injected with Escherichia coli (ATCC 15746). The role of eHsp72 was investigated by Hsp72-immunoneutralization at the inflammatory site. The potential contribution of T cells was examined by testing male athymic (rnu/rnu) nude rats lacking mature αβ T cells and heterozygous thymic intact control (rnu/+) rats. The results were that stressor exposure increased plasma concentrations of eHsp72 and facilitated recovery from bacterial inflammation. Immunoneutralization of eHsp72 at the inflammatory site attenuated this effect. Stressor exposure impacted bacterial inflammation and eHsp72 equally in both athymic and intact control rats. These results support the hypothesis that eHsp72 at the site of inflammation, and not αβ T cells, contributes to the effect of stressor exposure on subcutaneous bacterial inflammation.

Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1) expression under heat stress: influence of exercise intensity

Extracellular heat-shock protein 72 (eHsp72) expression during exercise-heat stress is suggested to increase with the level of hyperthermia attained, independent of the rate of heat storage. This study examined the influence of exercise at various intensities to elucidate this relationship, and investigated the association between eHsp72 and eHsp27. Sixteen male subjects cycled to exhaustion at 60% and 75% of maximal oxygen uptake in hot conditions (40°C, 50% RH). Core temperature, heart rate, oxidative stress, and blood lactate and glucose levels were measured to determine the predictor variables associated with eHsp expression. At exhaustion, heart rate exceeded 96% of maximum in both conditions. Core temperature reached 39.7°C in the 60% trial (58.9min) and 39.0°C in the 75% trial (27.2min) (P < 0.001). The rate of rise in core temperature was 2.1°Ch(-1) greater in the 75% trial than in the 60% trial (P < 0.001). A significant increase and correlation was observed between eHsp72 and eHsp27 concentrations at exhaustion (P < 0.005). eHsp72 was highly correlated with the core temperature attained (60% trial) and the rate of increase in core temperature (75% trial; P < 0.05). However, no common predictor variable was associated with the expression of both eHsps. The similarity in expression of eHsp72 and eHsp27 during moderate- and high-intensity exercise may relate to the duration (i.e., core temperature attained) and intensity (i.e., rate of increase in core temperature) of exercise. Thus, the immuno-inflammatory release of eHsp72 and eHsp27 in response to exercise in the heat may be duration and intensity dependent.

Plasma heat shock protein 72 as a biomarker of sarcopenia in elderly people

Sarcopenia is a geriatric syndrome in which there is a decrease of muscle mass and strength with aging. In age-related loss of muscle strength, there are numerous observations supporting the assertion that neural factors mediate muscle strength. A possible contributing cause may be that aging changes systemic extracellular heat shock protein (eHsp)72 activity. The present study was designed to assess the plasma levels of eHsp72 in elderly people and to investigate its potential interaction with components of sarcopenia. A total of 665 men and women participated in an official medical health examination and an integrated health examination, including psychological and physical fitness tests. Blood samples were assayed for levels of plasma Hsp72, serum C-reactive protein, interleukin 6, tumor necrosis factor α, and regular biomedical parameters. We found that higher Hsp72 in plasma is associated with lower muscle mass, weaker grip strength, and slower walking speed, and may be a potential biomarker of sarcopenia in elderly people. This finding was supported by other results in the present study: (1) older age and shrinking body and lower hemoglobin levels, all of which characterize sarcopenia, were related to higher eHsp72 tertiles and (2) the ORs of the highest tertile of eHsp72 for the lowest tertiles of muscle mass, grip strength, and walking speed were 2.7, 2.6, and 1.8, respectively. These ORs were independent of age, sex, and the incidence of related diseases. Our results would reveal that eHsp72 in plasma is linked to sarcopenia factors and is a potential biomarker or predictor of sarcopenia.