Extracellular Hsp70 Research Articles

Targeting Extracellular Heat Shock Protein 70 Ameliorates Doxorubicin-Induced Heart Failure Through Resolution of Toll-Like Receptor 2-Mediated Myocardial Inflammation.

BackgroundHeart failure (HF) is one of the most significant causes of morbidity and mortality for the cardiovascular risk population. We found previously that extracellular HSP70 (heat shock protein) is an important trigger in cardiac hypertrophy and fibrosis, which are associated with the development of heart dysfunction. However, the potential role of HSP70 in response to HF and whether it could be a target for the therapy of HF remain unknown.Methods and ResultsAn HF mouse model was generated by a single IP injection of doxorubicin at a dose of 15 mg/kg. Ten days later, these mice were treated with an HSP70 neutralizing antibody for 5 times. We observed that doxorubicin treatment increased circulating HSP70 and expression of HSP70 in myocardium and promoted its extracellular release in the heart. Blocking extracellular HSP70 activity by its antibody significantly ameliorated doxorubicin‐induced left ventricular dilation and dysfunction, which was accompanied by a significant inhibition of cardiac fibrosis. The cardioprotective effect of the anti‐HSP70 antibody was largely attributed to its ability to promote the resolution of myocardial inflammation, as evidenced by its suppression of the toll‐like receptor 2–associated signaling cascade and modulation of the intracellular distribution of the p50 and p65 subunits of nuclear factor‐κB.ConclusionsExtracellular HSP70 serves as a noninfectious inflammatory factor in the development of HF, and blocking extracellular HSP70 activity may provide potential therapeutic benefits for the treatment of HF.

Pro-inflammatory effects of extracellular Hsp70 on NCI-H292 human bronchial epithelial cell line.

Extracellular Hsp70 (eHsp70) exerts its biological actions via Toll-like receptors 2 and 4, and is increased in sera of chronic obstructive pulmonary disease (COPD) patients. The aim of this study was to explore the pro-inflammatory effects and cytotoxicity of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on NCI-H292 airway epithelial cells. NCI-H292 cells were treated with recombinant human Hsp70 protein (rhHsp70), LPS, LTA and their combinations for 4, 12, 24 and 48hours. IL-6, IL-8 and TNF-α levels were measured by an ELISA method. Cell viability was determined by the MTS method, and caspase-3/7, caspase-8 and caspase-9 assays. rhHsp70 induced secretion of IL-6 and IL-8 in a concentration- and time-dependent manner, with the highest secretion at 24hours. rhHsp70 combined with LTA had antagonistic and with LPS synergistic effect on IL-6 secretion, while the interactions between rhHsp70 and LPS or LTA on IL-8 were synergistic. TNF-α was not detected in the applied conditions. rhHsp70, LPS or LTA did not affect cell viability, and rhHsp70 even suppressed caspase-3/7 activities. We suggest that pro-inflammatory effects of eHsp70, together with other damaging molecules and/or COPD risk factors, might contribute to the aggravation of chronic inflammation in human bronchial epithelium.

Serum HSP70 level in patients with endometrial cancer with and without diabetes

Diabetes mellitus (DM) is associated with an increased risk of endometrial carcinoma (EC). Heat shock proteins have a role in the modulation of both diseases. The aim of this study was to investigate extracellular HSP70 (eHSP70) level alternations in patients with two different types of EC (endometrioid and non-endometrioid) with and without type 2 diabetes. In a case–control study, 88 participants were enrolled in four groups including: 18 EC patients with DM, 19 EC patients without DM, 29 patients with DM, and 22 healthy individuals. Blood samples were taken before surgery in cancer patients. Estradiol, eHSP70, sex hormone-binding globulin (SHBG), FBS, and HbA1c were assessed. Serum HSP70 level was higher in patients with diabetes (52.24 ± 14.2 ng/ml) compared to healthy controls (39.04 ± 6.96) (p < .05). It was lower in EC (26.05 ± 12.28) compared to healthy controls (39.04 ± 6.96) (p < .05). eHSP70 was also lower in endometrioid-type carcinoma (22.57 ± 11) compared to non-endometrioid type (31.55 ± 12.38) (p < .05). Further analysis showed increased levels of eHSP70 in patients having both endometrioid-type carcinoma and diabetes (27.23 ± 11.41) compared to the same patients without DM (17.08 ± 7.78) (p < .05). Presence of diabetes in patients with endometrioid type carcinoma resulted in an increase in eHSP70 approaching the level of eHSP70 in patients with non-endometrioid histology.

Co-chaperones TIMP2 and AHA1 Competitively Regulate Extracellular HSP90:Client MMP2 Activity and Matrix Proteolysis

The extracellular molecular chaperone heat shock protein 90 (eHSP90) stabilizes protease client the matrix metalloproteinase 2 (MMP2), leading to tumor cell invasion. Although co-chaperones are critical modulators of intracellular HSP90:client function, how the eHSP90:MMP2 complex is regulated remains speculative. Here, we report that the tissue inhibitor of metalloproteinases-2 (TIMP2) is a stress-inducible extracellular co-chaperone that binds to eHSP90, increases eHSP90 binding to ATP, and inhibits its ATPase activity. In addition to disrupting the eHSP90:MMP2 complex and terminally inactivating MMP2, TIMP2 loads the client to eHSP90, keeping the protease in a transient inhibitory state. Secreted activating co-chaperone AHA1 displaces TIMP2 from the complex, providing a "reactivating" mechanism for MMP2. Gene knockout or blocking antibodies targeting TIMP2 and AHA1 released by HT1080 cancer cells modify their gelatinolytic activity. Our data suggest that TIMP2 and AHA1 co-chaperones function as a molecular switch that determines the inhibition and reactivation of the eHSP90 client protein MMP2.

The Relationship of Serum Heat Shock Protein 70 Antibody Levels with the Inflammatory Factors and Serum Uric Acid Levels in Hemodialysis Patients

Background: Heat shock proteins are extracellular inflammatory intermediaries and intracellular cytoprotective molecules. Despite many studies on intracellular HSP70, the clinical association between inflammatory biomarkers and extracellular HSP70 antibody (anti-HSP70) levels is not well-studied. Objectives: The aim of this study was to investigate whether raised serum anti-HSP70 in hemodialysis (HD) patients are related to levels of serum inflammatory markers and uric acid, as the key players, in the pathogenesis of the disease. Methods: This cross-sectional study was carried out from January 2018 to July 2018, on patients referred by the nephrologists from the central outpatient dialysis center of a governmental university-affiliated hospital, in Ahvaz, Iran. Ninety HD patients enrolled based on the inclusion and exclusion criteria. Blood samples were collected directly from the arteriovenous fistula before a routine HD session. The circulating levels of anti-HSP70, highly sensitive C-reactive protein (hs-CRP), Interleukin-6 (IL-6), and endotoxin were measured by Enzyme-linked immunosorbent assay (ELISA). Results: The univariate regression analysis revealed a significant association between serum anti-HSP70 level and diabetes mellitus, hypertension, hemodialysis vintage, uric acid, hs-CRP, and IL-6 (P < 0.05). In a multiple regression model, after adjusting for confounders, the association between circulating anti-HSP70 and uric acid (B = 16.92, P = 0.001), hs-CRP (B = 11.77, P = 0.002), IL-6 (B = 2.87, P = 0.002), endotoxin (B = 0.14, P = 0.005), and hemodialysis vintage (B = 43.76, P = 0.002) was significant. Conclusions: Our findings suggest that the development of the excessive systemic inflammatory response and uric acid contribute to a higher serum anti-HSP70 leading to cardiovascular disease.

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status

IntroductionThe 72kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72). ObjectiveWe explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss. MethodsFemale 90 day-old Wistar rats (n=32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124dB SPL for 2h) and 14days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit. ResultsWe found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold. ConclusionsOur data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Effects of extracellular Hsp70 and cigarette smoke on differentiated THP-1 cells and human monocyte-derived macrophages

Extracellular Hsp70 (eHsp70) can act as pro-inflammatory mediator and is elevated in blood of chronic obstructive pulmonary disease (COPD) patients. Most of those patients are smokers, and it was suggested previously that cigarette smoke might induce Hsp70 secretion from the circulating cells. Therefore, we aimed to explore inflammation-associated effects of cigarette smoke extract (CSE) and its combinations with eHsp70 in monocyte-derived macrophages (MDMs) and THP-1 cell line, used as systemic component models of COPD. We hypothesized that eHsp70 induces inflammation, but that it can also modulate cigarette smoke extract (CSE)-stimulated inflammatory responses.We assessed IL-8 secretion, TLR2, TLR4 and Hsp70 expressions, MAPKs and NF-κB activation, and cytotoxicity after treating the cells with CSE (2.5 and 5%) and its combinations with low-endotoxin recombinant human (rh) Hsp70, used to mimic eHsp70 effects.CSE induced IL-8 secretion from both cell types, but its combinations with rhHsp70 increased IL-8 release compared to CSE alone only from MDMs. In THP-1, combinations of rhHsp70 with 2.5% CSE induced TLR2 and TLR4 mRNA, while 5% CSE decreased TLR2 expression. In MDMs, CSE alone attenuated TLR2, while rhHsp70 increased TLR2 and lowered TLR4 gene expression. Hsp70 mRNA expression was suppressed in THP-1 with rhHsp70 and CSE; however, the same treatments increased its level in MDMs. CSE had cytotoxic effect only on MDMs, but cytotoxicity was reduced in co-treatments with rhHsp70, which also triggered apoptosis. CSE and rhHsp70 activated p38 and JNK, while ERK was activated only by rhHsp70 in MDMs. In THP-1, 2.5% CSE activated ERK, and 5% CSE activated p38. Inhibition of NF-κB and JNK in MDMs, and ERK and JNK in THP-1 cells, attenuated IL-8 release after rhHsp70 treatment.In conclusion, rhHsp70 provoked pro-inflammatory effects and could also modulate inflammatory response to CSE on protein and gene expression levels in THP-1 cells and MDMs, which suggests that eHsp70 might be implicated in systemic inflammation induced by cigarette smoke.

Multifunctional Magnetic Nanoplatform Eliminates Cancer Stem Cells via Inhibiting the Secretion of Extracellular Heat Shock Protein 90.

Cancer stem cells (CSCs) are responsible for malignant tumor initiation, recurrences, and metastasis. Therefore, targeting CSCs is a promising strategy for the development of cancer therapies. A big challenge for CSC-based cancer therapy is the overexpression of therapeutic stress protein, heat shock protein 90 (Hsp90), which protects CSCs from further therapeutic-induced damage, leading to the failure of treatment. Thus, efficient strategies to target CSCs are urgently needed for cancer therapy. To this end, a multifunctional nanoparticle (MNP) for CSC-based combined thermotherapy and chemotherapy is reported. This strategy dramatically suppresses tumor growth in breast CSC xenograft-bearing mice. Furthermore, a new mechanism is present that the MNP exerts its striking effects on CSCs by inhibiting the secretion of extracellular Hsp90 (eHsp90), resulting in the interruption of several key signaling pathways. These findings open new perspectives on the use of an MNP for effective CSC-based cancer treatment by inhibiting the function of eHsp90.

Role of heat shock protein and cytokine expression as markers of clinical outcomes with glutamine-supplemented parenteral nutrition in surgical ICU patients

Nutrients, such as glutamine (GLN), have been shown to effect levels of a family of protective proteins termed heat shock proteins (HSPs) in experimental and clinical critical illness. HSPs are believed to serve as extracellular inflammatory messengers and intracellular cytoprotective molecules. Extracellular HSP70 (eHSP70) has been termed a chaperokine due to ability to modulate the immune response. Altered levels of eHSP70 are associated with various disease states. Larger clinical trial data on GLN effect on eHSP expression and eHSP70's association with inflammatory mediators and clinical outcomes in critical illness are limited. Explore effect of longitudinal change in serum eHSP70, eHSP27 and inflammatory cytokine levels on clinical outcomes such as pneumonia and mortality in adult surgical intensive care unit (SICU) patients. Further, evaluate effect of parenteral nutrition (PN) supplemented with GLN (GLN-PN) versus GLN-free, standard PN (STD-PN) on serum eHSP70 and eHSP27 concentrations. Secondary observational analysis of a multicenter clinical trial in 150 adults after cardiac, vascular, or gastrointestinal surgery requiring PN support and SICU care conducted at five academic medical centers. Patients received isocaloric, isonitrogenous PN, with or without GLN dipeptide. Serum eHSP70 and eHSP27, interleukin-6 (IL-6), and 8 (IL-8) concentrations were analyzed in patient serum at baseline (prior to study PN) and over 28 days of follow up. eHSP70 declined over time in survivors during 28 days follow-up, but non-survivors had significantly higher eHSP70 concentrations compared to survivors. In patients developing pneumonia, eHSP70, eHSP27, IL-8, and IL-6 were significantly elevated. Adjusted relative risk for hospital mortality was reduced 75% (RR=0.25, p=0.001) for SICU patients with a faster decline in eHSP70. The area under the receiver operating characteristic curve was 0.85 (95% CI: 0.76 to 0.94) for the final model suggesting excellent discrimination between SICU survivors and non-survivors. GLN-PN did not alter eHSP70 or eHSP27 serum concentrations over time compared to STD-PN. Our results suggest that serum HSP70 concentration may be an important marker for severity of illness and likelihood of recovery in the SICU. GLN-supplemented-PN did not increase eHSP70.

Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells.

The extracellular heat shock protein 90 (Hsp90) is known to participate in cell migration and invasion. Recently, we have shown that cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and anchoring of extracellular Hsp90 to the plasma membrane, but the biological relevance of this finding was unclear. Here, we demonstrated that the digestion of heparan sulfate (HS) moieties of HSPGs with a heparinase I/III blend and the metabolic inhibition of the sulfation of HS chains by sodium chlorate considerably impair the migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells stimulated by extracellular native Hsp90. Heparin, a polysaccharide closely related to HS, also reduced the Hsp90-stimulated migration and invasion of cells. Phorbol 12-myristate 13-acetate, an intracellular inducer of cell motility bypassing the ligand activation of receptors, restored the basal migration of heparinase- and chlorate-treated cells almost to the control level, suggesting that the cell motility machinery was insignificantly affected in cells with degraded and undersulfated HS chains. On the other hand, the downstream phosphorylation of AKT in response to extracellular Hsp90 was substantially impaired in heparinase- and chlorate-treated cells as compared to untreated cells. Taken together, our results demonstrated for the first time that cell surface HSPGs play an important role in the migration and invasion of cancer cells stimulated by extracellular Hsp90 and that plasma membrane-associated HSPGs are required for the efficient transmission of signal from extracellular Hsp90 into the cell.

Effects of severe whole-body hyperthermia on ovarian hormone and extracellular Hsp72 responses in young adult women

Background: Although acute thermal stress appears to be one of the most effective stressors that increase the intra- and extracellular concentrations of heat shock protein 72 (Hsp72), 17β-estradiol has been shown to inhibit heat-induced Hsp72 expression.Materials and Methods: To determine whether severe whole-body hyperthermia (increase in rectal temperature up to 39.5 °C) induced by lower-body heating is a sufficient stimulus to modulate hormonal (17β-estradiol, progesterone, prolactin, epinephrine, and norepinephrine) and extracellular Hsp72 responses, we investigated young adult women (21 ± 1 yr).Results and Conclusions: In the present study, we show that a severe whole-body hyperthermia (increase in rectal temperature of approximately 2.6 °C and heart rate of approximately 80 bpm from baseline) was sufficient to increase 17β-estradiol, progesterone, and prolactin and catecholamine norepinephrine concentration. Moreover, we show that the concentration of extracellular Hsp72 and catecholamine epinephrine were not affected by severe whole-body hyperthermia in young adult women. From the functional point of view, expression of ovarian hormones induced by passive heat stress may have therapeutic potential for young adult women in, for example, estrogen treatment and overall women’s health.

Extracellular Hsp70 modulates the inflammatory response of cigarette smoke extract in NCI-H292 cells.

What is the central question of this study? Does extracellular heat shock protein 70 (eHsp70) alter cigarette smoke extract (CSE)-induced inflammatory responses in NCI-H292 bronchial epithelial cells? What is the main finding and its importance? eHsp70 modulates inflammatory responses and TLR2, TLR4 and Hsp70 gene expression, and protects NCI-H292 cells against CSE-induced cytotoxicity. eHsp70 might be implicated in development of inflammatory diseases affected by cigarette smoke, such as COPD. One of the major risk factors for development of chronic obstructive pulmonary disease (COPD) is cigarette smoke. Extracellular Hsp70 (eHsp70) is increased in sera of COPD patients, and can act as damage-associated molecular pattern (DAMP). In this study, we explored inflammatory parameters (cytokine concentrations, Toll-like receptor (TLR) 2 and 4 and Hsp70 expression, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) activation, and cytotoxicity) after exposure of bronchial-epithelial NCI-H292 cells to cigarette smoke extract (CSE) alone (2.5 and 15%) and in combinations with recombinant human (rh) Hsp70 (0.3, 1 and 3μgml-1 ). We applied specific MAPKs, NF-κB and Hsp70 inhibitors to elucidate rhHsp70 inflammation-associated responses. CSE alone and combinations of 15% CSE with rhHsp70 stimulated IL-1α, IL-6 and IL-8 release. However, rhHsp70 applied with 2.5% CSE decreased secretion of cytokines indicating antagonistic effects. Individual and combined treatments with 2.5% CSE suppressed TLR2 expression. CSE at 15% induced TLR2 and TLR4 gene expression, whereas rhHsp70 abolished that effect. rhHsp70 and 15% CSE alone reduced, while their combination increased, intracellular Hsp70 mRNA level. CSE alone and in combination with rhHsp70 activated extracellular signal-regulated kinase and p38 MAPKs, while inhibition of MAPKs, NF-κB and Hsp70 attenuated IL-6 and IL-8 secretion. CSE at 15% reduced cell viability and induced apoptosis, as shown by MTS and caspases-3/7 assays. CSE at 2.5% alone stimulated lactate dehydrogenase release, but cellular membrane integrity remained intact in co-treatments with rhHsp70. rhHsp70 might modulate the inflammatory response of CSE and could also protect NCI-H292 cells against CSE cytotoxicity. Those effects are implemented via MAPK and NF-κB signalling pathways.

Pro-inflammatory effects of extracellular Hsp70 and cigarette smoke in primary airway epithelial cells from COPD patients

Extracellular Hsp70 (eHsp70) can activate immune cells via Toll-like receptors (TLR) 2 and 4, and induce cytokine synthesis. The aim of this study was to explore inflammation-associated effects of eHsp70 alone and in combination with cigarette smoke extract (CSE) in primary bronchial epithelial cells.We assessed IL-6 and IL-8 concentrations, TLR2, TLR4 and Hsp70 mRNA expressions, and mitogen-activated protein kinases (MAPKs) activation induced by recombinant human (rh) Hsp70, CSE or their combinations in normal human bronchial epithelial cells (NHBE) obtained commercially, and primary bronchial epithelial cells isolated from non-COPD lung donors (PBEC) or COPD patients (PBEC COPD).Baseline levels of IL-6 and IL-8 were significantly higher in PBEC COPD than in non-COPD PBECs. Upon rhHsp70 stimulation, IL-6 and IL-8 were significantly increased, with the strongest response in COPD-derived PBECs. CSE alone elevated cytokine secretion in all examined cells. rhHsp70 and CSE had antagonistic interactions on IL-8 release in PBECs from COPD patients, while the addition of rhHsp70 further increased CSE-induced IL-6 secretion in NHBE cells. rhHsp70 and CSE alone decreased TLR2 and TLR4 mRNA expression in COPD-derived PBECs. In non-COPD PBECs, combined treatments decreased only TLR2 mRNA expression. Hsp70 mRNA expression, as indicator of intracellular Hsp70, which may have anti-inflammatory effects, was reduced in COPD-derived cells upon exposure to CSE and rhHsp70 alone, but not with their combinations. Contrary to this, in PBECs from lung donors only combined treatments supressed Hsp70 gene expression. CSE activated JNK and p38 MAPKs, while rhHsp70 increased activation of c-Jun kinase in NHBE cells.Collectively, both eHsp70 and CSE induce pro-inflammatory responses in PBECs from non-COPD as well as COPD donors, but in combination antagonistic effects were observed in COPD-derived cells. These effects may be related to the regulation of TLR2/4 and might lead to modulation of inflammation with possible deleterious consequences for COPD patients.

Association of extracellular heat shock protein 70 and insulin resistance in type 2 diabetes; independent of obesity and C-reactive protein

Despite few studies on intracellular heat shock protein70, the clinical association between insulin resistance and extracellular heat shock protein70 (eHSP70) is not well studied. In the current study, we examined the association between homeostatic model assessment-insulin resistance (HOMA-IR) and eHSP70 in patients with type 2 diabetes (T2DM) and healthy controls. A total of 145 patients with T2DM and 41 matched healthy controls were selected. Patients and controls were divided based on waist circumference (WC) to two groups, and eHSP70 was compared between them. The association between HOMA-IR and eHSP70 was examined using regression models adjusted for age, high-sensitive C-reactive protein (hs-CRP), and central obesity as confounding factors. While eHSP70 and hs-CRP were significantly correlated with HOMA-IR in patients with T2DM (p = 0.032, 0.025, respectively), there was no correlation between eHSP70 and HOMA-IR in the control group. Extracellular HSP70 and hs-CRP were not correlated in healthy controls. But a significant association appeared between eHSP70 and hs-CRP in patients with T2DM (p = 0.05). Both BMI and WC were not correlated with eHSP70 in both groups. Extracellular HSP70 was positively associated with HOMA-IR in patients with T2DM, independent from hs-CRP and obesity. We also showed eHSP70 levels remained unchanged through increase in BMI or WC in patients with T2D and in healthy controls. Our findings suggest that eHSP70 may contribute to the pathogenesis of T2DM by increasing insulin resistance.

Detectable levels of eHSP72 in plasma are associated with physical activity and antioxidant enzyme activity levels in hypertensive subjects

Previous studies reported that extracellular HSP72 (eHSP72) correlates with poor prognosis, markers of vascular dysfunction, and the severity of cardiovascular diseases, associated with a systemic oxidative and inflammatory profile. On the other hand, eHSP72 may represent immune-regulatory signaling that is related to exercise benefits, but the association between physical activity levels and eHSP72 levels is not established. Thus, since regular physical activity may avoid oxidative stress and inflammation, we investigate whether detectable levels of eHSP72 in plasma are associated with physical activity and antioxidant enzyme activity levels in hypertensive subjects. Physical activity levels of hypertensive subjects (n = 140) were measured by tri-axial movement sensor pedometer for 24h during 5 consecutive days. One day after, blood was collected into heparinized tubes for oxidative stress analyses (catalase-CAT and superoxide dismutase-SOD activities and malondialdehyde levels) or in disodium EDTA tubes for eHSP72 assays. Thus, hypertensive subjects were classified as physically inactive (< 10,000 footsteps/day) or active (> than 10,000 footsteps/day) and according detectable or not detectable eHSP72 levels in plasma, performing the inactive/eHSP72-, active/eHSP72-, inactive/eHSP72+, and active/eHSP72+ groups. We found that detectable levels of eHSP72 in plasma were associated with physical activity levels and low oxidative stress profile (Higher CAT and SOD activities and low malondialdehyde levels). eHSP72 levels can be used as a biomarker of the amount of physical activity necessary to improve antioxidant defense and thus cardiovascular health in hypertensive subjects.

Abstract A46: Hsp90 regulates Twist1 expression through STAT3 to induce epithelial-mesenchymal transition in ovarian cancer

Abstract Background: Metastatic disease is the leading cause of death from ovarian cancer and its underlying mechanisms are poorly understood. Twist1 is a key driver of epithelial-mesenchymal transition (EMT) and metastasis. Understanding the function and regulation of Twist1 is a vital step in the development of effective treatments for metastatic ovarian cancer. Heat shock protein 90 (Hsp90) is a molecular chaperone that modulates multiple signaling networks, and recent studies have highlighted the roles of extracellular Hsp90 in promoting metastasis in cancer. The roles of Hsp90 in regulating intracellular pathways leading to EMT and metastasis remain largely unknown. Objective: In our study, we tested the hypothesis that Hsp90 promotes EMT in ovarian cancer through the regulation of Twist1 at the transcriptional level. Methods: We treated A2780 and SKOV3 with a Hsp90-specific inhibitor, 17-allylamino-17 demethoxygeldanamycin (17-AAG). The effects of Hsp90 inhibition on Twist1 mRNA expression and promoter activity were measured using quantitative PCR and luciferase reporter assays, respectively. Proximity ligation assays were performed to visualize the effects of 17-AAG on the interaction between Hsp90 and transcription factors, followed by chromatin immunoprecipitation to measure the binding of transcription factors to the Twist1 promoter. Results: Treatment with 17-AAG significantly downregulated Twist1 expression at the mRNA level in A2780, SKOV3, and three ovarian cancer patient-derived cell lines. Hsp90 overexpression substantially induced Twist1 promoter activity while treatment with 17-AAG significantly decreased the activity. Western blotting and immunofluorescent staining revealed the presence of 4 transcription factors known to be clients of Hsp90 and regulators of Twist1, which are β-catenin, signal transducer and activator of transcription 3 (STAT3), hypoxia-inducible factor 1-alpha (HIF-1α), and HIF-1β. We identified that Hsp90 interacts with β-catenin, STAT3, and HIF-1α in our cell lines. We observed that 17-AAG treatment dramatically impaired Hsp90-STAT3 interaction and the binding of STAT3 to the Twist1 promoter. Inhibition of Hsp90 was also shown to block interleukin-6 (IL-6) and transforming growth factor beta (TGF-β)-induced EMT. Conclusion: Taken together, our findings reveal that STAT3 is dependent on Hsp90 to activate Twist1 expression. Hsp90 plays a critical role in enabling STAT3 to bind to the Twist1 promoter and promote Twist1 transcription leading to EMT. We uncovered a previously unrecognized role of Hsp90, which cooperates with STAT3 in the transcriptional regulation of Twist1 in ovarian cancer cells. Inhibiting Hsp90 using small-molecule inhibitors such as 17-AAG may have potential as a therapeutic strategy to prevent EMT and metastasis in ovarian cancer. Citation Format: Kay Yi Chong, Francesca Garofalo, Oluwagbemisola Madarikan, Nicholas Pitruzzello, Cheng-Hsiu Tsai, Jamie Bingham, Yang Yang-Hartwich. Hsp90 regulates Twist1 expression through STAT3 to induce epithelial-mesenchymal transition in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A46.

Exercise cardiorespiratory and thermoregulatory responses in normoxic, hypoxic, and hot environment following 10-day continuous hypoxic exposure.

We examined the effects of acclimatization to normobaric hypoxia on aerobic performance and exercise thermoregulatory responses under normoxic, hypoxic, and hot conditions. Twelve men performed tests of maximal oxygen uptake (V̇O2max) in normoxic (NOR), hypoxic [HYP; 13.5% fraction of inspired oxygen (FiO2)], and hot (HE; 35°C, 50% relative humidity) conditions in a randomized manner before and after a 10-day continuous normobaric hypoxic exposure [FiO2 = 13.65 (0.35)%, inspired partial pressure of oxygen = 87 (3) mmHg]. The acclimatization protocol included daily exercise [60 min at 50% hypoxia-specific peak power output (Wpeak)]. All maximal tests were preceded by a steady-state exercise (30 min at 40% Wpeak) to assess the sweating response. Hematological data were assessed from venous blood samples obtained before and after acclimatization. V̇o2max increased by 10.7% (P = 0.002) and 7.9% (P = 0.03) from pre-acclimatization to post acclimatization in NOR and HE, respectively, whereas no differences were found in HYP [pre: 39.9 (3.8) vs. post: 39.4 (5.1) ml·kg-1·min-1, P = 1.0]. However, the increase in V̇O2max did not translate into increased Wpeak in either NOR or HE. Maximal heart rate and ventilation remained unchanged following acclimatization. Νo differences were noted in the sweating gain and thresholds independent of the acclimatization or environmental conditions. Hypoxic acclimatization markedly increased hemoglobin (P < 0.001), hematocrit (P < 0.001), and extracellular HSP72 (P = 0.01). These data suggest that 10 days of normobaric hypoxic acclimatization combined with moderate-intensity exercise training improves V̇o2max in NOR and HE, but does not seem to affect exercise performance or thermoregulatory responses in any of the tested environmental conditions.NEW & NOTEWORTHY The potential crossover effect of hypoxic acclimatization on performance in the heat remains unexplored. Here we show that 10-day continuous hypoxic acclimatization combined with moderate-intensity exercise training can increase maximal oxygen uptake in hot conditions.

Heat-induced extracellular HSP72 release is blunted in elderly diabetic people compared with healthy middle-aged and older adults, but it is partially restored by resistance training.

Recent evidence suggests that the anti-inflammatory heat shock response (HSR) is reduced in aging and diabetes. In this study we compared HSR between healthy middle-aged adults, healthy elderly and type 2 diabetic (T2DM) elderly, and tested whether resistance training (RT) could improve the HSR in T2DM group. Thirty sedentary participants volunteered for this study. HSR (assessed as the capacity to export HSP72 during heat stress) was measured in the blood and compared between the groups. HSR was similar between healthy middle-aged and healthy elderly volunteers, but diminished in elderly T2DM (p < 0.001). Hence, T2DM subjects (n = 12) were submitted to a 12-week RT program, because exercise is a physiological HSR inducer. HSR, cytokines, metabolic parameters and visceral adipose tissue (VAT) were measured before and after the RT. Remarkably, VAT was negatively correlated with HSR (r = - 0.49, p < 0.01) while RT improved the HSR and reduced inflammation [TNF-α: from 51.5 ± 9 to 40.7 ± 4 pg/mL and TNF-α/IL-10 ratio: from 1.55 ± 0.3 to 1.16 ± 0.2 (p < 0.001)], without affecting other parameters. All together, these findings confirm the hypothesis that the anti-inflammatory HSR is depressed in elderly diabetic people, but can be partially restored by RT.

Heat Shock Protein Response During a Prolonged Ultraendurance Cycling Event in Hot and Humid Conditions

The heat shock protein (HSP) response is critical to the cellular stress response. Circulating, extracellular stress‐inducible HSP72 is known to respond to exercise‐heat stress, but little is known about other, constitutively expressed and fundamental members of the HSP family. The aim of this study was to investigate the extracellular HSP27, HSP60, and HSP90α response during a prolonged cycling event in hot and humid conditions. Seventy‐six subjects (mean±SD: age, 51.64±10.47 years; height, 176.44±6.85 cm; mass, 86.78±13.93 kg) were recruited at the Hotter'n Hell 100 mile cycling event in Wichita Falls, Texas. Blood and urine samples, as well as data on body weight and gastrointestinal temperature (TGI) were recorded before (pre) and after (post) the event. Plasma was isolated from blood samples, stored at −80°C, and later analyzed with StressMarq Biosciences ELISAs for HSP27, HSP60, and HSP90α concentrations. Student's t‐test was used to test for significant differences of means. TGI significantly increased from pre to post (pre: 37.14±0.55 °C; post: 38.12±0.79 °C; p&lt;0.05). HSP27 and HSP90α were both significantly elevated post race (pre27: 2.73±0.51 ng × mL−1; post27: 3.46±0.73 ng × mL−1; pre90α: 18.03±3.27 ng × mL−1; post90α: 21.53±4.84 ng × mL−1; p &lt; 0.05). However, there was no significant difference in HSP60 plasma concentration from pre to post (pre: 44.05±7.38; post: 43.65±6.81; p=0.77). In conclusion, significant increases in circulating HSP27 and HSP90α suggest that chaperones required for thermotolerance and protein degradation or folding during heat stress increase in extracellular compartments post exercise‐heat stress. However, our data suggest that chaperones specifically involved in mitochondrial protein assembly or molecular danger signals like HSP60 are not elevated in blood fractions. Ongoing work will characterize cellular expression of these proteins in circulating peripheral blood mononuclear cells.Support or Funding InformationNew investigator start‐up, McNair Scholar ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A tumor-targeted Ganetespib-zinc phthalocyanine conjugate for synergistic chemo-photodynamic therapy

Therapeutic effects of photodynamic therapy (PDT) are limited by the selectivity of photosensitizer (PS). Herein, a novel tumor-targeted drug-PS conjugate (Gan-ZnPc) which integrated with zinc phthalocyanine (ZnPc) and Ganetespib has been developed. ZnPc is a promising PS with remarkable photosensitization ability. Ganetespib is a heat shock protein 90 (Hsp90) inhibitor with preferential tumor selectivity and conjugated to ZnPc as a tumor-targeted ligand. The multifunctional small molecule conjugate, Gan-ZnPc, could be bound to extracellular Hsp90 and then selectively internalized into the tumor cells, followed by the generation of abundant intracellular reactive oxygen species (ROS) upon irradiation. Besides, Gan-ZnPc can arrest cell proliferation and induce apoptosis by the inhibition of Hsp90. Herein, with combination of the inhibition of Hsp90 and the generation of cytotoxic ROS, Gan-ZnPc implements tumor selectivity, concentrated PDT and chemotherapy in a synergistic manner, which results in highly effective anti-tumor activity in vitro and in vivo.