Heat Shock Protein 27 Levels Research Articles

Abstract 17586: HSP27 Mediated Atheroprotection Requires GM-CSF Induction: Potential Role in Reverse Cholesterol Transport? (Best of Basic Science Abstract)

Introduction: Previously, we demonstrated that Heat Shock Protein 27 (HSP27) reduces experimental atherogenesis and patients with elevated serum HSP27 levels are relatively protected against future CV events. We previously reported that extracellular HSP27 activates the NF-kB pathway and increases the expression of GM-CSF. While the potential atheroprotective role of GM-CSF remains controversial, clinical reports highlight its effect on reducing serum cholesterol levels. We now hypothesize that GM-CSF plays an important role in HSP27-mediated atheroprotection, potentially by up-regulating reverse cholesterol transport (RCT). Methods / Results: 1. NF-KB signaling in vitro: Treatment of macrophages with recombinant HSP27 (rHSP27) activated NF-kB 15-fold via TLR-4 (NF-kB reporter gene assay; controls: truncated rHSP27 peptide and TLR-4 deficient cells; p<0.05). While rHSP27 substantially induced GM-CSF expression and secretion (300- and 400-fold respectively; p<0.05) both processes were blocked by NF-kB inhibitors. 2. In vivo role of GM-CSF induction in HSP27-mediated atheroprotection: Aortic atherogenesis in apoE null treated with rHSP27 was reduced by an expected 40%, while ApoE null / GM-CSF null did not respond to rHSP27 treatment. Moreover, apoE null / GM-CSF null macrophages expressed ~33% lower levels of the reverse cholesterol transporters, ABC-A1 and ABC-G1. 3. rHSP27 & Reverse Cholesterol Transport: THP-1 macrophages pre-treated with oxLDL and incubated with rHSP27 for 24h showed enhanced mRNA and protein expression of ABCA1 and ABCG1 (p<0.01). In a RCT assay, THP-1 macrophages labeled with NBD-cholesterol and treated with rHSP27 for 24h before being incubated with apoA1 or HDL showed increased cholesterol efflux compared to controls (p<0.0001 for both). Conclusion: HSP27 up regulates expression of GM-CSF that appears to play an important role in RCT-mediated atheroprotection.

Abstract 17326: Clues to Attenuating Plaque Inflammation: Heat Shock Protein-27 Augments Macrophage Histone Deacetylase-6 Activity and Cytoskeletal Migratory Capacity

Introduction: We recently demonstrated that elevated serum levels of Heat Shock Protein 27 (HSP27) are associated with reduced 5-year CV events and that extracellular HSP27 attenuates experimental atherogenesis and lesion progression. Indeed, HSP27 therapy reduces plaque macrophage and cholesterol content and enhances histological features associated with resilience to plaque rupture. Cell motility and adhesion involves dynamic microtubule acetylation/deacetylation, a process regulated by the enzyme histone deacetylase 6 (HDAC6), a major cytoplasmic α-tubulin deacetylase. We hypothesize that HSP27 enhances HDAC6 activity to promote favorable plaque remodeling by enhancing macrophage migration. Methods / Results: 1. The HDAC6 activity of THP-1 macrophages treated with recombinant HSP27 was assessed using a fluorescent plate reader ELISA. In a dose-dependent fashion, rHSP27 treatment resulted in a 100% increase in HDAC-6 activity compared to the control and was independent of HSP27 phosphorylation. Moreover, HDAC-6 activity was enhanced 100% in peritoneal macrophages harvested from rHsp27-treated ApoE-/- vs. control mice. With the addition of cycloheximide HDAC-6 activity was not prolonged, suggesting that HSP27 did not have an effect on the enzymatic half-life. 2. When HSP27 expression was knocked down in THP-1 cells transfected with a lentivirus carrying specific shRNA-HSP27 vector there was a reduction in HDAC6 activity and an increase in tubulin acetylation - and both of these effects were rescued with the addition of rHSP27. 3. Using a Boyden chamber cell migration assay, we found that rHSP27 treatment increased migration of THP-1 cells without and with ox-LDL loading by ~2- and ~1.5-fold; respectively - an effect that was blocked by either Tubacin (a specific inhibitor of HDAC6) or HSP27 knockdown. Conclusion: Extracellular HSP27 is capable of up-regulating HDAC6 activity in macrophages, an effect associated with enhanced macrophage migratory capacity. These data suggest that HSP27 may be important for potentially improving macrophage egress from plaques thereby resulting in reduced cholesterol content and more stable lesions - factors that may explain the proven predictive biomarker value of elevated serum HSP27 levels.

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

Lymphoma Monocyte Crosstalk Via Hsp27 to Promote Immune Suppression and Chemotherapy Resistance

Background: Immune editing is a major mechanism used by tumors to promote its survival. We have reported previously the presence of a novel phenotype of immunosuppressive monocytes (CD14+HLA-DRlow/neg) in a number of cancers. Increased presence of these cells was associated with decreased treatment response and OS. We have demonstrated that certain tumor cells can convert normal CD14+HLA-DR+ monocytes to CD14+HLA-DRlow/neg phenotype in an IL-10 independent and a tumor specific way. These CD14+HLA-DRlow/neg monocytes, in turn, protect tumors from cytotoxic killing from chemotherapy. Here we report up-regulation of heat shock protein-27 (HSP27) as one mechanism mediating this effect.Method: Monocytes from healthy donors were co-cultured with lymphoma cell lines (OCI-Ly3, Jeko-1, and Granta-519) with or without doxorubicin (DOX). Cultured cells were assessed for phenotype, viability and proliferation by flow cytometry. Lymphoma cells were isolated with anti-CD19 immunomagnetic beads and assayed by immunoblot for expressions of proteins regulating apoptosis. HSP27 levels in human plasma were measured by ELISA.Results: DOX incubation induced apoptosis and decreased viability of all three cell lines; and co-culture with monocytes improved the lymphoma cell survival (for example, untreated Granta-519 had a 2.1±0.45 fold expansion, that was reduced to 0.39±0.12 when treated with DOX and 0.81±0.27 after co-culture with monocytes with DOX. p<0.05, n=11.) Co-culture with monocytes induced increased HSP27 expression in lymphoma cells. HSP27 levels were further increased in co-culture with monocytes and DOX, with corresponding decrease in cleaved Caspase-3 levels. As tumor cells can secrete HSP27, we found detectable levels of HSP27 in plasma of lymphoma patients. Increased HSP27 in plasma correlated with increased proportion of CD14+HLA-DRlow/neg monocytes in blood.Conclusions: We have found that monocytes may promote lymphoma resistance to DOX killing by inducing increased HSP27. In turn, HSP27 from lymphoma patients may induce immune suppressive phenotype in monocytes. Together, this data demonstrates an active cross talk between monocytes and lymphoma resulting in multiple mechanisms of tumor resistance to chemo-immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Human myocardium releases heat shock protein 27 (HSP27) after global ischemia: the proinflammatory effect of extracellular HSP27 through toll-like receptor (TLR)-2 and TLR4.

The myocardial inflammatory response contributes to cardiac functional injury associated with heart surgery obligating global ischemia/reperfusion (I/R). Toll-like receptors (TLRs) play an important role in the mechanism underlying myocardial I/R injury. The aim of this study was to examine the release of small constitutive heat shock proteins (HSPs) from human and mouse myocardium after global ischemia and examine the role of extracellular small HSP in myocardial injury. HSP27 release was assessed by enzyme-linked immunosorbent assay. Anti-HSP27 was applied to evaluate the role of extracellular HSP27 in the postischemic inflammatory response and functional injury in mouse hearts. Isolated hearts and cultured coronary vascular endothelial cells were exposed to recombinant HSP27 to determine its effect on proinflammatory signaling and production of proinflammatory mediators. HSP27 levels were markedly elevated in coronary sinus blood of patients and in coronary effluent of mouse hearts after global ischemia. Neutralizing extracellular HSP27 suppressed myocardial nuclear factor (NF)-κB activation and interleukin (IL)-6 production and improved cardiac function in mouse hearts. Perfusion of HSP27 to mouse hearts induced NF-κB activation and IL-6 production and depressed contractility. Further, recombinant HSP27 induced NF-κB phosphorylation and upregulated monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 production in both human and mouse coronary vascular endothelial cells. TLR2 knockout (KO) or TLR4 mutation abolished NF-κB phosphorylation and reduced MCP-1 and ICAM-1 production induced by extracellular HSP27 in endothelial cells. In conclusion, these results show that the myocardium releases HSP27 after global ischemia and that extracellular HSP27 is proinflammatory and contributes to the inflammatory mechanism of myocardial functional injury. Both TLR2 and TLR4 are involved in mediating the proinflammatory effect of extracellular HSP27.

Serum Heat Shock Protein 27 Levels Represent a Potential Therapeutic Target for Atherosclerosis: Observations From a Human Cohort and Treatment of Female Mice

The aim of this study was to evaluate the potential of serum heat shock protein 27 (HSP27) as a therapeutic target in coronary artery disease. Expression of HSP27 in human coronary arteries diminishes with the progression of atherosclerosis, whereas ubiquitous HSP27 overexpression in apolipoprotein E(-/-) (ApoE(-/-)) mice attenuates atherogenesis. However, it remains unclear whether increasing serum HSP27 levels alone is sufficient for atheroprotection. Low- and intermediate-risk patients undergoing coronary or computed tomography angiography had serum HSP27 levels measured. Elevated serum HSP27 levels in female atheroprone ApoE(-/-) mice were achieved by transplantation with HSP27 overexpressing bone marrow or by administering recombinant HSP27. Patients with >50% stenosis in any major epicardial artery had lower HSP27 levels compared with those free of atherosclerosis (median [interquartile range]: 2,176 pg/ml [551-5,475] vs. 6,200 pg/ml [2,575-9,560]; p < 0.001). After a 5-year period of clinical follow-up, low serum HSP27 levels (<50th percentile) were predictive of subsequent major adverse cardiovascular events (hazard ratio: 2.93, 95% confidence interval: 1.06 to 8.12; p = 0.04). In experimental murine models of atherosclerosis, increasing serum HSP27 levels both reduced de novo atherosclerotic lesion formation and enhanced features of plaque stability. In humans, low serum HSP27 levels are associated with the presence of coronary artery disease and prognostic of future adverse clinical events. In mouse models of atherosclerosis, increasing HSP27 levels reduced lesion progression and promoted features of plaque stability. Serum HSP27 levels may represent a potential therapeutic target for atherosclerosis.

Proteomic Profiling Reveals That Resveratrol Inhibits HSP27 Expression and Sensitizes Breast Cancer Cells to Doxorubicin Therapy

The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4′,5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5′-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural alternative agents, as resveratrol, may be an effective adjuvant in breast cancer therapy.

Tu1928 Tissue Expression of Heat Shock Protein 27, 70 and 90 Might Be Utilized As a Diagnostic and Prognostic Biomarker in Patients With Gastric Adenocarcinoma

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflammation and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaperone, inhibit reactive oxygen species (ROS) accumulation. In previous reports have been suggested that HSP27 plays an important role in inflammation and carcinogenesis. We investigated HSP27 expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P= 0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased cell death and ROS accumulation that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated with an epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastric mucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastric cancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Tu1932 Endoscopic Features of Food Protein-Induced Proctocolitis in Neonates and Infants

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflammation and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaperone, inhibit reactive oxygen species (ROS) accumulation. In previous reports have been suggested that HSP27 plays an important role in inflammation and carcinogenesis. We investigated HSP27 expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P= 0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased cell death and ROS accumulation that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated with an epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastric mucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastric cancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Tu1927 Heat Shock Protein 27 Expression Is Inversely Correlated With Intraepithelial Neoplasia and Positively Correlated With Poor Differentiation of Gastric Cancer

Background and aim: Intestinal-type gastric carcinomas progress through chronic inflammation and atrophic gastritis. Heat shock protein 27(HSP27), a stress-induced molecular chaperone, inhibit reactive oxygen species (ROS) accumulation. In previous reports have been suggested that HSP27 plays an important role in inflammation and carcinogenesis. We investigated HSP27 expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. Methods:We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and in gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. Results: HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of those without gastric neoplasia(P= 0.004).In tumor necrosis factor -treated gastric cancer cell,HSP27 knockdown increased cell death and ROS accumulation that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiated of cancer cells is associated with an epithelial-mesenchymal transition signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. Conclusion: HSP27 expression in gastric mucosae is negatively correlated with intraepithelial neoplasia, a probable precursor to gastric cancer and HSP27 expression in cancer is positively correlated with poor differentiation.

Chronic Over-Expression of Heat Shock Protein 27 Attenuates Atherogenesis and Enhances Plaque Remodeling: A Combined Histological and Mechanical Assessment of Aortic Lesions

AimsExpression of Heat Shock Protein-27 (HSP27) is reduced in human coronary atherosclerosis. Over-expression of HSP27 is protective against the early formation of lesions in atherosclerosis-prone apoE−/− mice (apoE−/−HSP27o/e) - however, only in females. We now seek to determine if chronic HSP27 over-expression is protective in a model of advanced atherosclerosis in both male and female apoE−/− mice.Methods and ResultsAfter 12 weeks on a high fat diet, serum HSP27 levels rose more than 16-fold in male and female apoE−/−HSP27o/e mice, although females had higher levels than males. Relative to apoE−/− mice, female apoE−/−HSP27o/e mice showed reductions in aortic lesion area of 35% for en face and 30% for cross-sectional sinus tissue sections – with the same parameters reduced by 21% and 24% in male cohorts; respectively. Aortic plaques from apoE−/−HSP27o/e mice showed almost 50% reductions in the area occupied by cholesterol clefts and free cholesterol, with fewer macrophages and reduced apoptosis but greater intimal smooth muscle cell and collagen content. The analysis of the aortic mechanical properties showed increased vessel stiffness in apoE−/−HSP27o/e mice (41% in female, 34% in male) compare to apoE−/− counterparts.ConclusionsChronic over-expression of HSP27 is atheroprotective in both sexes and coincides with reductions in lesion cholesterol accumulation as well as favorable plaque remodeling. These data provide new clues as to how HSP27 may improve not only the composition of atherosclerotic lesions but potentially their stability and resilience to plaque rupture.

Heat Shock Protein 27 Expression is Inversely Correlated with Atrophic Gastritis and Intraepithelial Neoplasia

Intestinal-type gastric carcinomas progress through several sequential steps, including atrophic gastritis, intestinal metaplasia, dysplasia, and cancer. We investigated heat shock protein 27 (HSP27) expression in gastric neoplasia and background gastric mucosa to assess its involvement in gastric carcinogenesis. We used real-time quantitative polymerase chain reaction to examine HSP27 expression in gastric neoplasias and background gastric mucosae of 30 patients with intraepithelial neoplasias and in gastric mucosae of 30 patients without gastric neoplasia. Immunohistochemical staining was performed on 30 advanced gastric cancer tissues. HSP27 expression was negatively associated with atrophic gastritis. HSP27 expression in the background gastric mucosa of neoplasia-bearing patients was significantly lower than in the mucosa of those without gastric neoplasia. In tumor necrosis factor α-treated gastric cancer cells, HSP27 knockdown increased cell death and accumulation of the reactive oxygen species that link inflammation to cancer. Poorly differentiated tumors most frequently had high HSP27 levels. Dedifferentiation of cancer cells is associated with an epithelial-mesenchymal transition (EMT) signaling pathway. In gastric cancer MKN-1 cells, HSP27 knockdown upregulated E-cadherin and downregulated vimentin and smooth muscle actin, but this did not occur in MKN-74 cells. HSP27 expression in gastric mucosae is inversely correlated with intraepithelial neoplasia, a probable precursor to gastric cancer, and HSP27 expression in cancer is positively correlated with poor differentiation.

Serum HSP27 is associated with medullary perfusion in kidney allografts

Heat shock protein 27 (HSP27) is a small HSP up-regulated in response to stress in the kidney. The relationship between HSP27 and intrarenal oxygenation in patients with native and transplant kidney disease is unknown. We compared HSP27 levels, intrarenal oxygenation measured by blood oxygen-level dependent (BOLD) imaging using R(2)* values, and perfusion determined by arterial spin labeling (ASL) magnetic resonance imaging (MRI), between patients with native and transplant kidney disease (n=28). There were no statistical differences in mean age (53.9 vs. 47.1 years), kidney function (63.6 vs. 50.7 ml/min per 1.73 m(2)), mean arterial blood pressure (91.6 vs. 91.1 mm Hg), hematocrit (40.6% vs. 39.3%), diuretic or angiotensin-converting enzyme inhibitor use, serum or urine levels of hydrogen peroxide, nitric oxide, F(2) isoprostanes and HSP27 between native and transplant kidneys. BOLD-MRI studies demonstrated comparable patterns in intrarenal oxygen bioavailability (medullary R(2)* 18.1 vs. 18.3/s and cortical R(2)* 12 vs. 11.7/s, respectively). However, medullary perfusion was significantly lower in transplant kidneys (36.4 vs. 78.7 ml/100 g per minute, p=0.0002). There was a linear relationship between serum HSP27 concentrations and medullary perfusion in kidney allografts (HSP27 concentration [ng/mL] = 0.78 + 0.09 medullary perfusion, R(2)=0.43, p=0.01). Our study demonstrates that medullary perfusion is significantly lower in kidney allografts compared with native kidneys with comparable renal function. We further noted a direct association between serum HSP27 levels and medullary perfusion after transplantation. Additional studies are needed to examine the role of HSP27 as a biomarker of kidney disease progression.

Low molecular weight heat shock protein HSP27 is a prognostic indicator in rectal cancer but not colon cancer

ObjectiveThere are currently no biomarkers in routine clinical use for determining prognosis in rectal cancer. In a preliminary proteomic study, variation in the levels of heat shock protein 27 (HSP27)...

Abstract 5809: Recombinant HSP27 Therapy Reduces Serum Cholesterol Levels and Experimental Atherogenesis

Introduction: Recently we demonstrated that Heat Shock Protein 27 (HSP27) is a biomarker of atherosclerosis and apoE −/− mice that over-express HSP27 show a marked attenuation of aortic lesion size compared to apoE −/− mice (ATVB 20005; Circ Res 2008). Moreover, serum HSP27 levels are inversely related to atherosclerotic burden, estrogens mediate the release of HSP27 from cells and extracellular HSP27 binds to Scavenger Receptor-A where competitively inhibits acLDL uptake. Purpose/Methods: The goal of this study was to determine if recombinant HSP27 (rHSP27) administered to apoE −/− mice can attenuate atherogenesis. rHSP27 was produced in E.coli; (endotoxin concentration &lt;8EU/mg). In vitro aggregation and refolding assays revealed appropriate rHSP27 function. In vivo a series of subcutaneous dosing schedules were explored. Herein we report the results obtained for ApoE −/− mice maintained on a high fat diet and randomized to rHSP27 (100 μ g) vs. phosphate buffered saline (PBS) twice a day for 3 weeks. Results: All mice survived the study period without changes in body weight or adverse events. Total serum cholesterol levels were approximately 40% lower in rHSP27 treated mice. The aortic lesion areas were reduced by approximately 30% and 25% for en face and sinus cross-sectional analyses. In rHSP27 treated mice the abundance of aortic sinus lesion macrophages and apoptotic macrophages were reduced by approximately 45% and 80%, respectively. Conclusions: rHSP27 is a promising new therapeutic modality for reducing serum cholesterol levels as well as atherogenesis. Moreover, the lesions that form in mice treated with rHSP27 contain fewer inflammatory cells and undergo less apoptosis. Studies are ongoing to optimize the dose, schedule and form of rHSP27 that might be used not only for the prevention of atherogenesis but also regression of existing lesions.

Serum Heat Shock Protein 27 Is Increased in Chronic Pancreatitis and Pancreatic Carcinoma

A prior study suggested serum heat shock protein 27 (HSP27) as a potential marker for pancreatic carcinoma, but its accuracy in differentiating cancer from chronic pancreatitis was not evaluated. We aimed to analyze HSP27 levels in pancreatic carcinoma, chronic pancreatitis, and healthy subjects and assess its diagnostic efficacy. Pretreatment serums from 58 pancreatic carcinoma, 44 chronic pancreatitis, and 102 control subjects were collected. Serum HSP27 and carbohydrate antigen 19-9 (CA19-9) levels were analyzed using an enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Heat shock protein 27 levels were significantly higher in cancer and pancreatitis compared with control (P < 0.001 for both), but no significant difference was noted between cancer and pancreatitis (P = 0.978). By logistic regression, HSP27 was a significant predictor of differentiation between cancer and control (P < 0.0001) but not between cancer and pancreatitis (P = 0.885). At a cutoff of 1650 ng/L, the sensitivity and specificity for differentiating cancer from healthy control were 62.1% and 95.1%, respectively. Receiver operating characteristic analyses showed a greater area under curve for CA19-9 compared with HSP27 in differentiating between cancer and control (0.92 and 0.84, respectively, P = 0.014). Serum HSP27 is increased in both chronic pancreatitis and pancreatic carcinoma. It should not be recommended as a diagnostic marker for pancreatic carcinoma.

Anti–Heat Shock Protein 27 Antibody Levels and Diabetes Complications in the EURODIAB Study

OBJECTIVETo assess whether serum anti–heat shock protein 27 (HSP27) antibody levels are associated with micro- and macrovascular complications of type 1 diabetes.RESEARCH DESIGN AND METHODSAnti-HSP27 IgG antibody levels were measured in 531 type 1 diabetic subjects recruited as part of the cross-sectional analysis of the EURODIAB Prospective Complications Study. Case subjects (n = 363) were defined as individuals with one or more diabetes complications and control subjects (n = 168) as individuals with no evidence of any diabetes complication.RESULTSAnti-HSP27 levels were comparable in case and control subjects (19.6 arbitrary units/ml [11.3–32.7] vs. 20.4 arbitrary units/ml [11.7–35.3], geometric mean [interquartile range]), and there was no correlation between HSP27 and anti-HSP27 levels (r = 0.01, P = 0.81). In logistic regression analysis, anti-HSP27 was not associated with the presence of complications, even after adjustment for main risk factors.CONCLUSIONSAnti-HSP27 antibody levels are not a marker of vascular complications in type 1 diabetes.

Abstract 1948: Over-expression of HSP27 Provides Chronic Atheroprotection: Reductions in Foam Cell Content and Inflammatory Responses

Introduction: Heat Shock Protein 27 (HSP27) was recently shown by our laboratory to be a biomarker of atherosclerosis and after 4 weeks of a high fat diet (HFD) apoE −/− mice that over-express HSP27 (or apoE −/− HSP27 o/e mice) show a marked attenuation of aortic lesion size compared to apoE −/− mice - but only in females (ATVB 20005; Circ Res in press). Moreover, we demonstrated that serum HSP27 levels were inversely related to atherosclerotic burden, estrogens mediated the release of HSP27 from cells and extracellular HSP27 bound to Scavenger Receptor-A where it could competitively inhibit acLDL uptake. Purpose/Methods: The goal of this study was to determine if and how HSP27 over-expression persistently attenuates atherogenesis in apoE −/− mice fed a HFD for 12 weeks. Results: Compared to female apoE −/− mice, female apoE −/− HSP27 o/e mice showed persistent reductions in aortic (en face) lesion area and aortic sinus cross-sectional lesion area at 12 weeks (e.g., 38% and 39% respectively vs. 35% and 40% after 4 weeks of HFD). Although our original studies revealed that male apoE −/− HSP27 o/e mice had no protection from atherogenesis after 4 weeks of a HFD, in the current study we noted that 12 weeks of a HFD resulted in a 17% reduction in aortic (en face) lesion area and a 24% attenuation in aortic sinus cross-sectional lesion area with HSP27 over-expression. While lesions observed after 4 weeks of HFD consisted mainly of lipid-laden foam macrophages, after 12 weeks of HFD the histopathology was different and included a distinct foam cell layer (or FCL) luminal to a deeper necrotic layer (e.g., with cholesterol crystals). The FCL area was reduced in size in apoE −/− HSP27 o/e vs. apoE −/− mice (e.g., −63% in females, p=0.03; and −48% in males, p=0.02). In vitro, macrophages from apoE −/− HSP27 o/e mice showed a reduction in TNF-α expression compared to apoE −/− mice (e.g., 40% at baseline, and 55% after LPS provocation). Conclusions: Over-expression of HSP27 provides a persistent atheroprotective effect that benefits both sexes. Chronic lesions are not only smaller in size with HSP27 over-expression but show fewer foam cells and macrophages are less pro-inflammatory. These results highlight a potential clinical role for chronic HSP27 modulation for atheroprotection.

Extracellular Release of the Atheroprotective Heat Shock Protein 27 Is Mediated by Estrogen and Competitively Inhibits acLDL Binding to Scavenger Receptor-A

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ERbeta)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE-/- mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P < or =0.001) in atherosclerotic lesion area in apoE-/-HSP27o/e mice compared to apoE-/- mice, but primarily in females. Serum -HSP27levels were 10-fold higher in female apoE-/-HSP27o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r(2)=0.78, P < or =0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)--an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A-/- mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1beta and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.

Post-transcriptional regulation of TNF-induced expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells: An obligatory role for the p38 MAPK-MK2 pathway dissociated with HSP27

The tumor necrosis factor-alpha (TNF)-induced inflammatory response in human lung microvascular endothelial cells (MVECs) is an early event in acute lung injury. Studies have shown that p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase 2 (MK2) and heat shock protein 27 (HSP27) are involved in the expression of pro-inflammatory mediators in other cell types. However, their role in the TNF-induced inflammatory response in lung MVECs has not been determined. We evaluated the role of p38 MAPK, MK2 and HSP27 in regulating the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs. Inhibition of p38 MAPK reduced ICAM-1 and IL-8 expression without influencing NF-κB activation or ICAM-1 and IL-8 mRNA levels. TNF stimulation induced p38 MAPK-dependent phosphorylation of MK2 and HSP27. MK2 silencing reduced ICAM-1 and IL-8 expression without influencing NF-κB activation or ICAM-1 and IL-8 mRNA levels. HSP27 silencing reduced cellular HSP27 levels and HSP27 phosphorylation following TNF stimulation but had no effect on ICAM-1 and IL-8 expression. Our study demonstrates for the first time that MK2 mediates post-transcriptional regulation by p38 MAPK of the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs, and that this regulation by the p38 MAPK/MK2 pathway is dissociated from HSP27 phosphorylation.