Fig. enhancement of LDLR expression and is associated with marked reductions in inflammation. Graphical Abstract Introduction Small heat shock proteins, such as Heat Shock Protein 27 (HSP27), are intracellular chaperones that promote the proper reassembly of misfolded proteins and act as mediators of extracellular cellular signaling.1 HSP27 effectively preserves cellular homeostasis under various conditions of degenerative or inflammatory Oligomycin stress C including those common to the pathogenesis of atherosclerosis.2 Experiments from our group3 and four others using proteomic discovery Oligomycin approaches,4C7 show that serum HSP27 levels decline as human atherosclerosis develops with its tissue abundance inversely corelated with the degree of coronary artery plaque burden. In atherosclerosis-prone Apolipoprotein E null (mice? Third, what are the potential mechanisms by which the HSP27 immune complex (IC) alters the two key drivers of atherosclerosis: cholesterol and inflammation? Our results indicate that blood levels of HSP27 and AAbs are higher in health compared to CVD. Weekly rHSP25 vaccination boosts anti-HSP25 antibodies and is associated with reductions in plasma cholesterol as well as proprotein convertase subtilisin / kexin type 9 (PCSK9) levels,14 a key unfavorable regulator of low density lipoprotein receptor (LDLR) recycling. These anti-atherogenesis effects are associated with reduced plaque, circulating and hepatic biomarkers of inflammation. Finally, the biological effects of the HSP27 IC are novel, as it markedly upregulates LDLR expression impartial of intracellular cholesterol levels yet is usually reliant on activation of the NF-B pathway. Taken together, the discovery of HSP27-mediated upregulation of LDLR heralds a unique opportunity to develop HSP27 immuno-therapeutics for the treatment of atherosclerosis and hypercholesterolemia. Methods The data that support the findings of this study are available from the corresponding author upon affordable request. Please refer to the on-line Supplementary Information document for full methodological details. Briefly, this manuscript incorporates the results of three inter-related studies of: i) HSP27 and AAbs blood levels in healthy control and cardiovascular disease subjects, ii) a murine model of atherogenesis that assessments the efficacy of rHSP25 vaccination therapy to reduce plaque formation and lower cholesterol levels, and Oligomycin iii) studies that dissect out the molecular mechanisms by which the HSP27 IC upregulates the LDLR. Study Approval As part of a National Institute of Health sponsored project (#5P20MD002314C08; Sub-Project ID: 6374) designed to identify cardiovascular biomarkers for ischemic heart disease in a populace that is medically underserved, subjects were enrolled in a 5-12 months follow-up study that involved the collection of blood samples at various University of South Alabama-affiliated medical clinics. The study was approved by the Universitys Institutional Review Board on June 21, 2012 and complied with the Declaration of Helsinki. Written informed consent was obtained from participants prior to inclusion in the study. Similarly, all experimental murine procedures were approved by the Animal Care Committee of the University of Calgary (protocol: AC17C0015). Statistics a) Statistical analyses for Clinical Study For the clinical data, continuous variables are reported as mean standard deviation, with potential deviations from normality described as medians with interquartile ranges (25th and 75th percentiles). Categorical variables are reported as number (%) and were compared using 2 or Fishers exact assessments. Chi-square assessments were performed on nominal data sets: male sex, smoker, diabetes mellitus (defined by use of hypoglycemic brokers), hypertension (defined by of conventional anti-hypertensive therapy), statin use (defined by use of any HMG Co-A reductase medication), African American and Caucasian. All comparisons used two-tailed alpha levels of 0.05. Oligomycin In cases of multiple comparisons, one-way analysis of variance (ANOVA) and Tukeys method were used to correct for the family-wise error rate. The association of health vs. CVD status with blood concentrations of HSP27 and DLEU2 AAb was examined using multivariable linear regression, adjusting for age, sex, ethnicity, body mass index (BMI), diabetes mellitus, and active smoking status. Body mass index was log transformed in the multivariable regression model. Adjusted differential levels in HSP27 and AAb are reported with 95% CIs. All analyses were performed in a blinded fashion using GraphPad.