1Elevated Heat Shock Protein 27 levels predict relative freedom from cardiovascular events. In 2 ApoE -/mice HSP27 over-expression or twice daily subcutaneous injections reduce blood and 3 plaque cholesterol levels, inflammation and atherogenesis. While natural antibodies to HSP27 4 are present in human blood their role is unknown. Here, we show that blood levels of both 5 HSP27 and anti-HSP27 IgG antibodies are elevated in healthy controls compared to patients with 6 cardiovascular disease. ApoE -/mice vaccinated with recombinant HSP25 (murine ortholog) 7 develop elevated anti-HSP25 IgG antibodies and reduced levels of cholesterol, inflammation and 8 atherosclerosis. The effects on cholesterol metabolism were divergent: increased hepatic LDLR 9 expression and reduced plasma PCSK9 levels. In vitro, a polyclonal anti-HSP27 IgG antibody 10 combined with rHSP27 to upregulate hepatocyte LDLR expression via an NF-kB-dependent 11 pathway that is independent of SREBP2 expression and intracellular cholesterol levels. HSP27 12 immunotherapy represents a novel means of lowering not only cholesterol but also PCSK9. 13Small heat shock proteins, such as Heat Shock Protein 27 (HSP27), are intracellular chaperones 1 that promote the proper reassembly of misfolded proteins and act as mediators of extracellular 2 cellular signaling 1 . HSP27 effectively preserves cellular homeostasis under various conditions of 3 degenerative or inflammatory stress -including those common to the pathogenesis of 4 atherosclerosis 2 . There is overwhelming genetic, epidemiological and clinical evidence that 5 irrefutably establishes low density lipoprotein cholesterol (LDL-C) as causal for atherosclerosis 3, 6 4 . Experiments from our group 5 and four others using proteomic discovery approaches 6,7,8,9 show 7 that serum HSP27 levels decline as human atherosclerosis develops, with its tissue abundance 8 inversely corelated with the degree of coronary artery plaque burden. In atherosclerosis-prone 9 Apolipoprotein E null (ApoE -/-) mice augmentation of extracellular HSP27 levels via constitutive 10 over-expression, transplantation of bone marrow from mice that over-express HSP27, twice-11 daily subcutaneous administration of recombinant HSP27 (rHSP27; 100 µg) or estrogenic 12 therapy post-ovariectomy (that augment HSP27 blood levels) reduce both plasma and plaque 13 cholesterol content, resulting in the formation of more stable plaques that are less inflamed 10, 11, 14 12, 13 . Clinically, elevated HSP27 blood levels are associated with a lower 5-year risk of 15 myocardial infarction, stroke or cardiovascular death 11 . Interestingly, natural antibodies to 16 HSP27 (AAbs) are detectable in the blood, yet their biological significance is unclear 14, 15 . 18In this study we sought to address three questions. First, what is the correlation between blood 19 HSP27 and AAb abundance in human cardiovascular disease (CVD) patients compared to 20 healthy control subjects (CON)? Second, does augmenting levels of antibodies to HSP25 (the 21 murine ortholog of ...

Pancreatic islets adapt to insulin resistance of pregnancy by up regulating β-cell mass and increasing insulin secretion. Previously, using a transgenic mouse with global, heterozygous deletion of prolactin receptor (Prlr+/−), we found Prlr signaling is important for this adaptation. However, since Prlr is expressed in tissues outside of islets as well as within islets and prolactin signaling affects β-cell development, to understand β-cell-specific effect of prolactin signaling in pregnancy, we generated a transgenic mouse with an inducible conditional deletion of Prlr from β-cells. Here, we found that β-cell-specific Prlr reduction in adult mice led to elevated blood glucose, lowed β-cell mass and blunted in vivo glucose-stimulated insulin secretion during pregnancy. When we compared gene expression profile of islets from transgenic mice with global (Prlr+/−) versus β-cell-specific Prlr reduction (βPrlR+/−), we found 95 differentially expressed gene, most of them down regulated in the Prlr+/− mice in comparison to the βPrlR+/− mice, and many of these genes regulate apoptosis, synaptic vesicle function and neuronal development. Importantly, we found that islets from pregnant Prlr+/− mice are more vulnerable to glucolipotoxicity-induced apoptosis than islets from pregnant βPrlR+/− mice. These observations suggest that down regulation of prolactin action during pregnancy in non-β-cells secondarily and negatively affect β-cell gene expression, and increased β-cell susceptibility to external insults.

Type 1 diabetes is caused by autoimmune destruction of β-cells. Although immunotherapy can restore self-tolerance thereby halting continued immune-mediated β-cell loss, residual β-cell mass and function is often insufficient for normoglycemia. Using a growth factor to boost β-cell mass can potentially overcome this barrier and prolactin (PRL) may fill this role. Previous studies have shown that PRL can stimulate β-cell proliferation and up-regulate insulin synthesis and secretion while reducing lymphocytic infiltration of islets, suggesting that it may restore normoglycemia through complementary mechanisms. Here, we test the hypothesis that PRL can improve the efficacy of an immune modulator, the anticluster of differentiation 3 monoclonal antibody (aCD3), in inducing diabetes remission by up-regulating β-cell mass and function. Diabetic nonobese diabetic (NOD) mice were treated with a 5-day course of aCD3 with or without a concurrent 3-week course of PRL. We found that a higher proportion of diabetic mice treated with the aCD3 and PRL combined therapy achieved diabetes reversal than those treated with aCD3 alone. The aCD3 and PRL combined group had a higher β-cell proliferation rate, an increased β-cell fraction, larger islets, higher pancreatic insulin content, and greater glucose-stimulated insulin release. Lineage-tracing analysis found minimal contribution of β-cell neogenesis to the formation of new β-cells. Although we did not detect a significant difference in the number or proliferative capacity of T cells, we observed a higher proportion of insulitis-free islets in the aCD3 and PRL group. These results suggest that combining a growth factor with an immunotherapy may be an effective treatment paradigm for autoimmune diabetes.

Objective: Elevated HSP27 (heat shock protein 27) levels predict relative freedom from cardiovascular events. Over-expression or twice daily subcutaneous injections of human HSP27 in ApoE −/− mice reduces blood and plaque cholesterol levels, as well as inflammation and atherosclerotic plaque burden. Antibodies to HSP27 are present in human blood, and the purpose of the current studies is to explore their role. Approach and Results: Blood levels of both HSP27 and anti-HSP27 IgG antibodies are elevated in healthy controls compared with patients with cardiovascular disease. ApoE −/− mice fed a high-fat diet and vaccinated with recombinant HSP25 (rHSP25, murine ortholog) show increased levels of anti-HSP25 IgG antibodies and reductions in plasma cholesterol and atherogenesis. Moreover, rHSP25 vaccination markedly lowered serum amyloid A levels as well as hepatic macrophage abundance and inflammatory cytokine expression. The effects of the HPS25 vaccination on cholesterol metabolism are divergent: increased hepatic LDLR (low-density lipoprotein receptor) mRNA and protein expression and reduced plasma PCSK9 (proprotein convertase subtilisin/kexin type 9) levels—despite no effect on PCSK9 expression. In vitro, the HSP27 immune complex upregulates hepatocyte LDLR mRNA and protein expression independent of intracellular cholesterol levels and increases LDLR promoter activity. The increase in LDLR expression by the HSP27 immune complex is dependent upon activation of the NF-κB (nuclear factor κ light chain enhancer of activated B cells) pathway. Hepatocyte PCSK9 protein levels are reduced after HSP27 immune complex treatment in vitro despite only minor transient effects on gene expression. Conclusions: HSP27 immunotherapy represents a novel means of lowering cholesterol and PCSK9 levels, primarily due to augmentation of LDLR expression and is associated with marked reductions in inflammation.