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 ...

Blood levels of heat shock protein (HSP27) and natural IgG auto-antibodies to HSP27 (AAbs) are higher in healthy controls compared to cardiovascular disease patients. Vaccination of mice with recombinant HSP25 (rHSP25, murine ortholog of human rHSP27) increased AAb levels, attenuated atherogenesis and reduced plaque inflammation and cholesterol content. We sought to determine if the HSP27 immune complex (IC) altered MΦ inflammation signaling (Toll Like Receptor 4; TLR4), and scavenger receptors involved in cholesterol uptake (SR-AI, CD-36). Combining a validated polyclonal IgG anti-HSP27 antibody (PAb) with rHSP27 enhanced binding to THP-1 MΦ cell membranes and activation of NF-κB signaling via TLR4, competing away LPS and effecting an anti-inflammatory cytokine profile. Similarly, adding the PAb with rHSP27 enhanced binding to SR-AI and CD-36, as well as lowered oxLDL binding in HEK293 cells separately transfected with SR-AI and CD-36, or THP-1 MΦ. Finally, the PAb enhanced the uptake and internalization of rHSP27 in THP-1 MΦ. Thus, the HSP27 IC potentiates HSP27 cell membrane signaling with receptors involved in modulating inflammation and cholesterol uptake, as well as HSP27 internalization. Going forward, we will explore HSP27 Immune Complex Altered Signaling and Transport (ICAST) as a new anti-inflammatory therapeutic strategy in vivo.

AimsThe estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti-HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, we now compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis.Methods and ResultsApoE-/- mice subjected to ovariectomy (OVX) showed a 65% increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (−43%), a subcutaneous E2 slow release pellet (−52%) or a combination thereof (−82%). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52% higher in E2/OVX and 41% lower in rHSP25/OVX mice (p<0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148% with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303%, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64%.ConclusionThe reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women – are there effects beyond cholesterol?