Heat Shock Protein 27 Immune Complex Upregulates LDLR Expression Thereby Reducing Plasma Cholesterol and Atherogenesis

Abstract

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 ...

The Heat Shock Protein 27 Immune Complex Enhances Exosomal Cholesterol Efflux

“…Heat shock protein 27 (HSP27) is a member of the small HSP protein family and is involved in a variety of biological activities including ATP-independent molecular chaperoning, prevention of apoptosis, and more recently attenuating atherogenesis. Our laboratory reported that reduced serum HSP27 levels are associated with cardiovascular disease [17,18], and along with four independent laboratories demonstrated reduced arterial HSP27 expression with atherosclerosis [19][20][21][22]. Conversely, we showed that increasing HSP27 serum levels, by systemic over-expression, bone marrow transplantation or administration of the recombinant protein, not only reduced plaque burden, but also correlated with a lower tendency for experimental plaque rupture [23,24].…”

“…To study the in vitro role of exosomal HSP27 we will incorporate new information showing that anti-HSP27 IgG antibodies levels are more abundant in healthy subjects vs. CVD patients. To this end, we have generated and validated a polyclonal anti-HSP27 IgG antibody (PAb) that combines with HSP27 to form an immune complex (IC) that facilitates the biological effects of HSP27 (referred to as Immune Complex Altered Signaling and Transport (ICAST)) [18,26].…”

Heat shock protein 27 immune complex altered signaling and transport (ICAST): Novel mechanisms of attenuating inflammation

“…We used a validated rabbit anti-HSP27 polyclonal antibody (PAb) that shares the same epitope mapping pattern as human plasma-derived AAbs. 11 Herein, we show that compared to HSP27 alone, the HSP27 IC shows enhanced cell membrane interactions that result in altered signaling with key receptors involved in inflammation (Toll Like Receptor 4; TLR4) and oxLDL uptake (scavenger receptors, SR-AI and CD-36). Manassas, VA) and incubated at 37°C and 5% CO 2 in complete RPMI 1640 growth medium supplemented with 10 U/mL of penicillin and streptomycin, 1 mM of sodium pyruvate (11965092; Thermo Fisher Scientific, Burlington, ON) and heat inactivated 10% Fetal Bovine Serum (FBS; Gibco, Burlington, ON).…”

“…The production and validation of this PAb is previously described. 11 To verify the fidelity of this PAb, we compared its immunoreactivity with a commercial (goat) anti-HSP27 antibody using Western blotting of rHSP27 and rHSP25. As well, we used a spot blot epitope mapping technique to compare the immunoreactivity of the PAb vs naturally occurring anti-HSP27 antibodies found in human plasma.…”

“…9 Recently, we discovered that blood levels of natural IgG (but not IgM) auto-antibodies to HSP27 (AAbs) are higher in healthy control subjects compared to cardiovascular disease patients. 11 Vaccinating ApoE −/− mice with recombinant HSP25 (rHSP25; murine ortholog of human rHSP27) increased AAb levels, attenuated atherogenesis, reduced plasma cholesterol levels as well as the abundance of plaque inflammation and cholesterol content. As the role of these AAbs in athero-protection is not yet clear, we began our studies by trying to determine how the immune complex (IC) that forms between HSP27 and AAbs alters: (i) MΦ signaling, looking specifically at the NF-κB inflammation pathway, and (ii) the uptake of oxidized LDL (oxLDL).…”