Edward R. O'BRien

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.

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 are focusing on the development of HSP27 Immune Complex Altered Signaling and Transport (ICAST) as a means of modulating inflammation.

Previously, we demonstrated that Heat Shock Protein 27 (HSP27) reduces the inflammatory stages of experimental atherogenesis, is released by macrophage (MΦ) exosomes and lowers cholesterol levels in atherosclerotic plaques. Recently, we discovered that natural autoantibodies directed against HSP27 enhance its signaling effects, as HSP27 immune complexes (IC) interact at the cell membrane to modulate signaling. We now seek to evaluate the potential role of the HSP27 IC on MΦ exosomal release and cholesterol export. First, in human blood samples, we show that healthy control subjects have 86% more exosomes compared to patients with coronary artery disease (p < 0.0001). Treating human THP-1 MΦ with rHSP27 plus a validated anti-HPS27 IgG antibody increased the abundance of exosomes in the culture media (+98%; p < 0.0001) as well as expression of Flotillin-2, a marker reflective of exosomal release. Exosome cholesterol efflux was independent of Apo-A1. THP-1 MΦ loaded with NBD-labeled cholesterol and treated with the HSP27 IC showed a 22% increase in extracellular vesicles labeled with NBD and a 95% increase in mean fluorescent intensity. In conclusion, exosomal abundance and secretion of cholesterol content increases in response to HSP27 IC treatment, which may represent an important therapeutic option for diseases characterized by cholesterol accumulation.

Atherosclerosis is the primary cause of heart attacks, and while efforts to prevent its development or progression have historically focused largely on reducing cholesterol levels, there is now important proof-of-principle data that supports the role that inflammation plays in atherogenesis. Heat shock protein 27 (HSP27) is a novel biomarker of atherosclerosis that is also atheroprotective. Through a series of murine and in vitro experiments, an iterative narrative is emerging that demonstrates how HSP27 can act as an extracellular mediator that reduces plaque inflammation—either directly via transcriptional pathways, or indirectly via important effects on macrophage biology. While there is much more to learn about the biology of HSP27, we now review the strong foundation of knowledge that highlights the potential anti-inflammatory role of HSP27 as a novel therapeutic for not only atherosclerosis but potentially other inflammatory disorders.

Aims: The 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 Results: ApoE ^-/- 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 (pvs . 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%. Conclusion: The 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?

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 .

Elevated Heat Shock Protein 27 levels predict relative freedom from cardiovascular events. In ApoE ^-/- mice HSP27 over-expression or twice daily subcutaneous injections reduce blood and plaque cholesterol levels, inflammation and atherogenesis. While natural antibodies to HSP27 are present in human blood their role is unknown. Here, we show that blood levels of both HSP27 and anti-HSP27 IgG antibodies are elevated in healthy controls compared to patients with cardiovascular disease. ApoE ^-/- mice vaccinated with recombinant HSP25 (murine ortholog) develop elevated anti-HSP25 IgG antibodies and reduced levels of cholesterol, inflammation and atherosclerosis. The effects on cholesterol metabolism were divergent: increased hepatic LDLR expression and reduced plasma PCSK9 levels. In vitro , a polyclonal anti-HSP27 IgG antibody combined with rHSP27 to upregulate hepatocyte LDLR expression via an NF-kB-dependent pathway that is independent of SREBP2 expression and intracellular cholesterol levels. HSP27 immunotherapy represents a novel means of lowering not only cholesterol but also PCSK9.