HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Unlike estrogens that increase PCSK9 levels post-menopause HSP27 vaccination lowers cholesterol levels and atherogenesis due to divergent effects on PCSK9 and LDLR.

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, the rationale for this study is to 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 (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 %. 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?

Biophysical analyses and functional implications of the interaction between Heat Shock Protein 27 and antibodies to HSP27.

Heat Shock Protein 27 (HSP27) is a small molecular chaperone that reduces the development of atherosclerosis by lowering plasma cholesterol levels as well as inflammation. Human studies show an inverse correlation between atherosclerotic burden and HSP27 expression, and are supported by murine models in which augmenting HSP27 levels curbs experimental atherogenesis. Natural HSP27 auto-antibodies (AAb) are found in human plasma, however their role in modulating the athero-protective effects of HSP27 is unknown. The purpose of this study is to characterize the biophysical interaction between human recombinant HSP27 and AAb. A validated polyclonal anti-HSP27 IgG antibody (PAb) was used to mimic natural AAb. Homology modeling and secondary structure prediction tools facilitated the design of HSP27 truncation and phosphorylation mutants. Secondary structural changes were identified using Circular Dichroism (CD) and Dynamic Light Scattering (DLS). Similar to prior structural investigations of HSP27, there was a predominance of α-helical content in the N-terminal truncation and dephosphorylation ("AA") mutants. The α-crystallin domain (ACD) predominantly consists of ß-strands, with the addition of the N-terminal increasing helical content and the C-terminal maintaining ß structure. With increasing ratios of PAb to HSP27 ß structure abundance and particle size increased, with a similar trend observed with the N-terminus, C-terminus and ACD peptides but an opposite trend with the phosphorylation peptides. Taken together, these studies provide insights into the interaction of HSP27 and its AAb that ultimately may aid in optimizing the design of HSP27 peptidomimetics with anti-atherogenic potential.