Heat Shock Protein 25 Vaccination Attenuates Atherogenesis after Ovariectomy via Down-Regulation of Proprotein Convertase Subtilisin/Kexin Type 9 and Upregulation of Low Density Lipoprotein Receptor
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2019-01-25
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Abstract
Women are relatively spared from the development of atherosclerotic coronary artery disease (ASCAD) until after menopause when the frequency of ASCAD accelerates and surpasses that of men. From early observational studies, it was believed that ovarian production of estrogen delayed the onset of ASCAD in pre-menopausal women. However, surprisingly, randomized clinical trials of exogenous estrogen replacement administered to post-menopausal women failed to recapitulate this protection from ASCAD. This suggests that the mechanism for ASCAD protection in women remains poorly understood. In addition to natural menopause and the cessation of endogenous estrogen production, many women may undergo surgically-induced menopause which potentially places them at early increased risk for ASCAD. Approximately 15 years ago, the O’Brien laboratory discovered that heat shock protein 27 (HSP27) can attenuate atherogenesis by reducing cholesterol levels in the blood and artery wall. Furthermore, ovariectomy abolished atheroprotection in HSP27 over-expressing mice but could be restored with therapeutic supplementation of exogenous estrogen receptor modulators. More recently the O’Brien laboratory has discovered that natural antibodies to HSP27 appear to potentiate the beneficial biological effects of HSP27 on the vessel wall as well as on cholesterol metabolism and have developed a vaccination approach as an atheroprotective therapeutic. Based on these prior observations, I hypothesized that vaccination with HSP25 (the murine orthologue of the human HSP27) following ovariectomy would prevent atherogenesis in a mouse model by favorably regulating cholesterol metabolism. To address my hypothesis, two in vivo experiments were pursued in atherosclerosis-prone ApoE-/- mice subjected to ovariectomy (OVX) or sham surgery, fed a high fat diet and randomized to receive active treatment with rHSP25 vaccination, estradiol (E2) or control: i) An 8-week experiment with a supra-physiological dose of estrogen. ii) A 5-week experiment involving a physiological dose of estrogen. In addition, I pursued in vitro experiments looking specifically at the mechanism(s) by which rHSP25/27 may reduce atherogenesis. In the first in vivo experiment, using a previously-validated but supra-physiological dose of replacement estrogen, atherogenesis was attenuated by 21% and 72% in OVX mice either vaccinated with rHSP25 or receiving E2 replacement, respectively. Treatment with E2, but not rHSP25 vaccination, reduced total cholesterol levels. In the second in vivo experiment, using a physiological dose of estrogen, atherogenesis was attenuated by 19% and 33% in OVX mice receiving either rHSP25 vaccine or E2 and total cholesterol levels fell by 20% and 37%, respectively. However, expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a negative regulator of cholesterol metabolism, increased in E2-treated mice by 2.5-fold but was reduced by 62% when combined with rHSP25 vaccination. PCSK9 levels in mice vaccinated with rHSP25 alone were similar to those of sham and control-treated OVX mice. Hepatic LDLR transcript levels increased with rHSP25 vaccination, ~4-fold in rHSP25-treated sham-operated mice and ~3.2-fold in rHSP25-treated OVX mice, but low-density lipoprotein receptor (LDLR) mRNA levels were unchanged with E2 therapy or control treatment. In vitro, E2 increased PCSK9 promoter activity in a dose-dependent manner that, again, could be reduced by 41.2% by treating simultaneously with rHSP27. Both E2 and rHSP27 treatments increased LDLR promoter activity by 7.7- and 6.4-fold, respectively. Taken together, these studies demonstrate, in a murine model of surgical menopause, that rHSP25 vaccination can attenuate atherogenesis and, unlike E2 therapy, does not increase PCSK9 or cause off-target effects on estrogen-responsive tissues (i.e. the uterus and fallopian tubes). The upregulation of LDLR by rHSP25/27 is a unique observation that offers an excellent opportunity for the development of novel therapeutics. This thesis presents novel findings that elucidate key acquired mechanisms by which rHSP25, and its human orthologue rHSP27, prevent atherogenesis in ApoE-/- mice. The major finding is that rHSP25 vaccination is effective in reducing atherosclerotic plaque development following ovariectomy and the loss of endogenous estrogens. The protective actions of HSP25 on the vasculature are multifaceted and the mechanisms include: 1) lowering of total plasma cholesterol, 2) reduction of circulating VLDL-c and LDL-c, 3) reduction of plasma PCSK9, and 4) up-regulation of hepatic LDLR gene expression in vivo. In vitro mechanistic studies using the human hepatocyte cell line HepG2 confirmed that rHSP27 treatment downregulates PCSK9 promoter activity and upregulates LDLR promoter activity. These studies produced five main findings: 1) rHSP25 vaccination mediates athero-protection following OVX in ApoE-/- mice without adverse effects (Figure 4.2, Figure 4.8, Figure 4.13, Figure 4.17), 2) the combination therapy of rHSP25 vaccination plus physiological dose E2 yields synergistic atherogenesis prevention effects (Figure 4.13), 3) E2 upregulates PCSK9 promoter activity in vitro in a dose-dependent manner (Figure 4.22), 4) rHSP27/pAb complex treatment abolishes PCSK9 promoter activity in human hepatocytes (Figure 4.23), and 5) the rHSP27/pAb complex upregulates LDLR expression both in vivo and in vitro (Figure 4.24) (as evidenced by increased LDLR promoter activity). Taken together, these findings provide a novel and exciting evidence that the therapeutic actions of rHSP25/rHSP27 are independent of estrogen and that athero-protection appears to be mediated though the PCSK9/LDLR/cholesterol pathway. These observations support the working hypothesis of this thesis and support the possibility that rHSP27 is a novel therapeutic intervention for the prevention of post-menopausal ASCAD.
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Cardiovascular science
Citation
Maarouf, N. (2019). Heat shock protein 25 vaccination attenuates atherogenesis after ovariectomy via down-regulation of proprotein convertase subtilisin/kexin type 9 and upregulation of low density lipoprotein receptor (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.