Melissa McNulty

Extracellular Release of the Atheroprotective Heat Shock Protein 27 Is Mediated by Estrogen and Competitively Inhibits acLDL Binding to Scavenger Receptor-A

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ER&bgr;)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE−/− mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P≤0.001) in atherosclerotic lesion area in apoE−/−HSP27o/e mice compared to apoE−/− mice, but primarily in females. Serum HSP27 levels were >10-fold higher in female apoE−/−HSP27o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r2=0.78, P≤0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)—an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A−/− mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1&bgr; and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.

Heat Shock Protein 27 Protects Against Atherogenesis via an Estrogen-Dependent Mechanism Role of Selective Estrogen Receptor Beta Modulation

Objective—We recently identified HSP27 as an atheroprotective protein that acts extracellularly to prevent foam cell formation and atherogenesis in female but not male mice, where serum levels of HSP27 were increased and inversely correlated with degree of lesion burden. In the current study we sought to determine whether estrogens are required for the observed atheroprotective benefits of HSP27 as well as its extracellular release. Methods and Results—In vitro estrogens prompted the release of HSP27 from macrophages in an ERβ specific manner that involved exosomal trafficking. Ovariectomy nullified the previously recognized attenuation in aortic lesion area in HSP27o/eapoE−/− mice compared to apoE−/− mice. Supplementation with 17β-estradiol resulted in a >15× increase in uterine weight and attenuation of atherogenesis in all mice, although HSP27o/eapoE−/− had 34% less lesion burden compared to apoE−/− mice. Mice treated with the ERβ-specific agonist, DPN had no effect on uterine weight but a 28% decrease in aortic lesion area in HSP27o/eapoE−/− compared to apoE−/− mice. HSP27 serum levels showed a similar gradual increase with E2 and DPN replacement treatment but did not change in untreated mice. Conclusions—The extracellular release of and atheroprotection provided by HSP27 is estrogen dependent.

Chronic Over-Expression of Heat Shock Protein 27 Attenuates Atherogenesis and Enhances Plaque Remodeling: A Combined Histological and Mechanical Assessment of Aortic Lesions

Aims Expression of Heat Shock Protein-27 (HSP27) is reduced in human coronary atherosclerosis. Over-expression of HSP27 is protective against the early formation of lesions in atherosclerosis-prone apoE−/− mice (apoE−/−HSP27o/e) - however, only in females. We now seek to determine if chronic HSP27 over-expression is protective in a model of advanced atherosclerosis in both male and female apoE−/− mice. Methods and Results After 12 weeks on a high fat diet, serum HSP27 levels rose more than 16-fold in male and female apoE−/−HSP27o/e mice, although females had higher levels than males. Relative to apoE−/− mice, female apoE−/−HSP27o/e mice showed reductions in aortic lesion area of 35% for en face and 30% for cross-sectional sinus tissue sections – with the same parameters reduced by 21% and 24% in male cohorts; respectively. Aortic plaques from apoE−/−HSP27o/e mice showed almost 50% reductions in the area occupied by cholesterol clefts and free cholesterol, with fewer macrophages and reduced apoptosis but greater intimal smooth muscle cell and collagen content. The analysis of the aortic mechanical properties showed increased vessel stiffness in apoE−/−HSP27o/e mice (41% in female, 34% in male) compare to apoE−/− counterparts. Conclusions Chronic over-expression of HSP27 is atheroprotective in both sexes and coincides with reductions in lesion cholesterol accumulation as well as favorable plaque remodeling. These data provide new clues as to how HSP27 may improve not only the composition of atherosclerotic lesions but potentially their stability and resilience to plaque rupture.

Attenuation of atherogenesis via the anti-inflammatory effects of the selective estrogen receptor beta modulator 8β-VE2.

Background: Recent studies suggest that modulation of estrogen receptor β (ERβ) may play a crucial role in maintaining vascular homeostasis. We hypothesized that selective ERβ activation will attenuate atherogenesis via anti-inflammatory mechanisms. Methods and Results: Atherosclerosis-prone apoE−/− mice were ovariectomized and then fed a high-cholesterol diet with daily subcutaneous injections of the highly selective and potent ERβ agonist (8β-VE2) for 5 weeks. Compared with controls, treatment with 8β-VE2 reduced aortic arch atherosclerotic lesion areas by 34% of total and 75% of dense lesions, while not altering the serum lipid profile. We attribute these observed vascular effects solely to ERβ modulation as (1) treatment with the nonselective ER antagonist ICI 182,780 completely abrogated the beneficial vascular effects of 8β-VE2 and (2) uterine weight (a sensitive indicator of ERα modulation) did not change with 8β-VE2 treatment. Moreover, mice treated with 8β-VE2 had reduced serum interleukin 1β and tumor necrosis factor α levels. Finally, treatment of macrophages in vitro with 8β-VE2 blocked the uptake of acetylated low-density lipoprotein, suppressed the extracellular levels of the inflammatory cytokine tumor necrosis factor α, and enhanced the extracellular levels of the antiatherogenic/anti-inflammatory protein heat shock protein 27. Conclusions: Selective ERβ activation by 8β-VE2 attenuates atherogenesis and is associated with favorable modulation of vascular inflammation.

Back to the future: Rational maps for exploring acetylcholine receptor space and time☆

Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties.