Yong-Xiang Chen

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.

Modulation of Estrogen Signaling by the Novel Interaction of Heat Shock Protein 27, a Biomarker for Atherosclerosis, and Estrogen Receptor β: Mechanistic Insight Into the Vascular Effects of Estrogens

Objective—We sought to discover proteins that associate with estrogen receptor beta (ER&bgr;) and modulate estrogen signaling. Methods and Result—Using a yeast 2-hybrid screen, we identified heat shock protein 27 (HSP27) as an ER&bgr;-associated protein. HSP27 is a recently identified biomarker of atherosclerosis that is secreted at reduced levels from atherosclerotic compared with normal arteries. In vitro protein-binding assays confirmed the specific interaction of HSP27 with ER&bgr; and not ER&agr;. HSP27 expression was absent in coronary arteries with complex atherosclerotic lesions. Interestingly, HSP27 expression was also absent in 60% of coronary arteries from young males and females (27±6.5 years) with normal histology or nonobstructive fatty streaks/atheromas. Moreover, the absence of HSP27 in these normal or minimally diseased arteries coincided with the loss of ER&bgr; expression. Only 35% of arteries showed coexpression of HSP27 and ER&bgr;. Relative to controls, estradiol-mediated transcription was reduced 20% with overexpression of HSP27 and increased 44% when HSP27 protein levels were reduced with HSP27 siRNA. Conclusions—HSP27, an ER&bgr;-associated protein, shows attenuated expression with coronary atherosclerosis and modulates estrogen signaling.

Extracellular HSP27 acts as a signaling molecule to activate NF-κB in macrophages

Heat shock protein 27 (HSP27) shows attenuated expression in human coronary arteries as the extent of atherosclerosis progresses. In mice, overexpression of HSP27 reduces atherogenesis, yet the precise mechanism(s) are incompletely understood. Inflammation plays a central role in atherogenesis, and of particular interest is the balance of pro- and anti-inflammatory factors produced by macrophages. As nuclear factor-kappa B (NF-κB) is a key immune signaling modulator in atherogenesis, and macrophages are known to secrete HSP27, we sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling in macrophages. Treatment of THP-1 macrophages with rHSP27 resulted in the degradation of an inhibitor of NF-κB, IκBα, nuclear translocation of the NF-κB p65 subunit, and increased NF-κB transcriptional activity. Treatment of THP-1 macrophages with rHSP27 yielded increased expression of a variety of genes, including the pro-inflammatory factors, IL-1β, and TNF-α. However, rHSP27 also increased the expression of the anti-inflammatory factors IL-10 and GM-CSF both at the mRNA and protein levels. Our study suggests that in macrophages, activation of NF-κB signaling by rHSP27 is associated with upregulated expression and secretion of key pro- and anti-inflammatory cytokines. Moreover, we surmise that it is the balance in expression of these mediators and antagonists of inflammation, and hence atherogenesis, that yields a favorable net effect of HSP27 on the vessel wall.

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.

Comparison of processing and sectioning methodologies for arteries containing metallic stents.

The histological study of arteries with implanted metallic scaffolding devices, known as stents, remains a technical challenge. Given that the arterial response to stent implantation can sometimes lead to adverse outcomes, including the re-accumulation of tissue mass within the stent (or in-stent restenosis), overcoming these technical challenges is a priority for the advancement of research and development in this important clinical field. Essentially, the task is to section the stent-tissue interface with the least amount of disruption of tissue and cellular morphology. Although many methacrylate resin methodologies are successfully applied toward the study of endovascular stents by a variety of research laboratories, the exact formulations, as well as subsequent processing and sectioning methodology, remain largely coveted. In this paper, we describe in detail a methyl methacrylate resinembedding methodology that can successfully be applied to tungsten carbide blade, as well as saw and grinding sectioning methods and transmission electron microscopy. In addition, we present a comparison of the two sectioning methodologies in terms of their effectiveness with regard to morphological, histochemical, and immunohistochemical analyses. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Inhibition of endothelial progenitor cell glycogen synthase kinase-3β results in attenuated neointima formation and enhanced re-endothelialization after arterial injury

AIMS Endothelial progenitor cells (EPCs) are circulating pluripotent vascular cells capable of enhancing re-endothelialization and diminishing neointima formation following arterial injury. Glycogen synthase kinase (GSK)-3beta is a protein kinase that has been implicated in the regulation of progenitor cell biology. We hypothesized that EPC abundance and function could be enhanced with the use of an inhibitor of GSK-3beta (GSKi), thereby resulting in improved arterial repair. METHODS AND RESULTS Human EPCs were expanded ex vivo, treated with a specific GSKi, and then assessed for both yield and functional characteristics by in vitro assays for adherence, apoptosis, and survival. In vivo functionality of treated human EPCs was assessed in immune-tolerant mice subjected to femoral artery wire injury. Re-endothelialization was assessed at 72 h and neointima formation at 7 and 14 days following injury. GSKi treatment resulted in an improvement in the yield of EPCs and a reduction in apoptosis in cells derived from both healthy controls and patients with coronary artery disease. Treatment also increased vascular endothelial growth factor secretion, up-regulated expression of mRNA for the alpha-4 integrin subunit, and improved adhesion, an effect which could be abrogated with an alpha-4 integrin blocking antibody. EPCs without or with ex vivo GSKi treatment enhanced re-endothelialization 72 h following injury as well as reduced neointima formation at 7 days (e.g. endothelial coverage: 7.2 +/- 1.7% vs. 70.7 +/- 5.8% vs. 87.2 +/- 4.1%; intima to media ratios: 1.05 +/- 0.19 vs. 0.39 +/- 0.08 vs. 0.14 +/- 0.02; P < 0.05 for all comparisons), an effect that was persistent at 14 days. CONCLUSION GSKi improves the functional profile of EPCs and is associated with improved re-endothelialization and reduced neointima formation following injury.

Pre-Procedural Atorvastatin Mobilizes Endothelial Progenitor Cells: Clues to the Salutary Effects of Statins on Healing of Stented Human Arteries

Objectives Recent clinical trials suggest an LDL-independent superiority of intensive statin therapy in reducing target vessel revascularization and peri-procedural myocardial infarctions in patients who undergo percutaneous coronary interventions (PCI). While animal studies demonstrate that statins mobilize endothelial progenitor cells (EPCs) which can enhance arterial repair and attenuate neointimal formation, the precise explanation for the clinical PCI benefits of high dose statin therapy remain elusive. Thus we serially assessed patients undergoing PCI to test the hypothesis that high dose Atorvastatin therapy initiated prior to PCI mobilizes EPCs that may be capable of enhancing arterial repair. Methods and Results Statin naïve male patients undergoing angiography for stent placement were randomized to standard therapy without Atorvastatin (n = 10) or treatment with Atorvastatin 80 mg (n = 10) beginning three days prior to stent implantation. EPCs were defined by flow cytometry (e.g., surface marker profile of CD45dim/34+/133+/117+). As well, we also enumerated cultured angiogenic cells (CACs) by standard in vitro culture assay. While EPC levels did not fluctuate over time for the patients free of Atorvastatin, there was a 3.5-fold increase in EPC levels with high dose Atorvastatin beginning within 3 days of the first dose (and immediately pre-PCI) which persisted at 4 and 24 hours post-PCI (p<0.05). There was a similar rise in CAC levels as assessed by in vitro culture. CACs cultured in the presence of Atorvastatin failed to show augmented survival or VEGF secretion but displayed a 2-fold increase in adhesion to stent struts (p<0.05). Conclusions High dose Atorvastatin therapy pre-PCI improves EPC number and CAC number and function in humans which may in part explain the benefit in clinical outcomes seen in patients undergoing coronary interventions.

Delayed re-endothelialization with rapamycin-coated stents is rescued by the addition of a glycogen synthase kinase-3β inhibitor

AIMS Drug-eluting stents (DESs) reduce neointima area and in-stent restenosis but delay re-endothelialization. Recently, we demonstrated that pharmacological expansion and functional enhancement of endothelial progenitor cells (EPCs) can be achieved by treatment with a glycogen synthase kinase-3beta inhibitor (GSKi)-even for feeble cells derived from coronary artery disease patients. GSKi treatment enhanced EPC adhesion via up-regulated expression of the alpha-4 integrin, ameliorated re-endothelialization, and reduced neointima formation in denuded murine arteries. Hence, we hypothesized that GSKi-coated stents (GSs) will enhance EPC adhesion and attenuate delayed vascular healing associated with rapamycin, a key DES agent. METHODS AND RESULTS In vitro human EPCs adhered to GS with affinities that were 2x, 14x, and 13x greater than vehicle (VSs)-, rapamycin (RSs)-, and rapamycin plus GSKi (RGSs)-coated stents, respectively. Stents were inserted in rabbit carotid arteries, and at 14 days, neointima area was 45 and 49% lower in GSs compared with bare metal stents (BMSs) and VSs. Moreover, RSs had a 47% larger neointima area than GSs, but RGSs reduced neointima area to a level comparable to GSs. Seven days after stenting, GSs displayed re-endothelialization that was 40, 33, and 42% greater than BMSs, VSs, and RSs, respectively. Moreover, RGSs had 41% more re-endothelialization than RSs. At 14 days, the 7-day re-endothelialization patterns persisted. CONCLUSION GSKi efficiently ameliorates the vascular response to stent implantation and has an important redeeming effect on the deleterious endothelial effects of rapamycin-coated stents.

The interaction and cellular localization of HSP27 and ERβ are modulated by 17β-estradiol and HSP27 phosphorylation

Recently, we identified heat shock protein 27 (HSP27) as an estrogen receptor-β (ERβ) associated protein that acts as a co-repressor of estrogen signaling and serves as a biomarker of atherosclerosis. In this study, we sought to further characterize the subcellular interaction of HSP27 and ERβ, as well as explore the factors that may modulate this interaction. In vitro we determined that phosphorylated HSP27 is retained in the cytoplasm after treatment with 17β-estradiol and to a lesser extent with heat shock. Under all experimental conditions ERβ was found to be slightly more abundant in the cytoplasm compared to the nucleus. HSP27 and ERβ associate in both the cytoplasm and nucleus, however, co-localization studies reveal that in the presence of 17β-estradiol, a significant portion of this interaction occurs outside of the nucleus. These data highlight an extranuclear interaction between ERβ and HSP27 that may be of potential importance in modulating estrogen signaling.

Heat Shock Protein 27: Clue to Understanding Estrogen-Mediated Atheroprotection?

Although the use of estrogen replacement therapy for postmenopausal women has been dramatically curtailed due to an unfavorable risk-benefit profile, there remains strong experimental evidence that ovarian hormones have a favorable effect on vessel wall homeostasis. We recently discovered that release of heat shock protein 27 (HSP27) into the serum is atheroprotective and mediated by ovarian hormones, preferentially functioning via estrogen receptor-beta. HSP27 binds scavenger receptor-A, reduces cholesterol uptake in macrophages, and attenuates mediators of vascular inflammation. Therefore, it is attractive to consider HSP27 as the active foot soldier of estrogens and potentially a novel therapeutic opportunity for vascular disease.