Tara Seibert

Neuroimmune Guidance Cue Semaphorin 3E Is Expressed in Atherosclerotic Plaques and Regulates Macrophage Retention

Objective—The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques. Approach and Results—Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe–/– mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque. Conclusions—Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.

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.

Endothelial Expression of Guidance Cues in Vessel Wall Homeostasis Dysregulation Under Proatherosclerotic Conditions

Objective—Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues may regulate leukocyte trafficking into the vascular wall during atherogenesis. Approach and Results—We demonstrate that members of the netrin, semaphorin, and ephrin family of guidance molecules are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and semaphorin3A are expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes. Endothelial expression of these negative guidance cues is downregulated by proatherogenic factors, including oscillatory shear stress and proinflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show using intravital microscopy that inhibition of netrin-1 or semaphorin3A using blocking peptides increases leukocyte adhesion to the endothelium. Unlike netrin-1 and semaphorin3A, the guidance cue ephrinB2 is upregulated under proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines. Conclusions—The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking during atherogenesis.

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.

Serum Heat Shock Protein 27 Levels Represent a Potential Therapeutic Target for Atherosclerosis: Observations From a Human Cohort and Treatment of Female Mice

OBJECTIVES The aim of this study was to evaluate the potential of serum heat shock protein 27 (HSP27) as a therapeutic target in coronary artery disease. BACKGROUND Expression of HSP27 in human coronary arteries diminishes with the progression of atherosclerosis, whereas ubiquitous HSP27 overexpression in apolipoprotein E(-/-) (ApoE(-/-)) mice attenuates atherogenesis. However, it remains unclear whether increasing serum HSP27 levels alone is sufficient for atheroprotection. METHODS Low- and intermediate-risk patients undergoing coronary or computed tomography angiography had serum HSP27 levels measured. Elevated serum HSP27 levels in female atheroprone ApoE(-/-) mice were achieved by transplantation with HSP27 overexpressing bone marrow or by administering recombinant HSP27. RESULTS Patients with >50% stenosis in any major epicardial artery had lower HSP27 levels compared with those free of atherosclerosis (median [interquartile range]: 2,176 pg/ml [551-5,475] vs. 6,200 pg/ml [2,575-9,560]; p < 0.001). After a 5-year period of clinical follow-up, low serum HSP27 levels (<50th percentile) were predictive of subsequent major adverse cardiovascular events (hazard ratio: 2.93, 95% confidence interval: 1.06 to 8.12; p = 0.04). In experimental murine models of atherosclerosis, increasing serum HSP27 levels both reduced de novo atherosclerotic lesion formation and enhanced features of plaque stability. CONCLUSIONS In humans, low serum HSP27 levels are associated with the presence of coronary artery disease and prognostic of future adverse clinical events. In mouse models of atherosclerosis, increasing HSP27 levels reduced lesion progression and promoted features of plaque stability. Serum HSP27 levels may represent a potential therapeutic target for atherosclerosis.

Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE(-/-) mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (-34%; p<0.005) and uptake (-38%, p<0.05). rHSP27 reduced SR-A mRNA (-39%, p=0.02), total protein (-56%, p=0.01) and cell surface (-53%, p<0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p<0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE(-/-) and ApoE(-/-) SR-A(-/-) mice fed with a high fat diet were treated for 3weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE(-/-) mice by 39% and 36% (p<0.05), respectively, but not in ApoE(-/-)SR-A(-/-) mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.

Glycogen Synthase Kinase-3β Inhibition Augments Diabetic Endothelial Progenitor Cell Abundance and Functionality via Cathepsin B: A Novel Therapeutic Opportunity for Arterial Repair

Progenitor cell therapy is hindered in patients with diabetes mellitus (DM) due to cellular senescence. Glycogen synthase kinase-3β (GSK3β) activity is increased in DM, potentially exacerbating impaired cell-based therapies. Thus, we aimed to determine if and how GSK3β inhibitors (GSKi) can improve therapeutic efficacy of endothelial progenitor cells (EPC) from patients with DM. Patients with DM had fewer EPCs and increased rates of apoptosis. DM EPCs also exhibited higher levels of GSK3β activity resulting in increased levels of phosphorylated β-catenin. Proteomic profiling of DM EPCs treated with GSKi identified 37 nonredundant, differentially regulated proteins. Cathepsin B (cathB) was subsequently confirmed to be differentially regulated and showed 40% less baseline activity in DM EPCs, an effect reversed by GSKi treatment. Finally, in vivo efficacy of cell-based therapy was assessed in a xenotransplant femoral wire injury mouse model. Administration of DM EPCs reduced the intima-to-media ratio, an effect that was further augmented when DM EPCs were pretreated with GSKi yet absent when cathB was antagonized. In DM, increased basal GSK3β activity contributes to accelerated EPC cellular senescence, an effect reversed by small molecule antagonism of GSK3β, which enhanced cell-based therapy after vascular injury.