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Background: Inflammation plays a primordial role in atherosclerosis. Perivascular adipose tissue (PVAT) that surrounds the vasculature has been reported to undergo phenotypic changes in response to vascular inflammation. However, regulatory roles of PVAT in vascular inflammation and subsequent pathological remodeling remain unclear. Methods and Results: Endovascular injury was generated by wire insertion into the mice femoral artery. Here, we showed that vascular injury induced the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulated in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by adipose tissue-specific genetic ablation of PRDM16, a key regulator to beiging, exacerbated inflammation and vascular remodeling following injury. Local knockdown of Prdm16 in perivascular tissues of wild-type mice using small interfering RNA similarly exacerbated inflammation and vascular remodeling. Conversely, activation of PVAT beiging attenuated inflammation and pathological vascular remodeling. Single-cell RNA sequencing revealed that beige adipocytes abundantly expressed neuregulin 4 ( Nrg4 ) which critically regulated alternative macrophage activation. Furthermore, Nrg4 knockdown abolished the PVAT-mediated anti-inflammatory effects on macrophages. Importantly, significant beiging was observed in the diseased aortic PVAT in patients with acute aortic dissection. Conclusions: Endovascular injury induced the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitated alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrated the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis.

Background: Perivascular adipose tissue (PVAT) possibly plays a pivotal role in the development of atherosclerosis through its direct local action to the vessels. However, its physiological and molecular mechanisms are less understood. Visceral and subcutaneous white adipose tissues exert a brown adipose tissue-like phenotype under specific conditions such as cold exposure, i.e. browning. We here investigated the pathophysiological role of browning of PVAT against the development of atherosclerosis after endovascular injury. Methods and Results: Endovascular injury was generated by wire insertion into the femoral artery of C57BL/6 female mice. Transcriptome analysis revealed robust upregulation of brown adipose tissue markers such as Ucp1 , Elovl3 , Cox8b and Cidea in injured arteries. Notably, administration of an atheroprotective agent, 17-beta estradiol, significantly inhibited these changes; Ucp1 was the most down-regulated gene by 17-beta estradiol administration in the entire gene set (log2-fold change = -6.58, false discovery rate = 0.003). Consistently, the upregulation of browning markers after endovascular injury and these inhibitions by 17-beta estradiol were confirmed in PVAT by quantitative real-time PCR, western blot and immunohistochemical staining. The present study also demonstrated that vascular injury promotes macrophage infiltration in PVAT accompanied with upregulation of inflammatory cytokine production. Furthermore, we confirmed spatiotemporal synchronicity between browning and inflammation in PVAT by immunohistochemical staining. Conclusions: We observed that endovascular injury elicits browning in PVAT accompanied with exacerbated inflammation, and that atheroprotective 17-beta estradiol strongly inhibits this phenomenon. These findings may provide novel insights into pathogenesis of the atherosclerosis.

Background: Estrogen plays a significant role in cardiovascular benefits. As one of its beneficial pathways, ERα non-nuclear signaling is known to be protective for the vascular system, but its role for cardiac remodeling is unknown thus far. We previously reported cGMP pathways are one of the critical therapeutic targets for heart failure and ERα non-nuclear signaling played a pivotal role in cardioprotection and was indispensable to the therapeutic efficacy of cGMP-PDE5 inhibition. However, it is still remaining undetermined which cells are playing the key role in cardioprotective effects via ERα non-nuclear signaling. Methods: We generated genetically modified mouse lacking ERα in cardiomyocytes (ERα f/f /αMHC Cre/+ ) or endothelial cells (ERα f/f /Tie2 Cre/+ ) and assessed the effects of chronic pressure-overload (TAC) and the efficacy of PDE5 inhibitor (PDE5i) sildenafil after TAC. In addition, for the first time we generated endothelial cell specific genet-modified mouse lacking interactions between p85α and ERα which are critical for non-nuclear signaling (ERα KI/KI /Tie2 Cre/+ ). Cardiac functions were assessed for evaluating the effects of endothelial ERα non-nuclear singlaing to failing hearts. Results: Under physiological E2 status, PDE5i’s anti-remodeling effects after TAC were abrogated in ERα f/f /Tie2 Cre/+ (FS(%) ± SEM with vs without PDE5i 28.16 ± 3.43 vs 31.87 ± 2.30) but not in ERα f/f /αMHC Cre/+ (FS(%) ± SEM with PDE5i vs without PDE5i 54.57 ± 3.418 vs 43.3 ± 1.21) consistent with hemodynamic analysis. Assessment with ERα KI/KI /Tie2 Cre/+ showed decreased cardiac functions even with physiological E2. Hemodynamic analysis also supported the cardioprotective effects of ERα non-nuclear signaling in endothelial cells. Conclusion: ERα non-nuclear reaction in endothelial cells plays an important role in cardio-protective mechanism, and endothelial estrogen signals are indispensable to the therapeutic efficacy of cGMP-PDE5 inhibition. Our results clarified one of the cardioprotective signals of estrogen and highlighted the new strategy for heart failure treatment.

Introduction: Regulator of G protein signaling 2 (RGS2) is a negative regulator of G protein-coupled signaling, and is potentiated by cGMP-PKG. Our prior work using a global deletion model demonstrated the protective role for RGS2 against mal-adaptive cardiac remodeling, which is essential to the cGMP-PDE5 inhibitor efficacy; however, cell type-specific contribution remains to be determined, given the expression of RGS2 in myocytes, fibroblasts and vascular smooth muscle cells. We hypothesized that myocyte RGS2 might serve as a primary contributor to the cardio-protection and mediates cGMP-PKG benefits. Method: We generated cardiomyocyte-specific RGS2 knockout mice (cKO) on a C57BL/6 background. Pressure overload was produced by transverse aortic constriction (TAC) in male RGS2-cKO mice (RGS2cKO) and their littermate controls (WT). To investigate cGMP-PKG enhancing effects, a soluble guanylyl cyclase stimulator (riociguat) was orally administrated daily (3mg/kg/day). Invasive hemodynamics, anatomy, histology and molecular signaling were assessed at 1wk after TAC. Results: RGS2-cKO revealed exacerbated dilative cardiac remodeling (LVDD: cKO 3.57±0.36 vs WT 2.90±0.16) and severely impaired cardiac function (dPdt/IP: cKO 166±3.49 vs WT 195±7.40), while their basal phenotype was undistinguishable from WT. cKO-TAC hearts showed larger cardiomyocyte size and more fibrosis than WT-TAC hearts, associated with aberrant gene expression induction including Myh6, Myh7, Col1a2, and TGFβ . Intriguingly, concomitant riociguat treatment reduced LV chamber size and fibrosis but failed to improve cardiac performance in cKO-TAC hearts, while riociguat ameliorated all these in WT-TAC hearts. Conlusion: These results suggest that RGS2-PKG axis in cardiac myocytes critically contributes to cardiac functional performance and that cGMP-PKG exerts anti-fibrotic effects independently of myocyte conditions.

Background: Estrogen exerts complex physiological effects via its rapid (non-genomic) and genomic actions. In particular, rapid signaling of estrogen receptor alpha (ERα) has been implicated in the vasculo-protective effects, in which both endothelial and smooth muscle cells might be involved. However, no prior studies have determined the role of ERα rapid signaling in the endothelium. This study aims to clarify the impact of ERα rapid signaling in the vasculo-protection, using a novel mouse model lacking endotheilal-specific ERα rapid signaling. Methods and Results: In immunoblotting, p85α and pGSK3β, non-genomic pathway downstream signals, were reduced in ERα mutants RR259/260AA with estradiol (E2) stimulation at concentrations of 10 -11 and 10 -10 M . ERE-luciferase assay demonstrated E2 induced genomic pathway activity was preserved even in ERα mutants RR259/260AA. We generated a mouse model in which rapid signaling of ERα was ablated in the endothelium by crossing Tie2-Cre transgenic mice with floxed ERα mutants (RR 259/260 AA) in which p85α and ERα interaction was disrupted. In endothelial cells isolated from ERα KI/KI Tie2 cre/+ animals, E2 at a concentration of 10 -8 M failed to induce phosphorylation of Akt, confirming the absence of ERα rapid signaling. Baseline characteristics at 8 to 12 weeks of age were undistinguishable between the genotypes, including body weight (KI:19.2±0.366 g vs WT: 18.9±0.356 g ) systolic blood pressure (KI:106.1±3.336 vs WT: 103.5±2.133 mmHg) and echocardiographic fractional shortening (KI:54.4±0.751 vs WT: 55.19±0.947 %). We then assessed how vascular remodeling process was impacted in a carotid artery wire injury model. Histological analyses with Elastica van Gieson staining two weeks after induction of injury revealed that wall thickness and area of medial layer composed mainly of smooth muscle cells were significantly increased in ERα KI/KI Tie2 cre/+ mouse, as compared to wild types (KI:44.85±1.948 μm vs WT: 29.32±1.753 μm , KI:42,047±1,560 μm 2 vs WT: 32,815±1,211 μm 2 , respectively ). Conclusions: Our results demonstrate that the rapid signaling of ERα in the endothelium critically regulates vascular smooth muscle cell growth after vascular injury, suggesting the essential role to vascular remodeling.

Objective— The proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in vascular diseases, such as atherosclerosis and restenosis, after percutaneous coronary intervention. Many studies have shown that estrogen inhibits VSMC proliferation in response to vascular injury in the mouse carotid injury model. However, the mechanisms that mediate these effects remain unclear. Here, we investigated the mechanisms by which estrogen inhibits VSMC proliferation. Approach and Results— We established a novel transgenic mouse line, referred to as the disrupting peptide mice, in which rapid estrogen receptor (ER)–mediated signaling is abolished by overexpression of a peptide that prevents the ER from forming a signaling complex necessary for rapid signaling. Carotid artery VSMCs from disrupting peptide mice or littermate wild-type female mice were obtained by the explant method. In VSMCs derived from wild-type mice, estrogen significantly inhibited VSMC proliferation. Phosphorylation levels of Akt and extracellular regulated kinase induced by platelet derived growth factor were significantly inhibited by estrogen pretreatment. Estrogen enhanced complex formation between ERα and protein phosphatase 2A (PP2), and enhanced PP2A activity. The blockade of PP2A activity abolished the estrogen-induced antiproliferative effect on VSMCs. In contrast, none of these effects of estrogen observed in the wild-type VSMCs were observed in VSMCs derived from disrupting peptide mice. These results support that rapid, non-nuclear ER signaling is required for estrogen-induced inhibition of VSMC proliferation, and further that PP2A activation by estrogen mediates estrogen-induced antiproliferative effects. Conclusions— These findings support that PP2A activation via rapid, non-nuclear ER signaling may be a novel target for therapeutic approaches to inhibit VSMC proliferation, which plays a central role in atherosclerosis and restenosis.

We studied the neural activation associated with the expectancy of emotional stimuli using whole brain fMRI. Fifteen healthy subjects underwent fMRI scanning during which they performed a warned reaction task using emotional pictures carrying pleasant, unpleasant, or neutral content. The task involved an expected or unexpected condition. Data were analyzed by comparing the images acquired under the different conditions. In the expected condition, compared with the unexpected condition, significant activation was observed in the medial, inferior and dorsolateral prefrontal cortex. Whereas the expectancy of pleasant stimuli produced activation in the left dorsolateral and left medial prefrontal cortex as well as in the right cerebellum, the expectancy of unpleasant stimuli produced activation in the right inferior and right medial prefrontal cortex, the right amygdala, the left anterior cingulate cortex, and bilaterally in the visual cortex. These results suggest that the expectancy of emotional stimuli is mediated by the prefrontal area including the medial, inferior, and dorsolateral prefrontal cortex. Furthermore, our data suggest that left frontal activation is associated with the expectancy of pleasant stimuli and that right frontal activation is associated with the expectancy of unpleasant stimuli. Finally, our findings suggest that the amygdala and anterior cingulate cortex may play an important role in the expectancy of unpleasant stimuli and that the input of this negative information is modulated by these specific brain areas.

Objective— Granulocyte colony stimulating factor (G-CSF) has been reported recently to prevent cardiac remodeling and dysfunction after acute myocardial infarction through signal transducer and activator of transcription 3 (STAT3). In this study, we examined acute effects of G-CSF on the heart against ischemia-reperfusion injury. Methods and Results— Rat hearts were subjected to global 35-minute ischemia and 120-minute reperfusion in Langendorff system with or without G-CSF (300 ng/mL). G-CSF administration was started at the onset of reperfusion. Triphenyltetrazolium chloride staining revealed that G-CSF markedly reduced the infarct size. G-CSF strongly activated Janus kinase 2 (Jak2), STAT3, extracellular signal-regulated kinase (ERK), Akt, and endothelial NO synthase (NOS) in the hearts subjected to ischemia followed by 15-minute reperfusion. The G-CSF–induced reduction in infarct size was abolished by inhibitors of phosphatidylinositol 3-kinase, Jak2, and NOS but not of mitogen-activated protein kinase kinase (MEK). Conclusions— These results suggest that G-CSF acts directly on the myocardium during ischemia-reperfusion injury and has acute nongenomic cardioprotective effects through the Akt–endothelial NOS pathway.