High‐mobility group box protein 1 (HMGB1), a nonhistone nuclear protein and a cytokine mediator, is implicated in the pathogenesis of rheumatoid arthritis (RA). Extracellular HMGB1 binds to its receptors and triggers downstream signal cascade leading to the perpetuation of synovitis and local tissue invasion. Here, we investigated a novel role of HMGB1 in regulating hypoxia‐inducible factor (HIF)‐1α to mediate angiogenesis in RA synovium. HIF‐1α mRNA levels and activities in synovial fibroblasts from RA patients were enhanced by HMGB1. Pharmacological inhibition of TLR4 and NF‐kappaB activation blocked the HMGB1‐dependent upregulation of HIF‐1α mRNA expression and its activity, suggesting the involvement of transcriptional regulation. HMGB1 stimulated expression of vascular endothelial growth factor (VEGF), and inhibition of HIF‐1α attenuated HMGB1‐induced VEGF. Conditioned media derived from HMGB1‐stimulated synovial fibroblasts enhanced tube formation in human microvascular endothelial cells by upregulating HIF‐1α. In the joint tissues of mice with collagen‐induced arthritis, treatment with anti‐HMGB1 neutralizing antibody prevented blood vessel formation in association with decreased expression of HIF‐1α. These observations support the idea that increased HMGB1 induces an extension of inflamed synovium by accelerating angiogenesis in RA through enhancement of HIF‐1α activation. Therefore, inhibition of HMGB1 could prove beneficial for the treatment of angiogenesis in RA.

Macrophages are crucially involved in the pathogenesis of rheumatoid arthritis (RA). Macrophages of the M1 phenotype act as pro-inflammatory mediators in synovium, whereas those of the M2 phenotype suppress inflammation and promote tissue repair. SIRT1 is a class 3 histone deacetylase with anti-inflammatory characteristics. However, the role played by SIRT1 in macrophage polarization has not been defined in RA. We investigated whether SIRT1 exerts anti-inflammatory effects by modulating M1/M2 polarization in macrophages from RA patients. In this study, SIRT1 activation promoted the phosphorylation of an adenosine monophosphate-activated protein kinase (AMPK) α/acetyl-CoA carboxylase in macrophages exposed to interleukin (IL)-4, and that this resulted in the expressions of M2 genes, including MDC, FcεRII, MrC1, and IL-10, at high levels. Furthermore, these expressions were inhibited by sirtinol (an inhibitor of SIRT1) and compound C (an inhibitor of AMPK). Moreover, SIRT1 activation downregulated LPS/interferon γ-mediated NF-κB activity by inhibiting p65 acetylation and the expression of M1 genes, such as CCL2, iNOS, IL-12 p35, and IL-12 p40. Macrophages from SIRT1 transgenic (Tg)-mice exhibited enhanced polarization of M2 phenotype macrophages and reduced polarization of M1 phenotype macrophages. In line with these observations, SIRT1-Tg mice showed less histological signs of arthritis, that is, lower TNFα and IL-1β expressions and less severe arthritis in the knee joints, compared to wild-type mice. Taken together, the study shows activation of SIRT1/AMPKα signaling exerts anti-inflammatory activities by regulating M1/M2 polarization, and thereby reduces inflammatory responses in RA. Furthermore, it suggests that SIRT1 signaling be viewed as a therapeutic target in RA.

Vascular smooth muscle cells (VSMCs) are the major cell type in the blood vessel walls, and their phenotypic modulation is a key cellular event driving vascular remodeling. Although high mobility group box-1 (HMGB1) plays a pivotal role in inflammatory processes after vascular injuries, the importance of the links between VSMCs, HMGB1 and vascular inflammation has not been clarified. To prove the hypothesis that VSMCs might be active players in vascular inflammation by secreting inflammatory cytokines, we investigated the proinflammatory effects of HMGB1 and its intermediary signaling pathways in VSMCs. When cultured human VSMCs were stimulated with HMGB1 (10–500 ng/ml), IL-1β production was markedly increased. HMGB1 also increased the expression of NLRP3 inflammasome components including NLRP3, ASC and caspase-1. Among these components, HMGB1-induced expressions of NLRP3 and caspase-1 were markedly attenuated in TLR2 siRNA-transfected cells, whereas ASC and caspase-1 expressions were reduced in RAGE-deficient cells. In TLR4-deficient cells, HMGB1-induced caspase-1 expression was significantly attenuated. Moreover, IL-1β production in HMGB1-stimulated cells was significantly reduced in cells transfected with caspase-1 siRNA as well as in cells treated with monoclonal antibodies or siRNAs for TLR2, TLR4 and RAGE. Overall, this study identified a pivotal role for NLRP3 inflammasome and its receptor signaling involved in the production of IL-1β in VSMCs stimulated with HMGB1. Thus, targeting HMGB1 signaling in VSMCs offers a promising therapeutic strategy for treating vascular remodeling diseases.

Endothelial colony‐forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G‐protein‐coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA‐mediated silencing of FPR2, but not FPR3, abrogated WKYMVm‐induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC‐mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm‐induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2‐dependent mechanism. Stem Cells 2014;32:779–790

Background and Purpose– To clarify the role of vascular NAD(P)H oxidase in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), both the activity and/or activation mechanisms...