Tumor necrosis factor-alpha (TNF-alpha) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF-alpha inhibits the mineralization of osteoblasts, the effect of TNF-alpha on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF-alpha on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF-kappaB activation on TNF-alpha activity. TNF-alpha treatment dose-dependently increased osteogenic differentiation over the first 3 days of treatment. TNF-alpha activated ERK and increased NF-kappaB promoter activity. PDTC, an NF-kappaB inhibitor, blocked the osteogenic differentiation induced by TNF-alpha and TLR-ligands, but U102, an ERK inhibitor, did not. Overexpression of miR-146a induced the inhibition of IRAK1 expression and inhibited basal and TNF-alpha- and TLR ligand-induced osteogenic differentiation. TNF-alpha and TLR ligands increased the expression of transcriptional coactivator with PDZ-binding motif (TAZ), which was inhibited by the addition of PDTC. A ChIP assay showed that p65 was bound to the TAZ promoter. TNF-alpha also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF-alpha enhances osteogenic differentiation of hADSC via the activation of NF-kappaB and a subsequent increase of TAZ expression.

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.Keywords: Fibroblast r HIF-1α r angiogenesis r HMGB1 r Rheumatoid arthritis Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by hyperplastic synovium in association with immunemediated inflammatory synovitis involving the ingress of abundant Correspondence: Prof. Chi Dae Kim e-mail: chidkim@pusan.ac.kr leucocytes and enhanced angiogenesis. Furthermore, blood vessel growth supports the proliferation of inflammatory synovial pannus and ingress of inflammatory leukocytes into synovial tissues during the development and progression of arthritis [1].High-mobility group box protein 1 (HMGB1), a nuclear DNAbinding protein, is released by passive diffusion from necrotic cells and activated macrophages, and is found extracellularly in abundance in synovitis. Furthermore, HMGB1 levels are reportedwww.eji-journal.eu Eur. J. Immunol. 2015. 45: 1216-1227 Molecular immunology 1217 to be higher in RA synovial fluid than in the synovial fluid of osteoarthritis (OA) [2][3][4]. In addition, it has been demonstrated that released HMGB1 binds to its receptors, such as, TLR2, TLR4, and RAGE [5,6], and that these mediate inflammatory responses by inducing the productions of proinflammatory cytokines, such as, 8]. Furthermore, Lin et al. [9] have demonstrated HMGB1 initiates TLR4-dependent neovascularization in cornea. It has also been reported that hypoxia increases extracellular HMGB-1, which colocalizes with tissue h...

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 Gprotein-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 ECFCmediated 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

This study shows that 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2(1H)-quinolinone (cilostazol) suppresses the atherosclerotic lesion formation in the low-density lipoprotein receptor (Ldlr)-null mice. Ldlr-null mice fed a high cholesterol diet showed multiple plaque lesions in the proximal ascending aorta including aortic sinus, accompanied by increased macrophage accumulation with increased expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). Supplementation of cilostazol (0.2% w/w) in diet significantly decreased the plaque lesions with reduced macrophage accumulation and suppression of VCAM-1 and MCP-1 in situ. Increased superoxide and tumor necrosis factor-␣ (TNF-␣) production were significantly lowered by cilostazol in situ as well as in cultured human umbilical vein endothelial cells (HUVECs). TNF-␣-induced increased inhibitory B␣ degradation in the cytoplasm and nuclear factor-B (NF-B) p65 activation in the nuclei of HUVECs were reversed by cilostazol (1 ϳ 100 M) as well as by (E)-3[(4-t-butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085) (10 M), suggesting that cilostazol strongly inhibits NF-B activation and p65 translocation into the nuclei. Furthermore, in gel shift and DNA-binding assay, cilostazol inhibited NF-B/DNA complex and nuclear DNA-binding activity of the NF-B in the nuclear extracts of the RAW 264.7 cells. Taken together, it is suggested that the antiatherogenic effect of cilostazol in cholesterol-fed Ldlr-null mice is ascribed to its property to suppress superoxide and TNF-␣ formation, and thereby reducing NF-B activation/transcription, VCAM-1/MCP-1 expressions, and monocyte recruitments.Evidence accumulates that atherogenesis is closely related to the inflammatory and proliferative responses of the endothelium after injury (Ross, 1993). During early stages of the atherosclerosis, adhesion and chemoattractant molecules, including vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), are secreted by the activated endothelial cells in the atherosclerotic lesions, by which the immune cells and monocytes are recruited and migrated into the intimal area of the vascular wall (Reape and Groot, 1999). Reactive oxygen species and TNF-␣ are critically implicated not only in the induction of endothelial apoptosis (Dimmeler et al., 1998) but also in the development and progression of atherosclerotic lesions in humans (Meyer et al., 1999).Inactive NF-B resides in the cytoplasm bound by its inhibitory subunit, IB␣ (Pahl, 1999). Inflammatory stimuli including TNF-␣ and endotoxin lead to degradation of IB␣ by its phosphorylation pathway (Chen et al., 1995), which allows translocation of active NF-B into the nucleus, where it regulates gene expression and binds to the promoter of the target genes such as VCAM-1 and MCP-1.The low-density lipoprotein receptor (Ldlr)-null mouse is an animal model of homozygous familial hypercholesterolemia characterized by an absence of functional LDL receptors. Th...

This study shows cilostazol effect to prevent remnant lipoprotein particle (RLP)-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). Upon incubation of HUVECs with RLP (50 g/ml), adherent monocytes significantly increased by 3.3-fold with increased cell surface expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, E-selectin, and monocyte chemoattractant protein-1 (MCP-1). Cilostazol (ϳ1-100 M) concentration dependently repressed these variables as did (E)3-[(4-t-butylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7085) (10 M), a specific nuclear factor-B (NF-B) inhibitor. Cilostazol effects were significantly antagonized by iberiotoxin (1 M), a maxi-K channel blocker. RLP significantly increased expression of lectin-like receptor for oxidized low-density lipoprotein (LDL) (LOX-1) receptor protein. Upon transfection with antisense LOX-1 oligodeoxynucleotide (As-LOX-1), LOX-1 receptor expression was reduced, whereas HUVECs with sense LOX-1 oligodeoxynucleotide did express high LOX-1 receptor. RLP-stimulated superoxide and tumor necrosis factor-␣ levels were significantly lowered with decreased expression of VCAM-1 and MCP-1 by transfection with As-LOX-1 as did polyinosinic acid (10 g/ml, a LOX-1 receptor inhibitor). RLP significantly degraded inhibitory B␣ in the cytoplasm and activated nuclear factor-B (NF-B) p65 in the nucleus of HUVECs with increased luciferase activity of NF-B, all of which were reversed by cilostazol (10 M), BAY 11-7085, and polyinosinic acid. Together, cilostazol suppresses RLP-stimulated increased monocyte adhesion to HUVECs by suppression of LOX-1 receptorcoupled NF-B-dependent nuclear transcription via mediation of the maxi-K channel opening.Atherosclerosis is known as chronic inflammatory processes resulting from interaction between oxidized low-density lipoprotein (Ox-LDL), macrophages, lymphocytes, and other cellular elements of the arterial wall (Ross, 1999). Recent clinical evidence has suggested that endothelial dysfunction elicited by Ox-LDL is critically important in the pathogenesis of atherosclerosis, in that inflammation plays a central role in its development (Ross, 1999;Blake and Ridker, 2001). Since Nakajima et al. (1993) have developed a simple and rapid assay method for determination of remnant lipoprotein particles (RLP)-cholesterol, i.e., chylomicron and VLDL remnants, a number of reports have focused on the role of RLP, derived from VLDL and chylomicrons, as an atherogenic factor (Hodis, 1999). It has been shown that RLP elicit endothelial vasomotor dysfunction in human coronary arteries and in isolated rabbit aorta . Endothelium-derived reactive oxygen species initiate and propagate free radical chain reactions in polyunsaturated fatty acid in RLP (Doi et al., 2000). Ox-LDL elicits endothelial dysfunction by enhancing expresArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.077826. ABBREVIATIONS:Ox-LDL, oxidized low-density lipoprot...