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Monocyte chemoattractant protein-1 (MCP-1) plays an important role in initiating vascular inflammation; however, its cellular source in the injured vasculatures is unclear. Given the importance of high mobility group box 1 (HMGB1) in tissue injury, we investigated the role of vascular smooth muscle cells (VSMCs) in MCP-1 production in response to HMGB1. In primary cultured rat aortic VSMCs stimulated with HMGB1, the expression of MCP-1 and 5-lipoxygenase (LO) was increased. The increased MCP-1 expression in HMGB1 (30 ng/ml)-stimulated cells was significantly attenuated in 5-LO-deficient cells as well as in cells treated with zileuton, a 5-LO inhibitor. Likewise, MCP-1 expression and production were also increased in cells stimulated with exogenous leukotriene B4 (LTB4), but not exogenous LTC4. LTB4-induced MCP-1 expression was attenuated in cells treated with U75302, a LTB4 receptor 1 (BLTR1) inhibitor as well as in BLTR1-deficient cells, but not in 5-LO-deficient cells. Moreover, HMGB1-induced MCP-1 expression was attenuated in BLTR1-deficient cells or by treatment with a BLTR1 inhibitor, but not other leukotriene receptor inhibitors. In contrast to MCP-1 expression in response to LTB4, the increased MCP-1 production in HMGB1-stimulated VSMC was markedly attenuated in 5-LO-deficient cells, indicating a pivotal role of LTB4-BLTR1 signaling in MCP-1 expression in VSMCs. Taken together, 5-LO-derived LTB4 plays a key role in MCP-1 expression in HMGB1-exposed VSMCs via BLTR1 signaling, suggesting the LTB4-BLTR1 signaling axis as a potential therapeutic target for vascular inflammation in the injured vasculatures.

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.

Gomisin J (GJ) is a small molecular weight lignan found in Schisandra chinensis and has been demonstrated to have vasodilatory activity. In this study, the authors investigated the effect of GJ on blood pressure (BP) in angiotensin II (Ang II)-induced hypertensive mice. In addition, we determined the relative potencies of gomisin A (GA) and GJ with respect to vasodilatory activity and antihypertensive effects. C57/BL6 mice infused s.c. with Ang II (2 μg kg(-1) min(-1) for 2 weeks) showed an increase in BP and a decrease in plasma nitric oxide (NO) metabolites. In the thoracic aortas of Ang II-induced hypertensive mice, a decrease in vascular NO was accompanied by an increase in reactive oxygen species (ROS) production. Furthermore, these alterations in BP, plasma concentrations of NO metabolites and in the vascular productions of NO and ROS in Ang II-treated mice were reversed by the co-administration of GJ (1 and 3 μg kg(-1) min(-1)). In in vitro studies, Ang II decreased the cellular concentration of NO, which was accompanied by a reduction in phosphorylated endothelial nitric oxide synthase (eNOS) and an increase in ROS production. These eNOS phosphorylation and ROS production changes in Ang II-treated cells were also reversed by GJ pretreatment (0-3 μg ml(-1)). Interestingly, the vasodilatory and antihypertensive effects of GJ were more prominent than those of GA. Collectively, an increase in BP in mice treated with Ang II was markedly attenuated by GJ, which was attributed to the preservations of vascular NO bioavailability and eNOS function, and to the inhibition of ROS production in Ang II-induced hypertensive mice.

Taxifolin is a potent flavonoid that exerts anti-oxidative effect, and cilostazol increases intracellular cAMP levels by inhibiting phosphodiesterase 3 that shows antiinflammatory actions. BACE1 (β-site APP cleaving enzyme 1) is the rate-limiting enzyme responsible for the β-cleavage of amyloid precursor proteins to Aβ peptides. In this study, endogenous Aβ and C99 accumulation was explored in N2a Swe cells exposed to 1% FBS medium. Increased Aβ and C99 levels were significantly attenuated by taxifolin alone and in combination with cilostazol. Increased phosphorylated JAK2 at Tyr1007/1008 (P-JAK), phosphorylated STAT3 at Tyr 705 (P-STAT3) expressions and increased expressions of BACE1 mRNA and protein in the activated N2a Swe cells were significantly attenuated by taxifolin (10~50 μM), cilostazol (10~50 μM) alone and in combination at minimum concentrations. In these cells, decreased cytosol IκBα expression was elevated, and increased nuclear NF-κB p65 level and nuclear NF-κB p65 DNA binding activity were significantly reduced by taxifolin and cilostazol in a similar manner. Following STAT3 gene (~70% reduction) knockdown in N2a cells, Aβ-induced nuclear NF-κB and BACE1 expressions were not observed. Taxifolin, cilostazol, or resveratrol significantly stimulated SIRT1 protein expression. In SIRT1 gene-silenced (~50%) N2a cells, taxifolin, cilostazol, and resveratrol all failed to suppress Aβ1-42-stimulated P-STAT3 and BACE1 expression. Consequently, taxifolin and cilostazol were found to significantly decrease lipopolysaccharide (1-10 μg/ml)-induced iNOS and COX-2 expressions, and nitrite production in cultured BV-2 microglia cells and to increase N2a cell viability. In conclusion, taxifolin and cilostazol strongly inhibited amyloidogenesis in a synergistic manner by suppressing P-JAK2/P-STAT3-coupled NF-κB-linked BACE1 expression via the up-regulation of SIRT1.

A neuroprotective role of autophagy mediates the degradation of β-amyloid peptide (Aβ) in Alzheimer's disease (AD). The previous study showed cilostazol modulates autophagy by increasing beclin1, Atg5 and LC3-II expressions, and depletes intracellular Aβ accumulation. This study elucidated the mechanisms through which cilostazol modulates the autophagic degradation of Aβ in neurons. In N2a cells, cilostazol (10-30 μM), significantly increased the expression of P-AMPKα (Thr 172) and downstream P-ACC (acetyl-CoA carboxylase) (Ser 79) as did resveratrol (SIRT1 activator), or AICAR (AMPK activator), which were blocked by KT5720, compound C (AMPK inhibitor), or sirtinol. Furthermore, phosphorylated-mTOR (Ser 2448) and phosphorylated-P70S6K (Thr 389) expressions were suppressed, and LC3-II levels were elevated in association with decreased P62/Sqstm1 by cilostazol. Cilostazol increased cathepsin B activity and decreased p62/SQSTM 1, consequently decreased accumulation of Aβ1-42 in the activated N2aSwe cells, and these results were blocked by sirtinol, compound C and bafilomycin A1 (autophagosome blocker), suggesting enhanced autophagosome formation by cilostazol. In SIRT1 gene-silenced N2a cells, cilostazol failed to increase the expressions of P-LKB1 (Ser 428) and P-AMPKα, which contrasted with its effect in negative control cells transfected with scrambled siRNA duplex. Further, N2a cells transfected with expression vectors encoding pcDNA SIRT1 showed increased P-AMPKα expression, which mimicked the effect of cilostazol in N2a cells; suggesting cilostazol-stimulated expressions of P-LKB1 and P-AMPKα were SIRT1-dependent. Unlike their effects in N2a cells, in HeLa cells, which lack LKB1, cilostazol and resveratrol did not elevate SIRT1 or P-AMPKα expression, indicating cilostazol and resveratrol-stimulated expressions of SIRT1 and P-AMPKα are LKB1-dependent. In conclusion, cilostazol upregulates autophagy by activating SIRT1-coupled P-LKB1/P-AMPKα and inhibiting mTOR activation, thereby decreasing Aβ accumulation.

Mechanically stressed vascular smooth muscle cells (VSMCs) have potential roles in the development of vascular complications. However, the underlying mechanisms are unclear. Using VSMCs cultured from rat thoracic aorta explants, we investigated the effects of mechanical stretch (MS) on the cellular secretion of high mobility group box 1 (HMGB1), a major damage-associated molecular pattern that mediates vascular complications in stressed vasculature. Enzyme-linked immunosorbent assay (ELISA) demonstrated an increase in the secretion of HMGB1 in VSMCs stimulated with MS (0–3% strain, 60 cycles/min), and this secretion was markedly and time-dependently increased at 3% MS. The increased secretion of HMGB1 at 3% MS was accompanied by an increased cytosolic translocation of nuclear HMGB1; the acetylated and phosphorylated forms of this protein were significantly increased. Among various inhibitors of membrane receptors mediating mechanical signals, AG1295 (a platelet-derived growth factor receptor (PDGFR) inhibitor) attenuated MS-induced HMGB1 secretion. Inhibitors of other receptors, including epidermal growth factor, insulin-like growth factor, and fibroblast growth factor receptors, did not inhibit this secretion. Additionally, MS-induced HMGB1 secretion was markedly attenuated in PDGFR-β-deficient cells but not in cells transfected with PDGFR-α siRNA. Likewise, PDGF-DD, but not PDGF-AA, directly increased HMGB1 secretion in VSMCs, indicating a pivotal role of PDGFR-β signaling in the secretion of this protein in VSMCs. Thus, targeting PDGFR-β-mediated secretion of HMGB1 in VSMCs might be a promising therapeutic strategy for vascular complications associated with hypertension.

Monocyte-to-macrophage differentiation plays a central role in the pathophysiology of rheumatoid arthritis (RA)-associated inflammation because it results in the secretions of various inflammatory mediators in inflamed synovium, and thus, this differentiation is viewed as a clinical target. We aimed to determine whether SIRT1 inhibits the differentiation of monocytes from RA patients into macrophages by suppressing PU.1 phosphorylation. Monocytes from synovial fluid of RA patients (RAMCs), THP-1 monocytes, and mouse bone marrow-derived monocytes (BMDCs) were studied. The phorbol 12-myristate 13-acetate (PMA)-stimulated RA monocyte adherence was significantly inhibited by resveratrol (a SIRT1 activator), and this inhibition by resveratrol was prevented by pretreating cells with sirtinol (a SIRT1 inhibitor). Furthermore, resveratrol pretreatment inhibited PMA-induced expressions of macrophage surface markers (CD11b, CD14, and CD36) and PMA-induced NF-κB transcriptional activation and, thus, suppressed the secretions of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). In SIRT1 transgenic (Tg) mice, monocyte differentiation was inhibited and NF-κB transcriptional activity was suppressed and the expressions of TNF-α, IL-1β, and IL-6 were decreased at the protein and mRNA levels versus control C57BL/6 mice. Furthermore, SIRT1 activation by resveratrol suppressed PMA-induced phosphorylation and the nuclear translocation of PU.1 and, thus, inhibited monocyte differentiation. In conclusion, SIRT1 appears to inhibit monocyte to macrophage differentiation by suppressing PU.1 phosphorylation and inflammatory signaling, which suggests SIRT1 plays a critical role in the regulation of synovial inflammation in RA. Key message SIRT1 overexpression inhibits monocyte to macrophage differentiation. SIRT1 suppresses PU.1 phosphorylation. Inactivation of PU.1 phosphorylation inhibits monocyte differentiation. SIRT1 regulates inflammation by inhibiting NF-κB during monocyte differentiation.

SIRT1 signaling pathways modulate vascular inflammation; however, the precise role of SIRT1 in monocyte adhesion to the vascular endothelium, a key event initiating vascular inflammation, is unclear. Thus, this study investigated the roles and molecular interaction of SIRT1 and TLR2 in regulating monocyte adhesion to the vascular endothelium. In vitro, both Mac-1 expression and the endothelial adhesion of THP-1 cells stimulated with Pam3CSK4, a TLR2 ligand, were markedly increased in association with a decreased expression of SIRT1. In THP-1 cells stimulated with Pam3CSK4, the promoter activity and expression of SIRT1 were decreased. The TLR2-dependent suppression of SIRT1 expression in THP-1 cells was mediated by the transcription factors NF-κB and CREB, suggesting that the TLR2-mediated NF-κB and CREB signaling downregulated SIRT1 expression in monocytes. In peripheral blood monocytes (PBMCs) isolated from SIRT1 transgenic (TG) mice and THP-1 cells treated with recombinant SIRT1, both the increased Mac-1 expression and endothelial adhesion induced by Pam3CSK4 were significantly attenuated. In addition, the en face immunohistochemical study showed a marked increase in monocyte adhesion to the aortic endothelium of WT mice treated with Pam3CSK4, which was significantly attenuated in Pam3CSK4-treated SIRT1 TG mice. Moreover, a greater number of atherosclerotic plaques formed in WT mice fed a high-fat diet than in SIRT1 TG mice, indicating a pivotal role for SIRT1 in preventing vascular inflammation. Based on these results, SIRT1 might be a potential target for researchers aiming to develop therapeutic interventions for vascular inflammation, including atherosclerosis. Atherosclerosis: Intervening in the early stages of plaque formation Researchers have identified a possible new tool, the signaling molecule SIRT1, to fight vascular diseases such as atherosclerosis, hardening of the arteries. In atherosclerosis, plaque builds up in arteries, restricting blood flow and potentially causing heart attack or stroke. Early in the disease, white blood cells called monocytes stick to artery walls, triggering inflammation. Administering SIRT1 was known to decrease inflammation, but how it did so was unclear. Chi Dae Kim at Pusan National University in South Korea and co-workers investigated the signals that trigger monocytes to attach to artery walls, and how SIRT1 might prevent attachment. After identifying the signals, further testing in mice fed a high-fat diet showed that SIRT1 strongly decreased plaque formation. These findings illuminate the causes and progression of atherosclerosis and may help identify better treatments.

Retinal angiogenesis was delayed in VSMC-specific Akt1-deficient mice (Akt1 ∆SMC ) but not in Akt2 ∆SMC mice. The proliferation of ECs, recruitment of pericytes, and coverage of VSMCs to the endothelium were defective in Akt1 ∆SMC . The silencing of Akt1 in VSMCs led to the downregulation of angiopoietin 1 (Ang1) and the upregulation of Ang2. The activation of Notch3 in VSMCs was significantly reduced in the retinas of Akt1 ∆SMC mice. Silencing Akt1 suppressed the activation of Notch3. Moreover, the silencing of Notch3 downregulated Ang1, whereas the overexpression of Notch3 intracellular domain (NICD3) enhanced Ang1 expression. The nuclear localization and transcriptional activity of yes-associated protein (YAP) were affected by the expression level of Akt1. Silencing YAP downregulated Ang2 expression, whereas overexpression of YAP showed the opposite results. Ang1 antibody and Ang2 suppressed endothelial sprouting of wild-type aortic tissues, whereas the Ang2 antibody and Ang1 facilitated the endothelial sprouting of aortic tissues from Akt1 ∆SMC mice. Finally, severe hemorrhage was observed in Akt1 ∆SMC mice, which was further facilitated under streptozotocin (STZ)-induced diabetic conditions. Therefore, the Akt1-Notch3/YAP-Ang1/2 signaling cascade in VSMCs might play an essential role in the paracrine regulation of endothelial function. Blood vessel formation: Nearly identical proteins but different effects Proteins Akt1 and Akt2 are nearly identical, but they play different roles in angiogenesis, the formation of new blood vessels. Angiogenesis is crucial in wound healing and development, and also in tumor formation. Although Akt1’s role in angiogenesis is well studied, the role of other Akt isoforms—highly similar, related proteins—was unknown. Sun Sik Bae at Pusan National University School of Medicine, Yangsan, South Korea, and coworkers investigated how Akt1 and Akt2 influence angiogenesis in the retinas of mice. They found that despite having very similar sequences, Akt2 had almost no effect but Akt1 was crucial for vascular stability. Akt1 boosted levels of the blood vessel stabilization protein Ang1 and suppressed Ang2. Mice lacking Akt1 eventually showed blood vessel leakage. These results illuminate the mechanisms underlying blood vessel formation.

Molecular mechanisms underlying the decreased number of macrophages and T cells in the arteries of cholesterol-fed-rabbits following dexamethasone administration are unknown. We investigated the possibility that dexamethasone could affect activation of monocytic cells induced by oxygenated derivatives of cholesterol (oxysterols) using THP-1 monocyte/macrophage cells. 27-Hydroxycholesterol (27OHChol), an oxysterol elevated with hypercholesterolemia, enhanced production of CCL2, known as MCP1, chemokine from monocytes/macrophages and migration of the monocytic cells, but the CCL2 production and the cell migration were reduced by treatment with dexamethasone. Dexamethasone inhibited superproduction of CCL2 induced by 27OHChol plus LPS and attenuated transcription of matrix metalloproteinase 9 as well as secretion of its active gene product induced by 27OHChol. The drug downregulated cellular and surface levels of CD14 and blocked release of soluble CD14 without altering transcription of the gene. Dexamethasone also inhibited expression and phosphorylation of the NF-κB p65 subunit enhanced by 27OHChol. Collectively, these results indicate that dexamethasone inhibits activation of monocytes/macrophages in response to 27OHChol, thereby leading to decreased migration of inflammatory cells in milieu rich in oxygenated derivatives of cholesterol.