Background/Aims: The pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins (Angs) play a prominent role in synovial angiogenesis, an early and critical event in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-35 is an anti-inflammatory cytokine that attenuates collagen-induced arthritis, however, the mechanisms involved are not fully understood. Methods: The effects of IL-35 on endothelial cell migration, adhesion, and tube formation were examined using human umbilical vein endothelial cells (HUVEC) in vitro. The effects of IL-35 on vessel formation in vivo were examined using a murine Matrigel plugs model. MMP2/MMP9 and IL-6/IL-8 secretion were assessed by zymography and ELISA, respectively. The crosstalk between IL-35, VEGF, and Ang2 in HUVECs and RA synovial tissue explants was investigated. Results: IL-35 inhibited basal and VEGF-induced HUVEC migration and adhesion in vitro as well as tube formation in vitro and in vivo. VEGF increased Ang2 secretion by HUVECs and RA synovial tissue explants, and exogenous Ang2 promoted HUVEC migration, adhesion, and tube formation with similar potency to VEGF. Blocking the Ang/Tie2 pathway with a Tie2 kinase antibody inhibited the proangiogenic effects of exogenous Ang2 and VEGF in HUVECs. IL-35 inhibited basal and VEGF-induced Ang2 secretion by HUVECs and RA synovial tissue explants; it also antagonized the proangiogenic effects of exogenous Ang2 in HUVECs. Moreover, IL-35 reduced basal and VEGF/Ang2-induced MMP2/MMP9 and IL-6/IL-8 secretion. Conclusion: These results suggested that IL-35 restrains RA angiogenesis and inflammation by downregulating basal and VEGF-induced Ang2 secretion as well as disrupting Ang2/Tie2 signal transduction. Our findings extend current understanding of mechanisms regulating RA angiogenesis and may support development of novel angiogenesis-targeting therapeutics for RA treatment.

Glucose metabolism is a common target for cancer regulation and microRNAs (miRNAs) are important regulators of this process. Here we aim to investigate a tumor‐suppressing miRNA, miR‐33b, in regulating the glucose metabolism of non‐small cell lung cancer (NSCLC). In our study, quantitative real‐time polymerase chain reaction (qRT‐PCR) showed that miR‐33b was downregulated in NSCLC tissues and cell lines, which was correlated with increased cell proliferation and colony formation. Overexpression of miR‐33b through miR‐33b mimics transfection suppressed NSCLC proliferation, colony formation, and induced cell‐cycle arrest and apoptosis. Meanwhile, miR‐33b overexpression inhibited glucose metabolism in NSCLC cells. Luciferase reporter assay confirmed that miR‐33b directly binds to the 3′‐untranslated region of lactate dehydrogenase A (LDHA). qRT‐PCR and Western blot analysis showed that miR‐33b downregulated the expression of LDHA. Moreover, introducing LDHA mRNA into cells over‐expressing miR‐33b attenuated the inhibitory effect of miR‐33b on the growth and glucose metabolism in NSCLC cells. Taken together, these results confirm that miR‐33b is an anti‐oncogenic miRNA, which inhibits NSCLC cell growth by targeting LDHA through reprogramming glucose metabolism.

Vascular calcification is associated with atherosclerosis, but whether it mechanically affects plaque stability remains controversial. To assess the effect of mineralization on plaque vulnerability to mechanical shear stress, we applied fluid shear to cultures of calcifying vascular cells (CVC), a subpopulation of smooth muscle cells that spontaneously mineralize. CVC cultures containing nodules were treated for 10 days with vehicle control or beta-glycerophosphate (BGP) to accelerate mineralization. Cultures were placed in a parallel-plate flow system and were subjected to increasing fluid shear stress (4.9 dyn/cm(2)/min up to 400 dyn/cm(2)). The number of nodules remaining attached was recorded every 10 min. Results showed that control cultures and BGP-treated cultures, which contained significantly greater calcium mineral than control cultures, had similar detachment thresholds (50-100 dyn/cm(2)), with linear portions of their stress/detachment curves from 100 to 275 dyn/cm(2). Based on repeated measure analysis of variance, BGP-treated nodules were no more likely to detach at a given shear than controls, although they showed a trend toward greater stability. Thus, calcification does not appear to increase plaque vulnerability to fluid shear stress, although it may contribute to a slight stabilization. This model may represent the first in vitro model of mechanical rupture of atherosclerotic plaque.

Interleukin (IL)-27 is an IL-12 family cytokine and exerts a critical role in immune regulation in the context of infection, autoimmunity, and angiogenesis. In this study, we aimed to investigate the possible pathophysiological role of IL-27 and vascular endothelial growth factor (VEGF) in ankylosing spondylitis (AS). One hundred and forty AS patients and 90 healthy controls were included in the current study. The levels of IL-27 and VEGF in serum and synovial fluid (SF) samples were measured by enzyme-linked immunosorbent assay. Erythrocyte sedimentation rate, C-reactive protein, and human leukocyte antigen (HLA)-B27 were measured by standard laboratory techniques. Disease activity in AS was scored with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Hip involvement, peripheral arthritis, and eye involvement were also recorded. The serum levels of IL-27 were remarkably higher in AS patients than healthy groups and significantly correlated with serum levels of VEGF. Furthermore, the serum levels of IL-27 were correlated with BASDAI independent of other markers of inflammation. Elevated serum levels of IL-27 and VEGF were detected in AS patients with peripheral arthritis and HLA-B27 positive. The SF levels of IL-27 and VEGF were significantly higher than serum levels in AS patients with peripheral arthritis. By contrast, levels of IL-27 and VEGF were not increased in AS patients with hip involvement and eye involvement. IL-27 may regulate the immunological or inflammatory process of AS.

Rheumatoid arthritis (RA) is an inflammatory disorder of the joints that affects 0.5-1 % of adults. Excessive growth of the fibroblast-like synoviocytes (FLS) promotes hyperplasia of synovial tissues and causes its invasion into the bone and cartilage, which eventually causes deformity and dysfunction of affected joints. Interleukin 35 (IL-35) was shown to suppress the inflammatory responses to collagen-induced arthritis (CIA) via upregulation of T regulatory cells and suppression of T helper type 17 cells in a mouse model. To study the effects of IL-35 on the proliferation and apoptosis frequency of cultured FLS isolated from mice with CIA as well as to examine the effects of IL-35 on CIA in vivo. Thirty DBA/1 J mice, which are used as an animal model for RA, were divided randomly (ten mice per group) to a CIA group (collagen treatment), a CIA + IL-35 group (collagen and IL-35 treatments), and a control group (no treatment). Starting on the 24th day after collagen administration, IL-35 was injected intraperitoneally into mice of the CIA + IL-35 group once per day for 10 days. An arthritis index was calculated, and pathological analysis of synovial tissue was performed. FLS isolated from CIA mice were treated with various concentrations of IL-35 (12.5-100 ng/ml). The MTT assay was used to examine FLS proliferation, and apoptosis frequency of FLS was detected by flow cytometry. On day 24, the CIA mice began to exhibit arthritis symptoms, and the symptoms rapidly progressed with time. Treatment with IL-35 significantly alleviated arthritis symptoms and reduced the synovial tissue inflammation. In addition, IL-35 treatment inhibited proliferation and promoted apoptosis in cultured FLS from CIA mice in a dose-dependent manner. IL-35 could ameliorate the symptoms of arthritis in the CIA mouse model in vivo and inhibited FLS proliferation while promoting FLS apoptosis in vitro, thereby exhibited the potential in inhibiting the progression of RA.

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Within arterial bifurcations or branching points, oscillatory shear stress (OSS) induces oxidative stress mainly via the NADPH oxidase system. It is unknown whether 17β-estradiol (E 2 ) can regulate OSS-mediated low density lipoprotein (LDL) modifications. Bovine aortic endothelial cells (BAECs) were pre-treated with E 2 at 5 nmol/L, followed by exposure to OSS (0 ± 3.0 dynes/ cm 2 sec and 60 cycles/min) in a flow system. E 2 decreased OSS-mediated NADPH oxidase mRNA expression, and E 2 -mediated ·NO production was mitigated by the ·NO synthase inhibitor L-NAME. The rates of O 2 −· production in response to OSS increased steadily as determined by superoxide dismutase-inhibited ferricytochrome c reduction; however, pre-treatment with E 2 decreased OSS-mediated O 2 −· production (n=4, P<0.05). In the presence of native LDL (50 μg/ mL), E 2 also significantly reversed OSS-mediated LDL oxidation as determined by high performance liquid chromatography. In the presence of O 2 −· donor, xanthine oxidase (XO), E 2 further reversed XO-induced LDL lipid peroxidation (n=3, P<0.001). Mass spectra were acquired in the m/z 400-1800 range, revealing XO-mediated LDL protein nitration involving tyrosine 2535 in the α-2 domains, whereas pre-treatment with E 2 reversed this observation, consistent with the changes in nitrotyrosine intensities by the dot blots. E 2 plays an indirect antioxidative role. In addition to up-regulation of eNOS and down-regulation of Nox4 expression, E 2 influences LDL modifications via lipid peroxidation and protein nitration.

Real-time wall shear stress is difficult to monitor precisely because it varies in space and time. Microelectromechanical systems sensor provides high spatial resolution to resolve variations in shear stress in a 3-D bifurcation model for small-scaled hemodynamics. At low Reynolds numbers from 1.34 to 6.7 skin friction coefficients (C(f)) varied circumferentially by a factor of two or more within the bifurcation. At a Reynolds number of 6.7, the C(f) value at the lateral wall of the bifurcation along the 270 degree plane was 7.1, corresponding to a shear stress value of 0.0061 dyn/cm(2). Along the 180 degree plane, C(f) was 13 or 0.0079 dyn/cm(2), and at the medial wall along the 90 degree plane, C(f) was 10.3 or 0.0091 dyn/cm(2). The experimental skin friction coefficients correlated with values derived from the Navier-Stokes solutions.