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Transforming growth factor-β2 (TGF-β2) has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), due to its ability to stimulate the overproduction of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), and remodeling of the extracellular matrix (ECM). Although intravitreal triamcinolone acetonide (TA) is clinically useful in the treatment of PVR and PDR, its molecular mechanism has yet to be fully elucidated. The present study investigated whether TA treatment altered TGF-β2-driven biological effects on the behavior of cultured human retinal pigment epithelial (RPE) cells, in order to determine which signaling pathway may be essential for the pharmacological action of TA. The R-50 human RPE cell line was treated with TA in the presence of TGF-β2, followed by analyses of cell viability and contraction using cell viability and collagen gel contraction assays. VEGF mRNA expression and protein production were measured using reverse transcription-quantitative PCR and ELISA, respectively. The phosphorylation status of signaling mediators and the protein expression of type I collagen (COL1A1), α-smooth muscle actin (α-SMA), and ECM-remodeling enzymes, including MMP-2 and MMP-9, were analyzed using western blotting. The gelatinolytic activity of MMPs was detected using gelatin zymography. TA treatment exhibited no prominent cytotoxicity but markedly antagonized TGF-β2-induced cytostatic effects on RPE cell viability and TGF-β2-enhanced contractility in collagen gels. In the context of TGF-β2-related signaling, TA significantly attenuated TGF-β2-elicited Smad2, extracellular-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Moreover, TA markedly mitigated TGF-β2-induced VEGF upregulation through ablation of p38 signaling activity. TA also partially attenuated TGF-β2-elicted expression of COL1A1, α-SMA, MMP-2, and MMP-9, but only suppressed TGF-β2-induced MMP-9 gelatinolytic activity. Mechanistically, the MEK/ERK signaling pathway may have a critical role in the TGF-β2-induced upregulation of COL1A1, α-SMA and MMP-9. In conclusion, TA may be considered a useful therapeutic agent for treating TGF-β2-associated intraocular angiogenesis and tissue remodeling, the underlying mechanism of which may involve the ERK and p38 MAPK signaling pathways. [ABSTRACT FROM AUTHOR]

Hypoxia-induced retinal neovascularization plays a central role in the pathogenesis of diabetic retinopathy. This study aimed to investigate whether hypoxia leads to the release of nuclear high mobility group box 1 (HMGB1) peptides from cultured retinal pigment epithelial ARPE-19 cells, to determine the effect of HMGB1 on angiogenic cytokine production and elucidate the involved signaling pathways. A chemical hypoxia mimetic agent, cobalt chloride, induced SIRT1 downregulation, HMGB1 nucleocytoplasmic relocation and extracellular release from ARPE-19 cells, implicating its autocrine function. Resveratrol treatment significantly reduced secretion of HMGB1 from ARPE-19 cells exposed to hypoxia. Cell proliferation and cell cycle analyses demonstrated that exogenous HMGB1 caused significant growth suppression and G1 cell cycle arrest in ARPE-19 cells. Morphological observations showed that HMGB1 enhanced adhesion, but suppressed migration of ARPE-19 cells. More intriguingly, HMGB1 up-regulated expression of angiofibrogenic factors in ARPE-19 cells, including VEGF, bFGF, TGF-β2, and CTGF. Signal profiling characterization indicated that HMGB1 triggered hyperphosphorylation of Akt, p38 MAPK, and NF-κB, but not that of ERK, JNK, and Smad2, whereas inhibition of PI3K, MAPK, or NF-κB significantly attenuated the HMGB1-driven cytokine overproduction in ARPE-19 cells. Functional neutralization with anti-TLR4 and -RAGE antibodies confirmed that both receptors were involved in the cytokine overproduction. In conclusion, chemically-mimicked hypoxia induced nucleocytoplasmic relocation and release of HMGB1 peptides, which in turn up-regulated the production of angiofibrogenic factors in RPE cells, thereby contributing to the pathogenesis of hypoxia-associated diabetic retinopathies. Conversely, blockades of intraocular HMGB1 bioavailability or signal activation may prevent angiofibrogenesis in development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Aims Propofol (PPF), an intravenous anesthetic agent, is previously reported to attenuate oxidative stress- and inflammation-induced endothelial cell dysfunction. This study investigated its effect on endothelial cell biology. Main methods Cultured human umbilical vein endothelial cells (HUVECs) were treated with PPF and subject to measurements for nitric oxide (NO) production, autophagy flux, signal transduction, migration, and in vitro angiogenesis. Key findings Non-cytotoxic PPF treatment was found to significantly upregulate inducible nitric oxide synthase (NOS2) but downregulate constitutive NOS3 expression. It also potentiated LPS-induced ICAM-1 overexpression and NO overproduction. Mechanistically, the PPF-activated signal transduction in PI3K/Akt, ERK1/2, p38 MAPK, and JNK pathways were involved in the PPF-driven NO overproduction. PPF exhibited a stimulatory effect on autophagy flux by increasing expression of autophagy markers including mTOR, Beclin-1, Atg5, and LC3I/II, as well as a late endosomal indicator, Rab7. However, PPF appeared to antagonize the Rab7 upregulation by LPS. Functionally, PPF enhanced in vitro migratory and angiogenic capacities of HUVECs, but this enhancement was drastically abrogated by the presence of autophagy inhibitors, indicating a pro-angiogenic contribution of PPF-enhanced autophagy in cultured HUVECs. Significance Our findings support the notion that PPF enhances motility and angiogenic capacity of cultured HUVECs through an autophagy-involved regulatory mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Background: It has been reported that plasmin induces migration of endothelial cells. We hypothesized that tissue plasminogen activator (tPA) enhanced endothelial progenitor cell (EPC) mobilization from bone marrow (BM) into the circulation and angiogenesis in murine critical limb ischemia (CLI). Methods: Forty male B6 mice were randomly divided into group 1 (control), group 2 [control+recombinant tPA (intra-venous 4mg/kg)], group 3 (CLI) and group 4 (CLI+tPA). Results: The results demonstrated higher MMP-9 activity in BM and circulatory SDF-1α level in groups 2 and 3 than in group 1, and highest in group 4 (all p<0.01) at 18h after CLI. Compared with circulation, SDF-1α level in BM showed an opposite trend (all p<0.03). The circulating EPC (C-kit/CD31, Sca-1/KDR, CXCR4/CD34) levels at 18h after CLI were higher in group 2 than in groups 1 and 3, and highest in group 4 (all p<0.03). EPC (C-kit/CD31, Sca-1/KDR) levels in BM were lower in group 1 than in groups 2 to 4 (all p<0.03), whereas number of CXCR4/CD34-positive EPCs in BM did not differ among the four groups at 18h after CLI. Protein expressions of SDF-1α, CXCR4, eNOS, and VEGF, numbers of CD31+, CXCR4+, SDF-1α+, and vWF+ cells through immunofluorescent staining, numbers of small vessels (<15μm) and blood flow measured by Laser Doppler in an ischemic area were significantly higher in group 4 than in group 3 (all p<0.005) at day 14 after CLI. Conclusion: tPA treatment enhanced number of circulating EPCs, angiogenesis, and blood flow to ischemic tissue in a murine model of limb ischemia. [Copyright &y& Elsevier]

Abstract: An endemic peripheral vascular disorder due to chronic arsenic poisoning, named Blackfoot disease (BFD), occurs in Taiwan. BFD causes destruction of vascular endothelial cells, and an anti-endothelial cell IgG antibody was found in the sera of BFD patients. We studied the role of this IgG antibody (BFD-IgG) in modulating proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and found that a low concentration of BFD-IgG (200 μg/mL) stimulated endothelial cell growth and increased expressions of vascular cell adhesion molecule-1 (VCAM-1), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF). The apoptosis events appeared not altered by addition of BFD-IgG. An in vitro neoangiogenesis assay demonstrated that BFD-IgG promoted the formation of tube-like structures, which was completely abrogated by anti-VEGF neutralizing antibody and partially by NOS inhibitor, L-NAME. We conclude that BFD-IgG at 200 μg/mL results in cell proliferation and enhanced VEGF-dependent angiogenesis in vitro. Those results suggested that a low concentration of BFD-IgG plays a protective role in the pathogenesis or the progression of BFD. [Copyright &y& Elsevier]