Sulfated CXCR3 Peptide Trap Use as a Promising Therapeutic Approach for Age-Related Macular Degeneration.

BACKGROUND AND OBJECTIVES: Chemokines have various biological functions and potential roles in the development or progression of neuroinflammatory diseases. However, the specific pathogenic roles of chemokines in the major cause for vision loss among the elderly, the leading cause of blindness in older individuals, remain elusive. Chemokines interact with their receptors expressed in the endothelium and on leukocytes. The sulfation of tyrosine residues in chemokine receptors increases the strength of ligand-receptor interaction and modulates signaling. Therefore, in the present study, we aimed to construct a human recombinant sulfated CXCR3 peptide trap (hCXCR3-S2) and mouse recombinant sulfated CXCR3 peptide trap (mCXCR3-S2) to demonstrate in vivo effects in preventing choroidal neovascularization (CNV) and chemotaxis. MATERIALS AND METHODS: We generated expression vectors for mCXCR3-S2 and hCXCR3-S2 with GST domains and their respective cDNA sequences. Following overexpression in E. coli BL21 (DE3), we purified the fusion proteins from cell lysates using affinity chromatography. First, the impact of hCXCR3-S2 was validated in vitro. Subsequently, the in vivo efficacy of mCXCR3-S2 was investigated using a laser-induced CNV mouse model, a mouse model of neovascular age-related macular degeneration (AMD). RESULTS: hCXCR3-S2 inhibited the migration and invasion of two human cancer cell lines. Intravitreal injection of mCXCR3-S2 attenuated CNV and macrophage recruitment in neovascular lesions of mouse models. These in vitro and in vivo effects were significantly stronger with CXCR3-S2 than with wild-type CXCR3 peptides. CONCLUSION: These findings demonstrate that the sulfated form of the CXCR3 peptide trap is a valuable tool that could be supplemented with antivascular endothelial growth factors in AMD treatment.

Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice.

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C-C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

Salmonella­induced miR­155 enhances necroptotic death in macrophage cells via targeting RIP1/3.

Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella), a virulent pathogen, is known to induce host­cell death. Using reverse transcription­quantitative polymerase chain reaction, a 28­fold increase of microRNA (miR)­155 expression in RAW 264.7 macrophages was observed following infection with Salmonella for 24 h. This miR­155 upregulation increased macrophage cell death by up to 40% in 48 h following infection. Western blot analysis revealed that receptor interacting protein 1 (RIP1) and 3 (RIP3) were increased at 18 h following miR­155 transfection to macrophages, similar to Salmonella infection. In addition, inhibition of RIP1 by pre­incubating macrophages with necrostatin­1, a RIP1 specific inhibitor, increased the viability of Salmonella­infected cells and miR­155­transfected cells by up to 20%. The cleavage of poly (adenosine diphosphate­ribose) polymerase­1 (PARP­1) was also enhanced by miR­155 induction upon Salmonella infection. Therefore, it was suggested that RIP1/3­induced necroptosis and PARP­1­mediated necrosis caused by miR­155 induction may represent distinct routes of programmed necrotic cell death of Salmonella­infected macrophages.

Knockdown of RPL9 expression inhibits colorectal carcinoma growth via the inactivation of Id-1/NF-κB signaling axis.

Ribosomal protein L9 (RPL9), a component of the 60S subunit for protein synthesis, is upregulated in human colorectal cancer. In the present study, we investigated whether RPL9 gained extraribosomal function during tumorigenesis and whether targeting of RPL9 with small interfering (si) RNA could alter the course of colorectal cancer progression. Our results showed that siRNA knockdown of RPL9 suppresses colorectal cancer (CRC) cell growth and long-term colony formation through an increase in sub-G1 cell population and a strong induction of apoptotic cell death. To obtain insights into the molecular changes in response to RPL9 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that RPL9-specific knockdown led to dysregulation of 918 genes in HCT116 and 3178 genes in HT29 cells. Among these, 296 genes showed same directional regulation (128 upregulated and 168 downregulated genes) and were considered as a common RPL9 knockdown signature. Particularly, we found through a network analysis that Id-1, which is functionally associated with activation of NF-κB and cell survival, was commonly downregulated. Subsequent western blot analysis affirmed that RPL9 silencing induced the decrease in the levels of Id-1 and phosphorylated IκBα in both HCT116 and HT29 cells. Also, the same condition decreased the levels of PARP-1 and pro-caspase-3, accelerating apoptosis. Furthermore, inhibition of RPL9 expression significantly suppressed the growth of human CRC xenografts in nude mice. These findings indicate that the function of RPL9 is correlated with Id-1/NF-κB signaling axis and suggest that targeting RPL9 could be an attractive option for molecular therapy of colorectal cancer.

Anti-Inflammatory Effects of 3-(4'-Hydroxyl-3',5'-Dimethoxyphenyl)Propionic Acid, an Active Component of Korean Cabbage Kimchi, in Lipopolysaccharide-Stimulated BV2 Microglia.

We investigated the protective ability of 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA), an active principle in Korean cabbage kimchi, against the production of proinflammatory mediators and cytokines, and the mechanisms involved in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. HDMPPA significantly suppressed the production of nitric oxide (NO) and prostaglandin E2, along with the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV2 cells, at concentrations with no cytotoxicity. HDMPPA also attenuated the LPS-induced expression and secretion of proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1ß. Furthermore, HDMPPA inhibited LPS-induced nuclear factor-κB (NF-κB) activation, which was associated with the abrogation of IκB-α degradation and phosphorylation, and subsequent decreases in NF-κB p65 levels. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a downstream molecule of phosphatidylinositol-3-kinase (PI3K), in LPS-stimulated BV2 cells was suppressed markedly by HDMPPA. This effect was associated with a significant reduction in the formation of intracellular reactive oxygen species. The findings in this study suggest that HDMPPA may exert anti-inflammatory responses by suppressing LPS-induced expression of proinflammatory mediators and cytokines through blockage of NF-κB, MAPKs, and PI3K/Akt signaling pathways and oxidative stress in microglia.

Anti-inflammatory potential of newly synthesized 4-[(butylsulfinyl)methyl]-1,2-benzenediol in lipopolysaccharide-stimulated BV2 microglia.

In this study, we investigated the anti-inflammatory effects of newly synthesized 4-[(butylsulfinyl)methyl]-1,2-benzenediol (SMBD) in lipopolysaccharide (LPS)-stimulated BV2 microglia and the subsequent signaling events. Following stimulation with LPS, elevated production of nitric oxide (NO) and prostaglandin E2 (PGE2) was detected in BV2 cells; however, SMBD pretreatment inhibited the production of NO and PGE2 through suppressing gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, at non-toxic concentrations. LPS-stimulated gene expression and production of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were also significantly reduced by SMBD. The anti-inflammatory effects of SMBD were associated with suppression of LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB), and phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a phosphatidylinositol 3-kinase (PI3K) downstream effector. Therefore, the present results demonstrate that SMBD down-regulates inflammatory gene expression by inhibiting the activation of NF-κB through interference with the activation of MAPKs and PI3K/Akt signaling. Taken together, our data suggest that SMBD may have potential to be developed into an effective anti-inflammatory agent.