Interleukin-17 Weakens the NAFLD/NASH Process by Facilitating Intestinal Barrier Restoration Depending on the Gut Microbiota.

Interleukin-17 (IL-17) is associated with nonalcoholic fatty liver disease (NAFLD) and gut microbiota, and how IL-17 mediates the NAFLD/nonalcoholic steatohepatitis (NASH) process depending on the gut microbiota is unclear. We found that T helper 17 (TH17) cells were decreased in the small intestine in a methionine choline-deficient (MCD) diet-induced NASH model. IL-17-deficient (Il17-/-) mice showed alterations in intestinal microbiota, including the inhibition of probiotic growth and the overgrowth of certain pathogenic bacteria, and were prone to higher endotoxemia levels and more severe gastrointestinal barrier defects than wild-type (WT) mice. Furthermore, TH17 cells were responsible for restoring the intestinal barrier after administration of recombinant IL-17 to Il17-/- mice or injection of CD4+ T cells into a Rag1-/- mouse model. Additionally, transplantation of the microbiota from WT mice to Il17-/- mice restored the intestinal barrier. Notably, microbiota-depleted Il17-/- mice were resistant to MCD diet-induced intestinal barrier impairment. Fecal microbiota transplantation from Il17-/- mice to microbiota-depleted mice aggravated intestinal barrier impairment and then promoted the development of NASH. Collectively, this study showed that host IL-17 could strengthen intestinal mucosal barrier integrity and reduce dysbiosis-induced intestinal injury and secondary extraintestinal organ injury induced by a special diet. IMPORTANCE The morbidity of NASH has increased, with limited effective treatment options. IL-17 plays a protective role in the gut mucosa in high-fat-diet (HFD)-related metabolic disorders, and HFD-related microbiota dysbiosis is responsible for a decreased number of T helper 17 (TH17) cells in the lamina propria. The mechanism by which IL-17 mediates the NAFLD/NASH process depending on the gut microbiota is unclear. In our study, IL-17 originating from TH17 cells maintained intestinal barrier integrity and determined the outcomes of diet-related disease, which may be a target strategy for NAFLD/NASH.

MT-CO1 expression in nine organs and tissues of different-aged MRL/lpr mice: Investigation of mitochondrial respiratory chain dysfunction at organ level in systemic lupus erythematosus pathogenesis.

Objectives: This study aims to investigate the expression patterns of mitochondrially encoded cytochrome c oxidase 1 (MT-CO1) in different organs and tissues of MRL/lpr mice aged six and 18 weeks. Materials and methods: Six-week-old female MRL/lpr mice (n=10) were considered young lupus model mice, and 18-week-old MRL/lpr mice (n=10) were considered old lupus model mice. Additionally, six-week-old (n=10) and 39-week-old (n=10) female Balb/c mice were used as the young and old controls, respectively. The messenger ribonucleic acid (mRNA) and protein expression levels of MT-CO1 in nine organs/tissues were detected via quantitative polymerase chain reaction (qPCR) and Western blot. Malondialdehyde (MDA) levels were determined with thiobarbituric acid colorimetry. The correlation coefficient of MT-CO1 mRNA levels and MDA levels in each organ/tissue at different ages was analyzed by Pearson correlation analysis. Results: The results showed that most non-immune organs/tissues (heart, lung, liver, kidneys, and intestines) showed increased MT-CO1 expression levels in younger MRL/lpr mice (p<0.05) and decreased MT-CO1 expression in older mice (p<0.05). Expression of MT-CO1 in the lymph nodes was low in younger mice but high in older mice. In other immune organs (spleen and thymus), MT-CO1 expression was low in older MRL/lpr mice. Lower mRNA expression and higher MDA levels were observed in the brains of MRL/lpr mice. However, all MRL/lpr mice showed higher MDA levels than Balb/c mice in every organ no matter younger or older MRL/lpr mice. Conclusion: Our study results suggest that lymphoid mitochondrial hyperfunction at organ level may be an important intrinsic pathogenesis in systemic lupus erythematosus activity, which may affect mitochondrial dysfunction in non-immune organs.

A Comparative Study on the Incidence, Aggravation, and Remission of Lupus Nephritis Based on iTRAQ Technology.

AIM AND OBJECTIVE: Lupus nephritis (LN) is one of the major complications of systemic lupus erythematosus (SLE). The specific mechanisms of pathogenesis, aggravation, and remission processes in LN have not been clarified but is of great need in the clinic. Using isobaric tags for relative and absolute quantitation (iTRAQ) technology to screen the functional proteins of LN in mice. Especially under intervention factors of lipopolysaccharide (LPS) and dexamethasone. METHODS: Mrl-lps mice were intervened with LPS, dexamethasone, and normal saline (NS) using intraperitoneal injection, and c57 mice intervened with NS as control. The anti-ANA antibody enzyme-linked immunosorbent assay (ELISA) was used to verify disease severity. Kidney tissue is collected and processed for iTRAQ to screen out functional proteins closely related to the onset and development of LN. Western blot method and rt-PCR (real-time Polymerase Chain Reaction) were used for verification. RESULTS: We identified 136 proteins that marked quantitative information. Among them, Hp, Igkv8-27, Itgb2, Got2, and Pcx proteins showed significant abnormal manifestations. CONCLUSION: Using iTRAQ methods, the functional proteins Hp, Igkv8-27, Itgb2, Got2, and Pcx were screened out for a close relationship with the pathogenesis and development of LN, which is worth further study.

Proteomic analysis of stachyose contribution to the growth of Lactobacillus acidophilus CICC22162.

Stachyose is a functional oligosaccharide, acting as a potential prebiotic for colonic fermentation. To understand the mechanism of how stachyose promotes the growth of probiotic bacterium, we analyzed the differences of the proteome of Lactobacillus acidophilus grown on stachyose or glucose. By a combination of two-dimensional electrophoresis and mass spectrometry analysis, we observed 16 proteins differentially abundant under these two conditions and identified 9 protein spots. Six of these proteins were highly abundant when stachyose was used as the sole carbon source. They included the phosphotransferase system, the energy coupling factor (ECF) transporter and the mannose-6-phosphate isomerase, involved in the uptake and catabolism of stachyose in Lactobacillus acidophilus CICC22162. Supportively, these observations were validated by quantitative RT-PCR analysis and enzymatic activity determination. Positive correlation was found between the content of the proteins and their mRNA levels. Additionally, we explored the recognition mechanism for stachyose binding to the newly identified ECF transporter by MD simulations and free energy analysis. Taken together, these results provide new insights into the mechanism of stachyose in promoting the growth of probiotic bacterium.

Identification of Potent Chloride Intracellular Channel Protein 1 Inhibitors from Traditional Chinese Medicine through Structure-Based Virtual Screening and Molecular Dynamics Analysis.

Chloride intracellular channel 1 (CLIC1) is involved in the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has become an attractive new therapeutic target for several types of cancer. In this work, we aim to identify natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM) database using structure-based virtual screening and molecular dynamics (MD) simulation. First, structure-based docking was employed to screen the refined TCM database and the top 500 TCM compounds were obtained and reranked by X-Score. Then, 30 potent hits were achieved from the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis was used to refine the virtual hits. Six TCM compounds with top MM-GBSA scores and ideal-binding models were confirmed as the final hits. Our study provides information about the interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In addition, the top 6 natural products structural scaffolds could serve as building blocks in designing drug-like molecules for CLIC1 inhibition.

Proteomic analysis reveals that a global response is induced by subinhibitory concentrations of ampicillin.

In this study, a recipient-donor co-culture system was used to research the effect of subinhibitory concentrations of antibiotics on horizontal transmission in bacteria and the influence of antibiotics on protein expression. We employed two-dimensional gel electrophoresis combined with mass spectrometry to compare the protein expression profiles in systems with or without 0.5 × the minimum inhibitory concentration of ampicillin. RT-PCR was used to assess the transcriptional levels of the differentially expressed genes. Fifty-seven different proteins were induced or suppressed. The upregulated proteins were involved in transcription and translation, cell wall synthesis, bacterial SOS response, and detoxifying functions, and the downregulated proteins were involved in metabolism. These results indicated that a global response was induced in the recipient-donor co-culture system by the subinhibitory concentration of ampicillin. Further analysis revealed that a global regulatory network based on key pathways was induced in the system in response to the antibiotic pressure. These findings provide a new, more comprehensive view for research on antibiotic-resistance mechanisms in recipient-donor co-culture.

Potential molecular mechanisms for fruiting body formation of Cordyceps illustrated in the case of Cordyceps sinensis.

The fruiting body formation mechanisms of Cordyceps sinensis are still unclear. To explore the mechanisms, proteins potentially related to the fruiting body formation, proteins from fruiting bodies, and mycelia of Cordyceps species were assessed by using two-dimensional fluorescence difference gel electrophoresis, and the differential expression proteins were identified by matrix-assisted laser desorption/ionisation tandem time of flight mass spectrometry. The results showed that 198 differential expression proteins (252 protein spots) were identified during the fruiting body formation of Cordyceps species, and 24 of them involved in fruiting body development in both C. sinensis and other microorganisms. Especially, enolase and malate dehydrogenase were first found to play an important role in fruiting body development in macro-fungus. The results implied that cAMP signal pathway involved in fruiting body development of C. sinensis, meanwhile glycometabolism, protein metabolism, energy metabolism, and cell reconstruction were more active during fruiting body development. It has become evident that fruiting body formation of C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the possible mechanism provides insights into the culture of C. sinensis.

Carboxyl-terminal truncated HBx contributes to invasion and metastasis via deregulating metastasis suppressors in hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein (HBx), a trans-regulator, is frequently expressed in truncated form without carboxyl-terminus in hepatocellular carcinoma (HCC), but its functional mechanisms are not fully defined. In this report, we investigated frequency of this natural HBx mutant in HCCs and its functional significance. In 102 HBV-infected patients with HCC, C-terminal truncation of HBx, in contrast to full-length HBx, were more prevalent in tumors (70.6%) rather than adjacent non-tumorous tissues (29.4%) (p = 0.0032). Furthermore, two naturally-occurring HBx variants (HBxΔ31), which have 31 amino acids (aa) deleted (codons 123-125/124-126) at C-terminus were identified in tumors and found that the presence of HBxΔ31 significantly correlated with intrahepatic metastasis. We also show that over-expression of HBxΔ31 enhanced hepatoma cell invasion in vitro and metastasis in vivo compared to full-length HBx. Interestingly, HBxΔ31 exerts this function via down-regulating Maspin, RhoGDIα and CAPZB, a set of putative metastasis-suppressors in HCC, in part, by enhancing the binding of transcriptional repressor, myc-associated zinc finger protein (MAZ) to the promoters through physical association with MAZ. Notably, these HBxΔ31-repressed proteins were also significantly lower expression in a subset of HCC tissues with C-terminal HBx truncation than the adjacent non-tumorous tissues, highlighting the clinical significance of this novel HBxΔ31-driven metastatic molecular cascade. Our data suggest that C-terminal truncation of HBx, particularly breakpoints at 124aa, plays a role in enhancing hepatoma cell invasion and metastasis by deregulating a set of metastasis-suppressors partially through MAZ, thus uncovering a novel mechanism for the progression of HBV-associated hepatocarcinogenesis.

Proteomic analysis reveals that proteasome subunit beta 6 is involved in hypoxia-induced pulmonary vascular remodeling in rats.

BACKGROUND: Chronic hypoxia (CH) is known to be one of the major causes of pulmonary hypertension (PH), which is characterized by sustained elevation of pulmonary vascular resistance resulting from vascular remodeling. In this study, we investigated whether the ubiquitin proteasome system (UPS) was involved in the mechanism of hypoxia-induced pulmonary vascular remodeling. We isolated the distal pulmonary artery (PA) from a previously defined chronic hypoxic pulmonary hypertension (CHPH) rat model, performed proteomic analyses in search of differentially expressed proteins belonging to the UPS, and subsequently identified their roles in arterial remodeling. RESULTS: Twenty-two proteins were differently expressed between the CH and normoxic group. Among them, the expression of proteasome subunit beta (PSMB) 1 and PSMB6 increased after CH exposure. Given that PSMB1 is a well-known structural subunit and PSMB6 is a functional subunit, we sought to assess whether PSMB6 could be related to the multiple functional changes during the CHPH process. We confirmed the proteomic results by real-time PCR and Western blot. With the increase in quantity of the active subunit, proteasome activity in both cultured pulmonary artery smooth muscle cells (PASMCs) and isolated PA from the hypoxic group increased. An MTT assay revealed that the proteasome inhibitor MG132 was able to attenuate the hypoxia-induced proliferation of PASMC in a dose-dependent manner. Knockdown of PSMB6 using siRNA also prevented hypoxia-induced proliferation. CONCLUSION: The present study revealed the association between increased PSMB6 and CHPH. CH up-regulated proteasome activity and the proliferation of PASMCs, which may have been related to increased PSMB6 expression and the subsequently enhanced functional catalytic sites of the proteasome. These results suggested an essential role of the proteasome during CHPH development, a novel finding requiring further study.

Dynamic mobilization of PGC-1α mediates mitochondrial biogenesis for the protection of RGC-5 cells by resveratrol during serum deprivation.

Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1α; instead, resveratrol facilitated PGC-1α translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1α subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.

Identification of novel serological tumor markers for human prostate cancer using integrative transcriptome and proteome analysis.

The aim of this study was to identify novel serological tumor markers for human prostate cancer (PCa). We compared the gene expression profile of PCa tissues to adjacent benign tissues of prostate using gene expression microarray. 1207 genes that were consistently different from adjacent benign tissues of prostate (paired t test, P<0.05) were selected as differentially expressed genes (DEGs). Among them, 652 DEGs were upregulated in PCa, whereas 555 DEGs were downregulated in PCa. In addition, two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MS was performed to screen for candidate markers in the proteome of PCa and adjacent benign tissues of prostate. A total of 89 spots were significantly up-regulated (ratio≥2, P<0.01) in PCa samples, whereas 66 spots were down-regulated (ratio≤-2, P<0.01). Sixty gene products were identified among these spots. Moreover, 14 potential candidate markers, which were identified as differentially expressed molecules by both gene expression microarray and 2D-DIGE, were chosen for validation and analysis by ELISA. The serum levels of three proteins correlated well with the 2D-DIGE results. Furthermore, the increased serum level of Inosine monophosphate dehydrogenase II (IMPDH2) was significantly associated with the clinicopathological features of the patients with PCa, suggesting its potential as a serological tumor marker. These results demonstrated that integrative transcriptome and proteome analysis could be a powerful tool for marker discovery in PCa. We suggest IMPDH2 as a novel serological tumor marker for detection of early PCa and evaluation of tumor progression.

[Effectiveness of cultured Cordyceps sinensis combined with glucocorticosteroid on pulmonary fibrosis induced by bleomycin in rats].

OBJECTIVE: To study the treatment effects of cultured Cordyceps sinensis combined with glucocorticosteroid on experimental pulmonary fibrosis in rats induced by bleomycin. METHOD: Fifty rats were randomly divided into five groups, including control group, model group, cultured C. sinensis groups, prednisone group, cultured C. sinensis combined with prednisone group. On experimental day 0, the rats were respectively intratracheally instilled with bleomycin, and rats in the control group and model group with the same volume of normal saline. One day after the injection, cultured C. sinensis and glucocorticosteroid was respectively given to rats daily by gastric gavage, while the same volume of normal saline was given to those in the control group and model group. On 28th d, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Histological changes of the lungs were evaluated by HE stain, Masson's trichrome stain. Collagen content of the lung tissue was assessed by hydroxyprolin concentration. Lung expression of CTGF protein was assessed by immunohistochemistry. The level of TGF-beta1 protein was measured by ELISA. RESULT: Compared to model group, pulmonary fibrosis were alleviated in cultured C. sinensis and prednisone group, and CTGF expression, Hydroxyproline concentrations and protein TGF-beta1 were decreased. The combination effect of C. sinensis and prednisone group is augmented compared with using C. sinensis or prednisone group alone. CONCLUSION: The cultured C. sinensis and prednisone alleviates pulmonary fibrosis, and the combination use of both drugs has synergia effects in anti-fibrous degeneration.

[Research of differential expression of virulence-related proteins of T3SS in Pseudomonas aeruginosa].

OBJECTIVE: To analyze the difference of virulence-related protein concerned with type III secretion system (T3SS) in Pseudomonas aeruginosa during the changes of antibiotic sensitivity and interpret the clinical patient data to explore the relationship between the changes in resistance and variance of virulence. METHODS: The isolates of Pseudomonas aeruginosa was isolated from the respiratory tract of a same patient with an altered sensitivity of antibiotics. It turned out to be one clone. The homolog of isolates was determined by ERIC-PCR. The Kirby-Bauer antibiotic testing was employed to detect the sensitivity of antibiotics of isolates. PCR was used to detect the gene of T3SS and virulence of isolates and two-dimensional gel electrophoresis to compare the whole-cell proteins. The mass spectrometry was employed to analyze a variety of protein spots. The relevant information was retrieved from protein databases. Clinical record was collected to study the relationship of clinical features, bacteria resistance and virulence-associated protein. RESULTS: One subject was diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with pneumonia. Pseudomonas aeruginosa was isolated from his respiratory tract three times in one year. Sensitivity spectrum of isolates were as follows: sensitivity, multi-drug resistant (MDR) and pan-drug resistance (PDR). Virulence gene was exo U+/exo S-. Twenty-one differentially expressed proteins were revealed by two-dimensional gel electrophoresis in three isolates. The functions of 11 proteins were definite. Only 9 proteins were associated with basal bacterial metabolism. Disulfide oxidoreductase A (DsbA) corresponded to the variation of virulence and sensitivity spectrum. Clinical record revealed that the severe lung infection was caused by the PDR strain and the patient died within one month. CONCLUSIONS: The sensitivity spectrum and virulence of Pseudomonas aeruginosa may undergo changes when there is an alteration of eco-environment during colonization and infection over a long period of time. DsbA plays a key role in the variety of virulence.

[Differential expression of proteins in peripheral blood mononuclear cells from patients with pulmonary tuberculosis.].

OBJECTIVE: To screen the differential expressed proteins of peripheral blood mononuclear cells (PBMC) in patients with pulmonary tuberculosis by comparative proteomics. METHODS: PBMCs from 10 patients with tuberculosis and 10 healthy volunteers were collected. The total proteins were extracted and separated by two-dimensional gel electrophoresis (2-DE). The sites of differential expression were analyzed using ImageMaster 2D Elite 5.0 image analysis software, and the peptide mass finger-printing (PMF) identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The biological information of these proteins was searched in the database and confirmed by Western blot. RESULTS: Well reproducible 2-DE maps of PBMC from the patients with tuberculosis and normal controls were established. Forteen differential protein spots were found. Twelve proteins were analyzed and identified. Pleckstrin and Ras suppressor protein1 were decreased in patients with tuberculosis while the other 10 proteins were increased. The decreased expression of Pleckstrin in tuberculosis was confirmed by Western blot. CONCLUSIONS: Analysis of differential protein expression in PBMC from patients with pulmonary tuberculosis by comparative proteomics identified 12 proteins. The results suggest that Pleckstrin and Coronin-1A may have an important role in the pathogenesis of pulmonary tuberculosis through regulating the phagocytosis of mononuclear macrophages.

Establishment of a lung cancer biobank of a southern chinese population.

There is an increasing need for establishment of biobanks for human cancer, which may facilitate basic and clinical researches as well as the development of novel approaches to early diagnostics, prevention and treatment, including personalized medicine. Herein we report the establishment of a lung cancer biobanks using biological samples from a lung cancer patient population in Southern China. Since 2007, we have collected lung cancer tissue from 1,054 patients with lung cancer, from whom 11,895 frozen tumor and normal matched tissues, 4,899 tissue paraffin blocks, and 3,562 blood serum samples were accumulated. The information on clinical manifestations, laboratory tests, and follow-up was maintained by an independent information management system, including three data sets. The primary data set included frozen tissue specimens, formalin-fixed paraffin-embedded tissues, blood specimens and clinical and long-term follow-up data. The secondary data set included DNA, RNA and proteins extracted from corresponding tissue specimens. And the tertiary data set contained improved genome, RNA and proteomics correlation analysis with relevant clinical and long-term follow-up data. The lung cancer biobanks is accessible to academic research and public services. To our best knowledge, this biobanks represents the first lung cancer tissue reservoir in China and should facilitate the basic and clinical research of this disease and developing diagnostic markers and novel therapeutic modalities.

Attenuation of bleomycin-induced lung fibrosis by oxymatrine is associated with regulation of fibroblast proliferation and collagen production in primary culture.

There is no satisfactory treatment for pulmonary fibrosis, which is characterized by altered control of proliferation of mesenchymal fibroblasts and extracellular matrix production. Oxymatrine is an alkaloid extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with capacities of anti-inflammation, inhibition of immune reaction, antivirus, protection against acute lung injury and antihepatic fibrosis. In this study, the effect of oxymatrine on pulmonary fibrosis was investigated using a bleomycin-induced pulmonary fibrosis mouse model. The results showed that bleomycin challenge provoked severe pulmonary fibrosis with marked increase in hydroxyproline content of lung tissue and lung fibrosis fraction, which was prevented by oxymatrine in a dose-dependent manner. In addition, bleomycin injection resulted in a marked increase of myeloperoxidase activity and malondialdehyde level that was attenuated by oxymatrine. Administration of oxymatrine inhibited the proliferation of murine lung fibroblasts, arrested the cells at G(0)/G(1) phase and reduced the expression of cell cycle regulatory protein, cyclin D1 in vitro. Furthermore, the steady-state production of collagen and the expression of alpha1(I) pro-collagen and alpha2(I) pro-collagen mRNA in fibroblasts were inhibited by oxymatrine in a dose-dependent manner. These results suggested that oxymatrine may attenuate pulmonary fibrosis induced by bleomycin in mice, partly through inhibition of inflammatory response and lipid peroxidation in lung induced by bleomycin and reduction of fibroblast proliferation and collagen synthesis.

CXCR4 down-regulation by small interfering RNA inhibits invasion and tubule formation of human retinal microvascular endothelial cells.

The progressive alterations to the retinal microvasculature in diabetic retinopathy are known to cause vision loss. Chemokines are characterized by their ability to induce cell invasion, adhesion and migration. In this study, we used double siRNA transfection to transiently and selectively decrease the level of the endogenous CXCR4 in human retinal microvascular endothelial cells (HRMECs). The functional consequences of silencing CXCR4 expression in HRMECs were investigated using an endothelial cell migration assay and tubule formation in Matrigel. When CXCR4 expression was decreased with siRNA, HRMECs were less invasive and also resulted in markedly diminished vascular networks on Matrigel as compared to the controls. Additionally, hypoxia and VEGF, the factors affecting microvascular, regulate the expression level of CXCR4 in HRMECs, respectively, which have synergistic, additive effect in the HRMECs. As such, CXCR4 antagonists may become a therapeutic target for the treatment of retinal angiopathies.