Pyxinol Fatty Acid Ester Derivatives J16 against AKI by Selectively Promoting M1 Transition to M2c Macrophages.

Acute kidney injury (AKI) is a common, multicause clinical condition that, if ignored, often progresses to chronic kidney disease (CKD) and end-stage kidney disease, with a mortality rate of 40-50%. However, there is a lack of universal treatment for AKI. Inflammation is the basic pathological change of early kidney injury, and inflammation can exacerbate AKI. Macrophages are the primary immune cells involved in the inflammatory microenvironment of kidney disease. Therefore, regulating the function of macrophages is a crucial breakthrough for the AKI intervention. Our team chemically modified pyxinol, an ocotillol-type ginsenoside, to prepare PJ16 with higher solubility and bioavailability. In vitro, using a model of macrophages stimulated by LPS, it was found that PJ16 could regulate macrophage function, including inhibiting the secretion of inflammatory factors, promoting phagocytosis, inhibiting M1 macrophages, and promoting M1 transition to the M2c macrophage. Further investigation revealed that PJ16 may shield renal tubular epithelial cells (HK-2) damaged by LPS in vitro. Based on this, PJ16 was validated in the animal model of unilateral ureteral obstruction, which showed that it improves renal function and inhibits renal tissue fibrosis by decreasing inflammatory responses, reducing macrophage inflammatory infiltration, and preferentially upregulating M2c macrophages. In conclusion, our study is the first to show that PJ16 resists AKI and fibrosis by mechanistically regulating macrophage function by modulating the phenotypic transition from M1 to M2 macrophages, mainly M2c macrophages.

Current trends of clinical trials involving CRISPR/Cas systems.

The CRISPR/Cas9 system is a powerful genome editing tool that has made enormous impacts on next-generation molecular diagnostics and therapeutics, especially for genetic disorders that traditional therapies cannot cure. Currently, CRISPR-based gene editing is widely applied in basic, preclinical, and clinical studies. In this review, we attempt to identify trends in clinical studies involving CRISPR techniques to gain insights into the improvement and contribution of CRISPR/Cas technologies compared to traditional modified modalities. The review of clinical trials is focused on the applications of the CRISPR/Cas systems in the treatment of cancer, hematological, endocrine, and immune system diseases, as well as in diagnostics. The scientific basis underlined is analyzed. In addition, the challenges of CRISPR application in disease therapies and recent advances that expand and improve CRISPR applications in precision medicine are discussed.

Distinct molecular basis for endothelial differentiation: gene expression profiles of human mesenchymal stem cells versus umbilical vein endothelial cells.

The capacity for endothelial differentiation has been described in mesenchymal stem cells (MSC) from human bone marrow. To identify genes associated with the endothelial differentiation potential of this cell-type, and search for the optimal regulatory factors, the expression profile of MSC was compared with cDNA from primary human umbilical vein endothelial cells as controls, using cDNA chips with 4096 genes. The data were corroborated by quantitative PCR, Western blotting, and immunocytochemistry. Among the 3948 effective genes, ∼84% (3321) were co-expressed in both cell-types, and 627 were differentially expressed more than twofold in MSC versus EC. MSC highly expressed numerous stem-cell-like genes. Early development genes of endothelial cells, though not up-regulated, had a high expression in MSC, such as EDF1, MDG1, and EDG2. In contrast, mature endothelial growth and signal pathway genes, like VEGF, CXCR4, and CTNNB1, were down-regulated in MSC. In conclusion, human MSC have a distinct molecular basis for endothelial differentiation.

[Differential expression of proteins in rat mesenchymal stem cells undergoing endothelial differentiation].

OBJECTIVE: To screen and identify differentially expressed proteins of mesenchymal stem cells (MSC) during endothelial differentiation. METHODS: MSCs were induced to endothelial differentiation with vascular endothelial growth factor (VEGF) and epithelial growth factor (EGF) mixture. The whole cell proteins were extracted and isolated by two-dimensional gel electrophoresis. After gel was analyzed by Imagemaster 5.0 software, differentially expressed proteins were partially selected and identified by MALDI-TOF-MS. RESULTS: The differentiated MSC highly expressed endothelial cells related markers, CD31, CD34 and FVIIIAg were 56.8%, 38.8% and 14.5% respectively by flow cytometer. Compared with the primary cultured MSC, the differentiated cells differentially expressed 91 proteins. Among the 19 identified proteins, 11 up-regulated and 8 down-regulated, which include cytoskeletal proteins, such as myosin, filamin, vimentin and vinculin; cell metabolism enzymes, such as ORP-150, ERO1-α, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, protein disulfide-isomerase A3, FAS and enolase 3; nuclear factors, such as TAR DNA binding protein, guanine nucleotide binding protein and hypoxia up-regulated protein 1; VEGF receptors, such as KDR and so on. CONCLUSIONS: Cytoskeletal proteins, metabolism enzymes and KDR were all involved in endothelial differentiation of MSC. These proteins may be the regulatory targets for endothelial differentiation of MSC.

Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression.

BACKGROUND: Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells. METHODS: To further confirm WTH3's drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the WTH3 promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the p53 transcription factor relative to WTH3 expression. To do so, the in vitro methylation method was utilized to examine the p53 transgene's influence on either the methylated or non-methylated WTH3 promoter. RESULTS: The results generated from the gene knockdown strategy showed that reduction of WTH3 expression increased MDR1 expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of p53 on WTH3 promoter activity. CONCLUSION: Taken together, our studies provided further evidence that WTH3 played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized p53's positive impact on WTH3 expression.

Differential methylation of TSP50 and mTSP50 genes in different types of human tissues and mouse spermatic cells.

Earlier studies identified human TSP50 as a testis-specific gene that encoded a threonine protease. Most importantly, TSP50 could be a cancer/testis antigen since there was a high frequency of reactivation in breast cancer biopsies. It was also found to be negatively regulated by the p53 gene. To further characterize this gene, we recently examined the DNA methylation patterns of the TSP50 gene promoter in normal human testis, as well as breast tissue and a testicular embryonic carcinoma cell line (HTECCL). Bisulfite genomic sequencing results demonstrated that the promoter exhibited mixed DNA methylation patterns in normal human testis, mainly non-methylation versus slight methylation, which could be attributed to the different stages spermatic cells go through during spermatogenesis. In contrast, it was methylated to a much greater extent in both breast tissue and HTECCL. To find out whether DNA methylation status was related to spermatogenesis stages, we analyzed DNA methylation patterns of the mTSP50 (the mouse ortholog of TSP50) promoter in spermatocytes and spermatozoa isolated from sexually mature mice. The results clearly demonstrated that each group of cells exhibited its preferential DNA methylation pattern that apparently was consistent with the gene expression status observed before. Taken together, our findings suggested that DNA methylation might regulate the TSP50 and mTSP50 gene expressions in different types of tissues and spermatic cells.

[Research on induced differentiation of human bone marrow mesenchymal stem cells into vascular endothelial cells].

AIM: To analyze gene expression difference between human mesenchymal stem cells and umbilical vein endothelial cells, to discuss the feasibility of inducing hMSCs to differentiate into endothelial cells through in vitro gene transfection as well as the use and prospective as a seeding cell source of vascular tissue engineering. METHODS: hMSCs and hUVECs were isolated, expanded in culture, and characterized by flow-cytometry, immunocytochemistry, immunofluorescence and transmission electron microscopy (TEM). Differential analysis of gene expression profiles between them was performed by Biostar H-40 cDNA microarrays. The properties of VEGF 165 transfected were also detected with RT-PCR, ELISA et al. RESULTS: Nearly 86% genes were coexpressed in both cells and hMSCs expressed typical endothelial antigen marker of EC. After VEGF165 transfection, hMSCs (or committed hMSCs) were positive for CD31. To different extent, the expression of CD44 was down regulated and CD34, FVIIIAg, Flt-1 up regulated. CONCLUSION: Generation of functional EC or tissue engineered blood vessels from human MSCs is feasible utilizing an in vitro environment gene transfection.

[Cloning, expression of soluble VEGFR2 fragment and its effect on tumor angiogenesis].

OBJECTIVE: To study the anti-tumor angiogenesis effect of soluble VEGF receptor fragment by blocking the combination of VEGF and its receptor in vivo and in vitro. METHODS: RT-PCR technique was used to amplify Flk-1/KDR fragment from embryo mouse liver, which was recombinated to expression vector pET-28b(+) and retrovirus vector PLXSN, which was induced to be expressed, purified and identified with EcoR I and Hind III. Mouse endothelial cells were separated, cultured and identified by immunocytochemistrical staining using VIII factor-related antigen antibody. The expressed product was analyzed about its effect on endothelial cell's growth in vitro with MTT method. The retrovirus vector was transfected to tumor cell lines S180 and B16 by liposome method to observe the biological specificity in vitro after gene transfection. RESULTS: 1000 bp size sVEGFR fragment was amplify from E9, E11 embryo mouse liver tissues, which was recombinated to TA clone vector and identified by sequence analysis. This fragment was cloned to expression vector pET-28b(+), the expressed product was purified and identified correctly. The in vitro study showed this expressed product can effectively inhibit endothelial cell(s), growth and proliferation. The fragment was then cloned to retrovirus vector PLXSN and transfected to tumor cell lines S180 and B16 successfully with RT-PCR and SDS-PAGE. The experiments in vivo showed that the weight of tumor smaller, the size decreased significantly, the microvessel density was fewer and Flk1 protein expression were higher in the group of gene transfection than that of control. CONCLUSION: Soluble VEGFR fragment is a kind of effective gene engineer product for anti-tumor angiogenesis gene therapy and the development of anti-tumor drug.

[Comparative study on activated immunocytes of human bone marrow and peripheral blood by cytokines].

To study immunophenotype and cytotoxicity of the immunocytes in bone marrow and peripheral blood after activation by combined cytokines, mononuclear cells (MNC) of bone marrow and peripheral blood were activated by IFN-gamma, IL-1, IL-2 and McAb-CD3 in vitro. The cell amount and morphology during culture were observed. Cytochemical staining and immunophenotype analysis were done before and after culture in two groups of MNC. Cytotoxicity was tested by MTT method. The results showed that the cell number of two groups increased obviously in culture (P < 0.05), while the peripheral blood mononuclear cells increased more markedly (P < 0.05). The cytochemical staining showed POX decrease, but PAS increase in two groups. The positive ratios of CD3(+), CD56(+) and CD38(+) cells in two groups increased obviously after culture (P < 0.05), but there was no significant difference between those two groups. CD3(+) CD56(+) cells increased obviously in peripheral blood mononuclear cells activated by cytokines (P < 0.05), but CD3(+) CD56(+) cells did not increase in bone marrow mononuclear cells. There was no significant difference between two groups' cytotoxicity. It was concluded that IFN-gamma, IL-1, IL-2 and McAb-C D3 increased cell number and cytotoxicity of both bone marrow and peripheral blood mononuclear cells that can be used in cell immunotherapy.

[The expression of Fas, FasL and Bcl-2 on RMA cells during the process of apoptosis induced by chemotherapeutic drugs].

The objective of the study is to explore the effect of Fas, FasL and Bcl-2 on the process of apoptosis induced by chemotherapeutic drugs through detecting the expression of Fas, FasL and Bcl-2 on murine lymphoma cell line RMA. Dexamethasone(DEX), etoposide (VP-16), arsenic trioxide As(2)O(3) and all trans-retinoic-acid (ATRA) were added to the RMA cells as well as to the cells preincubated with interleukin-2 (IL-2), interleukin-6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. The effect on apoptosis was observed and the expression of Fas and FasL mRNA as well as the expression of Fas and Bcl-2 antigen were measured. DEX and VP-16 could promote apoptosis of RMA cells while upregulating the expression of Fas and FasL without affecting the expression of Bcl-2. ATRA downregulated the expression of Bcl-2 without any change of Fas and FasL, and no apoptosis of RMA cells induced by ATRA was observed. Although As(2)O(3) induced apoptosis of RMA cells, it did not affect the expression of Fas, FasL and Bcl-2, which suggested that different drugs induce apoptosis of the same kind of cells by different signal transduction system and apoptosis induced by Fas system needed the coexistence of Fas and FasL. Although IL-2, IL-6 and GM-CSF upregulated the expression of Fas protein when adding to RMA cells separately, none of them induced apoptosis. Apoptosis could be induced by combination of IL-2 and IL-6 along with the upregulation of Fas and FasL. The cytokines facilitated the apoptotic action of chemotherapeutic drugs, the drug concentration for inducing apoptosis decreased and the time period of starting apoptosis shortened. Apoptosis could be observed without the expression of FasL when anti-Fas-antibody was added to RMA cells. The results demonstrated that there was synergistic effect of chemotherapeutic drugs and some cytokines for induction of apoptosis. Fas-FasL system participated in the apoptosis induced by DEX and VP-16; different drugs induce apoptosis by different pathway of signal transduction.

[Synergistic Induction of Apoptosis by Chemotherapeutic Drugs and Cytokines in Mouse T-Lymphoma Cell Line]

The objective of the study is to find out the synergistic effect on apoptosis resulting from the combination of chemotherapeutic drugs and some cytokines. Dexamethasone (DEX), etoposide (VP16), arsenic trioxide (As(2)O(3)) and alltrans-retinoic acid (ATRA) were added to the murine T lymphoma cell line RMA as well as to the cells preincubated with IL-2, IL-6 or GM-CSF, respectively. The effect on apoptosis was observed. All four chemotherapeutic drugs inhibited the cell proliferation. DEX, VP16 or As(2)O(3), except ATRA, singly induced apoptosis of RMA cells. The DEX concentration of inducing apoptosis was reduced when it was added together with ATRA. IL-2, IL-6 and GM-CSF did not induce apoptosis when the cytokines were added to RMA separately, however, apoptosis could be induced by combination of IL-2 and IL-6. The cytokines facilitated the apoptotic action of chemotherapeutic drugs, the drug concentration for inducing apoptosis decreased and the time period of starting apoptosis shortened. The results demonstrated that there was synergistic effect of chemotherapeutic drugs and some cytokines for induction of apoptosis. It raised an experimental basis for combination of chemotherapeutic drugs and some cytokines, especially IL-2, in the treatment of malignant lymphoma.