PrA inhibited IP-10 expression in infiltrating monocytes of cardiac allografts.

<p>Immunofluorescence staining of heart allograft sections stained with Abs to CD68 (FITC, green) and IP-10 (TMRITC, red). Nuclei were counterstained with DAPI (blue). The IP-10 expression in CD68 positive infiltrating monocytes was reduced after PrA treatment. The results are representative of three independent experiments (n = 3).</p

The migrating capacity of naïve T cells was slightly affected by PrA.

<p>The naïve T cells isolated from rat spleen were cultured with or without PrA (20 nM) for 72 h. A chemotactic migration assay was used to assess the migration of naïve T cells in the presence or absence of PrA towards different doses of either recombinant IP-10 or Mig. (A) PrA-conditioned T cells exhibited decreased migration in presence of 25 ng/mL of IP-10, but not at 50 ng/mL or 100 ng/mL, compared with those of non-condition T cells. (B) The migration of PrA-conditioned T cells was decreased at 50 ng/mL and 100 ng/mL of Mig, but not at 200 ng/mL, as compared with those of non-conditioned T cells. The result suggests that the capacity of T cell migration was partially affected by PrA <i>in vitro</i>. * indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA reduced T cell migration to PBMC supernatant through inhibition of IP-10-CXCR3 receptor interaction.

<p>IP-10 (50 ng/ml), Mig (100 ng/ml), or SDF-1 (100 ng/ml) was added to the PBMC supernatant. T cells were incubated with the isotype control Ab (20 µg/ml) or anti-CXCR3 Ab (20 µg/ml) prior to chemotactic migration assays (n = 3 in each group). (A) IP-10 addition to the supernatant of PrA-exposed PBMCs improved the migration of PrA-treated recipients T cells. Furthermore, addition of anti-CXCR3 Ab restored PrA inhibition of IP-10-induced T cell migration, but isotype Ab did not have the same effect. (B) The addition of Mig and SDF-1 did not significantly increase the migration of PrA-treated recipients T cells. However the addition of Mig, but not SDF-1 addition increased the migration of non PrA-treated recipients T cells. *indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA conditioning reduced PBMC IP-10 secretion <i>in vitro</i>.

<p>PBMCs isolated from control recipient rats on day four were cultured with different doses of PrA for 72(n = 3 per group). (A) PBMC viability was measured by MTT assay. The highest dose induced a significant decrease in viable cell count, therefore, only 5 nM and 20 nM concentrations were used in subsequent experiments. (B) Culture supernatants of PBMC with or without PrA condition were harvested and analyzed for the production of IP-10 by ELISA. * indicates <i>p</i><0.05 when comparing treatment condition to control.</p

PrA prevented CXCR3⁺T cell infiltration into allografts.

<p>(A) The heart allograft was harvested on day seven post heart transplantation, sectioned, and stained for TCR (TMRITC, red) and CXCR3 (FITC, green). The extent of colocalization (yellow) of TCR (T cell marker) and CXCR3 indicating CXCR3⁺ T cell infiltration into allografts was significantly reduced after PrA administration. The results are representative of three independent experiments. (B) The relative quantitative analysis to determine the percentage of CXCR3⁺TCR⁺ cells within infiltrating cells of allografts further showed that PrA treatment inhibited the CXCR3⁺ T cell infiltration into allografts. *indicates <i>p</i><0.05 when comparing treatment to control.</p

PrA inhibited recipient T cell migration towards PBMC supernatant.

<p>PBMC and spleen T cells were isolated from recipient control and PrA-treated rats on day seven posttransplantation. The migration of T cells toward PBMC supernatant was tested using a chemotactic migration assay. In the presence of PBMC supernatant from control recipients, the migration of T cells from PrA-treated recipients was not impaired compared with those from control recipients. However, in the presence of PBMC supernatant from PrA-treated recipients, the migration of T cells was significantly impaired when cells were harvested from either PrA-treated or control recipients. * indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA depressed IP-10 mRNA level in allografts.

<p>(A) The effect of PrA administration on IP-10 mRNA expression in heart allografts of recipient rats was assessed by relative qRT-PCR on day 0, 3, 7, and 9 posttransplantation. The IP-10 mRNA level decreased with PrA addition (n = 8) as compared to controls (n = 8) on day seven and nine. (B) The gene expression of Mig, ITAC and RANTES was assessed in cardiac allografts on day 7 posttransplantation. However, no significant difference between control (n = 8) and PrA treated group (n = 8) was detected. * indicates <i>p</i><0.05 when comparing treatment to control.</p

PrA treatment reduced IP-10 secretion from PBMCs in recipient rats.

<p>Control and PrA-treated PBMCs from recipient rats (n = 8) were separately isolated on day seven posttransplantation and cultured at a concentration of 2×10⁶ cells/ml for 4 h. Culture supernatant was harvested and analyzed for the IP-10 secretion by ELISA. * indicates <i>p</i><0.05 when comparing treatment to control.</p

ANGPTL4 influenced expression of integrin β1 in MSCs.

<p>(<b>A to C</b>) mRNA and protein levels of integrin β1 molecules were analyzed by qRT-PCR and western blot analysis as described in the methods. (Each column represents the mean ± SD of three independent experiments; *<i>P</i><0.05 vs. control; ▴<i>P</i><0.05 vs. hypoxia/SD). (<b>D</b>) Expression levels of integrin β1 on the MSCs surfacewere analyzed by immunofluorescence assay.</p

Angiopoietin-Like 4 Confers Resistance to Hypoxia/Serum Deprivation-Induced Apoptosis through PI3K/Akt and ERK1/2 Signaling Pathways in Mesenchymal Stem Cells

<div><p>Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured <i>in vitro</i>. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.</p></div