Fig 2: Plg is critical for MSC-mediated tissue repair after HI.HI was induced in Plg+/+ and Plg−/− mice by ligation of the femoral artery, followed by MSC transplantation or saline injection in the ischemic limbs. (A and B) Mice were subjected to analysis by laser Doppler scanning. (A) Representative images are shown. (B) Blood perfusion rates in ischemic limbs were quantified and expressed as the percentage of normal limbs (n = 6–8, mean ± SEM). Statistical analysis by 2-way ANOVA (comparing MSC-treated Plg+/+ and Plg−/− mice). (C) Gastrocnemius muscle in normal or ischemic limbs was collected day 7 after the surgery and subjected to H&E staining. Arrows or left area of dashed line indicate newly regenerated muscles. Original magnification, ×100. (D) Gastrocnemius muscle in ischemic limbs was collected at day 7 after HI surgery. Tissue sections were probed with eMHC antibody. Upper, representative images. Bottom, quantitative analysis (n = 3, mean ± SEM). Statistical analysis by unpaired Student’s t test.

Fig 3: Plg is required for blood vessel formation mediated by MSC in ischemic tissues.HI was induced in Plg+/+ and Plg−/− mice by ligation of the femoral artery, followed by MSC transplantation or saline injection in the ischemic limbs. MSCs were harvested from Col2-Cre Rosa26-LSL-tdTomato mice. Gastrocnemius muscle in ischemic limbs was collected at day 7 after HI surgery. Tissue sections were probed with (A) anti-CD31 antibody, (B) anti-tdTomato and anti–NG-2 antibodies, or (C) anti-tdTomato and anti–α-SMA antibodies, followed by immunofluorescence imaging. (A) Left, representative images. Right, quantitative analysis of CD31+ area for mice treated with MSCs (n = 3, mean ± SEM). Statistical analysis by unpaired Student’s t test. (B and C) Representative images of ischemic tissues from MSC-treated mice are shown. Original magnification, ×100.

Fig 4: Plg promotes MSC proliferation and migration under normoxia and improves MSC survival under hypoxia.(A) MSCs were cultured with serum-free medium and treated with or without Plg under normoxia. Cell proliferation was determined by MTS-based assay (n = 4–8, mean ± SEM). Statistical analysis by 1-way ANOVA (compared with control). (B) MSCs were suspended in serum-free medium supplemented with or without Plg and seeded on Transwell membranes precoated with Matrigel. Plg-depleted FBS was used as the attractant to induce the migration through Matrigel. Cell migration was determined after 4-hour incubation. Top, representative images. Bottom, quantified results (n = 12, mean ± SEM). Statistical analysis by unpaired Student’s t test. (C) MSCs were cultured with serum-free medium and treated with or without Plg under hypoxia (2% O2). Cell proliferation was determined by MTS-based assay (n = 3, mean ± SEM). Statistical analysis by 1-way ANOVA (compared with control). (D) MSCs were cultured with serum-free medium and treated with or without Plg under hypoxia (2% O2) for 24 hours. Cell apoptosis was analyzed by using FITC-annexin V–based flow cytometry. Left, representative sorting. Right, quantified results (n = 3, mean ± SEM). Statistical analysis by 1-way ANOVA. Experiments were repeated 3 times and representative results are shown.

Fig 5: Plg-activated Cyr61 is critical for angiogenesis in vivo and blood reperfusion after HI.(A and B) Matrigel supplemented with PBS, control CM (CM derived from MSCs), or Plg CM (CM derived from MSCs treated with Plg) plus IgG, or Plg CM plus anti-Cyr61 antibody, was implanted subcutaneously in Plg−/− mice. Sections of Matrigel plugs were probed with anti-CD31 antibody, followed by immunofluorescence analysis. (A) Representative images. (B) Quantitative analysis (n = 4–8, mean ± SEM). Statistical analysis by 1-way ANOVA. (C–E) MSCs were lentivirally transduced with Lenti-actCyr61 or empty vector (EV), followed by transplantation into ischemic limbs of Plg+/+ and Plg−/− mice immediately after HI surgery. Blood perfusion was analyzed by laser Doppler scanning. (C) Representative images are shown. (D) Blood perfusion rates in ischemic limbs were quantified and expressed as the percentage of normal limbs (n = 3–6, mean ± SEM). Statistical analysis by 2-way ANOVA. (E) Cell lysates from transduced MSCs were immunoblotted. L (low), M (medium), and H (high) indicate different doses of virus used in lentiviral transduction.