MSC Transplantation Attenuates Inflammation, Prevents Endothelial Damage and Enhances the Angiogenic Potency of Endogenous MSCs in a Model of Pulmonary Arterial Hypertension

Figure 1. Experimental design.

Figure 2. Characterization and differentiation of chicken bone marrow-derived MSCs. (A and B) Morphology of MSCs. The cells exhibit spindle-like morphology during the early growth in DMEM ((A), passage 0, day 3). In later passages, the cells display various morphologies including star-like (arrow), spindle-shaped (arrowhead), and flattened morphology (*) ((B), passage 1, d 2). (C) Reverse transcription (RT)‐ PCR products of surface markers of cells at passage 2. β actin was used as reference gene. Fragments were fractioned on a 1.3% agarose gel and stained with Goldview reagent. Note the lack of CD34, CD45 and CD31 expression. Images are representative of three independent experiments. (D) Representative immunofluorescence staining showing the expression of α-SMA (upper) and CD133 (down) of cells at passage 2. The cell nuclei were labeled with DAPI. Images are representative of three independent experiments. (E) Representative photomicrographs of Oil Red O stain (left) showing intracytoplasmic lipid droplets (red) and of Alizarin Red S stain (right) showing calcium deposits (orange) of cells at passage 2. (F) The “s-shape” cell growth curves show that the chicken bone marrow-derived MSCs…

Figure 3. Detection of transplanted MSCs in the lung. (A) Representative image showing 4’,6-diamidino-2-phenylindole (DAPI)-labelled MSCs. (B) DAPI-labelled MSCs (2×106) (upper panel) were injected via the wing vein of broiler chickens. The unlabeled MSCs (lower panel) were served as a control. At d 6 post implantation, lung was collected and embedded in optimal cutting temperature (OCT) compound to obtain 15-μm-thick sections for determination of DAPI-positive cells (arrow). Images were acquired with fluorescence microscopy. Bright field and DAPI-fluorescence are shown.

Figure 4. Right-to-total ventricle ratio (RV/TV) of birds treated with MSCs (MSC group) or PBS (mock group) at weeks 1–4 after MSC implantation. Data are expressed as mean ± SEM of 6 birds.

Figure 5. Regulatory effect of MSC transplantation on the production of inflammatory mediators in the lung. Lung samples of birds treated with MSCs (MSC group) or PBS (mock group) were collected at weeks 1, 2 and 3 post transplantation, homogenized and subjected to Western blot analysis with anti-tumor necrosis factor (TNF)-α (A), anti-interleukin (IL)-6 (B) and anti- IL-1β (C). Tubulin was used as the equal loading control. Results are expressed as mean ± SEM of at least 5 birds. The data are representative of 2 separate experiments.

Figure 6. Transplantation of MSCs alleviated the histologic features of PAH. (A–E) Representative photographs of small pulmonary arteries from birds treated with PBS (mock group) showing (A) loss of endothelial cells, (B) eccentric intimal thickening (arrow), (C) endothelial proliferation, (D) an immature lesion, and (E) a mature glomeruloid-like plexiform lesion (arrowhead) with perivascular inflammatory infiltrates (arrow). (F) A small pulmonary artery from a bird treated with MSCs (MSC group) showing intact vascular endothelium. (G) Percentage of arterioles with normal endothelium. At least 20 arterioles with an outer diameter < 50 μm were randomly selected in each slide for analysis. Data are expressed as mean ± SEM of six birds. (H) Plexiform lesion density in the lung. Data are expressed as mean ± SEM of at least three birds.

Figure 7. Expression of angiogenic factors in the lung. Lung samples of birds treated with MSCs (MSC group) or PBS (mock group) at weeks 3 post transplantation were subjected to qPCR analysis for measuring the mRNA levels of transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF) and angiopoietin (Ang)-1 and its receptor Tie-2. Results are expressed as mean ± SEM of 3 birds. The data are representative of 2 separate experiments.

Figure 8. Transplantation of MSCs enhanced endothelial differentiation of endogenous MSCs. Blood samples were collected from normal (A), PBS- (B) and MSC-treated (C) broiler chickens at 3 weeks post transplantation. Cells at Passage 1 were suspended in DEME and seeded in 96-well plates pre-coated with Matrigel matrix at 5 × 104/well with triplicate. Tubular-like structures were observed udder phase-contrast microscopy within 4 h of incubation.