Fig 2: Brain endothelial specific signature.(A) Heat map representation of differentially upregulated genes identified by comparing brain ECs to lung and heart ECs at baseline. The blue to white to red gradient represents increasing expression of the pathway with blue representing minimal expression while the red represents high expression of the pathway. Individual gene expression values can be visualized at www.rehmanlab.org/ribo (B) The GSEA results of enriched GO terms from RiboTag brain ECs at baseline. (C) Top RiboTag brain EC signature markers ranked in order of logFC. (D) Top RiboTag brain EC cell surface markers identified using the Cell Surface Protein Atlas. (E) Confocal analysis was performed after brain, lung, and heart ECs were processed on a cytospin to assess brain EC PTN (Pleotrophin) specificity. A scale bar of 20 µm is included on all images.

Fig 3: Astrocyte-derived Pleiotrophin is required for recovery from acute neuroinflammation. (A) Schematic of lentiviral vector (Top). EAE progression in mice transduced with Gfap-Scrmbl (n = 5) or Gfap-Ptn (n = 5). Representative of two independent experiments. Data shown as mean ± S.E.M. Two-way repeated measures ANOVA. ****P < 0.0001. (B) Representative scatterplots (entire group concatenated) and quantification (C) of cellular abundance in the CNS (brain and spinal cord) of Gfap-Scrmbl (black, n = 4) and Gfap-Ptn (red, n = 5) mice according to CD45 and CD11b expression at day 28 post immunization. Data shown as mean ± SD. Sidak’s multiple comparisons test. **P < 0.01, ****P < 0.0001 (D) Abundance of CD45hiCD11b+ myeloid cells and quantification of their cytokine production (E, F) in CNS (brain and spinal cord) of Gfap-Scrmbl (black, n = 4) and Gfap-Ptn (red, n = 5) mice at day 28 post immunization. Data shown as mean ± SD. Sidak’s multiple comparisons test. *P < 0.05, ***P < 0.001. (G) Abundance of CD3+CD4+ T cell subsets in CNS (brain and spinal cord) of Gfap-Scrmbl (black, n = 4) and Gfap-Ptn (red, n = 5) mice at day 28 post immunization. Data shown as mean ± SD. Sidak’s multiple comparisons test. (H) Abundance of CNS-resident cell populations (brain and spinal cord) in Gfap-Scrmbl (black, n = 4) and Gfap-Ptn (red, n = 5) mice at day 28 post immunization. Data shown as mean ± SD. *P < 0.05.

Fig 4: Triad1 inhibited the MDM2-mediated PTN ubiquitinationin HEK-293 cells.A, PTN ubiquitination in HEK-293 cells when Flag-PTN and His-ubiquitin were cotransfected with HA-MDM2 or not was detected by Co-IP. B, in the presence or absence of MG132, PTN ubiquitination in HEK-293 cells when Flag-PTN and His-ubiquitin were cotransfected with HA-MDM2 or not was determined by Co-IP. C and D, the protein expressions of PTN and Triad1 in astrocytes transfected with plasmids overexpressing MDM2 and Triad1 were detected by Western blot. E, in the presence or absence of MG132, PTN ubiquitination in HEK-293 cells when Flag-PTN, His-ubiquitin, and HA-MDM2 were cotransfected with Myc-Triad1 or not was detected by Co-IP. (∗∗∗p < 0.001, versus NC; ###p < 0.001, versus OE-MDM2; ˆˆp < 0.01, versus OE-Triad1). Co-IP, coimmunoprecipitation; MDM2, murine double minute 2; NC, negative control; OE, overexpressed; PTN, pleiotrophin; Triad1, two RING fingers and DRIL 1.

Fig 5: T-MSCs produce pleiotrophin (PTN) and promote BM engraftment. PTN expression levels in (A) whole-cell lysates and (B) conditioned media of BM-, AT-, or T-MSCs were determined by western blot; 1 ng of rhPTN was loaded in parallel. (C) Secreted levels of PTN in CM of BM-, AT-, or T-MSCs were quantified by ELISA. (D) BMT was performed in the presence of CM, rhPTN, or CM + anti-PTN antibody, and mice were sacrificed on day 10 post-BMT (n = 5). Body weight changes are indicated. (E) The number of circulating RBC and WBC were counted. (F) Histological BM changes were determined by H&E staining of mouse femurs (100× magnification) and (G) BM cellularity was measured from more than eight different fields using ImageJ software. Data are presented as mean ± S.E.M. and were analyzed using one-way ANOVA (** p < 0.01, *** p < 0.001).