Pericytes die rapidly in the acute phase and replenish in the subacute and chronic phases after stroke.

A.Immunoflurescence staining shows Tunel+ pericytes(white) and Tunel+ ECs(yellow) after MCAO at RP2D and quantitative the proportion of Tunel+ cells in pericytes and ECs(n=3, 20 slices/mouse). B.Immunoflurescence staining shows CD13+ soma after MCAO at RP2D(n=3,20 slices/mouse). C.Immunoflurescence staining shows CD13+ soma after MCAO at RP7D and RP34D, quantitative the ratio of CD13+ soma (n=3,20 slices/mouse). D.Flow cytometry analysis of the proportion of pericytes and ECs after MCAO at RP2D, RP7D and RP34D(n=6). E. Quantitative the proportion of pericytes and ECs at different reperfusion times after stroke(n=6). F.Schematic diagram displaying the time course for EdU injection and analysis time points. G.Maximum EdU signal in the ischemic area at different EdU injection times(n=3). H.Immunoflurescence staining shows EdU+ pericytes (white) and EdU+ ECs(yellow) after MCAO at RP34D and quantitative the proportion of EdU+ cells in pericytes and ECs(n=3,20 slices/mouse). I.Flow cytometry analysis of the proportion of EdU+ pericytes and EdU+ ECs after MCAO at RP14D (n=4). J.Flow cytometry analysis of the proportion of EdU+ pericytes and EdU+ ECs after MCAO at RP7D, RP14 and RP34D(n=4).

scRNA-seq is used to explore the fate of ECs after stroke.

A.Samples were obtained from mouse ischemic brains at Sham, RP7D and RP34D. Single cells were processed using Chromium 10x 3′DEG chemistry. B.Uniform manifold approximation and projection (UMAP) embedding of all cells and marker genes. C.UMAP analysis of individual Sham, RP7D and RP34D cell transcriptomes showed 10 clusters. D.Heatmap displays an expression of the top 50 upregulated genes in each cluster. The scale bar represents the z-score of average gene expression (log). E.Relative proportion of major cell types in different reperfusion times. F.Differential genes expression variance over pseudotime of ECs differentiation trajectory branches. G.Differential transcription factors expression variance over pseudotime of ECs differentiation trajectory branches. H.Pseudotime trajectory of ECs differentiation trajectory branches. The arrows show the direction of pseudotime trajectories.

Endothelial-to-pericytic transition can replenish pericytes and undergo an intermediate fibroblast-like cell state after stroke.

A.Immunoflurescence staining of CD31 and CD13 expression in Cdh5CreERT2;Ai47 mice with MCAO at RP34D and quantitative the proportion of CD13+ cells(n=5,20 slices/mouse). B.Immunoflurescence staining of CD31 and PDGFRβ expression in Cdh5CreERT2;Ai47 mice with MCAO at RP34D and quantitative the proportion of PDGFRβ+ cells(n=5,20 slices/mouse). C.Immunoflurescence staining of CD31 and NG2 expression in Cdh5CreERT2;Ai47 mice with MCAO at RP34D and quantitative the proportion of NG2+ cells(n=5,20 slices/mouse). D.Immunoflurescence staining of CD31 and PDGFRα expression in Cdh5CreERT2;Ai47 mice with MCAO at RP34D and quantitative the proportion of PDGFRα+ cells (n=5,20 slices/mouse). E.Quantitative the proportion of EGFP+ pericytes on blood vessel/pericytes on blood vessel(n=5,20 slices/mouse). F.Quantitative the proportion of EGFP+ pericytes on blood vessel/EGFP+ pericytes (n=5,20 slices/ mouse). G.Quantitative the proportion of EGFP+ pericytes on blood vessel/ migrating EGFP+ pericytes(n=5, 20 slices/mouse). H.Quantitative the proportion of EGFP+ pericytes/all pericytes(n=5, 20 slices/ mouse). I.Flow cytometry analysis of the proportion of EGFP+&CD31- cells after MCAO at different times and quantitative the proportion of EGFP+&CD31- cells(n=4). J.Immunoflurescence staining of CD31 and PDGFRα expression in Cdh5CreERT2;Ai47 mice with MCAO at RP8D(n=5,20 slices/mouse). K.Immunoflurescence staining of CD31 and vimentin expression in Cdh5CreERT2;Ai47 mice with MCAO at RP8D (n=5,20 slices/mouse). L.GO analysis of upregulated DEGs in EGFP+&CD31- cells subgroup, compared with EGFP+&CD31+ cells from contralateral. M.KEGG pathway analysis of upregulated DEGs in EGFP+&CD31- cells subgroup, compared with EGFP+&CD31+ cells from contralateral.

E-Perciytes deletion by AAV2/9-BI30-DIO-NG2-promotor-DTA virus aggravates BBB leakage after stroke.

A.Schematic diagram displaying Cdh5CreERT2 injection with AAV2/9-BI30-DIO-NG2-promotor -DsRed or DTA virus to kill the cell and the time course for tamoxifen, MCAO and analysis time points. B.Immunoflurescence staining of CD13 expression in Cdh5CreERT2 injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed A virus(n=3). C.Flow cytometry analysis the proportion of DsRed+&CD13+/ DsRed+ cells and quantitative the proportion (n=3). D.Flow cytometry analysis the proportion of E-Pericytes cells in Cdh5CreERT2 injection with AAV2/9-BI30-DIO-NG2-promotor-DTA virus and quantitative the proportion (n=5). E.Image showing the leakage of Evans blue in WT mice at different times after MCAO and quantitative the leakage of Evans blue(n=3). F.Image showing the leakage of Evans blue in Cdh5CreERT2 injection with AAV2/9-BI30-DIO-NG2-promotor-DTA virus at RP34D after MCAO , quantitative the leakage of Evans blue(n=5) and the brain atrophy volume (n=5). G.Image showing the leakage of trypan blue in Cdh5CreERT2 injection with AAV2/9-BI30-DIO-NG2-promotor-DTA virus at RP34D after MCAO and quantitative the leakage of trypan blue(n=3).

E-Pericytes deletion by AAV2/9-BI30-DIO-NG2-promotor-DTA virus exacerbates neurological deficit after stroke.

A. Graph showing the survival rate of mice in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO at RP34D(n=15). B.Graph showing rotarod test in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO(n=9-10). C.Graph showing beam walking test in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO(n=9-10). D.Graph showing corner test in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO (n=9-10). E.Graph showing adhesive movement test in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO(n=9-10). F.Immunoflurescence staining of NeuN expression in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-Long-DsRed or DTA virus after MCAO at RP34D and quantitative the number in the unit area(n=4,20 slices/mouse). G.The heat map showing promotion neuron survival and growth genes expression in all groups (n=5). H.Image showing the change of CBF in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO at RP34D and quantitative the change of CBF(n=5). I.Immunoflurescence staining of CD13 and CD31 expression in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO at RP34D and quantitative the percentage (n=3,20 slices/mouse). J.Flow cytometry analysis of the proportion of CD13+ cells and CD31+ cells in Cdh5CreERT2 mice injection with AAV2/9-BI30-DIO-NG2-promotor-DsRed or DTA virus after MCAO at RP34D and quantitative the percentage (n=3).

Endothelial-specific loss and reinforcement of Tgfbr2 gene expression affect EndoMT and E-Pericytes.

A.Immunoflurescence staining of p-SMAD3 expression in Cdh5CreERT2;Ai47 mice at RP2D, RP8D and RP34D and quantitative the proportion and intensity(n=3,20 slices/mouse). B.Immunoflurescence staining of CD31 and p-SMAD3 expression in Cdh5CreERT2;Ai47 mice at RP8D (white:EGFP+&p-SMAD3+&CD31-; yellow:EGFP+&p-SMAD3-&CD31+; n=3). C.Immunoflurescence staining of EndoMT markers (p-SMAD3, KLF4 and α-SMA) expression in Cdh5CreERT2; Ai47;Tgfbr2fl/fl mice at RP2D and quantitative the proportion and intensity(n=3,20 slices/mouse). D.Immunoflurescence staining of CD31 expression in Cdh5CreERT2; Ai47;Tgfbr2fl/fl mice at RP34D and quantitative the number in the unit area (n=3,20 slices/mouse). E.Flow cytometry analysis the proportion of E-Pericytes in Cdh5CreERT2; Ai47;Tgfbr2fl/fl mice at RP34D and quantitative the proportion(n=5). F.Immunoflurescence staining of EndoMT markers(p-SMAD3+ and KLF4+) expression in Cdh5CreERT2; Ai47 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs at RP2D and quantitative the proportion of EndoMT markers(n=3,20 slices/mouse). G.Immunoflurescence staining of CD31 and CD13 expression in Cdh5CreERT2;Ai47 mice with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs at RP34D and quantitative the number in unit area(n=3,20 slices/mouse). H.Flow cytometry analysis of the proportion of E-Pericytes in Cdh5Cre ERT2; Ai47 mice with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs at RP34D and quantitative the proportion of E-Pericytes(n=3).

Infiltrating myeloid cells produce dominant TGFβ1 in mouse brain after stroke.

A.Immunoflurescence staining of CD45 and Tgfb1 gene expression in WT mice with MCAO at RP2D and quantitative the proportion of Tgfb1+ cells(n=4,10 slices/mouse). B.scRNA-seq of CD45+ cells isolated from the ipsilateral brain with MCAO:2H and RP1.5D. Dimensionality reduction and identification of clusters of transcriptionally similar cells were performed in an unsupervised manner(n=1), monocyte-derived macrophage (MDM). C.Tgfb1 gene expression in dimensionality reduction and identification of clusters(n=1). D.The percentage of Tgfb1 gene expression in different clusters(n=1). E.TGFβ1 protein expression in contralateral brain with MCAO at RP2D(n=3,20 slices/mouse). F.TGFβ1 protein expression in the ipsilateral brain with MCAO at RP2D, RP4D and RP8D (n=3,20 slices/mouse). G.Immunoflurescence staining of CD45 and Tgfb1 gene expression in WT mice injection with anti-Ly6C/Ly6G after MCAO at RP2D and quantitative the proportion of CD45+&Tgfb1+ cells in the unit area(n=4,10 slices/mouse). H.Immunoflures -cence staining of CD45 and Tgfb1 protein expression in WT mice injection with anti-Ly6C/Ly6G after MCAO at RP2D.

Infiltrating myeloid cells promote BBB renovation and neurological recovery after stroke.

A.Immunoflurescence staining of CD13 and CD31 expression in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the number in the unit area(n=4,20 slices/mouse). B.Flow cytometry analysis the proportion of EGFP+&CD31+ cells in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the percentage(n=4). C.Image showing the leakage of Evans blue in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the leakage of Evans blue and brain atrophy volume(n=5). D.Immunoflurescence staining of NeuN expression in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the number in the unit area(n=4,20 slices/mouse). E.Graph showing the survival rate of mice in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO(n=10). F.Graph showing rotarod test in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO(n=7-10). G.Graph showing beam walking test in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO(n=7-10). H.Graph showing corner test in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO(n=7-10). I.Graph showing adhesive movement test in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO(n=7-10).

E-Pericytes deletion by specific endothelial cells knockout the Tgfbr2 gene aggravates BBB leakage and neurologic deficit after stroke.

A.Flow cytometry analysis of the proportion of E-Pericytes synchronous injection with AAV2/9-BI30-DIO-NG2-promotor DTA virus and AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at 14D (n=4). B.Image showing the leakage of Evans blue in iECs;Tgfbr2fl/fl mice at different times after MCAO and quantitative the leakage of Evans blue(n=3). C.Graph showing the leakage of Evans blue in iECs;Tgfbr2fl/fl mice at different times after MCAO and quantitative the leakage of Trypan blue(n=3). D.Graph showing the percentage of pericyte and ECs, the leakage of Evans blue in iECs;Tgfbr2fl/fl mice at different times after MCAO(n=3-6). E.Immunoflurescence staining of NeuN expression in iECs;Tgfbr2fl/fl mice at RP34D after MCAO and quantitative the number of neurons in the unit area(n=3,20 slices/mouse). F.Graph showing the rotarod test in iECs;Tgfbr2fl/fl mice after MCAO at RP34D(n=8-10). G.Graph showing the beam walking test in iECs;Tgfbr2fl/fl mice after MCAO at RP34D(n=8-10). H.Graph showing the corner test in iECs;Tgfbr2fl/fl mice after MCAO at RP34D(n=8-10). I.Graph showing the adhesive movement test in iECs; Tgfbr2fl/fl mice after MCAO at RP34D (n=8-10).

EC-specific overexpression of the Tgfbr2 gene reinforces BBB function and neurological recovery after stroke.

A. Image showing the leakage of Evans blue in Cdh5CreERT2 injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus at RP34D after MCAO and quantitative the leakage of Evans blue(n=5). B.Immunoflurescence staining of NeuN expression in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at RP34D and quantitative the number in the unit area(n=3,20 slices/mouse). C.Graph showing rotarod test in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO(n=8-10). D.Graph showing beam walking test in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRedWPREs virus after MCAO(n=8-10). E.Graph showing corner test in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO(n=8-10). F.Graph showing adhesive movement test in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG -P2A-DsRed-WPREs virus after MCAO(n=8-10).

Cdh5CreERT2 mice specifically recombine parenchymal endothelial cells, related to Figure 2.

A.Schematic diagram displaying Cdh5CreERT2 mice label ECs. B.Immunoflurescence staining show with or without Tamoxifen in Cdh5Cre-ERT2; Ai47 mice(n=3,10 slices/mouse). C.Flow cytometry analyzes the proportion of EGFP+ cells and quantitatively the proportion of EGFP+ cells(n=4). D.Immunoflurescence staining of CD31 expression in Cdh5CreERT2;Ai47 mice and quantitative the proportion of EGFP+ &CD31+/ CD31+ or EGFP+ blood vessel(n=8,10 slices/mouse). E.Flow cytometry analysis of the proportion of EGFP+ &CD31+ /EGFP+ cells and quantitative the proportion(n=5). F.Immunoflurescence staining of CD31 expression in Cdh5CreERT2; Ai14 mice(n=3,20 slices/mouse). G. Immunofluorescence staining ECs markers (ERG, GLUT1 and VE-Cadherin) expression in Cdh5CreERT2; Ai47 mice (n=3-27, 10 slices/mouse). H.Immunoflurescence staining pericyte markers(CD13, PDGFRβ ,α-SMA and NG2) expression in Cdh5CreERT2; Ai47 mice(n=3-6, 20 slices/mouse). I.Quantitative the proportion of F, G and H. J.Flow cytometry analysis of the proportion of PDGFRβ+ cells in EGFP+ cells and quantitative the proportion (n=4).

EGFP+ cells transcriptomic dataset quality.

A.Violin plots showing the distribution of the number of total UMI counts per cell (nCount), genes detected per cell (nFeature), and percentage of mitochondrial genes (percent.mt) per identified cell type. B.UMAP plots depicting expression of individual marker genes for ECs (Pecam1), Arterial ECs (Gkn3), Venous ECs (Slc38a5), Capillary ECs (Rgcc), SMCs (Cnn1), Pericytes (Pdgfrb), Fibroblasts (Pdgfra), Microglia (Iba1) and Ependymal cells (Dynlrb2). Scale bars represent the log of normalized gene expression. C.Statistical table showing the percentage and number for each cell type.

Different means prove that ECs can give rise to pericyte-like cells after stroke, related to Figure 3.

A.Schematic diagram displaying the time course for MCAO, tamoxifen and analysis time points. B.Laser speckle displaying CBF change after stroke(ipsilateral/contralateral) and quantitative the proportion (n=7). C.Immunoflurescence staining of ECs markers (ERG, GLUT1 and VE-Cadherin) expression in Cdh5CreERT2; Ai47 mice with MCAO after RP34D(n=3,20 slices/mouse). D.Immunoflurescence staining of CD31 and vimentin expression in Cdh5CreERT2;Ai47 mice with MCAO at RP34D. E.Immunoflurescence staining of CD68 and Iba1 expression in Cdh5CreERT2;Ai47 mice with MCAO at RP34D. F.Quantitative the number of EGFP+&CD31-cells in unit area at different reperfusion times after stroke(n=2-6). G.Immunoflurescence staining of CD31 and CD13 expression in Cdh5CreERT2;Ai47 mice with MCAO at RP8D. H.Immunoflurescence staining of CD31 and NG2 expression in Cdh5CreERT2;Ai47 mice with MCAO at RP8D. I.Quantitative the percentage of EGFP+&CD31-cells in core and penumbra after stroke at RP34D (n=4,20 slices/mouse). J.Immunoflurescence staining of EdU expression in EGFP+ pericytes after MCAO at RP34D and quantitative the proportion of EdU+ cells(n=4,20 slices/mouse). K.Immunoflurescence staining of CD31 and CD13 expression in Cdh5CreERT2 mice injection with AAV2/9-CAG-DIO-EGFP virus after MCAO at RP34D and quantitative the proportion of CD13+ cells(n=4,20 slices/mouse). L.Immunoflurescence staining of CD31 and CD13 expression in Tie2Dre;Mfsd2aXER;Ai47 mice with MCAO at RP34D and quantitative the proportion of CD13+ cells(n=3,20 slices/mouse). M.Immunoflurescence staining of CD31 and CD13 expression in Ai47 mice infected with AAV-BI30-Cre virus after MCAO at RP34D and quantitative the proportion of CD13+ cells (n=3,20 slices/ mouse).

ECs give rise to cells with a similar pericyte transcriptome profile after stroke, related to Figure 3.

A.The gate of sorting ECs in contralateral(C-ECs), ECs in ipsilateral(I-ECs) and EGFP+&CD31- cells in ipsilateral(EGFP+ non-ECs). B.Principal component analysis (PCA) of the variance-stabilized estimated raw counts of differentially expressed genes. C.The heat map shows 100 genes in all groups. D.The heat map showing endothelial cell transcription factors genes expression in all groups(n=2-5). E.The heat map showing endothelial enriched transmembrane receptor genes expression in all groups(n=2-5). F.The heat map showing endothelial enriched ligand genes expression in all groups(n=2-5). G.The heat map showing pericytic transcription factors genes expression in all groups(n=2-5). H.The heat map showing pericytic-enriched transmembrane receptor gene expression in all groups(n=2-5). I.The heat map showing pericytic enriched ligand genes expression in all groups(n=2-5). J.Volcano plot showing differential expression of genes in EGFP+ non-ECs/C-ECs(n=5). K.GO functional enrichment analysis from up-regulation genes in DEG. L.KEGG functional enrichment analysis from up-regulation genes in DEG.

Systemic inhibition of TGFR2 reduces EndoMT and E-Pericytes, related to Figure 6.

A.Immunofluorescence staining of KLF4 expression in Cdh5CreERT2;Ai47 mice at RP2D, RP8D and RP34D and quantitative the proportion and intensity(n=3,20 slices/mouse). B.Immunoflurescence staining of ɑ-SMA expression in Cdh5CreERT2;Ai47 mice and at RP2D, RP8D and RP34D and quantitative the proportion and intensity(n=3,20 slices/mouse). C.Flow cytometry analysis the proportion of ɑ-SMA+&CD31+ ECs in Cdh5CreERT2; Ai47 mice at RP2D and quantitative the proportion(n=3). D. Heatmap depiction of different EndoMT marker genes in ECs at RP2D(n=4). E.Immunoflurescfence staining of EndoMT markers (p-SMAD3, KLF4 and α-SMA) expression in Cdh5CreERT2; Ai47 mice injection with TGFβR2 inhibitors at RP2D and quantitative the proportion(n=3,20 slices/mouse). F.Flow cytometry analysis the proportion of ɑ-SMA+&CD31+ ECs in Cdh5CreERT2; Ai47;Tgfbr2fl/fl mice at RP2D and quantitative the proportion (n=3). G.Immunoflurescence staining of CD31 expression in Cdh5CreERT2; Ai47 mice injection with TGFβR2 inhibitors at RP34D and quantitative the number in the unit area (n=3,20 slices/ mouse). H.Flow cytometry analysis of the proportion of E-Pericytes in Cdh5CreERT2; Ai47 mice injection with TGFβR2 inhibitors at RP34D and quantitative the proportion(n=3). I.Immunoflurescence staining of CD31 and TGFBR2 expression in Cdh5CreERT2; Ai47 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs and quantitative the proportion and intensity(n=3,20 slices/mouse). J.Flow cytometry analysis of the proportion of ɑ-SMA+&CD31+ ECs in Cdh5CreERT2; Ai47 mice with AAV2/9-BI30-EF1α -DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs at RP2D and quantitative the proportion.

Myeloid cells are leading immune cells in the mouse brain after stroke at RP2D, related to Figure 7.

A.Immunoflurescence staining of CD45 and Tgfb1 gene expression in WT mice with MCAO at RP1D and quantitative the proportion of Tgfb1+ cells(n=4,10 slices/mouse). B.Immunoflurescence staining of Iba1 and Tgfb1 gene expression in WT mice with MCAO at RP2D and quantitative the proportion of Tgfb1+ cells(n=4,10 slices/mouse). C.Immunoflurescence staining of CD13 and Tgfb1 gene expression in WT mice with MCAO at RP2D and quantitative the proportion of Tgfb1+ cells(n=4,10 slices/mouse). D.Flow cytometry analysis the proportion of CD45hi cells from ipsilateral brain with MCAO:2H and RP1.5D. E.Flow cytometry analysis the proportion of myeloid cells from the ipsilateral brain with MCAO:2H and RP1.5D. F.Quantitative the proportion of CD45hi cells (D) and monocyte(E)(n=5). G.Flow cytometry analysis the proportion of myeloid cells from bone marrow, blood and ipsilateral brain after mice injection with anti-Ly6C/Ly6G at RP2D(n=4). H. Quantitative the proportion of monocyte and neutrophil in(J)(n=4).

Infiltrating myeloid cells promote vascular function recovery after stroke, related to Figure 8.

A.Flow cytometry analysis the proportion of myeloid cells from bone marrow, blood and ipsilateral brain after mice injection with anti-Ly6C/Ly6G at RP34D(n=4). B.Quantitative the proportion of monocyte and neutrophil in(A)(n=4). C.Image showing the change of CBF in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the change of CBF(n=5). D.Flow cytometry analysis the proportion of CD13+ cells and CD31+ cells in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the percentage(n=3). E.Immunofluorescence analysis the proportion of CD13+ cells and CD31+ cells in WT mice injection with anti-Ly6C/Ly6G at RP34D after MCAO and quantitative the percentage(n=3).

E-Pericytes deletion by specific endothelial cells knockout the Tgfbr2 gene aggravates BBB leakage small molecule, related to Figure 9.

A.Image showing the leakage of Trypan blue in iECs;Tgfbr2fl/fl mice at RP34D after MCAO and quantitative the leakage of Trypan blue(n=3). B.Image showing the leakage of dextran-rhodamine B in iECs;Tgfbr2fl/fl mice at RP34D after MCAO(n=3,10 slices/mouse). C.Image showing the leakage of Texas-Ted in iECs;Tgfbr2fl/fl mice at RP34D after MCAO (n=3,20 slices/mouse).

ECs-specific overexpression of the Tgfbr2 gene reinforces CBF and vessel length, related to Figure 10.

A.Image showing the change of CBF in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO -Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at RP34D and quantitative the change of CBF(n=5). B.Graph showing the change of CBF using Doppler test in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at RP34D and quantitative the change percentage (n=5). C.Image showing FITC-Dextran in gelatin from Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO -Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at RP34D and quantitative the percentage(n=3). D.Immunoflurescence staining of CD31 expression in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at RP34D and quantitative the percentage, FITC-Dextran in gelatin injection by cardiac perfusion (n=3,20 slices/mouse). E.Flow cytometry analysis of the proportion of PDGFR+ cells and CD31+ cells in Cdh5CreERT2 mice injection with AAV2/9-BI30-EF1α-DIO-Tgfbr2-3XFLAG-P2A-DsRed-WPREs virus after MCAO at RP34D and quantitative the percentage(n=5).