sca-1 c-kit / flk-1 isotype controls sca-1 ObR gating R2 sca-1 +, ObR +, flk-1 + sca-1 +, ObR +, c-kit + supplemental Material: Figure I Suppl. Figure I. Flow cytometry analysis after triple staining with fluorescence-conjugated antibodies was used to determine the percentage of sca-1 +, c-kit + and sca-1 +, flk-1 + bone marrow cells also expressing ObR. (A) First, data after cell staining for sca-1 and c-kit or sca-1 and flk-1, respectively, were plotted and double-positive cells (upper right quadrant in A) were gated (R2). (B) Next, cells in R2 were examined for ObR expression and triple-positive cells (upper right quadrant in B) were quantified. Representative dot plots, the gating strategy and the mean±SEM of n=3 separate experiments are shown. R2 A B specific antibodies
B supplemental Material: Figure II A Suppl. Figure II. (B) Western Blot analyses revealed increased phosphorylation (p-) of AKT, PKC and STAT3 in bone marrow cells, isolated from WT, db/db or NOX2 -/- mice, in response to leptin (100 ng/mL for 5 min; white bars; n=3-10 mice per group). *P<0.05 and **P<0.01, ***P<0.001 vs. unstimulated controls (black bars). Representative findings are also shown. Suppl. Figure II. (A) Quantitative real time PCR analysis confirmed significantly increased MMP9 mRNA levels in bone marrow of leptin-treated WT mice, whereas the effect of leptin was absent in WT mice transplanted with bone marrow from db/db mice (db/db), or in NOX2 knockout mice (NOX2 -/- ; n=3-6 mice per group). *P<0.05 vs. control. p-AKT AKT WT NOX2 -/- db/db controlleptin controlleptincontrolleptin p-PKC controlleptin controlleptincontrolleptin GAPDH WT NOX2 -/- db/db WT p-STAT3 STAT3 NOX2 -/- db/db controlleptin controlleptin controlleptin
supplemental Material: Figure III A B C Suppl. Figure III. (A) Systemic administration of leptin significantly enhanced the number of acLDL +, lectin + progenitors after cultivation of spleen-derived cells for 7 days under endothelial cell-specific conditions (please see Methods for details). **P<0.01 vs. cells from vehicle-treated mice (control). In the representative images, acLDL uptake is indicated by the red signal, lectin binding by the green signal, and double-positive cells by the yellow signal (B, C) Angiogenic properties of spleen-derived progenitor cells ex vivo: systemic administration of leptin for 5 days increased the number of cultivated spleen-derived progenitor cells (red fluorescent signal) adherent to tubular networks (B) or sprouts (C) provided by mature endothelial cells. ***P<0.001. Size bars represent 100 µm. control leptin controlleptin control leptin
A B Suppl. Figure IV. The effects of leptin on the angiogenic properties of bone marrow-derived progenitor cells was examined after systemic administration of recombinant leptin (0.6 µg/g body weight) for 5 days into WT mice, previously transplanted with bone marrow from GFP transgenic mice, followed by the harvest of mononuclear cells and cultivation under endothelial cell-specific conditions for 7 days. Leptin treatment in vivo increased the number of spleen-derived GFP-positive progenitor cells (green) integrated into endothelial cell networks (A) or adherent to endothelial cell sprouts (B) provided by HUVEC. ***P<0.001 vs. cells from vehicle-treated mice (control). Representative images are shown. Size bars represent 100 µm. supplemental Material: Figure IV control leptin control
supplemental Material: Figure V Suppl. Figure V. The effects of recombinant leptin (i.p. injection once daily for 5 consecutive days) in wildtype mice fed by normal chow (NC) or high fat diet (HFD) on the number of circulating flk-1 + / sca1 + progenitor cells was examined. Data were expressed as % increase of findings in mice injected with vehicle alone (NaCl). **P<0.01 and ***P<0.001 vs. vehicle- treated controls; # P<0.05 and ##P<0.01 vs. WT mice fed NC. Summarized findings in 5-12 mice per group are shown.
supplemental Material: Figure VI Suppl. Figure VI. Schematic diagram depicting possible mechanisms involved in mediating the effects of leptin on flk-1 +, sca-1 + vascular progenitor cell (VPC) mobilization. Our findings suggest that leptin, following the interaction with its receptor (ObR) on bone marrow cells, activates NOX2-based NADPH oxidase (NOX2) resulting in the formation of reactive oxygen species (ROS), activation of transcription factors (TF) and MMP9 expression and subsequently, proteolytic release of membrane-bound Kit ligand (mKitL). Soluble KitL then may mediate the mobilization of VPC into the circulation from where they are recruited to sites of ischemia and participate in new blood vessel formation. bone marrow stromal cell mKitL ROS sKitL PKC / AKT ObR leptin progenitor cell niche in bone marrow sKitL c-Kit VPC NOX2 blood vessel P VPC STAT3 P redox-sensitive TF activation MMP9 redox-sensitive gene expression sKitL