VEGF Receptor 1 Promotes Hypoxia-Induced Hematopoietic Progenitor Proliferation and Differentiation

Figures

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  Figure 1. Hypoxia induces inflammation. C57BL/6 mice were placed under either normoxic or hypoxic conditions for 21 days. Lungs, blood and bone marrow were collected. (A) Il1b, Il6, Il18 and Tnfa expression was assessed by RT-qPCR.(B) Il-6, TNF-α and IL1-β protein expression was evaluated in the whole lung and serum by ELISA. (C) The numbers of blood monocytes, neutrophils, B and T cells in hypoxic versus normoxic mice were determined by flow cytometry. (D) The numbers of lung infiltrating neutrophils and interstitial macrophages in these mice were quantified by flow cytometry. n = 5 mice per condition. Data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.001.

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  Figure 2. Hypoxia drives HSPC into the cell cycle. C57BL/6 mice were placed under either normoxic or hypoxic conditions for 21 days. Bone marrow was collected. The frequencies and numbers of the hematopoietic progenitors in the bone marrow were determined by flow cytometry (A) and whole mount confocal microscopy (B). The frequencies of HSC (C), LSK (D) and GMP (E) in the cell cycle stages were assessed using intracellular staining of Ki-67 and PI. (F) KikGR-GFP+ (Kikume Green Red) HSPC were adoptively transferred into mice housed in either normoxic or hypoxic conditions for seven days. GFP+ progenies were quantified four days later. n = 5 mice per condition. Data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

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  Figure 3. VEGFr1 expression is increased in HSPC in hypoxia. (A–C) C57BL/6 mice were placed under either normoxic or hypoxic conditions for 21 days. Bone marrow was collected. (A) Vegfa and Vegfr 1 expression in the whole bone marrow was assessed by RT-qPCR. B–C) Vegfr 1 expression was measured in bone marrow LSK and HSC in normoxic conditions (B) and GMP in normoxic and hypoxic conditions (C) by flow cytometry. The results are represented as mean fluorescent intensity (MFI). (D) Vegfr 1 gene expression was measured in sorted GMP by RT-qPCR. (E–G) HSPC were sorted from bone marrow of C57BL/6 mice and cultured in complete SFEM media in either normoxic or hypoxic conditions. (E) Heatmap representing expression of the genes downstream to Vegf/Vegfr 1 in HSPC cultured under normoxic and hypoxic conditions. (F) Schematic depicting HIF-1α binding sites on both murine and human VEGFr 1 promoter regions. (G) Confirmation of the binding of HIF-1α to the murine Vegfr 1 promoter region assessed by ChIP qPCR. (H, I) VEGFr 1 expression assessed in human (H) and murine (I) HSPC transfected with either siCTL or siHIF1A in hypoxic conditions. (J) Heatmap showing the expression of the cell cycle genes in…

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  Figure 4. Vegfr inhibition decreased hematopoietic progenitor proliferation in hypoxia. (A) Heatmap depicting expression of the cell cycle genes in mouse HSPC treated with increasing amounts of a Vegfr inhibitor (SU5416, 10-100nM) or vehicle. (B-D) C57BL/6 mice were treated with either SU5416 or vehicle diluent and placed in either normoxia or hypoxia for 21 days. Bone marrow cells were analyzed using flow cytometry to assess GMP proliferation (B, C) and number (D). n = 5 replicates or mice per condition. Data are shown as mean ± s.e.m. *P < 0.05, **P <. 0.01, ***P < 0.001, ****P < 0.001.

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  Figure 5. Vegfr inhibition decreased inflammation in hypoxia. (A) Mouse HSPC were treated with increasing amounts of a Vegfr inhibitor (SU5416, 10-100nM) or vehicle diluent (no treatment). Lineage commitment of HSPC was assessed by flow cytometry. B&C) C57BL/6 mice were treated with either a Vegfr inhibitor (SU5416) or vehicle diluent and placed in hypoxia for 21 days. Bone marrow and blood leukocytes were analyzed using flow cytometry. Percentages of blood (B) and bone marrow (C) monocytes and neutrophils were ascertained. n = 5 replicates or mice per condition. Data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 6. Patients with hypoxia have elevated numbers of inflammatory leukocytes. A restrospective chart review was performed to enumerate inflammatory leukocytes in peripheral blood of patients with OSA and COPD. n = 26 (OSA), 10 (COPD) and 22 (Healthy controls). Data are shown as mean ± s.e.m. *P < 0.05.