Loss of myeloid cell-derived vascular endothelial growth factor accelerates fibrosis

Abstract

Tissue injury initiates a complex series of events that act to restore structure and physiological homeostasis. Infiltration of inflammatory cells and vascular remodeling are both keystones of this process. However, the role of inflammation and angiogenesis in general and, more specifically, the significance of inflammatory cell-derived VEGF in this context are unclear. To determine the role of inflammatory cell-derived VEGF in a clinically relevant and chronically inflamed injury, pulmonary fibrosis, we deleted the VEGF-A gene in myeloid cells. In a model of pulmonary fibrosis in mice, deletion of VEGF in myeloid cells resulted in significantly reduced formation of blood vessels; however, it causes aggravated fibrotic tissue damage. This was accompanied by a pronounced decrease in epithelial cell survival and a striking increase in myofibroblast invasion. The drastic increase in fibrosis following loss of myeloid VEGF in the damaged lungs was also marked by increased levels of hypoxia-inducible factor (HIF) expression and Wnt/beta-catenin signaling. This demonstrates that the process of angiogenesis, driven by myeloid cell-derived VEGF, is essential for the prevention of fibrotic damage.

Fig. 1.

Deletion of VEGF in myeloid cells leads to increased fibrosis and collagen deposition after pulmonary injury. (A) Trichrome staining of lungs from mice treated i.p. with bleomycin (10 mg/kg twice a week) after 28 days. VEGF Mut mice lacking VEGF in myeloid cells show aggravated histopathological signs of fibrosis. (B) Collagen assay from the entire right lung of Mut mice and littermate WT mice 28 days after i.p. bleomycin injection or treatment with saline (n = 5–7). (C) Assessment of oxygen uptake (VO₂) at the anaerobic threshold of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline (n = 5). Error bars show SEM. (Scale bars: 100 μm.)

Fig. 2.

Loss of myeloid cell-derived VEGF results in higher numbers of fibroblasts and myofibroblasts as well as a decrease in type II pneumocytes after pulmonary injury. (A) Detection of fibroblasts positive for the marker FSP in lungs from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline. (B) Quantitative analysis of the area in pixels covered by FSP within lung sections from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline (n = 4). (C) Simultaneous immunodetection of myofibroblasts and type II pneumocytes with the specific markers SMA and TTF-1 in lungs from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline. (D) Quantitative analysis of the area in pixels covered by SMA and TTF-1 within lung sections from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline (n = 4). Error bars show SEM. (Scale bars: 100 μm.)

Fig. 3.

Ablation of VEGF in myeloid cells results in reduced levels of VEGF, VEGFR2 phosphorylation, and impaired angiogenesis in fibrotic lungs. (A) Immunoblotting for VEGF from entire left lung lysates of VEGF Mut and littermate WT mice 21 days after i.p. bleomycin injection or treatment with saline. (B) Immunoblotting for VEGFR2 and phosphotyrosine (p-Tyr) after immunoprecipitation of VEGFR2 from entire left lung lysates of Mut and littermate WT mice 21 days after i.p. bleomycin injection or treatment with saline. (C) Quantitative analysis of VEGF signals from entire left lung lysates of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as measured by photon emission (n = 5). (D) Ratio of p-Tyr to VEGFR2 signal from entire left lung lysates of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as a measure of receptor activation (n = 5). (E) Immunodetection of pulmonary vasculature with the marker CD34 on sections from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline. (F) Quantitative analysis of the area in pixels covered by CD34 within lung sections from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline (n = 4). Error bars show SEM. (Scale bars: 100 μm.)

Fig. 4.

Increased activation of HIFs and increased Wnt/β-catenin signaling in fibrotic lungs from animals lacking VEGF in myeloid cells. (A) Immunoblotting for HIF-1α and HIF-2α from entire left lung nuclear extracts of VEGF Mut or littermate WT mice 21 days after i.p. bleomycin injection or treatment with saline. (B) Quantitative analysis of HIF-1α signals from entire left lung nuclear extracts of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as measured by photon emission (n = 5). (C) Quantitative analysis of HIF-2α signals from entire left lung nuclear extracts of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as measured by photon emission (n = 5). (D) Simultaneous immunodetection of CD34 and CA-IX in lung sections from Mut and WT mice 28 days after i.p. bleomycin injection or treatment with saline. (E) Immunoblotting for β-catenin and cyclin D1 from left lung nuclear extracts and E-cadherin from left lung cytoplasmic extracts of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline. (F) Quantitative analysis of β-catenin signals from entire left lung nuclear extracts of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as measured by photon emission (n = 5). (G) Quantitative analysis of cyclin D1 signals from entire left lung nuclear extracts of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as measured by photon emission (n = 5). (H) Quantitative analysis of E-cadherin signals from entire left lung cytoplasmic extracts of Mut and WT mice 21 days after i.p. bleomycin injection or treatment with saline as measured by photon emission (n = 5).