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A2108 - Bone Morphogenic Protein 2 Is an Important Regulator of Angiogenesis in Clonally Enriched Lung Endothelial Cells
Author Block: A. Bhagwani1, S. Hultman1, D. Farkas1, R. L. Heise2, M. C. Yoder3, M. Clauss4, R. J. Freishtat5, L. Farkas6; 1Virginia Commonwealth University, Richmond, VA, United States, 2Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, United States, 3Indiana University School of Medicine, Indianapolis, IN, United States, 4IU School of Medicine, Indianapolis, IN, United States, 5Pediatrics, Children's National Med Ctr, Washington, DC, United States, 6Virginia Commonwealth Univ, Richmond, VA, United States.
Rationale: Recently, populations of highly proliferative endothelial cells (ECs) have been identified in the adult lungs of humans, mice and rats. Some of these ECs were able to generate clonal colonies over several expansion steps, but the detailed mechanism driving extensive angiogenesis, self-renewal and proliferation in these EC clones has not been identified. Both hypoxia and bone morphogenic protein 2 (BMP2) regulate pulmonary angiogenesis. In this study we aimed to characterize CD117+ EC clones and assess the role of hypoxia and BMP2 signaling for their proliferation and angiogenesis.
Methods: CD117+ ECs were isolated from adult Sprague Dawley rats by positive selection for CD117 and EC markers, and exclusion of hematopoietic cells. CD117- ECs were isolated as control cells. CD117+ ECs were then enriched by multi-step clonal selection, yielding highly proliferative clones. The cells were characterized by flow cytometry and assays of EC function. The cells were exposed to hypoxia and BMP2 was inhibited by a neutralizing antibody. Changes in gene expression were evaluated by qRT-PCR. Human and rat lung tissue sections were evaluated by double immunofluorescence and confocal microscopy.
Results: CD117+ EC clones and CD117- ECs both expressed lung EC markers and showed typical EC function. Yet only CD117+ ECs were able to expand clonally. Using qRT-PCR, we detected a 60-fold elevation of BMP2 expression in CD117+ EC clones. In human and rat lung tissue sections, we found isolated BMP2(high) ECs in the microvasculature, indicating that high BMP2 expression in specific ECs may be physiological. We tested the role of hypoxia and BMP2 in these CD117+ EC clones and found that both exposure to chronic hypoxia and inhibition of BMP2 inhibited angiogenic tube formation in vitro. Exposure to hypoxia resulted in a significant decline in expression of EC markers, BMP receptor 2 and BMP downstream target Id1. In contrast, expression of the TGFβ downstream target Serpin1 was induced. The phenotype of the cells changed towards an elongated, fibroblast-like phenotype, indicating that mesenchymal transition may occur in these cells.
Conclusions: Our data indicate that a distinct population of BMP2(high) ECs exist in the lung microvasculature, which is important for angiogenesis and regeneration. The mechanistic connection between CD117 expression and BMP2/BMPR2 signaling is currently subject of ongoing studies.