Delineating the angiogenic gene expression profile prior to pulmonary vascular remodeling in a lamb model of congenital heart disease

Disordered angiogenesis is implicated in the pulmonary vascular remodeling secondary to congenital heart disease (CHD). However, the underlying genes are not well delineated. We have previously shown that an ovine model of CHD with increased pulmonary blood flow (PBF, Shunt) has an angiogenesis burst between 1-4 weeks of age. Thus, we hypothesized that the increased pulmonary blood flow elicited a pro-angiogenic gene expression profile prior to the onset of vessel growth. To test this we utilized microarray analysis to identify genes that could be responsible for the angiogenic response. Total RNA was isolated from the lungs of Shunt and Control lambs at 3 days of age and hybridized to Affymetrix GeneChips for microarray analyses (n=8 for each group). A total of 89 angiogenesis-related genes were found to be up-regulated and 26 angiogenesis-related genes down-regulated in Shunt lungs compared to control (cutting at 1.2 fold difference, P<0.05). We then confirmed the up-regulation of pro-angiogenic genes: FGF2, Angiopoietin2 (Angpt2), and Birc5 at both mRNA and protein levels and the up-regulation of ccl2 at mRNA level in the 3-day shunt lungs. Further, we found that pulmonary arterial endothelial cells (PAEC) isolated from juvenile lambs exhibited increased expression of FGF2, Angpt2, Birc5, and ccl2 as well as enhanced angiogenesis when exposed to elevated shear stress (35 dyn/cm2) compared to cells exposed to more physiological level shear stress (20 dyn/cm2). Finally, we demonstrated that blocking FGF2, Angpt2, or Birc 5 signaling, using neutralizing antibodies, significantly decreased the angiogenic response induced by shear stress. In conclusion, we have identified a pro-angiogenic gene expression profile in a lamb model of CHD with increased PBF that precedes the onset of pulmonary vascular remodeling. Further, our data indicate that FGF2, Angiopoietin2, Birc5, and ccl2 may play important roles in the angiogenic response.

16