Role of epidermal growth factor receptor in ovine fetal pulmonary vascular remodeling following exposure to high altitude long-term hypoxia

Abstract

High altitude long-term hypoxia (LTH) in the fetus may result in pulmonary vascular smooth muscle cell (PVSMC) proliferation and pulmonary vascular remodeling. Our objective was to determine if epidermal growth factor receptor (EGFR) is involved in hypoxia induced PVSMC proliferation or in pulmonary vascular remodeling in ovine fetuses exposed to high altitude LTH. Fetuses of pregnant ewes that were held at 3820-m altitude from *30 to 140 days (LTH) gestation and sea level control pregnant ewes were delivered near term. Morphometric analyses and immunohistochemistry were done on fetal lung sections. Pulmonary arteries of LTH fetuses exhibited medial wall thickening and distal muscularization. Western blot analyses done on protein isolated from pulmonary arteries demonstrated an upregulation of EGFR. This upregulation was attributed in part to PVSMC in the medial wall by immunohistochemistry.Proliferation of fetal ovine PVSMC after 24 h of hypoxia (2% O2) was attenuated by inhibition of EGFR with 250 nmol tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), a specific EGFR protein tyrosine kinase inhibitor, when measured by [3H]-thymidine incorporation. Our data indicate that EGFR plays a role in fetal ovine pulmonary vascular remodeling following long-term fetal hypoxia and that inhibition of EGFR signaling may ameliorate hypoxia-induced pulmonary vascular remodeling.

FIG. 1.

Ovine fetal pulmonary arteries and their accompanying airways. Representative photomicrographs of pulmonary arteries from sea-level controls (A) and ovine fetuses in high altitude long-term hypoxia (B). Four-μm tissue sections stained for elastin. There is increased muscularity in the hypoxic vessel.

FIG. 2.

EGFR protein in pulmonary arteries of ovine fetuses. Western blot analysis was done of protein isolates from pulmonary arteries of ovine fetuses exposed to high altitude long-term hypoxia and sea-level controls, n = 3. Top panel is representative blots, bottom panel is densitometry, expressed in arbitrary units and normalized with actin. ^*p < 0.01.

FIG. 3.

EGFR expression in fetal ovine pulmonary arteries. Compared with sea-level control (A), there is upregulation of EGFR in the pulmonary artery exposed to high altitude longterm hypoxia (B) at 400 ×. Bar = 20 μm. Immunohistochemistry of 4-mm sections stained with EGFR antibody and then counterstained with hematoxylin.

FIG. 4.

Proliferative cell nuclear antigen (PCNA) in ovine fetal pulmonary arteries. Western blot analysis of PCNA, a nuclear marker of cell proliferation, in protein isolates from pulmonary arteries of ovine fetuses exposed to high altitude long-term hypoxia and sea-level controls, ^*p = 30.05. Top panel is representative blots and bottom panel is densitometry, expressed in arbitrary units, and normalized with actin.

FIG. 5.

Proliferative cell nuclear antigen (PCNA) and α-smooth muscle actin in fetal ovine pulmonary arteries. PCNA (brown) and alpha-smooth-muscle actin (red) by immunohistochemistry using DakoCytomation Envision Doublestain Kit (Dako, Carpinteria, CA, USA) at 400× in ovine fetuses from sea-level control (A) and high altitude long-term hypoxia (B). Bar = 20 μm.

FIG. 6.

Hypoxia-induced pulmonary vascular smooth-muscle cell (PVSMC) proliferation and the effect of a specific EGFR inhibitor (AG1478). PVSMC exposed to 2% O₂ for 24 h. Cell proliferation measured by [³H]-thymidine incorporation and expressed as percent of vehicle-treated control in normoxia. ^#,*p < 0.05 for hypoxia-induced proliferation in both vehicle and AG1478 treatment groups, as well as for diminished hypoxia-induced cell proliferation in the AG1478 treatment group compared with the vehicle group. The difference in proliferation between the two normoxia groups was not significant.