Submission Number: LAW-4-35-38

Abstract Number: 603

CO-IMMUNOLOCALIZATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE (ENOS) AND VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) IN ENDOMETRIAL VASCULATURE OF ESTRADIOL-TREATED OVARIECTOMIZED EWES.

LP Reynolds*, DR Arnold*, KC Kraft and DA Redmer*

Dept of Animal & Range Sci, North Dakota State University, Fargo, ND ¹

Abstract:
Estradiol (E2) stimulates expression of eNOS and VEGF in endometrial tissues. This study was conducted to determine: 1) if E2 regulates expression of eNOS and VEGF in endometrial vascular tissues; and 2) if eNOS and VEGF are co-localized in these tissues. Cross-sections of uterine horns from long-term ovariectomized ewes (n=5/group) were obtained at 0, 2, 4, 24, 48, or 72 h after receiving a subcutaneous E2 implant (100 mg in Silastic), fixed in Carnoy's solution, and embedded in paraffin. For co-immunolocalization, we used specific antibodies against eNOS (Transduction Lab.) and VEGF (Red-1) and the ABC method. Regardless of time after E2 implant, endometrial arteriolar endothelial cells exhibited strong eNOS staining. At 0 h, eNOS localized only to arteriolar endothelial cells, and no VEGF staining was detected. By 2 h, although eNOS continued to localize to the arteriolar endothelial cells, light eNOS staining began to appear in the capillary endothelial cells; light VEGF staining also was observed in arteriolar smooth muscle cells. By 4 h, strong staining for eNOS was seen in both arteriolar and capillary endothelial cells, and strong VEGF staining was detected in the arteriolar smooth muscle cells. By 8 h, co-localization was strongest in the arterioles (eNOS in endothelial cells and VEGF in vascular smooth muscle cells), but capillary staining for both eNOS (endothelial cells) and VEGF (pericytes) had increased in intensity. By 24 h, co-localization of eNOS and VEGF was very intense in both the arterioles and the capillary beds. By 48 h, VEGF staining in the capillary pericytes had decreased in intensity, but eNOS staining in the capillary endothelial cells remained strong. By 72 h, the intensity of VEGF staining was decreased in both capillary pericytes and arteriolar smooth muscle cells, whereas the intensity of eNOS staining was decreased in the capillary endothelial cells but remained strong in the arterioles. These data suggest that E2 regulates eNOS and VEGF protein expression in endometrial vascular tissues. In addition, because eNOS can stimulate VEGF expression, the co-localization of eNOS in endothelial cells and VEGF in peri-endothelial cells (vascular smooth muscle and pericytes) suggests a paracrine mechanism to maximize endometrial vasodilation and angiogenesis in response to E2.

Keywords: VEGF, NOS, endothelial cell, pericyte, vascular smooth muscle, estradiol, ewe

This abstract is being presented at: 8:00 AM in session:
Uterus/Oviduct II