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Angiogenic factor expression in pericytes isolated from ovine corpus luteum.

The corpus luteum (CL) is a highly vascularized tissue, in which 50-70% of the tissue comprises microvascular pericytes and endothelial cells. Angiogenesis, the formation of new blood vessels, requires several key processes that are mediated by growth factors. Capillary pericytes express one of the major angiogenic growth factors, vascular endothelial growth factor (VEGF). Angiogenesis is associated with vasodilation, or relaxation of the vascular wall, a process that is mediated by nitric oxide (NO). In the CL, the microvascular endothelium has been shown to express endothelial NO synthase (eNOS), the enzyme responsible for the production of NO. Our previous research has demonstrated strong expression of VEGF in the capillary pericytes of the developing CL (Redmer et al, Biol. Reprod. 65:879-889,2001). VEGF targets endothelial cells to initiate angiogenesis and has also been shown to stimulate NO production through eNOS. Conversely, NO has been shown to upregulate VEGF expression by perivascular cells, including vascular smooth muscle cells and pericytes.

In order to investigate the relationship between angiogenic factors and eNOS, microvascular pericytes and endothelial cells were isolated from CL collected from superovulated ewes (n=5) nine days following the estrus cycle. The CL from each ewe were pooled and digested with collagenase. Endothelial cells were isolated by using BS-1 lectin-coated magnetic beads, and cultured in a selective medium containing DMEM, endothelial cell growth supplement, D-valine, heparin, and plasma-derived horse serum. Pericytes were isolated using Percoll gradients and were cultured in DMEM containing fetal bovine serum. Cell types were identified by using immunofluorescent staining for the pericyte cell marker smooth muscle cell alpha actin (SMCA), the endothelial cell marker Factor VIII, and the fibroblast cell marker Type IV collagen, as well as morphological comparisons with pure cultures of aortic vascular smooth muscle cells (AVSM) and aortic endothelial cells. Cell lines for luteal endothelial cells and luteal pericytes/vascular smooth muscle cells were successfully established.

Isolated pericytes were incubated with or without doses ranging from 0 to 10 mM of the NO-donor stock solution (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate (DETA-NO) or a control of diethylenetriamine (DETA) for 8 hr. Expression of mRNA for VEGF, fibroblast growth factor-2 (FGF-2), soluble guanylate cyclase (sGC, NO receptor), and the angiopoietins (ANG) 1 and 2 by these luteal pericytes was evaluated using real time RT-PCR. NO caused a twelve to thirteen fold increase (p<0.001) in VEGF expression, a five-fold increase (p<0.01) in FGF-2 expression, and a twelve to thirteen fold increase (p<0.06) in ANG-2 expression over the DETA control treatments. The expression of sGC and ANG-1 in luteal pericytes was not affected by NO treatment. Our work using a microvascular model of luteal development, along with work by others using various tissues, indicates that there is a potentially complex set of paracrine interactions that occur between the endothelium and pericytes, perhaps including a series of positive feedback loops between NO and VEGF, NO and FGF-2, FGF-2 and VEGF, and VEGF and ANG-2. Combined, these data provide strong evidence that NO plays a role in the regulation of angiogenic factor expression by luteal microvascular pericytes.

Supported by USDA Grant 2002-35203-12246 to Redmer, D.A. and Reynolds, L.P. and a NASA Undergraduate Research Grant to Beckman, J.D.

Joan D. Beckman (1), Larry P. Reynolds (1,2), Anna T. Grazul-Bilska (1,2), James D. Kirsch (1), Kim C. Kraft (1), Kimberly D. Petry (1), Corrie B. Redmer (1), Mary Lynn Johnson (1), and Dale A. Redmer (1,2)

(1) Department of Animal & Range Sciences and (2) Cell Biology Center, North Dakota State University, Fargo, ND
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Title Annotation:A. Rodger Denison Student Research Competition: COMMUNICATIONS: UNDERGRADUATE DIVISION
Author:Beckman, Joan D.; Reynolds, Larry P.; Grazul-Bilska, Anna T.; Kirsch, James D.; Kraft, Kim C.; Petry
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2004
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