Differential Effects of Thrombin and Hypoxia on Endometrial Stromal and Glandular Epithelial Cell Vascular Endothelial Growth Factor Expression

Abstract

Ovarian steroids and/or premenstrual endometrial hypoxia are thought to restore the endometrial vasculature shed during menstruation by elevating endometrial vascular endothelial growth factor (VEGF) levels. During the luteal phase, VEGF levels peak, progesterone induces estradiol (E(2))-primed human endometrial stromal cells (HESCs) to decidualize and express tissue factor (TF), and endometrial vascular permeability is enhanced. The latter would present circulating clotting factors to decidual cell-expressed TF to form local thrombin. HESCs were incubated in serum-supplemented medium containing vehicle (control) or 10(-8) M E(2) or 10(-7) M medroxyprogesterone acetate (MPA) or E(2) + MPA for 7 d to induce decidualization, while monolayers of human endometrial glandular epithelial cells (HEGECs) formed during 4-d incubation of glands. The medium was exchanged for a defined medium containing corresponding vehicle or steroids +/- thrombin under normoxia or hypoxia (0-1% O(2)). Hypoxia enhanced secreted immunoreactive VEGF levels by severalfold in HESCs and HEGECs, but the steroids did not affect VEGF output in either cell type under normoxia or hypoxia. In E(2) + MPA-decidualized HESCs, VEGF levels were elevated by 0.1 U/ml of thrombin, and 0.5-2.5 U/ml of thrombin elicited maximum effects. The addition of 0.5 U/ml of thrombin evoked a time-dependent enhancement of VEGF levels and about an 8-fold increase at 48 h (P < 0.02; n = 6). Northern blotting indicated that E(2) + MPA-decidualized HESCs expressed VEGF(121), VEGF(165), and VEGF(189) mRNA, which were enhanced severalfold during 5- to 20-h incubation with thrombin. Moreover, TRAP, a synthetic peptide activator of the constitutively expressed protease activated receptor-1 thrombin receptor in decidualized HESCs, also elevated secreted VEGF levels. By contrast, HEGECs were unresponsive to thrombin added alone or with ovarian steroids. These results suggest that thrombin formed by progestin-augmented TF levels acts as an autocrine enhancer of VEGF expression in decidualized HESCs. Because angiogenesis occurs in a matrix of decidualized HESCs, these in vitro results provide a novel mechanism to account for both the peak in VEGF and angiogenesis in luteal phase human endometrium.

Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

“…We found that hypoxia markedly enhanced VEGF synthesis in primary cultures of both glandular epithelial and stromal cells. Consistent with the absence of estrogen and progestin response elements from the VEGF gene promoter,27 the VEGF output by glandular epithelial or stromal cells was unaffected by incubation of glandular epithelial or stromal cell with E 2 , a progestin, or E 2 plus progestin either under normoxia or under hypoxia 36. Hypoxia-induced VEGF expression is a putative regulator of an initial wave of angiogenesis, which occurs in the perimenstrual period prior to the elevation in circulating E 2 levels and involves early follicular phase repair of endometrial vessels 37…”

Regulation of endometrial vascular remodelling: role of the vascular endothelial growth factor family and the angiopoietin–TIE signalling system

“…In several studies, VEGFA mRNA and protein expression is increased in endometrial cells in response to oestradiol17b (E 2 ) treatment (Charnock-Jones et al 1993, Shifren et al 1996, Huang et al 1998, Classen-Linke et al 2000. In contrast, other studies report no effect of E 2 or progesterone treatment on endometrial epithelial or stromal cell VEGFA expression (Sharkey et al 2000, Lockwood et al 2002.…”

“…In artificially-cycling macaques, VEGFA mRNA was upregulated in the glands and stroma of superficial endometrial tissues 1-2 days following progesterone withdrawal (Nayak & Brenner 2002). VEGFA expression and secretion is also increased in endometrial epithelial and stromal cells exposed to hypoxic conditions in vitro (Popovici et al 1999, Sharkey et al 2000, Lockwood et al 2002.…”

Thrombin impairs human endometrial endothelial angiogenesis; implications for progestin-only contraceptive-induced abnormal uterine bleeding

“…Significantly higher CSPG4 immunostaining in stromal cells and intense superficial microvascular endothelial staining in the endometrium from women post-vs. pre-DMPA use complements our in later angiogenesis stages. Previously, we observed reciprocal thrombin-enhanced secretion of vascular endothelial growth factor (VEGF) [37] and inhibited secretion of angiopoietin (Ang)-1, a blood vessel stabilizing and maturing agent, derived from HESCs [38] as well as increased expression of Ang-2, a proangiogenic vessel-branching and permeability factor, derived from HEECs [39]. Extending these proangiogenic effects, the current study demonstrates that thrombin directly disrupts in vitro HEEC tube formation, branches and meshwork.…”