Uterine artery leptin receptors during the ovarian cycle and pregnancy regulate angiogenesis in ovine uterine artery endothelial cells†

Abstract

Leptin regulates body weight, reproductive functions, blood pressure, endothelial function, and fetoplacental angiogenesis. Compared to the luteal phase, the follicular phase and pregnancy are physiological states of elevated estrogen, angiogenesis, and uterine blood flow (UBF). Little is known concerning regulation of uterine artery (UA) angiogenesis by leptin and its receptors. We hypothesized that (1) ex vivo expression of leptin receptors (LEPR) in UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm) is elevated in pregnant versus nonpregnant (Luteal and Follicular) sheep; (2) in vitro leptin treatments differentially modulate mitogenesis in uterine artery endothelial cells from pregnant (P-UAECs) more than in nonpregnant (NP-UAECs) ewes; and (3) LEPR are upregulated in P-UAECs versus NP-UAECs in association with leptin activation of phospho-STAT3 signaling. Local UA adaptations were evaluated using a unilateral pregnant sheep model where prebreeding uterine horn isolation (nongravid) restricted gravidity to one horn. Immunolocalization revealed LEPR in UAendo and UAvsm from pregnant and nonpregnant sheep. Contrary to our hypothesis, western analysis revealed that follicular UAendo and UAvsm LEPR were greater than luteal, nongravid, gravid, and control pregnant. Compared to pregnant groups, LEPR were elevated in renal artery endothelium of follicular and luteal sheep. Leptin treatment significantly increased mitogenesis in follicular phase NP-UAECs and P-UAECs, but not luteal phase NP-UAECs. Although UAEC expression of LEPR was similar between groups, leptin treatment only activated phospho-STAT3 in follicular NP-UAECs and P-UAECs. Thus, leptin may play an angiogenic role particularly in preparation for the increased UBF during the periovulatory period and subsequently to meet the demands of the growing fetus.

Keywords: angiogenesis; blood flow; estrogen; leptin; pregnancy; sheep; uterine artery.

Figure 1.

LEPR immunolocalization in (A) luteal, (B) follicular, (C) pregnant and (D) IgG negative control uterine arteries (40× magnification images are representative of n = 4). E = endothelium; L = lumen; TI = tunica intima; and VSM = vascular smooth muscle. Bar = 50 μm. Reddish staining indicates positive staining. Representative LEPR_L western blot analysis comparing relative ex vivo levels for UAendo and UAvsm of luteal, follicular, nongravid, gravid, and pregnant (E and F) groups are illustrated above the values that are presented as relative protein expression from optical density analysis of UAendo luteal; n = 3, follicular; n = 3, nongravid; n = 3, gravid; n = 3, and pregnant; n = 3, and for UAvsm luteal; n = 3, follicular; n = 2, nongravid; n = 4, gravid; n = 4, and pregnant; n = 3. Data are presented as Mean ± SEM fold of luteal. Different letters denote significant differences (P < 0.05).

Figure 2.

Representative LEPR_L western blots are shown in the upper portion of each panel comparing relative ex vivo levels for (A) OAendo, (B) OAvsm, (C) RAendo, and (D) RAvsm obtained from luteal (n = 4), follicular (n = 4), and unilateral gravid (n = 8), and control pregnant (n = 7) sheep. Densitometry values are presented in the lower portions of each panel. Data are presented as Mean ± SEM fold of luteal. Different letters denote differences (P < 0.05).

Figure 3.

Concentration-dependent in vitro proliferative responses by (A) luteal, (B) follicular, and (C) pregnant UAECs obtained from sheep in response to leptin treatment; 24 and 48 h responses were combined. Compared to luteal NP-UAECs, leptin treatment significantly increased cell proliferation in UAECs from follicular phase (treatment effect, *P < 0.05; biphasic dose response) and late pregnant (treatment effect, *P < 0.05; typical dose response) sheep. *Increase (P < 0.05; n = 8) in follicular and pregnant UAECs proliferation compared with luteal UAEC (n = 8) and untreated control. Data are presented as Mean±SEM fold of untreated control.

Figure 4.

Representative LEPR under in vitro basal culture conditions, and phosphorylation of STAT3 western blots. (A) LEPR_S, and LEPR_L under in vitro untreated basal culture conditions, and (B) phosphorylation of STAT3 in response to 1ng/ml leptin treatment from passage 4 luteal (n = 4), follicular (n = 4), and control pregnant (n = 4) UAECs. Densitometry values are presented for (A) below the panel and (B) is presented to the left of the right panel as relative protein expression. Data are presented as Mean ± SEM fold of luteal. *Denotes differences from control (P < 0.05).