Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription.

Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.

The impact of a human IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction.

Enhancing placental insulin-like growth factor (IGF) availability appears to be an attractive strategy for improving outcomes in fetal growth restriction (FGR). Our approach was the novel use of [Leu(27)]IGF-II, a human IGF-II analog that binds the IGF-II clearance receptor IGF-IIR in fetal growth-restricted (FGR) mice. We hypothesized that the impact of [Leu(27)]IGF-II infusion in C57BL/6J (wild-type) and endothelial nitric oxide synthase knockout (eNOS(-/-); FGR) mice would be to enhance fetal growth and investigated this from mid- to late gestation; 1 mg·kg(-1)·day(-1) [Leu(27)]IGF-II was delivered via a subcutaneous miniosmotic pump from E12.5 to E18.5. Fetal and placental weights recorded at E18.5 were used to generate frequency distribution curves; fetuses <5th centile were deemed growth restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum was collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu(27)]IGF-II treatment halved the number of FGR fetuses, reduced fetal(P = 0.028) and placental weight variations (P = 0.0032), and increased the numbers of pups close to the mean fetal weight (131 vs. 112 pups within 1 SD). Mixed-model analysis confirmed litter size to be negatively correlated with fetal and placental weight and showed that [Leu(27)]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional (14C)MeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu(27)]IGF-II-treated WT animals (P < 0.01). In eNOS(-/-) mice, [Leu(27)]IGF-II reduced the number of FGR fetuses(1 vs. 5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS(-/-) litters suggests the use of this analog as a means of standardizing and rescuing fetal growth, preferentially in the smallest offspring.

Human uterine and placental arteries exhibit tissue-specific acute responses to 17ß-estradiol and estrogen-receptor-specific agonists.

The discrete regulation of vascular tone in the human uterine and placental circulations is a key determinant of appropriate uteroplacental blood perfusion and pregnancy success. Humoral factors such as estrogen, which increases in the placenta and maternal circulation throughout human pregnancy, may regulate these vascular beds as studies of animal arteries have shown that 17ß-estradiol, or agonists of estrogen receptors (ER), can exert acute vasodilatory actions. The aim of this study was to compare how acute exposure to ER-specific agonists, and 17ß-estradiol, altered human placental and uterine arterial tone in vitro. Uterine and placental arteries were isolated from biopsies obtained from women with uncomplicated pregnancy delivering a singleton infant at term. Vessels were mounted on a wire myograph, exposed to the thromboxane receptor agonist U46619 (10(-6) M), and then incubated with incremental doses (5 min, 0.03-30 µM) of either 17ß-estradiol or agonists specific for the ERs ERα (PPT), ERß (DPN) or the G-protein-coupled estrogen receptor GPER-1 (G1). ERα and ERß mRNA expression was assessed. 17ß-estradiol, PPT and DPN each relaxed myometrial arteries (P < 0.05) in a manner that was partly endothelium-dependent. In contrast, 17ß-estradiol or DPN relaxed placental arteries (maximum relaxation to 42 ± 1.1 or 47.6 ± 6.53% of preconstriction, respectively) to a lesser extent than myometrial arteries (to 0.03 ± 0.03 or 8.0 ± 1.0%) and in an endothelial-independent manner whereas PPT was without effect. G1 exposure did not inhibit the constriction of myometrial nor placenta arteries. mRNA expression of ERα and ERß was greater in myometrial arteries than placental arteries. ER-specific agonists, and 17ß-estradiol, differentially modulate the tone of uterine versus placental arteries highlighting that estrogen may regulate human uteroplacental blood flow in a tissue-specific manner.

Differential effect of insulin like growth factor-I on constriction of human uterine and placental arteries.

CONTEXT: Discrete regulation of the uterine and placental vasculatures is an important feature of uteroplacental perfusion and pregnancy success because appropriate maternal/fetal exchange of nutrients and gases is crucial for normal fetal growth. Placental vasculature lacks autonomic innervation so tone is controlled by locally derived vasoactive factors. IGF-I, which is produced by the placenta, is critical for normal fetal growth and studies of animal vascular systems have shown that IGF-I regulates vasomotor tone. OBJECTIVE: The objective of the study was to determine whether IGF-I directly alters human placental and myometrial arterial tone in vitro. PARTICIPANTS: Women with uncomplicated pregnancy delivering a singleton infant at term participated in the study. SETTING: The study was conducted at university hospital laboratories. MAIN OUTCOME MEASURE(S): Comparison of arterial tension measured before and after exposure to IGF-I. DESIGN: Placental and myometrial arteries were mounted on a wire myograph, exposed to the constrictor U46619 (10(-10) to 10(-5) m), returned to baseline tension, and then incubated with IGF-I (0-500 ng/ml) for various time points before performing a second dose-response curve to U46619. IGF-I receptor protein expression was assessed. RESULTS: IGF-I did not acutely alter the response of placental arteries to U46619. Exposure of myometrial arteries to IGF-I caused a rightward shift of U46619 dose-response curves (P < 0.05); EC(50) data were significantly increased at 30 (15.5 ± 2.8 vs. 133 ± 44 nm, before and after IGF treatment, respectively) and 60 min (10.9 ± 1.9 vs. 146 ± 47 nm). Placental and myometrial arteries had a similar IGF-I receptor expression profile. CONCLUSIONS: IGF-I acutely modulates the vasomotor tone of human myometrial, but not placental, arteries, suggesting that IGF-I regulates the delivery of maternal blood to the placenta.

Hoxa3 promotes the differentiation of hematopoietic progenitor cells into proangiogenic Gr-1+CD11b+ myeloid cells.

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair, but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization, but show altered recruitment/retention kinetics in the diabetic environment. Moreover, diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative, chemotaxis, adhesion, and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response, enhanced neovascularization, and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing.