Jill M. Koch

Estradiol-17β and Its Cytochrome P450- and Catechol-O-Methyltransferase–Derived Metabolites Stimulate Proliferation in Uterine Artery Endothelial Cells. Role of Estrogen Receptor-α Versus Estrogen Receptor-β

Estradiol-17β and its metabolites which are sequentially synthesized by cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) to form 2 and 4-Hydroxyestradiol (2-OHE2 and 4-OHE2) and 2- and 4-Methoxestradiol (2-ME2, and 4-ME2) are elevated during pregnancy. We investigated whether CYP450s and COMT are expressed in uterine artery endothelial cells (UAECs) and if E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β and play roles in physiologic uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant (P-UAECs) and nonpregnant (NP-UAECs) ewes were treated with 0.1-100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, and 4-ME2. ER-α or ER-β specificity was tested using ICI 182,780, ER-α-specific MPP, ER-β –specific PHTPP antagonists and their respective agonists ER-α-specific PPT and ER-β –specific DPN. Angiogenesis was evaluated using BrdU Proliferation Assay. Utilizing confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4 and COMT expression in UAECs; however, expressions were similar between NP-UAECs and P-UAECs. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in P-UAECs, but not NP-UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of P-UAECs to E2β were solely mediated by ER-β, whereas responses to E2β metabolites were neither ER-α nor ER-β mediated. We demonstrate an important vascular role for E2β, its CYP450- and COMT-derived metabolites and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders.

Proteomic Profile of Uterine Luminal Fluid from Early Pregnant Ewes

Embryonic development is a time-sensitive period that requires a synchronized uterine environment, which is created by the secretion of proteins from both the embryo and uterus. Numerous studies have identified uterine luminal proteins and related these to specific adaptations during early pregnancy (EP). However, no study has yet utilized LC-MS/MS to identify the signature profile of proteins in the uterine lumen during EP. In this study, uterine luminal fluid from nonpregnant (NP; n = 3) and EP (n = 3; gestational day 16) ewes were analyzed by LC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid; 15 proteins related to specific aspects of embryonic development including growth and remodeling, immune system regulation, oxidative stress balance, and nutrition were significantly altered (up to 65-fold of NP) in EP profile. Specific uterine remodeling proteins such as transgelin (P = 0.008) and placental proteins like PP9 (P = 0.02) were present in EP luminal fluid but were barely detectable in the NP flushings. Direct correlations (R(2) = 0.84, P = 0.01) were observed between proteomics and immunoblotting. These data provide information on dynamic physiological processes associated with EP at the level of the uterus and conceptus and may potentially demonstrate a signature profile associated with embryonic well-being.

Estradiol-17beta and its cytochrome P450- and catechol-O-methyltransferase-derived metabolites stimulate proliferation in uterine artery endothelial cells: role of estrogen receptor-alpha versus estrogen receptor-beta.

Estradiol-17β and its metabolites which are sequentially synthesized by cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) to form 2 and 4-Hydroxyestradiol (2-OHE2 and 4-OHE2) and 2- and 4-Methoxestradiol (2-ME2, and 4-ME2) are elevated during pregnancy. We investigated whether CYP450s and COMT are expressed in uterine artery endothelial cells (UAECs) and if E2β and its metabolites modulate cell proliferation via ER-α and/or ER-β and play roles in physiologic uterine angiogenesis during pregnancy. Cultured ovine UAECs from pregnant (P-UAECs) and nonpregnant (NP-UAECs) ewes were treated with 0.1-100 nmol/L of E2β, 2-OHE2, 4-OHE2, 2-ME2, and 4-ME2. ER-α or ER-β specificity was tested using ICI 182,780, ER-α-specific MPP, ER-β –specific PHTPP antagonists and their respective agonists ER-α-specific PPT and ER-β –specific DPN. Angiogenesis was evaluated using BrdU Proliferation Assay. Utilizing confocal microscopy and Western analyses to determine enzyme location and levels, we observed CYP1A1, CYP1A2, CYP1B1, CYP3A4 and COMT expression in UAECs; however, expressions were similar between NP-UAECs and P-UAECs. E2β, 2-OHE2, 4-OHE2, and 4-ME2 treatments concentration-dependently stimulated proliferation in P-UAECs, but not NP-UAECs; 2-ME2 did not stimulate proliferation in either cell type. Proliferative responses of P-UAECs to E2β were solely mediated by ER-β, whereas responses to E2β metabolites were neither ER-α nor ER-β mediated. We demonstrate an important vascular role for E2β, its CYP450- and COMT-derived metabolites and ER-β in uterine angiogenesis regulation during pregnancy that may be dysfunctional in preeclampsia and other cardiovascular disorders.

Bilateral administration of autologous CD133+ cells in ambulatory patients with refractory critical limb ischemia: lessons learned from a pilot randomized, double-blind, placebo-controlled trial

BACKGROUND AIMS CD133+ cells confer angiogenic potential and may be beneficial for the treatment of critical limb ischemia (CLI). However, patient selection, blinding methods and end points for clinical trials are challenging. We hypothesized that bilateral intramuscular administration of cytokine-mobilized CD133+ cells in ambulatory patients with refractory CLI would be feasible and safe. METHODS In this double-blind, randomized sham-controlled trial, subjects received subcutaneous injections of granulocyte colony-stimulating factor (10 μg/kg per day) for 5 days, followed by leukapheresis, and intramuscular administration of 50-400 million sorted CD133+ cells delivered into both legs. Control subjects received normal saline injections, sham leukapheresis and intramuscular injection of placebo buffered solution. Subjects were followed for 1 year. An aliquot of CD133+ cells was collected from each subject to test for genes associated with cell senescence. RESULTS Seventy subjects were screened, of whom 10 were eligible. Subject enrollment was suspended because of a high rate of mobilization failure in subjects randomly assigned to treatment. Of 10 subjects enrolled (7 randomly assigned to treatment, 3 randomly assigned to control), there were no differences in serious adverse events at 12 months, and blinding was preserved. There were non-significant trends toward improved amputation-free survival, 6-minute walk distance, walking impairment questionnaire and quality of life in subjects randomly assigned to treatment. Successful CD133+ mobilizers expressed fewer senescence-associated genes compared with poor mobilizers. CONCLUSIONS Bilateral administration of autologous CD133+ cells in ambulatory CLI subjects was safe, and blinding was preserved. However, poor mobilization efficiency combined with high CD133+ senescence suggests futility in this approach.

Ovine Surgical Model of Uterine Space Restriction: Interactive Effects of Uterine Anomalies and Multifetal Gestations on Fetal and Placental Growth

Intrauterine growth restriction (IUGR) is observed in conditions with limitations in uterine space (e.g., uterine anomalies and multifetal gestations). IUGR is associated with reduced fetal weight, organ growth, and a spectrum of adult-onset diseases. To examine the interaction of uterine anomalies and multifetal gestations, we developed a surgical uterine space restriction model with a unilateral uterine horn ligation before breeding (unilateral surgery). Placentas and fetuses were studied on Gestational Day (GD) 120 and GD 130 (term = 147 days). Unilateral surgery decreased placentome numbers in singleton and twin pregnancies (25% and 50%, respectively) but not unilateral triplets. Unilateral surgery decreased total placentome weight in twin pregnancies (decreased 24%). Fetuses categorized as uterine space restricted (unilateral twin and both groups of triplets) had 51% fewer placentomes per fetus and a 31% reduction in placentomal weight per fetus compared to the nonrestricted group (control singleton, unilateral singleton, and control twin). By GD 130, uterine space-restricted fetuses exhibited decreased weight, smaller crown-rump, abdominal girth, and thoracic girth as well as decreased fetal heart, kidney, liver, spleen, and thymus weights. Lung and brain weights were unaffected, demonstrating asymmetric IUGR. At GD 130, placental efficiency (fetal weight per total placentomal weight) was elevated in uterine space-restricted fetuses. However, fetal arterial creatinine, blood urea nitrogen, and cholesterol were elevated, suggesting insufficient placental clearance. Maternal-to-fetal glucose and triglycerides ratios were elevated in the uterine space-restricted pregnancies, suggesting placental nutrient transport insufficiency. This model allows for examination of interactive effects of uterine space restriction-induced IUGR on placental adaptation and fetal organ growth.

Periconceptional growth hormone treatment alters fetal growth and development in lambs.

Research in the area of fetal programming has focused on intrauterine growth restriction. Few studies have attempted to examine programming mechanisms that ultimately lead to lambs with a greater potential for postnatal growth. We previously demonstrated that treatment of ewes with GH at the time of breeding led to an increase in birth weight. Therefore, the objective of this study was to determine the effects of a single injection of sustained-release GH given during the periconceptional period on fetal growth and development and to determine if the GH axis would be altered in these offspring. Estrus was synchronized using 2 injections of PGF(2alpha); at the time of the second injection, ewes assigned to treatment were also given an injection of sustained-release GH. A maternal jugular vein sample was taken weekly to analyze IGF-I as a proxy for GH to estimate the duration of the treatment effect. In ewes treated with GH, IGF-I increased (P < 0.05) by wk 1 and remained elevated until wk 4 postinjection. Lambs were weighed, crown-rump length and abdominal girth were determined, and a plasma sample was collected. In a subset of male lambs, liver, heart, and brain weights were obtained, as well as left and right ventricular wall thicknesses. On postnatal d 100, a subset of ewe lambs were weighed and challenged with an intravenous injection of GHRH. Lambs from treated ewes had increased (P < 0.05) birth weight and abdominal girth compared with control lambs; however, there was no difference in crown-rump length. Expression of GH receptor and IGF-I were increased (P < 0.05) in lambs gestated by GH-treated ewes compared with control ewes. The left ventricular wall was thinner (P < 0.05) from lambs in the GH-treated group compared with control lambs. On postnatal d 100, those ewe lambs born to ewes treated with GH continued to be heavier (P < 0.05) and had no IGF-I response to GHRH challenge. In conclusion, treating ewes with a single injection of GH appeared to alter fetal growth and development. Lambs born to ewes treated with GH were larger at birth and had altered organ development, which may indicate that early maternal GH treatment may lead to permanent changes in the developing fetus. The ewe lambs maintained their growth performance to at least 100 d of postnatal life and appeared to have an altered GH axis, as demonstrated by the altered response to GHRH.

Local Effects of Pregnancy on Connexin Proteins That Mediate Ca2+-Associated Uterine Endothelial NO Synthesis

Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. Hypothesis: local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and NO production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43, and P635eNOS protein levels in uterine artery and systemic artery (omental and renal) endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy, uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P635eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P635eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37–specific Gap peptides, abrogated uterine artery endothelial ATP-induced Ca2+-mediated NO production. Thus, uterine artery endothelial connexin 43, but not connexin 37, regulates Ca2+-mediated NO production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

Ovine uterine space restriction alters placental transferrin receptor and fetal iron status during late pregnancy

Background:Fetal growth restriction is reported to be associated with impaired placental iron transport. Transferrin receptor (TfR) is a major placental iron transporter in humans but has not been studied in sheep. TfR is regulated by both iron and nitric oxide (NO), the molecule produced by endothelial nitric oxide synthase (eNOS). We hypothesized that limited placental development downregulates both placental TfR and eNOS expression, thereby lowering fetal tissue iron.Methods:An ovine surgical uterine space restriction (USR) model, combined with multifetal gestation, tested the extremes of uterine and placental adaptation. Blood, tissues, and placentomes from non–space restricted (NSR) singletons were compared with USR fetuses at gestational day (GD) 120 or 130.Results:When expressed proportionate to fetal weight, liver iron content did not differ, whereas renal iron was higher in USR vs. NSR fetuses. Renal TfR protein expression did not differ, but placental TfR expression was lower in USR fetuses at GD130. Placental levels of TfR correlated to eNOS. TfR was localized throughout the placentome, including the hemophagous zone, implicating a role for TfR in ovine placental iron transport.Conclusion:Fetal iron was regulated in an organ-specific manner. In USR fetuses, NO-mediated placental adaptations may prevent the normal upregulation of placental TfR at GD130.

Concomitant changes in progesterone catabolic enzymes, cytochrome P450 2C and 3A, with plasma insulin concentrations in ewes supplemented with sodium acetate or sodium propionate*

Progesterone is essential for maintaining pregnancy, and several authors have suggested that low peripheral concentrations of progesterone may be responsible for high rates of embryonic loss. The primary organ involved in the catabolism of progesterone is the liver, and cytochrome P450 2C and 3A sub-families account for a large proportion of this catabolism. Elucidating a mechanism to decrease progesterone catabolism, thereby increasing embryonic and uterine exposure to progesterone, seems a logical approach to ameliorate high rates of embryonic loss. The objectives of the current experiment were to determine the pattern of insulin secretion after supplementing feed with either sodium acetate or sodium propionate and to determine any association between the differential patterns of insulin secretion with the hepatic activity of cytochrome P450 2C and 3A and progesterone clearance. Sixteen ovariectomized ewes were fed 3 kg/day for 10 days of a diet consisting of 50% corn silage, 38% triticale haylage, 12% soybean meal and 600 ml of 3.5 M sodium acetate (energy control; n = 8) or 2.0 M sodium propionate (gluconeogenic substrate; n = 8). Equal portions of the ration (1 kg as-fed basis along with 200 ml of 3.5 M sodium acetate or 2.0 M sodium propionate) were offered three times daily at 0600, 1400 and 2200 h. Concentrations of insulin in plasma were determined immediately before feeding and at 15, 30, 60, 90, 120, 180, 240 and 300 min after feeding. Progesterone clearance from peripheral circulation (ng/ml per min) was measured by giving a 5 mg injection of progesterone into the left jugular vein and collecting blood via the right jugular vein at 0, 2, 4, 6, 8, 10, 15, 20 and 30 min afterwards. Liver biopsies were taken 1 h after feeding to determine cytochrome P450 2C and 3A activities. Insulin concentrations in ewes supplemented with sodium propionate were elevated at 15, 30 and 60 min after feeding compared to the sodium acetate group. Cytochrome P450 2C and 3A activities were decreased 1 h after feeding in the sodium propionate-treated ewes relative to sodium acetate. Insulin appears to down-regulate cytochrome P450 activity, which could be used to decrease the catabolism of progesterone during early gestation, thereby increasing peripheral concentrations of progesterone and, consequently, embryonic exposure to progesterone.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice

BACKGROUND AIMS Existing treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. METHODS Induced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 10(6) cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. RESULTS Blood flow in the MCA-derived MSC-treated animals was higher at each day (P <0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P <0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. DISCUSSION MCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.