Showing papers by "Asif Ahmed published in 2000"

Angiopoietin-1 and Angiopoietin-2 Activate Trophoblast Tie-2 to Promote Growth and Migration during Placental Development

Abstract: Human placental development involves coordinated angiogenesis and trophoblast outgrowth that are compromised in intrauterine growth restriction (IUGR). As Tie-2(−/−) mice exhibit growth retardation and vascular network malformation, the expression of Tie-2 and its ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), were investigated in human placenta from normal pregnancies and those complicated by severe IUGR. Ribonucleotide protection assays showed no significant change in the expression of Ang-2 mRNA between gestationally matched normal and IUGR placentas; however, immunoblots revealed that Ang-2 protein was significantly decreased in IUGR, suggesting that this may contribute to the abnormal development of the villous vasculature. In situ hybridization studies showed that Ang-1 and Tie-2 were detected in the cyto/syncytiotrophoblast bilayer in first-trimester placenta, whereas Ang-2 mRNA was restricted to the cytotrophoblast, suggesting their role in trophoblast function. At term, Ang-1 mRNA and immunoreactive protein were restricted to the paravascular tissues of the primary stem villi, supporting its role in vessel maturation. In contrast, Ang-2 was expressed throughout the term villous core, perhaps to permit the developing placental vascular network to remain in a state of fluidity. As these studies also revealed that trophoblast, in addition to endothelial cells, expressed Tie-2 receptors, we investigated the potential role of Ang-1/Ang-2 on trophoblast proliferation, migration, and the release of NO. Using spontaneously transformed first-trimester trophoblast cell lines that exhibit cytotrophoblast-like (ED27) and extravillous trophoblast-like (ED77) properties, we show that the addition of Ang-2 (250 ng/ml) stimulated DNA synthesis in ED27 trophoblast cells and triggered the release of NO. Ang-1 stimulated trophoblast (ED77) migration in a dose-dependent manner that was inhibited by recombinant Tie-2-FC. These data thus imply, for the first time, a specific role for angiopoietins as regulators of trophoblast behavior in the development of the utero/fetoplacental circulation, an action independent of their well-established roles in vascular endothelium.

Induction of placental heme oxygenase-1 is protective against TNFalpha-induced cytotoxicity and promotes vessel relaxation

Abstract: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNFα) induced placental damage and on feto-placental circulation was studied. We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNFα-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p < 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

Angiogenesis and intrauterine growth restriction.

Abstract: Human placental development involves co-ordinated angiogenesis and trophoblast outgrowth that are compromised in intrauterine growth restriction. Adaptive angiogenesis in IUGR placental villi is a result of an imbalance in the orderly progression of the expression profile of vascular endothelial growth factor, placenta growth factor and angiopoietin during placental development. VEGF receptors and the angiopoietin receptor Tie-2 are expressed on trophoblast, and their activation leads to trophoblast proliferation, migration and production of nitric oxide. Thus, these vascular factors act as autocrine regulators of trophoblast behaviour in the development of the utero-/feto-placental circulation, an action independent of their well-established roles in vascular endothelium.

Activation of platelet-activating factor (PAF) receptor stimulates nitric oxide (NO) release via protein kinase C-alpha in HEC-1B human endometrial epithelial cell line.

Abstract: Impairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC). Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, NG-monomethyl-l-arginine (l-NMMA) and Ro 31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCα hr. Before agonist stimulation, in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation. PAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by NG-monomethyl-l-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCα, βII and ι were the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of both PKCα and PKCι within the time frame of NO release. The translocation of PKCα, but not PKCι, was susceptible to inhibition by Ro 31-8220 that also inhibited PAF-evoked NO release, suggesting that PKCα is the principal isozyme involved in this process and that eNOS may be a substrate for PKCα. Kinase assays performed using immunoprecipitated PKCα showed that PAF (1 nM) activated PKCα that was inhibited by co-incubation with Ro 31-8220 and Ca2+-free medium. This study demonstrates that PAF-stimulated NO release via PKCα in epithelial cells might regulate endometrial functions such as implantation and menstruation.

Growth Factor Regulators of Placental Angiogenesis

Abstract: The human placenta is a highly vascularised organ that mediates gas and nutrient exchange between the maternal and fetal circulations In this chapter we discuss the processes of vasculogenesis, angiogenesis and the factors influencing them in the formation of the placental vasculature Also described are the effects of growth factors on the specialised structures of the mature intermediate and terminal villi, which are the main site of gas exchange We have identified VEGF to be a key agent in early placental angiogenesis, acting in concert with a related factor, placenta growth factor (PlGF) via two high affinity receptors VEGFR-1 and VEGFR-2 to stimulate endothelial cell proliferation and vessel formation A second class of receptor tyrosine kinases includes the Tie-2 receptor Its newly identified ligands angiopoietin-1 and a natural antagonist angiopoietin-2 are thought to play a role in vessel maturation, acting downstream to VEGF