Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.
C Wulff, M Weigand, R Kreienberg and HM Fraser
Normal embryonic development is dependent upon a sufficient oxygen, nutrient and waste exchange through the placenta. In primates including humans, this exchange is attained by successful haemochorial placentation which requires the transformation of maternal intramyometrial spiral arterioles by trophoblast invasion to gain uteroplacental circulation, and establishment and maintenance of a competent fetoplacental vasculature. Thus, trophoblast and endothelial cell differentiation, proliferation and invasion occurring during placentation have to be tightly regulated. This review focuses on the diverse developmental steps during haemochorial placentation in humans and other primates and the possible involvement of angiogenic growth factors (vascular endothelial growth factor (VEGF) and angiopoietins (Ang)) in these processes, highlighting the importance of specific actions of angiogenic ligand-receptor pairs. It is hypothesized that VEGF/VEGF-R1 and Ang-1/Tie receptor 2 (Tie-2) may regulate trophoblast differentiation and invasion; VEGF/VEGF-R2 and Ang-1/Tie-2 may promote fetoplacental vascular development and stabilization; and Ang-2/Tie-2 may be involved in maternal vascular remodelling. The importance of a tight regulation of angiogenic factors and their endogenous antagonists for normal development of the placenta is demonstrated by failure of this system, resulting in abnormal placenta vascularization and trophoblast invasion associated with intrauterine growth retardation or pre-eclampsia.
D Herr, M Rodewald, H M Fraser, G Hack, R Konrad, R Kreienberg and C Wulff
This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin–angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.