Elsevier

Placenta

Volume 25, Issues 2–3, February–March 2004, Pages 208-217
Placenta

Umbilical Vein and Placental Vessels from Newborns with Hereditary Haemorrhagic Telangiectasia Type 1 Genotype are Normal despite Reduced Expression of Endoglin

https://doi.org/10.1016/S0143-4004(03)00181-4Get rights and content

Abstract

Hereditary haemorrhagic telangiectasia, HHT, is an autosomal dominant disorder that affects approximately 1 in 8000 people. HHT1 is associated with mutations in the ENG (Endoglin) gene and with haploinsufficiency. The disorder is characterized by focally dilated vessels, which can lead to arteriovenous malformations and serious complications even in young children. In the current study, umbilical cord and placenta samples from newborns with ENG mutations were analyzed to estimate the level of corresponding protein and look for potential vascular dysplasia. We confirmed, using metabolic labelling and flow cytometry, that endoglin levels were significantly reduced to median values of 47 per cent (range 32–56 per cent) and 58 per cent (46–90 per cent), respectively, in human umbilical vein endothelial cells derived from newborns with ENG mutations (HHT1 group; n=18) relative to samples from newborns shown not to have the familial mutation (non-HHT group). We also quantified the relative expression of endoglin by estimating the endoglin/PECAM-1 staining ratio in tissue sections. We observed significantly lower values in the HHT1 group, compared to the non-HHT group for the umbilical vein (n=9; median 0.6 vs 0.9; ranges 0.2–1.0 and 0.5–1.5) and for placental stem villus vessels (n=9 and 10; median 0.42 vs 0.93; ranges 0.24–0.58 and 0.56–1.18). No differences in the estimated umbilical vein cross-sectional area and in the proportion of vessels present in placental villi were observed in sections from the HHT1 group relative to the non-HHT group. Thus, blood vessels from HHT1 individuals are maintained intact in the umbilical vein and placenta during pregnancy and delivery, despite a significant reduction in endoglin expression.

Introduction

Hereditary Haemorrhagic Telangiectasia (HHT) is inherited as an autosomal dominant disorder and affects 1 in 8000 people, worldwide. It is a heterogenous disease, in terms of age of onset and severity, both within and between families, suggesting a complex disorder. Part of the clinical heterogeneity can be explained by at least two different loci. The first gene, responsible for HHT type 1 (HHT1) was mapped to chromosome 9q33 [1], [2]and identified as ENG (endoglin) [3]. The second gene, ACVRL1 (activin receptor-like kinase I) or ALK1[4], mapped to chromosome 12 [5], [6]and is mutated in HHT type 2 (HHT2). Both ENG and ACVRL1 gene products are highly expressed on endothelial cells and are receptors for the TGF-β superfamily of growth factors [7], [8], [9], [10]. A third as yet unidentified gene, was shown to be unlinked to either the ENG or ACVRL1 loci in an HHT family with pulmonary arteriovenous malformations (AVMs) [11].

The most common manifestation of HHT is the telangiectasia, which is a small raised red lesion made of dilated and convoluted fragile capillaries that can easily bleed [12]. Telangiectases are the cause of frequent nosebleeds observed in most patients and of gastrointestinal bleeding in older patients. They are also present in the liver. A morphometric analysis of hepatic sections from an HHT patient has revealed that arteries were on average more dilated than in control samples; dilated portal veins were also observed [13]. Sonographic measurements have also revealed a dilated hepatic artery at early stages of HHT, and in more advanced cases, multiple dilated and tortuous vessels including markedly dilated vena cava and hepatic vein [14]. In a murine model of HHT1, we have also reported the presence of dilated vessels in several organs [15].

We showed previously that endoglin is a transmembrane glycoprotein highly expressed on the syncytiotrophoblast of human placenta throughout pregnancy [16], [17], [18]. It is however absent from blood vessels within the villi during the first trimester, while present on all placental vessels at term [17], [18]. Endoglin appears to mediate the effects of TGF-β1 and TGF-β3, which are potent regulators of early placental development and more specifically of the differentiation of extravillous trophoblasts needed to establish fetal-maternal interactions [18], [19]. Since endoglin and TGF-β play an important role in placental development, we argued that mutations in the ENG gene might be associated with placental and/or vascular abnormalities at the time of delivery.

We previously demonstrated that clinically affected individuals with an ENG mutation and thus of HHT1 genotype, express reduced amounts of endoglin protein, as measured by metabolic labelling and immunoprecipitation of activated peripheral blood monocytes [20], [21], [22]. Newborns with an ENG mutation, irrespective of the type or position of the mutation, also showed reduced levels of endoglin on their HUVEC (human umbilical vein endothelial cells) [20], [22], [23], [24]. This implies that the mutant copy of the gene is not expressed and therefore cannot interfere in a dominant negative fashion with the functional protein. HHT1 is then due to haploinsufficiency, the expression of one rather than two copies of the gene.

In this study, we analyzed sections from the umbilical cord and placenta derived from newborns of HHT families. We observed reduced expression of endoglin in the HHT1 group by immunostaining of the umbilical cord and placental vessels, and by metabolic labelling and flow cytometry of the endothelial cells isolated from the umbilical vein. We did not see abnormally dilated vessels nor altered placental structures in the HHT1 group, suggesting no pathological signs in these tissues following pregnancy and delivery. Our data showing normal vessels in fetal extracorporal tissues support previous reports of no increase in the rate of spontaneous abortion or placental complications in HHT patients [25], [26].

Section snippets

Clinical evaluation and patient samples

Informed consent was obtained from the expecting parents and all experimental procedures involving the use of placenta and umbilical cord samples were reviewed and approved by the Research Ethics Board of the Research Institute of the Hospital for Sick Children, Toronto, Canada. All members of families with HHT are designated with the prefix H (for HHT), followed by an assigned patient number. A positive HHT clinical diagnosis is based on the established criteria [27].

Placenta and umbilical

Reduced levels of endoglin on HUVEC from newborns with ENG mutations as determined by metabolic labelling and flow cytometry

Eighteen cases of HUVEC from 16 HHT families had reduced levels of endoglin, as measured by metabolic labelling and immunoprecipitation (Figure 1A). A median value of 47 per cent, expressed relative to the value observed for a normal control ran in each experiment, was estimated with a range of 32 per cent to 56 per cent. As endoglin is constitutively expressed at high levels on HUVEC, a decreased protein level allowed us to predict the newborns with an HHT1 genotype and perform the appropriate

Discussion

We have demonstrated that endoglin levels are reduced on the endothelium of the umbilical vein and placental vessels in newborns with ENG mutations, as demonstrated by several methods. We have also shown that there is no apparent dysplasia in these vessels at the time of delivery and that no significant dilatation was observed relative to non-HHT samples.

In HUVEC, levels of endoglin were significantly reduced when measured either by metabolic labelling/immunoprecipitation or flow cytometry,

Acknowledgements

This research was supported by grants from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Canada and the March of Dimes Foundation (ML). ML is a member of the Heart and Stroke Richard Lewar Centre of Excellence. We are very grateful to Adonna Greaves and Marie-Eve Paquet for their technical assistance.

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