Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction.

During early human pregnancy extravillous cytotrophoblasts invade the uterus and spiral arteries transforming them into large vessels of low resistance. Failure of trophoblast invasion and spiral artery transformation occurs in preeclampsia and fetal growth restriction (FGR); these processes are not well understood. Recent studies have suggested that cytotrophoblasts that invade spiral arteries mimic the endothelial cells they replace and express PECAM-1. It was also reported that in preeclampsia, cytotrophoblasts fail to express PECAM-1 and that failure to express endothelial cell adhesion molecules may account for failed trophoblast invasion. Despite the possible importance of adhesion molecules in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of invasion, particularly in the myometrial segments where the key failure occurs. There are no studies on PECAM-1 expression in the placental bed in FGR. We have examined the expression of PECAM-1 in placental bed biopsies and placentas from 8 to 19 weeks of gestation and in the placenta and placental bed in the third trimester in cases of preeclampsia, FGR, and control pregnancies. PECAM-1 was expressed on endothelium of vessels in the placenta and placental bed but not by villous or extravillous trophoblasts in normal or pathological samples. These findings do not support a role for PECAM-1 in normal invasion or in the pathophysiology of preeclampsia or FGR.

Relationship of cell adhesion molecule expression to endothelium-dependent relaxation in normal pregnancy and pregnancies complicated with preeclampsia or fetal growth restriction.

OBJECTIVES: To determine the degree of endothelium-dependent relaxation in myometrial and omental resistance arteries from normal pregnancies and pregnancies complicated with preeclampsia or fetal growth restriction (FGR) (compromised pregnancy group), and to correlate the results with the endothelial surface expression of cell adhesion molecules (CAMs) in the same vessels. METHODS: Parallel wire myography was used to assess the relaxation of omentum or myometrial vessels obtained from nonpregnant women (n = 3), women with normal pregnancies (n = 11), and women with pregnancies complicated by preeclampsia or fetal growth restriction (n = 10). These resistance vessels were constricted with incremental concentrations of vasopressin (10(-10) mol/L to 3.3 x 10(-8) mol/L) prior to the addition of incremental concentrations of bradykinin (10(-10) mol/L to 3.3 x 10(-6) mol/L). Immunohistochemistry was used to assess the endothelial expression of the CAMs E-selectin, ICAM-1, VCAM-1, and PECAM. RESULTS: A significant reduction in endothelium-dependent relaxation of myometrial vessels was found in the compromised pregnancy group when compared with both the normotensive pregnant group and the nonpregnant group. This reduction was not noted with omental vessels. All vessels in the nonpregnant group, normal pregnant group, and compromised pregnancy group expressed PECAM and ICAM-1 on the endothelium. There was no difference in intensity of immunostaining between the groups. None of the vessels in any of the groups expressed VCAM-1 or E-selectin. CONCLUSIONS: We found no evidence that impaired relaxation responses to bradykinin are linked to altered expression of CAMs in preeclampsia and FGR. These results suggest that increased CAM expression occurs in a vascular bed separate from those investigated in the present study. Possible sites for this would be in the microcirculation of organs such as the kidney.

Human trophoblast invasion and spiral artery transformation: the role of nitric oxide.

During early human pregnancy extravillous cytotrophoblasts invade the uterus and also migrate up the spiral arteries, transforming them into large vessels of low resistance. Failure of transformation has been described in pre-eclampsia, fetal growth restriction, and miscarriage. Recent evidence suggests that some maternal vessels undergo structural changes without interaction with cytotrophoblasts. The possibility arises that local vasoactive mediators such as nitric oxide result in spiral artery dilatation before their invasion. In support of this, a recent histological study in the guinea pig suggested that cytotrophoblasts expressed nitric oxide synthase (NOS) as they surrounded vessels. This study tested the hypothesis that invading cytotrophoblasts express NOS and therefore have the potential to induce vasodilatation by releasing nitric oxide. The expression of NOS on extravillous cytotrophoblasts was studied in placental bed biopsies, obtained, using a transcervical sampling technique, from normal human pregnancies between 8 to 19 weeks of gestation and in the third trimester. Whereas eNOS was expressed by syncytiotrophoblast, neither eNOS or iNOS was expressed by extravillous cytotrophoblasts at any time during invasion. The mechanisms controlling spiral artery transformation are pivotal to understanding normal and abnormal placentation. These results suggest that trophoblast-derived nitric oxide is unlikely to contribute to spiral artery dilatation.

TGFbeta1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice.

TGFbeta1 has been implicated in cell cycle control and carcinogenesis. To address the exact function of TGFbeta1 in skin carcinogenesis in vivo, mice with TGFbeta1 expression targeted to keratinocytes were subjected to long-term chemical carcinogenesis treatment. TGFbeta1 showed biphasic action during multistage skin carcinogenesis, acting early as a tumor suppressor but later enhancing the malignant phenotype. The transgenics were more resistant to induction of benign skin tumors than controls, but the malignant conversion rate was vastly increased. There was also a higher incidence of spindle cell carcinomas, which expressed high levels of endogenous TGFbeta3, suggesting that TGFbeta1 elicits an epithelial-mesenchymal transition in vivo and that TGFbeta3 might be involved in maintenance of the spindle cell phenotype. The action of TGFbeta1 in enhancing malignant progression may mimic its proposed function in modulating epithelial cell plasticity during embryonic development.

Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of haemophilia B.

It has previously been suggested that keratinocytes might provide a suitable target cell for delivery of factor IX to the systemic circulation for gene therapy of haemophilia B. Here, an investigation of the use of cellular gene promoters specific for keratinocytes was undertaken to examine whether factor IX could be passed from the epidermis to the systemic circulation. Utilizing two bovine cytokeratin gene promoters, BKIII and BKVI, three lines of transgenic mice were generated with targeted expression of human factor IX in the epidermis. All three transgenic mouse lines secreted epidermally derived human factor IX into the blood system. Most effective factor IX expression (46 ng/ml steady-state levels of circulating human factor IX) was obtained utilizing the BKVI gene promoter, the human homologue of K10, which is expressed exclusively in differentiated keratinocytes, localized distal to the basement membrane. This report demonstrates, for the first time, that human factor IX can be efficiently synthesized and secreted from keratinocytes in situ, and can cross the epidermal basement membrane to reach the systemic circulation. The transgenic mouse model will provide a good in vivo system with which to optimize the efficiency of different keratin gene promoter constructs for delivery of therapeutic gene products to the serum, especially for those promoters, such as K10, which are not effectively expressed in vitro.

Concerted action of TGF-beta 1 and its type II receptor in control of epidermal homeostasis in transgenic mice.

Transforming growth factor-beta 1 (TGF-beta 1) is a modulator of cellular proliferation, differentiation, and extracellular matrix deposition. It is a potent epithelial growth inhibitor and can alter the differentiative properties of keratinocytes, in vitro, but little is known about its normal physiological function in the epidermis in vivo. Transgenic mice were generated using a keratin 10 (K10) gene promoter to drive constitutive expression of TGF-beta 1 in the suprabasal keratinocyte compartment. Surprisingly, these mice showed a two- to threefold increase in epidermal DNA labeling index over control mice, in the absence of hyperplasia. The transgene, however, acted in the expected fashion, as a negative regulator of cell growth, when hyperplasia was induced by treatment by 12-tetradecanoyl-phorbol-13-acetate (TPA). Epidermal TGF-beta type I and II receptor (T beta RI and T beta RII) levels were examined in control and transgenic mice during induction of hyperplasia by TPA. Whereas T beta RI levels remained relatively constant, T beta RII expression was strongly induced in TPA-treated skins, prior to the induction of the growth inhibitory response to TGF-beta 1, and its level of expression correlated with growth sensitivity to TGF-beta 1 in vivo and in vitro. These results suggest that TGF-beta 1 and its type II receptor are part of the endogenous homeostatic regulatory machinery of the epidermis.

Lack of transforming growth factor-beta 1 expression in benign skin tumors of p53null mice is prognostic for a high risk of malignant conversion.

Expression of transforming growth factor beta 1 (TGF beta 1) protein was examined in chemically induced benign skin tumors with genetically defined empirical risks for malignant conversion. Benign tumors induced in mice which have both alleles of the p53 gene deleted have a malignant conversion frequency of approximately 50%, whereas similar tumors induced in wildtype and heterozygous p53 mice have conversion probabilities of 3 and 8%, respectively (Kemp et al., Cell, 74: 813-822, 1993). The TGF beta 1 antibody, anti-CC (1-30-1), was shown to stain either the proliferative keratinocyte compartment of the tumor or the tumor stroma, whereas another TGF beta 1 antibody, anti-LC (1-30-1), stained highly differentiated granular cells of the tumors. A strong correlation was found between staining of the proliferative keratinocyte compartment of tumors with the anti-CC (1-30-1) antibody and tumor genotype. Only 18% (6 of 32) of homozygous p53 null tumors showed any basal keratinocyte staining with this antibody, whereas over 80% (32 of 38) of heterozygous and wild-type tumors showed positive staining. Additionally, in most tumors examined, the spatial distribution of staining for the proliferating cell nuclear antigen appeared to be mutually exclusive with that of TGF beta 1 on adjacent serial sections. This suggests that, in these cases, tumor keratinocytes are sensitive to negative growth regulation by TGF beta. TGF beta 1 protein staining in benign tumors is thus prognostic for a low probability of malignant conversion, and its expression may be mechanistically involved in limiting malignant conversion since, at the benign tumor stage examined, keratinocytes are still sensitive to growth inhibition by TGF beta 1.

RNA and protein localisations of TGF beta 2 in the early mouse embryo suggest an involvement in cardiac development.

We have performed a detailed analysis of the localisations of RNAs for TGF beta 2 and beta 3, and of TGF beta 2 protein in mouse embryos from 6.5 to 9.5 days post coitum, using in situ hybridisation and immunohistochemistry on serial sections, and whole-mount in situ hybridisation to complete embryos. TGF beta 3 RNA was not seen in any of the tissue sections, but very low levels of the RNA were seen by whole-mount in situ hybridisation around the outflow tract of the heart at 8.5 days post coitum. TGF beta 2 RNA is expressed at high levels in all cells with the potential to differentiate into cardiomyocytes. Additionally, the foregut endoderm, juxtaposed to the heart, and the neuroepithelium at the rostral extremity of the foregut, express very high levels of TGF beta 2 RNA, between 8.5 and 9.5 days post coitum. As cardiomyogenesis proceeds, TGF beta 2 RNA levels diminishes within the myocytes, with a concomitant increase in staining for TGF beta 2 protein. TGF beta 2 protein staining of cardiomyocytes persists throughout development and in the adult, in the absence of detectable levels of the corresponding RNA. Superimposed upon this myocardial pattern of expression, there is an upregulation of TGF beta 2 RNA in the myocardium of the outflow tract and atrioventricular canal between 8.5 and 9.5 days post coitum, which returns to low levels by 11.5 days post coitum. The results are discussed in terms of a potential role of TGF beta 2 in controlling cardiomyogenesis and in inductive interactions leading to cardiac cushion tissue formation.

Discordant transforming growth factor beta 1 RNA and protein localization during chemical carcinogenesis of the skin.

Transforming growth factor beta (TGF-beta) inhibits proliferation of normal keratinocytes, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta 2, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 12-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal keratinocytes, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 12-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal keratinocyte compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative growth regulation by altered translational and/or posttranslational processing mechanisms of this growth factor. Levels of TGF-beta 2 and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.

TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis.

The tissue distribution of TGF beta-1 RNA was examined within whole mouse embryos from implantation to 10.5 days gestational age and, in the developing heart, up to 8 days postpartum. The earliest high level expression of TGF beta-1 RNA is at 7.0 days postcoitum (p.c.) in the cardiac mesoderm. At 8.0 days gestational age, cardiac TGF beta-1 RNA expression is limited to endocardial cells. By 9.5 days p.c., this expression pattern becomes regionalized to those cells that overlie cardiac cushion tissue. High TGF beta-1 RNA levels continue to persist in endothelial cells of the heart valves until approximately one week postpartum. The TGF beta-1 RNA distribution was compared with the extracellular distributions of polypeptides for TGF beta and J1/tenascin. As previously reported, endothelial expression of TGF beta-1 RNA is correlated with mesenchymal expression of TGF beta polypeptide, suggesting a paracrine mode of action for this growth factor in cardiac development. Minor discrepancies in the distributions of TGF beta-1 RNA and the extracellular form of the TGF beta polypeptide suggest that translational or post-translational control of protein levels occurs and/or the possibility that the antibody used may also recognise other members of the TGF beta polypeptide family. A correlation between endothelial TGF beta-1 expression and distribution of J1/tenascin in the mesenchyme gives further support to the proposition that the biological effects of TGF beta-1 may, in part, be mediated by J1/tenascin.