Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction

Early placental development occurs in an environment of relative hypoxia. Hypoxia promotes angiogenesis and up-regulates vascular endothelial growth factor (VEGF) expression while it down-regulates placenta growth factor (PIGF) that possess 53% homology with VEGF. Morphological studies show poor placental vascular development and an increase in the mitotic index of cytotrophoblasts in intrauterine growth restriction (IUGR). We hypothesized that the reported relatively high oxygen level in the intervillous space in contact with IUGR placental villi will limit angiogenesis by changes in VEGF and PIGF expression and function. Western immunoblot analysis demonstrates a diametric expression of PIGF and VEGF proteins throughout pregnancy with PIGF levels increasing and VEGF levels decreasing, consistent with placental oxygenation. In IUGR placentae, the ratio of PIGF/GAPDH mRNA was increased by 2.3-fold (p < 0.03) and PIGF protein levels were also increased, (p < 0.05) as compared with gestationally-matched normal placentae. PIGF mRNA and protein were localized to the trophoblast bilayer and villous mesenchyme of the human placenta throughout gestation. In vitro studies demonstrated that increasing oxygen tension (hyperoxia) up-regulated PIGF protein in term placental villous explants, whereas hypoxic culture of a term trophoblast choriocarcinoma cell line (BeWo) down-regulated PIGF mRNA and protein and VEGFR-1 (Flt-1) autophosphorylation. The addition of PIGF-1 to a spontaneously transformed first trimester cytotrophoblast cell line stimulated DNA synthesis while PIGF-2 had little effect. VEGF and PIGF exert their biological actions by means of a common receptor VEGFR-1. In the first trimester trophoblast cells, PIGF-1 increased the association of phosphorylated extracellular signal-related kinase (ERK) with VEGFR-1 immunoprecipitates while both PIGF-1 and PIGF-2 also potentiated endogenous VEGF mediated association of phosphorylated extracellular related kinase (ERK) with VEGFR-2 (KDR). More importantly, the addition of PIGF-1 had little effect while PIGF-2 inhibited cell growth in cultured endothelial cells derived from human umbilical vein. Nitric oxide (NO) is reported to promote angiogenesis and PIGF-2 inhibited the basal release of NO from the first trimester trophoblast. The tissue expression and functional studies support the hypothesis of "placental hyperoxia" in early-onset IUGR because hypoxia down-regulates trophoblast PIGF levels, PIGF expression is increased in IUGR, and PIGF-2 inhibits endothelial cell growth. Taken together, these changes provide a cellular explanation for the observed poor angiogenesis in the pathogenesis of IUGR and show that the two PIGF isoforms may modulate trophoblast and endothelial cell function differently, possibly through potentiation of VEGF mediated activation of VEGF-2.     

血管内皮生长因子及其受体在子宫内膜癌中的表达

目的探讨血管内皮生长因子(VEGF)及其受体fms样酪氨酸受体-1 (flt-1)和含插入区的激酶受体(KDR)在子宫内膜癌血管生成中的作用及其与内膜癌分化程度的关系.方法采用免疫组织化学及原位杂交方法对23例子宫内膜癌及6例正常绝经期子宫内膜中VEGF、flt-1、KDR蛋白质及其mRNA进行检测,并对少数病例行Western印迹分析,以检测VEGF亚型在内膜癌组织的分布,用内皮细胞标志Ⅷ因子标记内膜癌组织中的微血管密度.结果 VEGF、flt-1、KDR蛋白质及其mRNA主要分布在子宫内膜癌组织血管内皮细胞及癌细胞胞质内.VEGF蛋白质在中分化(G2)、低分化(G3)内膜癌血管内皮细胞及癌细胞上的表达高于高分化内膜癌(G1)及正常绝经期子宫内膜(P<0.05), VEGF mRNA在不同分化程度内膜癌组织的表达差异无显著性意义(P>0.05),但均大于正常绝经期子宫内膜(P<0.05);flt-1蛋白质及flt-1mRNA在G3内膜癌血管内皮细胞的表达高于G1、G2及正常绝经期子宫内膜(P<0.05),在癌细胞的表达差异无显著性意义(P>0.05) ,但均高于正常绝经期子宫内膜(P<0.05);KDR蛋白质在子宫内膜癌组织血管内皮细胞及癌细胞上的表达较强,但不随分化程度发生变化,其mRNA在中分化(G2)、低分化(G3)内膜癌血管内皮细胞及癌细胞上的表达高于正常绝经期子宫内膜(P<0.05).G3子宫内膜癌组织的血管密度(48个±12个)高于G1(27个±14个)、G2(26个±16个)及正常绝经期子宫内膜(26个±11个,P<0.05).结论 VEGF、flt-1、KDR及mRNA在子宫内膜癌中的表达形式提示其与癌组织血管生成及血管通透性相关,VEGF及其受体是与子宫内膜癌旺盛生长相关的因子之一.     

Antiangiogenic effect of soluble vascular endothelial growth factor receptor-1 in placental angiogenesis.

Differential splicing of the flt-1 mRNA generates soluble variant of vascular endothelial growth factor (VEGF) receptor-1 (sVEGFR-1, also known as sFlt-1). The action of VEGF is antagonized by sVEGFR-1. Soluble VEGFR-1 binds to VEGF with a high affinity and therefore works to modulate VEGF and VEGF signaling pathway. In this study, the authors tested the hypothesis that VEGF-mediated endothelial cell angiogenesis is tightly modulated by the release of sVEGFR-1 and placental expression of sVEGFR-1 is upregulated by hypoxia. Immunolocalization studies showed progressively intense staining for sVEGFR-1 and VEGF in the trophoblast of placental villous explants throughout gestation. Endothelial cell migration studies using a modified Boyden's chamber showed a significant increase in cell migration in response to VEGF that was significantly attenuated in the presence of exogenous sVEGFR-1. Furthermore, stimulation of endothelial cells with VEGF led to a dose-dependent increase in the release of sVEGFR-1 as determined by enzyme-linked immunosorbent assay (ELISA). Exposure of normal placental villous explants to hypoxia (1% pO2) increased trophoblast expression of sVEGFR-1 when compared with tissue normoxia (5% pO2). In addition, conditioned media from hypoxia treated placental villous explants induced a significant increase in endothelial cell migration that was significantly reduced in presence of sVEGFR-1. Our study demonstrates that hypoxia positively regulates sVEGFR-1 protein expression in ex vivo trophoblasts, which control VEGF-driven angiogenesis.     

Endothelial nitric oxide synthase in placental villous tissue from normal, pre-eclamptic and intrauterine growth restricted pregnancies

Nitric oxide (NO) regulates blood flow in the human placenta. As increased resistance to blood flow is seen in the fetal-placental vasculature in pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR), we examined expression of endothelial nitric oxide synthase (eNOS) in these placentas. Placental villous tissue sections were obtained from normotensive control (n = 5), IUGR alone (n = 5) or pre-eclamptic (with or without IUGR (n = 9) patients, immunostained for eNOS and scored for localization, type (punctate or diffuse) and intensity of eNOS staining in syncytiotrophoblast and placental vessels. The significance of differences was calculated using the Mann-Whitney U-test. No differences in intensity or type of immunostaining in syncytiotrophoblast were seen. Placentas from patients with pre- eclampsia with or without IUGR had a significantly more basal distribution of eNOS in syncytiotrophoblast. eNOS immunostaining was absent in terminal villous capillary and faint in stem villous vessel endothelium of normal placentas, but was intense in the endothelium of both of these types of vessels in the IUGR and pre-eclampsia groups, with significantly greater staining seen in stem vessels of patients with IUGR alone. This increased eNOS expression and hence increased NO production in the fetal-placental vasculature may be an adaptive response to the increased resistance and poor perfusion in these pathological pregnancies.      

Gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma

BACKGROUND: Angiogenesis is a feature of airway remodeling in bronchial asthma. The mechanism responsible for this angiogenesis is unknown. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cells, which may contribute to chronic inflammation and angiogenesis. OBJECTIVE: We sought to investigate the molecular mechanisms underlying increased vascularity, and we examined the mRNA expression of VEGF and its receptors (flt-1 and flk-1) within bronchial biopsy specimens from asthmatic patients and normal control subjects. METHODS: Endobronchial biopsy specimens were examined immunocytochemically by staining with anti-type IV collagen mAb to evaluate vessel density by using computer-assisted image analysis. Specimens were also analyzed for the presence of the mRNAs of VEGF and its receptors with in situ hybridization. RESULTS: The extent of airway vascularity was increased in asthmatic subjects compared with that in control subjects (P <.01). Asthmatic subjects exhibited a greater expression of VEGF, flt-1, and flk-1 mRNA(+) cells in the airway mucosa compared with that in control subjects (P <.001 for each comparison). The degree of vascularity was associated with the number of VEGF, flt-1, and flk-1 mRNA(+) cells. Numbers of cells expressing VEGF mRNA inversely correlated with airway caliber (r = -0.83, P <.01) and airway hyperresponsiveness (r = -0.97, P <.001). Colocalization studies showed that macrophages, eosinophils, and CD34(+) cells were the major sources of VEGF; CD34(+) cells, macrophages, and T cells expressed both flt-1 and flk-1. CONCLUSION: These findings provide evidence that VEGF may play an important role in angiogenesis and subsequent airway remodeling in bronchial asthma.     

Expression of IGR-IR and VEGF and trophoblastic proliferative activity in placentas from pregnancies complicated by IUGR

Intrauterine growth retardation (IUGR) is recognized as an important cause of low birth weight and elective preterm delivery. IUGR is associated with multiple causative factors, including placental dysfunction. The aim of this prospective study was to investigate the role of trophoblastic proliferative activity and type I insuline-like growth factor receptor (IGF-IR) and vascular endothelial growth factor (VEGF) expressions in the pathogenesis of IUGR. Immunohistochemistry using VEGF, IGF-IR, and Ki-67 antibodies was performed on formalin-fixed placental tissues of third-trimester pregnancies complicated by IUGR (n = 19) and pregnancies with appropriately grown fetuses (n = 27). In addition, histopathological examination of the placentas was performed, and histological findings were categorized into three groups: utero-placental vascular pathologies (UPVP), coagulation-related pathologies, and chronic inflammation. Statistical analysis revealed that villous trophoblastic IGF-IR immunostaining was significantly weaker in placentas with IUGR (p < 0.001), whereas trophoblastic Ki-67 proliferative index and VEGF immunoscoring did not show any significant difference. Histologically, UPVP and chronic inflammation were significant findings in placentas with IUGR (p = 0.04 and p = 0.04, respectively). In addition, placentas were significantly smaller in the IUGR group (p < 0.001). We conclude that villous trophoblastic IGF-IR expression may play a significant role in the pathogenesis of IUGR, and histopathological examination of placentas in pregnancies complicated by IUGR may yield significant findings. In contrast, based on our findings, trophoblastic proliferation and VEGF expression are unlikely to be significant parameters in the pathogenesis of IUGR.     

血管内皮生长因子及其受体mRNA在输卵管妊娠蜕膜组织的表达

目的:检测血管内皮生长因子(VEGF)及其受体(KDR、Flt-1)在输卵管妊娠蜕膜组织的表达,探讨其在输卵管妊娠中的作用。方法:采用半定量RT-PCR方法,检测输卵管妊娠蜕膜组织中的VEGF、KDR、Flt-1 mRNA的表达,并与正常输卵管黏膜及正常宫内早孕子宫蜕膜组织比较。结果:半定量结果表明,VEGF、KDR、Flt-1 mRNA在输卵管妊娠蜕膜组织中的表达强于正常输卵管组织,但弱于正常宫内早孕蜕膜组织,差异均有显著性。结论:输卵管妊娠时,VEGF及其受体mRNA水平的增高是使滞留在输卵管的胚泡着床于输卵管的重要因素。     

Intra-uterine growth restriction downregulates the hepatic toll like receptor-4 expression and function

Maternal starvation is a significant cause of intrauterine growth restriction (IUGR) in the world and increases the risk of infection in the neonate. We examined the effect of maternal starvation on Toll like receptor (TLR)4 expression in hepatic, splenic and intestinal tissues obtained from the adult IUGR offspring of prenatal calorie restricted rats. The hepatic TLR4 protein concentration was undetectable in the IUGR rats that had restricted milk intake during the suckling period (SM/SP; n = 4. p < 0.05) as compared to the normal growth controls (CM/CP; n = 4), and access to ad lib milk intake during the sucking period partially corrected the hepatic TLR4 expression (SM/CP; n = 4). IUGR had no effect on the splenic (n = 4) or intestinal (n = 4) TLR4 mRNA levels. In the liver, IUGR led to a 20% increase in baseline tumor necrosis factor (TNF)-alpha mRNA expression (p < 0.03) and a 70% increase in interleukin-1beta (IL-1beta) mRNA expression (p < 0.008) as compared to the control rats (CM/CP; n = 7). LPS-induced hepatic TNF-alpha release was significantly higher in SM/SP as compared to CM/CP. We propose that IUGR dysregulates TLR4 expression and function in the offspring, which may help explain the increased risk of Gram-negative sepsis and inflammatory diseases in this population.     

Hypoxia and cobalt stimulate vascular endothelial growth factor receptor gene expression in rats

 This study aimed to examine the influence of acute tissue hypo-oxygenation on the expression of the vascular endothelial growth factor (VEGF) receptor genes. To this end male Sprague-Dawley rats were exposed to different hypoxic conditions such as 10% or 8% oxygen, 0.1% carbon monoxide and cobalt chloride (60 mg/kg) for 6 h and the abundance of flt-1, flt-4 and flk-1 mRNA in lungs and livers was determined by RNase protection assay. The relative proportions of flt-1, flt-4 and flk-1 were 10 : 2.5 : 1 and 10 : 10 : 2 in normoxic lungs and livers, respectively. It was found that 8% but not 10% oxygen increased flt-1 mRNA two- to threefold in both organs, whilst flt-4 and flk-1 mRNA were not changed by acute inspiratory hypoxia. Carbon monoxide inhalation also increased flt-1 mRNA but not flt-4 or flk-1 mRNA in both organs. Subcutaneous cobalt administration increased flt-1 mRNA in the livers only, whilst flt-4 and flk-1 mRNA remained unchanged. These findings show that acute tissue hypo-oxygenation is a rather selective stimulus for flt-1 gene expression. The efficiency of the different manoeuvres applied to stimulate flt-1 gene expression is rather similar to the stimulation of erythropoietin gene expression. It is not unreasonable to assume, therefore, that the oxygen-dependent regulation of both genes at the cellular level has significant similarities.     

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.     

VEGF-B expression in human primary breast cancers is associated with lymph node metastasis but not angiogenesis

Angiogenesis is essential for tumour growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently VEGF-B, a new VEGF family member that binds to the tyrosine kinase receptor flt-1, has been identified. Although the importance of VEGF has been shown in many human tumour types, the contribution of VEGF-B to tumour neovascularization is unknown in any tumour type. This study therefore measured the mRNA level of VEGF-B and its receptor flt-1 by ribonuclease protection assay and the pattern of VEGF-B expression by immunohistochemistry in 13 normal breast samples and 68 invasive breast cancers. Flt-1 expression was significantly higher in tumours than in normal breast (p=0.02) but no significant difference was seen in VEGF-B between normal and neoplastic breast (p=0.3). There was a significant association between VEGF-B and node status (p=0.02) and the number of involved nodes (p=0.01), but not with age (p=0.7), size (p=0.6), oestrogen receptor (ER) (p=0.2), grade (p=0.5) or vascular invasion (p=0.16). No significant relationship was present between VEGF-B and flt-1 (p=0.2) or tumour vascularity (p=0.4). VEGF-B was expressed mostly in the cytoplasm of tumour cells, although occasional stromal components including fibroblasts and endothelial cells were also positive. No difference in VEGF-B expression was observed adjacent to regions of necrosis, in keeping with this VEGF family member not being hypoxically regulated. These findings suggest that VEGF-B may contribute to tumour progression by a non-angiogenic mechanism, possibly by increasing plasminogen activators and hence metastasis, as has been described in vitro. Measurement of VEGF-B together with other angiogenic factors may identify a poor prognostic patient group, which may benefit from anti-VEGF receptor therapy targeted to flt-1 (VEGFR1) as well as kdr (VEGFR2).     

Ontogeny of Hepatocyte Growth Factor (HGF) and Its Receptor (c-met) in Human Placenta : Reduced HGF Expression in Intrauterine Growth Restriction

Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.     

Altered cell kinetics in cultured placental villous explants in pregnancies complicated by pre-eclampsia and intrauterine growth restriction

Placentae in pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are characterized by morphological variations, apoptosis, and syncytial shedding, features that are linked to inappropriate oxygen and inflammatory cytokines. Cell turnover within the placenta is dynamic. In this study, these cellular events have been investigated longitudinally using a placental explant model. Intrinsic variations between normal (n = 14), PE (n = 16) and IUGR pregnancies (n = 11), and their responses to oxygen (3% and 17%) and exogenous tumour necrosis alpha (TNFalpha), were recorded. Placental explants were assessed for apoptotic morphology, immunolocalization of MIB-1 (a proliferation marker), lactate dehydrogenase (a necrosis marker), and human chorionic gonadotrophin (hCG, a marker of cytotrophoblast differentiation). Explants under TNFalpha and 17% O2 revealed progressive degeneration of syncytiotrophoblast (ST) followed by restoration of hCG, localized to newly differentiated cytotrophoblasts. This differentiation was significantly enhanced in PE and IUGR. Responses to 3% O2 were similar between groups: a sharp decline in hCG and failure to recover thereafter. Exaggerated cell death was recorded in PE and IUGR explants exposed to TNFalpha and 3% O2. All significant changes in apoptosis were confined to ST and stromal compartments. Enhanced cell death was predominantly apoptotic in PE and necrotic in IUGR. 3% O2 promoted cell proliferation in normal placentae but this response was not reciprocated in PE and IUGR. Elevated hCG in PE and IUGR explants may represent a placental predisposition to differentiation in vivo. In addition, the increased susceptibility of villous components to cell death, in the absence of stimulated proliferation, may provide a powerful mechanism for aberrant or adaptive placental cell turnover in utero.     

Expression of a flt-4 (VEGFR3) splicing variant in primary human prostate tumors. VEGF D and flt-4t(Delta773-1081) overexpression is diagnostic for sentinel lymph node metastasis

Utilizing a cDNA expression library established from human prostate PC-3ML tumor cells, we have cloned a truncated flt-4 gene, termed flt-4t(Delta773-1081). We have then utilized RNase protection and ELISA to measure the relative levels of VEGF B, C, D and flt-1, KDR, flt-4 and flt-4t(Delta773-1081) expression in freshly isolated benign prostatic hyperplasia or BPH tissue (n=21), primary prostate cancers (n=82) and matching sentinel lymph node metastases from stage T2a-T2b/T3 tumors (n=52). Comparisons of the primary tumors with BPH showed that there was a significant upregulation of VEGF-B (P=0.003), VEGF D (P=0.005), flt-1 (P=0.003), KDR (P=0.002), flt-4 (P=0.007), and flt-4t(Delta773-1081) (P=0.001), but not VEGF-C (P=0.543). There was no correlation between VEGF-B and its receptor flt-1 (P=0.545), or VEGF-C and flt-4 (P=0.16) and KDR (P=0.23) receptor expression in tumor specimens. Conversely, there was no significant relationship between VEGF-D and the flt-4t(Delta773-1081) receptor (P=0.516) expression. Statistical analysis further showed that there was no significant correlation between VEGF-B, VEGF-C, VEGF-D, flt-1, KDR, flt-4 and flt-4t(Delta773-1081) with patient age (P>0.10), stage (P>0.10), PSA value (P>0.15) or tumor size (P>0.15). Likewise, there was no significant correlation between VEGF-B, VEGF-C, flt-1, KDR, and flt-4 with Gleason score (P>0.15). In comparison, flt-4t(Delta773-1081) levels clearly increased significantly in Gleason score 7 and Gleason score 8-10 tumors as well as in stage T2a-T2b/T3 tumors. The studies were extended to compare gene expression profiles in T2a-T2b and T3 tumors with (n=26) and without (n=26) matching sentinel lymph node metastases. The data showed that VEGF D and flt-4t(Delta773-1081) expression levels were significantly elevated in primary tumors with sentinel lymph node involvement compared to those lacking lymph node involvement (P>0.0022 and 0.006, respectively). These data suggest that targeting VEGF D and flt-4t(Delta773-1081) receptors may be particularly effective in the prevention of lymph node metastases.     

Renal Angiotensin receptor type 1 and 2 upregulation in intrauterine growth restriction of newborn piglets

Epidemiological and experimental studies suggest that intrauterine growth restriction (IUGR) is associated with abnormalities in kidney development which is thought to be linked with alterations causing adult cardiovascular diseases. The renin-angiotensin system (RAS) plays an important role in the development of renal vascular and tubular structures, and is known to be altered by experimentally induced IUGR. These experimental models of IGUR have been criticized because they may have a more severe impact on intrauterine development than that which is normally encountered in humans. Therefore, we asked whether naturally occurring small-for-gestational-age newborn piglets exhibit features of altered RAS activity. We investigated the regional renal expression of angiotensin II type 1 (AT1) and AT2 receptors in normal-weight and IUGR piglets. The AT1 receptor mRNA expression was markedly enhanced in IUGR piglets, in the renal cortex by 64% and in the renal medulla by 52% (p < 0.05, compared with normal littermates). In contrast, mRNA expression for the AT2 receptor was similar in both the normal-weight and IUGR piglets. A significantly higher AT1 receptor protein expression was found in the IUGR piglets (p < 0.05) in the glomeruli, in the proximal and distal tubules, as well as in the collecting ducts by immunohistochemistry. Furthermore, AT2 receptor protein expression was significantly higher in the IUGR piglets (p < 0.05) in the subcapsular nephrogenic zone and in the distal tubules and collecting ducts. Thus, IUGR is accompanied by an upregulation of angiotensin II receptor expression in the kidneys of newborn piglets. This may indicate an alteration of the RAS in newborns suffering from naturally occurring IUGR.     

The role of estrogen receptors,erbB receptors,vascular endothelial growth factor and its receptors,and vascular endothelial growth inhibitor in the development of the rat mammary gland

We identified the localization and distribution of cell-specific epidermal growth factor receptors (EGFRs: erbB-1, erbB-2, erbB-3, erbB-4), vascular endothelial growth factor (VEGF), VEGF receptors [VEGFRs: VEGF-R1 (flt-1), VEGF-R2 (flk-1/KDR), VEGF-R3 (flt-4)], vascular endothelial growth inhibitor (VEGI), and estrogen receptor (ER), and determined whether or not these growth factors in rat mammary glands are functional. Thirty-five adult female Spraque-Dawley rats were randomly divided into five groups, each of which were at the 7th, 14th, and 21st day of pregnancy; 7th day post-delivery; and 7th day after weaning. It was determined that erbB, VEGF and its receptors, VEGI, and ER stained at different intensities. Intense staining was observed, in particular, in erbB receptors during pregnancy and involution, and also in VEGF and its receptors during lactation, while ER stained during the last periods of pregnancy and lactation. In conclusion, the expression of erbB, VEGF and its receptors, and ER were determined at varying intensities at different sites of the mammary gland during pregnancy, lactation, and involution periods.