Differences in local environment determine the site of physiological angiogenesis in rat skeletal muscle.

The specificity in location of angiogenesis to either glycolytic or oxidative fibre types, or muscle regions, was examined in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of rat. Angiogenesis was induced by mechanical means either with (chronic muscle stimulation) or without (muscle stretch by overload) changes in blood flow, treatments which invoked only minor changes in fibre type and fibre size. Proliferation estimated by PCNA labelling of cells co-localised with capillaries was very rare in control muscles, where it occurred mainly in the glycolytic regions, but was increased in both models of angiogenesis. However, when labelled capillaries were scored according to the type of surrounding fibres, only muscle stimulation significantly accentuated proliferation of capillaries surrounded by glycolytic fibres. We conclude that while mechanical stimuli are important for proliferation in glycolytic regions in both models, capillary growth occurs specifically around glycolytic fibres in that region when the angiogenic stimulus includes increased blood flow and/or increased metabolic demand.

Expression of thyroid receptor isoforms in the human fetal central nervous system and the effects of intrauterine growth restriction.

BACKGROUND: Congenital hypothyroidism is known to be associated with mental retardation which, if recognized promptly, is largely prevented by thyroid hormone replacement. Intrauterine growth restriction (IUGR) is a major cause of perinatal mortality and morbidity, and is also associated with neurodevelopmental delay. Fetuses with IUGR have reduced circulating concentrations of free thyroxine (T4) and free triiodothyronine (T3), leading to the hypothesis that a reduction in the tissue effects of thyroid hormones in the central nervous system (CNS) may contribute to neurodevelopmental morbidity. Since thyroid hormone effects are mediated through binding to specific nuclear thyroid hormone receptors (TRs), we have defined the pattern of TR isoform expression in the CNS throughout normal human development and have compared TR expression in the CNS of normal fetuses with those affected by IUGR. METHODS: Samples of normal human fetal brain from first and second trimesters were obtained at surgical termination of pregnancy. Appropriately grown and third trimester fetuses affected by Intrauterine growth restriction (IUGR) were also investigated after unexplained stillbirth at post mortem examination. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine the expression of TR isoform mRNAs in frozen cerebral cortex from 10 to 16 weeks gestation. TR protein expression in human fetal brains (both cerebral hemispheres and cerebellum) was also examined in formalin fixed sections and expression of TR alpha1, alpha2, beta1 and beta2 isoforms being defined using semiquantitative immunocytochemistry. RESULTS: RT-PCR revealed the presence of mRNAs encoding TR alpha1, beta1 and beta2 isoforms and the nonfunctional TRalpha2 variant in the fetal cerebral cortex from week 10 of human pregnancy. Immunostaining of the fetal brain revealed TR alpha1 and alpha2 protein from week 11 of human gestation. Expression of all TR isoform proteins was largely confined to the pyramidal neurones of the cerebral cortex and the Purkinje cells of the cerebellum with increasing receptor expression evident with gestational age. Semiquantitative observer scoring showed that by the second trimester, there was a marked increase in the proportion of pyramidal and Purkinje cells expressing TR isoforms, while by the third trimester, all these cells immunostained. Comparison of TR immunostaining in the cerebral cortex and cerebellum from IUGR fetuses (n = 18) matched for gestational age to normal fetuses revealed a lower intensity of expression of all the TR isoforms confirmed by observer scoring and quantification using TR protein immunofluoresence (P<0.01). CONCLUSIONS: Our findings indicate both pre- and post-translational expression of TR alpha and beta isoforms in the cerebral cortex of first trimester fetuses. These findings support the view that the transplacental passage of thyroid hormone in early gestation may be critical to neurological development. Our finding that in severe IUGR the expression of TR isoforms in the human fetal cerebral cortex and cerebellum was significantly reduced, in association with reduced circulating thyroid hormone concentrations indicate that changes in free ligand and receptors may affect CNS development. These findings should prompt further investigation of the potential therapeutic role of peripartum thyroid hormone treatment.

Circulating thyroid hormone concentrations and placental thyroid hormone receptor expression in normal human pregnancy and pregnancy complicated by intrauterine growth restriction (IUGR).

Thyroid hormones are critical to growth and development of the human fetus. Abnormal placental development, a major cause of intrauterine growth restriction (IUGR), is associated with a high perinatal mortality and morbidity. Thyroid status has been postulated to play a role in the pathogenesis of such morbidity. In the present study, we have investigated fetal thyroid function and placental expression of thyroid hormone receptor (TR) alpha and beta variants during normal human pregnancy and in pregnancy associated with IUGR. Measurement of free thyroid hormones and TSH concentrations revealed significant rises in free T4 and free T3 between the second and third trimesters of normal pregnancy. Serum concentrations of free T4 and free T3 were lower in fetuses affected by IUGR, although serum TSH levels were not significantly different. Immunocytochemistry demonstrated the presence of TR alpha1, alpha2, and beta1 proteins within the nuclei of trophoblast and stromal placental cells. Immunostaining for these TR variants increased with increasing gestation in normal placenta. Comparison of IUGR placental samples with normal samples revealed greater immunostaining for TR alpha1, alpha2, and beta1 variants in IUGR. Examination of pretranslational expression of TR alpha1, alpha2, beta1, and beta2 variants by semiquantitative RT-PCR revealed increasing expression of TR alpha1, alpha2, and beta2 messenger RNAs with increasing gestation in normal pregnancy, which "mirrored" post-translational expression. However, and in contrast, there were no significant differences in expression of TR messenger RNAs in normal and IUGR placenta. The present findings of reduction in serum free thyroid hormones and increased expression of TR alpha and beta proteins in association with IUGR highlight the potential importance of thyroid status in influencing long-term fetal outcome in this condition.

Retinoid X receptor expression in the normal pituitary and clinically 'non-functioning' pituitary tumours.

OBJECTIVE: The glycoprotein hormone common alpha-subunit is frequently expressed in clinically 'non-functioning' tumours (NFTs) of the anterior pituitary, despite normal levels of T3 and gonadal steroids. This observation suggests abnormal negative-feedback regulation of the alpha-subunit by T3 and gonadal steroids in NFTs. We have previously documented reduced expression of thyroid hormone receptor (TR) variants in NFTs compared to normals and proposed that this observation may, in part, explain the defective negative regulation. Due to the important role of retinoid X receptors (RXRs) in transactivating TR-mediated transcriptional regulation, via heterodimer formation, we hypothesize that aberrant RXR isoform expression in NFTs may contribute to the defective negative regulation of the alpha-subunit by T3. DESIGN: Comparison of RXR isoform protein and mRNA expression in NFTs and normal pituitaries. PATIENTS AND TUMOURS: Twenty clinically non-functioning pituitary tumours and 27 normal pituitaries were obtained for analysis. MEASUREMENTS: Immunocytochemistry and semiquantitative RT-PCR was performed on tumours and normal pituitaries to determine the relative levels of expression of RXR isoform proteins and mRNAs, respectively. RESULTS: RXR alpha was expressed in a similar proportion (approximately 50%) of both normal human pituitaries and NFTs, while RXR beta and gamma were each observed in 26% of normals but were undetectable in NFTs. The application of semiquantitative RT-PCR revealed similar levels of mRNAs encoding the RXR alpha and RXR beta isoforms in normals and NFTs but significantly reduced expression of RXR gamma mRNA was observed in NFTs. CONCLUSIONS: We propose that abnormal RXR isoform expression in clinically 'non-functioning' pituitary tumours may contribute to abnormal T3-mediated negative regulation of alpha-subunit production.

Immunohistochemical localization of type 1 11beta-hydroxysteroid dehydrogenase in human tissues.

Two isozymes of 11beta-hydroxysteroid dehydrogenase (11betaHSD) catalyze the interconversion of hormonally active cortisol to inactive cortisone. Activity and messenger ribonucleic acid studies indicate that type 1 11betaHSD (11betaHSD1) is expressed in glucocorticoid target tissues such as liver, gonad, and cerebellum, where it regulates the exposure of cortisol to glucocorticoid receptors. To further understand the role of 11betaHSD1 in human tissues, we have studied the localization of this isozyme using an antibody raised in sheep against amino acids 19-33 of human 11betaHSD1. Western blot analyses indicated that the immunopurified antibody recognized a band of approximately 34 kDa in human liver and decidua. Immunoperoxidase studies on liver, adrenal, ovary, decidua, and adipose tissue indicated positive cytoplasmic staining for 11betaHSD1. 11BetaHSD1 immunoreactivity was observed more intensely around the hepatic central vein, with no staining around the portal vein, hepatic artery, or bile ducts. No staining for 11betaHSD1 was observed in the adrenal medulla, but 11betaHSD1-immunoreactive protein was observed in all three zones of the adrenal cortex, with the most intense staining in the zona reticularis > zona glomerulosa > zona fasciculata. In the human ovary, immunoreactivity was observed in the developing oocyte and the luteinized granulosa cells of the corpus luteum. No staining was observed in granulosa cells, thecal cells, or ovarian stroma, which contrasted with the marked expression of 11betaHSD2 in the granulosa cell layer. Sections of human decidua showed high expression of 11betaHSD1 in decidual cells. In omental adipose tissue, 11betaHSD1 immunoreactivity was observed in both stromal and adipocyte cells. Immunohistochemical localization of 11betaHSD1 in human liver, adrenal, ovary, decidua, and adipose tissue using this novel antiserum provides us with a tool to investigate the role of this isozyme in modulating glucocorticoid hormone action within these tissues.

Fibroblast growth factors 1 and 2 and fibroblast growth factor receptor 1 are elevated in thyroid hyperplasia.

We have previously reported increased expression of fibroblast growth factor (FGF-1 and FGF-2) in benign and malignant human thyroid neoplasia. To determine the role of these factors in thyroid hyperplasia we have examined their expression in multinodular goiter and compared findings with those in normal thyroid tissue. Because the effects of FGF-1 and FGF-2 are predominantly mediated through the FGF receptor-1 (FGFR-1), its expression has also been examined. Immunocytochemistry was performed on sections from multinodular goiters (n = 18) and normal thyroid (n = 7). Cytoplasmic staining for FGF-1, FGF-2, and FGFR-1 was scored on a scale of 0 (no staining) to 3 (heavy staining) and expressed as a percentage of total cells stained. Confocal microscopy of immunofluorescent staining for FGF-1, FGF-2, and FGFR-1 in sections of multinodular goiter (n = 3) and normal thyroid (n = 3) provided quantitation of immunostaining. FGF-1 expression was significantly increased in multinodular goiter when compared with normal. A mean of 74% of follicular cells in multinodular goiter compared with 9% of follicular cells in normal thyroid expressed FGF-1 (P < 0.0001). When expression of FGF-2 was examined, 77% of the follicular cells in multinodular goiter compared with 5% in normal thyroids were immunopositive (P < 0.0001). Confocal microscopy revealed that the intensity was 160 times greater in follicular cells in sections of multinodular goiters when compared with normal. When expression of FGFR-1 was analyzed, 89% of the follicular cells in multinodular goiter stained positively, compared with 15% of follicular cells in sections of normal thyroid. Confocal microscopy revealed a 6-fold increase in intensity of FGFR-1 expression in follicular cells of multinodular goiter (P < 0.05). In addition, there was significant nuclear expression of FGFR-1 in multinodular goiter contrasting with negligible expression in normal thyroid. These data show that enhanced expression of FGF-1, FGF-2, and FGFR-1 accompany thyroid hyperplasia and are not exclusively associated with the neoplastic state. These factors may be involved in the pathogenesis of uncontrolled thyroid growth observed in these conditions.

An abnormality of thyroid hormone receptor expression may explain abnormal thyrotropin production in thyrotropin-secreting pituitary tumors.

Thyrotropin (TSH)-secreting pituitary adenomas cause hyperthyroxinemia in the presence of "inappropriately" elevated concentrations of TSH. TSH production under these circumstances escapes the normal negative feedback effect of thyroid hormone. We propose that this defective negative feedback is mediated by an abnormality of thyroid hormone receptor (TR) expression. Two TSH-secreting pituitary adenomas were analyzed by immunocytochemistry for TR isoform protein expression and by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) for TR isoform mRNA expression. The results obtained from these tumors were compared with the findings from six normal human pituitaries. Neither tumor examined expressed detectable levels of nuclear TRalpha or TRbeta proteins, in contrast to the normal pituitaries studied, which expressed all TR isoforms. Application of RT-PCR, however, revealed mRNAs encoding each TR isoform in all tumorous and normal tissues examined. Semiquantitative RT-PCR revealed similar levels of expression of TRalpha and TRbeta isoform mRNAs in tumors and normal tissue, in contrast to the observed difference in TR proteins. Absent TRalpha and TRbeta protein expression, in association with normal mRNA levels, implies a post-transcriptional defect in TR mRNA processing in TSH-secreting adenomas. Reduced TR expression in these tumors may explain defective negative feedback of thyroid hormone on TSH production, and may also contribute to uncontrolled tumor growth.

Thyroid hormone and estrogen receptor expression in normal pituitary and nonfunctioning tumors of the anterior pituitary.

Nonfunctioning tumors (NFTs) of the anterior pituitary often express elevated levels of the glycoprotein hormone alpha-subunit, which, under normal physiological conditions, is under negative feedback control by thyroid and gonadal steroid hormones. We postulate that inappropriately elevated levels of expression of alpha-subunit in the face of normal levels of these target organ hormones may reflect an abnormality of thyroid hormone receptors (TRs) and/or gonadal steroid receptors in NFTs. Using immunocytochemistry and Western blotting we have examined TR and estrogen receptor (ER) protein expression in normal human anterior pituitary glands and NFTs. Pretranslational expression of these receptors was examined using semiquantitative reverse transcriptase-PCR. Expression of all TR variant and ER proteins was reduced in pituitary tumors compared with that in normal pituitaries. The expression of messenger ribonucleic acids encoding the TR beta1 and TR beta2 isoforms and ER was also significantly reduced in tumors compared with normal tissues, although there was no difference between tumors and normals in the level of expression of TR alpha1 and alpha2 messenger ribonucleic acids. We suggest that reduced expression of TRs and ER may account for inappropriate expression of the glycoprotein hormone alpha-subunit gene in some NFTs and may contribute to uncontrolled tumor growth.