Detection of endothelin immunoreactivity and mRNA in pulmonary tumours

Paraffin sections of 66 surgically resected lung tumours were immunostained with antisera to human endothelin-1 and to the C-terminal peptide of big endothelin. With both antisera, strong immunoreactivity was demonstrated in 11 of 15 squamous cell carcinomas and 11 of 16 adenocarcinomas. Focal immunoreactivity was seen in small cell carcinoma (2/12), large cell carcinoma (2/5), and carcinoid tumours (2/11). Four lymphomas and three sarcomas did not show endothelin immunoreactivity. Cryostat sections of 22 of the 66 tumours were hybridized with radiolabelled complementary RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization demonstrated the presence of endothelin mRNAs in 4 of 7 squamous cell carcinomas and in 5 of 8 adenocarcinomas, in a pattern similar to that shown by immunocytochemistry. No hybridization signals were obtained from the other types of tumours. In lung tissue adjacent to the tumours, endothelin-like immunoreactivity and mRNA were detected in pulmonary endocrine cells and, in some cases, other epithelial cells, and in alveolar capillary endothelial cells. This study demonstrates the expression of endothelin in a number of pulmonary tumours and suggests a possible role for this peptide in the growth and/or differentiation of these tumours.     

Chromogranin A and B messenger ribonucleic acids in pituitary and other normal and neoplastic human endocrine tissues

The distribution of the messenger ribonucleic acids (mRNAs) for chromogranin A and B was analyzed by in situ hybridization in normal and neoplastic endocrine tissues using frozen and paraffin tissue sections. Combined in situ hybridization and immunochemical staining was also done on tissue sections from the same cases using a monoclonal antibody against chromogranin A (LK2H10). Most endocrine tumors expressed chromogranin A and B mRNAs as well as chromogranin A protein. Normal pituitary expressed chromogranin A and B mRNAs and chromogranin A protein in the anterior pituitary gland. Most of these cells were gonadotropic hormone-producing cells. Prolactinomas (5/5) did not express chromogranin A mRNA or protein, but contained chromogranin B mRNA. Null cell or nonfunctional adenomas (8/8) expressed chromogranin A and B mRNAs and reacted with antibody LK2H10. In some tumors such as Merkel cell carcinomas, insulinomas, and parathyroid adenomas, a stronger signal for chromogranin A mRNA was detected than for the immunoreactive proteins. These results indicate that in situ hybridization complements immunochemical techniques in the analysis of endocrine cells and neoplasms. The gene products for chromogranin A and B are widely distributed in many endocrine cells and tumors, but some neoplasms such as prolactinomas have a differential distribution of chromogranin A and B mRNA and proteins.     

Type I and III collagen protein precursors and mRNA in the developing human lung

Extracellular matrix proteins have a prominent role in both ontogenesis and fibrogenesis in the human lung. The aim of this study was to analyse the expression of newly formed precursor proteins and mRNA of collagen types I and III in developing human lung tissues from 12 to 40 weeks of gestation, and also in neonatal disorders such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Lung tissues were obtained at autopsy from 60 non-malformed cases. All tissues were analysed by immunohistochemistry and 24 were also investigated by mRNA in situ hybridization. The precursor proteins and mRNA of both collagens were expressed in abundance in pulmonary arteries and veins during all developmental periods. In RDS and BPD, precursor proteins and mRNAs of both collagen types were increased within alveolar walls. The cells in these locations showed alpha-smooth muscle actin, vimentin, and variable desmin immunoreactivity. Collagen I and III precursor proteins and mRNA were also observed in pleura, bronchi, bronchioles, and around chondrocytes during all developmental periods and in diseased lung. In conclusion, collagens I and III were expressed in a similar way in and around various cell types in the developing lung and their expression was increased within alveolar walls in RDS and BPD. Myofibroblast-type cells appeared to produce mRNA for both types of collagen in alveoli.     

Nitric oxide synthase immunoreactivity in human bladder carcinoma.

AIMS: To study the expression of the endothelial and inducible isoforms of nitric oxide synthase (eNOS and iNOS, respectively) in human bladder carcinoma and schistosomal bladder disease, and to compare it with normal adult and fetal urothelium. Nitric oxide is thought to play a complex role in human carcinogenesis, but has only recently been investigated in bladder cancer. METHODS: Immunohistochemistry was performed on paraffin wax embedded sections of 33 human bladder carcinomas and five bladder carcinoma cell lines; in addition, seven schistosomal bladder cases and normal and fetal urothelium were investigated. In the cell lines enzymatic activity was examined by the NADPH diaphorase reaction. RESULTS: Immunoreactivity for eNOS was present in most cells of all 31 cases examined. Immunoreactivity for iNOS was less abundant and was seen in 23 of 25 cases. Similar findings were noted in schistosomal bladder cancer. In the normal bladder mucosa, eNOS immunoreactivity was found only in the superficial cell layer and iNOS was not expressed, whereas in the fetal urothelium immunoreactivity for both isoforms was seen in all cell layers. Enzymatic activity and immunoreactivity for eNOS and iNOS were evident in the five bladder carcinoma cell lines. CONCLUSIONS: It is possible that NOS plays a role in the differentiation of the transitional epithelium in fetal life, has a biological function in the adult bladder mucosa, and is involved in bladder carcinogenesis. eNOS and iNOS immunoreactivity do not differ in schistosomal and non-schistosomal bladder carcinoma, but resemble the pattern of expression typical of fetal urothelium.     

Cellular fibronectins are differentially expressed in human fetal and adult kidney

The localization of cellular forms of fibronectin (cFn) was studied in fetal and adult kidneys. We used monoclonal antibodies reacting with the extradomains A and B in cFN (EDAcFn, EDBcFn) as well as with a differentially glycosylated fetal form of the protein (Onc-cFn). In adult human kidney EDAcFn was present in glomerular mesangium and in the walls of larger blood vessels, whereas a polyclonal rabbit fibronectin antiserum widely reacted also with interstitial areas. Immunoreactivity for EDBcFn and Onc-cFn, however, was not found in adult kidney. In the basement membranes and interstitial areas of developing tubules and glomeruli the immunoreactivity for EDAcFn was distinct and detectable in the earlier stages also for EDBcFn. In developing glomeruli, EDAcFn and EDBcFn were detected in teh mesangial areas, but in more mature fetal glomeruli, the mesangial immunoreactivity only persisted for EDAcFn. Both EDAcFn and EDBcFn were found in the basement membranes in the medullary area of all developing kidneys. In fetal kidney, immunoreactivity for EDAcFn and EDBcFn was seen also in small blood vessels, including the capillaries. Immunoreactivity for Onc-cFn was found in mesangial cells of fetal glomeruli as well as in the intima of larger blood vessels but not in the basement membranes. The results show that the three forms of cFn are present in the fetal kidney and have certain differences in their distribution. Conversely, only the EDAcFn was detected in the adult kidney. The different, partially age-related distributions of the three types of cFns suggest that they may also differ in their functions.     

Immunohistochemical localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp

Summary The distribution in oral tissues of endothelin, a multifunctional peptide originally identified within endothelial cells, and subsequently in some epithelial cells, neurons and neuroendocrine cells, has not been investigated yet. We have studied the localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp by immunohistochemical techniques. Such immunoreactivity was detected only within endothelial cells in both mature dental pulp and developing tooth. Arteries and veins of various sizes as well as small thin vessels displayed endothelin-like immunoreactivity. In the tooth germ, the cells of the enamel organ or the precursors of the odontoblasts were found unreactive. In the mature pulp, no cells of the stroma or nerves displayed endothelin-like immunoreactivity. These findings suggest that vascular endothelium may be the only source of endothelin in human dental tissues. It is tentatively proposed that endothelin released in mature tooth pulp may participate in the regulation of the pulpal blood flow. Although the possible role of endothelin in developing tissues is far from being clear, the mitogenic effects and the proto-oncogenes expression induced by endothelin in some cells raise the possibility that this peptide might also play a role during tooth development.     

Cell- and tissue-specific expression of a 34,000-molecular-weight peptide growth factor in humans

A 34,000-dalton peptide growth factor that we originally identified in human placental trophoblasts and in certain carcinomas was shown to be expressed in several normal human tissues. A highly specific antibody to the trophoblast-derived growth factor was used in an immunoperoxidase staining technique to identify the immunoreactive peptide in tissue sections. Immunoreactivity was seen in the adrenal cortex, Leydig cells of the testes, and follicular cells of the thyroid. In addition, strong staining was seen in the ducts and terminal ductules of the pancreas, in glandular epithelium of the prostate, in the chief cells of the stomach, and in the columnar epithelium of the trachea and bronchus of the lung. Certain tissues were negative for the peptide, including the adrenal medulla, liver, esophagus, small intestine, colon, bladder, lymph node, spleen, bone marrow, and thymus. Thus, the expression of the peptide depends on cell lineage and the state of differentiation; tissues of hematopoietic lineage are devoid of the 34,000-dalton peptide, whereas some of the major hormone-secreting tissues that are under pituitary control show the highest immunoreactivity.     

Aromatase in human endometrial carcinoma and hyperplasia. Immunohistochemical, in situ hybridization, and biochemical studies.

The expression of P450 aromatase and other steroidogenic enzymes were evaluated in 42 endometrioid endometrial carcinomas, 23 endometrial hyperplasias, and 7 normal endometrial specimens. These findings were correlated with clinicopathological findings to elucidate the possible biological significance of in situ estrogen production in the development of human endometrial carcinoma. Only weak aromatase immunoreactivity was observed in vascular walls and myometrial cells. In contrast, strong aromatase stromal immunoreactivity was observed in 28 of 42 (66.7%) endometrial carcinomas. However, no stromal immunoreactivity was seen in normal or hyperplastic endometrial specimens. Immunoreactivity in the carcinoma stromal cells was significantly increased at sites of invasion. These aromatase-positive cells were immunohistochemically negative for other steroidogenic enzymes involved in estrogen biosynthesis. In situ hybridization studies revealed aromatase mRNA hybridization signals in stromal cells but not in carcinoma cells. The distribution of aromatase mRNA correlated well with the immunohistochemical localization of aromatase enzyme. Quantitation of aromatase activity demonstrated 8.75 +/- 2.75 pmol/hour/mg of protein for endometrial carcinomas (22 specimens) and 0.98 +/- 1.95 pmol/hour/mg of protein for normal endometrial specimens (4 specimens). Aromatase activity was found in both estrogen receptor-positive and -negative endometrial carcinomas. Aromatase did not vary with respect to the menopausal status of patients with endometrial carcinoma. These results suggest that estrogen is produced in situ in endometrial carcinoma but not in benign endometrial lesions. Such locally synthesized estrogen may act on carcinoma cells in a paracrine fashion to promote tumor growth. Additional investigations are necessary, but increased aromatase expression in the stromal cells of endometrial carcinoma may therefore play an important role in the development of human endometrioid endometrial carcinoma.     

hASH1 expression is closely correlated with endocrine phenotype and differentiation extent in pulmonary neuroendocrine tumors.

The human homolog 1 of the Drosophila neurogenic achaete-scute genes, hASH1, is specifically expressed in fetal pulmonary neuroendocrine cells and in some neuroendocrine tumor cell lines. However, no data have been gathered regarding its in vivo expression in tumors. hASH1 mRNA expression was investigated by in situ hybridization in 238 surgically resected lung carcinomas, and the correlations between hASH1 expression status and immunostaining results of neuroendocrine markers chromogranin A, neural cell adhesion molecule, gastrin-releasing peptide and calcitonin, and clinical outcome were analyzed. hASH1 expression was detected in 2/20 (10%) adenocarcinomas, 4/30 (13.3%) typical carcinoids, 11/13 (84.6%) atypical carcinoids, 38/67 (56.7%) large-cell neuroendocrine carcinomas and 56/78 (71.8%) small-cell carcinomas, respectively, but not in any squamous cell carcinoma (0/21) or large-cell carcinoma (0/9). The 2 hASH1+ adenocarcinomas also expressed multiple neuroendocrine markers. Thus, hASH1 expression was restricted to lung cancers with neuroendocrine phenotypes. However, not all neuroendocrine tumors expressed hASH1. Within the entities of large-cell neuroendocrine carcinoma and small-cell carcinoma, hASH1 expression correlated very closely with chromogranin A, gastrin-releasing peptide and calcitonin expression (P<0.0001, r=0.852), but was not related to neural cell adhesion molecule expression (P=0.8892), suggesting that hASH1 expression, at least in lung cancer, is associated with endocrine phenotype expression other than 'neuroendocrine differentiation' in a broad sense. The fact that hASH1 was virtually absent in almost fully differentiated typical carcinoids, but was expressed in most, if not all, less differentiated atypical carcinoids as well as large-cell neuroendocrine carcinomas and small-cell carcinomas, suggests that hASH1 expression in lung cancer imitates its early and transient expression in fetal development, and that hASH1 is instrumental in the establishment, but not in the maintenance, of a cellular endocrine phenotype. Finally, hASH1 expression correlated with a significantly shortened survival in small-cell carcinoma patients (P=0.041).     

Distribution and mRNA expression of tenascin-C in developing human lung

Tenascin-C is an extracellular matrix glycoprotein that is spatially expressed during organogenesis, in inflammatory and fibrotic disorders, and in neoplasms. The aim of this study was to analyze its expression in developing human lung tissues during pseudoglandular, canalicular, saccular, and alveolar periods corresponding to Weeks 12 to 40. Lung tissues were obtained at autopsy from 34 nonmalformed cases. An immunohistochemical analysis and a messenger RNA (mRNA) in situ hybridization method combined with light microscopy were used. The extent of tenascin-C immunoreactivity was scored as absent, low, moderate, or strong in and around different types of pulmonary cells. The immunohistochemical expression for tenascin-C was strong beneath the airway epithelium, especially at the sites of airway subdivision during Weeks 12 to 23, whereas its expression was moderate or weak underneath alveolar and bronchiolar epithelia between Weeks 24 and 40. The expression for tenascin-C was strong in the intima of veins, especially in the canalicular period, i.e., Weeks 17 to 28. A moderate or strong immunoreactivity for tenascin-C was also observed around chondrocytes in every case studied during all periods. The increased expression of tenascin-C mRNA was most often seen in the cells below the airway epithelium. Taken together, tenascin-C is expressed in human lung during all developmental periods, and its expression is especially strong below the airway epithelium at the sites of airway subdivision.     

Gastrin-releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas.

Gastrin-releasing peptide (GRP; mammalian bombesin) is present in the neuroendocrine cells of human fetal lung and in small cell lung carcinomas (SCLCs), where it may act as a growth factor. Considering the potential importance of GRP as a tumor marker, we have conducted a retrospective immunohistochemical analysis of 176 lung tumors for markers of GRP gene expression, as well as several other markers of neuroendocrine cell differentiation: chromogranin A, neuron-specific enolase, and calcitonin. The majority of carcinoids contained mature GRP, in contrast to only a minority of SCLCs and large cell lung carcinomas (LCLCs). However, a majority of SCLCs and LCLCs contained proGRP immunoreactivity. In situ hybridization did not add any information beyond what was obtained using proGRP antisera. In spite of sharing these neuroendocrine cell markers, SCLCs are associated with a graver prognosis than LCLCs. No prognostic significance was associated with immunostaining for GRP or several other markers of neuroendocrine cell differentiation.     

Expression of cathepsins B, H, K, L, and S during human fetal lung development.

Cathepsins are involved in lysosomal protein degradation, proenzyme activation, antigen processing, and hormone maturation. They are secreted by tumor cells and macrophages and catalyze the remodeling of extracellular matrix proteins. To gain insight into the expression pattern of cathepsins during fetal lung development, the expression of cathepsins B, H, K, L, and S at protein and mRNA levels were evaluated by using immunohistochemistry and in situ hybridization. Early expression of cathepsins B, H, and K was found in epithelial cells of the branching presumptive bronchi (<12th week of gestation). The most intense cathepsin K-specific immunoreactivity was found in developing airways with a lumen. Cathepsin K was found in epithelial cells only, whereas in contrast, cathepsins B and H were detected both in epithelial and interstitial cells. During fetal maturation, interstitial cells displayed cathepsin L immunoreactivity and, in the saccular phase (>26th week of gestation), both cathepsin L and S immunoreactivities. A continuous decline in the proportion of cathepsin H-positive interstitial CD68-positive cells was observed. These discrete temporal and spatial variations in cathepsin expression during organogenesis of the human lung indicate different physiological roles for the individual enzymes in different cell types and developmental stages.     

In situ hybridization study of pro-opiomelanocortin (POMC) gene expression in human pituitary corticotrophs and their adenomas

Summary Pro-opiomelanocortin (POMC) mRNA was detected on paraffin sections by in situ hybridization (ISH) in corticotrophs of 12 nontumorous pituitaries, 11 functioning corticotroph, and 11 silent pituitary adenomas. ISH combined with immunocytochemistry for adrenocorticotrophic hormone (ACTH), a POMC-derived peptide, was also performed. ACTH immunoreactive cells of the anterior lobes and those invading the posterior lobe showed a high or moderate level of POMC mRNA that was not correlated with the intensity of ACTH immunoreactivity. Variable levels of POMC gene expression were present in Crooke's cells, corticotrophs suppressed by glucocorticoid excess. Most functioning corticotroph adenomas and silent subtype 1 adenomas had an intense hybridization signal and ACTH immunoreactivity. In silent subtype 2 and 3 adenomas, POMC mRNA had a diffuse low level or was absent; in these adenomas ACTH immunoreactivity was diffuse, restricted to some cells, or negative. The results indicate that POMC gene is expressed in both normal and suppressed nontumorous corticotrophs. Intense signals for POMC mRNA are found in most functioning corticotroph adenomas. The difference between POMC gene expression in silent 1 and silent 2 and 3 adenomas suggests that different mechanisms are responsible for the lack of endocrine activity.     

Insulin-like growth factor-II in endocrine pancreatic tumours. Immunohistochemical, biochemical and in situ hybridization findings

In earlier studies a high-molecular-weight (HMW) insulin-like growth factor-II (IGF-II) peptide was identified in adult human pancreas and localized to the insulin-producing B-cells. This peptide has now been investigated in neoplastic insulin cells. Forty endocrine pancreatic tumours and 17 pancreatic adenocarcinomas of ductal type were included in the study. All cases were investigated with immunohistochemical techniques using antibodies to IGF-II, insulin, pro-insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin and vasoactive intestinal peptide (VIP). Frozen tissue from nine tumours and two normal pancreatic glands was extracted, gel separated, and quantified using radioimmunoassay. The tumours were also investigated by in situ hybridization. IGF-II-immunoreactive cells were found in nearly all the 18 insulin-producing tumours (16/18), in a minority of the other endocrine tumours, but not in pancreatic adenocarcinomas. All extracts from the endocrine tumours showed varying amounts of IGF-II and had different molecular-weight forms. The immunohistochemical and radioimmunoassay findings are both based on immunological binding and were further confirmed by Northern blot and in situ hybridization. These results show that IGF-II is expressed in insulin-producing tumours as well as in pancreatic tumours producing other peptides, in contrast to normal pancreatic islets where IGF-II is found exclusively in insulin-producing cells.     

Big endothelin-1 but not endothelin-1 is present in the smooth muscle stroma of the prostate gland of the rat

Immunohistochemical techniques were employed to localize the presence of endothelins in the mature rat prostate gland. Immunoreactivity for big endothelin‐1 but not endothelin‐1 was observed in the fibromuscular stroma of the rat prostate gland. No immunoreactivity was seen in the glandular epithelium. Double staining procedures showed big endothelin‐1 immunoreactivity to be co‐localized with α‐actin immunoreactivity. The stroma of the prostate gland also contained nerve fibres coursing through it which are immunopositive for tyrosine hydroxylase. These results suggest that big endothelin‐1 but not endothelin‐1 is co‐localized with α‐actin in the smooth muscle cells of the rat prostate gland. This implies that endothelin‐1 is synthesized on demand from big endothelin‐1 in the fibromuscular stroma of the rat prostate.     

Expression of neuropeptides and neuropeptide mRNAs in spinal cord after axotomy in the rat,with special reference to motoneurons and galanin

The extent to which the plasticity in peptide expression observed in developing spinal motoneurons occurs following proximal peripheral axotomy in the adult rat was examined using in situ hybridization and immunohistochemical techniques to visualize the changes. Transient upregulation of galanin, vasoactive intestinal polypeptide (VIP) and substance P messenger ribonucleic acids (mRNAs) was observed within subpopulations of motoneurons ipsilateral to lesion for periods lasting 2–3 weeks after injury. In contrast, the axotomy-induced heterogenous increases in somatostatin and neuropeptide tyrosine mRNA expression in ipsilateral motoneurons remained elevated, or, in the case of somatostatin, continued to increase for the time period studied (1 month). Immunohistochemical analysis agreed with the in situ hybridization results, showing some motoneurons within the injured ventral horn to contain galanin-, VIP-or somatostatin-like immunoreactivity. In some instances, galanin-immunoreactive motoneurons colocalized with calcitonin gene-related peptide immunoreactivity. Most of the neurons expressing the injury-induced peptides appeared large, presumably alpha-motoneurons, but there were also many small neurons expressing galanin in the ventral horn ipsilateral to lesion. This may represent evidence for peptide synthesis in gamma-motoneurons. The only peptide mRNA studied to be downregulated in response to axotomy was enkephalin. The results show that peptide expression in injured motoneurons is dramatically altered, the significance of which remains to be determined.     

Endothelin. Immunohistologic localization in aorta and biosynthesis by cultured human aortic endothelial cells.

BACKGROUND: Endothelin-1 (ET-1) has been shown to exist in many organs and to have various biologic functions including vasoconstriction. However, an exact location of ET gene expression of the tissues is not fully investigated. Human aortic tissue was examined to elucidate the exact location of ET gene expression. EXPERIMENTAL DESIGN: Human aortas were obtained at autopsy and fixed in either conventional 10% formalin or 3% paraformaldehyde. The aortic thin sections were subjected to examinations of an immunohistochemistry and in situ hybridization of ET-1. Human aortic endothelial cells were cultured by a previously reported method. ET-1 released in the supernatant from the cultured endothelial cells was radioimmunoassayed. RESULTS: Immunohistologic study of ET-1 revealed a linear staining of the endothelial monolayer and diffuse staining in the intimal and medial smooth muscle cells on human aorta except for fetal aorta. In situ hybridization signals were intense in the endothelial cells from the elderly as well as younger subjects as examined with 35S-labeled anti-sense probe RNA. Fetal aortic endothelial cells revealed the least signals that meant developing but still immature gene translation. Smooth muscle cells showed positive but weak in situ hybridization signals. Control immunohistologic and hybridization studies were negative. ET-1 biosynthesis by cultured human aortic endothelial cells was invariably low in the subjects under the age of 50, ranging from 0.23 to 0.40 pmol/1 x 10(5) cells for 3 days. On the other hand, endothelial cells from the elderly subjects generally synthesized a greater amount of endothelin in vitro. CONCLUSIONS: These findings indicate that ET-1 is most highly expressed in endothelial cells, although not as highly but certainly, expressed in intimal and medial smooth muscle cells. This fact gives a new insight into the biophysiologic and pathologic roles of ET. In addition, these methods are applicable to investigate the gene expression of ET-1 in all organs and tissues.