Stage-dependent expression of an angiogenic agent and vascular organization in experimental skin tumor development

Increased angiogenesis and expression of antibodies to vascular endothelial growth factor (VEGF), an angiogenic agent, have been shown in the tumor development of many tissues. Areas of skin expressing VEGF and total volume of vessels expressing laminin in the wall were measured in chemical carcinogen-exposed mice using CAS-200 morphometry apparatus having a sensitivity exceeding 99% and reproducibility exceeding 99%. The area of VEGF expression was increased in carcinogen-exposed skin, dysplasia and in well-differentiated squamous cell carcinomas, but decreased in squamous cell carcinomas with decreased degree of differentiation. The vessel volume increased prior to the formation of tumors in carcinogen-exposed skin as well as in highly malignant neoplasms. In well-differentiated squamous cell carcinomas with an expansive growth pattern, the vessels were parallel to the basal membrane, in moderately differentiated tumors the vessels were in the direction of tumor invasion, and in poorly differentiated tumors, active angiogenesis consisted of numerous, enlarged vessels within the tumor. This study showed increased VEGF expression and number of vessels occurring in early stages of skin tumor development, pointing to a role of angiogenesis in chemical risk assessment and in cancer prevention. Altered vessel structure and vessel arrangement were distinct in later stages of tumor growth and in malignant neoplasms, pointing to the utility of detailed vessel analysis in neoplasm characterization.     

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.     

Immunohistochemical localization of syndecan-1 in normal and pathological human uterine cervix

Expression of syndecan-1, a cell surface proteoglycan that binds growth factors and extracellular matrix components, was studied in normal and pathological human uterine cervix using immunohistochemical methods. Normal cervical squamous epithelium showed positive staining for syndecan-1 in all cell layers, except the basal cell layer, whereas endocervical columnar epithelium stained weakly. In non-neoplastic reactive lesions, metaplastic squamous cells were positive for syndecan-1, whereas columnar cells showed weak or negative staining. In cervical condylomas, cells showing koilocytotic atypia were positive for syndecan-1. The progression of cervical intraepithelial neoplasia (CIN) grade I to grade III was associated with reduced syndecan-1 expression and localization of syndecan-1 to more superficial cell layers. In squamous cell carcinomas (SCCs), syndecan-1 expression correlated with histological differentiation, being absent from most poorly differentiated tumours. The results suggest that loss of syndecan-1 from atypical cells is an early event during cervical carcinogenesis and show a close association of syndecan-1 expression with preserved epithelial morphology and differentiation.     

Non-linearity of neoplastic conversion induced in rat liver by low exposures to diethylnitrosamine

Neoplastic conversion induced in rat liver by diethylnitrosamine(DEN) was quantified by measuring preneoplastic and neoplasticlesions over a 34 week period in the beginning of which thecarcinogen was given at three dose levels and two dose ratesfor the first 10 weeks, after which animals were maintainedfor 24 weeks with either no further exposure or were fed phenobarbital(PB) to promote neoplastic development of cells converted byDEN. DEN was injected s.c. inmale F344 rats at weekly or biweeklyintervals for total doses of 1, 2 or 4 mmol/kg body wt and thenthe rats were maintained on basal diet alone or diet containing0.05% PB. At the end of exposure, DEN had produced a dose-relateddecrease in centrilobular glutamine synthetase-expressing (GS⁺)hepatocytes which is indicative of mild cytotoxicity. All dosesinduced foci that were     

Plasma renin substrate and oestrogens in normal pregnancy

Relationship between concentrations of serum oestrogens, plasma renin substrate and plasma renin activity were studied in six women throughout pregnancy. There was a significant positive correlation between serum oestradiol-17β and plasma renin substrate concentrations (r=0.60). Serum oestriol concentrations also correlated significantly with plasma renin substrate concentrations (r=0.68). Correlation coefficients calculated separately for each subject throughout pregnancy were higher than those for the whole group. Also, there was much individual variation in dose-response of serum oestrogens to plasma renin substrate concentrations. There was no significant correlation between serum oestrogens and plasma renin activity.

Our results support the view that oestrogens cause the increase in plasma renin substrate concentration during pregnancy, and emphasize the individual variation in response of renin substrate concentration to serum level of oestrogens.     

Activated mast cells increase the level of endothelin-1 mRNA in cocultured endothelial cells and degrade the secreted Peptide

Subendothelial mast cells have been implicated in the pathogenesis of allergic inflammation, in atherosclerosis, and in the regulation of vascular tone. Because endothelin-1 (ET-1) is an important regulator of vascular tone and has also been implicated in the pathogenesis of atherosclerosis, we studied the role of mast cells in the metabolism of endothelial cell-derived ET-1. In mast cell-endothelial cell cocultures, activation of the mast cells with ensuing degranulation was accompanied by the increased expression of ET-1 mRNA in the endothelial cells, yet the immunoreactive ET-1 protein in the coculture medium disappeared almost completely during the 24-hour coculture. Activation of the mast cells with the ensuing degranulation resulted in proteolytic degradation of ET-1 by the 2 neutral proteases, chymase and carboxypeptidase A, of the exocytosed mast cell granules. With synthetic ET-1 and purified mast cell granule enzymes, efficient degradation of ET-1 by chymase and carboxypeptidase A was verified. These in vitro results imply a novel role for mast cell-derived neutral proteases in ET-1 metabolism and suggest that activated subendothelial mast cells are important local regulators of ET-1 metabolism.     

Basement membrane structures in tumors of the ovary

The location and amount of the basement membrane (BM) components collagen IV and laminin were studied in ovarian epithelial, sex cord-stromal and germ cell tumors. BM structures were found in the epithelial stromal interface of benign surface epithelial tumors and, though discontinuous, around well-differentiated tumor islets, being less well developed in invasive undifferentiated neoplasms. The stromal components in Müllerian mixed tumors had less distinct BM structures, a finding useful for the classification of these neoplasms. Thecomas and fibromas had scanty collagen IV and laminin; granulosa cell tumors contained large amounts of BM material. A fine diffuse BM-positive pattern occurred in dysgerminomas and endodermal sinus tumors; BM structures in cystic teratomas were distinct. Collagen IV and laminin were well-developed in benign and slow-growing tumors with epithelial components and in their metastases, but less distinct in stromal tumors and highly malignant undifferentiated tumors, showing the usefulness of this method for the clinical and biological classification of such tumors.     

Automated image analysis of proliferating cells in carcinoma of the larynx

OBJECTIVE: To determine the role of cell proliferation neoplastic progression in the larynx and possibly derive criteria of clinical significance using automated quantitative image analysis. MATERIAL AND METHODS: In a retrospective study involving archival material, the occurrence and location, size, shape and staining intensity of proliferating cell nuclear antigen (PCNA)-positive cells (12,538 cells in total) were analyzed in squamous cell carcinoma (SCC), as well as in pre- and non-neoplastic conditions, using computer-assisted morphometry with reproducibility and sensitivity exceeding 99%. RESULTS: Immunohistochemically detectable PCNA-positive cells were located in the basal layer in non-neoplastic states, in well-differentiated SCCs in layers adjacent to the basal membrane and in poorly differentiated neoplasms in the neoplastic epithelial islets. An increased degree of dysplasia was associated with an increased number of PCNA-immunoreactive cells of increased nuclear size and staining intensity. There was a significant difference between carcinomas and dysplasia in terms of altered nuclear shape. With increasing malignancy of SCCs, nuclear shape alterations and PCNA staining intensity increased, whilst nuclear size decreased. CONCLUSIONS: Automated image analysis of cell populations allowed the identification of populations of malignant cells and provided information on the severity of preneoplastic and neoplastic conditions of use in studies of tumor behavior and with potential clinical application.     

Hypertension,cardiac state,and the role of volume overload during peritoneal dialysis

The cardiac state and the prevalence of high blood pressure (BP) were analyzed in 21 pediatric patients (mean age 5.3±5.3 years) on chronic peritoneal dialysis (CPD), the aim being to specify the impact of hypervolemia in the etiology of hypertension. C- and N-terminal atrial natriuretic peptide (ANP-C, ANP-N) were measured as possible additional markers of hypervolemia. Baseline investigations were carried out 0.2 years after initiation of PD, and repeated after 0.9±0.2 years. Fifty-two percent of the patients had high BP, and in 40% the nocturnal BP decline was decreased. Left ventricular hypertrophy was present in 45%, but the systolic and diastolic functions of the heart were not impaired. Left ventricular mass correlated significantly with the severity of hypertension and with ANP-N (r=0.79, P<0.01 and r=0.66, P<0.01, Spearman rank correlation). Significant correlations were also found between the severity of hypertension and ANP-N and ANP-C (r=0.82, P<0.01 and r=0.66, P<0.01, Spearman rank correlation). High BP and cardiac impairment were more frequent in the younger and nephrectomized patients in whom volume overload seemed to be the most-important etiological factor. Our results suggest further that an ANP-N over 3.0 nmol/l combined with hypertension is strongly indicative of volume overload in patients on PD. Received: 31 May 2000 / Revised: 22 December 2000 / Accepted: 22 December 2000