ABSTRACT
OBJECTIVE: To determine the expression of VEGF in the placental tissue from pregnancies complicated by hypertension disorders of different clinical severity. DESIGN: Prospective cohort study. SETTING: Polyclinic of Careggi, University of Florence, Italy. SAMPLE: Placentas from women with gestational hypertension (n= 20), pre-eclampsia (n= 20) and pre-eclampsia with HELLP syndrome (n= 20) and from normotensive women (n= 20), as control group (gestational age comprised between 35 and 38 weeks). METHODS: An immunohistochemical technique and a quantitative analysis to measure mRNA levels (RT-PCR) were employed. MAIN OUTCOME MEASURES: Intensity of immunoreactivity and mRNA levels in the placental components. Differences between the data. RESULTS: VEGF immunoreactivity was observable in all the placental components in the gestational hypertension cases as in the control ones. In the cases with pre-eclampsia and pre-eclampsia with HELLP syndrome, some placental components were not immunoreactive. However, the VEGF positive components of all the pathological groups showed a higher intensity of reactivity with respect to that of the control group. The levels of VEGF mRNA were higher in the gestational hypertension cases and lower in the cases of pre-eclampsia with HELLP syndrome with respect to the control ones; in the cases of pre-eclampsia, the levels were the same as the control ones. CONCLUSION: The different expression of VEGF in the placenta of the pathological cases is probably related to haemodynamic changes that take place in these disorders, in order to attempt restoration of a normal uteroplacental flow.
Subject(s)
Hypertension/metabolism , Placenta/metabolism , Pregnancy Complications, Cardiovascular/metabolism , Vascular Endothelial Growth Factor A/metabolism , Adult , Cohort Studies , Female , HELLP Syndrome/metabolism , Humans , Pre-Eclampsia/metabolism , Pregnancy , Prospective Studies , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
Since PSA is supposed to play an active role in the progression of prostate cancer, we applied a quantitative RT-PCR to measure the absolute levels of prostate-specific antigen (PSA) mRNA expression in benign and malignant prostatic tissue. Consecutive fine needle prostate biopsy material from 59 patients (43 with prostate adenocarcinoma and 16 with benign prostatic hyperptrophy; BPH) was used for the measurement of PSA mRNA expression. In addition, we evaluated the correlation between PSA synthesis and PSA circulating levels in the same patients. The relationship between PSA mRNA expression and histological grade was also evaluated. PSA mRNA was measured with a quantitative RT-PCR, based on the use of fluorogenic probes, according to the TaqMan reaction system. The mRNA expression for PSA in prostate adenocarcinoma biopsies was highly variable, ranging from 2 x 10(4) to 2.1 x 10(8) molecules/microg total RNA with a mean value of 2.5 x 10(7) and significantly higher (p = 0.006) than that found in BPH patients (mean: 1.3 x 10(6) and range: 6.9 x 10(2) to 8 x 10(6)). The mRNA PSA expression in needle biopsy material did not seem to be related to PSA circulating levels in prostate cancer patients (r = 0.281), whereas in BPH patients the two parameters correlated significantly (r = 0.667, p < 0.01). A reduction of PSA mRNA expression in samples with a lower grade of differentiation (Gleason score 9-10) was also observed. Even though a mean increase of PSA expression was demonstrated in cancer samples, this small difference does not confirm a significant role of PSA proteolytic activity in prostate cancer progression. In conclusion, the assay procedure we proposed represents a reliable basis for more extensive study of PSA physiopathology in prostate cancer.
Subject(s)
Adenocarcinoma/genetics , Prostate-Specific Antigen/genetics , Prostatic Hyperplasia/genetics , Prostatic Neoplasms/genetics , RNA, Messenger/analysis , Adenocarcinoma/blood , Aged , Aged, 80 and over , Biopsy, Needle , DNA Primers/chemistry , Humans , Male , Middle Aged , Predictive Value of Tests , Prostatic Hyperplasia/blood , Prostatic Neoplasms/blood , RNA, Neoplasm/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and SpecificityABSTRACT
Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.
