Mice that did not show a durable response to castration were used as a source of prostate tumor tissue representing androgenindependent disease.Approximately onehalf of each prostate specimen was used for histological analysis and subsequent pathological grading according to a previously described scheme. Remaining tissues were stored at C and used for protein analysis.B, lowgrade PIN in TRAMP demonstrates presence of intraductal vasculature.F, quantification of IMVD during tumor progression.Confirmation of the sequence and insert orientation was determined by sequence analysis. Before hybridization, paraffin sections were hydrated in xylene and graded alcohol and equilibrated into PBS.Slides were treated with proteinase K and were allowed to hybridize with RNA probes at C overnight in hybridization solution.Emulsion was developed after days, antisense and sense of each probe were developed after equal time of emulsion, and slides were counterstained with HE.Slides were then dehydrated through graded alcohol into xylene and mounted under glass coverslips.To elucidate the sequence of molecular events intricate with angiogenesis and the initiation and progression prostate cancer, we first examined the temporal and spatial pattern of PECAMCD expression as a marker of vascularization in cohorts of nontransgenic and TRAMP mice.In contrast, we observed two distinct and different types of vascularization in the PIN lesions of TRAMP mice.In lowgrade PIN, the vasculature was predominantly interductal; however, vasculature was observed to become increasingly arching the edge of the duct and progressively intraductal within epithelial clusters concomitant with the appearance of the highgrade PIN lesions. In the moderately and poorly differentiated TRAMP tumors, the vasculature was observed to be scattered within the tumor, with the most obvious blood vessels appearing in the most poorly differentiated tumors. These observations demonstrate that an early angiogenic event temporally Vitamin B6 correlates with the transition between low and highgrade PIN.A, expression of VEGF isoform was restricted to poorly differentiated and androgenindependent tumors was determined by immunoassay analysis.Furthermore, the increase in IMVD was found to be a function of progression.The poorly differentiated tumors had a significant increase in IMVD when compared with lowgrade PIN, highgrade PIN, and welldifferentiated tumors. In addition, the androgenindependent tumors derived from castrated mice demonstrated a significant increase in IMVD compared with all of the other stages, including the poorly differentiated tumors isolated from intact mice. To further characterize changes in the VEGF axis at the molecular level during progression of prostate cancer, in situ hybridization analysis was performed, in which the VEGF riboprobe was designed across the first exons to detect all known isoforms of VEGF.In contrast, VEGFB mRNA was readily detectable in all of the samples examined. Transcripts encoding VEGFC were detected in only of poorly differentiated tumors. However, these isoforms were not detected in the extracts Enalaprilat Dihydrate prepared from dorsolateral or ventral prostate of adult mice nor in the samples prepared from PIN lesions or from welldifferentiated or moderately differentiated tumors.In contrast, VEGF was readily detectable in samples prepared from the poorly differentiated tumors of intact and castrated mice.VEGFR mRNA was observed to be expressed only in the poorly differentiated tumor; inset, large vessel that expresses VEGFR.