Identification of vascular surface proteins by in vivo biotinylation: a method sufficiently sensitive to detect changes in rat liver 2 weeks after partial hepatectomy.

We have developed a methodology to selectively isolate and identify proteins associated with the luminal surface of blood vessels using in vivo biotinylation, streptavidin-affinity chromatography, and SDS-PAGE/LC-MS/MS. This had sufficient sensitivity to identify 32 proteins with changed expression in rat livers at 2 weeks or 5 weeks after partial hepatectomy, well after the 7 day tissue remodeling period. This method could be adapted to study other angiogenic tissues including tumors.

Enhancement of cisplatin efficacy by thalidomide in a 9L rat gliosarcoma model.

With the aim of improving the treatment of glioblastoma multiforme, we investigated the potential of thalidomide to enhance the effectiveness of cisplatin chemotherapy in a rat glioma model. Female F344 rats were implanted with 9L gliosarcoma tumors either intracranially or subcutaneously and treated with 1 mg/kg cisplatin injected i.p. or with 1% thalidomide in the food or with these treatments combined. Cisplatin in combination with thalidomide significantly reduced both the subcutaneous tumor volume at 30 days to 22 +/- 5% (mean +/- SEM, P < 0.001) and the intracranial tumor volume at 18 days to 44 +/- 15% (P < 0.05) of that with cisplatin alone. Thalidomide selectively increased the cisplatin concentration 10-fold in intracranial tumors (P < 0.05) and 2-fold in the subcutaneous tumors (P < 0.05) without increasing its concentration in major organs including brain and kidney. Cisplatin combined with thalidomide caused a significant decrease in vascular endothelial growth factor (VEGF) levels by 73% in intracranial tumors (P < 0.05) and by 50% in subcutaneous tumors (P < 0.05) and caused the level of active hepatic growth factor (a-HGF) to double in both the subcutaneous and intracranial tumors (P < 0.05), suggesting this treatment altered the vasculature in these tumors. We conclude the increased efficacy of cisplatin in the presence of thalidomide was due to the selective increase in cisplatin concentration within the tumors and speculate that this is the result of thalidomide or the cisplatin/thalidomide combination, selectively altering the tumor vasculature. Based on the selective effects of thalidomide on tumor cisplatin concentrations and the resulting increase in efficacy, thalidomide may also increase the efficacy of other drugs that are presently considered ineffective against glioma.

Sertoli and germ cell development in hypogonadal (hpg) mice expressing transgenic follicle-stimulating hormone alone or in combination with testosterone.

We recently created a novel transgenic (tg) model to examine the specific gonadal actions of FSH, distinct from LH effects, by expressing tg-FSH in gonadotropin-deficient hypogonadal (hpg) mice. Using this unique in vivo paradigm, we now describe the postnatal cellular development in seminiferous tubules selectively stimulated by tg-FSH alone or combined with testosterone (T). In the alphabeta.6 line, tg-FSH stimulated the maturation and proliferation ( approximately 2-fold) of Sertoli cells in hpg testes. Total Sertoli cell numbers were also significantly increased (1.5-fold) independently of FSH effects by T treatment alone. Selective FSH activity in alphabeta.6 hpg testes increased total spermatogonia numbers 3-fold, which established a normal spermatogonia/Sertoli cell ratio. FSH also elevated meiotic spermatocyte numbers 7-fold, notably at pachytene (28-fold), but induced only limited numbers of postmeiotic haploid cells (absent in hpg controls) that arrested during spermatid elongation. In contrast, T treatment alone had little effect on postnatal spermatogonial proliferation but greatly enhanced meiotic progression with total spermatocytes increased 12-fold (pachytene 53-fold) relative to hpg testes, and total spermatid numbers 11-fold higher than tg-FSH hpg testes. Combining tg-FSH and T treatment had no further effect on Sertoli or spermatogonia numbers relative to FSH alone but had marked additive and synergistic effects on meiotic cells, particularly pachytene (107-fold more than hpg), to establish normal meiotic germ cell/Sertoli cell ratios. Furthermore, tg-FSH had a striking synergistic effect with T treatment on total spermatid numbers (19-fold higher than FSH alone), although spermatid to Sertoli cell ratios were not fully restored to normal, indicating elevated Sertoli cell numbers alone are insufficient to establish a maximal postmeiotic germ cell capacity. This unique model has allowed a detailed dissection of FSH in vivo activity alone or with T and provided compelling evidence that FSH effects on spermatogenesis are primarily via Sertoli and spermatogonial proliferation and the stimulation of meiotic and postmeiotic germ cell development in synergy with and dependent on T actions.

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice.

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.