Research On The Effect And Mechanism Of Dihydrotestosterone And Estradiol On Endothelial Cell Proliferation

Objective:1. To examine the direct effect of dihydrotestosterone (DHT) on cell proliferation and cell migration in different source of endothelial cells.2. To explore the mechanism of DHT actions on endothelial cell proliferation.3. To investigate the differential modulation of DHT action by estradiol.Method:1. Human arotic endothelial cells (HAECs) and murine microvascular endothelial cells (MECs) were treated with different doses of DHT for24hr and48hr, respectively. The number of viable cells was determined by MTS assay. To analyse the effect of AR on DHT-induced HAECs cell proliferation, HAECs were treated with DHT, AR siRNA or Casodex alone or in combination for48hr, MTS was used to test determine viable cells number.2. TCMs collected from LAPC-4or LNCaP cells at24,48, and72hr of incubation were administrated to MECs at various concentrations and the number of viable MECs was determined at24or48hr of treatment. LAPC-4or LNCaP cells were treated with vehicle control or various doses of DHT for48hr, then TCMs were collected to treat MECs for48hr, MTS assay was used to test the viable cell number. To analyse the role of VEGF/VEGFR pathway in DHT-induced MECs cell proliferation, RT-PCR and MDS assay was used to test VEGF mRNA in cells and VEGF-A concentration in TCMs. Furthermore, MECs were treated with corresponding TCM and SU5416alone or in combination at doses indicated for48hours, and the viable cell number was determined.3. Cell culture study:HAECs and MECs were treated with DHT,17a-estradiol or17β-estradiol alone or in combination at doses indicated for each experiment, the number of viable cells was determined by using MTS assay. To assess whether17a-estradiol and17β-estradiol can attenuate DHT-enhanced MEC cell growth through a paracrine mechanism by acting on tumor cells, MECs cells were treated with TCMs from LAPC-4cells treated with DHT,17a-estradiol or17β-estradiol alone or in combination for48hr, endothelial cell number was determined by MTS assay.Animal study:Xenograft animal models with LAPC-4or LNCaP prostatic tumor were prepared, monitored and treated with a placebo,17a-estradiol or17β-estradiol pellet for4weeks, At the end of the4weeks, animals were euthanized and tumor tissues were collected, immunohistochemical analysis was applied to test CD31expression in tissues.Result:1. To explore the effect of DHT on cell proliferation in different source of endothelial cells, HAECs and MECs were treated with DHT at doses ranging from1nM to50nM in different time period. DHT produced a time-and dose-dependent cell proliferation in HAECs. While no significant effect was observed in MECs. To explore the effect of DHT on cell migration in HAECs, HAECs were pre-treated with DHT10nM, the migration cell number was increased (p<0.01)To determine the role of AR in DHT-induced HAECs cell proliferation, AR siRNA and casodex was administrated to silence AR gene expression and block the combination of DHT and AR, respectively. The results showed that viable cell number was decreased significantly when treated with casodex (1μM～10μM) in combination with DHT10nM(p<0.01), or treated with DHT10nmol/L after siRNA transfection, the viable cell number was significantly decreased in cells transfected with AR siRNA (P<0.01), while no effect was observed in cells transfected with non-specific siRNA.2. Tumor cell conditioned media (TCM) collected from LAPC-4or LNCaP cells produced a time-and concentration-dependent induction of cell growth in MECs. This TCM-induced cell growth in MECs was enhanced by the treatment of tumor cells with DHT. VEGF concentration in TCM collected at24hours and48hours was12.43ng/ml and28ng/ml, respectively. No significant difference was observed in the VEGF concentration between DHT-TCM and control-TCM. Both the TCM-stimulation and DHT-enhancement effects in MECs were completely blocked by SU5416, a specific VEGF receptor antagonist.3. In the model of DHT-induced HAECs cell proliferation, co-administration of17β-estradiol with DHT partly attenuated DHT-induced HAECs cell proliferation, but no effect was observed in17a-estradiol. Furthermore, co-administration of17a-estradiol or17P-estradiol with DHT in prostatic cells completely inhibited the DHT-enhancement effect, while treatment with DHT,17a-estradiol or17β-estradiol did not produce any significant direct effect in MECs. Moreover, administration of17a-estradiol or17β-estradiol in xenograft animals with LAPC-4or LNCaP tumor significantly decreased the micro vessel number in the tissues.Conclusion:1. Androgen produced different effect on cell proliferaion in different source of endothelial cells.2. Androgen promotes endothelial cell proliferation via androgen receptor or microenvironment. VEGFR pathway in the miroenvironment is an important pathway involving in DHT-induced endothelial cell proliferation.3. The androgen-induced endothelial cell growth is modulated by estrogen differentially.17β-estradiol, but not17a-estradiol, can inhibit the effect of DHT-induced HAECs cell proliferation. Both17a-estradiol and17β-estradiol is able to modulate DHT-induced MECs cell proliferation via tumor cells.