Study Of The Pathogenetic Mechanisms Underlying Pituitary Adenoma Apoplexy

PartⅠImmunohistochemical studies of human pituitary adenoma tissues on the pathogenetic mechanisms underlying pituitary adenoma apoplexyBackground and Objectives:Pituitary apoplexy is a potentially life-threatening syndrome resulting from hemorrhage and/or infarction of a pre-existing pituitary adenoma. Recently, accumulating evidence has suggested that many factors play pivotal physiopathological roles in various hemorrhagic events, such as VEGF, MMPs, cytokines, apoptosis, thrombus and vasospasm. Whether these factors abovementioned are involved in the pathogenesis of pituitary adenoma apoplexy has not been clarified. In this study, using immunohistochemical and western blot method, we explored the possible pathogenetic role of above factors in pituitary adenoma tissues with and without apoplexy. Since hemorrhagic incite has been reported to have impact on the expression levels of some abrove proteins, we also determined the effect of blood injection on expression levels of these proteins in xenograft tumors in nude mice in order to eliminate potential fake positive results.Methods:A series of 60 surgically removed pituitary adenomas were obtained from patients undergoing transsphenoidal surgery. Among these tissues, there were 30 pituitary apoplexy adenomas and 30 pituitary adenomas without apoplexy. The VEGF, MMP9, HIF-1α, PCNA, TNFα, IL-6, IL-10, TF protein expression levels and apoptosis rate were examined in these pituitary adenoma tissues via immunohistochemistry, Western blot and Tunel staining. Moreover, we injected whole blood (500μL) or normal saline into subcutaneous xenograft tumors of MMQ cells, respectively. Two days after injection, the mice were sacrificed and tumors were collected. Western blot was used to study the expression levels of VEGF, MMP9, HIF-1α, PCNA, TNFα, IL-6, IL-10 and TF proteins in xenograft tumors with and without blood injection.Results:Immunohistochemistry and Western blot showed that expression levels of VEGF, MMP9, HIF-1α, PCNA, TNFα, and TF proteins in hemorrhagic pituitary adenomas were higher than non-hemorrhagic ones. The expression levels of IL-6 and IL-10 in pituitary adenomas with and without apoplexy displayed no significant difference. Pituitary adenomas with apoplexy display elevated apoptosis. Furthermore, blood injection increased the expression levels of MMP9 and TF proteins. However, the increase rate of MMP9 level in xenograft tumors in response to blood injection was much lower than that in pituitary adenomas in response to apoplexy. Moreover, the blood injection and the blood incite displayed similar increase rates of TF level in xenograft tumors and human pituitary adenomas.Conclusions:Pituitary apoplexy adenomas displays higher expression levels of VEGF, MMP9, HIF-1α, PCNA, TNFαproteins and apoptosis levels compared to the non apoplexy pituitary adenomas. These factors may be involved in the development of pituitary adenoma apoplexy. The increased TF levels in apoplectic pituitary adenomas may be an effect rather than a cause of hemorrhagic incite. PartⅡTNF-αpromotes hemorrhagic transformation in pituitary adenomas Background and Objectives:The results of partⅠhad shown that TNF-α, VEGF and MMP-9 may play a role in the development of intratumoral hemorrhage in pituitary adenomas. Since TNF-αinduces VEGF and MMP-9 expression, recent papers have reported that TNF-αis a critical cytokine mediating various hemorrhagic events, but whether it correlate with the development of pituitary apoplexy. Here we employed MMQ pituitary adenoma cell xenografts to determine the involvement of TNF-αsignaling in intratumoral hemorrhage. We also carried out in vitro experiment exploring the mechanism underlying the role of TNF-αin hemorrhagic transformation in pituitary adenomas.Methods:Mice were implanted with MMQ cells. After two weeks, mice were treated with saline, rhTNF-α, rhTNF-αplus bevacizumab, rhTNF-αplus GM6001, rhTNF-αplus bevacizumab and GM6001. Five mice in each group. rhTNF-α(10000 U/kg i.m.), bevacizumab (10 mg/kg i.p.) and GM6001 (10 mg/kg i.p.) were implemented twice per week. Both bevacizumab and GM6001 were administrated one week before and during TNF-αtreatment. Tumors were harvested after one week rhTNF-αtreatment. The hemorrhagic transformation, VEGF and MMP9 protein expression in xenografts was evaluated via HE and Western blot. Cells were treated with TNF-α(20 ng/ml) or pretreated with bevacizumab (5 mg/ml) for 72 h and an additional 24 h with TNF-α(20 ng/ml), and then VEGF and MMP-9 expression were determined via Western blot analysis, real-time RT-PCR and zymography.Results:TNF-a (5/5) administration caused hemorrhagic transformation. Blockers of VEGF (2/5) or MMP-9 (3/5), either alone or in combination (1/5) attenuated but not abrogated TNF-a mediated hemorrhagic transformation in xenografts. TNF-αenhanced VEGF and MMP-9 expression in MMQ pituitary adenoma cell xenografts in mice. Either Bevacizumab or GM6001 significantly inhibited TNF-a mediated VEGF or MMP9 up-regulation, respectively. TNF-αinduced higher protein and mRNA levels of VEGF and MMP-9 expression. Bevacizumab attenuated TNF-αmediated VEGF expression but had no effect on TNF-αmediated MMP-9 expression.Conclusions:TNF-a may play a role in the development of intratumoral hemorrhage in pituitary adenomas via up-regulation of VEGF and MMP-9. PartⅢHypoxia induces hemorrhagic transformation in pituitary adenomas Background and Objectives:The results of partⅠhad shown that apoplectic pituitary adenomas displayed higher protein expression levels of HIF-1α, VEGF, PCNA, BNIP3 and apoptosis rate than non apoplectic ones, indicating that these above factors may be involved in the development of intratumoral hemorrhage in pituitary adenomas. Recent papers have shown that, under tumor hypoxia, HIF-1αmay promote the development of various hemorrhagic events via HIF-1α-VEGF and HIF-1α-BNIP3 signaling pathways. The biological role of HIF-1αin the hemorrhagic transformation of pituitary adenomas remains unknown. Here we used apoplectic and non-apoplectic pituitary adenomas to determine the involvement of HIF-1αsignaling in intratumoral hemorrhage. We employed HIF-1αoverexpression/knockdown strategies to examine the association between HIF-1αsignaling and hemorrhagic presentation in vitro and in vivo.Methods:We established stable MMQ cell line overexpressing HIF-1α(MMQ/HIF-1α) and stable MMQ cell line transfected with pCDNA3 vector (MMQ/C) to serve as control. Western blot analysis, real-time RT-PCR and flow cytometry was performed to address whether increased level of HIF-1αcould modulate apoptosis rate and its target genes VEGF and BNIP3 in pituitary adenoma cells. We knockdown HIF-1αin the MMQ cells using siRNA and measured apoptosis levels, VEGF and BNIP3 expression. We implanted the nude mice subcutaneously with MMQ/HIF-1αcells, MMQ/C cells and MMQ cells, respectively. After four weeks, tumors were harvested. The hemorrhagic transformation, apoptosis rate, VEGF and BNIP3 protein expression in xenografts was evaluated via HE staining, Western blot analysis, real-time RT-PCR and TUNEL staining.Results:HIF-1αoverexpression activated its downstream genes, VEGF and BNIP3, in MMQ cells and this upregulation was attenuated by HIF-1 siRNA. In vivo studies of MMQ cell xenografts in nude mice showed that HIF-1αoverexpression significantly promoted hemorrhagic transformation and induced apoptosis, VEGF and BNIP3 expression.Conclusions:Our study indicates that tumor hypoxia, following rapid tumor growth, may promote hemorrhagic transformation in pituitary adenomas. HIF-1α-VEGF and HIF-1α-BNIP3 apoptosis pathways may be involved in hemorrhagic transformation of MMQ cell xenografts.