p27kip1 overexpression regulates VEGF expression, cell proliferation and apoptosis in cell culture from eutopic endometrium of women with endometriosis

Abstract

We hypothesized that p27(kip1) overexpression can regulate endometriosis cell proliferation, apoptosis and vascular endothelial growth factor (VEGF) expression in the endometrium. The overexpression of p27(kip1) was obtained by transduction of p27(kip1) in primary cultures of endometrium obtained from women with endometriosis tissue with gene therapy technology. First generation bicistronic adenovirus: AdCMVhp27IRESEGFP (Adp27) and AdCMVNull (AdNull) were engineered in order to induce p27(kip1) expression in endometrial cells primary culture. The effect of p27(kip1) overexpression was elucidated through the cell proliferation evaluation and the expression of the cell cycle-related proteins p16, p21, p27, and p53. Cell cycle and apoptosis in endometrial cells from women with and without endometriosis were also evaluated. The VEGF levels were evaluated 1 and 7 days after transduction. The experiments were performed using Immunofluorescence stainings and flow cytometry technique. The cell proliferation statistically diminished markedly following p27(kip1) overexpression in the endometriosis group. This process was accompanied, however, by a statistically significant modulation of the cell cycle-related proteins p16, p21, p27 and p53 markedly increase following p27(kip1) overexpression in the endometriosis group (p < 0.001) and an increase in apoptotic cells was observed. In the endometriosis group, significant downregulation of VEGF expression was observed 7 days after p27(kip1) overexpression, attaining levels strikingly similar to those observed in the control endometrial cells. The findings of this study showed a link between the cell cycle control protein (p27(kip1)) and angiogenesis (VEGF). Our results, also reinforces the background of endometrial dysfunction as part of the origin of endometriosis. We believe that better knowledge of endometrium milieu and the establishment of the link between different, previously describe, altered pathways in this tissue can facilitate future genetic cell therapy.

Inflammatory cytokine profile of co‑cultivated primary cells from the endometrium of women with and without endometriosis

“…In accordance to previous research ( 18 , 19 , 21 ,18), the findings of the present study suggest that the soluble factors secreted by endometriotic cells may have an important role in the disruption of the cell cycle and in the establishment of the peritoneal environment of women with endometriosis. The endometriotic cell pool may be responsible for the establishment of an inflammatory peritoneal environment favorable to the initiation and progression of endometrial cell adhesion and clustering.…”

“…For example, despite the presence of healthy endometrial cells in retrograde menstruation, the presence of endometriotic cells may be decisive in the secretory profile of the cell pool ( 18 , 21 , 37 ). The endometrial cells regurgitated into peritoneal cavity may be responsible for the induction of the aberrant cytokine profile observed in the peritoneal fluid and eutopic endometrium of women with endometriosis ( 18 , 19 , 21 ,18). The variations observed in the CD146 + population support the putative role of eMSCs in the pathogenesis of endometriosis.…”

“…Interleukin (IL)-1β has a proliferative effect on endometriotic cells that does not occur in healthy endometrial cells ( 13 ). IL-1β stimulates the production of IL-6 and IL-8 in endometriotic cell cultures, which further induce proliferation ( 14 , 15 ) and reduce the apoptotic rate ( 16 – 19 ). Additionally, IL-1β increases the shedding of intercellular adhesion molecule (ICAM)-1 from peritoneal mesothelial cells, indicating a role in the neovascularization mediated by IL-6 and vascular endothelial growth factor (VEGF) ( 20 ).…”

TGF-β1/FGF-2 signaling mediates the 15-HETE-induced differentiation of adventitial fibroblasts into myofibroblasts

“…p27 kip1 is a kind of CDK inhibitor different from the p53 or p16. p27 kip1 negatively regulates the cell cycle through the inhibition of cyclin enzyme activity, preventing the cells’ G1/S phase transition, and contributes to depressing the cell proliferation [ 25 ]. Cyclin E, a regulation actor of CDK2 subunits, mediates the cell cycle G1/S transition.…”

Endometriosis: current challenges in modeling a multifactorial disease of unknown etiology

“…Cell-culture has helped our understanding of gene and protein expression [114], the roles of hormones in endometriosis [115] and also the possibility to recreate early stages of endometriosis in a "Petri-dish" [116]. Three-dimensional models offer the opportunity to study aspects of endometrial stromal and epithelial cells communication and paracrine cross-talk between stromal and epithelial cells [117].…”