Impact of bazedoxifene on endothelial health markers

Resumen

Atherosclerosis and its most significant clinical repercussion, cardiovascular disease (CVD), is the leading cause of morbidity and mortality in postmenopausal women, with a substantial global public health impact as a consequence. Vascular endothelial dysfunction, which is potentiated by decreasing estrogen levels in the menopausal transition period, is an early hallmark of developing CVD. The action of estrogens has been widely investigated in many critical areas of atherogenesis, due to the presence of estrogen receptors in the vascular system. Estrogens can have both positive and negative effects on the vascular wall depending on the stage of atherosclerosis disease. The estrogen response is mediated by different mechanisms, involving either genomic pathways requiring ligand-activated transcription with posterior modulation of protein synthesis, or non-genomic pathways, mainly affecting enzyme activity and rapid signaling cascades. The vigorous debate surrounding the risk/benefit profile of estrogen in the development and timing of CVD in postmenopausal women is still ongoing. Evidence that the progestin component of hormone therapy increases breast cancer risk or even attenuates the potential beneficial effects of estrogens has been paralleled by a decline in the use of hormone therapy in postmenopausal women in recent years. This data, along with problems associated with long-term use of bisphosphonates, has shifted the focus of recent research to other molecules capable of activating estrogen receptors, leading to transcriptional activity in some tissues but not in others. Selective estrogen receptor modulators (SERMs) and more recently, tissue selective estrogen complex (TSEC) have been developed as alternatives to hormonal replacement therapy (HRT). Previous experimental studies have shown the capacity of estrogens and second-generation SERM raloxifene to promote endothelial cell regrowth and improve cell survival rate in the early stages of atherosclerosis disease. Clinical trials involving bazedoxifene or conjugated estrogens plus bazedoxifene have shown cardiovascular safety in healthy postmenopausal women. However, one research group working with an atherosclerotic animal model in monkeys demonstrated that the therapeutic application of bazedoxifene had no effect on atherosclerosis. What is more, bazedoxifene completely impeded the antiatherosclerotic effects of estrogen treatment in the mentioned study. To date, no report has been published of bazedoxifene’s effect on vascular cell proliferation and the mechanisms involved using an in vitro model. This study was designed to evaluate the effect of bazedoxifene on the proliferation of human umbilical arterial endothelial cells (HUAEC) compared with estradiol, raloxifene and the combination of bazedoxifene plus estradiol. Additionally, we assessed whether the process was associated with changes in the protein and gene expression of key cell cycle regulators such as cyclin A, cyclin B, cyclin D1 and p27Kip1 in both fresh and cryopreserved endothelial cells. HUAEC were obtained from umbilical cords from healthy women at the time of delivery in a Hospital Clinico of Valencia maternity ward. Umbilical cords were processed under specific conditions in the laboratory of the Pediatrics, Obstetrics, and Gynecology Department in the Faculty of Medicine. Primary culture and subcultures were carried out until sufficient endothelial cells were obtained for the study. Cells were synchronized at the G0 and G1 cell cycle phases, and active treatments were applied. Cell proliferation was measured using the Colorimetric Cell-Proliferation Kit II (XTT). Western blot experiments measured changes in protein expression and quantitative real-time polymerase chain reaction tested for gene expression alterations. At increasing doses bazedoxifene alone showed moderately enhanced increased cell proliferation, a similar increase to estradiol and raloxifene. No significant results on proliferation were found after concurrent cell treatment with bazedoxifene plus estradiol. Immunoblotting assays detected an isolated increase in cyclin A and p27Kip1 expression after treatment with estradiol, even though no significant changes in gene expression were associated with bazedoxifene, estradiol, raloxifene, or bazedoxifene plus estradiol for any cyclins. Analysis of the previous cryopreserved status of endothelial cells revealed positive changes in the protein content of almost all cyclins compared to the fresh endothelial cells. In contrast, no significant trend was observed in their RNA profile. From a broader perspective, the results of this study show for the first time the capacity of bazedoxifene to promote endothelial cell proliferation in an in vitro model. This beneficial effect has been corroborated in the literature for its predecessors estradiol and raloxifene, as is also confirmed in our work. The bazedoxifene-induced improvement in cell proliferation may facilitate endothelial regeneration and repair and thus contribute to the recovery of vascular functions during the early stages of atherosclerosis development. This observation turns the spotlight on bazedoxifene as a potential therapeutic target in CVD prevention when administered to women during the initial postmenopausal period. Although our results were inconclusive for the gene and protein expression of cell cycle key regulators such as cyclin A, cyclin B, cyclin D1 and p27Kip1 associated with bazedoxifene treatment, further experimental studies are nonetheless warranted. These should include the full panel of key regulators in cell cycle machinery to confirm whether other regulatory proteins play a role in the proliferative process.