Gender, age and oestrogen metabolism

优先领域：基础生物科学支撑健康关键词：LC-MS，肺循环，雌激素代谢摘要：性别对血管健康和健康老龄化有着深远的影响。女性患心肺功能障碍的风险更大，这可能会增加绝经后的风险。然而，很少有研究直接考察性别和年龄可能会导致正常肺血管功能和雌激素代谢的变化，这可能会使女性对血管危险因素产生预感。在这里，我们将探讨肺血管正常功能的性别差异，特别是雌激素、雌激素代谢和雌激素代谢产物的作用和影响。虽然有几篇关于雌激素对血管系统影响的论文和综述，但雌激素代谢产物对正常衰老血管系统的影响研究很少。同样，性别对正常的增殖信号通路的影响在很大程度上还没有得到充分的研究。我们对人肺动脉平滑肌细胞(HPASMCs)的初步数据表明，信号通路存在性别差异&雌激素可能是性别差异的原因。我们最近已经证明雌激素本身可以通过BMPR2途径减少hPASMCs中的信号转导，增加MAPK信号转导，因此雌性hPASMCs的增殖大于雄性细胞。我们已经证明，hPASMCs中microRNA的表达可以受到性别和雌激素的影响。例如，来自女性肺的hPASMC中的microRNA96减少，这导致5-羟色胺通过5-HT1B受体诱导的增殖增加。雌激素代谢产物对正常血管功能的影响可能比雌激素本身更大。这些研究的一个限制是我们实际测量雌激素代谢和血管组织中的代谢物的能力。在过去的两年里，我们开发了一种新的高效液-液-质联用‘类固醇’方法来评估hPASMCs的雌激素代谢。我们现在可以应用这项技术来了解雌激素和雌激素代谢在肺动脉的正常功能和衰老中的作用。1-2年级。学生将帮助开发LC-MS技术来分析hPASMCs和血浆中的雌激素代谢物。我们已经确定了一些积累在PASMCs中的代谢物&具有促增殖或抗增殖作用&首先我们将检查这些代谢物(例如16-OHE1/2，4-OHE1/2，2-OHE1/2，2和4-MeOHE1/2)。随后，将在月经周期的第7天、第14天、第21天和第28天对正常健康志愿者进行血浆测量。类似的分析也将在绝经后女性和年龄匹配的男性的样本中进行。学生还将研究这些样本中关键microRNAs的表达，特别是那些可能与雌激素代谢或作用相互作用的microRNAs(例如，miRNA-22、miRNA-206、miRNA-27b)。二至四年级。HPASMC将来自健康的男性和女性&这些细胞将根据年龄进行分组。这将与尼克·莫雷尔(剑桥)合作。将确定正常衰老和性别对基础和刺激雌激素代谢的影响。雌激素合成和代谢酶对此的影响将通过应用芳香化酶抑制剂(如阿那曲唑)和CYP1B1抑制剂(如TMS)和/或通过siRNA技术来沉默这些酶来确定。此外，还将确定关键信号通路(BMPR2、PERK、PAKT、活性氧等)的活性和表达。雌激素的合成将通过检测芳香酶的表达&通过芳香酶活性测定来确定。这名学生还将重复对其他类型的肺细胞的关键实验，如成纤维细胞，以及来自人类阻力动脉的血管平滑肌细胞(来自臀部活检材料)。如果学生愿意，也可以进行活体技能的高保真培训。

Priority area: Basic Bioscience Underpinning HeathKeywords: LC-MS, pulmonary circulation, oestrogen metabolism Abstract:Gender exerts profound influences on vascular health and 'healthy ageing'. Women are more at risk of developing cardio-pulmonary dysfunction and this may increase post-menopause. Few studies have, however, directly examined the possibility that gender and age may induce changes on the normal pulmonary vascular function and oestrogen metabolism that might pre-dispose women to vascular risk factors. Here we will determine gender differences in the normal function of the pulmonary vasculature, in particular the role & influence of oestrogen, oestrogen metabolism & oestrogen metabolites.Whilst there are several papers and reviews concerning the influence of oestrogen on the vasculature, the influence of oestrogen metabolites on the normal ageing vasculature is very under-researched. Likewise, the influence of gender on normal proliferative signalling pathways is largely under-investigated. Our preliminary data on human pulmonary artery smooth muscle cells (hPASMCs) suggests there are gender differences in signalling pathways & that oestrogen may be the reason for the gender differences. We have recently demonstrated that oestrogen itself can decrease signalling in hPASMCs through the BMPR2 pathway increase MAPK signalling; hence proliferation of female hPASMCs is greater than in male cells. We have shown that microRNA expression in hPASMCs can be influenced by gender and oestrogens. For example, microRNA96 is decreased in hPASMCs from female lung & this causes an increase in serotonin-induced proliferation via the 5-HT1B receptor. It is emerging that oestrogen metabolites may play a more influential role on normal vasculature function than oestrogen itself. One limitation to these investigations is our ability to actually measure oestrogen metabolism and metabolites in vascular tissue. Over the last two year we have developed a novel HPLC/LC-MS 'steroidomic' method for assessing oestrogen metabolism in hPASMCs. We can now apply this technology to understand the role of oestrogen & oestrogen metabolism in the normal function and ageing of pulmonary arteries. Year 1-2. The student would assist the development of LC-MS techniques to analyse oestrogen metabolites in hPASMCs & plasma We have already identified some metabolites that accumulate in PASMCs & that have either pro- or anti-proliferative effects & at first we will examine these (e.g. 16-OHE1/2, 4-OHE1/2, 2-OHE1/2, 2 and 4-MeOHE1/2). Following this, measurements will be made in plasma at days 7, 14, 21 and 28 of the menstrual cycle from normal healthy volunteers. Similar analysis will be made in samples from post-menopausal women and age-matched men. The student will also examine the expression of key microRNAs in these samples, especially those that may interact with oestrogen metabolism or action (e.g. miRNA-22, miRNA-206, miRNA-27b). Year 2-4. hPASMCs will be derived from healthy men & women & these will be grouped according to age. This will be in collaboration with Nick Morrell (Cambridge). The effects of normal ageing and gender on basal and stimulated oestrogen metabolism will be determined. The influence of oestrogen synthesis & metabolising enzymes on this will be determined by applying aromatase inhibitors such as anastrozole and CYP1B1 inhibitors such as TMS and /or by siRNA techniques to silence these enzymes. In addition, the activity and expression of key signalling pathways will be determined (BMPR2, pERK, pAkt, reactive oxygen species etc). The synthesis of oestrogen will be determined by examining aromatase expression & via aromatase activity assay. The student will also repeat key experiments on other pulmonary cell types such as fibroblasts & also vascular smooth muscle cells from human resistance arteries (from gluteal biopsy material). High fidelity training in in vivo skills is also available if the the student wishes this.