Vascular Endothelial Dysfunction in Preeclampsia

描述（由申请人提供）：虽然已知内皮功能障碍是子痫前期（PE）的一部分，但内皮功能障碍的分子基础尚不清楚。目前相关高血压的治疗仍主要针对血管平滑肌的松弛，但这还不足以完全控制这种疾病。需要基于新的治疗干预方法的新策略。只有在明确确定新的治疗靶点并在内皮功能水平上进行筛选的情况下，这才有可能实现。这种大规模的筛选方法需要大量高纯度、特征明确的PE受试者细胞。然后，在进行药物改良和人体试验之前，可以在抵抗水平血管中进行第二轮实际规模的测试。问题仍然是，正常妊娠和PE妊娠的人子宫动脉组织不足以进行初始筛选，较小的血管活检样本不足以产生足够高纯度或数量的细胞进行药物小组筛选。我们的初步数据表明，虽然正常妊娠期间子宫动脉血管内皮功能确实最大化，但在其他血管床（包括胎儿胎盘血管，如脐静脉）中也观察到相同的机制并最大化。此外，这些信号事件和正常脐带内皮中相关的NO产生似乎在体育运动受试者中也失败了。因此，为了确定是否有可能使用PE受试者的HUVEC作为这种药物筛选的合适细胞，我们提出：西班牙1：假设：观察到正常妊娠完整UV Endo中对ATP反应的持续[Ca2+]i爆发是由于TRPC3和CX43的激活，而与TRPC3或CX43抑制策略相关的Ca2+爆发的损失复制了PE受试者中看到的较低/钝化的Ca2+和NO反应。西班牙文献2（向文献3过渡）：假设：与正常受试者相比，PE衍生的UV Endo或PE衍生的HUVEC（文献3）的PE相关功能障碍不是由于P2Y2受体、G蛋白、PLC、IP3受体、TRPC通道或连接蛋白的相对表达水平的简单变化。西班牙3：假设：在短期培养中，由于CX43增强IP3敏感的TRPC3激活，正常妊娠HUVEC中持续的[Ca2+]i对ATP的爆发反应被保留，并且在PE受试者的细胞中缺乏。此外，抑制正常受试者HUVEC中IP3介导的TRPC3激活或CX43介导的细胞间通讯，复制了PE受试者HUVEC中所见的功能丧失，但不会进一步抑制PE受试者HUVEC的功能。如果取得成功，现成的脐带和来自PE受试者的大量UV Endo细胞意味着未来可以进行大规模的药物测试，甚至额外关注目标/弱势公众亚群。这可以让新药或现有药物的新应用更有选择性地干预这一信号通路，从而减轻先兆子痫的破坏性影响。

DESCRIPTION (provided by applicant): While endothelial dysfunction is known to be a part of preeclampsia (PE), the molecular basis for endothelial dysfunction remains unclear. Current therapies for the associated hypertension are still largely targeted to relaxation of vascular smooth muscle and yet this is not sufficient to fully control the disorder. New strategies are needed based on novel approaches to therapeutic intervention. This will only be possible given a clearly identified new therapeutic target and a means to screen it at the level of endothelial function. Such large scale screening approaches require large numbers of high purity, well characterized cells from PE subjects. Second round testing in resistance level vessels can then be undertaken on a realistic scale before moving towards drug refinement and trials in human subjects. The problem remains that human uterine artery tissue from normal and PE pregnancy are not sufficiently available to undertake the initial screen, and smaller vessel biopsy samples are insufficient to yield cells of high enough purity or quantity for drug panel screening. Our preliminary data suggests that while vascular endothelial function is indeed maximized in the uterine artery in normal pregnancy, these same mechanisms are also observed and maximized in other vascular beds including fetoplacental vessels such as the umbilical vein. Further, these signaling events and associated NO production in the endothelium of normal cords also appear to have failed in PE subjects. Thus, in order to establish if it is possible to use HUVEC from PE subjects as suitable cells in such a drug screen we propose: SpAim 1: Hypothesis: The observed sustained [Ca2+]i bursts in responses to ATP in intact UV Endo from normal pregnancy are due to TRPC3 and CX43 activation, and losses of Ca2+ burst associated with strategies aimed at TRPC3 or CX43 inhibition replicate the lower/blunted Ca2+ and NO responses seen in PE subjects. SpAim 2 (Transitional to Aim 3): Hypothesis: PE associated dysfunction in PE derived UV Endo or PE derived HUVEC (passage 3) compared to that from normal subjects is not due to simple changes in the relative levels of expression of P2Y2 receptors, G proteins, PLC, IP3 receptors, TRPC channels, or connexins. SpAim 3: Hypothesis: In short term culture, the sustained [Ca2+]i burst responses to ATP in HUVEC from normal pregnancy due to CX43 potentiation of IP3 sensitive TRPC3 activation are retained, and are lacking in cells derived from PE subjects. Further, inhibition of either IP3 mediated TRPC3 activation or CX43 mediated cell-cell communication in HUVEC from normal subjects replicates the loss of function seen in HUVEC from PE subjects, but does not further inhibit the function of HUVEC from PE subjects. Given success, the ready availability of cords and large yields of UV Endo cells from PE subjects means that future large scale drug testing could be performed, even with additional focus on targeted/disadvantaged subpopulations of the public. This could allow more selective intervention with new drugs or new applications of existing drugs that impact on this signaling pathway, and so relieve the devastating effects of preeclampsia.