Ferrochelatase & guanylate cyclase regulation in vascular dysfunction

 描述(申请人提供)：这个项目的总体假设是，线粒体产生的超氧化物增加会导致铁络合酶(FECH)破坏血管平滑肌血红素的生物合成，导致失去可溶性鸟苷环化酶(SGC)有益的血管调节作用。已知的是，sGC血红素的氧化促进了一氧化氮(NO)调节的丧失和sGC的蛋白质降解。虽然线粒体血红素生成酶FECH具有稳定所必需的铁-硫簇，这是超氧化物破坏的潜在目标，但文献似乎证实了FECH功能障碍对任何生物系统中的SGC调节对血红素依赖的后果的证据。由于血管紧张素II(AngII)在多种疾病中促进人类血管功能障碍的潜在重要性，以及先前证据表明，包括线粒体在内的亚细胞部位超氧化物生成增加，以及NO及其相关的血红素依赖的调节sGC/cGMP介导的血管扩张的机制被破坏，我们假设并发现了Angii破坏FECH活性以及FECH控制sGC的表达和NO介导的激活的证据。目标1的研究集中于显示Angii如何调节线粒体超氧化物增加导致FECH对血红素生物合成的干扰，从而削弱sGC对用Angii和siRNA或调节超氧化物和FECH亚细胞来源的机械探针处理的离体鼠和牛冠状动脉的血管调节。血管紧张素转换酶1型受体(AT1R)、铜锌超氧化物歧化酶(SOD1)、线粒体基质SOD2、FECH、NOX1和NOX2氧化酶缺陷小鼠的动脉将在这些研究中用于确定AngiI对胞浆和线粒体超氧化物歧化的调节如何影响sGC和FECH系统的调节。目的2研究新的初步观察结果表明，促进原卟啉IX(原卟啉IX，sGC的激活剂)和血红素从δ-氨基酮戊酸(ALA)中获得似乎可能通过cGMP介导的线粒体超氧化物抑制来保护铁络合酶和sGC调节，这可能涉及cGMP阻止sD2的耗竭。目标3的研究重点是利用血管紧张素转化酶的小鼠渗透模型，确定铁络合酶和血红素相关的sGC对血管功能的调节和ALA提供的保护的体内重要性。将评估血压、超声心动图的无线电遥测监测，以及评价骨骼肌微循环中孤立动脉和活体小动脉功能的变化。亚细胞来源的作用 在AT1R、SOD1、SOD2、NOX1和NOX2基因缺陷的血管紧张素转换酶输注小鼠中，也将评估超氧化物在铁络合酶破坏中的作用，以及一种专门针对线粒体超氧化物的治疗。这些研究有望证明线粒体超氧化物歧化铁络合酶在sGC相关的血管功能障碍中具有重要作用，这可以通过有益的ALA治疗来调节sGC的活性、表达和无刺激。

 DESCRIPTION (provided by applicant): The overall hypothesis for this project is that increased mitochondrial generation of superoxide causes a disruption of vascular smooth muscle heme biosynthesis by ferrochelatase (FECH), resulting in a loss of the beneficial vascular regulatory effects of soluble guanylate cyclase (sGC). It is known that oxidation of the sGC heme promotes loss of regulation by nitric oxide (NO) and sGC degradation by proteolysis. While the mitochondrial heme generating enzyme FECH has an iron-sulfur cluster essential for its stability, which is a potential target for disruption by superoxide, the literature appears to ack evidence for the consequences of FECH dysfunction on the heme-dependence of sGC regulation in any biological system. Due to the potential importance of angiotensin II (AngII) in promoting human vascular dysfunction in multiple diseases and previous evidence for mechanisms associated with increased superoxide generation in subcellular sites including mitochondria, and disruption of NO and its associated heme-dependent regulation of sGC/cGMP-mediated vasodilation, we hypothesized and found evidence for both FECH activity being disrupted by AngII and for FECH controlling the expression and NO-mediated activation of sGC. Studies in Aim 1 focus on showing how AngII regulation of increases in mitochondrial superoxide cause a disruption in heme biosynthesis by FECH that impairs vascular regulation by sGC in isolated mouse and bovine coronary arteries treated with AngII and siRNA or mechanistic probes modulating subcellular sources of superoxide and FECH. Arteries from mice deficient in AngII type-1 receptor (AT1R), Cu, Zn-SOD (SOD1), mitochondrial matrix SOD2, FECH, Nox1 and Nox2 oxidase will be used in these studies to define how AngII regulation of cytosolic and mitochondrial superoxide influences regulation by the sGC and FECH systems. Aim 2 investigates new preliminary observations suggesting how promoting availability of protoporphyrin IX (PpIX, an activator of sGC) and heme from δ-aminolevulinic acid (ALA) appears to protect ferrochelatase and sGC regulation potentially through a cGMP-mediated inhibition of mitochondrial superoxide that may involve cGMP preventing the depletion of SOD2. Studies in Aim 3 focus in defining the in vivo importance of the disruption of ferrochelatase and heme- associated sGC regulation of vascular function and protection provided by ALA, using a mouse model of osmotic minipump delivery of AngII. Radiotelemetry monitoring of blood pressure, echocardiography, and evaluation of changes in isolated arteries and in vivo arteriolar function in the skeletal muscle microcirculation will be evaluated. The role of subcellular sources of superoxide in the disruption of ferrochelatase will also be assessed in AngII-infused mice deficient in AT1R, SOD1, SOD2, Nox1 and Nox2, together with a therapy specifically targeting mitochondrial superoxide. These studies are expected to document that mitochondrial superoxide disruption of ferrochelatase has an important role in sGC-associated vascular dysfunction, which can be targeted with a beneficial ALA therapy regulating the activity, expression and NO-stimulation of sGC.