Role of Cytoglobin in the Regulation of Vascular Tone

摘要 内皮源性舒张因子（endothelial-derived relaxing factor，ET）是调节血管张力的重要介质，被称为一氧化氮（nitric oxide，NO） 还有血压NO通过激活可溶性鸟苷酸环化酶（sGC）介导血管舒张， 平滑肌而NO是由内皮细胞中一种特异的NO合酶合成的， 内皮型一氧化氮合酶（eNOS）是血管NO降解和代谢的一个重要机制。它是假设 血管壁中的NO降解由O2依赖性NO双加氧酶（NOD）介导， 硝酸盐。然而，作为O2-的主要体内调节剂的特异性NO双加氧酶的身份是未知的。 血管平滑肌中依赖NO的降解仍然是难以捉摸的。细胞珠蛋白（Cgb）是近年来发现的一种 发现在平滑肌中表达的功能未知的珠蛋白。根据我们的初步数据， 我们观察到：1）Cgb是平滑肌中表达的主要珠蛋白; 2）Cgb的敲除大大增加了平滑肌中表达的珠蛋白的量， NO衰变，增加血管舒张，降低血压和全身血管阻力， 我们推测Cgb是调节O2依赖的NO代谢速率的主要血红素蛋白 在血管壁中，进而深刻地调节血管张力。在这份补助金中，我们有三个目标， 依次检验这一假设，首先在血管平滑肌细胞中，然后在离体血管中，最后在 在体心血管系统的实验测量和计算建模。后者将 使我们能够确定Cgb介导的O2依赖性NO代谢过程在多大程度上解释了 测量的血管NO代谢沿着能够预测调节Cgb表达的作用 NOD功能。由于Cgb的O2依赖性NOD功能受其还原速率控制，研究 本研究旨在阐明Cgb在平滑肌细胞、血管和平滑肌细胞中减少的过程和机制， 以及在小鼠体内利用分子敲低、药理学抑制或这些的其它操作， 减少路径。我们将确定Cgb的NOD功能的调节如何调节血管张力， 正常生理学和血管疾病，在血管和具有不同Cgb表达水平的小鼠中进行研究 以及抑制其还原的每一个主要途径或特异性抑制其NOD功能。这些 将在正常小鼠和患有血管紧张素诱导的高血压的小鼠中进行研究。在这种情况下， 研究将进行测量和分解超氧化物（O2-.）来确定氧气依赖性 NO与Cgb的反应与O2-的反应相比。特别关注内皮细胞的作用， eNOS在NO代谢中的偶联状态。本研究计划的完成将阐明 Cgb在血管NO代谢中重要作用的分子机制， 对正常生理学和心血管疾病中血管张力调节的重要见解 疾病这项工作将照亮道路，导致发展新的治疗方法，以扭转 通过调节NO的降解过程，改善血管功能障碍和继发性疾病。

Abstract Endothelium-derived relaxing factor, identified as nitric oxide (NO), is a key mediator regulating vascular tone and blood pressure. NO mediates vascular relaxation through activation of soluble guanylate cyclase (sGC) in the smooth muscle. While NO is synthesized by a specific well characterized NO synthase in the endothelium (eNOS), the process of vascular NO degradation and metabolism is poorly understood. It is hypothesized that NO degradation in the vessel wall is mediated by an O2-dependent NO dioxygenase (NOD) that oxidizes NO to nitrate. However, the identity of the specific NO dioxygenase that serves as the main in vivo regulator of O2- dependent NO degradation in vascular smooth muscle has remained elusive. Cytoglobin (Cgb) is a recently discovered globin expressed in smooth muscle with unknown function. Based on our preliminary data where we observe that: 1) Cgb is the major globin expressed in smooth muscle; and 2) knockout of Cgb greatly prolongs NO decay, increases vascular relaxation, lowers blood pressure and systemic vascular resistance, we hypothesize that Cgb is the major heme protein regulating the rate of O2-dependent NO metabolism in the vessel wall, in turn profoundly modulating vascular tone. In this grant, there are 3 aims in which we sequentially test this hypothesis, first in vascular smooth muscle cells, then in isolated vessels and finally in the in vivo cardiovascular system with experimental measurements and computational modeling. The latter will enable us to determine how well the process of Cgb-mediated O2-dependent NO metabolism accounts for the measured vascular NO metabolism along with enabling prediction of the effects of modulating Cgb expression and NOD function. Since the O2-dependent NOD function of Cgb is controlled by its rate of reduction, studies will be performed to elucidate the process and mechanisms of Cgb reduction in smooth muscle cells, vessels and in vivo in mice utilizing molecular knockdown, pharmacological inhibition, or other manipulation of these reducing pathways. We will determine how modulation of the NOD function of Cgb modulates vascular tone in normal physiology and vascular disease with studies in vessels and mice with varying Cgb expression levels and inhibition of each of the major pathways of its reduction or specific inhibition of its NOD function. These studies will be performed in normal mice and mice with angiotensin-induced hypertension. In these settings, studies will be performed to measure and scavenge superoxide (O2-.) to determine how the O2-dependent reaction of NO with Cgb compares to that of O2-. and specific attention paid to the role of the endothelium and the coupling state of eNOS in NO metabolism. Accomplishment of this research plan will elucidate the molecular mechanism underlying the important role of Cgb in vascular NO metabolism and provide important insights into the regulation of vascular tone in normal physiology and cardiovascular disease. This work will illuminate the path leading to development of novel therapeutic approaches to reverse vascular dysfunction and ameliorate secondary disease through modulation of the process of NO degradation.