Magnetic nanoparticles assisted modulation of the vascular cGMP system

在第一个资助期，我们专注于使用磁性纳米颗粒（MNP）来增强和靶向血管系统中的慢病毒（LV）基因转移。研究了广谱MNP以优化体外和离体LV基因转移。此外，还进行了首次体内研究。基于这项工作，我们现在将重点研究一氧化氮（NO）/cGMP通路，这是血管稳态所必需的。在本提案中，我们将研究控制NO和cGMP产生的新机制。主要关注cGMP依赖性蛋白激酶（蛋白激酶G，PKG）及其介导NO/cGMP/PKG效应的下游靶点。我们有初步的数据表明，迄今未知的途径，涉及PKG底物和小GTd-Rac调节细胞cGMP浓度。此外，我们将研究cGMP/PKG信号和NO的生产在内皮细胞之间的联系。在不应用MNP和磁梯度场的情况下，不可能在流动条件下对血管进行有效和靶向的转导。因此，我们将使用优化的LV/MNP复合物来研究血管系统中信号通路的生理效应。除了离体灌注系统，也允许力的测量作为一个重要的生理读数，我们将进一步开发LV/MNP系统，使用含MNP的微泡（MB）的目的是实现有效的基因转移到血管系统中的血管在体内。

In the first funding period, we focused on the use of magnetic nanoparticles (MNPs) to enhance and target lentiviral (LV) gene transfer in the vascular system. A broad spectrum of MNPs was studied to optimize LV gene transfer in vitro and ex vivo. In addition, first studies in vivo were performed. Based on this work, we will now focus on the study of the nitric oxide (NO)/cGMP pathway, which is essential for vascular homeostasis. In the present proposal, we will study novel mechanisms that control NO and cGMP production. The major focus will be on cGMP-dependent protein kinase (protein kinase G, PKG) and its downstream targets that mediate the NO/cGMP/PKG effects. We have preliminary data suggesting that a so far unknown pathway involving a PKG substrate and the small GTPase Rac regulate cellular cGMP concentrations. Moreover, we will study the link between cGMP/PKG signaling and NO production in the endothelium. Without the application of MNPs and magnetic gradient field an efficient and targeted transduction of vessels under flow conditions is not possible. Therefore, we will use optimized LV/MNP complexes to study the physiological effects of the signaling pathways in the vascular system. Apart from ex vivo perfusion systems that also allow for force measurements as an important physiological read-out, we will further develop the LV/MNP system using MNP-containing microbubbles (MBs) with the aim to achieve efficient gene transfer to vessels in the vascular system in vivo.