Investigating the Pathophysiology of Pulmonary Arterial Hypertension with Organ-on-Chip Technology

肺动脉高压（PAH）是一种罕见的心肺疾病，预后差，治疗方法很少。目前的治疗是基于血管扩张剂，可以减缓，但不能逆转疾病的进展。肺毛细血管的丧失（毛细血管稀疏）最近被认为是PAH的重要病理机制，并且与肺周细胞从毛细血管转移到重塑动脉（ol）有关。然而，这种（以及其他）多环芳烃血管重构过程的机制仍然没有得到充分的了解，主要是因为现有的体内和体外模型对血管重构动力学、潜在的多细胞串扰和局部生物力学背景的作用提供了有限的见解。因此，IPACHIP项目的目标是基于器官芯片（OOC）技术开发相关的多环芳烃肺微血管体外模型，以解决多环芳烃的基本机制问题。两种OOC模型将被开发：VASC-1是基于我们现有的微血管自组装的微流体平台，VASC-2是一个更复杂的远端肺血管芯片模型，除了一个自组装的毛细血管网络外，还包括流入和流出血管。在VASC-2中，相关的生物机械和化学信号（剪切应力、呼吸运动、缺氧）将进一步复制和控制。我们将采用两种策略来模拟PAH：由原代PAH患者细胞生成的模型将与由健康供体细胞制成的模型进行比较。由健康供体细胞生成的模型将暴露于模拟多环芳烃的刺激（慢性缺氧和/或VEGFR拮抗剂SU5416）。通过共聚焦和电子显微镜、免疫组织化学、流式细胞术、分子生物学技术和单细胞RNA测序，将开发出用于纵向评估血管网络参数、细胞反应和潜在分子机制和信号通路的适当读数。通过这些工具，我们将评估周细胞和内皮细胞之间的动态相互作用及其在PAH肺微血管网络不稳定和毛细血管稀疏中的作用，重点关注：a)周细胞从PAH毛细血管的潜在脱离，其潜在机制和对网络完整性的功能后果；b) PAH中微血管向毛细血管前动脉（ol）的周细胞募集；C)通过间隙连接的周细胞-内皮细胞通讯的作用。这项工作有望产生一套独特的新型芯片血管模型，用于分析肺血管重塑及其潜在机制。这些模型的应用将对驱动肺毛细血管稀薄的机制产生重要的见解，特别强调内皮-周细胞的相互作用。这些多功能平台可以进一步用于筛选新的或重新利用的药物来停止甚至逆转重塑。

Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disease with poor prognosis and few therapeutic options. Current therapies are based on vasodilators that can slow down, but not reverse disease progression. Loss of pulmonary capillaries (capillary rarefaction) has recently emerged as important pathomechanism of PAH, and has been linked to a translocation of lung pericytes from capillaries to remodeling arteri(ol)es. Yet, mechanisms underlying this (as well as other) process(es) of vascular remodeling in PAH remain insufficiently understood, largely because existing in vivo and in vitro models provide limited insight into the dynamics of vascular remodeling, the underlying multicellular crosstalk, and the role of the local biomechanical context. The objective of the IPACHIP project is thus to develop relevant in vitro models of the PAH lung microvasculature based on organs-on-chip (OOC) technologies, to address fundamental mechanistic questions in PAH. Two OOC models will be developed: VASC-1 is based on our existing microfluidic platform for microvascular self-assembly and VASC-2 is a more complex distal lung vasculature-on-chip model that in addition to a self-assembling capillary network includes inflow and outflow vessels. In VASC-2 relevant bio-mechanical and -chemical cues (shear stress, respiratory motions, hypoxia) will further be reproduced and controlled. Two strategies will be used to simulate PAH: 1. Models generated from primary PAH patient cells will be compared to models made from healthy donor cells, and 2. Models generated from healthy donor cells will be exposed to PAH-simulating stimuli (chronic hypoxia and/or the VEGFR antagonist SU5416). Appropriate read-outs for longitudinal assessment of vascular network parameters, cellular responses and underlying molecular mechanisms and signaling pathways will be developed by confocal and electron microscopy, immunohistochemistry, flow cytometry, molecular biology techniques, and single cell RNA sequencing. With these tools, we will assess the dynamic interaction between pericytes and endothelial cells and their role in lung microvascular network destabilization and capillary rarefaction in PAH, by focusing on:a) a potential detachment of pericytes from PAH capillaries, its underlying mechanisms and functional consequences for network integrity;b) pericyte recruitment from microvessels to precapillary arteri(ol)es in PAH;c) the role of pericyte-endothelial communication via gap junctions.The proposed work is expected to generate a unique set of novel vasculature-on-chip models for the analysis of lung vascular remodeling and its underlying mechanisms. Application of these models will yield important insights into the mechanisms driving lung capillary rarefaction with a specific emphasis on endothelial-pericyte interactions. These versatile platforms may further be used to screen for novel or repurposed drugs to halt or even reverse remodeling.