Microengineered arterial wall-on-a-chip to model atherosclerosis

• Main Author: Su, Chengxun
• Other Authors: Hou Han Wei
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Engineering::Mechanical engineering; Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/155569
• Institution: Nanyang Technological University

Vascular complication is the most common cause of death and morbidity in Type 2 Diabetes Mellitus (T2DM), with cardiovascular diseases (CVD) being the major contributor of mortality. The main pathological cause of CVD is atherosclerosis, a multifaceted disease that arises from the accumulation of lipoprotein particles in the arterial wall and progresses through the ensuing inflammatory responses and endothelial dysfunction. Phenotypic switching of smooth muscle cells (SMC) also plays a significant role in atherosclerosis progression by over-proliferation and migration into the intima layer of the arterial wall. Animal models and 2D tissue culture systems are widely used to study atherosclerosis. However these models have limited accuracy in predicting human responses due to inter-species differences and the lack of complex cellular microenvironment, respectively. Organ-on-a-chip is a burgeoning technology that combines advances in microfluidics and cell biology to create physiologically relevant in vitro models for study of organ functions and human diseases. Current blood vessel-on-a-chip models primarily focus on establishing endothelial cells (EC) functions and lack well-defined arterial wall structure, particularly the microarchitecture and function of the smooth muscle layer. Hence there is an unmet need to develop organ-on-a-chip platforms for reconstitution of the arterial wall structure and biology to model atherosclerosis and its manifestation in T2DM. In this thesis, the overarching aims are to 1) engineer a novel 3D human arterial wall-on-a-chip (AWoC) for study of EC and SMC phenotypic changes in atherosclerosis, and 2) develop a “personalized’ AWoC model using patient-derived circulating extracellular vesicles. Firstly, a versatile and scalable microfluidic-based hydrogel patterning technique was developed to create continuous cell-extracellular matrix (ECM) interfaces using channel stepped height features. This technique was subsequently applied to create the AWoC model by co-culturing human aortic EC (HAoEC) and human aortic SMC (HAoSMC) with a cell-free subendothelial layer in a dual-lane hydrogel chip. Using an optimized ECM composition, the SMC was retained in a quiescent and contractile state, characteristic of a healthy artery. To model atherosclerosis, the AWoC was stimulated with cytokines and oxidized low-density lipoprotein (oxLDL) to recapitulate various atherogenic events including endothelial inflammation, SMC migration, EC/SMC lipoprotein uptake, and monocyte-EC adhesion. It was shown that high oxLDL alone could induce SMC migration, and oxLDL acted in concert with inflammatory stimulus to enhance oxLDL uptake in EC. As a proof-of-concept for drug screening using the AWoC, the atheroprotective effects of vitamin D and metformin were evaluated on chip, which showed significant mitigation of cytokine-induced monocyte–EC adhesion and SMC migration. Extracellular vesicles (EV) are nanoscale (~ 50 to 1000 nm) cell-derived vesicles that contain bioactive molecules for cell-cell communication and are implicated in diabetes pathophysiology. Circulating EV including medium-sized EV (mEV) and small EV (sEV) were first isolated using ultracentrifugation from blood samples of healthy (n = 5) and T2DM subjects (n = 5), following which the profile of EV was characterized using nanoparticle tracking analysis and flow cytometry. Increased levels of immune cells and inflamed EC-derived mEV were detected in T2DM subjects, possibly suggesting chronic vascular inflammation. It was further observed that diabetes sEV treatment resulted in a significant upregulation of endothelial ARG-1, an enzyme known for inhibiting generation of nitric oxide which further leads to endothelial and SMC dysfunction. Finally, patient-derived circulating EV were used to establish a novel form of ‘personalized’ AWoC model. Consistent with the transcriptomics study, diabetes sEV induced a higher level of SMC migration on-chip. This atherogenic phenotype was most apparent in T2DM individuals with abnormal clinical measurements of vascular functions. In summary, the thesis reports the development of a novel microengineered 3D arterial wall model (the AWoC) to study key pathological EC and SMC changes in atherosclerosis. The thesis also provides new findings on the use of patient-derived circulating EV as novel mediators to induce disease phenotype in vascular models. This not only serves as a cost-effective and rapid approach for “personalized” organ-on-chip platforms, but also opens up exciting translational applications for EV-based cardiovascular risk stratification, as well as testing of anti-inflammatory drugs in patients with T2DM and other metabolic disorders.