An endothelial-smooth muscle cell bilayer 3D microvessel to study human arteriole inflammation

Introduction: Arterioles are small arteries connecting arteries and capillaries. Common disorders of arterioles include arteriolosclerosis and thrombosis, leading to cardiovascular diseases, a major cause of death worldwide. Early stage of arteriole disorders is hallmarked by inflammatory conditions in the intima of the artery, causing the accumulation of monocytes, lymphocytes and platelets and initiating plague formation. Without interventions, the plague narrows the artery and blocks blood flow, and the burst of the plague can eventually lead to critical situations of human body. Hence, elucidating the structure and physiology of arterioles is pivotal for drug development. Although numerous in vitro artery models have been demonstrated to recapitulate the structure and functions of large artery, few studies have reported the construction of arteriole due to the difficulties of maintaining confluent monolayers of both smooth muscle cells (SMCs) and endothelial cells (ECs) in microscale. Here, we demonstrate the first 3D tissue-engineered arteriole microvessel constructed with co-cultured confluent monolayers of SMCs and ECs with the diameter of 150 µm. We then validate the structural integrity and barrier function of the arteriole with permeability assay. We apply the pulsatile flow through the arteriole model to recapitulate the physiological arteriole dilation. Finally, we study the inflammatory response of the arteriole model by monitoring immune cells and platelets adhesion on the vessel wall under endothelium activation.

Materials and

Methods: The arteriole microvessels were fabricated and maintained in collagen hydrogel (Fig. A). 150 µm diameter microchannels were created in 7 mg/mL genipin-crosslinked collagen I hydrogel housed in a PDMS microdevice. The microchannels were coated with collagen IV and fibronectin, followed by SMC seeding at a cell density of 40×10^6 cells/mL. After the formation of confluent monolayer, the SMC microvessel were perfused with serum-free maintenance medium (SFMM) for 7 days. ECs were then seeded on the SMC microvessel at a cell density of 60×10^6 cells/mL to form an EC monolayer on the SMC microvessel. The co-cultured bilayer of ECs and SMCs were maintained in SFMM for further analysis. Epi-fluorescence microscopy, confocal microscopy and transmission electron microscopy (TEM) were used to construct the 3D bilayered microvessel and the cellular structure of the arteriole model. Sinusoidal flow (mean wall shear stress (WSS) of 20 dyne/cm^2, WSS amplitude of 5 to 20 dyne/cm^2, and frequency of 1 Hz) was applied on the microvessel to monitor the dilation of arteriole under pulsatile flow. Fluorophore-labeled 70 kDa dextran was perfused through the microvessel to derive the molecular permeability across the arteriole model. Endothelium activation of the arteriole was triggered by Tumor Necrosis Factor alpha (TNF-α) overnight perfusion at 1 ng/mL. Immune cell adhesion was performed by perfusing THP-1 and HL-60 cells through the arteriole model. Intercellular Adhesion Molecule 1 (ICAM-1) expression level was measured by immunocytochemistry. Human platelet adhesion on the complete and incomplete endothelium was conducted by perfusing human platelet-rich plasma through the arteriole model.

Results, Conclusions, and Discussions: The structural integrity of co-cultured bilayer EC-SMC microvessel was visualized by microscopy (Fig. B-H). Confocal microscopy images of equatorial plane of the live microvessel showed EC (labeled with GFP) monolayer was formed on the inner wall of SMC (labeled with CellTracker Deep Red) monolayer. The 3D structure of the microvessel were reconstructed by confocal z-stacks, showing the cross-section of the bilayer structure of arteriole model. Immunocytochemistry visualized the α-SMA (an SMC marker) and VE-Cadherin (an EC marker) of the polar plane of the microvessel, confirming the characteristics of SMCs and ECs were conserved in the co-cultured bilayer microvessel. Furthermore, TEM images of a cross section of the arteriole model revealed the cellular structure of microvessel with EC, SMC and ECM, recapitulating tunica intima, tunica media and tunica adventitia tri-layer of arteriole. The permeability assay of 70 kDa dextran indicates a comparable permeability between SMC-EC and EC only microvessel, demonstrating the barrier integrity of the co-culture bilayer microvessel (Fig. I-J). Together, the microscopy characterization and permeability assay demonstrate the structural and barrier integrity of the arteriole model.
We applied sinusoidal flow through the microvessel to simulate the dilation of arteriole under pulsatile flow (Fig. K-N). The periodic contraction and dilation of the microvessel were recorded. The dilation of the microvessel increased with the increasing of WSS amplitude in both crosslinked and uncrosslinked (data not shown) collagen hydrogel. Endothelium activation was triggered by TNF-α treatment, confirmed with elevated ICAM-1 expression level. The adhesion number of immune cells (THP-1 and HL-60) was significantly increased on the activated endothelium (Fig. O-Q), indicating the microvessel can model the inflammatory response of arteriole. Endothelium injury was simulated with incomplete seeding of ECs on the SMC microvessel (Fig. R-S), and the increased adhesion of platelets on the incomplete endothelium indicates the physiological response of wound healing on the arteriole model.
Taken together, we demonstrated a co-cultured bilayer SMC-EC microvessel with unprecedented structural and functional similarities to model human arteriole in vitro. This arteriole model provides a promising platform for investigating the mechanisms of inflammatory response, paving the way for drug development targeting arteriole disorders.

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