Modeling Oxidative Stress in Small Arteries In Vitro

Introduction: : Oxidative stress is an imbalance between reactive oxygen species and antioxidants that can cause tissue damage and is present in various cardiovascular diseases. Oxidative stress is also a high-risk space stressor that affects several organs in astronauts. Space flight is shown to dysregulate free radical normalization in the body, including downregulation of the antioxidant gene nuclear factor erythroid 2-related factor 2 (Nrf2). In the vasculature, the effects of spaceflight-induced oxidative stress include endothelial dysfunction, arteriosclerosis, and arterial stiffness and wall thickening.

In vitro models of the vasculature provide a reproducible and controlled environment to explore various pathologies. The more physiologically accurate the model, the better that structure and function are properly mimicked, thus reducing errors in results attributed to modeling in vitro. However, producing stable vessels less than 3 mm in diameter that can be challenging compared to more common microvascular network or large vessel models. When modeling blood vessels, the selected endothelial cell (EC) phenotype should closely match the application due to the unique roles they serve and the variation in phenotypes.

Our lab has created an artery-on-chip (ARTOC) model made of electrospun fibrin hydrogel microfibers that are seeded with a monolayer of iPSC-derived ECs (iEC) with an arterial phenotype, thus forming a stable in vitro replica of a small blood vessel less than 3 mm in diameter. Here, we hypothesize that the ARTOC can be used to study the effects of oxidative stress in small arteries when using Nrf2 knockout (KO) stem cells to genetically induce oxidative stress.

Materials and

Methods: : Wild type (WT) induced pluripotent stem cells (iPSC) and Nrf2 KO iPSCs are differentiated into arterial iECs using published protocols. Differentiation efficiency is measured using flow cytometry to find the percentage of cells expressing endothelial markers VE-Cadherin and CD31 at the end of a differentiation. Arterial iEC identity is verified by testing for expression of VE-Cadherin, CD31, and arterial marker SOX17 by using flow cytometry and by immunofluorescence imaging after magnetic-activated cell sorting. Functional assays are performed consisting of (1) network formation by seeding iECs in 3D collagen gels for three days and examining immunofluorescent images for the ability of iECs to form networks and performing quantitative analysis using Imaris, and (2) inflammatory response by exposing iECs to bacterial lipopolysaccharide (LPS) for 24 hours and comparing expression of inflammatory markers ICAM-1 and VCAM-1 to non exposed iECs via immunofluorescent staining. Transendothelial electrical resistance (TEER) is used to assess barrier function of the endothelial monolayer using published protocols for six days. Fibrin grafts are generated by following published protocol, where fibrinogen is electrospun into a thrombin bath and then rolled in layers around mandrels, creating grafts that are 0.6 mm in diameter and 10 mm in length. iECs are injected into fibrin grafts and rotated while settling to create a monolayer of cells on the luminal graft surface. ARTOCs are cultured for 48 hours and then examined for proper seeding and growth of iECs by immunofluorescent staining of endothelial markers and F-actin.

Results, Conclusions, and Discussions:: Arterial iECs were successfully differentiated from WT and Nrf2 KO iPSCs. The differentiation efficiency between WT and Nrf2 KO was not significantly different, but both iEC types expressed arterial marker SOX17 and endothelial markers VE-Cadherin and CD31 (Fig. 1A-B). Both iECs formed networks in collagen gels, but there was no significant difference in network length, area, or volume (Fig. 1C, 1D). Both iECs upregulated inflammatory markers ICAM-1 and VCAM-1 in response to inflammation with bacterial LPS (Fig. 1E). There was no significant difference in TEER values between WT and Nrf2 KO iECs for all six days of measurement (Fig. 1F). Stable fibrin grafts 0.5 mm in diameter were successfully electrospun and supported the attachment and proliferation of iECs, forming a biomimetic ARTOC (Fig. 1G). Studies comparing the attachment and function of WT versus Nrf2 KO iECs seeded in grafts are upcoming.

The in vitro model of small arteries produced is highly physiologically relevant, where the ARTOCs are accurately sized and the iECs exhibit an arterial phenotype. By using iPSCs, this experiment can be performed with people of diverse backgrounds and studied in different organ systems. Additionally, studies can be personalized to specific patients, since people are affected differently by oxidative stress. Genetic factors, such as variations in antioxidant enzyme genes, may influence susceptibility to oxidative damage. Future studies will involve incorporating pulsatile flow through the ARTOCs, coculture with induced smooth muscle cells on the basal side of the graft, and quantitative measurements of oxidative stress levels and oxidative damage. Overall, the model produced in this study can greatly increase our understanding of how oxidative stress affects small arteries.

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