(L-474) iPSC-derived Tissue Engineered Blood Vessel Model Reveals Radiation-Induced Senescence and Inflammatory Phenotype in Vascular Smooth Muscle

Introduction:: Cardiovascular disease (CVD), the leading cause of death worldwide, is known to have a myriad of causes. Ionizing radiation is a particular concern for many groups of people exposed to it: astronauts are exposed to cosmic rays, cancer patients are exposed to clinical photon and proton rays, and many others are exposed to radioactive isotopes from material spills or weapons. In these groups, radiation exposure is associated with significantly higher rates of CVD and even death due to cardiovascular events. Rodent models and adherent cell culture systems have historically been used to better understand the biological phenomena occurring in vascular cells after radiation exposure, but many aspects of the disease pathways are still poorly understood. Many studies focus solely on endothelial cells as the primary radiation targets that initiate the inflammatory cascade and trigger longer term disease in the vasculature, while smooth muscle cells, the mediators of many chronic CVDs, are often overlooked. Our 3D model of a small artery, using tubular electrospun fibrin scaffolds cultured with human stem cell-derived vascular smooth muscle cells (iSMCs) in a bioreactor, emulates the structure and forces in the human vasculature to study direct radiation-induced damage of SMCs and resulting abnormal changes in vascular tissue. We hypothesize that exposing our tissue engineered blood vessels (TEBVs) to ionizing radiation will reveal dose-dependent changes in SMC phenotype and protein expression different from iSMCs cultured in 2D, and that activated pathways can be targeted with novel countermeasures to prevent pathological changes leading to CVD.


Materials and Methods:: Smooth muscle cells are derived from human induced pluripotent stem cells (iPSCs) using established protocols [1]. For our TEBV model, the iSMCs are removed as a single sheet and wrapped around a 0.6 mm ID tubular electrospun fibrin scaffold [2]. The cellularized graft is sutured into a bioreactor system and cultured with pulsatile flow through the lumen (Fig 1A). The bioreactors are exposed to either 0.1, 0.5 or 10 Gray of 160 KeV x-ray, gamma ray, or simulated Galactic Cosmic Radiation (GCR) at the Nasa Space Radiation Laboratory. At either 24 hours (acute timepoint) or 8 days (chronic timepoint) of continued culture after irradiation, samples are harvested and digested for protein and RNA isolation, after which proteomic and transcript analysis is performed. For iSMCs cultured in 2D at 8 days post-exposure, an array of 88 gene transcripts, known to regulate ECM secretion and inflammatory activation, are analyzed via reverse transcription quantitative real-time PCR (RT-qPCR). Additionally, protein expression is quantified via immunofluorescent confocal microscopy: samples are stained for reactive oxygen species abundance and DNA damage markers, in addition to phenotypic markers for proliferation (Ki67), senescence (p21 and p16), contractility (smooth muscle myosin heavy chain (SM-MHC), smooth muscle actin and calponin), and structural extracellular matrix (ECM) including collagens, elastin, and elastin assembly proteins. Senescence (p21, p16) and DNA damage (gamma-H2AX) marker expression is further confirmed via Western Blot at 24 hr and 8 days post-exposure.


Results, Conclusions, and Discussions:: In 2D cell culture, irradiated iSMCs show decreased Ki67 expression at 24 hours and 8 days after exposure to 0.5 and 10 Gray x-rays (Fig 1B). At 10 Gray x-rays, contractile marker and collagen 1 expression, and mature elastin abundance is increased in 2D cell culture only after the extended 8 day period, but not after 24 hours. These structural phenotypic changes also occur in iSMC-TEBVs, where we see increased contractile marker expression and altered collagen architecture in irradiated TEBVs only after 8 days post-exposure (Fig 1C). RT-qPCR analysis of SMCs at 8 days post-exposure showed a significant upregulation in genes associated with the SMC Senescence-Associated Secretory Phenotype (SASP) such as CD44, while ITGA7 (integrin alpha 7) was downregulated, which is associated with a shift to a synthetic or migratory phenotype in SMCs (Fig 1D). To further investigate a potential senescent secretory phenotype, expression of senescent markers p21 and p16 was quantified, and irradiated TEBVs exhibit increased p21 expression at 24 hrs post-exposure, while irradiated 2D iSMCs show increased p21 and p16 expression at both 24 hrs and 8 days post-exposure (Fig 1E).

These results indicate that photon radiation (x-rays) may induce a chronic pro-inflammatory senescent phenotype in SMCs. The combination of decreased proliferation, changes in production and architecture of ECM proteins, and increased senescent marker expression all indicate a senescent phenotypic shift. Additionally, upregulation of genes involved in the smooth muscle SASP, and downregulation of integrins indicate a shift to a potentially more inflammatory and migratory state. This direct phenotypic shift that is induced by ionizing radiation could implicate a larger primary role for vascular SMCs in radiation-induced CVD. By understanding the mechanisms of direct radiation damage in SMCs, we can expand the understanding of disease pathways that have been previously unexplored in the many studies focusing only on endothelial cell radiation response. Ongoing work focuses on examining different types and doses of radiation to determine the specific metabolic pathways being activated in each case, in addition to identifying potential factors that can be therapeutically targeted.


Acknowledgements (Optional): : Funding Agencies: NSF Graduate Research Fellowship Program, and Translational Research Institute for Space Health

Edwin Yu and Roy Chen assisted in data collection and processing throughout this work.

All schematics were created using Biorender.com.

References (Optional): : [1] Yang L, et al. (2016) Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols. PLoS ONE, 11(1): e0147155. doi:10.1371/journal.pone.0147155

[2] Elliot M, et al. (2019) ‘Regenerative and durable small-diameter graft as an arterial conduit’, Proceedings of the National Academy of Sciences, 116(26):12710. doi:10.1073/pnas.1905966116