Mechanisms of radiation-induced endothelium damage: Emerging models and technologies

Abstract

Radiation-induced endothelial/vascular injury is a major complicating factor in radiotherapy and a leading cause of morbidity and mortality in nuclear or radiological catastrophes. Exposure of tissue to ionizing radiation (IR) leads to the release of oxygen radicals and proteases that result in loss of endothelial barrier function and leukocyte dysfunction leading to tissue injury and organ damage. Microvascular endothelial cells are particularly sensitive to IR and radiation-induced alterations in endothelial cell function are thought to be a critical factor in organ damage through endothelial cell activation, enhanced leukocyte-endothelial cell interactions, increased barrier permeability and initiation of apoptotic pathways. These radiation-induced inflammatory responses are important in early and late radiation pathologies in various organs. A better understanding of mechanisms of radiation-induced endothelium dysfunction is therefore vital, as radiobiological response of endothelium is of major importance for medical management and therapeutic development for radiation injuries. In this review, we summarize the current knowledge of cellular and molecular mechanisms of radiation-induced endothelium damage and their impact on early and late radiation injury. Furthermore, we review established and emerging in vivo and in vitro models that have been developed to study the mechanisms of radiation-induced endothelium damage and to design, develop and rapidly screen therapeutics for treatment of radiation-induced vascular damage. Currently there are no specific therapeutics available to protect against radiation-induced loss of endothelial barrier function, leukocyte dysfunction and resulting organ damage. Developing therapeutics to prevent endothelium dysfunction and normal tissue damage during radiotherapy can serve as the urgently needed medical countermeasures.

Keywords: Endothelial cells; Inflammatory response; Ionizing radiation; Microphysiological systems; Permeability; Radioprotective agents.

Figure 1:

Overview of vascular endothelium and mechanisms by which IR impacts endothelial cell regulation. Top panel: Normal tissue - Endothelial cells act as a semipermeable barrier that regulates the delivery of nutrients and oxygen to tissue and the removal of carbon dioxide and waste products. Normal endothelial cells have basal levels of some adhesion molecules. Bottom panel: Irradiated tissue – Ionizing radiation increases the production of ROS leading to DNA and mitochondrial damage and increased apoptosis. IR also alters endothelial permeability by acting on tight and adherens junctions allowing excess extravasation of proteins to cross into the extracellular tissue. Radiation exposure also increases the release of proinflammatory cytokines and chemokines and upregulation of adhesion molecules resulting in increased leukocyte-endothelial cell interaction and trafficking to vital organs.

Figure 2:

Multi-step process of neutrophil recruitment that includes rolling, adhesion and transmigration. On endothelial cells, selectins (e.g., P- & E-selectin) are responsible for neutrophil capture and rolling, while adhesion molecules ICAM-1, VCAM-1 and PECAM-1 are critical regulators of neutrophil firm attachment and migration.

Figure 3:

Use of microphysiological systems in radiobiological research: (A) micro engineered 3D scaffolds; (B) a novel in vitro biomimetic microfluidic assay (bMFA) developed to study radiation-induced endothelium damage; C) map of microvascular networks in animals obtained using intravital microscopy; D) vascular network reproduced on polydimethylsiloxane device; E) the bMFA includes vascular channels that are connected to the tissue compartment through a 3 μm barrier; F) EC are aligned in the direction of flow in the bMFA (scale bar 250 μm); (G) confocal microscopy demonstrates that EC form a complete 3D lumen in the vascular channel. F-actin is labeled in green, and nuclei are labeled in red. [Figures 3C–3G: reproduced with permission from reference [41]]

Figure 4:

PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage; (A) Neutrophil migration across irradiated human EC increases over time by up to 20-fold at 60 minutes. PKCδ-TAT inhibitor (PKCδ-i) at 24 hours post-IR significantly reduces neutrophil migration by up to 82% after 60 minutes; (B) Dextran permeability of EC exposed to irradiation is significantly increased. Treatment of cells with PKCδ-i restores permeability to control levels (0 Gy). Data are normalized with respect to the permeability of EC with no treatment; (Mean±SEM, n=3/group, * p<0.05, ** p<0.01, *** p<0.001). (C) EC are aligned in the direction of flow under control conditions; (D) whereas in response to 5Gy IR, they are not as well aligned and denuded (solid arrows); (E) PKCδ-i 24 hrs post-IR prevents denuding of EC which align in the direction of flow (open arrow); green: VE-cadherin (adherens junction); red: phalloidin (actin filament); blue: Hoechst 33342 (cell nucleus). [Reproduced with permission from reference [41]]; (F) all control mice whole body irradiated with 7Gy treated with PBS died between days 11 and 12 post-IR, while 80% of mice whole body irradiated with 7Gy treated with PKCδ-i lived to days 12-16 post-IR, with one mouse living for >60 days (→) post-IR when it was euthanized as required by our animal protocol (n=5/group).