Ischemia-Reperfusion

Microvascular Dysfunction

The vascular endothelium is an early target of the IRI storm. The endothelium provides an anti-thrombotic barrier for blood flow and protects against permeability, aided by the endothelial glycocalyx. The endothelial cell is particularly vulnerable to the deleterious effects of IRI. During ischemia, the lack of oxygen depletes energy stores and alters the endothelial membrane potential leading to cellular stress and, if not corrected, death. The release of proinflammatory mediators disturbs both tight junctions (occludins and claudin) and adherens junctions (cadherins). The resulting increased vascular permeability and altered vascular tone sets the stage for the systemic spread of IRI. With reperfusion, nitric oxide production decreases and reactive oxygen species (ROS) production increases, contributing to an activated and pro-thrombotic endothelium. The end response to IRI is reduced blood flow and subsequent perfusion throughout capillary beds and the initiation of robust inflammatory responses.

Arterioles

In IRI, arterioles have impaired nitric oxide (NO)-mediated vasodilation and an increased reactivity leading to vasoconstriction. In a 1987 landmark study NO was found to be responsible for impaired vasodilation during IRI. Endothelial cells contain both constitutively expressed endothelial NO synthase (eNOS) and inducible NOS (iNOS). In IRI, increased arginase activity exhausts the l -arginine pool critical to NO synthesis. Impaired vasodilation contributes to vascular smooth muscle impairment and further vasoconstriction. The resultant sluggish flows reduce shear forces within the microvasculature creating a vicious cycle of malperfusion. ^, This transmural vascular dysfunction can be systemic involving key organ systems (lung, liver, intestines) and large volumes of tissues (skeletal muscle and skin).

Capillaries

Endothelial barrier dysfunction in capillaries is occlusive in nature, either through narrowing or leukocyte plugging. The vessel narrows in response to interstitial edema. Leukocyte plugging occurs in response to platelets and leukocytes attaching to the activated endothelium. Leukocytes are not deformable and can become trapped in the narrowed capillary channels. Some promising therapeutic targets have been discovered in mice that are genetically deficient in leukocyte or endothelial adhesion factors. Stall dynami, arising from the plugging of capillaries with leukocytes, can be modulated by the injection of anti-Ly6 antibodies that target neutrophils and improve penumbral blood flow. Additionally, animals that express superoxide dismutase in excess demonstrate improved microvascular perfusion in the capillaries post-IRI. Reduced damage at the capillary level may attenuate the inflammatory response both upstream in the arterioles and downstream in the venules, making this a therapeutically attractive target. This location is also the origin of the no-reflow phenomenon.

Venules

The canonical inflammatory response associated with IRI peaks in the venules. As in the capillaries, the endothelial glycocalyx is damaged early, promoting neutrophil extravasation. This is mediated by xanthine oxidase and MMP-2 and -9. Mast cells and macrophage residing in the interstitial spaces are activated by inflammation and reperfusion and migrate toward the venular endothelium, eliciting leukocyte migration and venule permeability, followed by platelet adhesion and aggregation. ^, Systemically, diffuse edema contributes to hemodynamic instability and volume depletion.

Leukocytes and Endothelial Interactions

Activated neutrophils play a major role in innate immune system–mediated tissue damage as a part of IRI. They are a major source of ROS, generated through two main pathways: (1) the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and (2) the production of myeloperoxidase from azurophilic granules. In addition to producing damaging O2− and hypochlorous acid, neutrophils produce membrane-degrading proteases, including matrix metalloproteinases (MMPs), that severely damage the basement membrane and other functional barrier proteins. This inflammatory response, termed “sterile inflammation” because of the lack of microorganisms, is essential for wound repair, but in IRI it is a major source of proinflammatory stimuli and tissue damage. ^,

Transendothelial migration occurs primarily during reperfusion and the restoration of oxygen and nutrients to the tissues. This coordinated passage is accompanied by a significant amount of plasma fluid and protein leakage, attributing to the mechanical disruption of the endothelial barrier. Upregulation of ICAM-1, VCAM-1 and E-selectin allow for margination and rolling, followed by tight adhesion and diapedesis. Once across the barrier and inside the tissues, chemotactic factors guide the migration process. TNF-alpha (TNF-α) is one of the best studied endothelial-barrier cytokines. It directly modulates endothelial permeability by regulating the expression of proinflammatory agonist-1 (PAR-1) and the TRPC1 Ca2+ channel. IL-1, along with TNF-α, is a NF-κB dependent proinflammatory cytokine that works to upregulate E-selectin and the ICAM-1 pathway of leukocyte extravasation.

The chemokine arsenal that neutrophils secrete is stored in azurophilic and specific granules. Azurophilic granules are lysosome-like organelles that contain myeloperoxidase (MPO), elastase, glycoproteins and neutral proteases. MPO catalyzes hydrogen peroxide and chloride ions to produce hypochlorous acid (HOCl), which reacts to form singlet oxygen ( ¹ O ₂ ). ROS initiate lipid peroxidation and leaky membrane bilayers. HOCl also produces hydroxyl radicals through a reaction with superoxide and ferrous iron via the Fenton reaction. These extremely destructive radicals modify DNA through strand breaks and base modifications. Specific granules are released from neutrophils during an inflammatory response and proteases, like MMP-8, are released from these organelles further degrading the extracellular membrane and remodel connective tissues. Neutrophils are known to cause remote organ injury, with increased levels seen after aortic occlusion and SIRS.