Reactive species-induced microvascular dysfunction in ischemia/reperfusion

Abstract

Vascular endothelial cells line the inner surface of the entire cardiovascular system as a single layer and are involved in an impressive array of functions, ranging from the regulation of vascular tone in resistance arteries and arterioles, modulation of microvascular barrier function in capillaries and postcapillary venules, and control of proinflammatory and prothrombotic processes, which occur in all segments of the vascular tree but can be especially prominent in postcapillary venules. When tissues are subjected to ischemia/reperfusion (I/R), the endothelium of resistance arteries and arterioles, capillaries, and postcapillary venules become dysfunctional, resulting in impaired endothelium-dependent vasodilator and enhanced endothelium-dependent vasoconstrictor responses along with increased vulnerability to thrombus formation, enhanced fluid filtration and protein extravasation, and increased blood-to-interstitium trafficking of leukocytes in these functionally distinct segments of the microcirculation. The number of capillaries open to flow upon reperfusion also declines as a result of I/R, which impairs nutritive perfusion. All of these pathologic microvascular events involve the formation of reactive species (RS) derived from molecular oxygen and/or nitric oxide. In addition to these effects, I/R-induced RS activate NLRP3 inflammasomes, alter connexin/pannexin signaling, provoke mitochondrial fission, and cause release of microvesicles in endothelial cells, resulting in deranged function in arterioles, capillaries, and venules. It is now apparent that this microvascular dysfunction is an important determinant of the severity of injury sustained by parenchymal cells in ischemic tissues, as well as being predictive of clinical outcome after reperfusion therapy. On the other hand, RS production at signaling levels promotes ischemic angiogenesis, mediates flow-induced dilation in patients with coronary artery disease, and instigates the activation of cell survival programs by conditioning stimuli that render tissues resistant to the deleterious effects of prolonged I/R. These topics will be reviewed in this article.

Keywords: Angiogenesis; Arterioles; Capillaries; Capillary no-reflow; Cell survival programs; Connexins; Endothelial permeability; Endothelium; Endothelium-dependent vasodilators; Inflammasome; Ischemia; Leukocyte adhesion; Microvesicles; Mitochondrial fission; Pannexins; Reactive species; Reperfusion; Venules.

Figure 1.

Ischemia/reperfusion (I/R) induces the generation of damaging levels of reactive species (RS) from a number of cellular sources in the vasculature (and parenchymal cells) and mitochondria. In addition to producing cell damage, reactive species derived from these sources can also induce RS formation by mitochondria and vice versa, a phenomenon designated as RS-induced RS release (RIRR). Neutrophils, other immunocytes, and platelets represent other major sources of reactive species in I/R. RS produced from these sources cause damage to all biomolecules in cells. The enhanced RS flux induced by I/R overwhelms cellular antioxidant systems, which results in oxidative stress. In the microcirculation, postischemic RS produces vasomotor dysfunction and thrombus formation in arterioles, elicits impaired nutritive perfusion (no-reflow) and increased fluid filtration in capillaries, and enhances fluid and protein efflux and provokes adhesive interactions between blood cells (e.g., platelets, neutrophils, lymphocytes) and the endothelium of postcapillary venules.

Figure 2.

Ischemia/reperfusion (I/R) causes the formation of reactive species (RS) that causes impaired vasomotor responses in resistance arteries and arterioles by several mechanisms. Endothelium-dependent vasodilator dysfunction results from impaired eNOS protein expression, reduced bioavailability of eNOS-derived nitric oxide (NO), eNOS uncoupling, oxidation of soluble guanylyl cyclase (sGC), mitochondrial permeability transition (MPT) pore opening, increased arginase expression and activity, and reductions in cofactors required for eNOS function. Postischemic RS production also impairs arteriolar vasoconstrictor responses to norepinephrine (NEPI), angiotensin II (AII), and vasopressin (AVP). On the other hand, I/R results in enhanced arteriolar production and responsiveness to endothelin, which produces paradoxical coronary vasoconstriction by direct receptor activation and indirectly by induction of RS formation, which reduces NO bioavailability.

Figure 3.

Ischemia/reperfusion (I/R)-induced formation of reactive species (RS) elicits capillary noreflow, which impairs nutritive perfusion, even though feed arteries were successfully recanalized. Mechanisms induced by dysfunction arising in endothelial cells relate primarily to promotion of adhesive interactions with leukocytes and platelets, disruption of the endothelial glycocalyx, as well swelling and formation of blebs. Contributions attributable to leukocytes include the formation of neutrophil extracellular traps (NETs) and disruption of endothelial barrier function secondary to leukocyte transmigration. Contraction of pericytes localized at capillary orifices and along the length of capillaries compress luminal diameter, thereby impairing nutritive perfusion. Disruption of endothelial barrier function leads to increased microvascular fluid filtration from the blood to the tissue space. The excessive accumulation of fluid in the tissue spaces (interstitial edema) raises tissues pressure and physically compresses capillaries and postcapillary venules, an effect exacerbated by endothelial cell swelling and cell edema in the parenchyma). Postischemic fluid efflux across microvessels also increases microvascular hematocrit, which in turn reduces blood fluidity, thereby increasing vascular resistance to blood flow.

Figure 4.

Mechanisms underlying I/R-induced, RS-mediated, Drp1-dependent mitochondrial fission causes mitochondrial permeability transition and apoptosis to evoke microvascular dysfunction (decreased eNOS activity, capillary no-reflow, and endothelial barrier disruption). See text for further explanation. Abbreviations: I/R = ischemia/reperfusion, BI1 = Bax inhibitor, XO = xanthine oxidase, RS = reactive species, NRF4A1 = nuclear receptor subfamily 4 group A member 1, CK2α = serine/threonine kinase casein kinase2α (CK2α), p-Mff = phosphorylated mitochondrial fission factor, Drp1 = dynamin-related guanosine triphosphatase protein 1, FLNa = actin binding protein filamin A, MitoRS = mitochondrial RS, VDAC1 = voltage-dependent anion channel 1, HK2 = hexokinase 2, MPTP = mitochondrial permeability transition pore, eNOS = endothelial nitric oxide synthase.

Figure 5.

Ischemia/reperfusion (I/R) causes the expression of growth factors (eg, vascular endothelial growth factor (VEGF)) to provoke angiogenesis days after reperfusion is initiated. VEGF interacts with VEGF receptor 2 (VEGFR2) to directly provoke the formation of reactive species (RS) by NADPH oxidases isoform 4 (NOX4). This induces NOX2-dependent RS formation, which can be enhanced by VEGF-initiated, Rac1-dependent signaling. VEGF-induced NOX4/NOX2 signaling leads to phosphorylation of p66^Shc, a RS source in mitochondria. This RS-induced RS release signaling mechanism results in phosphorylation of VEGF (p-VEGF), which activates VEGFR2-dependent downstream effectors to promote angiogenesis.

Figure 6.

Antecedent ethanol ingestion evokes the appearance of an anti-inflammatory phenotype in postcapillary venules by a mechanism initiated by reactive species (RS) derived from NADPH oxidase (NOX) and xanthine oxidase. These RS interact with nitric oxide formed by endothelial nitric oxide synthase (eNOS) to form reactive nitrogen oxide species which activate transient receptor potential dependent vanilloid 1 (TRPV1) channels on sensory neurons. Subsequent neuronal release of calcitonin gene-related peptide (CGRP) activates CD4⁺ T cells to provoke the release of tumor necrosis factor-α (TNFα). Stimulation of resident and/or emigrated neutrophils by TNFα results in matrix metalloproteinase (MMP) release and activation. MMP-dependent cleavage of extracellular matrix proteins exposes matricryptins such as αvβ3 ligands that ligate and activate large conductance, calcium-activated potassium channels (BKCa), which form RS and activate Nrf2/ARE-dependent heme oxygenase-1 (HO-1) expression and activity. The enzymatic products of HO-1 are anti-adhesive and exert anti-oxidant effects to prevent postischemic RS production, leukocyte adhesion, and microvascular barrier function, thereby producing an anti-inflammatory phenotype and limit I/R injury. Conditioning with short bouts of I/R (ischemic conditioning) or a variety of pharmacologic agents, such as BKCa agonists TNF receptor agonists, also activate cell survival programs by RS-dependent signaling.