**3.1. Activation of the complement system**

Posthypoxic vasoconstriction, in response to vasoconstrictors, and endothelium-independ‐ ent vasodilation, induced by direct vasodilators (direct action on VSMCs), are slightly af‐ fected by I/R, demonstrating the relative resistance of VSMCs. [8]-[10] In contrast, endothelium-dependent dilatation is deeply affected. Despite the fact that endothelial cells seem relatively more resistant than other cells types (cardiomyocytes, neurons, renal tubular cell), I/R modifies their phenotype: diminution of their anticoagulant properties, increased vascular permeability, increased leukoadhesivity and establishment of a proin‐

The production of some bioactive agents decreases (e.g., prostacyclin, nitric oxide), while that of others increases during I/R (e.g., endothelin, thromboxane A2). [1],[11]-[16] These endothe‐ lial modifications are called endothelial dysfunction and are widely described in human and animals studies.[15],[17]-[21] IR-related endothelial dysfunction is mainly characterized by the loss of NO availability and seems to be related to the reperfusion more than to ischemia. [10] In normal situations, NO acts in numerous pathways: direct vasodilation, indirect vasodilation by inhibiting the influences of vasoconstrictors (e.g., inhibiting angiotensin II and sympathetic vasoconstriction), inhibiting platelet adhesion to the vascular endothelium (anti-thrombotic effect), inhibiting leukocyte adhesion to vascular endothelium (anti-inflammatory effect), and inhibiting smooth muscle hyperplasia by scavenging superoxide anion (anti-proliferative

Multiple hypotheses have been proposed to explain postischemic endothelial dysfunction: massive ROS production by mitochondria, activation of immune cells, activation of xanthine oxidase and NADPH2 oxidase by the ceramide/sphingosine kinase pathway, the depletion of dihydrobiopterin (an essential cofactor of nitric oxide synthase), increased arginine consump‐ tion in other intracellular pathways, the production of chemokines and cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-1, -6, and -8) or the activation of the complement

In normoxic conditions, the endothelium permits only restricted diffusion. During hypoxia, the modifications of the cytoskeleton of endothelial cells, induced by hypoxia and low intracellular cyclic adenosine monophosphate phosphate (cAMP) concentration, increase vascular permea‐ bility, leading to capillary leakage and perivascular interstitial edema.[1] Complement system activation, leukocyte endothelial adhesion and platelet-leukocyte aggregation increase after re‐ perfusion.[1],[32] A clinical example is the acute respiratory failure with hypoxia and pulmona‐ ry edema observed in several surgeries. Acute respiratory distress syndrome is caused by heart

Ischemia-reperfusion induces a vigorous inflammatory reaction including activation of the complement system; activation of the innate and adaptive immune systems; increased ROS, cytokine, chemokine and other proinflammatory metabolite production; and activation of programmed cell death. If inflammation concerns mainly ischemic organs, its effects will

effect). The diminution of NO concentration jeopardizes these functions.

failure but also by a disruption of the alveolar-capillary barrier.[33]-[36]

flammatory state in the endovascular milieu.

20 Artery Bypass

system (C3a fraction, C5b-9 fraction). [21]-[31]

**3. The inflammatory response**

Reperfusion injury is characterized by autoimmune responses, including natural antibodies recognizing neoantigens and subsequent activation of the complement system (auto-im‐ munity). 1 Locally produced and activated, the complement system amplifies inflammation during ischemia and reperfusion through complement-mediated recognition of damaged cells and anaphylatoxin release. The anaphylatoxins C3a, C4a and C5a lead to the recruit‐ ment and stimulation of immune cells, which promotes cell-cell interactions by increasing the expression of adhesion molecules (vascular cell adhesion molecule-1, ICAM-1, E-selectin and P-selectin) on the surface of the endothelial cells and neutrophils. [12],[39] Moreover, C5a is a chemotactic factor that directly stimulates leukocytes to synthesize and secrete cyto‐ kines such as interleukin (IL)-1, IL-6, monocyte chemoattractant protein-1 (MCP-1) and TNF-α. iC3b is implicated in neutrophil-endothelium interactions. C5b-9, known as the final cytolytic membrane attack complex complement, is a powerful chemotactic agent that caus‐ es direct lesions to the endothelial cells, stimulates the endothelial production of IL-8, MCP-1, and ROS and inhibits endothelium-dependent vasodilatation. [12],[39]

### **3.2. Cell-cell interactions during reperfusion**

### *3.2.1. Neutrophil–endothelium interaction*

During reperfusion, neutrophils play a central part in the inflammatory response and in the genesis of the I/R injuries. Activated neutrophils produce high amounts of cytokines, che‐ mokines, and ROS in the vascular lumen but also in the parenchyma that directly contacts cells. These neutrophils and endothelial cells activated by cytokines (e.g., IL-6, TNF-α, IL-8, IL-1β) and other proinflammatory mediators (e.g., platelet-activating factor, ROS) promote a close interaction between these cell types that will result in a significant concentration of ac‐ tivated neutrophils in the interstitium. [1],[13],[15],[17],[32],[40]-[43] This complex process can be summarized in four steps: chemoattraction, weak neutrophil adhesion to the endo‐ thelium, followed by a stronger adhesion and, finally, neutrophil migration (Figure 1). Three families of sarcoplasmic adhesion molecules are implicated in the neutrophil-endothelium interaction: selectins, β2-integrins and immunoglobulins.

**•** Chemoattraction:

Upon reperfusion, the endothelium, parenchyma and resident immune cells (mainly macro‐ phages and neutrophils) release cytokines such as IL-1, TNF-α and chemokines, inducing the production of selectins by endothelial and immune cells. Circulating leukocytes are concen‐ trated towards the site of injury by the concentration gradient of chemokines.

**•** Rolling adhesion

Endothelial L-selectin interacts with the P-selectin and the E-selectin-specific ligand-1 (ESL-1) expressed by neutrophils. [44],[45] The activation of TLR-2, ROS production, the complement system and thrombin and a high intracellular calcium concentration promotes the expression of endothelial P-selectin from the Weibel–Palade bodies. Its peak of expression occurs 10–20 min after the beginning of reperfusion.[40],[46] P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) expressed by neutrophils. These interactions are weak and reversible, providing transitory neutrophil adherence, slowing down leukocytes and allowing them to "roll" along the endothelial surface. During this rolling motion, transitory bonds are formed and broken between selectins and their ligands. This phase prepares the neutrophils and the endothelium for the following stage.

**•** Migration into the interstitium or diapedesis

of injury, where it causes considerable damage.

its granules directly near the cell. [54],[55]

jeopardize the quality of the microcirculation. 60

**3.3. Reactive oxygen species or oxygen free radicals**

Reactive oxygen species, such as superoxide anion (O2 <sup>−</sup>

*3.2.2. Neutrophil-platelet interaction*

hydroxyl radical (OH<sup>−</sup>

Intercellular adhesion molecule-1 (ICAM-1) and platelet-endothelium adhesion molecule-1 (PECAM-1) are sarcoplasmic adhesion molecules belonging to the superfamily of the immu‐ noglobulins. They are implicated in the transfer of neutrophils towards the interstitium, termed diapedesis. Leukocytes extravasation comprises many stages, which are not fully understood. Nevertheless, it seems that PECAM-1, found on neutrophil and endothelial cell membranes, is necessary for diapedesis. [1],[49] It interacts with several sarcoplasmic pro‐ teins of neutrophils. The cytoskeleton of the neutrophil is reorganized to allow the projec‐ tion of pseudopodia between endothelial cells. This transfer is facilitated by inflammatory mediators, the CD11/CD18–ICAM-1 interaction and ROS, which combine to decrease the ex‐ pression of cadherin and induce the phosphorylation vascular endothelial-cadherin and cat‐ enin, components of the intercellular junctions. [50]-[53] There is controversy concerning the mechanisms underlying this transfer through the basal membrane of the endothelium. Once into the interstitium, the neutrophil migrates along a chemotactic gradient towards the site

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The neutrophil-related injuries in the interstitium are mainly related to the massive ROS production, proteases from the intracellular neutrophilic granules and the metabolites of arachidonic acid (PAF and LTB4). PAF and LTB4 are powerful chemoattractants that stimu‐ late neutrophil degranulation. The neutrophil granules contain proteases, collagenases, ela‐ stases, lipoxygenases, phospholipases and myeloperoxidases that digest the protein network of the extracellular matrix. For example, elastase digests substrates such as collagen types III and IV, immunoglobulins, fibronectin and proteoglycans. Several cells, such as cardiomyo‐ cytes, stimulated by IL-6, express ICAM-1. The neutrophil binds to its receptor and empties

The role of platelets in ischemia-reperfusion injuries is unclear. However, it seems that they participate directly and indirectly in posthypoxic endothelial injury. [32],[56] Platelets affect neutrophil activation by releasing thromboxane A2, platelet-derived growth factor, seroto‐ nin, lipoxygenase products, proteases and adenosine. During reperfusion, approximately 25% of the fixed platelets are directly bound to the endothelium and the remaining 75% to neutrophils linked to the endothelium. [32],[57] This platelet-neutrophil interaction potenti‐ ates the neutrophils' capacity to produce superoxide and platelet-activating factor. [58],[59] Moreover, the neutrophil-platelet aggregates contribute to the no-reflow phenomenon and

•), hydrogen peroxide (H2O2) and

), are highly reactive and able to oxide all cellular constituents, includ‐

**•** Tight adhesion

At the same time, chemokines released by endothelial and immune cells activate the rolling neutrophils. Stimulated by ROS, platelet-activating factor (PAF), IL-1, TNF-α and leukotriene B4 (LTB4), neutrophils present CD11a/CD18, CD11b/CD18 and CD11c/CD18 from intracellu‐ lar granules. These sarcoplasmic proteins interact with the iC3a fraction of the complement system and ICAM-1, an endothelial protein whose expression is reinforced by TNF-α and IL-1. [47],[48] This interaction switches from a low-affinity link to a high-affinity state and firmly attaches the neutrophil to the surface of the endothelial cell, despite the shear forces of the blood flow.

**Figure 1.** Ischemia–reperfusion-induced neutrophils accumulation in the interstitium is a mechanism described in three phases implicating specific complementary proteins. CD11b/CD18, sarcoplasmic neutrophil integrin; CO2, car‐ bon dioxide; ESL-1, E-selectin-specific ligand-1; I/R, ischemia– reperfusion; O2, oxygen; PECAM, platelet–endothelial cell adhesion molecule-1; PSGL-1, P-selectin glycoprotein ligand-1; Rec IL-8, neutrophil IL-8 receptor; ROS, reactive oxygen species; TNF-α, tumour necrosis factor-a; WPB, Weibel–Palade body.

**•** Migration into the interstitium or diapedesis

system and thrombin and a high intracellular calcium concentration promotes the expression of endothelial P-selectin from the Weibel–Palade bodies. Its peak of expression occurs 10–20 min after the beginning of reperfusion.[40],[46] P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) expressed by neutrophils. These interactions are weak and reversible, providing transitory neutrophil adherence, slowing down leukocytes and allowing them to "roll" along the endothelial surface. During this rolling motion, transitory bonds are formed and broken between selectins and their ligands. This phase prepares the neutrophils and the

At the same time, chemokines released by endothelial and immune cells activate the rolling neutrophils. Stimulated by ROS, platelet-activating factor (PAF), IL-1, TNF-α and leukotriene B4 (LTB4), neutrophils present CD11a/CD18, CD11b/CD18 and CD11c/CD18 from intracellu‐ lar granules. These sarcoplasmic proteins interact with the iC3a fraction of the complement system and ICAM-1, an endothelial protein whose expression is reinforced by TNF-α and IL-1. [47],[48] This interaction switches from a low-affinity link to a high-affinity state and firmly attaches the neutrophil to the surface of the endothelial cell, despite the shear forces of the

**Figure 1.** Ischemia–reperfusion-induced neutrophils accumulation in the interstitium is a mechanism described in three phases implicating specific complementary proteins. CD11b/CD18, sarcoplasmic neutrophil integrin; CO2, car‐ bon dioxide; ESL-1, E-selectin-specific ligand-1; I/R, ischemia– reperfusion; O2, oxygen; PECAM, platelet–endothelial cell adhesion molecule-1; PSGL-1, P-selectin glycoprotein ligand-1; Rec IL-8, neutrophil IL-8 receptor; ROS, reactive

oxygen species; TNF-α, tumour necrosis factor-a; WPB, Weibel–Palade body.

endothelium for the following stage.

**•** Tight adhesion

22 Artery Bypass

blood flow.

Intercellular adhesion molecule-1 (ICAM-1) and platelet-endothelium adhesion molecule-1 (PECAM-1) are sarcoplasmic adhesion molecules belonging to the superfamily of the immu‐ noglobulins. They are implicated in the transfer of neutrophils towards the interstitium, termed diapedesis. Leukocytes extravasation comprises many stages, which are not fully understood. Nevertheless, it seems that PECAM-1, found on neutrophil and endothelial cell membranes, is necessary for diapedesis. [1],[49] It interacts with several sarcoplasmic pro‐ teins of neutrophils. The cytoskeleton of the neutrophil is reorganized to allow the projec‐ tion of pseudopodia between endothelial cells. This transfer is facilitated by inflammatory mediators, the CD11/CD18–ICAM-1 interaction and ROS, which combine to decrease the ex‐ pression of cadherin and induce the phosphorylation vascular endothelial-cadherin and cat‐ enin, components of the intercellular junctions. [50]-[53] There is controversy concerning the mechanisms underlying this transfer through the basal membrane of the endothelium. Once into the interstitium, the neutrophil migrates along a chemotactic gradient towards the site of injury, where it causes considerable damage.

The neutrophil-related injuries in the interstitium are mainly related to the massive ROS production, proteases from the intracellular neutrophilic granules and the metabolites of arachidonic acid (PAF and LTB4). PAF and LTB4 are powerful chemoattractants that stimu‐ late neutrophil degranulation. The neutrophil granules contain proteases, collagenases, ela‐ stases, lipoxygenases, phospholipases and myeloperoxidases that digest the protein network of the extracellular matrix. For example, elastase digests substrates such as collagen types III and IV, immunoglobulins, fibronectin and proteoglycans. Several cells, such as cardiomyo‐ cytes, stimulated by IL-6, express ICAM-1. The neutrophil binds to its receptor and empties its granules directly near the cell. [54],[55]

#### *3.2.2. Neutrophil-platelet interaction*

The role of platelets in ischemia-reperfusion injuries is unclear. However, it seems that they participate directly and indirectly in posthypoxic endothelial injury. [32],[56] Platelets affect neutrophil activation by releasing thromboxane A2, platelet-derived growth factor, seroto‐ nin, lipoxygenase products, proteases and adenosine. During reperfusion, approximately 25% of the fixed platelets are directly bound to the endothelium and the remaining 75% to neutrophils linked to the endothelium. [32],[57] This platelet-neutrophil interaction potenti‐ ates the neutrophils' capacity to produce superoxide and platelet-activating factor. [58],[59] Moreover, the neutrophil-platelet aggregates contribute to the no-reflow phenomenon and jeopardize the quality of the microcirculation. 60

### **3.3. Reactive oxygen species or oxygen free radicals**

Reactive oxygen species, such as superoxide anion (O2 <sup>−</sup> •), hydrogen peroxide (H2O2) and hydroxyl radical (OH<sup>−</sup> ), are highly reactive and able to oxide all cellular constituents, includ‐ ing proteins, DNA, phospholipids and other biological structures. During reperfusion, PAF, TNF-α, IL-6, IL-1β, granulocyte-macrophage colony-stimulating factor, complement fraction C5a and the ROS themselves stimulate endothelial and neutrophil ROS production. [49], [61],[62] On the other hand, ROS activate nuclear factor-κB, promote cytokine production (e.g., TNF-α, IL-6, PAF), and induce the synthesis and expression of endothelial and leuko‐ cyte adhesion molecules. [15],[41],[63]

**4. Integration of different aspects of ischemia-reperfusion**

vasopressive drugs in the postextracorporeal circulation. [83 ],[84]

According to the level of the vascular system considered (small arteries, capillaries and post‐

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The principal manifestation of I/R in arterioles is a loss of the vasodilatation-dependent en‐ dothelium and the appearance of spasms. [78] Widespread endothelial lesions decrease the production of nitric oxide and do not counterbalance the arterioles' tendency toward vaso‐ constriction. This tendency is highlighted in several tissues, such as skeletal muscle, heart, lung and brain. [79]-[82] The combined effects of IR and inflammation on arteriolar vasomo‐ tricity are well documented. The increase in the contractile response of the pulmonary and mesenteric microcirculation after cardiac surgery predisposes the patient to the develop‐ ment of pulmonary shunt or mesenteric ischemia, particularly during the administration of

The posthypoxic recovery of an organ depends on the quality of its microcirculation and the re‐ sultant nutrient delivery and gaseous exchange. However, the microcirculation is the site of a paradoxical phenomenon called "no reflow", characterized by a major reduction in the capilla‐ ry density. Despite the reestablishment of complete blood flow, an incomplete and heterogene‐ ous perfusion of microcirculation persists. [85],[86] The capillaries are blocked by the parenchymatous and endothelial edema and the adhesion of the neutrophils and platelets to the surface of the endothelium, aided by the reduction in the production of nitric oxide. [15],[81], [85]-[87] Increased ROS and the depletion of ATP modify the cytoskeleton and the intercellular junctions, contributing to the loss of liquid from the vascular bed towards the interstitium. [88],

The postcapillary veins are the sites of the inflammatory reaction. The margination and ex‐ travasation of the leukocytes are facilitated by the slower blood flow. Venous blood, arriving from the reperfused zones, is rich in proinflammatory mediators and activated neutrophils. These cause lesions both directly and indirectly through their interactions with platelets. [15],[90] Endothelial lesions prevent the intravascular oncotic pressure from recovering the excess liquid from the interstitium, thereby increasing the edema and contributing to the

In pulmonary transplantation surgery, I/R-induced lung injury is characterized by non‐ specific alveolar damage, lung edema and hypoxemia. The most severe form may lead to

[89] The phenomenon of no reflow persists several weeks after reperfusion. [85]

capillary veins), the repercussions of I/R are identical, but the clinical pictures differ.

**4.1. Blood vessel**

*4.1.1. At the arteriolar level*

*4.1.2. At the capillary level*

*4.1.3. At the postcapillary vein level*

phenomenon of "no reflow".

**4.2. Organs**

In the reperfused tissue, the principal sources of ROS are neutrophil NADPH-oxidase, xan‐ thine oxidase, mitochondria and the arachidonic acid pathways. [64]-[66] The massive ROS production quickly exceeds the capacity of cellular defense systems (catalase, superoxide dismutase, glutathione peroxidase and vitamins C and E). ROS directly cause much struc‐ tural damage, increase the susceptibility to the opening of the mitochondrial permeability transition pore, activate immune and endothelial cells and induce apoptosis. [67]

ROS can also be produced by monoamine oxidase (MAO) of the outer mitochondrial mem‐ brane. MAO transfers electrons from amine compounds with oxygen to produce hydrogen peroxide. [68] p66Shc, a cytosolic adaptor protein for tyrosine kinase receptors that has been implicated in signal transduction, translocates to the mitochondrial matrix during reperfu‐ sion and oxidizes the reduced cytochrome *c*, which generates oxygen peroxide. [67],[69]

#### **3.4. Ischemia-reperfusion-induced apoptosis**

Reperfusion is vital for the functional recovery of an ischemic organ but also initiates the apoptosis pathways. [70],[71] Apoptosis is an active mechanism of cellular death, is geneti‐ cally programmed, consumes energy, requires the expression or activation of specific en‐ zymes, and can be induced by the oxidative stress of reperfusion. Reperfusion-induced apoptosis occurs in many organs, including heart, brain, kidney and liver. The reperfusion of an organ can induce apoptosis in other, distant organs. For example, reperfusion of a low‐ er limb or the small bowel can induce apoptosis of cardiomyocytes or lung cells, respective‐ ly. [72],[73] The TNF-α production by the reperfused organ seems to play a crucial part in the induction of apoptosis. [70],[74]-[76] TNF-α initiates a receptor-dependent death path‐ way by activating downstream caspases. [70],[76],[77] Other causes of reperfusion-induced apoptosis are also important: mitochondrial depolarization, high intracellular calcium, mPTP opening and the release of some mitochondrial proteins into the cytoplasm, such as cytochrome *c*. When this protein is released from mitochondria into the cytoplasm, it inter‐ acts with apoptotic protease activating factor-1 (Apaf-1) and ATP to form the apoptosome, a large oligomeric protein complex that can activate caspase 9, which activates the caspase-de‐ pendent apoptosis pathway.

Endothelial cell apoptosis precedes and influences the apoptosis of the subjacent parenchymal cells. For example, a reduction in endothelial apoptosis decreases the apoptosis of subjacent cardiomyocytes. This suggests that signals emanating from the endothelium during apoptosis can induce or reinforce that of the cardiomyocytes.
