Turn Up the HETE on Septic Shock

Sepsis is life-threatening organ dysfunction caused by a dysregulated response to infection.1 Annually, sepsis affects 1.7 million adults in the United States and is responsible for 270,000 deaths.2–4 The global burden exceeds 19 million cases/yr and 5 million deaths.5 As summarized in Figure 1, the pathogenesis of inflammatory shock begins with the digestion of gram-negative bacterium by macrophages and the release of lipopolysaccharide (LPS). This potent endotoxin interacts with an LPS binding protein, CD14, and TLR4 Toll-like receptors to stimulate the release of inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, and interferon-γ) by monocytes, macrophages, and neutrophils. The increase in circulating cytokines trigger the release of numerous inflammatory mediators from cells throughout the body and activates the blood-clotting cascade.2 One of the inflammatory mediators, prostaglandin E1, acts on the hypothalamus to cause fever and elevate tissue metabolism and oxygen demand. Lipoxygenase metabolites of arachidonic acid and cysteinyl leukotrienes are released, which augment vascular leakage and the inflammatory response. Finally, LPS and inflammatory cytokines increase levels of histamine, bradykinin, serotonin, nitric oxide (NO), cyclooxygenase, and cytochrome P450 (CYP) metabolites of arachidonic acid that promote vasodilation, vascular leakage, and edema formation.4 These mediators initially produce a compensated hyperdynamic state characterized by fever, elevated heart rate, and peripheral vasodilation, which is sufficient to meet the increased oxygen demands of the tissues. However, the high vascular permeability causes loss of intravascular volume that eventually reduces venous return and cardiac output. The decrease in cardiac output, along with the vasodilation associated with the release of inflammatory mediators, causes hypotension refractory to vasoconstrictors, followed by reflex vasoconstriction, disseminated intravascular coagulation, and endothelial dysfunction. This decompensatory cascade ultimately leads to tissue hypoperfusion below the level necessary to sustain cellular metabolism, resulting in cell death and multiorgan deficiency, primarily in the liver, kidney, and small intestines. In addition, pulmonary vascular resistance is elevated, leading to pulmonary edema, respiratory distress, and cardiac dysfunction and failure.2

F1FIGURE 1.: Central role of iNOS and 20-HETE deficiency in septic shock. Lipopolysaccharide (LPS) is released from bacterial cell membranes by macrophages. LPS stimulates the release of various cytokines, including tumor necrosis factor-TNFα, interferon-gamma (IFN-γ), interleukins-1, 6, and 12 (IL1, IL6, and IL12). These cytokines activate coagulation pathways leading to disseminated intravascular coagulation (DIC), upregulation of the expression of inducible nitric oxide synthase (iNOS), formation of nitric oxide (NO), and increased formation of leukotriene B4 (LTB4) and prostaglandins (PGs) that promote fever, inflammation, and vasodilation. NO binds to heme in cytochrome P4504A—CYP4A enzymes and inhibits the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) in arteries and arterioles. The fall in 20-HETE levels reduces the activity of the newly discovered G protein 75/20-HETE receptor (GPR75). This increases the activity of the large-conductance, Ca2+-activated potassium channel (KCa), causing membrane hyperpolarization and vasodilation. The vasodilation and LTB4-induced capillary leakage reduce venous return and cardiac output. Reduced cardiac output in conjunction with peripheral vasodilation causes severe refractory hypotension and tissue hypoperfusion leading to multiorgan dysfunction, especially in the liver, kidney, heart, and gastrointestinal tract. Further details regarding the downstream GPR75/20-HETE signaling pathways can be found in the current study of Tunctan et al1 and previous studies by others.20,21

Despite intensive investigation and numerous clinical trials, there are still no effective pharmacological treatments for septic shock. Current management relies on supportive care, including the use of antibiotics to control the infection, fluid resuscitation to maintain intravascular volume, corticosteroids to reduce inflammation, and the maintenance of mean arterial pressure (MAP) >60 mm Hg with pressor agents to ensure adequate tissue perfusion.2,4,5

INHIBITORS OF NITRIC OXIDE SYNTHESIS FOR THE TREATMENT OF SEPTIC SHOCK

Previous studies have shown that the expression of inducible nitric oxide synthase (iNOS) is markedly enhanced in many tissues by LPS, which is the most potent microbial signaling molecule in septic shock. NO is a potent vasodilator that causes hypotension resistant to vasopressors and tachycardia similar to that seen following the administration of LPS or in septic shock models. These observations naturally led to the study of NO synthesis inhibitors to treat septic shock. Administration of broad-spectrum NO synthesis inhibitors or aminoguanidine, a more selective iNOS inhibitor, attenuated the fall in blood pressure following administration of LPS or induction of septic shock.6 Similarly, the knockout of iNOS attenuated the fall in MAP and the lethal effects of LPS. However, considerable evidence concerning the detrimental effects of NO inhibitors in septic shock emerged. Inhibitors of NO increase pulmonary vascular resistance and promote pulmonary edema, respiratory distress, and heart failure. They also increase intravascular thrombosis and ischemic injury in the intestines, liver, and kidney, compromise cardiac function, and have cytotoxic effects. The high incidence and severity of these side effects led to the abandonment of NO inhibitors to treat septic shock.6 Others advocated the use of methylene blue, an inhibitor of guanylyl cyclase. Methylene blue is relatively well tolerated and increases peripheral vascular resistance and MAP in shock patients allowing for a reduction in the use of pressor agents. However, although methylene blue improves hemodynamics, it does not reduce mortality, perhaps because of its short-duration action.7

INHIBITION OF 20-HETE PRODUCTION CONTRIBUTES TO THE VASODILATOR EFFECTS OF NO

NO mediates the effects of endothelium-dependent vasodilators and contributes to the loss of vascular reactivity and hypotension seen in septic shock.2,8 It is widely accepted that the vasodilator effects of NO in the aorta and large arteries are due to the activation of guanylyl cyclase that elevates cGMP and activates cGMP-dependent protein kinase (PKG) to increase the conductance of the large-conductance, Ca-sensitive, K+ (KCa) channel. The opening of this channel hyperpolarizes vascular smooth muscle cells (VSMCs) and blocks calcium influx. NO and PKG also interact with actin and myosin cross-bridge formation to attenuate the contractile response to elevations in cytoplasmic Ca2+ in VSMCs.9–11 However, the vasodilator response to NO in several vascular beds was found to be partially cGMP independent. These reports triggered a search for alternative pathways by which NO promotes vasodilation. Several studies reported that NO binds to iron in heme-containing proteins and inhibits CYP enzymes responsible for hepatic drug metabolism and the formation of 20-HETE in the microcirculation.4,12,13 20-HETE is an ω-hydroxylation product of arachidonic acid that is produced by CYP enzymes of the 4A and 4F families in the kidney, heart, liver, brain, lung, and vasculature. 20-HETE augments vascular tone by activating PKC, MAPK, tyrosine kinase, and Rho kinase, which promotes Ca2+ entry through depolarization of VSMCs secondary to blockade of the KCa channel and by activating transient receptor potential canonical 6 and L-type calcium channels.10,11,14 Elevations in transmural pressure increase the formation of 20-HETE in renal, cerebral, and mesenteric arterioles. Inhibitors of the formation of 20-HETE attenuate the myogenic response of these vessels in vitro and autoregulation of renal and cerebral blood flow in vivo. The formation of 20-HETE in VSMCs is stimulated by angiotensin II, norepinephrine, and endothelin, and 20-HETE inhibitors attenuate the constrictor responses to the compounds by 50%.10,11

Given the importance of 20-HETE in the regulation of KCa channel activity, several studies examined whether a fall in vascular 20-HETE levels might contribute to the vasodilator response to NO. The addition of an NO donor to renal arterioles and microsomes increased absorption at 440 nm, characteristic of the formation of an iron–nitrosyl complex in the heme-binding site of CYP enzymes, and it completely inhibited the formation of 20-HETE.15 Similar results were reported for the effects of NO donors on the formation of 20-HETE in renal and cerebral arterioles, isolated glomeruli, and renal microsomes.9,12,13,16 NO donors activate the KCa channel in VSM cells isolated from renal or cerebral arterioles of rats. Blockade of guanylyl cyclase had no effect on the response of KCa channels to NO donors, but this effect was entirely blocked by preventing the fall in 20-HETE levels by adding it to the bath. Inhibitors of the formation of 20-HETE mimicked the effects of NO on the KCa channel and vascular tone and attenuated the subsequent vasodilator responses to NO donors or the endothelial-dependent vasodilators acetylcholine and bradykinin by 70%. Pretreatment of rats with a 20-HETE inhibitor reduced the fall in MAP and the renal vasodilator response to an NO donor in vivo by >50%. It also attenuated the increase in MAP and fall in renal blood flow caused by an NOS inhibitor.10,11 These studies indicate that 20-HETE inhibitors attenuate the hemodynamic responses to NO, especially in the peripheral microcirculation.

EVIDENCE THAT A 20-HETE MIMETIC PREVENTS SEPTIC SHOCK BY ACTIVATING THE NEWLY DISCOVERED GPR75/20-HETE RECEPTOR

Given the importance of 20-HETE in the control of vascular tone and that it contributes to the vasodilator effects of NO in the microcirculation, a group from the Mersin University in Turkey, headed by Bahar Tunctan, published a remarkable series of studies to determine if preventing the fall in 20-HETE levels prevents septic shock in rats and mice.1,4,17–19 They first demonstrated that administration of LPS to rats produces a fall in MAP and increased heart rate (HR) over a 4-hour course of their experiments.18 This was associated with an increase in iNOS expression and a decrease in plasma 20-HETE levels.4,19 They found that administration of a stable synthetic analog of 20-HETE, N-[20-hydroxyeicosa-5(Z), 14(Z)-dienoyl]glycine (5,14-HEDGE) prevented LPS-induced vascular hyporeactivity, hypotension, and tachycardia in endotoxemic rats, as well as LPS-induced mortality in mice. Decreased levels of proinflammatory cytokines (ie, TNF-α, IL-1β, and IL-8), neutrophil infiltration, and lipid peroxidation were also observed.4,19 Moreover, a competitive antagonist of 20-HETE reversed the effects of 5,14-HEDGE on vascular reactivity, MAP, and HR, as well as changes in the expression/activity of CYP4A1, iNOS, PKG, COX-2, and NADPH in various tissues in the LPS rat model of septic shock.4 These studies advanced the concept that 20-HETE agonists might be a novel therapeutic agent for the treatment of septic shock. However, these findings are not well recognized because LPS and/or NO have yet to be directly shown to block vascular 20-HETE production in septic shock, and the mechanisms for the loss of vascular reactivity and hypotension have not been clearly established. For example, it remains determined if inhibitors of NO synthesis prevent the fall in vascular 20-HETE production. Moreover, the signaling pathways and mechanisms by which 20-HETE mimetics prevent the loss of vascular reactivity and hypotension in septic shock, and the cell types involved are unknown.

In this regard, Garcia et al recently identified the first 20-HETE receptor.20,21 They found that 20-HETE affects vascular function by binding to the Gαq protein–coupled receptor, GPR75, which was previously identified as a receptor for the chemokine, CCL5.20,22 In human umbilical vein endothelial cells expressing GPR75, 20-HETE caused dissociation of the Gαq/11 subunit and recruited a scaffolding protein GPCR-kinase interacting protein-1 (GIT1), which facilitated Src-mediated transactivation of the epidermal growth factor receptor (EGFR). EGFR transactivation leads to the inhibition of eNOS activity, increased formation of reactive oxygen species, and endothelial dysfunction. In rat aortic VSMCs, GPR75 activation was associated with Gαq/11/PKC and Src-mediated phosphorylation of the KCa channel and enhanced the response of rat renal interlobar arteries to phenylephrine.20,21 The present study by Tunctan et al1 investigated whether the activation of GPR75-mediated signaling in various vascular beds is responsible for the ability of 5,14-HEDGE to prevent vascular hyporeactivity, hypotension, and tachycardia in the LPS-induced rat model of septic shock. Recent studies have shown that GPR75 is highly expressed in the brain, cerebral arterioles, and pericytes,23,24 but very little is currently known about its expression in the aorta and other vessels throughout the rest of the body.1,21 Moreover, studies have yet to demonstrate whether the effects of 20-HETE on KCa channel activity or vascular tone are mediated by its effects on the GPR75 receptor.21 Endogenous production of 20-HETE has to have a significant influence on basal vascular tone throughout the body to explain how a loss of 20-HETE production could be involved in septic shock. To address these issues, Tunctan et al first validated a new GPR75 antibody (GTX55193; GeneTex, Hsinchu City, Taiwan) and showed that it is expressed in the aorta mesenteric, renal, and pulmonary arteries. Then, they performed immunoprecipitation studies and demonstrated that LPS dissociated Gαq/11 and GIT1 from the GPR75 receptor and that 5,14-HEDGE prevented this effect.1 LPS also decreased the coupling of GIT1 and PKC and increased the association of PKC with c-Src. Finally, LPS reduced the association of PKC with the KCa channel, and these effects were prevented by 5,14-HEDGE.1 This is the first study to indicate that the vascular hyporeactivity, hypotension, and tachycardia in the LPS-induced septic shock model are mediated by induction of iNOS that inhibits CYP4A enzymes and the production of 20-HETE in both conduit arteries and peripheral arterioles. This leads to a reduction in GPR75 signaling that increases in KCa channel activity, membrane hyperpolarization, and vascular hyporeactivity to pressor agents. The very exciting take-home message is that preventing the fall in 20-HETE levels using a novel 20-HETE mimetic prevented LPS-induced vascular hyporeactivity, hypotension, and tachycardia in rats by activating the newly discovered GPR75/20-HETE receptor.1 Unlike its effects in the peripheral vasculature, 20-HETE is a vasodilator in the pulmonary circulation.10,14 Thus, a fall in 20-HETE levels may also be responsible for the increase in pulmonary vascular resistance, pulmonary hypertension and edema, and cardiac dysfunction in septic shock.2

Overall, these results support the hypothesis that inhibition of the formation of 20-HETE and decreased GPR75 signaling contribute to vascular hyporeactivity, reflex tachycardia, hypotension, and tissue hypoxia in septic shock. Moreover, pretreatment of the rats with a 20-HETE mimetic attenuated the development of septic shock, suggesting that it may be effective during the systemic inflammatory response syndrome of sepsis and possibly during the early compensated hyperdynamic stage of septic shock. However, it remains to be determined if 20-HETE mimetics will improve outcomes in the later refractory hypoperfusion stage of septic shock, and their potential adverse effects are unknown. In this regard, previous studies established that inhibitors and the knockout of COX-2 improved outcomes in animal models of septic shock. However, human clinical trials failed to establish any significant benefit of nonsteroidal anti-inflammatory drugs in sepsis.25 Nevertheless, the current study of Tunctan et al1 is a significant step forward and provides a very compelling case for the further development of 20-HETE mimetics in clinical trials for the treatment of septic shock.

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