Inhibition of myeloperoxidase decreases vascular oxidative stress and increases vasodilatation in sickle cell disease mice

Abstract

Activated leukocytes and polymorphonuclear neutrophils (PMN) release myeloperoxidase (MPO), which binds to endothelial cells (EC), is translocated, and generates oxidants that scavenge nitric oxide (NO) and impair EC function. To determine whether MPO impairs EC function in sickle cell disease (SCD), control (AA) and SCD mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC). SCD humans and mice have high plasma MPO and soluble L-selectin (sL-selectin). KYC had no effect on MPO but decreased plasma sL-selectin and malondialdehyde in SCD mice. MPO and 3-chlorotyrosine (3-ClTyr) were increased in SCD aortas. KYC decreased MPO and 3-ClTyr in SCD aortas to the levels in AA aortas. Vasodilatation in SCD mice was impaired. KYC increased vasodilatation in SCD mice more than 2-fold, to ∼60% of levels in AA mice. KYC inhibited MPO-dependent 3-ClTyr formation in EC proteins. SCD mice had high plasma alanine transaminase (ALT), which tended to decrease in KYC-treated SCD mice (P = 0.07). KYC increased MPO and XO/XDH and decreased 3-ClTyr and 3-nitrotyrosine (3-NO₂Tyr) in SCD livers. These data support the hypothesis that SCD increases release of MPO, which generates oxidants that impair EC function and injure livers. Inhibiting MPO is an effective strategy for decreasing oxidative stress and liver injury and restoring EC function in SCD.

Although polymorphonuclear neutrophils (PMN) are well recognized for playing important roles in inflammation in sickle cell disease (SCD), direct evidence linking myeloperoxidase (MPO) to impaired vascular endothelial cell (EC) function in SCD remains lacking. Over the last 25 years, only a handful of studies have been published suggesting that MPO may be involved. One of the first reports, published in 1986, showed that vaso-occlusive crises increased PMN stickiness, an index of activation, in SCD patients (1). In 1993, plasma levels of MPO and C3d were reported to be higher in SCD patients than in controls, and these biomarkers inversely correlated with red cell hemoglobin (2). In 1996, PMN were shown to interact directly with the sickle red blood cell (RBC), and this interaction notably enhanced PMN activation (3). Later in 2002, it was reported that activated PMN increased sickle RBC retention in isolated rat lungs (4) and that MPO and 3-nitrotyrosine colocalized in sections of lungs from patients who died from complications associated with SCD (5). Evidence that stress increases MPO in SCD comes from studies showing that exercise induced a dual increase in plasma MPO and percentage of sickled cells in SCD patients (6). Finally, evidence linking the coagulation pathway to inflammation in sickle cell anemia comes from studies showing that inhibiting tissue factor decreased MPO in the lungs of SCD mice (7). Although these reports and others (8–10) provide strong support for the idea that PMN activation and MPO play important roles in SCD, direct proof of MPO increasing oxidative stress, vascular EC dysfunction, and liver injury in SCD remains lacking.

In the present report, control (AA) and SCD mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC), a novel tripeptide inhibitor of MPO that dose-dependently inhibits MPO HOCl production, MPO-mediated LDL oxidation, and MPO-dependent EC injury and death (11). Our studies show that KYC decreases soluble L-selectin (sL-selectin), oxidative stress, and liver injury and that it improves vascular EC function in SCD mice.

MATERIALS AND METHODS

Humans

Informed consent was obtained from patients and/or guardians of children. All patients in the study had SCD as documented by quantitative hemoglobin (Hb) electrophoresis or high-performance liquid chromatography (n = 6). Patients attended the Wisconsin Sickle Cell Disease Comprehensive Center in Milwaukee, WI. Controls consisted of patients without SCD (n = 6). Human studies were approved by the Institutional Review Board committees of the Medical College of Wisconsin (MCW) and Children's Hospital of Wisconsin.

Mice

Both male and female Berkeley SCD and AA mice were used for the studies. The genetics and hematological characteristics of MCW Berkeley SCD and AA mice have been described previously (12). All of the mice used for the studies were approximately 12–14 weeks old. Mice were housed in sterile autoclavable microisolation cages. Standard mouse chow and water were provided ad libitum. SCD and AA mice were treated with KYC (0.3 mg/kg/day via intraperitioneal or subcutaneous injection) or with an injection of an equal volume of PBS for 3 weeks. All research involving the mice was conducted in conformity with PHS policy. The Institutional Animal Care and Use Committee (IACUC) of the MCW approved all protocols.

Materials

The MPO antibody (ab65871) was from Abcam (Cambridge, MA). The 3-ClTyr antibody (HP5002) was from Cell Science (Canton, MA). The XO/XDH antibody (Cat# MS-474-P) was from NeoMarkers (Freemont, CA). The 3-NO₂Tyr antibody (Cat# 10189540) was from Cayman Chemical (Ann Arbor, MI). The β-actin antibody, a loading control, was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CD45 and anti-CD64 antibodies were from Santa Cruz Biotechnology. All other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO).

Peptide synthesis

KYC was synthesized using Fmoc [N-(9-fluorenyl)methoxycarbonyl] chemistry and purified by the Protein, Nucleic Acid Core Laboratory of the Medical College of Wisconsin or was made by Biomatik USA, LLC (Wilmington, DE). Purity was confirmed by HPLC analysis (Agilent, Model 1200) using a C-18 column (2.3 × 150 mm) and a CH₃CN gradient over 30 min (5-50%, 0.1% TFA, flow rate 0.22 ml/min). Structural authenticity of the tripeptide was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Plasma sL-selectin and MPO ELISA

sL-selectin levels in plasma were quantified using ELISA kits purchased from R & D Systems (MLS00 for mouse and BBE4B for human; Minneapolis, MN). Plasma MPO protein levels were quantified using ELISA kits from Hycult Biotech, Inc. (HK210 for mouse and HK324 for human; Plymouth Meeting, PA). All assays were performed in duplicate.

Immunofluorescence studies

Thoracic aortas were surgically removed and rinsed free of blood elements with ice-cold PBS. Livers were isolated from perfused SCD mice. The aortas and livers were fixed in 10% PBS-formalin, imbedded in paraffin, and sectioned. The slides were deparaffinized using a descending alcohol-row. After a single wash with PBS (5 min), the slides were incubated with primary antibodies against MPO (0.5 mg/ml), 3-ClTyr (1:50 dilution), 3-NO₂Tyr or XO/XDH for 30 min at 37°C. Aortic sections were stained for anti-CD45 and anti-CD64 to detect leukocytes and PMN, respectively. The slides were washed with PBS (2 × 5 min) followed by incubation with the appropriate secondary antibody labeled with Alexa 488 (Invitrogen, Carlsbad, CA) for 30 min at 37°C. Finally, the slides were washed with PBS (2 × 5 min) followed by a 3 min incubation with To-Pro3 (1 µg/ml, Invitrogen), and then sealed with an aqueous mounting media and stored at −20°C until analysis by confocal microscopy.

Vasodilatation

Mice were anesthetized and then euthanized by exsanguination. The facialis arteries were isolated by microdissection, cannulated, and pressurized. Physiological responses to increasing concentrations of acetylcholine (ACh) were determined in the absence and presence of L-nitroargininemethylester (L-NAME) by videomicroscopy as previously described (13).

Effects of KYC on MPO-mediated oxidation of EC protein

MPO (10 ng/ml) and H₂O₂ (20 µM) were added to confluent human umbilical vein endothelial cell (HUVEC) cultures ± KYC (25 µM) in HBSS for 15 min at 37°C. Excess buffer was removed, cells were lysed, and cell proteins were separated by SDS-PAGE. EC proteins were transferred to nitrocellulose membranes, and membranes were immunoblotted for 3-ClTyr and endothelial nitric oxide synthase (eNOS) as described (14).

Plasma alanine transaminase measurements

Alanine transaminase (ALT) activity in plasma was measured using EnzyChrom™ Alanine Transaminase Assay Kit (BioAssay Systems, Cat# EALT-100; Hayward, CA) following the manufacturer's protocol. Briefly, 20 µl of plasma was mixed with lactate dehydrogenase (LDH), the cosubstrate, NADH, in assay buffer and then incubated at room temperature. Water was substituted for plasma to generate NADH standards while plasma and NADH were both replaced with water to generate reagent blanks. Absorbance at 340 nm was measured after incubation at 5 min and 10 min. ALT activity was calculated per manufacturer's equation: ALT = 381 × [(ODsample,5min − ODsample,10min) − (ODstd,5min − ODstd,10min)] / (ODstd,5min − ODblank,5min) (U/l). All assays were performed in duplicate.

Liver immunofluorescence studies and 3-ClTyr and 3-NO₂Tyr immunoblots

To assess the effects of SCD and KYC on oxidative stress in liver, livers were perfused in situ and then removed. One lobe was fixed in paraffin, and sections were cut for immunofluorescence studies. The other lobe was homogenized, and the homogenates were centrifuged to remove cell debris. The liver proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were immunoblotted for MPO, XO/XDH, 3-ClTyr, and 3-NO₂Tyr as described (14).

Plasma malondialdehyde

Plasma malondialdehyde (MDA), a highly reactive di-aldehyde generated from free radical oxidation of polyunsaturated fatty acids, was measured using the TBARS Assay Kit from Cayman Chemical, Inc. (Cat# 10009055; Ann Arbor, MI) following the manufacturer's published protocol with some modifications. Briefly, BHT was added to plasma samples at a final concentration of 0.05%. The BHT-treated samples were incubated with TBA reagent at 100°C for 1 h. The reaction was halted by cooling the mixture on ice. MDA-TBA adducts in the samples and standards were extracted into n-butanol. MDA levels were calculated by comparing the fluorescence (λ_Ex/λ_Em = 530/550 nm) intensity in the samples to concentration-dependent increases in fluorescence in external MDA standards.

Statistical analysis

Statistical analysis was by the Student t-test for comparisons between two groups. Vasodilatation data were analyzed using ANOVA and Boniferroni analysis for post-hoc comparisons. All data were presented as mean ± SEM unless otherwise indicated.

RESULTS

SCD patients have higher plasma MPO than controls (Fig. 1A, left). SCD mice have higher plasma MPO than AA mice. KYC had no effect on plasma MPO in SCD mice (Fig. 1A, right). SCD patients have higher plasma sL-selectin than controls (Fig. 1B, left). SCD mice have higher sL-selectin than AA mice. KYC decreased sL-selectin in SCD mice to the levels in AA mice. As sL-selectin is a biomarker of leukocyte activation and interactions with the vessel wall (10), these data suggest that KYC treatments decrease leukocyte activation and interactions with the vessel wall.

Fig. 1.

Plasma MPO and sL-selectin in humans and mice. A: Humans with SCD have increased plasma concentrations of MPO compared with apparently healthy humans (left bar graph, P < 0.05, n = 6). SCD mice also have increased plasma concentrations of MPO compared with the concentrations in AA mice (right bar graph, gray versus white bar). KYC was dissolved in PBS and administered by intraperitoneal or subcutaneous injection. KYC treatments had no effect on plasma MPO concentrations in AA (right bar graph, hatched versus white bar) or SCD mice (hatched and gray versus gray bar). Plasma MPO concentrations were increased in KYC-treated SCD mice compared with KYC-treated AA mice (right bar graph, hatched gray versus hatched bar, *P < 0.05, **P < 0.01, n = 6). B: Humans with SCD have increased plasma sL-selectin levels than apparently healthy humans (left bar graph, **P < 0.01, n = 6). SCD mice have increased plasma levels of sL-selectin compared with AA mice, which are reduced by KYC intraperitoneal or subcutaneous treatments (right bar graph, hatched gray versus hatched bar, ***P < 0.001, ****P < 0.0001, n = 6).

Low levels of MPO (green, Fig. 2A, far left) and 3-ClTyr (green, Fig. 2B, far left) were observed in sections of thoracic aortas isolated from PBS-treated AA mice. KYC treatments reduced immunostaining for MPO (Fig. 2A, second left) and 3-ClTyr (Fig. 2B, second left) below the levels observed in sections of aortas isolated from PBS-treated AA mice.

Fig. 2.

Effects of KYC on vascular MPO, 3-ClTyr, cell composition, and vessel structure. Representative images of MPO and 3-ClTyr immunofluorescence (green) relative to nuclear staining for To-Pro3 (blue) in sections of thoracic aortas isolated from control AA and SCD mice; (A) anti-MPO, (B) anti-3-ClTyr, and (C) H&E stains of sections of thoracic aortas from control AA and SCD mice treated with either PBS or KYC (n = 6).

In contrast to AA mice, high levels of MPO (green, Fig. 2A, middle right) and 3-ClTyr (green, Fig. 2B, middle right) were observed in sections of thoracic aortas isolated from PBS-treated SCD mice. MPO and 3-ClTyr staining appeared as diffuse punctuate spots throughout the smooth muscle cell layers in thoracic aortas isolated from PBS-treated SCD mice. In some images, intense MPO and 3-ClTyr staining was observed on the endothelium of aortas isolated from PBS-treated SCD mice. Control studies indicate that the adventitia have increased nonspecific staining for MPO and 3-ClTyr (data not shown). KYC reduced immunostaining for MPO (Fig. 2A, middle right) and 3-ClTyr (Fig. 2B, middle right) below that observed in sections of thoracic aortas isolated from PBS-treated SCD mice. H&E sections showed no differences in aortic structure. Few if any cells in the aortic sections stained positive for CD45 or CD64 in thoracic aorta from AA and SCD mice regardless of treatments (data not shown), confirming a lack of leukocyte or monocyte/macrophage infiltration in the aortic wall as a means of increasing vascular oxidative stress (Fig. 2C).

Although KYC treatments slightly decreased eNOS-dependent vasodilatation in AA mice, the differences between means were not statistically significant (Fig. 3A). KYC increased vasodilatation in SCD mice (black triangle for KYC group and black square for PBS group, Fig. 3B, P < 0.001). L-NAME inhibited vasodilatation of vessels isolated from the KYC treated group (white triangle) (Fig. 3B, P < 0.001), suggesting that the mechanism by which KYC improved vasodilatation was, for the most part, eNOS-dependent. Overall, KYC treatments improved ACh-induced vasodilatation by more than 2-fold (Fig. 3C).

Fig. 3.

Effects of KYC on vasodilatation . A: Acetylcholine (ACh)-induced relaxation responses for pressurized facialis arteries isolated from PBS-treated SCD mice (black squares) are impaired compared with those for arteries isolated from PBS-treated control AA mice (black triangles). KYC treatments (0.3 mg/kg/day via intraperitoneal or subcutaneous injections for 3 weeks) increased ACh-induced relaxation responses in arteries isolated from the SCD mice (inverted white triangles) to essentially the same levels as for arteries isolated from KYC-treated AA mice (white diamonds). B: ACh-induced relaxation responses for arteries isolated from KYC-treated AA mice (black triangles) tended to be slightly less than for arteries isolated from PBS-treated AA mice (black squares). However, the differences in the curves were not significant (NS). Relaxation responses of arteries isolated from PBS- and KYC-treated AA mice (black squares and triangles, respectively) were inhibited by pretreatment with L-NAME (100 µM, white squares and triangles, respectively), confirming that the mechanism of relaxation was eNOS-dependent. C: eNOS-dependent vasodilatation in arteries isolated from KYC-treated SCD mice (difference in area under the curves, black triangles minus white triangles) increased nearly 3-fold over levels in PBS-treated SCD mice (difference in area under the curves black squares minus white squares). These data demonstrate that KYC improves ACh-induced, EC- and eNOS-dependent vasodilatation in SCD mice (n = 17, **P < 0.01, ***P < 0.005, ****P < 0.001).

To determine whether KYC protects EC against MPO-dependent oxidation, we exposed EC cultures to MPO/H₂O₂ in the absence and presence of KYC. Fig. 4 shows that KYC (25 µM) reduced MPO-dependent chlorination of tyrosine in EC proteins compared with the levels in EC incubated with MPO/H₂O₂ in the absence of KYC. As the anti-3-ClTyr antibody easily discriminates between low and high concentrations of 3-ClTyr (supplementary Fig. I-A), these data clearly demonstrate that KYC inhibits MPO-dependent oxidative stress. Data in Figs. 1–4 demonstrate that KYC is highly effective at inhibiting MPO-dependent oxidation of EC in vitro and in vivo, which begins to explain how this tripeptide is able to protect and improve vascular EC function even in environments characterized by high levels of MPO-dependent oxidative stress.

Fig. 4.

Effects of KYC on MPO-dependent 3-ClTyr formation in EC. The immunoblots for 3-ClTyr and β-actin, a loading control, in lysates isolated from EC cultures treated with MPO (20 ng/ml) and H₂O₂ (25 µM) in HBSS ± KYC (25 µM) show that where MPO/H₂O₂ systems increase chlorination of tyrosine in EC proteins, adding KYC effectively reduces 3-ClTyr formation in the EC cultures to the levels observed in control EC cultures.

Many consider plasma ALT to be a biomarker of tissue injury and, more specifically, liver injury. SCD increased ALT concentrations by more than 6-fold over the concentrations in control AA mice (Fig. 5). Although KYC treatments tended to reduce ALT in SCD mice by more than 30%, the differences between the means were not of statistical significance, even though a one-tailed Student t-test yielded a P value of 0.07. Although these data are consistent with the idea that SCD induces liver injury, more sensitive and specific assays may be required to assess oxidative injury. To explore mechanisms mediating protection against oxidative injury, livers from SCD mice ± KYC treatments (0.3 mg/kg/day for 3 weeks via intraperitoneal or subcutaneous injection) were examined histologically and biochemically for changes in MPO, XO/XDH, 3-ClTyr, and 3-NO₂Tyr. KYC treatments slightly increased immunoreactivity for MPO, slightly decreased 3-ClTyr, while markedly decreasing 3-NO₂Tyr and increasing XO/XDH in SCD liver sections (Fig. 6). For a more quantitative assessment of protection, immunoblots for MPO, XO/XDH, 3-ClTyr, and 3-NO₂Tyr were performed on liver homogenates. KYC treatments significantly increased the expression of MPO and XO/XDH in SCD livers while reducing formation of 3-ClTyr and 3-NO₂Tyr in SCD liver proteins (Fig. 7).

Fig. 5.

Effects of KYC on plasma ALT. KYC treatments (0.3 mg/kg/day via intraperitoneal or subcutaneous injections for 3 weeks) had little effect on plasma ALT concentrations in AA mice (hatched versus white bar). SCD mice had higher ALT than AA mice (gray versus white bar, **P < 0.01). Although KYC treatments tended to decrease ALT in SCD mice (hatched gray versus gray bar), the differences between the means did not achieve statistical significance (P = 0.07).

Fig. 6.

Effects of KYC on expression of MPO and XO/XDH and formation of 3-ClTyr and 3-NO2Tyr in SCD livers. SCD mice were injected with KYC (0.3 mg/kg/day via intraperitoneal or subcutaneous for 3 weeks) or an equivalent volume of PBS. Immunofluorescence for MPO, XO/XDH, 3-ClTyr, and 3-NO₂Tyr indicated KYC treatments had little effect on MPO but markedly increase XO/XDH expression and decrease 3-ClTyr and 3-NO₂Tyr formation in the livers of SCD mice. H&E staining revealed no significant structural differences in the livers of KYC-treated and PBS-treated SCD mice.

Fig. 7.

Effects of KYC on expression of MPO and XO/XDH and formation of 3-ClTyr and 3-NO2Tyr in SCD livers. A: Representative immunoblots for MPO, XO/XDH, and β-actin (loading control) show that KYC treatments (0.3 mg/kg/day via intraperitoneal or subcutaneous injections for 3 weeks) increase liver MPO and XO/XDH. B: Summary data for immunoblots in A. Band densities were normalized to expression levels in livers isolated from PBS-treated SCD mice. (n = 6, *P < 0.05). C: 3-ClTyr immunoblots of homogenates of livers isolated from PBS- and KYC-treated SCD mice. D: 3-NO₂Tyr immunoblots of homogenates of livers isolated from PBS- and KYC-treated SCD mice.

To determine whether inhibiting MPO decreased oxidative stress with respect to lipids, we measured plasma MDA in SCD mice treated with PBS or KYC. Fig. 8 shows that KYC treatments decreased plasma MDA levels in SCD mice.

Fig. 8.

Effects of KYC on plasma MDA in SCD mice. The bar graph shows that KYC treatments (0.3 mg/kg/day via intraperitoneal and/or subcutaneous for 3 weeks) reduced plasma MDA levels in SCD mice (n = 6 per group, *P < 0.05). Plasma MDA levels in C57BL/6J mice were ∼1.2 µM (unpublished observations).

DISCUSSION

Our findings demonstrate that MPO plays a central role in the mechanisms by which SCD impairs vascular function. This conclusion is based on data showing that KYC decreases sL-selectin, a biomarker of leukocyte activation; decreases vascular levels of MPO and 3-ClTyr, a finger print biomarker of MPO activity; improves EC- and eNOS-dependent vasodilatation; decreases plasma MDA levels; and protects EC from MPO-dependent oxidation. At the same time KYC prevents formation of 3-ClTyr and 3-NO₂Tyr formation in SCD liver while increasing liver MPO and XO/XDH levels, suggesting that KYC, by decreasing oxidative stress, reduces liver injury and enzymatic leakage from the livers of SCD mice.

sL-selectin studies demonstrate that KYC effectively inhibits leukocyte-vascular interactions in SCD mice. Immunofluorescence studies demonstrate that KYC not only inhibits MPO binding and uptake by the vessel wall but also HOCl-mediated oxidative stress. Vasodilatation studies demonstrate that KYC dramatically improves endothelial- and eNOS-dependent vascular function in SCD mice. The fact that KYC inhibited 3-ClTyr formation in EC cultures treated with MPO and activated PMN provides additional support for the idea that KYC inhibits MPO-dependent oxidative stress to protect vascular EC function in SCD. The decrease in plasma ALT in KYC-treated SCD mice directly correlates with notable decreases in 3-ClTyr and 3-NO₂Tyr in SCD liver homogenates, confirming that KYC protects SCD livers against oxidative stress and injury.

Our findings are consistent with observations by Freeman and associates who postulated that activated leukocytes release MPO, which subsequently enters the vessel wall, increases inflammation, and generates oxidants that impair vasodilatation (15, 16). Interestingly, KYC decreased not only sL-selectin but also 3-ClTyr, a fingerprint biomarker of MPO-dependent oxidative stress in the vessel wall of the SCD mice. In addition, immunofluorescence data showed that MPO was taken up into the vessel wall. Possibly, MPO uptake by the vessel wall is also regulated in the same way as MPO is reported to regulate PMN activation and degranulation (17). As sL-selectin and MPO are released from activated leukocytes by two separate mechanisms (10), our data suggest that KYC decreases leukocyte activation and interactions with the vessel wall but not necessarily degranulation. This result seems to run counter to the report suggesting that MPO enhances PMN activation and degranulation (17). ELISA data in this initial study suggest that plasma MPO is not reduced in KYC-treated SCD mice. Possibly higher KYC concentrations are needed to decrease MPO in the SCD mice. Regardless, additional studies will be required to determine whether KYC treatments in SCD mice modulate the function of PMN, the most abundant cellular source of MPO. However, such studies are beyond the scope of the present article and will be pursued in a separate study.

KYC is a potent inhibitor of MPO activity. KYC decreases MPO-dependent HOCl production; LDL and protein oxidation; stimulated PMN-dependent MPO activity; and MPO-dependent EC injury and cell death (11). Cell culture studies reveal KYC is not cytotoxic even when used at concentrations as high a 4 mM (11). Using LC/MS/MS to quantify plasma KYC, we observed that after subcutaneous injection, KYC followed a biphasic pattern of decay. In the early phase, in the first 30 min, its plasma half-life was ∼10 min. In the late phase, after 1 h, its plasma half-life was ∼130 min (supplementary Fig. I-B). KYC decreased ALT in SCD mice (P = 0.07). This trend is consistent with other data showing that KYC protects SCD livers against MPO-dependent oxidative stress. Immunofluorescence studies and immunoblot analyses confirm that KYC increases MPO and XO/XDH expression in SCD livers and inhibits 3-ClTyr and 3-NO₂Tyr formation in SCD liver proteins. Such increases in MPO and XO/XDH can be explained by the fact that healthy livers retain MPO and XDH more than injured livers and the fact that injured livers leak enzymes.

In conclusion, KYC, is an effective, nontoxic inhibitor of MPO activity (11) that decreases MPO-dependent oxidative stress with respect to plasma lipid peroxidation and oxidation of vascular tissues, improves vascular function, and decreases oxidative injury in SCD livers. Accordingly, KYC represents a new tool that may be useful for not only gaining a better understanding of the mechanisms by which MPO increases oxidative stress and impairs vascular function in SCD but also as a possible therapy for preventing MPO-dependent inflammation and injury in SCD.