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• W2788732368 abstract "Patients with diabetes are at higher risk of developing carotid artery stenosis and resultant stroke. Arachidonoyl phospholipids affect plaque inflammation and vulnerability, but whether diabetic patients have unique carotid artery phospholipidomic profiles is unknown. We performed a comprehensive paired analysis of phospholipids in extracranial carotid endarterectomy (CEA) plaques of matched diabetic and nondiabetic patients and analyzed mass spectrometry-derived profiles of three phospholipids, plasmenyl-phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylinositol (PI), in maximally (MAX) and minimally (MIN) diseased CEA segments. We also measured levels of arachidonic acid (AA), produced by pPE hydrolysis, and choline-ethanolamine phosphotransferase 1 (CEPT1), responsible for most pPE de novo biosynthesis. In paired analysis, MIN CEA segments had higher levels than MAX segments of pPE (P < 0.001), PS (P < 0.001), and PI (P < 0.03). MIN diabetic plaques contained higher levels than MAX diabetic plaques of arachidonoyl pPE38:4 and pPE38:5 and CEPT1 was upregulated in diabetic versus nondiabetic plaques. AA levels were relatively greater in MIN versus MAX segments of all CEA segments, and were higher in diabetic than nondiabetic plaques. Our findings suggest that arachidonoyl phospholipids are more likely to be abundant in the extracranial carotid artery plaque of diabetic rather than nondiabetic patients. Patients with diabetes are at higher risk of developing carotid artery stenosis and resultant stroke. Arachidonoyl phospholipids affect plaque inflammation and vulnerability, but whether diabetic patients have unique carotid artery phospholipidomic profiles is unknown. We performed a comprehensive paired analysis of phospholipids in extracranial carotid endarterectomy (CEA) plaques of matched diabetic and nondiabetic patients and analyzed mass spectrometry-derived profiles of three phospholipids, plasmenyl-phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylinositol (PI), in maximally (MAX) and minimally (MIN) diseased CEA segments. We also measured levels of arachidonic acid (AA), produced by pPE hydrolysis, and choline-ethanolamine phosphotransferase 1 (CEPT1), responsible for most pPE de novo biosynthesis. In paired analysis, MIN CEA segments had higher levels than MAX segments of pPE (P < 0.001), PS (P < 0.001), and PI (P < 0.03). MIN diabetic plaques contained higher levels than MAX diabetic plaques of arachidonoyl pPE38:4 and pPE38:5 and CEPT1 was upregulated in diabetic versus nondiabetic plaques. AA levels were relatively greater in MIN versus MAX segments of all CEA segments, and were higher in diabetic than nondiabetic plaques. Our findings suggest that arachidonoyl phospholipids are more likely to be abundant in the extracranial carotid artery plaque of diabetic rather than nondiabetic patients. Phospholipid content in the arterial wall is altered by metabolic disorders such as diabetes and is an important contributor to the development and progression of atherosclerotic disease (1.Falk E. Pathogenesis of atherosclerosis.J. Am. Coll. Cardiol. 2006; 47: C7-C12Crossref PubMed Scopus (834) Google Scholar, 2.Edsfeldt A. Duner P. Stahlman M. Mollet I.G. Asciutto G. Grufman H. Nitulescu M. Persson A.F. Fisher R.M. Melander O. et al.Sphingolipids contribute to human atherosclerotic plaque inflammation.Arterioscler. Thromb. Vasc. Biol. 2016; 36: 1132-1140Crossref PubMed Scopus (99) Google Scholar, 3.Miyazawa T. Nakagawa K. Shimasaki S. Nagai R. Lipid glycation and protein glycation in diabetes and atherosclerosis.Amino Acids. 2012; 42: 1163-1170Crossref PubMed Scopus (79) Google Scholar). The “phospholipidomic code” representing the pattern of phospholipids in the setting of health and disease is becoming an area of interest in an effort to identify unique biochemical signatures that can influence atherosclerotic disease prevention and treatment in diabetic patients (3.Miyazawa T. Nakagawa K. Shimasaki S. Nagai R. Lipid glycation and protein glycation in diabetes and atherosclerosis.Amino Acids. 2012; 42: 1163-1170Crossref PubMed Scopus (79) Google Scholar, 4.Nakagawa K. Oak J.H. Higuchi O. Tsuzuki T. Oikawa S. Otani H. Mune M. Cai H. Miyazawa T. Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes.J. Lipid Res. 2005; 46: 2514-2524Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5.Demirkan A. van Duijn C.M. Ugocsai P. Isaacs A. Pramstaller P.P. Liebisch G. Wilson J.F. Johansson A. Rudan I. Aulchenko Y.S. et al.Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations.PLoS Genet. 2012; 8: e1002490Crossref PubMed Scopus (143) Google Scholar). In particular, ether lipids are known to have diverse biological effects on cellular and intracellular functions, and have an impact on tissue homeostasis and inflammation, as well as progression of cardiovascular disease (6.Annibal A. Riemer T. Jovanovic O. Westphal D. Griesser E. Pohl E.E. Schiller J. Hoffmann R. Fedorova M. Structural, biological and biophysical properties of glycated and glycoxidized phosphatidylethanolamines.Free Radic. Biol. Med. 2016; 95: 293-307Crossref PubMed Scopus (14) Google Scholar, 7.Braverman N.E. Moser A.B. Functions of plasmalogen lipids in health and disease.Biochim. Biophys. Acta. 2012; 1822: 1442-1452Crossref PubMed Scopus (644) Google Scholar). Ubiquitously expressed phosphatidylethanolamine (PE) accounts for approximately 20% of mammalian phospholipids (8.Vance J.E. Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids.J. Lipid Res. 2008; 49: 1377-1387Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). The majority of PEs are synthesized in the endoplasmic reticulum (ER) by the CDP-ethanolamine enzymatic pathway (Kennedy pathway) (9.Gibellini F. Smith T.K. The Kennedy pathway–de novo synthesis of phosphatidylethanolamine and phosphatidylcholine.IUBMB Life. 2010; 62: 414-428Crossref PubMed Google Scholar, 10.Kennedy E.P. Weiss S.B. The function of cytidine coenzymes in the biosynthesis of phospholipides.J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). In the majority of mammalian tissue, the final step of PE synthesis is catalyzed by the essential enzyme, choline-ethanolamine phosphotransferase 1 (CEPT1) (11.Henneberry A.L. Wistow G. McMaster C.R. Cloning, genomic organization, and characterization of a human cholinephosphotransferase.J. Biol. Chem. 2000; 275: 29808-29815Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Ether lipids derived from PE, such as plasmenyl-PE (pPE), are also synthesized by CEPT1 and are highly concentrated in vascular tissue and inflammatory cells (6.Annibal A. Riemer T. Jovanovic O. Westphal D. Griesser E. Pohl E.E. Schiller J. Hoffmann R. Fedorova M. Structural, biological and biophysical properties of glycated and glycoxidized phosphatidylethanolamines.Free Radic. Biol. Med. 2016; 95: 293-307Crossref PubMed Scopus (14) Google Scholar, 7.Braverman N.E. Moser A.B. Functions of plasmalogen lipids in health and disease.Biochim. Biophys. Acta. 2012; 1822: 1442-1452Crossref PubMed Scopus (644) Google Scholar, 12.Farooqui A.A. Yang H.C. Horrocks L.A. Plasmalogens, phospholipases A2 and signal transduction.Brain Res. Brain Res. Rev. 1995; 21: 152-161Crossref PubMed Scopus (88) Google Scholar). Although initial reports link pPE and CEPT1 to vascular tissue inflammation (3.Miyazawa T. Nakagawa K. Shimasaki S. Nagai R. Lipid glycation and protein glycation in diabetes and atherosclerosis.Amino Acids. 2012; 42: 1163-1170Crossref PubMed Scopus (79) Google Scholar, 13.Zieseniss S. Zahler S. Muller I. Hermetter A. Engelmann B. Modified phosphatidylethanolamine as the active component of oxidized low density lipoprotein promoting platelet prothrombinase activity.J. Biol. Chem. 2001; 276: 19828-19835Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14.Ivanova P.T. Milne S.B. Brown H.A. Identification of atypical ether-linked glycerophospholipid species in macrophages by mass spectrometry.J. Lipid Res. 2010; 51: 1581-1590Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), how they are associated with human arterial plaque biology is largely unknown. Diabetic patients are known to have more vulnerable carotid artery plaques that lead to a higher risk of resultant ischemic stroke (15.Gregg E.W. Li Y. Wang J. Burrows N.R. Ali M.K. Rolka D. Williams D.E. Geiss L. Changes in diabetes-related complications in the United States, 1990–2010.N. Engl. J. Med. 2014; 370: 1514-1523Crossref PubMed Scopus (1155) Google Scholar). Prior reports show that the content of specific lipid subgroups is altered in the homogenates of heterogeneous whole carotid endarterectomy (CEA) plaques (2.Edsfeldt A. Duner P. Stahlman M. Mollet I.G. Asciutto G. Grufman H. Nitulescu M. Persson A.F. Fisher R.M. Melander O. et al.Sphingolipids contribute to human atherosclerotic plaque inflammation.Arterioscler. Thromb. Vasc. Biol. 2016; 36: 1132-1140Crossref PubMed Scopus (99) Google Scholar, 3.Miyazawa T. Nakagawa K. Shimasaki S. Nagai R. Lipid glycation and protein glycation in diabetes and atherosclerosis.Amino Acids. 2012; 42: 1163-1170Crossref PubMed Scopus (79) Google Scholar, 14.Ivanova P.T. Milne S.B. Brown H.A. Identification of atypical ether-linked glycerophospholipid species in macrophages by mass spectrometry.J. Lipid Res. 2010; 51: 1581-1590Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), as well as in plaques from other arterial beds (4.Nakagawa K. Oak J.H. Higuchi O. Tsuzuki T. Oikawa S. Otani H. Mune M. Cai H. Miyazawa T. Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes.J. Lipid Res. 2005; 46: 2514-2524Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16.Ujihara N. Sakka Y. Takeda M. Hirayama M. Ishii A. Tomonaga O. Babazono T. Takahashi C. Yamashita K. Iwamoto Y. Association between plasma oxidized low-density lipoprotein and diabetic nephropathy.Diabetes Res. Clin. Pract. 2002; 58: 109-114Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 17.Huang Y.S. Horrobin D.F. Manku M.S. Mitchell J. Ryan M.A. Tissue phospholipid fatty acid composition in the diabetic rat.Lipids. 1984; 19: 367-370Crossref PubMed Scopus (74) Google Scholar). In this study, we build upon these prior observations by providing the first phospholipidomic analysis between maximally (MAX) and minimally (MIN) diseased CEA segments in diabetic and nondiabetic subjects with high-grade carotid artery stenosis. Forty-nine subjects with >70% stenosis in the extracranial carotid artery bifurcation participated in this study, which was approved by the local Human Research Protection Office. Using an IRB-approved protocol, all subjects provided research consent to allow collection of intraoperative CEA plaque specimens at the time of their CEA. In the operating room, CEA plaques were removed en bloc from the subject's carotid bifurcation, and immediately sectioned into MAX (the segment that is at the level of the carotid bifurcation) and MIN diseased segments (the segment that is at the plaque periphery distal edge in the internal carotid artery; Fig. 1A). The CEA plaque segments were independently inspected to confirm that MAX diseased segments had American Heart Association type IV–VIII atherosclerotic plaques, and MIN diseased segments had American Heart Association type I–III atherosclerotic plaques (Fig. 1) (18.Stary H.C. Chandler A.B. Dinsmore R.E. Fuster V. Glagov S. Insull Jr., W. Rosenfeld M.E. Schwartz C.J. Wagner W.D. Wissler R.W. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1512-1531Crossref PubMed Scopus (978) Google Scholar). Plaque segments were immersed in cold hypotonic nondetergent-based lysis buffer [1 M NaHCO3, 1 M sucrose, 1.5 M NaN3, 0.1 M PMSF, and Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA)] for 30 min. All specimens were then homogenized with a high-speed rotational power tissue homogenizer (Glas-Col, Terre Haute, IN). Homogenized samples underwent centrifugation (4,697 g for 5 minutes), and the supernatants were collected and standardized relative to protein concentration using a colorimetric Bradford protein concentration assay (Bio-Rad, Hercules, CA). Supernatant aliquots were obtained for each sample and a set quantity of homologous nonnaturally occurring phospholipid internal standard species was added to each aliquot. This internal standard cocktail included 1,2-dimyristoyl-sn-glycero-3-phosphocholine [phosphatidylcholine (PC) 14:0/14:0], dimyristoyl-sn-glycero-3-phosphoethanolamine (PE 14:0/14:0), dimyristoyl-sn-glycero-3-phosphochoserine [phosphatidylserine (PS) 14:0/14:0], dimyristoyl-sn-glycero-3-phosphoglycerol [phosphatidylglycerol (PG) 14:0/14:0], dipalmitoyl-sn-glycero-3-phosphoinositol [phosphatidylinositol (PI) 16:0/16:0], and N-lauroyl-D-erythro-sphingosine [ceramide (Cer) d18:1/12:0]. Samples were mixed with lipid extraction buffer [2:2 (v/v) chloroform/methanol] and the organic phase was collected. Extracts were then dried under nitrogen and reconstituted in methanol with 0.25% NH4OH (29%) (19.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42681) Google Scholar). Lipid extracts from MAX and MIN diseased samples obtained from 17 patients (10 diabetic and 7 nondiabetic; supplemental Table S1) were analyzed by direct injection electrospray ionization mass spectrometry using a Thermo Vantage triple-quadruple mass spectrometer (San Jose, CA) and an Accela 1250 UPLC system operated via an Xcalibur operating system. From each extract, 10 ul were loop injected into the electrospray ion source. The mass spectrometer skimmer was set at ground potential; the electrospray needle was set at 3.0 kV for positive-ion and 2.5 kV for negative-ion mode operation; and the temperature of the heated capillary was set at 300°C. Argon was used as the collision gas for linked scan collision-induced dissociation tandem mass spectrometry. Precursor-ion scan (PIS) and neutral loss scan (NLS) were used to profile PC (PIS of 184) as [M+H]+ ions, and PE (PIS of 196), PI (PIS of 241), PG (PIS of 153), PS (NLS of 87), and Cer (NLS of 256) as [M-H]− ions, under each optimal collision energy and collision gas pressure. Supplemental Figs. S1 and S2 provide an example of two sets of linked scan spectra of the phospholipid families analyzed in MAX and MIN diseased CEA segments from a single nondiabetic human subject. Structures for all phospholipids were identified using a multiple-stage linear ion-trap that was operated at a low energy collision-induced dissociation and high-resolution mass spectrometry. Phospholipid structural assignments were made as previously described (20.Hsu F-F. Turk J. Electrospray ionization with low-energy collisionally activated dissociation tandem mass spectrometry of glycerophospholipids: Mechanisms of fragmentation and structural characterization.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2673-2695Crossref PubMed Scopus (243) Google Scholar, 21.Hsu F.F. Turk J. Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: the fragmentation processes.J. Am. Soc. Mass Spectrom. 2003; 14: 352-363Crossref PubMed Scopus (260) Google Scholar, 22.Hsu F-F. Complete structural characterization of ceramides as [M-H]- ions by multiple-stage linear ion trap mass spectrometry.Biochimie. 2016; 130: 63-75Crossref PubMed Scopus (34) Google Scholar, 23.Hsu F.F. Kuhlmann F.M. Turk J. Beverley S.M. Multiple-stage linear ion-trap with high resolution mass spectrometry towards complete structural characterization of phosphatidylethanolamines containing cyclopropane fatty acyl chain in Leishmania infantum.J. Mass Spectrom. 2014; 49: 201-209Crossref PubMed Scopus (15) Google Scholar, 24.Hsu F.F. Turk J. Structural determination of sphingomyelin by tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2000; 11: 437-449Crossref PubMed Scopus (118) Google Scholar, 25.Hsu F.F. Turk J. Tandem mass spectrometry with electrospray ionization of sphingomyelins.in: Part A. Gross M.L. Caprioli R. The Encyclopedia of Mass Spectrometry, Vol. III. Applications in Biochemistry, Biology, and Medicine. Elsevier Science, New York2005: 430-447Google Scholar, 26.Hsu F-F. Turk J. Charge-driven fragmentation processes in diacyl glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal.J. Am. Soc. Mass Spectrom. 2000; 11: 797-803Crossref PubMed Scopus (97) Google Scholar, 27.Hsu F-F. Turk J. Studies on phosphatidylserine by tandem quadrupole and multiple stage quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes.J. Am. Soc. Mass Spectrom. 2005; 16: 1510-1522Crossref PubMed Scopus (109) Google Scholar, 28.Hsu F-F. Turk J. Zhang K. Beverley S. Characterization of inositol phosphorylceramides from Leishmania major by tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2007; 18: 1591-1604Crossref PubMed Scopus (41) Google Scholar, 29.Hsu F.F. Kuhlmann F.M. Turk J. Beverley S.M. Multiple-stage linear ion-trap with high resolution mass spectrometry towards complete structural characterization of phosphatidylethanolamines containing cyclopropane fatty acyl chain in Leishmania infantum.J. Mass Spectrom. 2014; 49: 201-209Crossref PubMed Scopus (21) Google Scholar), and as summarized in supplemental Tables S2–S10. Supplemental Figs. S3–S8 provide product-ion spectra that were used to produce structural determinations for each phospholipid group. The mass spectrometry-derived lipid mass spectrum for each sample was averaged over time, and the average background spectrum was subtracted from it. The net sample spectrum were described as a set of signal intensities of the predefined phospholipid species whose isotopologue distribution patterns for m+0 through m+4 natural abundance isotopologues were determined using a custom algorithm in MATLAB (Mathworks, Inc., Natick, MA). Absolute lipid concentration quantitation was obtained by deriving the ratio of the signal intensity of each species against the known quantity of the homologous nonnaturally occurring internal standard species added to the lipid extract samples. This analysis included 50 PC-related species [including 23 PCs (supplemental Table S2), 16 alkyl ether PCs (aPCs; supplemental Table S3), and 11 SMs (supplemental Table S4)], 25 PS species (supplemental Table S5), 22 PE-related species [including 11 PEs (supplemental Table S6) and 11 pPEs (supplemental Table S7)], 13 PG species (supplemental Table S8), 16 PI species (supplemental Table S9), and 7 Cer species (supplemental Table S10). Lysed tissue homogenates from MAX and MIN diseased samples (from the matched 10 diabetic and 7 nondiabetic human subjects that underwent mass spectrometry lipidomic analysis; supplemental Table 1) were separated using a gradient-gel SDS-PAGE, and transferred to a PVDF membrane for Western blotting using anti-human CEPT1 antibody (Abcam). Lysed tissue homogenates from an additional 11 subjects (5 diabetic and 6 nondiabetic, age and comorbidity matched; supplemental Table S11) were similarly blotted for human anti-cytosolic phospholipase A2 (cPLA2) antibody (Abcam, ab198898), anti-calcium-independent PLA2 (iPLA2) antibody (Abcam, ab103258), and anti-GAPDH antibody (Cell Signaling, #2118). Bands were quantified by densitometry. Using the same 11 subject samples evaluated for cPLA2 protein expression, the levels of nonesterified arachidonic acid (AA) in MAX and MIN diseased segments were evaluated using a competitive ELISA assay (Aviva Systems Biology, OKEH02583) according to manufacturer's instructions. Freshly collected MAX and MIN diseased CEA segments from 23 subjects (8 diabetic and 15 nondiabetic, age and comorbidity matched; supplemental Table S12) were submerged in liquid nitrogen and crushed into a fine powder using a mortar and pestle. Crushed samples were then placed in 1 ml TRIZOL (Thermo Fisher), and RNA was extracted using an RNeasy Mini kit (Qiagen). Following extraction, RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer RNA Nano kit (Agilent) with RIN <8. One microgram of RNA per sample was converted to cDNA using an iScript cDNA Synthesis kit (Bio-Rad). cDNA concentration was evaluated using NanoDrop (Thermo Fisher ND-ONE-W) and adjusted to a standard concentration across all samples. Quantitative PCR was carried out using the SYBR Green PCR Master Mix (Applied Biosystems) one-step RT-PCR protocol using cept1 forward (5′-AGG TGG TCC TCCAAT CAC TG-3′) and reverse (5′-TGG CAA ACG TAT GTT TCT GG-3′) primers. Amplification was performed using StepOnePlus real-time PCR system (Thermo Fisher) to determine relative changes in amplification cycle between different MAX and MIX diseased CEA specimens. MAX and MIN diseased segments were serially dehydrated in 15% and 30% sucrose solutions. CEA segments were then embedded in OCT and sectioned at 10 μm sections. Following fixation with 4% paraformaldehyde, tissue sections were denatured, permeablized with 1% Triton/PBS solution, and blocked with 1% BSA, 0.2% milk powder, 0.3% Triton X-100 solution. CEA sections were then stained with Pentachrome, as previously described (30.Carr S. Farb A. Pearce W.H. Virmani R. Yao J.S. Atherosclerotic plaque rupture in symptomatic carotid artery stenosis.J. Vasc. Surg. 1996; 23: 755-765Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar), or with anti-CEPT1 antibody (1:50; Proteintech, Rosemont, IL), followed by labeling with Alexa Fluor 594-conjugated secondary antibody (1:250; Invitrogen, Waltham, MA). Tissue sections were counterstained with DAPI and with Alexa Fluor 488-conjugated Griffonia simplicifolia isolectin-1-B4 (1:100; Invitrogen). Representative images of CEPT1-stained CEA tissue sections were obtained at 60× magnification using a Nikon A1Rsi confocal microscope. Using a DFC 3000 G Leica Microsystems inverted fluorescent microscope, 30 random 20× images were obtained of the superficial intima of MAX and MIN diseased specimens of three diabetic and three nondiabetic patients. Quantitative assessments of CEPT1- and isolectin-1-B4-positive cells in the superficial intima were performed using Image J software. A nonparametric Mann-Whitney U test was used to evaluate differences in age, demographics, and medications between diabetic and nondiabetic subjects who provided CEA plaque specimens. Mass spectrometry descriptive analysis was performed to evaluate heat map relative fold differences in phospholipid mean absolute quantities. A nonparametric Wilcoxon two-sample test was used to evaluate differences between phospholipid groups between MAX and MIN diseased segments, as well as diabetic and nondiabetic patient groups. Further subgroup analysis was performed using a univariate signed-rank test to evaluate differences in phospholipid classes between MAX and MIN diseased segments in all patients combined, or selectively among diabetic and nondiabetic patients. Similarly, a univariate signed-rank test was used to evaluate differences in specific phospholipid species in all patients combined, or selectively among diabetic and nondiabetic patients. A two-tailed Student's t-test was used to evaluate relative differences in specific protein and mRNA expression in MAX and MIN diseased CEA segments from diabetic and nondiabetic patients. For all statistical tests, P < 0.05 was considered statistically significant and was not adjusted for multiple comparisons. Biochemical and molecular analysis of MAX and MIN diseased human CEA plaque segments was performed using specimens collected from 49 human subjects (21 diabetic and 28 nondiabetic). Comparative analysis of subject demographics and medication profiles revealed no difference in age groups, cardiovascular risk factors, or prevalence of symptomatic carotid artery stenosis prior to CEA (Table 1). As expected, diabetic subjects were more likely to be clinically obese (BMI >30; P < 0.001) and taking medications such as β-blockers, anti-platelets, and insulin (P < 0.05; Table 1). Matched human subjects were used for each subgroup analysis (supplemental Tables S2–S4).TABLE 1Demographics of patients in cohort 1NondiabeticDiabeticPAge50–60 (%)18100.4261–70 (%)29480.1871–80 (%)43330.5181–90 (%)11100.94DemographicsGender (n)M28/F8M21/F80.49BMI ≥30 (%)1467<0.001aSignificance with a nonparametric Mann-Whitney U test. CAD, coronary artery disease.Current Smoker (%)11290.11Hypertension (%)79950.10Hyperlipidemia (%)82900.42CAD (%)43480.75Stroke (%)32290.79MedicationsAntiplatelet (%)100760.02aSignificance with a nonparametric Mann-Whitney U test. CAD, coronary artery disease.Beta-blocker (%)2576<0.001aSignificance with a nonparametric Mann-Whitney U test. CAD, coronary artery disease.Statin (%)82810.92Insulin (%)033<0.001aSignificance with a nonparametric Mann-Whitney U test. CAD, coronary artery disease.a Significance with a nonparametric Mann-Whitney U test. CAD, coronary artery disease. Open table in a new tab Our group recently demonstrated that skeletal muscle CEPT1 affects insulin sensitivity and lipid metabolism in diet-induced diabetes (31.Funai K. Lodhi I.J. Spears L.D. Yin L. Song H. Klein S. Semenkovich C.F. Skeletal muscle phospholipid metabolism regulates insulin sensitivity and contractile function.Diabetes. 2016; 65: 358-370Crossref PubMed Scopus (69) Google Scholar). To determine whether CEPT1 is similarly altered in the carotid arteries of diabetic human subjects, we evaluated CEA plaques (Fig. 1A, B) of 17 subjects and observed a 53% increase in CEPT1 protein expression in diabetic specimens (P < 0.05; Fig. 1C). Immunofluorescent microscopy detected scattered CEPT1 tissue staining in the intima of MAX and MIN diseased segments (Fig. 1D). There was more CEPT1 staining in the superficial intima of MAX and MIN segments of diabetic subjects (Fig. 1E; P < 0.001), and even more staining in MAX segments compared with diseased MIN segments. Expression patterns were further confirmed in an additional 23 human subjects (7 diabetic and 16 nondiabetic), with higher cept1 mRNA expression in both the MAX and MIN diseased CEA segments of diabetic subjects (P < 0.001; Fig. 1F). The terminal enzyme of the Kennedy pathway is CEPT1, and it is responsible for the de novo biosynthesis of the majority of phospholipids in mammalian tissue, including pPEs (9.Gibellini F. Smith T.K. The Kennedy pathway–de novo synthesis of phosphatidylethanolamine and phosphatidylcholine.IUBMB Life. 2010; 62: 414-428Crossref PubMed Google Scholar, 10.Kennedy E.P. Weiss S.B. The function of cytidine coenzymes in the biosynthesis of phospholipides.J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). Because we observed higher cept1 expression in CEA plaque of diabetic subjects, we next evaluated the potential consequences of this on tissue phospholipid content. Paired mass spectrometry analysis of all major phospholipid groups revealed a significant relative increase in the absolute quantities of pPEs and PSs in MIN segments of diabetic subjects (Table 2; P = 0.001 and P < 0.001, respectively). The relative absolute quantity of PIs was only increased in MIN segments of nondiabetic subjects (P = 0.03), and pPEs were only increased in the MIN segments of diabetic subjects (Table 2; P = 0.01). No significant differences were observed in the relative absolute quantities of PC, aPC, SM, PE, Cer, or PG groups between MAX and MIN segments of diabetic and nondiabetic subjects (Table 2).TABLE 2Paired phospholipid analysis between MAX and MIN diseased CEA segments in matched nondiabetic and diabetic subjectsLipid FamilyAllNondiabeticDiabeticMAX (nmol/ug)MIN (nmol/ug)PMAX (nmol/ug)MIN (nmol/ug)PMAX (nmol/ug)MIN (nmol/ug)PPC0.630.750.240.670.970.110.610.591aPC0.30.310.850.330.440.30.280.210.28SM1.511.570.681.572.020.381.461.261PE0.230.240.640.250.230.810.210.240.43pPE0.30.470.001aSignificance with a univariate signed-rank test.0.30.420.110.290.510.01aSignificance with a univariate signed-rank test.PI0.140.190.05aSignificance with a univariate signed-rank test.0.140.220.03aSignificance with a univariate signed-rank test.0.140.170.56Cer0.060.050.490.060.070.940.050.030.32PS0.190.44<0.001aSignificance with a univariate signed-rank test.0.170.380.03aSignificance with a univariate signed-rank test.0.20.5<0.01aSignificance with a univariate signed-rank test.PG0.020.020.420.020.020.30.020.020.97a Significance with a univariate signed-rank test. Open table in a new tab To determine which specific pPE and PS species were most notably altered, we performed a phospholipid subgroup heat map analysis in MAX and MIN diseased segments of diabetic versus nondiabetic subjects (Fig. 2A, C). This revealed a >20% relative change in the absolute quantities of specific pPEs (pPE36:4, pPE38:4, pPE38:5, and pPE40:7; Fig. 2A) and PSs (PS38:3, PS38:5, PS40:4, PS40:5, PS40:6, PS48:4, and PS48:6; Fig. 2C). Compared with MAX, the MIN segments of diabetic subjects demonstrated higher amounts of arachidonoyl species, 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (pPE38:4; 32%" @default.
• W2788732368 created "2018-03-06" @default.
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• W2788732368 title "Diabetes adversely affects phospholipid profiles in human carotid artery endarterectomy plaques" @default.
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