Tg6F ameliorates the increase in oxidized phospholipids in the jejunum of mice fed unsaturated LysoPC or WD
2016; Elsevier BV; Volume: 57; Issue: 5 Linguagem: Inglês
10.1194/jlr.m064352
ISSN1539-7262
AutoresA. Chattopadhyay, Mohamad Navab, Greg Hough, Víctor Grijalva, Pallavi Mukherjee, Hannah R. Fogelman, Lin Hwang, Kym F. Faull, Aldons J. Lusis, Srinivasa T. Reddy, Alan M. Fogelman,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoMouse chow supplemented with lysophosphatidylcholine with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1) increased LysoPC 18:1 in tissue of the jejunum of LDL receptor (LDLR)-null mice by 8.9 ± 1.7-fold compared with chow alone. Western diet (WD) contained dramatically less phosphatidylcholine 18:1 or LysoPC 18:1 compared with chow, but feeding WD increased LysoPC 18:1 in the jejunum by 7.5 ± 1.4-fold compared with chow. Feeding LysoPC 18:1 or feeding WD increased oxidized phospholipids in the jejunum by 5.2 ± 3.0-fold or 8.6 ± 2.2-fold, respectively, in LDLR-null mice (P < 0.0004), and 2.6 ± 1.5-fold or 2.4 ± 0.92-fold, respectively, in WT C57BL/6J mice (P < 0.0001). Adding 0.06‥ by weight of a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) decreased LysoPC 18:1 in the jejunum of LDLR-null mice on both diets (P < 0.0001), and prevented the increase in oxidized phospholipids in the jejunum in LDLR-null and WT mice on both diets (P < 0.008). Tg6F decreased inflammatory cells in the villi of the jejunum, decreased dyslipidemia, and decreased systemic inflammation in LDLR-null and WT mice on both diets. We conclude that Tg6F reduces diet-induced inflammation by reducing the content of unsaturated LysoPC and oxidized phospholipids in the jejunum of mice. Mouse chow supplemented with lysophosphatidylcholine with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1) increased LysoPC 18:1 in tissue of the jejunum of LDL receptor (LDLR)-null mice by 8.9 ± 1.7-fold compared with chow alone. Western diet (WD) contained dramatically less phosphatidylcholine 18:1 or LysoPC 18:1 compared with chow, but feeding WD increased LysoPC 18:1 in the jejunum by 7.5 ± 1.4-fold compared with chow. Feeding LysoPC 18:1 or feeding WD increased oxidized phospholipids in the jejunum by 5.2 ± 3.0-fold or 8.6 ± 2.2-fold, respectively, in LDLR-null mice (P < 0.0004), and 2.6 ± 1.5-fold or 2.4 ± 0.92-fold, respectively, in WT C57BL/6J mice (P < 0.0001). Adding 0.06‥ by weight of a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) decreased LysoPC 18:1 in the jejunum of LDLR-null mice on both diets (P < 0.0001), and prevented the increase in oxidized phospholipids in the jejunum in LDLR-null and WT mice on both diets (P < 0.008). Tg6F decreased inflammatory cells in the villi of the jejunum, decreased dyslipidemia, and decreased systemic inflammation in LDLR-null and WT mice on both diets. We conclude that Tg6F reduces diet-induced inflammation by reducing the content of unsaturated LysoPC and oxidized phospholipids in the jejunum of mice. Our laboratory, together with the University of California, San Diego group, pioneered the now widely accepted concept that oxidized phospholipids formed in the subendothelial space of the artery wall are responsible for initiating the early inflammatory response that is critical to the development of atherosclerosis (1Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. et al.The yin and yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 831-842Crossref PubMed Scopus (610) Google Scholar, 2Navab M. Hama S.Y. Cooke C.J. Anantharamaiah G.M. Chaddhaz M. Jin L. Subbanagounder G. Faull K.F. Reddy S.T. Miller N.E. et al.Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1.J. Lipid Res. 2000; 41: 1481-1494Abstract Full Text Full Text PDF PubMed Google Scholar, 3Navab M. Hama S.Y. Anantharamaiah G.M. Hassan K. Hough G.P. Watson A.D. Reddy S.T. Sevanian A. Fonarow G.C. Fogelman A.M. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3.J. Lipid Res. 2000; 41: 1495-1508Abstract Full Text Full Text PDF PubMed Google Scholar, 4Navab M. Anantharamaiah G.M. Reddy S.T. Van Lenten B.J. Ansell B.J. Fonarow G.C. Vahabzadeh K. Hama S. Hough G. Kamranpour N. et al.The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL.J. Lipid Res. 2004; 45: 993-1007Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). The recognition of oxidized phospholipids in the artery wall was established by using antibodies to oxidized phospholipids (5Palinski W. Horkko S. Miller E. Steinbrecher U.P. Powell H.C. Curtiss L.K. Witztum J.L. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma.J. Clin. Invest. 1996; 98: 800-814Crossref PubMed Scopus (501) Google Scholar) and confirmed by our laboratory using MS (6Watson A.D. Leitinger N. Navab M. Faull K.F. Horrkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. et al.Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induces monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar). Oxidized phospholipids have been implicated in both pro-inflammatory and anti-inflammatory processes acting through multiple signaling pathways (7Lee S. Birukov K.G. Romanoski C.E. Springstead J.R. Lusis A.J. Berliner J.A. Role of phospholipid oxidation products in atherosclerosis.Circ. Res. 2012; 111: 778-799Crossref PubMed Scopus (155) Google Scholar). We previously reported that adding 1 μg of unsaturated (but not saturated) lysophosphatidic acid (LPA) to each gram of standard mouse chow induced dyslipidemia and systemic inflammation in LDL receptor (LDLR)-null mice similar to that seen when the mice were fed a Western diet (WD) (8Navab M. Hough G. Buga G.M. Su F. Wagner A.C. Meriwether D. Chattopadhyay A. Gao F. Grijalva V. Danciger J.S. et al.Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid.J. Lipid Res. 2013; 54: 3403-3418Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Remaley A.T. Tomatoes, lysophosphatidic acid, and the small intestine: new pieces in the puzzle of apolipoprotein mimetic peptides?.J. Lipid Res. 2013; 54: 3223-3226Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). More recently, we demonstrated that the LPA-mediated dyslipidemia resulted in aortic atherosclerosis in LDLR-null mice that was similar in cellular characteristics to that seen on feeding these mice WD (10Navab M. Chattopadhyay A. Hough G. Meriwether D. Fogelman S.I. Wagner A.C. Grijalva V. Su F. Anantharamaiah G.M. Hwang L.H. et al.Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis.J. Lipid Res. 2015; 56: 871-887Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Additionally, we presented evidence that in the enterocytes of the small intestine, unsaturated lysophosphatidylcholine (LysoPC) is converted to unsaturated LPA by the action of lysophospholipase D (autotaxin) (10Navab M. Chattopadhyay A. Hough G. Meriwether D. Fogelman S.I. Wagner A.C. Grijalva V. Su F. Anantharamaiah G.M. Hwang L.H. et al.Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis.J. Lipid Res. 2015; 56: 871-887Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Because the levels of unsaturated LPA in the tissues and plasma significantly increased on feeding WD [but not as much as after feeding LysoPC with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1)], we hypothesized that unsaturated LysoPC was likely generated from dietary unsaturated phosphatidylcholine (PC) acted upon in the duodenum by pancreatic phospholipase group 1B (10Navab M. Chattopadhyay A. Hough G. Meriwether D. Fogelman S.I. Wagner A.C. Grijalva V. Su F. Anantharamaiah G.M. Hwang L.H. et al.Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis.J. Lipid Res. 2015; 56: 871-887Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Here, we demonstrate that is not likely the case, because the content of unsaturated PC and unsaturated LysoPC in WD is dramatically lower than the content of standard mouse chow. Despite the lower content of unsaturated PC and LysoPC in WD compared with chow, we report here that feeding WD results in levels of unsaturated LysoPC in the tissue of the jejunum and in the plasma that are comparable to the levels seen after supplementing standard mouse chow with 1 mg per gram of chow of unsaturated LysoPC. In 2012, we hypothesized that apoA-I mimetic peptides may reduce systemic inflammation by modulating intestinal oxidized lipid metabolism (11Navab M. Reddy S.T. Van Lenten B.J. Buga G.M. Hough G. Wagner A.C. Fogelman A.M. High-density lipoprotein and 4F peptide reduce systemic inflammation by modulating intestinal oxidized lipid metabolism. Novel hypotheses and review of literature.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2553-2560Crossref PubMed Scopus (54) Google Scholar). Following clinical trials in humans that suggested that plasma apoA-I mimetic peptide levels did not predict efficacy, we came to focus on the intestine as a major site of action for these peptides. These studies in mice were designed to understand why clinical trials that administered relatively high peptide doses orally were successful, despite achieving low plasma peptide levels (12Bloedon L.T. Dunbar R. Duffy D. Pinell-Salles P. Norris R. DeGroot B.J. Movva R. Navab M. Fogelman A.M. Rader D.J. Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients.J. Lipid Res. 2008; 49: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 13Dunbar R.L. Bloedon L.T. Duffy D. Norris R.B. Movva R. Navab M. Fogelman A.M. Rader D.J. Daily oral administration of the apolipoprotein A-I mimetic peptide D-4F in patients with coronary heart-disease or equivalent risk improves high-density lipoprotein anti-inflammatory function.J. Am. Coll. Cardiol. 2007; 49(Suppl A): 366AGoogle Scholar), in contrast to clinical trials using low peptide doses administered intravenously or by subcutaneous injection that failed to achieve efficacy, despite achieving very high plasma peptide levels (14Watson C.E. Weissbach N. Kjems L. Ayalasomayajula S. Zhang Y. Chang I. Navab M. Hama S. Hough G. Reddy S.T. et al.Treatment of patients with cardiovascular disease with L-4F, an apoA-1 mimetic, did not improve select biomarkers of HDL function.J. Lipid Res. 2011; 52: 361-373Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). These studies in mice demonstrated conclusively that plasma peptide levels did not predict efficacy, but the levels of peptide in the intestine did (15Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Navab M. Reddy S.T. Anantharamaiah G.M. Hough G. Buga G.M. Danciger J. Fogelman A.M. D-4F-mediated reduction in metabolites of arachidonic and linoleic acids in the small intestine is associated with decreased inflammation in low-density lipoprotein receptor-null mice.J. Lipid Res. 2012; 53: 437-445Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These studies also suggested that high doses of peptide were required, which made the use of the original apoA-I mimetic peptide [Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (4F)] problematic, because it requires blocked end groups for biologic activity; these end groups can only be added by chemical synthesis, which makes the cost of production of such high doses prohibitive. Consequently, we searched for and found an apoA-I mimetic peptide that did not require blocked end groups [D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F (6F)]. We successfully expressed the 6F peptide in transgenic tomatoes and demonstrated that adding 2.2‥ by weight of freeze-dried transgenic tomato powder expressing the 6F peptide to WD ameliorated dyslipidemia and systemic inflammation in LDLR-null mice (17Chattopadhyay A. Navab M. Hough G. Gao F. Meriwether D. Grijalva V. Springstead J.R. Palgunachari M.N. Namiri-Kalantari R. Su F. et al.A novel approach to oral apoA-I mimetic therapy.J. Lipid Res. 2013; 54 ([Erratum. 2013. J. Lipid Res. 54: 3220.]): 995-1010Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 18Getz G.S. Reardon C.A. ApoA-I mimetics: tomatoes to the rescue.J. Lipid Res. 2013; 54: 878-880Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). We also demonstrated that this dose of the freeze-dried tomato powder containing the 6F peptide ameliorated aortic atherosclerosis that was induced by either adding unsaturated LPA to standard mouse chow, or by feeding WD to LDLR-null mice (8Navab M. Hough G. Buga G.M. Su F. Wagner A.C. Meriwether D. Chattopadhyay A. Gao F. Grijalva V. Danciger J.S. et al.Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid.J. Lipid Res. 2013; 54: 3403-3418Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Remaley A.T. Tomatoes, lysophosphatidic acid, and the small intestine: new pieces in the puzzle of apolipoprotein mimetic peptides?.J. Lipid Res. 2013; 54: 3223-3226Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). To deliver similar doses of the freeze-dried tomato powder expressing the 6F peptide to humans would require approximately 3 cups, three times daily. To reduce the volume, we sought a method to concentrate the 6F peptide. We found that extracting the freeze-dried tomato powder overnight in ethyl acetate containing 5‥ acetic acid resulted in a 37-fold reduction in the weight of transgenic tomato required for biologic activity in mouse models of dyslipidemia and cancer (19Chattopadhyay A. Grijalva V. Hough G. Su F. Mukherjee P. Farias-Eisner R. Anantharamaiah G.M. Faull K.F. Hwang L.H. Navab M. et al.Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer.Pharmacol. Res. Perspect. 2015; 3: e00154Crossref PubMed Scopus (16) Google Scholar). Here, we demonstrate the efficacy of this tomato concentrate containing the 6F peptide (Tg6F) in both LDLR-null and WT C57BL/6J mice. This work also provides evidence in support of our hypothesis that Tg6F reduces systemic inflammation by modulating intestinal oxidized lipid metabolism (11Navab M. Reddy S.T. Van Lenten B.J. Buga G.M. Hough G. Wagner A.C. Fogelman A.M. High-density lipoprotein and 4F peptide reduce systemic inflammation by modulating intestinal oxidized lipid metabolism. Novel hypotheses and review of literature.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2553-2560Crossref PubMed Scopus (54) Google Scholar). We report that feeding mice standard mouse chow supplemented with unsaturated LysoPC, or feeding the mice WD results in: i) increased formation of oxidized phospholipids in the villi of the jejunum; and ii) an increase in inflammatory cells in the villi of the jejunum. We further report that addition to the diet of 0.06‥ by weight of Tg6F (19Chattopadhyay A. Grijalva V. Hough G. Su F. Mukherjee P. Farias-Eisner R. Anantharamaiah G.M. Faull K.F. Hwang L.H. Navab M. et al.Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer.Pharmacol. Res. Perspect. 2015; 3: e00154Crossref PubMed Scopus (16) Google Scholar) ameliorates the increased formation of oxidized phospholipids in the villi, and ameliorates the increase in inflammatory cells in the villi of the jejunum. Our data also provide evidence that one mechanism by which Tg6F may mitigate these processes is by decreasing the levels of unsaturated LysoPC in the jejunum. Transgenic tomato plants expressing the control marker protein, β-glucuronidase (EV), or expressing the 6F peptide were constructed and grown at the Donald Danforth Plant Science Center in Saint Louis, MO; the fruit was harvested, the seeds removed, and the pulp and skin were quick frozen and shipped frozen overnight to the University of California, Los Angeles (UCLA) where the pulp and skins were freeze-dried, powdered, and stored as previously described (8Navab M. Hough G. Buga G.M. Su F. Wagner A.C. Meriwether D. Chattopadhyay A. Gao F. Grijalva V. Danciger J.S. et al.Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid.J. Lipid Res. 2013; 54: 3403-3418Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17Chattopadhyay A. Navab M. Hough G. Gao F. Meriwether D. Grijalva V. Springstead J.R. Palgunachari M.N. Namiri-Kalantari R. Su F. et al.A novel approach to oral apoA-I mimetic therapy.J. Lipid Res. 2013; 54 ([Erratum. 2013. J. Lipid Res. 54: 3220.]): 995-1010Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Concentrates of the freeze-dried powdered tomatoes were prepared and stored as described (19Chattopadhyay A. Grijalva V. Hough G. Su F. Mukherjee P. Farias-Eisner R. Anantharamaiah G.M. Faull K.F. Hwang L.H. Navab M. et al.Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer.Pharmacol. Res. Perspect. 2015; 3: e00154Crossref PubMed Scopus (16) Google Scholar) except that after removal of the ethyl acetate, the concentrate was resuspended in water and freeze-dried again, resulting in a more homogenous preparation. Antibody that recognizes oxidized phospholipids (E06) was obtained from Avanti Polar Lipids (catalog #330001S). Anti-F4/80 antibody for immunohistochemistry studies was purchased from Serotec (catalog #MCA497B), anti-F4/80 antibody for flow cytometry studies was purchased from eBiosciences (catalog #48-4801-82), polyclonal rabbit anti-rat immunoglobulins/HRP were purchased from Dako (catalog #P0450), DakoEnVision+System-HRP labeled polymer anti-rabbit was purchased from Dako (catalog #K4003). Anti-CD68 antibody was purchased from Bio-Rad (catalog #MCA1957) and EDTA solution (pH 8) was purchased from Invitrogen (catalog #005501). Anti-Ly6G antibody was purchased from BD Pharmingen (catalog #551459). Anti-CD8 antibody was purchased from Thermo Scientific (catalog #MA1-145). Anti-CD103 antibody was purchased from BD Pharmingen (catalog #553699). The lamina propria isolation kit (catalog #130-097-410) was purchased from Miltenyi Biotec (San Diego, CA). The interleukin 6 (IL-6) ELISA kit (catalog #KMC0062) was purchased from Life Technologies, Grand Island, NY. LysoPC with stearic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:0) and LysoPC 18:1 with the fatty acids at the sn-1 position, and all other materials were purchased from sources previously described (10Navab M. Chattopadhyay A. Hough G. Meriwether D. Fogelman S.I. Wagner A.C. Grijalva V. Su F. Anantharamaiah G.M. Hwang L.H. et al.Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis.J. Lipid Res. 2015; 56: 871-887Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). C57BL/6J mice were purchased from Jackson Laboratories. LDLR-null mice originally purchased from Jackson Laboratories on a C57BL/6J background were obtained from the breeding colony of the Department of Laboratory and Animal Medicine at the David Geffen School of Medicine at UCLA. The gender and age of the mice are stated in the legend to each figure. The mice were maintained on standard mouse chow (Ralston Purina) before being switched to either standard mouse chow supplemented with LysoPC or WD (Teklad, Harlan, catalog #TD88137). Addition of LysoPC to standard mouse chow was performed as previously described (10Navab M. Chattopadhyay A. Hough G. Meriwether D. Fogelman S.I. Wagner A.C. Grijalva V. Su F. Anantharamaiah G.M. Hwang L.H. et al.Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis.J. Lipid Res. 2015; 56: 871-887Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Concentrates of EV or Tg6F were added to the diets at 0.06‥ by weight, as described (19Chattopadhyay A. Grijalva V. Hough G. Su F. Mukherjee P. Farias-Eisner R. Anantharamaiah G.M. Faull K.F. Hwang L.H. Navab M. et al.Efficacy of tomato concentrates in mouse models of dyslipidemia and cancer.Pharmacol. Res. Perspect. 2015; 3: e00154Crossref PubMed Scopus (16) Google Scholar). At the end of the treatment periods, the mice were fasted overnight in clean cages with new bedding, and following blood collection for plasma determinations, and prior to harvesting of organs, the mice were perfused under anesthesia to remove all blood (8Navab M. Hough G. Buga G.M. Su F. Wagner A.C. Meriwether D. Chattopadhyay A. Gao F. Grijalva V. Danciger J.S. et al.Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid.J. Lipid Res. 2013; 54: 3403-3418Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17Chattopadhyay A. Navab M. Hough G. Gao F. Meriwether D. Grijalva V. Springstead J.R. Palgunachari M.N. Namiri-Kalantari R. Su F. et al.A novel approach to oral apoA-I mimetic therapy.J. Lipid Res. 2013; 54 ([Erratum. 2013. J. Lipid Res. 54: 3220.]): 995-1010Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and organs were harvested and washed as described previously (8Navab M. Hough G. Buga G.M. Su F. Wagner A.C. Meriwether D. Chattopadhyay A. Gao F. Grijalva V. Danciger J.S. et al.Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid.J. Lipid Res. 2013; 54: 3403-3418Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 17Chattopadhyay A. Navab M. Hough G. Gao F. Meriwether D. Grijalva V. Springstead J.R. Palgunachari M.N. Namiri-Kalantari R. Su F. et al.A novel approach to oral apoA-I mimetic therapy.J. Lipid Res. 2013; 54 ([Erratum. 2013. J. Lipid Res. 54: 3220.]): 995-1010Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). All mouse studies were approved by the Animal Research Committee at UCLA. Prior to removal of the jejunum, the mice were extensively perfused with cold saline to remove blood. Five to six mice (chosen at random) from each treatment group were used for each marker assessed, and three to five segments of jejunum from each mouse were analyzed. Immunohistochemistry was performed by the Immunohistochemistry Core in the Department of Pathology at David Geffen School of Medicine at UCLA. The jejunum segments were fixed in 10‥ neutral buffered formalin (pH 7.4) at physiological pressure at room temperature overnight. The fixed segments were thoroughly washed with distilled water and transferred to 70‥ ethanol followed by embedding in paraffin and sectioning. Photomicrographs of the sections were captured using an Olympus BX51 microscope and the application Q Capture 7.0 (Q Imaging, Inc.). Randomly selected fields were quantified for each sample and the ratio of the stain signal to the villus area of the jejunum was determined using Image Pro Plus 7 (Media Cybernetics). To detect oxidized phospholipids, after processing the slides to remove paraffin, the slides were rinsed in PBS containing 0.05‥ Tween-20 (PBST) and then incubated at room temperature for 45 min with anti-E06 antibody (Avanti Polar Lipids, catalog #330001S) at a 1:50 dilution. The slides were rinsed in PBST and the signals were amplified using VECTSTAIN Elite ABC kit (Vector Laboratories, catalog #PK-6100) following the recommended procedures. After a rinse with PBST, the slides were incubated with 3,3′-diaminobenzidine for visualization. The slides were washed in tap water and counterstained with Harris Hematoxylin, followed by dehydration in ethanol and mounting. Controls consisted of sections exposed to secondary-only antibodies. To detect F4/80, after processing the slides to remove paraffin, the slides were rinsed in tap water followed by PBST, and then incubated at 4°C overnight with anti-F4/80 antibody (Serotec, catalog #MCA497B) at a dilution of 1:50. The slides were rinsed with PBST and were incubated with polyclonal rabbit anti-rat immunoglobulins/HRP (Dako, catalog #P0450) at a dilution of 1:200 at room temperature for 30 min. The slides were rinsed with PBST and were incubated with DakoEnVision+System-HRP labeled polymer anti-rabbit (Dako, catalog #K4003) at room temperature for 30 min. After another rinse with PBST, the slides were incubated with 3,3′- diaminobenzidine for visualization. The slides were washed in water, counterstained with Harris Hematoxylin, dehydrated in ethanol, and mounted. To detect CD68, the slides were processed as described for F4/80, but incubated overnight at 4°C with anti-CD68 antibody (Bio-Rad, catalog #MCA1957) at a dilution of 1:100 instead of being incubated with anti-F4/80 antibody. To detect Ly6G, the slides were washed in distilled water and then incubated for 2 min in EDTA solution (pH 8) (Invitrogen, catalog #005501) at 95°C using a digital programmable pressure system (Decloaking Chamber; NexGen). The slides were brought to room temperature, rinsed in PBST, and incubated at room temperature for 1 h with anti-Ly6G antibody (BD Pharmingen, catalog #551459) at a dilution of 1:1,500. To detect CD8, the slides were incubated at room temperature for 45 min with anti-CD8 antibody (Thermo Scientific, catalog #MA1-145) at a dilution of 1:25 to 1:50. To detect CD103, the slides were incubated at room temperature for 1 h with anti-CD103 antibody (BD Pharmingen, catalog #553699) at a dilution of 1:100. After an overnight fast, mice were anesthetized; enterocytes from the jejunum were isolated by the method of Iqbal and Hussain (20Iqbal J. Hussain M.M. Evidence for multiple complementary pathways for efficient cholesterol absorption in mice.J. Lipid Res. 2005; 46: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). After isolation, the enterocytes were checked with trypan blue and more than 99‥ of the cells were found to exclude trypan blue. Cell pellets from each mouse were resuspended and incubated at 37°C with 5‥ CO2 in 4 ml DMEM alone (control) or with 25 μg/ml of either LysoPC 18:0 or LysoPC 18:1 for zero time, 1 h, or 2 h. The cells at each time point were separated by centrifugation and the cell pellets and supernatants were frozen and stored at −80°C until the day of assay. On the assay day, the cell pellets and supernatants were thawed on ice and the cell pellets were resuspended in 2.7 ml saline; the supernatants (4 ml) and the cell pellets were sonicated, as described below, for the E06 ELISA assay; 200 μl of sonicate was added to each of 12 replicate wells and analyzed for E06 reactive material by ELISA, as described below. The mice were fasted overnight in clean cages with new bedding, bled and extensively perfused with ice-cold saline to remove blood prior to the harvest of the jejunum as described above. The lumen of the jejunum was washed with ice-cold saline, the jejunum was everted, and the tissue was placed in PBS containing 50 μg/ml of either LysoPC 18:1 or LysoPC 18:0. Zero time points were obtained by rapidly removing the segments of jejunum without incubation. The remaining segments were incubated at 37°C with gentle mixing for 1, 2, or 3 h. The jejunum was removed after incubation and was washed with PBS, cut into five pieces, which were washed with distilled water and placed in 10‥ neutral buffered formalin overnight at room temperature. The tissue was thoroughly washed with distilled water, placed in 70‥ ethanol, and embedded in paraffin. Five micrometer sections were prepared and processed and E06 staining was performed, and quantification of the area staining positively for E06 in the villi of the jejunum was performed as described above. After an overnight fast in clean cages with new bedding, the mice were bled, and after the mice were perfused to remove residual blood, the jejunum was harvested and everted, washed with ice-cold saline, weighed, and a 100 mg wet weight sample was placed in a glass tube containing ice-cold saline and sonicated twice, each for 10 s, at 5 watts using a Fisher 60 sonic dismembrator. A volume of the sonicate solution equivalent to 7.14 mg wet weight of the jejunum was placed in each of 12 wells of a 96-well plate (Nunc Polysorp, Thermo Fisher Scientific) and the plates were kept at 4°C overnight. The solution was aspirated; the wells were washed three times with PBS-E (PBS containing sodium EDTA at 0.27 mM). The plates were dried in a chamber at 30" vacuum and the wells treated with 1‥ BSA-PBS-E for 1 h at room temperature. The wells were then washed once with PBS-E. Primary antibody E06 (at a concentration of at 1 μg/ml) was added and incubated for 2 h at 20°C. Plates were then washed three times with PBS-E. The secondary antibody conjugate, HRP-conjugated goat anti-mouse IgM (Southern Biotech 1020-05), was used at 1:5,000, and incubated for 1 h at 20°C. The substrate, 3,3′,5,5′-tetramethylbenzidine (KPL, catalog #50-76-00), was then added at 200 μl per well. Plates were placed on an Orbitron Rotator II model 260250 (Boekel Scientific, Feasterville, PA) at 30 rpm and with tilt. After 20 min, 100 μl of the supernatant was transferred into a 96-well plate and the absorption spectrum of the well contents was measured using a Molecular Devices reader at 650 nm. To stop
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