Abstract Background: Ovarian function suppression (OFS) combined with tamoxifen (TAM) or an aromatase inhibitor (AI) is standard for premenopausal (PM) ER+, HER2- breast cancer (BC). However, > 40% PM patients (pts) are intolerant of OFS. For these pts, TAM is the only FDA approved option. In the neoadjuvant endocrine setting, pts with endocrine sensitive disease (ESD) (Ki-67 ≤ 10% at 4 weeks) have 5yr dDFS > 97%; however, only 45% of TAM pts vs >75% with AI+OFS achieve 4 wk ESD (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent anti-estrogen superior to TAM and AI in xenograft models and with antitumor activity in endocrine-resistant postmenopausal pts. ENDX additionally targets protein kinase C beta 1 (PKCβ1) at concentrations >500 ng/mL, resulting in AKT inhibition and apoptosis (Jayaraman npj Breast Cancer 2023). We hypothesize that ENDX dual targeting of ERα and PKCβ1 will obviate the need for OFS and be non-inferior to AI plus OFS for PM pts with ER+/HER2- BC. Methods: EVANGELINE (NCT05607004) is an ongoing phase 2 multicenter neoadjuvant study with pharmacokinetic (PK) run-in assessing ENDX in PM women with ER+/HER2- BC. The primary objective for the PK run-in is to identify a dose (40 or 80 mg/day) resulting in ENDX steady state concentrations (Css) of 500-1000 ng/mL (to target both ERα and PKCβ1) without significant toxicity. Following PK run-in, the randomized phase II goal is to assess whether the ESD rate with ENDX is non-inferior to exemestane plus goserelin. Women with ESD at wk4 continue treatment for 24 weeks followed by surgery. Here we report the results from the 40 mg/day PK run-in. Results: Seven PM women (6 White, 1 Asian) aged 28-51 (median 46) received ENDX 40 mg/day. Pt characteristics on study were: ER > 90% (all pts), median Ki-67 = 13 (range 4-33%), cTstage (cT2: 6 pts, cT3: 1 pt), and tumor grade (G1: 1 pt, G2: 6 pts). The median ENDX 28 day Css (ng/mL) was 263.6 (range 180.3-376.6). One pt discontinued due to wk4 Ki-67 remaining > 10%. The remaining 6 had ESD, with either wk4 Ki-67 remaining ≤ 10% (3 pts) or decreasing to ≤ 10% (3 pts) and after 24 wks underwent surgery with surgical Ki-67 ≤ 3% (range 0-3%). MRI central review (wk12 and wk24) demonstrated target lesion decreases in all pts with 1 CR, 1 PR and 4 SD (RECIST). Treatment related toxicities included grade 3 headache (n=1), grade 2 amenorrhea (n=1), and grade 2 hot flashes (n=1). The median (range) baseline estrone (n=5) was 54 pg/mL (19-114) with median (range) fold increase from baseline of 9.0 (1.3-23.2) at wk4 and 4.7 (0.4 - 25.9) at wk24. The median baseline estradiol level (n=5) was 29 pg/mL (19-209) with median fold increases from baseline of 17.9 (0.4-57.0) at wk4 and 8.1 (0.04 - 56.6) at wk24. Additional surgical and blood biomarker data will be presented at the meeting. Conclusions: ENDX (40 mg/day) exhibits promising antitumor activity for PM ER+/HER2- BC but with ENDX Css below target. Enrollment is ongoing to the 80 mg/day dose level. Citation Format: Matthew P. Goetz, Vera J. Suman, Heather Fraser, Lida Mina, Pooja Advani, Roberto Leon-Ferre, Karthik Giridhar, Felipe Batalini, Katie N. Hunt, Swaathi Jayaraman, James Jakub, Patricia Cronin, Mara Piltin, Amy Degnim, James N. Ingle, Judy C. Boughey, Sarah Buhrow, Joel Reid, Matthew Schellenberg, John Hawse, Steven Quay. Neoadjuvant (Z)-endoxifen in premenopausal ER+, HER2- breast cancer: Evaluation of the first pharmacokinetic cohort of the EVANGELINE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT205.
A previous study in our laboratory showed that streptozotocin (STZ) induced diabetic, deoxycorticosterone acetate (DOCA) induced hypertensive rats exhibited significantly lower levels of plasma glucose than did normotensive diabetic animals. The present experiments further investigate the effects of DOCA treatment on fasting levels of plasma glucose and insulin and on their changes after oral glucose challenge in nondiabetic and STZ-diabetic rats. It was found that, in nondiabetic rats, DOCA-induced hypertension was associated with normal glucose levels and glucose tolerance but with significantly lower levels of plasma insulin. DOCA-treated diabetic animals showed significantly lower levels of plasma glucose, but their plasma insulin concentrations were not significantly different from those of the DOCA vehicle treated diabetic rats. DOCA-treated diabetic rats also had significantly higher plasma levels of cholesterol and triglycerides. It is suggested that DOCA may have a direct or indirect action on the assimilation, production, or utilization of glucose, perhaps leading to an improvement in insulin sensitivity and subsequently a decrease in insulin secretion.Key words: glucose metabolism, insulin, deoxycorticosterone acetate, streptozotocin.
Effects of salt loading by drinking 0.9% NaCl solution on the myocardial performance in nondiabetic and diabetic Wistar rats were studied using the isolated working heart apparatus. Body weight and fluid and food intakes of these animals were monitored. Blood pressure and plasma levels of glucose, insulin, cholesterol, and triglycerides were also measured. Diabetes was induced by intravenous injection of streptozotocin (60 mg/kg). Diabetic rats were found to develop myocardial dysfunction at 8 weeks after STZ injection, accompanied by significant increases in food and fluid intakes, slowed body weight gain, hyperglycemia, hypoinsulinemia, and hyperlipidemia but without significant changes in blood pressure. Salt loading did not cause significant changes in any of the parameters studied in nondiabetic rats. However, in streptozotocin-diabetic rats given saline to drink, the impaired myocardial function was significantly improved and was associated with a significant reduction in hyperphagia and hyperlipidemia. Plasma glucose levels significantly decreased at weeks 1–3 but increased to the levels of untreated diabetic animals at weeks 4–7. There was an increase in fluid intake, but neither blood pressure nor plasma insulin levels were significantly affected. It is suggested that the improvements in cardiac function and hyperlipidemia in diabetic rats by salt loading may be related to each other; however, the mechanisms for these effects are not clear but are unlikely to be due to changes in glycemic control.Key words: streptozotocin diabetes, salt loading, cardiac dysfunction.
Alterations in myocardial glucose metabolism are a key determinant of ischemia-induced depression of left ventricular mechanical function. Since myocardial glycogen is an important source of endogenous glucose, we compared the effects of ischemia on glucose uptake and utilization in isolated working rat hearts in which glycogen content was either replete (G replete, 114 μmol/g dry wt) or partially depleted (G depleted, 71 μmol/g dry wt). The effects of low-flow ischemia (LFI, 0.5 ml/min) on glucose uptake, glycogen turnover (glycogenolysis and glycogen synthesis), glycolysis, adenosine 5′-monophosphate-activated protein kinase (AMPK) activity, and GLUT4 translocation were measured. Relative to preischemic values, LFI caused a time-dependent reduction in glycogen content in both G-replete and G-depleted groups due to an acceleration of glycogenolysis (by 12-fold and 6-fold, respectively). In G-replete hearts, LFI (15 min) decreased glucose uptake (by 59%) and did not affect GLUT4 translocation. In G-depleted hearts, LFI also decreased initially glucose uptake (by 90%) and glycogen synthesis, but after 15 min, when glycogenolysis slowed due to exhaustion of glycogen content, glucose uptake increased (by 31%) in association with an increase in GLUT4 translocation. After 60 min of LFI, glucose uptake, glycogenolysis, and glycolysis recovered to near-preischemic values in both groups. LFI increased AMPK activity in a time-dependent manner in both groups (by 6-fold and 4-fold, respectively). Thus, when glycogen stores are replete before ischemia, ischemia-induced AMPK activation is not sufficient to increase glucose uptake. Under these conditions, an acceleration of glycogen degradation provides sufficient endogenous substrate for glycolysis during ischemia.
We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.
Cardioprotection by adenosine A 1 receptor activation limits infarct size and improves post‐ischaemic mechanical function. The mechanisms responsible are unclear but may involve alterations in myocardial glucose metabolism. Since glycogen is an important source of glucose during ischaemia, we examined the effects of N 6 ‐cyclohexyladenosine (CHA), an A 1 receptor agonist, on glycogen and glucose metabolism during ischaemia as well as reperfusion. Isolated working rat hearts were perfused with Krebs‐Henseleit solution containing dual‐labelled 5‐ 3 H and 14 C glucose and palmitate as energy substrates. Rates of glycolysis and glucose oxidation were measured directly from the production of 3 H 2 O and 14 CO 2 . Glycogen turnover was measured from the rate of change of [5‐ 3 H and 14 C]glucosyl units in total myocardial glycogen. Following low‐flow (0.5 ml min −1 ) ischaemia (60 min) and reperfusion (30 min), left ventricular minute work (LV work) recovered to 22% of pre‐ischaemic values. CHA (0.5 μ M ) improved the recovery of LV work 2 fold. CHA altered glycogen turnover in post‐ischaemic hearts by stimulating glycogen synthesis while having no effects on glycogen degradation. CHA also partially inhibited glycolysis. These changes accelerated the recovery of glycogen in CHA‐treated hearts and reduced proton production. During ischaemia, CHA had no measurable effect on glycogen turnover or glucose metabolism. Glycogen phosphorylase activity, which was elevated after ischaemia, was inhibited by CHA, possibly in response to CHA‐induced inhibition of AMP‐activated protein kinase activity. These results indicate that CHA‐induced cardioprotection is associated with alterations of glycogen turnover during reperfusion as well as improved metabolic coupling of glycolysis to glucose oxidation. British Journal of Pharmacology (1999) 128 , 197–205; doi: 10.1038/sj.bjp.0702765
Secretory phospholipase A2 (sPLA2) activity promotes foam cell formation, increases proinflammatory bioactive lipid levels, decreases HDL levels, increases atherosclerosis in transgenic mice, and is an independent marker of cardiovascular disease. The effects of the sPLA2 inhibitor A-002 (varespladib) and pravastatin as monotherapies and in combination on atherosclerosis, lipids, and paraoxonase (PON) activity in apoE−/− mice were investigated. Male apoE−/− mice were placed on a 12-week high-fat diet supplemented with A-002 alone or combined with pravastatin. Atherosclerotic lesions were examined for size and composition using en face analysis, Movat staining, anti-CD68, and anti-α actin antibodies. Plasma lipids and PON activity were measured. A-002 decreased atherosclerotic lesion area by ∼75% while increasing fibrous cap size by over 200%. HDL levels increased 40% and plasma PON activity increased 80%. Pravastatin monotherapy had no effect on lesion size but when combined with A-002, decreased lesion area 50% and total cholesterol levels 18% more than A-002 alone. A-002, a sPLA2 inhibitor, acts synergistically with pravastatin to decrease atherosclerosis, possibly through decreased levels of systemic inflammation or decreased lipid levels. A-002 treatment also resulted in a profound increase in plasma PON activity and significantly larger fibrous caps, suggesting the formation of more stable plaque architecture. Secretory phospholipase A2 (sPLA2) activity promotes foam cell formation, increases proinflammatory bioactive lipid levels, decreases HDL levels, increases atherosclerosis in transgenic mice, and is an independent marker of cardiovascular disease. The effects of the sPLA2 inhibitor A-002 (varespladib) and pravastatin as monotherapies and in combination on atherosclerosis, lipids, and paraoxonase (PON) activity in apoE−/− mice were investigated. Male apoE−/− mice were placed on a 12-week high-fat diet supplemented with A-002 alone or combined with pravastatin. Atherosclerotic lesions were examined for size and composition using en face analysis, Movat staining, anti-CD68, and anti-α actin antibodies. Plasma lipids and PON activity were measured. A-002 decreased atherosclerotic lesion area by ∼75% while increasing fibrous cap size by over 200%. HDL levels increased 40% and plasma PON activity increased 80%. Pravastatin monotherapy had no effect on lesion size but when combined with A-002, decreased lesion area 50% and total cholesterol levels 18% more than A-002 alone. A-002, a sPLA2 inhibitor, acts synergistically with pravastatin to decrease atherosclerosis, possibly through decreased levels of systemic inflammation or decreased lipid levels. A-002 treatment also resulted in a profound increase in plasma PON activity and significantly larger fibrous caps, suggesting the formation of more stable plaque architecture. The secretory phospholipase A2 (sPLA2) family of enzymes catalyze the production of fatty acids and lysophospholipids by the hydrolysis of phospholipids on cell membranes and circulating lipoproteins (1Dennis E.A. Diversity of group types, regulation, and function of phospholipase A2.J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). sPLA2 activity has been shown to increase the ability of LDL to aggregate, promote foam cell formation in vitro (2Wooton-Kee C.R. Boyanovsky B.B. Nasser M.S. de Villiers W.J. Webb N.R. Group V sPLA2 hydrolysis of low-density lipoprotein results in spontaneous particle aggregation and promotes macrophage foam cell formation.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 762-767Crossref PubMed Scopus (97) Google Scholar), increase proinflammatory bioactive lipid levels (3Leitinger N. Watson A.D. Hama S.Y. Ivandic B. Qiao J.H. Huber J. Faull K.F. Grass D.S. Navab M. Fogelman A.M. et al.Role of group II secretory phospholipase A2 in atherosclerosis: 2. Potential involvement of biologically active oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1291-1298Crossref PubMed Scopus (144) Google Scholar), decrease HDL levels, and increase atherosclerosis in transgenic mice (4Ivandic B. Castellani L.W. Wang X.P. Qiao J.H. Mehrabian M. Navab M. Fogelman A.M. Grass D.S. Swanson M.E. de Beer M.C. et al.Role of group II secretory phospholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1284-1290Crossref PubMed Scopus (224) Google Scholar). Additional mouse studies have shown more specifically that overexpression of human sPLA2 groups IIa and V enzymes resulted in increased lesion size (5Bostrom M.A. Boyanovsky B.B. Jordan C.T. Wadsworth M.P. Taatjes D.J. de Beer F.C. Webb N.R. Group v secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 600-606Crossref PubMed Scopus (113) Google Scholar, 6Tietge U.J. Pratico D. Ding T. Funk C.D. Hildebrand R.B. Van Berkel T. Van Eck M. Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress.J. Lipid Res. 2005; 46: 1604-1614Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), while human group IIa overexpression increased oxidative stress. Human studies have shown that circulating sPLA2 levels and activity are associated with increased risk of coronary artery disease (CAD) in healthy individuals (7Mallat Z. Benessiano J. Simon T. Ederhy S. Sebella-Arguelles C. Cohen A. Huart V. Wareham N.J. Luben R. Khaw K.T. et al.Circulating secretory phospholipase A2 activity and risk of incident coronary events in healthy men and women: the EPIC-Norfolk study.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1177-1183Crossref PubMed Scopus (96) Google Scholar) and those with existing disease (8Kugiyama K. Ota Y. Takazoe K. Moriyama Y. Kawano H. Miyao Y. Sakamoto T. Soejima H. Ogawa H. Doi H. et al.Circulating levels of secretory type II phospholipase A(2) predict coronary events in patients with coronary artery disease.Circulation. 1999; 100: 1280-1284Crossref PubMed Scopus (234) Google Scholar). Expression of sPLA2 enzymes (groups IIa, IId, IIe, IIf, III, V, and X) in atherosclerotic lesions increases with the development of atherosclerosis (9Kimura-Matsumoto M. Ishikawa Y. Komiyama K. Tsuruta T. Murakami M. Masuda S. Akasaka Y. Ito K. Ishiguro S. Morita H. et al.Expression of secretory phospholipase A2s in human atherosclerosis development.Atherosclerosis. 2008; 196: 81-91Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Additional human studies have identified single nucleotide polymorphisms within the sPLA2 group V gene associated with increased levels of oxidized lipids and CAD risk (10Wootton P.T. Arora N.L. Drenos F. Thompson S.R. Cooper J.A. Stephens J.W. Hurel S.J. Hurt-Camejo E. Wiklund O. Humphries S.E. et al.Tagging SNP haplotype analysis of the secretory PLA2-V gene, PLA2G5, shows strong association with LDL and oxLDL levels, suggesting functional distinction from sPLA2-IIA: results from the UDACS study.Hum. Mol. Genet. 2007; 16: 1437-1444Crossref PubMed Scopus (34) Google Scholar). Statins are a class of compounds (HMG-CoA reductase inhibitors) known to significantly decrease the risk of CAD by their ability to beneficially alter lipid levels and decrease systemic inflammation (11Monetti M. Canavesi M. Camera M. Parente R. Paoletti R. Tremoli E. Corsini A. Bellosta S. Rosuvastatin displays anti-atherothrombotic and anti-inflammatory properties in apoE-deficient mice.Pharmacol. Res. 2007; 55: 441-449Crossref PubMed Scopus (68) Google Scholar). However, statins are estimated to address only approximately 1/3 of coronary events (12Sever P.S. Dahlof B. Poulter N.R. Wedel H. Beevers G. Caulfield M. Collins R. Kjeldsen S.E. Kristinsson A. McInnes G.T. et al.Prevention of coronary and stroke events with atorvastatin in hypertensive patients who have average or lower-than-average cholesterol concentrations, in the Anglo-Scandinavian Cardiac Outcomes Trial–Lipid Lowering Arm (ASCOT-LLA): a multicentre randomised controlled trial.Lancet. 2003; 361: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (3354) Google Scholar). A significant need exists to identify additional therapeutic approaches to decrease the risk of CAD while working in association with statins. The majority of mouse studies using statins in apoE−/− mice show a dose-response effect on prevention of atherosclerosis while the effect on lipid levels is less pronounced (13Zadelaar S. Kleemann R. Verschuren L. de Vries-Van der Weij J. van der Hoorn J. Princen H.M. Kooistra T. Mouse models for atherosclerosis and pharmaceutical modifiers.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1706-1721Crossref PubMed Scopus (436) Google Scholar); this model has also been used to determine synergism between statins and an apolipoprotein A-I mimetic peptide with cardioprotective properties (14Navab M. Anantharamaiah G.M. Hama S. Hough G. Reddy S.T. Frank J.S. Garber D.W. Handattu S. Fogelman A.M. D-4F and statins synergize to render HDL antiinflammatory in mice and monkeys and cause lesion regression in old apolipoprotein E-null mice.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1426-1432Crossref PubMed Scopus (143) Google Scholar). A-002 (varespladib methyl/LY333013/S-3013) is the oral prodrug of A-001 (varespladib sodium/LY315920/S-5920), which was discovered using structural-based drug design using sPLA2 IIa structure (15Mihelich E.D. Schevitz R.W. Structure-based design of a new class of anti-inflammatory drugs: secretory phospholipase A(2) inhibitors, SPI.Biochim. Biophys. Acta. 1999; 1441: 223-228Crossref PubMed Scopus (54) Google Scholar) and takes advantage of the unique catalytic site His-Asp dyad not present on the related cPLA2 or Lp-PLA2 enzymes to specifically and selectively inhibit the target enzyme (16Murakami M. Kudo I. Secretory phospholipase A2.Biol. Pharm. Bull. 2004; 27: 1158-1164Crossref PubMed Scopus (110) Google Scholar). A-001 inhibits both and murine sPLA2 activity with IC50s between 9 and 15 nM when measured using recombinant forms of the group IIa, V, or X enzymes, or when measured in serum from various species (17Snyder D.W. Bach N.J. Dillard R.D. Draheim S.E. Carlson D.G. Fox N. Roehm N.W. Armstrong C.T. Chang C.H. Hartley L.W. et al.Pharmacology of LY315920/S-5920, [[3-(aminooxoacetyl)-2-ethyl-1- (phenylmethyl)-1H-indol-4-yl]oxy] acetate, a potent and selective secretory phospholipase A2 inhibitor: a new class of anti-inflammatory drugs, SPI.J. Pharmacol. Exp. Ther. 1999; 288: 1117-1124PubMed Google Scholar). The oral or parenteral administration of A-001 abolished PLA2 activity in a dose-dependent fashion in a transgenic mouse overexpressing human sPLA2 IIa(17) and in sepsis patients expressing high levels of sPLA2(17). Additional in vivo studies in several animal models of lung injury in rats and rabbits (18Koike K. Yamamoto Y. Hori Y. Ono T. Group IIA phospholipase A2 mediates lung injury in intestinal ischemia-reperfusion.Ann. Surg. 2000; 232: 90-97Crossref PubMed Scopus (46) Google Scholar) demonstrated that administration of A-001 or A-002 inhibited sPLA2 activity, lowered the production of eicosanoids downstream of sPLA2, and prevented injury, which are consistent with the specific inhibition of sPLA2 enzymes rather than downstream enzymes or cPLA2. Based on the combined animal and human data implicating sPLA2 enzymes in atherosclerosis and the known protective effect of statins in human CAD, the potential synergistic effect between A-002 and pravastatin was explored in an apoE−/− mouse model of atherogenesis. In addition to atherosclerotic lesions, plasma lipids and paraoxonase (PON) activity were also measured. There was a significantly larger decrease in atherosclerosis and total cholesterol levels with the combined treatment compared with either treatment alone, and there was a significant increase in PON activity associated with A-002 treatment. Thus, the combination of the sPLA2 inhibitor, A-002, with statins is an effective strategy to increase the antiatherosclerotic potential of statin therapy. ApoE−/− null male mice on a C57BL/6J genetic background were purchased from the Jackson Laboratory (Bar Harbor, MI). At approximately 8 weeks of age the mice were divided into six groups (n = 12 per group) and placed on a 12-week ad libitum Western-type diet (TD 88137, Teklad, Madison, WI) in which the diet was supplemented with A-002 and/or pravastatin. See Supplemental Methods for more details. Animals were fasted overnight before being bled from the retroorbital sinus. Plasma was collected using heparin as an anticoagulant and used to determine total cholesterol, HDL cholesterol levels, and PON activity. Plasma lipids were determined as described previously (19Hedrick C.C. Castellani L.W. Warden C.H. Puppione D.L. Lusis A.J. Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice.J. Biol. Chem. 1993; 268: 20676-20682Abstract Full Text PDF PubMed Google Scholar). See Supplemental Methods for more details. Methods for the quantitation of atherosclerotic lesions in the aortic root were as previously reported (20Shaposhnik Z. Wang X. Weinstein M. Bennett B.J. Lusis A.J. Granulocyte macrophage colony-stimulating factor regulates dendritic cell content of atherosclerotic lesions.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 621-627Crossref PubMed Scopus (79) Google Scholar). See Supplemental Methods for more details. Data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using ANOVA (StatView, SAS Institute, Cary, NC) except for comparisons of lesion area, where a nonparametric Kruskal-Wallace test was used to determine differences followed by a Mann-Whitney test to identify synergistic effects of A-002 and pravastatin on lesion area. The anti-inflammatory and antiatherosclerotic potential of a specific small molecule sPLA2 inhibitor, A-002, was tested alone and in combination with the widely used statin, pravastatin. Further, a dose-response effect of A-002 and the potential for a synergistic effect of the sPLA2 inhibitor combined with pravastatin was evaluated. The pravastatin dose used was previously demonstrated to not show an effect on lesion size or lipoprotein profiles in apoE−/− mice when administered as monotherapy (14Navab M. Anantharamaiah G.M. Hama S. Hough G. Reddy S.T. Frank J.S. Garber D.W. Handattu S. Fogelman A.M. D-4F and statins synergize to render HDL antiinflammatory in mice and monkeys and cause lesion regression in old apolipoprotein E-null mice.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1426-1432Crossref PubMed Scopus (143) Google Scholar) but was effective when combined with a complementary anti-inflammatory agent. Male apoE−/− mice were placed for 12 weeks on Western-type diets formulated to deliver 2 mg/kg per day of pravastatin and 15 mg/kg (low dose A-002) or 150 mg/kg (high dose A-002) per day of A-002. The high dose was selected to maintain serum levels of A-002 at least 10-fold over the concentration required to inhibit sPLA2 enzyme activity by 50% (IC50s for murine sPLA2 groups V and X are 17 nM and 1.8 nM, respectively; data not shown). The low dose was selected to partially inhibit sPLA2 enzyme activity based on earlier efficacy studies using transgenic mice overexpressing human sPLA2 group IIa that showed that doses in the 0.3–3 mg/kg range exhibited dose-dependent inhibition of sPLA2 activity (data not shown). The formulations were based on an empirically determined average food intake of 2.5 g/day per mouse. No significant differences were detected between any of the groups with respect to behavior, the rate of weight gain or food intake (see Supplemental Results). A-002 decreased atherosclerotic lesion formation in a dose-dependant manner and demonstrated synergistic effects when combined with pravastatin. As previously described, pravastatin alone demonstrated no effect on lesion size, either in the aortic root or in the ascending and thoracic aorta. The low dose of A-002 decreased lesions 40% (P = 0.019) while the high dose decreased lesions by 75% (P = 0.0006), as measured by en face analysis. When the low dose of A-002 was combined with pravastatin, en face measured lesions decreased 75%, a significant decrease as compared with the low dose A-002 alone (P = 0.048) (Fig. 1A). Although the high dose of A-002 combined with pravastatin suppressed en face measured lesion area 86% to levels about half as much as high dose A-002 alone, this was not significantly smaller than the 74% reduction in lesion size of mice treated only with high dose A-002. Aortic root lesions decreased more modestly, by 23% (P = 0.049), with low dose A-002 and 27% (P = 0.02) with high dose A-002 (Fig. 1B). There were no significant differences in aortic root lesion area between any of the other groups of mice. Lesion composition was examined in the aortic root by staining sections for macrophage content, smooth muscle cell (SMC) content, and by Movat staining, which allows visualization of the fibrous cap as well as collagen and proteoglycans. The most striking effect of A-002 was a substantial increase in the size of the fibrous cap (Fig. 2A–D) (see supplementary Figure IIA–D). Movat staining revealed that mice treated with the high dose of A-002 exhibited approximately a 2- to 3-fold increase in the percentage of lesion area consisting of fibrous caps (Fig. 2D). There was no change in fibrous cap size in mice treated with low dose A-002. Increased fibrous cap area was also observed after examining the SMC content of the fibrous cap (Fig. 2B). No change was detected in the overall percentage of lesion area consisting of collagen and proteoglycans (Fig. 2C); and there was no effect of pravastatin alone on any parameter of lesion composition or any synergistic effect of treating mice with pravastatin and A-002. Macrophages, which constituted approximately 70% of the aortic root lesion area across all groups, did not significantly decrease as a percentage of lesion area (Fig. 2E). A small increase of approximately 10% in the macrophage positive area was noted in low dose A-002 treated mice. This increase was not noted in any other treatment group and is not statistically different from the statin-treated group. Also, this treatment group had dramatically smaller lesion area when examined by an en face approach. It appears to be an anomaly not indicative of any larger significant trends. The sum of the macrophage and collagen/proteoglycan area appears to exceed 100% because the matrix components overlap with the macrophage positive area and are not mutually exclusive markers of plaque composition. Overexpression of sPLA2 in mice has been shown to modify LDL and HDL, presumably by hydrolysis of phospholipids. Therefore, various parameters of plasma lipids and lipoprotein particle size distribution were measured. A synergistic effect of combining A-002 and pravastatin was observed in decreasing total cholesterol levels 18% (P = 0.015) in the high dose A-002 plus pravastatin group as compared with the control group (Table 1). HDL cholesterol levels trended higher in A-002 treated mice and increased 20% to 40% in both groups treated with A-002 and pravastatin. There was no effect of pravastatin or a synergistic effect between pravastatin and A-002 on HDL cholesterol levels.TABLE 1Plasma lipid parametersLow A-002 (n = 11)High A-002 (n = 12)Pravastatin (n = 12)Low A-002 + Pravastatin (n = 10)High A-002 + Pravastatin (n = 10)Placebo (n = 12)Total cholesterol (mg/dl)1,355 ± 1031,301 ± 461,405 ± 391,399 ± 971,219 ± 70aP < 0.05.1,478 ± 68HDL cholesterol (mg/dl)21 ± 1.1bP < 0.0001.17 ± 1.014 ± 0.921 ± 1.4bP < 0.0001.18 ± 1.0aP < 0.05.15 ± 0.7Triglycerides (mg/dl)100 ± 495 ± 690 ± 3100 ± 874 ± 684 ± 5Relative PON activity46 ± 5aP < 0.05.46 ± 1aP < 0.05.33 ± 3aP < 0.05.45 ± 3aP < 0.05.38 ± 2aP < 0.05.24 ± 2PON, paraoxonase.a P < 0.05.b P < 0.0001. Open table in a new tab PON, paraoxonase. Fast protein liquid chromatograpy analyses did not show that the sizes and relative levels of the various lipoprotein classes were substantially affected by A-002 (Fig. 3). There were no significant differences in glucose (data not shown) or triglyceride levels (Table 1) between any groups. PON1 is an enzyme carried on HDL that exerts antioxidant effects. Plasma PON activity was elevated approximately 90% (P < 0.0001) in both groups treated with A-002 and in the group treated with low dose A-002 plus pravastatin (Table 1). Pravastatin treatment alone increased PON activity approximately 38% (P = 0.02) but did not enhance the ability of A-002 to increase PON activity. The ability of the specific small molecule sPLA2 inhibitor A-002 to inhibit the development of atherosclerosis in apoE−/− mice on a Western type diet was tested. A-002 treatment resulted in a substantial decrease in atherosclerotic lesion size and a remodeling of the plaque toward a lesion with a more prominent fibrous cap. These effects of A-002 were enhanced by pravastatin in a synergistic manner with respect to atherosclerosis lesion content and plasma total cholesterol. Lipoprotein metabolism was altered, resulting in elevated HDL levels and an even larger increase in plasma antioxidant PON activity. Profound effects of the combined sPLA2/statin treatment were observed, particularly on lesion area in the ascending and thoracic aorta as measured by en face. The low dose of A-002 decreased lesion size by 40% alone and further decreased lesion size by 75% in the presence of pravastatin. A similar trend was observed with the higher dose of A-002, which decreased lesion area 75% alone. This decreased further to an 86% reduction with the addition of pravastatin (P = NS). These data strongly suggest synergy of A-002 with pravastatin on inhibition of atherosclerotic lesion size. In contrast to en face data, a maximum 27% lesion size reduction was observed when examining aortic root atherosclerotic lesions from mice treated with high dose A-002 with no significant difference in lesion size noted in mice treated with A-002 and pravastatin. This observation is similar to results reported from LDLR−/− mice that received group V sPLA2 deficient bone marrow and demonstrated no significant difference in aortic root lesion size but a 36% reduction in lesion area within the aortic arch and thoracic aorta (5Bostrom M.A. Boyanovsky B.B. Jordan C.T. Wadsworth M.P. Taatjes D.J. de Beer F.C. Webb N.R. Group v secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 600-606Crossref PubMed Scopus (113) Google Scholar). In light of large variability in plaque size and the number of mice examined in this study, we estimated that we could reliably detect a 40–50% difference in lesion size. Smaller differences, as observed in this study, can be detected, but not in all cases. Therefore, the lack of our ability to detect an effect in the groups receiving both agents could be due to a lack of power in our study to detect modest changes in plaque size. Also, variation in lesion formation between these two compartments could be due to sPLA2 having a more dramatic effect on amount of aortic surface area covered by plaque by promoting lateral plaque growth rather than the total amount of plaque volume. Human studies (21Barnett P.A. Spence J.D. Manuck S.B. Jennings J.R. Psychological stress and the progression of carotid artery disease.J. Hypertens. 1997; 15: 49-55Crossref PubMed Scopus (336) Google Scholar) have indicated that carotid plaques grow approximately 2.5 times more rapidly in width than in thickness along the vessel in the direction of flow. Overall, these results suggest that sPLA2 has a much larger effect on lesion size in the ascending and thoracic aorta compared with the aortic root. Increased sPLA2 expression has been demonstrated to enhance arterial collagen deposition (22Ghesquiere S.A. Gijbels M.J. Anthonsen M. van Gorp P.J. van der Made I. Johansen B. Hofker M.H. de Winther M.P. Macrophage-specific overexpression of group IIa sPLA2 increases atherosclerosis and enhances collagen deposition.J. Lipid Res. 2005; 46: 201-210Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). While no clear mechanistic explanation was provided or careful examination of the fibrous cap conducted, Oestvang and Johansen (23Oestvang J. Johansen B. PhospholipaseA2: a key regulator of inflammatory signalling and a connector to fibrosis development in atherosclerosis.Biochim. Biophys. Acta. 2006; 1761: 1309-1316Crossref PubMed Scopus (46) Google Scholar) speculate that this increased collagen formation could be due to some alteration in eicosanoid production as a result of sPLA2 activity. However, because the mice used in this study are deficient in group II sPLA2, no direct comparisons can be made to those results. Work by Bostrom et al. (5Bostrom M.A. Boyanovsky B.B. Jordan C.T. Wadsworth M.P. Taatjes D.J. de Beer F.C. Webb N.R. Group v secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 600-606Crossref PubMed Scopus (113) Google Scholar) examining the effect of macrophage specific group V sPLA2 expression on plaque formation is more relevant to this study. That report noted increased collagen content only within the ascending aorta and not within the aortic sinus, the region where we examined lesion size and composition. Although plaque size and sPLA2 distribution was also examined in mice deficient in macrophage specific group V sPLA2, no mention was made of collagen content within the intima. No change was detected in collagen content within the lesions of the A-002 treated mice using a Movat staining method but a 2- to 3-fold increase in fibrous cap area and lesional SMC content was observed, suggesting increased smooth muscle proliferation and/or decreased protease or matrix metalloproteinase activity within the lesion. It is still possible that increased sPLA2 activity could enhance collagen deposition while sPLA2 inhibition would have no effect on collagen deposition. Also it is possible that a decreased inflammatory state exists in this model as a result of sPLA2 inhibition resulting in increased SMC content, which is manifested in the larger fibrous caps. Extended treatment of SMCs with oxidized lipids led to the formation of oxidized adducts of the platelet-derived growth factor β receptor, decreasing cell proliferation (24Vindis C. Escargueil-Blanc I. Elbaz M. Marcheix B. Grazide M.H. Uchida K. Salvayre R. Negre-Salvayre A. Desensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers.Circ. Res. 2006; 98: 785-792Crossref PubMed Scopus (63) Google Scholar). Platelet-derived growth factor is an important factor regulating SMC proliferation in vitro and in the fibrous cap (25Sano H. Sudo T. Yokode M. Murayama T. Kataoka H. Takakura N. Nishikawa S. Nishikawa S.I. Kita T. Functional blockade of platelet-derived growth factor receptor-beta but not of receptor-alpha prevents vascular smooth muscle cell accumulation in fibrous cap lesions in apolipoprotein E-deficient mice.Circulation. 2001; 103: 2955-2960Crossref PubMed Scopus (146) Google Scholar). sPLA2 inhibition could be suppressing the formation of such oxidized adducts and result in increased SMC proliferation and larger fibrous caps. A 3-fold increase in lesional SMC content was observed in mice deficient in the serine protease Serp-1 gene, suggesting that altered protease activity may also have a role in the fibrous cap size increase (26Bot I. von der Thusen J.H. Donners M.M. Lucas A. Fekkes M.L. de Jager S.C. Kuiper J. Daemen M.J. van Berkel T.J. Heeneman S. et al.Serine protease inhibitor Serp-1 strongly impairs atherosclerotic lesion formation and induces a stable plaque phenotype in ApoE−/−mice.Circ. Res. 2003; 93: 464-471Crossref PubMed Scopus (59) Google Scholar). However, due to the fact that there was no change in the proteoglycan or collagen content of the lesion, it seems more likely that sPLA2 inhibition has specifically altered SMC proliferation or survival and not changed the matrix degrading potential of macrophages or other cells within the lesion. No change was detected in the macrophage content of the lesions except for the group receiving low dose A-002. It appears that treatment with the sPLA2 inhibitor A-002 promotes the development of a smaller and more stable lesion encased in a thicker fibrous cap. It is interesting to speculate on the role of group V sPLA2 in atherosclerosis. Group V sPLA2 is most frequently expressed by macrophages in mouse lesions but is also expressed in aortic SMC (5Bostrom M.A. Boyanovsky B.B. Jordan C.T. Wadsworth M.P. Taatjes D.J. de Beer F.C. Webb N.R. Group v secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 600-606Crossref PubMed Scopus (113) Google Scholar). We did observe a large increase in the fibrous cap size with A-002 treatment, indicating that perhaps group V enzyme activity negatively regulates SMC proliferation or migration. The group V enzyme has approximately a 3-fold greater enzymatic activity with purified HDL as a substrate compared with LDL and can modify lipoproteins in the presence of serum, properties that the group IIa enzyme lacks. Also, the group V gene is induced by a high-fat Western diet while the group IIa gene is not affected (27Rosengren B. Peilot H. Umaerus M. Jonsson-Rylander A.C. Mattsson-Hulten L. Hallberg C. Cronet P. Rodriguez-Lee M. Hurt-Camejo E. Secretory phospholipase A2 group V: lesion distribution, activation by arterial proteoglycans, and induction in aorta by a Western diet.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1579-1585Crossref PubMed Scopus (78) Google Scholar). This suggests that in this study A-002 could be inhibiting the group V enzyme's ability to modify plasma LDL into a more inflammatory species and to prevent the degradation of plasma HDL function that normally occurs in apoE−/− mice on a high-fat diet. sPLA2's effects on lipid metabolism have been studied primarily in transgenic mice. Such mice were more prone to atherosclerosis and showed increased plasma levels of apoB-containing lipoproteins combined with suppressed HDL cholesterol and PON activity (4Ivandic B. Castellani L.W. Wang X.P. Qiao J.H. Mehrabian M. Navab M. Fogelman A.M. Grass D.S. Swanson M.E. de Beer M.C. et al.Role of group II secretory phospholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1284-1290Crossref PubMed Scopus (224) Google Scholar). A-002 did not significantly change the VLDL/LDL fraction of lipoproteins based on the fast protein liquid chromatograpy analysis, but there were increased levels of HDL cholesterol. In fact, plasma HDL cholesterol increased up to 40% with A-002 treatment. It is interesting to note that sPLA2 expression appears to increase HDL catabolism while simultaneously increasing hepatic SR-BI cholesterol ester uptake (28Tietge U.J. Maugeais C. Cain W. Grass D. Glick J.M. de Beer F.C. Rader D.J. Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I.J. Biol. Chem. 2000; 275: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Thus, A-002 may increase both HDL production and turnover, but the former to a greater extent. A-002 treatment not only increased plasma HDL concentration but also mediated up to a 90% increase in plasma PON activity, suggesting that the increased HDL is functionally active and perhaps more significantly anti-inflammatory on a per-particle basis than prior to treatment. PON1 expression can be suppressed in HepG2 liver cells by the addition of oxidized-LDL or inflammatory cytokines and atherogenic diets reduce PON activity in mice (29Ng C.J. Shih D.M. Hama S.Y. Villa N. Navab M. Reddy S.T. The paraoxonase gene family and atherosclerosis.Free Radic. Biol. Med. 2005; 38: 153-163Crossref PubMed Scopus (251) Google Scholar). It is therefore possible that inhibiting sPLA2 activity results in decreased oxidized lipid or inflammatory cytokine production, as indicated by previous studies (3Leitinger N. Watson A.D. Hama S.Y. Ivandic B. Qiao J.H. Huber J. Faull K.F. Grass D.S. Navab M. Fogelman A.M. et al.Role of group II secretory phospholipase A2 in atherosclerosis: 2. Potential involvement of biologically active oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1291-1298Crossref PubMed Scopus (144) Google Scholar, 4Ivandic B. Castellani L.W. Wang X.P. Qiao J.H. Mehrabian M. Navab M. Fogelman A.M. Grass D.S. Swanson M.E. de Beer M.C. et al.Role of group II secretory phospholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1284-1290Crossref PubMed Scopus (224) Google Scholar) that in turn leads to increased expression of hepatic PON1 and the subsequent increase in plasma PON activity. PON1−/− apoE−/− mice showed a 2-fold increase in lesion size (30Shih D.M. Xia Y.R. Wang X.P. Miller E. Castellani L.W. Subbanagounder G. Cheroutre H. Faull K.F. Berliner J.A. Witztum J.L. et al.Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis.J. Biol. Chem. 2000; 275: 17527-17535Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar) that was attributed to increased lipid oxidation and production of bioactive phospholipids while human PON1 transgenic mice on an apoE−/− background showed a 20% reduction in aortic root lesions in the context of no increased HDL levels (31Tward A. Xia Y.R. Wang X.P. Shi Y.S. Park C. Castellani L.W. Lusis A.J. Shih D.M. Decreased atherosclerotic lesion formation in human serum paraoxonase transgenic mice.Circulation. 2002; 106: 484-490Crossref PubMed Scopus (384) Google Scholar). Therefore, treatment with the sPLA2 inhibitor A-002 could be inhibiting lesion formation by decreasing the levels of bioactive oxidized phospholipids through increased PON activity. Increased PON activity could also be important in regulating fibrous cap size by suppressing the formation of oxidized adducts of the platelet-derived growth factor β receptor. A link between elevated HDL levels and substantially increased SMC content was previously demonstrated within lesions from apoA1 transgenic mice (32Rong J.X. Li J. Reis E.D. Choudhury R.P. Dansky H.M. Elmalem V.I. Fallon J.T. Breslow J.L. Fisher E.A. Elevating high-density lipoprotein cholesterol in apolipoprotein E-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content.Circulation. 2001; 104: 2447-2452Crossref PubMed Scopus (192) Google Scholar), again supporting the idea that a component of HDL can increase SMC proliferation and plaque stability by decreasing inflammation. Overall, the sPLA2 inhibitor A-002 can synergize with statins to decrease lesion size via different mechanisms. A-002 treatment results in certain changes in lipid metabolism that lead to elevated HDL levels and increased HDL protective capacity. This perhaps leads to a suppression of inflammatory lipid generation, decreased systemic inflammation, and/or increased reverse cholesterol transport. Interestingly, we detected a synergistic effect between high dose A-002 and pravastatin on decreasing total cholesterol levels. These data also suggest that inhibiting sPLA2 in human disease, particularly when combined with the activity of statins, could retard atherogenesis. Unlike most direct interventions that target one vulnerable plaque at a time, A-002 promises to broadly improve plaque stability and decrease CAD by increasing the size of the fibrous cap and decreasing plaque size while increasing plaque SMC content. Download .pdf (1.0 MB) Help with pdf files
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