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LipoCardium: Endothelium-directed cyclopentenone prostaglandin-based liposome formulation that completely reverses atherosclerotic lesions  Paulo I. Homem.

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Presentation on theme: "LipoCardium: Endothelium-directed cyclopentenone prostaglandin-based liposome formulation that completely reverses atherosclerotic lesions  Paulo I. Homem."— Presentation transcript:

1 LipoCardium: Endothelium-directed cyclopentenone prostaglandin-based liposome formulation that completely reverses atherosclerotic lesions  Paulo I. Homem de Bittencourt, Denise J. Lagranha, Alexandre Maslinkiewicz, Sueli M. Senna, Angela M.V. Tavares, Lisiane P. Baldissera, Daiane R. Janner, Joelso S. Peralta, Patrícia M. Bock, Lucila L.P. Gutierrez, Gustavo Scola, Thiago G. Heck, Maurício S. Krause, Lavínia A. Cruz, Dulcinéia S.P. Abdalla, Cláudia J. Lagranha, Thais Lima, Rui Curi  Atherosclerosis  Volume 193, Issue 2, Pages (August 2007) DOI: /j.atherosclerosis Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

2 Fig. 1 Structures of CP-PGs of A2 and J2 families. Asterisks denote the electrophilic carbons that are susceptible to Michael addition reaction with nucleophiles, such as reactive sulfhydryls present in GSH molecules and cysteine residues of cellular proteins: C-11 in PGA2 ring, C-9 in PGJ2 ring, and C-9 and C-13 in both Δ12-PGJ2 and 15-d-PGJ2 molecules. Atherosclerosis  , DOI: ( /j.atherosclerosis ) Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

3 Fig. 2 PG effects on lipogenesis and cholesterol metabolism in oxLDL-elicited foamy macrophages. [1-14C]acetate incorporations (24h) into both cell (lipogenesis) and supernatant (exporting rates) lipid fractions were assessed in the presence or absence of test PGs: (A) phospholipids, (B) free cholesterol, (C) free fatty acids, (D) triacylgycerols and (E) cholesteryl esters. (F) [4-14C]Cholesterol uptake and intracellular distribution into free cholesterol or cholesteryl esters. The influence of PGs on cholesterol export towards the extracellular space (G) in [4-14C]cholesterol-labeled cells was assessed after washing and further cultivating cells for additional 24h; (H), cholesterol moieties remaining in the cells. (I) [2,3-3H]cholesterol oleate uptake and distribution (hydrolysis and re-esterification). The effects of PGs on exporting capacity and fates of cholesteryl esters were assessed by measuring [2,3-3H]cholesterol moieties exported (J) from or remaining (K) in the cells cultured for additional 24h. Data are the means±S.E.M. of three different preparations, each in double. Significant differences: (*) between each test group and the oxLDL controls by the Student's t-test; (†) between control (untransformed) and oxLDL controls (one-way ANOVA, Bonferroni's test); P values are in the text. Expected routes for: (L) [1-14C]acetate-derived acetyl units, (M) [4-14C]cholesterol and (N) [2,3-3H]cholesterol fluxes. In this inset: black arrows represent lipid incorporation into cell membrane; A and H letters indicate reactions catalyzed by ACAT and cholesteryl ester hydrolases respectively; shaded arrows indicate lipid export from the cell. CHOL, cholesterol; CEST, cholesteryl ester; PL, phospholipid; TAG, triacylglycerol; OxCHOL, oxysterols derived from the oxidation reactions over cholesterol molecules; CHOL-FA, cholesteryl esters of fatty acids. Atherosclerosis  , DOI: ( /j.atherosclerosis ) Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

4 Fig. 3 Effect of PGA2 on cholesterol accumulation in foamy macrophages. OxLDL-elicited foamy macrophages were treated with PGA2 for 72h and assayed for cholesterol and cholesteryl ester contents (mass). Data are the means±S.E.M. of three different preparations, each in duplicate. Significant differences: (*) Student's t-test, between each test group and control (no oxLDL) values; (†) between each PGA2 test group and oxLDL controls (one-way ANOVA, Bonferroni's test). Individual P values are given in the text. Atherosclerosis  , DOI: ( /j.atherosclerosis ) Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

5 Fig. 4 Effects of PGA2 and 15-PGJ2 on gene expression. OxLDL-transformed human U937 foamy macrophages were treated (24h) with PGA2, 15-PGJ2 or ethanol diluent and assayed for expression of mRNA encoding HMG-CoA reductase (A), PPARγ (B), CD36 (C), caspase-3 (D), p53 (E) and bcl-xL (F). Rat-derived foamy macrophages were identically treated and assayed for hsp70 protein expression (G). Data are the means±S.E.M. of three different experiments in duplicate. Significant differences: (*) Student t-test, between each test group and its control (oxLDL or no oxLDL); (†) between control (untransformed) and oxLDL controls (one-way ANOVA, Bonferroni's test); P values are in the text. (H) Interference photomicropgraphies of Sudan-III stained rat macrophages. Control cells (1) were oxLDL-transformed into foam cells (2) and showed the characteristic high-lipid “fatty-islet” shape. The cells were 24-h treated with 1μM PGA2 (3) still in the presence of oxLDL (added 15min after PGA2 addition). Atherosclerosis  , DOI: ( /j.atherosclerosis ) Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

6 Fig. 5 LipoCardium in vivo effects. Male ldlr−/− mice under a high-fat diet during 4 months, commencing from the adulthood (3 months), showed marked evidence of cardiovascular disturbances such as lost of extremities (A) due to poor peripheral blood supply, loss of hair and of hair brightness (B), prostration, blindness and generalized sickness (C). Then, the animals were daily treated (i.p.) with LipoCardium (or EDCPL placebo injections containing no PGA2) for 14 days when they were killed to be analyzed for anatomical-pathological alterations or maintained under high-lipid diet (with no LipoCardium treatment) until aging. A 14-day treated mouse is shown (D). The effects of LipoCardium treatment (or placebo) on the thoracic aorta (E–H), renal artery (I and J) and coronary artery (K and L) slices are shown in tissues stained with Sudan black dye (which evidences neutral lipids). Thickness-to-diameter (T/D) ratios were calculated as follows: T/D=c/[(a+b)/2], where c is the average thickness (measured in three different points along the artery perimeter), and a and b are the smallest and the largest internal diameters respectively (M). Mean values±S.E.M. (expressed as percentage values) of six different animals are shown for thoracic aorta (N), renal (O) and coronary artery (P) preparations. *P= for the difference between control and LipoCardium-treated groups (Student's t-test). Q and R: hematoxylin–eosin stained slices of thoracic aortas. Arrows: subendothelial cell proliferation and neoitimal hyperplasia (Q1), which is absent in LipoCardium-treated animals (R1), and the typical basophilic cytoplasmic staining of infiltrating cells (characteristic of foamy macrophage subendothelial occupation, Q2), which is absent in LipoCardium-treated groups (R2). Atherosclerosis  , DOI: ( /j.atherosclerosis ) Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions

7 Fig. 6 Proposed mechanism for LipoCardium action. Cardiovascular risk factors determine endothelial injury which triggers inflammation via NF-κB-induced genes, including vascular cell adhesion molecule-1 (VCAM-1), which drives the firm adhesion of circulating monocytes that invade subendothelial space becoming foamy macrophages, the pivotal cell type that perpetuates atherosclerotic lesions (1). The anti-VCAM-1 antibodies of LipoCardium specifically direct PGA2 molecules to injured cells (2). After uptake and lysosomal disassembly, PGA2 may influence atheroma plaque progression (3) through its tetravalent effect: (a) anti-inflammatory, (b) antiproliferative, (c) anticholesterogenic and (d) cytoprotective. In view of PGA2-mediated vanish of inflammatory signals brought about endothelial cells and foamy macrophages, no further inflammatory cells transmigrate from the blood stream towards the subendothelial space. Atherosclerosis  , DOI: ( /j.atherosclerosis ) Copyright © 2006 Elsevier Ireland Ltd Terms and Conditions


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