Highly induced concentrations of the pro-inflammatory cytokines MCP-1 and KC (murine IL-8) in serum and increased splenic neutrophil content in ABCA1 KO→LDLr KO mice.

<p>Serum was obtained from WTD fed WT→LDLr KO and ABCA1 KO→LDLr KO mice. Serum MCP-1 and KC levels were determined by ELISA (A, B). n≥4 mice ± SEM per group. After red blood cell lysis, splenic neutrophil content (CD11b⁺GR-1⁺ cells) was analyzed by FACS (C, left panel). Values represent the percentage of total spleen cells. n≥6 mice ± SEM per group. Representative spleen sections were stained with an anti-Ly6G antibody (original magnification 100x; C, right panels). *p<0.05, **p<0.01.</p

Increased levels of circulating neutrophils in splenectomized ABCA1 KO→LDLr KO mice.

<p>At 18 weeks after BMT, blood was drawn from WT→LDLr KO and ABCA1 KO→LDLr KO mice. After red blood cell lysis, neutrophil content (CD11b⁺GR-1⁺ cells) was analyzed by FACS. Values are the percentage of total white blood cell counts. n≥3 mice ± SEM per group. *p<0.05 compared to splenectomized controls.</p

Increased VLDL cholesterol and triglycerides in LDLr KO mice receiving ORP8 KO bone marrow.

<p>Blood was isolated from LDLr KO mice after transplantation with ORP8 KO bone marrow (BM) or Wild Type (WT) BM. Lipoproteins were fractioned by size using FPLC and cholesterol and triglyceride contents were measured in each fraction. A) 17 weeks after transplantation and after 9 weeks of WTD feeding ORP8 KO BM recipients exhibit increased VLDL cholesterol (* P<0.05) B) Similarly, an increase in triglycerides in the VLDL fraction in blood of ORP8 KO BM recipients on WTD was found (* P<0.05). Significance was determined by Student t-test. N = 7, data are expressed as mean ±SEM.</p

Orp8 Deficiency in Bone Marrow-Derived Cells Reduces Atherosclerotic Lesion Progression in LDL Receptor Knockout Mice

<div><p>Introduction</p><p>Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown.</p><p>Methods and Results</p><p>LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity.</p><p>Conclusions</p><p>Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.</p></div

ORP8 expression detected in murine atherosclerotic plaques correlates with expression of CD68.

<p>mRNA was isolated from collar-induced plaques located in the carotid artery of LDLr KO mice. Samples were taken at 2 week intervals from week 2 baseline until week 12. Expression of ORP8 and the monocyte/macrophage marker CD68 was measured at each timepoint by microarray analysis and normalized for expression at baseline. A) ORP8 was found to be upregulated during plaque progression, as was CD68 (* P<0.05; $$<0.01, respectively). N = 3, values are expressed as mean ± SEM. Significance was assessed by ANOVA with Dunnetts post-test. B) Expression of ORP8 in murine atherosclerotic plaques correlated with expression of CD68 (P<0.001). Significance was assessed by linear regression.</p

Serum TC, TG and anti (α)-oxLDL antibody levels were measured at 10 and/or 18 weeks after BMT.

<p>Data represent mean ± SEM of ≥10 mice. *p<0.05, **p<0.01, compared to respective WT. n.d. indicates not determined.</p

Independent effects on atherosclerosis for both leukocyte ABCA1 deficiency and splenectomy.

<p>Quantification of atherosclerotic lesion sizes of WT→LDLr KO and ABCA1 KO→LDLr KO mice after 8 weeks of WTD feeding (left panel). Representative oil red-O stained cross-sections (original magnification 50x; right panels). Values represent the means of the average lesion size in 10 consecutive aortic root sections per mouse. n≥8 mice ± SEM per group. *p<0.05 compared to respective WT controls; #p<0.05 compared to sham operated controls.</p

No effect on blood leukocyte counts in LDLr KO mice receiving ORP8 KO bone marrow.

<p>At 17 weeks after transplantation and after 9 weeks of WTD feeding, blood was isolated from bone marrow recipient animals. Blood leukocytes were counted using a hematology analyzer, and different subpopulations were quantified as percentage of total leukocytes. A) No difference between the ORP8 KO BM recipients and the wildtype (WT) BM recipients in the amount of circulating leukocytes. B–D) Similarly, there was no difference in the percentage of lymphocytes, neutrophils and monocytes of total leukocytes. N = 15, results are expressed as mean ±SEM and significance was assessed by Student T-test.</p

After 6 and 9 weeks of WTD feeding no difference in blood cholesterol levels was found between LDLr KO mice receiving ORP8 KO BM or WT BM.

<p>After 6 and 9 weeks of WTD feeding no difference in blood cholesterol levels was found between LDLr KO mice receiving ORP8 KO BM or WT BM.</p

ORP8 KO BMDMs have reduced excretion of pro-inflammatory cytokines and reduced expression of co-stimulatory factors.

<p>Bone Marrow-Derived Macrophages (BMDMs) were generated from bone marrow of ORP8 KO mice or WT controls and stimulated with LPS (100 ng/mL). A) A decrease in secretion of IL-6 by LPS stimulated ORP8 KO BMDMs was measured using ELISA. (** P<0.01, N = 4) B) Similarly, TNFα secretion was also decreased in LPS-stimulated ORP8 KO BMDMs. (* P<0.05, N = 4). Expression of co-stimulatory factors CD40 and CD80 on BMDMs from ORP8 BM and WT BM was measured using FACS C) The percentage of unstimulated BMDMs expressing CD40 was decreased in the ORP8 KO group. (** P<0.01, N = 5) D) After stimulation with LPS, the percentage of BMDMs expressing CD80 was decreased in the ORP8 KO group (* P<0.05, N = 5). Significance of ELISA results was determined by Student T-test. Significance of FACS results was assessed by two-way ANOVA with Bonferroni post-tests. Data are expressed as mean ± SEM.</p