Impacts of ORP manipulation on SGBS cell adipogenic differentiation.

<p>ORP8 or ORP3 were overexpressed by infecting cells on day 10 with control (Blank) or ORP (Ad-ORP8, Ad-ORP3) adenoviral vectors, and collected for analysis on day 13. SGBS preadipocytes were transduced with an ORP11 shRNA (Sh-ORP11) or non-targeting (Sh-NT) shRNA lentivirus, followed by differentiation for 22 days. A. Western blots of total cell protein (10 µg/lane); ORP11, after 22 days of differentiation; ORP8 and ORP3, after 72 h of adenoviral transduction. The blots were probed with anti-β-actin as a loading control. B. The impacts of ORP11 silencing or ORP8/ORP3 overexpression on the mRNA levels of adipocyte differentiation markers adiponectin, aP2, leptin and PPARγ. In cells with ORP11 stably silenced also ORP8 and ORP3 mRNAs were quantified. The results are shown as fold changes relative to cells infected with the corresponding control viruses, and represent mean ± S.E., n = 3; *p<0.05. C. The cellular triglyceride concentration was measured by using an enzymatic assay. The results were normalized for total cell protein and are presented relative to cells infected with the corresponding control viruses (mean ± S.E., n = 3; *p<0.05, **p<0.01).</p

Time course of ORP11, ORP3, and ORP8 mRNA changes upon SGBS cell adipogenic differentiation.

<p>A. Phase contrast images of SGBS preadipocytes (0 D) and differentiating adipocytes on days 10, 14, and 22. Bars, 200 µm. B. The ORP mRNA levels were quantified at the differentiation time points indicated, as fold changes relative to preadipocytes (day 0). The data represents mean ± S.E., n = 3.</p

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

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

Decreased plaque size in ORP8 KO BM recipients and increased plaque macrophage content after 9 weeks WTD feeding.

<p>A) Plaque size was quantified in Oil Red O stained sections. After 9 weeks of WTD feeding following BMT, a decrease in plaque size could be measured. No difference was observed after 6 weeks of dietary challenge. (N = 13 * P<0.05, data was analyzed by two-way ANOVA with Bonferroni post-tests) B) Plaque macrophages were visualized using MoMa2 staining and quantified as a percentage of total plaque area. After 9 weeks of WTD feeding, an increase in the MoMa2 positive fraction was detected in the ORP8 KO BM recipients. (N = 13 * P<0.05, data was analyzed by two-way ANOVA with Bonferroni post-tests) C) Collagen was visualized by staining plaques from ORP8 BM and WT BM recipients with a Masson's Trichrome (MTC) kit, but no difference was found between the groups (N = 15, significance was assessed by Student T test). D) Apoptotic cells in atherosclerotic plaques of ORP8 KO BM recipients and WT BM controls were visualized by TUNEL staining. No difference in the amount of apoptotic cells was detected between treatment groups (N = 8, significance was assessed by Student T test). E) Aortic root sections from ORP8 KO BM recipients and WT BM controls were stained for CD3 to detect T cells. No difference between the groups was detected (N = 8, significance was assessed by Student T test). F) Representative images of Oil-Red O (ORO), MoMa2, TUNEL, CD3 and MTC stainings performed on atherosclerotic plaques of mice receiving ORP8 KO BM or WT BM. Images of ORO, Moma2 and MTC stainings, original magnification 5×. Images of TUNEL and CD3 stainings, original magnification 20×.</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

Adipogenic differentiation of SGBS cells.

<p>Haematoxylin and Oil Red O staining of SGBS preadipocytes (A) and adipocytes differentiated for 22 days (B). C. Relative mRNA levels of the adipocyte differentiation markers adiponectin, aP2, leptin and PPARγ in SGBS adipocytes as compared to preadipocytes. The bars indicate fold-change, mean ± S.E. (n = 3).</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

Copy numbers of OSBP/ORP mRNAs in human adipose tissues and SGBS adipocytes.

<p>The mRNAs were quantified by qPCR using the corresponding cDNAs as calibrators (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045352#s2" target="_blank">Methods</a>), and the mRNA quantities are presented on a log10 scale as copies/ng total RNA. A. Subcutaneous adipose tissue; B. Visceral adipose tissue. The data represents mean ± S.E., n = 4. C. Simpson-Golabi-Behmel syndrome (SGBS) adipocytes after 22-day differentiation. The mean from a single experiment carried out in triplicate is shown.</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