SR-BI -/- apoER61 h/h

1. SR-BI-/-apoER61+/+ SR-BI+/+apoER61h/h SR-BI-/-apoE-R61h/h Merged Bead Flourescence Oil Red O staining Bead fluorescence Donor : SR-BI-/-ApoEh/h SR-BI-/-apoER61h/h Donor : SR-BI+/+ApoEh/h Donor: SR-BI-/- apoER61h/h SR-BI-/- apoE +/+ SR-BI+/+ apoE-R61h/h Donor: SR-BI-/- apoE-R61h/h SR-BI-/- apoE+/+ SR-BI+/+ apoE-R61h/h Donor: SR-BI-/-apoE-R61h/h SR-BI-/-apoE +/+ SR-BI+/+apoE-R61h/h Expression of SR-BI in bone marrow derived cells protects against diet-induced coronary artery atherosclerosis in SR-BI-/-apoE-R61-hypomorphic miceYing Pei, Pei Yu, Xing Chen, Mark Fuller, Bernardo TrigattiAtherothrombosis Research Group, Department of Biochemistry and Biomedical Sciences, McMaster University and Thrombosis and Atherosclerosis Research Institute, Hamilton Health Sciences Abstract Results Mice deficient in the HDL receptor SR-BI with mutant apolipoproteinE (apoE) develop occlusive coronary artery atherosclerosis, myocardial infarction and premature death. They can be rescued by transplantation of wild type bone marrow. However, it is unknown if the protection of wild type bone marrow depends on the contributions of SR-BI, apoE or both. Here, we used bone marrow transplantation into SR-BI-/-apoE-R61h/h recipients to test if restoration of either SR-BI or ApoE alone was sufficient to protect against either aortic sinus or diet-induced CA atherosclerosis. Transplantation of bone marrow from SR-BI+/+apoE-R61h/h mice into SR-BI-/-apoE-R61h-h recipients generated mice expressing SR-BI exclusively in bone marrow-derived cells. This prevented the development of diet-induced occlusive CA atherosclerosis and myocardial infarction. In contrast, mice transplanted with autologous bone marrow or with bone marrow from SR-BI-/-/apoE+/+ mice (allowing restoration of normal apoE expression in bone marrow-derived cells) were not protected from occlusive coronary artery atherosclerosis and myocardial infarction. To test the mechanism involved, we compared the recruitment of leukocytes into the atherosclerotic vessels in SR-BI-/-apoE-R61h/h and SR-BI+/+apoE-R61h/h mice. Our results indicate that restoration of SR-BI in bone marrow-derived cells is sufficient to prevent occlusive coronary artery atherosclerosis in SR-BI-/-apoE-R61h/h mice and indicate an important role for SR-BI in BM-derived cells in protection against atherosclerosis. Fig. 2. Bone marrow transplantation timeline Fig. 1. Genotypes of recipients and donors Fig. 3. Restoration of SR-BI, but not apoE in bone marrow derived cells protects against atherosclerosis in the aortic sinus of HFCC diet fed SR-BI -/- apoE-R61 h/h mice SR-BI -/- apoE-R61 h/h mice were transplanted with BM from either SR-BI+/+apoE-R61h/h or control SR-BI -/-apoE-R61h/h donors as previously described. 4 weeks later, mice were fed HFCC diet for 18 days. Then monocytes were labeled in circulation by intravenous injection of fluorescent beads. Mice were sacrificed 1 day later. Aortic sinus sections were stained with oil red O and hematoxylin and examined by fluorescence microscopy in the green channel (left) for to visualize fluorescent bead bearing monocytes, or bright-field (right) to visualized atherosclerotic plaques. Representative images are shown. Numbers of beads normalized to the area of atherosclerotic plaques were determined for several sections from each mouse and are ;potted on the right. * SR-BI -/- apoE-R61 h/h mice were transplanted with BM from from either SR-BI+/+apoE-R61h/h, SR-BI -/-apoE+/+ donors or control SR-BI -/-apoE-R61h/h donors. 4 weeks later, mice were fed HFCC diet for 4 weeks. Representative oil red O stained and hematoxylin counter stained sections of the aortic sinus sections were observed. Data in the panel on the right are averages +/- SD of n=15, 6 or 10 mice transplanted with control autologous BM (SR-BI-/-apoEh/h) or BM from SR-BI-/-apoE+/+ or SR-BI+/+apoEh/h donors. * signifies P<0.05 compared to control BM. Introduction HDLs protect against atherosclerosis partly through reverse transport of cholesterol from peripheral cells to the liver for disposal. They are also known to suppress inflammatory signalling in endothelial cells, and may play a similar role in macrophages, which may also contribute to atheroprotection. The HDL receptor SR-BI is central to these processes. Mice that lack SR-BI, and have a deficiency in apolipoprotein E (apoE), develop occlusive coronary artery (CA) atherosclerosis either spontaneously at 6-8 weeks of age (SR-BI-/- mice)1 or after 3-6 weeks of being fed HFCC diet (SR-BI-/-apoE-R61 hypomorphic mice)2. Transplantation of wild type bone marrow into SR-BI -/- apoE -/- mice rescues the occlusive CA atherosclerosis phenotype3. It is not clear whether this is due to restoration of SR-BI, apoE, or both. We provide evidence here that restoration of SR-BI expression in BM but not of normal apoE in BM derived cells prevents diet induced occlusive CA atherosclerosis development in SR-BI -/-apoE-R61 hypomorphic mice. Preliminary studies suggest reduced monocyte recruitment into atherosclerotic plaques in mice transplanted with SR-BI+/+ BM. Conclusions • Restoration of SR-BI but not apoE in bone marrow derived-cells is sufficient to prevent occlusive coronary artery atherosclerosis in SR-BI-/-apoE-R61h/h mice. • This demonstrates directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis. • This may at least in part be due to reduced recruitment of monocytes expressing SR-BI Fig. 4. Restoration of SR-BI, but not apoE in bone marrow derived cells protects against the coronary artery disease in HFCC diet fed SR-BI -/- apoE-R61 h/h mice References 1) Braun, A., Trigatti, B.L., Post, M.J., Sato, K., Simons, M., Edelberg, J.M., Rosenberg, R.D., Schrenzel, M., Krieger, M. (2002) Circulation Research 90, 270-276. 2) Zhang S, Picard MH, Vasile E, Zhu Y, Raffai RL, Weisgraber KH, Krieger M. (2005) Circulation. 111:3457-64. 3) Yu, H., Zhang, W., Yancey, P.G., Koury, M.J., Zhang, Y, Fazio S., Linton M.F. (2006) Arterioscler. Thromb. Vasc. Biol. 26:150-156. 4) Frank, T., David, A., Theodore, J. K., Claudia, J., Rainer, S., Jaime, L., Alexandre, G., Jianhua, L., Matthias, M., Nico, V. R., Sergio, A. L., Andreas, J. H., Gwendalyn, J. R. (2007) J. Clin. Invest. 117: 185-194 Mice as described in the legend to Fig 3. Representative sections through CA’s are shown on the left. Numbers of CA’s that exhibited plaques occluding greater than 50 % of the lumen were determined in several cross sections through the hearts of each mouse and are plotted on the right as a percentage of total CA cross sections. Numbers of mice indicated in the legend to Fig 3. * signifies P<0.05 compared to control BM. Acknowledgements This research was funded by a grant from the Canadian Institute of Heath Research, Institute of Genetics to BT. The Atherothrombosis Research Group is funded by a program grant from the Heart and Stroke Foundation of Ontario. Ying Pei was funded by Heart & Stroke Foundation Research fellowship. Pei Yu is a recipient of a graduate scholarship from the Chinese Scholarship Council MF was funded by John D. Shultz Summer Studentship, and graduate scholarships from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research Fig. 5. Restoration of SR-BI, but not apoE, in bone marrow derived cells protects against cardiac fibrosis in HFCC diet fed SR-BI -/- apoE-R61 h/h mice Brief Methods • After BMT recovery, mice were fed a high fat (15 %), high cholesterol (1.25 %), cholate (0.5 %) containing (HFCC) diet. • Atherosclerosis in the aortic sinus and coronary arteries was assessed histologically in oil red O stained, 10µm cryosections • Cardiac fibrosis was assessed by trichrome staining of 10µm cryosections • Recruitment of monocytes labeled in vivo with fluorescent beads4 was analyzed by fluorescence microscopy. Fig. 6. A trend towards reduced monocyte recruitment into growing atherosclerotic plaques is observed in SR-BI -/-ApoEh/h mice transplanted with BM from SRBI+/+ApoEh/h . Mice as described in the legend to Fig 3. Cross sections through hearts were stained with Trichrome which stains health myocardium and collagen rich fibrotic areas blue. Substantial fibrosis was seen in hearts from SR-BI -/- apoE-R61 h/h mice transplanted with BM from either SR-BI-/-apoE-R61h/h or SR-BI -/-apoE+/+ donors but not in hearts from SR-BI -/- apoE-R61 h/h mice transplanted with BM from SR-BI+/+ apoE-R61h/h donors.