Protein Kinase Cθ Via Activating Transcription Factor 2-Mediated CD36 Expression and Foam Cell Formation of Ly6Chi Cells Contributes to Atherosclerosis

Abstract

Background: Although the role of thrombin in atherothrombosis is well studied, its role in the pathogenesis of diet-induced atherosclerosis is not known.

Methods: Using a mouse model of diet-induced atherosclerosis and molecular biological approaches, here we have explored the role of thrombin and its G protein-coupled receptor signaling in diet-induced atherosclerosis.

Results: In exploring the role of G protein-coupled receptor signaling in atherogenesis, we found that thrombin triggers foam cell formation via inducing CD36 expression, and these events require Par1-mediated Gα12-Pyk2-Gab1-protein kinase C (PKC)θ-dependent ATF2 activation. Genetic deletion of PKCθ in apolipoprotein E (ApoE)^-/- mice reduced Western diet-induced plaque formation. Furthermore, thrombin induced Pyk2, Gab1, PKCθ, and ATF2 phosphorylation, CD36 expression, and foam cell formation in peritoneal macrophages of ApoE^-/- mice. In contrast, thrombin only stimulated Pyk2 and Gab1 but not ATF2 phosphorylation or its target gene CD36 expression in the peritoneal macrophages of ApoE^-/-:PKCθ^-/- mice, and it had no effect on foam cell formation. In addition, the aortic root cross-sections of Western diet-fed ApoE^-/- mice showed increased Pyk2, Gab1, PKCθ, and ATF2 phosphorylation and CD36 expression as compared with ApoE^-/-:PKCθ^-/- mice. Furthermore, although the monocytes from peripheral blood and the aorta of Western diet-fed ApoE^-/- mice were found to contain more of Ly6C^hi cells than Ly6C^lo cells, the monocytes from Western diet-fed ApoE^-/-:PKCθ^-/- mice were found to contain more Ly6C^lo cells than Ly6C^hi cells. It is interesting to note that the Ly6C^hi cells showed higher CD36 expression with enhanced capacity to form foam cells as compared with Ly6C^lo cells.

Conclusions: These findings reveal for the first time that thrombin-mediated Par1-Gα12 signaling via targeting Pyk2-Gab1-PKCθ-ATF2-dependent CD36 expression might be playing a crucial role in diet-induced atherogenesis.

Keywords: CD36; G protein-coupled receptor signaling; PKCθ; atherosclerosis; foam cell; macrophages; thrombin.

Figure 1.. Thrombin induces CD36 expression and foam cell formation via Par1 activation.

A. Quiescent RAW264.7 cells were treated with or without thrombin (0.5 U/ml) for the indicated time periods and analyzed for foam cell formation. B. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for SR-A1, SR-B1 and CD36 levels and normalized to β-tubulin. C. Cells were transfected with siControl or siCD36 (100 nM), quiesced, treated with and without thrombin for 4 hrs and analyzed for foam cell formation. D. Cells were transfected with siControl, siPar1, siPar3 or siPar4 (100 nM), quiesced, treated with and without thrombin for 1 hr and analyzed by Western blotting for CD36 levels and the blot was reprobed for Par1, Par3, Par4 or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. E. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) for 4 hrs and analyzed for foam cell formation. F. Cells were transfected with siControl or siPar1 (100 nM), quiesced, treated with and without thrombin for 4 hrs and analyzed for foam cell formation. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl; **, p < 0.05 vs Thrombin or siControl + Thrombin. Scale bar is 50 μm.

Figure 2.. Gα12, Pyk2 and Gab1 mediate thrombin-induced CD36 expression and foam cell formation.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were immunoprecipitated with anti-Par1 antibodies and the immunocomplexes were analyzed by Western blotting for Gαq, Gα11, Gα12 or Gα13 levels and the blot was normalized to Par1. B. Cells were transfected with siControl or siGα12 (100 nM), quiesced, treated with and without thrombin for 1 hr and analyzed by Western blotting for CD36 levels and the blot was reprobed for Gα12 or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. C. Cells were transfected with siControl or siGα12 (100 nM), quiesced, treated with and without thrombin for 4 hrs and analyzed for foam cell formation. D. Extracts of control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for pPyk2 levels and the blot was normalized to its total levels. E. Quiescent cells were treated with and without thrombin in the presence and absence of PF431396 (5 μM) for 1 hr and analyzed by Western blotting for CD36 levels and the blot was normalized to β-tubulin levels. F. Quiescent cells were treated with and without thrombin in the presence and absence of PF431396 for 4 hrs and analyzed for foam cell formation. G. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) for 1 min and analyzed by Western blotting for pPyk2 levels and the blot was normalized to its total levels. H. All the conditions were same as in Panel B except that after quiescence the cells were treated with and without thrombin for 1 min and analyzed by Western blotting for pPyk2 levels and the blot was reprobed for Pyk2 or Gα12 levels to show the effect of the siRNA on its target and off target molecules levels. I. Extracts of control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for pGab1 levels and the blot was normalized to its total levels. J & K. Cells were transfected with siControl or siGab1 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for Gab1 or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. L. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) for 1 min and analyzed by Western blotting for pGab1 levels and the blot was normalized to its total levels. M. Cells were transfected with siControl or siGα12 (100 nM), quiesced, treated with and without thrombin for 1 min and analyzed for pGab1 levels and the blot was reprobed for Gab1 or Gα12 levels to show the effect of the siRNA on its target and off target molecules levels. N. Quiescent cells were treated with and without thrombin in the presence and absence of PF431396 (5 μM) for 1 min and analyzed for pGab1 levels and the blot was normalized for its total levels. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl; **, p < 0.05 vs Thrombin or siControl + Thrombin. Scale bar is 50 μm.

Figure 2.. Gα12, Pyk2 and Gab1 mediate thrombin-induced CD36 expression and foam cell formation.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were immunoprecipitated with anti-Par1 antibodies and the immunocomplexes were analyzed by Western blotting for Gαq, Gα11, Gα12 or Gα13 levels and the blot was normalized to Par1. B. Cells were transfected with siControl or siGα12 (100 nM), quiesced, treated with and without thrombin for 1 hr and analyzed by Western blotting for CD36 levels and the blot was reprobed for Gα12 or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. C. Cells were transfected with siControl or siGα12 (100 nM), quiesced, treated with and without thrombin for 4 hrs and analyzed for foam cell formation. D. Extracts of control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for pPyk2 levels and the blot was normalized to its total levels. E. Quiescent cells were treated with and without thrombin in the presence and absence of PF431396 (5 μM) for 1 hr and analyzed by Western blotting for CD36 levels and the blot was normalized to β-tubulin levels. F. Quiescent cells were treated with and without thrombin in the presence and absence of PF431396 for 4 hrs and analyzed for foam cell formation. G. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) for 1 min and analyzed by Western blotting for pPyk2 levels and the blot was normalized to its total levels. H. All the conditions were same as in Panel B except that after quiescence the cells were treated with and without thrombin for 1 min and analyzed by Western blotting for pPyk2 levels and the blot was reprobed for Pyk2 or Gα12 levels to show the effect of the siRNA on its target and off target molecules levels. I. Extracts of control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for pGab1 levels and the blot was normalized to its total levels. J & K. Cells were transfected with siControl or siGab1 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for Gab1 or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. L. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) for 1 min and analyzed by Western blotting for pGab1 levels and the blot was normalized to its total levels. M. Cells were transfected with siControl or siGα12 (100 nM), quiesced, treated with and without thrombin for 1 min and analyzed for pGab1 levels and the blot was reprobed for Gab1 or Gα12 levels to show the effect of the siRNA on its target and off target molecules levels. N. Quiescent cells were treated with and without thrombin in the presence and absence of PF431396 (5 μM) for 1 min and analyzed for pGab1 levels and the blot was normalized for its total levels. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl; **, p < 0.05 vs Thrombin or siControl + Thrombin. Scale bar is 50 μm.

Figure 3.. Lack of a role of p115 RhoGEF, Rac1, RhoA and Pak2 in thrombin- induced CD36 expression and foam cell formation.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were immunoprecipitated with anti-p115 RhoGEF or anti-Pak2 antibodies and immunocomplexes were analyzed by Western blotting using anti-PY20 or anti-pSer/Thr antibodies, respectively. For GTP-Rac1 or GTP-RhoA levels, equal amounts of protein from control and the indicated time periods of thrombin- treated cells were incubated with GST-Pak1 or GST-Rhotekin-conjugated glutathione sepharose 4B beads and the pull down proteins were analyzed by Western blotting for Rac1 and RhoA levels, respectively. The same cell extracts were also analyzed by Western blotting for total Rac1 and RhoA levels. B & C. Cells were transfected with siControl or sip115 RhoGEF(100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 expression and foam cell formation, respectively. The CD36 blot was reprobed for p115 RhoGEF or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. D & E. Cells were transfected with siControl or siRac1 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for Rac1 or β-tubulin to show the effect of the siRNA on its target and off target molecules levels. F & G. Cells were transfected with siControl or siRhoA (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for RhoA or β-tubulin to show the effect of the siRNA on its target and off target molecules levels. H & I. Cells were transfected with siControl or siPak2 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for Pak2 or β-tubulin to show the effect of the siRNA on its target and off target molecules levels. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl. Scale bar is 50 μm.

Figure 4.. PKCθ mediates thrombin-induced CD36 expression and foam cell formation.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were analyzed for phosphorylation of the indicated PKC isoform either by Western blotting using their phosphospecific antibodies or immunoprecipitation with pSer/Thr antibody followed by immunoblotting with the indicated PKC isoform antibody. B & C. Cells were transfected with siControl or siPKCθ siRNA, quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation. The CD36 blot was reprobed for PKCθ and β-tubulin to show the effect of the siRNA on its target and off target molecules levels. D & F. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) or PF431396 (5 μM) for 1 hr and analyzed by Western blotting for pPKCθ and normalized to its total levels. E & G. Cells were transfected with siControl or siGα12 siRNA, quiesced, treated with and without thrombin for 1 hr and analyzed for pPKCθ and the blots were reprobed for PKCθ, Gα12 or Gab1 levels to show the effects of the siRNAs on their target and off target molecules levels. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl; **, p<0.05 vs Thrombin or siControl + Thrombin. Scale bar is 50 μm.

Figure 5.. Par1, Gα12, Pyk2, Gab1, PKCα and ATF2 mediate thrombin-induced CD36 promoter activity.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were immunoprecipitated with anti-PPARγ antibodies and the resulting immunocomplexes were analyzed by Western blotting for pSer/Thr or acetyl lysine antibodies and the blot was normalized to PPARγ levels. B. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for pATF2 levels and the blot was normalized to its total levels. C & D. Cells were transfected with siControl or siATF2 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation. The CD36 blot was reprobed for ATF2 and β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. E & G. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) or PF431396 (5 μM) for 1 hr and analyzed by Western blotting for pATF2 levels and normalized to its total levels. F, H & I. Cells were transfected with siControl, siGα12, siGab1 or siPKCθ (100 nM), quiesced, treated with and without thrombin and analyzed by Western blotting for pATF2 levels. The blots were reprobed for ATF2, Gα12, Gab1 or PKCθ to show the effects of the siRNAs on their target and off target molecules levels. J. CD36 promoter encompassing from -662 nt to +147 nt was cloned, sequenced and analyzed by TRANSFAC for transcriptional factors binding elements. K. The CD36 promoter encompassing from −662 nt to +147 nt was cloned into pGL3 vector and the RAW264.7 cells were transfected with the empty vector or pGL3-mCD36 promoter plasmids, growth-arrested, treated with and without thrombin for 6 hrs and the luciferase activity was measured. L. The RAW264.7 cells were transfected with empty vector, pGL3-mCD36 or pGL3-mCD36m (mutant for ATF2-binding site) plasmids, growth-arrested, treated with and without thrombin for 6 hrs and the luciferase activity was measured. M. All the conditions were the same as in panel K except that after transfection and quiescence, cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM), PF431396 (5 μM) for 6 hrs and the luciferase activity was measured. N. All the conditions were the same as in panel K except that after transfection with the plasmids, cells were again transfected with siGab1, siPKCθ or siATF2 (100 nM), quiesced, treated with and without thrombin for 6 hrs and analyzed for luciferase activity. O. Nuclear extracts of control and various time periods of thrombin treated cells were analyzed by EMSA for ATF2 binding using ATF2 binding site at −107 nt as a biotin labeled oligonucleotide probe. P. Nuclear extracts of control and thrombin-treated cells were analyzed for the presence of ATF2 in the protein-DNA complexes by supershift EMSA. Q. Control and thrombin-treated cells were analyzed for ATF2 binding to CD36 promoter by ChIP assay. R. Primary peritoneal macrophages from WT mice were treated with and without thrombin for the indicated time periods and analyzed by Western blotting for pPKCθ pATF2 or CD36 levels and normalized to their total levels or β-tubulin. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl or vector; **, p < 0.05 vs Thrombin, siControl + Thrombin or pGL3-mCD36p + Thrombin. Scale bar is 50 μm.

Figure 5.. Par1, Gα12, Pyk2, Gab1, PKCα and ATF2 mediate thrombin-induced CD36 promoter activity.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were immunoprecipitated with anti-PPARγ antibodies and the resulting immunocomplexes were analyzed by Western blotting for pSer/Thr or acetyl lysine antibodies and the blot was normalized to PPARγ levels. B. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were analyzed by Western blotting for pATF2 levels and the blot was normalized to its total levels. C & D. Cells were transfected with siControl or siATF2 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation. The CD36 blot was reprobed for ATF2 and β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. E & G. Quiescent cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM) or PF431396 (5 μM) for 1 hr and analyzed by Western blotting for pATF2 levels and normalized to its total levels. F, H & I. Cells were transfected with siControl, siGα12, siGab1 or siPKCθ (100 nM), quiesced, treated with and without thrombin and analyzed by Western blotting for pATF2 levels. The blots were reprobed for ATF2, Gα12, Gab1 or PKCθ to show the effects of the siRNAs on their target and off target molecules levels. J. CD36 promoter encompassing from -662 nt to +147 nt was cloned, sequenced and analyzed by TRANSFAC for transcriptional factors binding elements. K. The CD36 promoter encompassing from −662 nt to +147 nt was cloned into pGL3 vector and the RAW264.7 cells were transfected with the empty vector or pGL3-mCD36 promoter plasmids, growth-arrested, treated with and without thrombin for 6 hrs and the luciferase activity was measured. L. The RAW264.7 cells were transfected with empty vector, pGL3-mCD36 or pGL3-mCD36m (mutant for ATF2-binding site) plasmids, growth-arrested, treated with and without thrombin for 6 hrs and the luciferase activity was measured. M. All the conditions were the same as in panel K except that after transfection and quiescence, cells were treated with and without thrombin in the presence and absence of SCH79797 (10 μM), PF431396 (5 μM) for 6 hrs and the luciferase activity was measured. N. All the conditions were the same as in panel K except that after transfection with the plasmids, cells were again transfected with siGab1, siPKCθ or siATF2 (100 nM), quiesced, treated with and without thrombin for 6 hrs and analyzed for luciferase activity. O. Nuclear extracts of control and various time periods of thrombin treated cells were analyzed by EMSA for ATF2 binding using ATF2 binding site at −107 nt as a biotin labeled oligonucleotide probe. P. Nuclear extracts of control and thrombin-treated cells were analyzed for the presence of ATF2 in the protein-DNA complexes by supershift EMSA. Q. Control and thrombin-treated cells were analyzed for ATF2 binding to CD36 promoter by ChIP assay. R. Primary peritoneal macrophages from WT mice were treated with and without thrombin for the indicated time periods and analyzed by Western blotting for pPKCθ pATF2 or CD36 levels and normalized to their total levels or β-tubulin. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl or vector; **, p < 0.05 vs Thrombin, siControl + Thrombin or pGL3-mCD36p + Thrombin. Scale bar is 50 μm.

Figure 6.. Genetic deletion of PKCα gene reduces the diet-induced atherosclerotic burden.

A. Representative en face staining of aortas from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice fed with WD for 16 wks are shown and the bar graph shows the plaque area as lesion %. B. Representative Oil red O staining of the aortic root sections of the mice described in panel A are shown and the bar graph represents the quantification of the area positive for lipid staining. C. Equal amounts of protein from peritoneal macrophages of ApoE^-/- and ApoE^-/-:PKCθ^-/- mice fed with CD or WD for 16 wks were analyzed by Western blotting for pPyk2, pGab1, pPKCθ, pATF2 and CD36 levels and the blots were normalized to their total levels. The CD36 blot was normalized to β-tubulin. D. The peritoneal macrophages isolated from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were treated with and without thrombin for the indicated time periods and analyzed by Western blotting for pPyk2, pGab1, pPKCθ, pPKCα/βII, pATF2 and CD36 levels and the blots were normalized to their total levels. The CD36 blot was normalized to β-tubulin. For Gα12 activation, the cell extracts were immunoprecipitated with anti-Par1 antibodies and the immunocomplexes were analyzed by Western blotting for Gα12 levels and the blot was normalized to Par1 levels. E. The macrophages from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were also analyzed for thrombin-induced foam cell formation. F & G. Peritoneal macrophages from ApoE^-/- mice were transfected with siControl or siATF2 (100 nm), quiesced, treated with and without thrombin for 1 hr, 4 hr or 24 hr and analyzed for CD36 expression, foam cell formation and proliferation, respectively. H. The aortic root cryosections of the mice described in panel B were analyzed by double immunofluorescence staining for pPyk2, pGab1, pPKCθ, pATF2 or CD36 in combination with Mac3. I. Aortic SMCs from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were treated with and without thrombin for the indicated time periods and analyzed by Western blotting for pPKCθ pATF2 or CD36 levels and normalized to their total levels or β-tubulin. The bar graphs represent Mean ± S.D. values of three experiments or 7 animals. *, p < 0.05 vs ApoE^-/-, or control; **, p < 0.05 vs ApoE^-/- + Thrombin. Scale bars are 200 μm (panel B), 20 μm (panels E & F) and 100 μm (panel H).

Figure 6.. Genetic deletion of PKCα gene reduces the diet-induced atherosclerotic burden.

A. Representative en face staining of aortas from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice fed with WD for 16 wks are shown and the bar graph shows the plaque area as lesion %. B. Representative Oil red O staining of the aortic root sections of the mice described in panel A are shown and the bar graph represents the quantification of the area positive for lipid staining. C. Equal amounts of protein from peritoneal macrophages of ApoE^-/- and ApoE^-/-:PKCθ^-/- mice fed with CD or WD for 16 wks were analyzed by Western blotting for pPyk2, pGab1, pPKCθ, pATF2 and CD36 levels and the blots were normalized to their total levels. The CD36 blot was normalized to β-tubulin. D. The peritoneal macrophages isolated from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were treated with and without thrombin for the indicated time periods and analyzed by Western blotting for pPyk2, pGab1, pPKCθ, pPKCα/βII, pATF2 and CD36 levels and the blots were normalized to their total levels. The CD36 blot was normalized to β-tubulin. For Gα12 activation, the cell extracts were immunoprecipitated with anti-Par1 antibodies and the immunocomplexes were analyzed by Western blotting for Gα12 levels and the blot was normalized to Par1 levels. E. The macrophages from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were also analyzed for thrombin-induced foam cell formation. F & G. Peritoneal macrophages from ApoE^-/- mice were transfected with siControl or siATF2 (100 nm), quiesced, treated with and without thrombin for 1 hr, 4 hr or 24 hr and analyzed for CD36 expression, foam cell formation and proliferation, respectively. H. The aortic root cryosections of the mice described in panel B were analyzed by double immunofluorescence staining for pPyk2, pGab1, pPKCθ, pATF2 or CD36 in combination with Mac3. I. Aortic SMCs from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were treated with and without thrombin for the indicated time periods and analyzed by Western blotting for pPKCθ pATF2 or CD36 levels and normalized to their total levels or β-tubulin. The bar graphs represent Mean ± S.D. values of three experiments or 7 animals. *, p < 0.05 vs ApoE^-/-, or control; **, p < 0.05 vs ApoE^-/- + Thrombin. Scale bars are 200 μm (panel B), 20 μm (panels E & F) and 100 μm (panel H).

Figure 7.. PKCθmediates diet-induced monocyte differentiation towards Ly6C^hi phenotype in ApoE^-/- mice.

A. Blood was collected from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice fed with WD for 16 wks, peripheral blood mononuclear cells were collected by density-gradient centrifugation, washed with RBC lysis buffer, resuspended into FACS buffer, blocked with mouse serum, washed with FACS buffer and incubated with fixable viability stain 450, anti-CD90-PE, anti-CD45R(B220)-PE, anti-CD49b-PE, anti-NK1.1-PE, anti-Ly6G-PE, anti-CD11b-APC, anti-Ly6C-FITC, anti-F4/80-PerCP-Cy5.5, anti-I-Ab-PerCP-Cy5.5 and anti-CD11c-PerCP-Cy5.5 antibodies. After washings, the cells were resuspended into sorting buffer and subjected to FACS analysis. The gating strategy is indicated as follows. Live cells (selected based on viability stain and higher forward scatter and lower side scatter) were gated as PE^– cells (in gate I). CD11b monocytes were gated as APC⁺ cells from PE^– cells (in gate II). CD11b⁺ monocytes were gated as Ly6C^hiF4/80^loCD11c^loI-Ab^lo, Ly6C^hiF4/80^hiCD11c^hiI-Ab^hi, Ly6C^loF4/80^hiCD11c^hiI-Ab^hi and Ly6C^loF4/80^loCD11c^loI-Ab^lo. All gates were set using full-minus-one (FMO) controls. The percentages of each CD11b⁺ monocyte subpopulations for all mice are indicated in the respective gate. B. Aortas from ApoE^-/- and ApoE^-/-:PKCθ^-/- mice fed with WD for 16 wks were collected, digested with a mixture of collagenase I, collagenase XI, Dnase I and hyaluronidase, washed with Hank’s balanced salt solution, resuspended in FACS buffer and subjected to FACS analysis as described in panel A. The percentages of each CD11b⁺ monocyte subpopulations for all mice are indicated in the respective gate. C & D. The bar graphs represent the number of retrieved Ly6C^hi and Ly6C^lo cells in the blood and aorta of WD-fed ApoE^-/- mice versus ApoE^-/-:PKCθ^-/- mice. E. The Ly6C^hi and Ly6C^lo cells were retrieved and analyzed for CD36 mRNA levels by RT-PCR or subjected to foam cell formation. F. Plasma from WD-fed ApoE^-/- and ApoE^-/-:PKCθ^-/- mice were analyzed for cytokines levels using mouse Multi-Analyte ELISArray Kit. Data were presented as Mean ± S.D. values of three experiments *, p < 0.01 vs ApoE^-/- mice (n = 6 with 3 mice per group per experiment). Scale bar is 20 μm.