Supplemental Figure 1. Loss of Cdkn2b promotes atherosclerosis in both genders and at serial timepoints. Loss of Cdkn2b also increases growth of the

Transcription

1 Supplemental Figure. Loss of Cdkn2b promotes atherosclerosis in both genders and at serial timepoints. Loss of Cdkn2b also increases growth of the necrotic core and increases early apoptosis. (A) Relative to male Cdkn2b +/+,ApoE -/- control mice, male Cdkn2b -/-,ApoE -/- mice develop advanced atherosclerotic plaques as soon as 8 weeks after initiating Western diet, with these changes persisting over time (4x magnification). (B) A similar change is observed in female mice (P =0.04, 4x magnification). (C) Additional examples of the large acellular necrotic cores observed in Cdkn2b -/-,ApoE -/- mice relative to Cdkn2b +/+,ApoE -/- control animals (H&E staining on top, Masson Trichrome on bottom at various magnifications). (D) Because very little apoptosis was observed at the terminal endpoint of the chronic model provided in Figure, an additional 0 Cdkn2b +/+,ApoE -/- and Cdkn2b -/-,ApoE -/- who had only received 4 weeks of high-fat diet were infused with Angiotensin II by osmotic minipump for 72 hours prior to sacrifice to induce acute vascular inflammation. In this case, Cdkn2b -/-,ApoE -/- displayed a 97% increase in TUNEL positive cells per section relative to controls, of borderline significance (P=0.06). (E) Compensation of other 9p2.3 locus genes in aortic tissue from Cdkn2b -/-,ApoE -/- relative to Cdkn2b +/+,ApoE -/- control mice. As previously described, there is significant upregulation of Cdkn2a in Cdkn2b knockout animals. In the current studies, no difference in the expression of the pro-apoptotic gene p9/arf was observed at the terminal endpoint. (F) No difference in lipid levels or fasting glucose was observed between genotypes after 2 weeks of Western diet. 43

2 Suppl. Fig. Time course A Week 2 ** Atherosclerosis area (mm2) Week 8 Cdkn2b-/ + Baseline B * TUNEL positive cells E F Cdkn2b-/-/ApoE-//ApoE-/mg/dL Apoptosing cells () Cdkn2b-/- Week 2 20x Magnification 0x Magnification 4x Magnification 4x Magnification Cdkn2b-/ Week 8 Cdkn2b-/- Relative expression Atherosclerosis area (mm2) Cdkn2b-/ D C Female mice Cdkn2b-/ Cd kn 2b p 9A rf Cd kn 2a MT AP To ta lc ho Tri g les lyc ter eri HD ol LD de L-C s ho les L- ter Ch Glu ol co ole se ste rol

3 Supplemental Figure 2. Bioinformatics analysis of human atherosclerosis samples and evaluation of additional efferocytosis genes in HCASMC. (A) Topological relationship between and genes involved in efferocytosis in human coronary artery segments. Color bars correspond to module assignment. (B) Gene coexpression network cluster dendogram from human coronary artery segments. The network adjacency was calculated from topological overlap between all gene pairs represented in the expression data set. The dynamic tree cut algorithm was used to iteratively choose stable cluster sizes and partition the network into modules. (C) Correlation between expression and genes involved in efferocytosis in human coronary artery segments: (a) All samples; (b) samples without atherosclerotic lesions; (c) samples with atherosclerotic lesions. (D) Relative expression of several additional candidate efferocytosis genes in si HCASMC compared to sicont HCASMC at baseline (D) and during apoptosis (E). (F) Compared to control Cdkn2b +/+,ApoE -/- specimens, aortas from female heterozygous Cdkn2b +/-,ApoE -/- had an intermediate 62.% reduction in Calr expression (P < 0.03) compared to the 90.8% reduction in Calr expression observed in Cdkn2b -/-,ApoE -/- animals (P < 0.02, top panel). These findings correlated with the in vivo reduction of aortic Cdkn2b expression observed in herterozygous animals (bottom panel). (G) Co-expression analysis in human carotid artery atherosclerotic plaques confirm that and CALR are positively associated, as observed in human coronary artery atherosclerotic plaque (r 2 = 0.74, P <0.000). 44

4 A rho B Cluster Dendrogram Suppl. Fig. 2 C ELMO PPARG CD36 TIMD4 ABCA6 AKT CALR GAS6 MFGE8 CX3CL CD4 CQB MERTK GULP DOCK CD47 ANXA PROS LRP APOH BAI SIRPA LRPAP MBL2 NRH3 ICAM3 PANX TGFB ELMO PPARG CD36 TIMD4 ABCA6 AKT CALR GAS6 MFGE8 CX3CL CD4 CQB MERTK GULP DOCK CD47 ANXA PROS LRP APOH BAI SIRPA LRPAP MBL2 NRH3 ICAM3 PANX TGFB a. b. c. Height Modules d hclust (*, "average") Correlation coefficient Correlation coefficient Correlation coefficient D E Eat-Me ligand expression Eat-Me ligand expression C ALR M FGE 8 CX3CL ABCA6 ICAM3 AS6 G A PO H PROS CQB ANXA D47 RP C L M BL 2 S IRP A 3 NRH PPARG LRPAP TGFB AI B T IMD C D 4 M ERT K C D3 6 E LMO DOCK A KT P ANX GULP HCASMC - Baseline CD47 ICAM-3 Gas-6 Thrombospondin Lactadherine Cq APOH ABC- HCASMC - Apoptosis C ALR M FGE 8 CX3CL ABCA6 ICAM3 AS6 CD47 ICAM-3 Gas-6 Thrombospondin Lactadherine Cq APOH ABC- G A PO H PROS CQB ANXA D47 RP C L M BL 2 S IRP A 3 NRH PPARG LRPAP TGFB AI B T IMD C D 4 M ERT K C D3 6 E LMO DOCK A KT P ANX GULP F Aortic calreticulin expression (mrna, %) Aortic Cdkn2b expression (mrna, %) C ALR M FGE 8 CX3CL ABCA6 ICAM3 AS6 G A PO H PROS CQB ANXA D47 RP C L M BL 2 S IRP A 3 NRH PPARG LRPAP TGFB AI B T IMD C D 4 M ERT K C D3 6 E LMO DOCK A KT P ANX GULP Heterozygous aortas * Cdkn2b -/- / Cdkn2b +/- / Cdkn2b +/+ / ApoE -/- ApoE -/- ApoE -/- Heterozygous aortas Cdkn2b -/- / Cdkn2b +/- / Cdkn2b +/+ / ApoE -/- ApoE -/- ApoE -/- G Calreticulin Carotid plaque expression correlation var2 95% CI Fitted values var

5 Supplemental Figure 3. Additional analysis of the CALR promoter. (A) Publically-available data from the UCSC genome browser reveals that the CALR promoter has an open chromatin pattern, DNase hypersensitivity sites, a consistent AoSMC DNA methylation pattern and published ChIP-seq data all of which suggest that E2F4 could regulate CALR expression in human SMCs. (B) Untransfected HCASMC increased their expression of in response to exogenous TGF- β stimulation in a pattern consistent with the luciferase reporter data shown in Figure 3. (C) CALR promoter oligo sequences and ChiP primers employed in the EMSA and immunoprecipitation studies, respectively, described in Figure 3. (D) Positive and negative control reactions employed in the EMSA experiments for Figure 3. 45

6 A B Fold Change (mrna) TGF-B: Suppl. Fig. 3 0 ng/ml 2 ng/ml 0.5 ng/ml None C D A

7 Supplemental Figure 4. does not alter the phagocytic capacity of the neighboring cell. (A) Control gates employed for all efferocytosis studies. (B) Although loss of rendered apoptotic cells resistant to efferocytosis, loss of in the neighboring, non-apoptotic HCASMC had no impact on its phagocytic capacity. Note that was undetectable in professional phagocytes such as macrophages, in culture. (C) Apoptotic primary Cdkn2b -/- aortic smooth muscle cells resist phagocytic clearance by RAW macrophages relative to apoptotic primary Cdkn2b +/+ aortic smooth muscle cells (P < 0.0), in keeping with findings provided for -deficient HCASMC in Figure 4. (D) Heterozygous Cdkn2b +/- aortic smooth muscle cells show an intermediate efferocytosis phenotype. (E) Additional examples of failed efferocytosis in vivo in Cdkn2b -/-,ApoE -/- mice. Electron micrographs at various magnifications again reveal more frequent apoptosis and necrosis in Cdkn2b -/-,ApoE -/- animals, with condensed chromatin and interruption of plasma membrane integrity (black arrows), along with a high burden of extracellular debris and ABs not associated with an adjacent macrophage (black arrowheads). Conversely, Cdkn2b +/+,ApoE -/- control mice displayed smoothly outlined nuclei with normal heterochromatin patterns, normal sized mitochondria and intact plasma membranes (white arrowheads). These plaques also routinely displayed macrophages which had ingested numerous ABs, suggestive of intact efferocytosis. 46

8 si sicontrol Efferocytosis Resistance Assay - Primary Mouse Aortic Smooth Muscle Cells Cdkn2b-/- 200x 200x 00 FITC Green RAW MΦ 00 FITC Green RAW MΦ 2500x 6000x D Efferocytosis rate (%) Cdkn2b-/ Efferocytosis rate (%) sicontrol Efferocytosis rate (%) si C Cdkn2b-/ Efferocytosis Capacity Assay B E Orange ABs Green Phagocytes Unstained ABs Unstained SMC A Suppl. Fig. 4 Efferocytosis Gates and Single Positive Controls Heterozygous cells Cdkn2b-/- Cdkn2b+/- 500x 2500x 3000x 800x

9 Supplemental Figure 5. Macrophages co-cultured with Cdkn2b +/+ AB (grey bar) upregulated Abca relative to baseline (white bar). This homeostatic pathway was significantly blunted when macrophages were co-cultured with Cdkn2b -/- AB (black bar), as observed for the experiments with -deficient HCASMCs provided in Figure 5. Supplemental Table. Summary of topological characteristics of gene coexpression modules identified in human coronary artery segments. Supplemental Table 2. Summary of gene coexpression module assignment in human coronary artery segments. ktotal = global network connectivity, kwithin = intramodular network connectivity, kout = extramodular network connectivity, kdiff = difference between extramodular and intramodular connectivity, MAR = maximum adjacency ratio, kme0 kmen = correlation between gene expression and module eigengene expression for modules 0 n. Supplemental Table 3. Primers and probes used in these studies. 47

10 A Abca expression (mrna %) Macrophages + + Cdkn2b +/+ AB + Cdkn2b -/- AB Unstimulated Suppl. Fig. 5