Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE–/– mice (original) (raw)
RAGE expression in apoE–/– mouse aorta. To establish the role of RAGE in the vasculature of apoE–/– mice (14, 15) fed rodent chow, we began by evaluating the time course of RAGE expression in atherosclerosis-prone vasculature. In the aortas of apoE–/– mice, a sustained, time-dependent increase in RAGE antigen was detected (Figure 1A). Beginning at age 10 weeks, a statistically significant increase in RAGE expression versus 6 weeks was noted, and by age 24 weeks, an approximately 6-fold increase in expression of RAGE versus 6 weeks was observed (P < 0.001; Figure 1A). Of note, at age 24 weeks, RAGE antigen was detected as 2 bands. Previous studies illustrated that RAGE immunoreactivity occasionally revealed more than one band, due either to alternative splicing or to differences in N-glycosylation (16, 17).
Expression of RAGE in the aortas of apoE–/– mice. (A) Western blotting. At the indicated ages, aortas were retrieved from apoE–/– mice and subjected to Western blotting with anti-RAGE IgG followed by anti–β-actin IgG. RAGE/β-actin antigen relative densitometry units were calculated. *P = 0.006, #P = 0.0001, ^P < 0.0001 versus 6 weeks. (B–H) Immunohistochemistry. Aorta tissue was subjected to immunohistochemistry to detect RAGE antigen (B and F). Sites of prominent RAGE expression were confirmed to be endothelial cells, based on colocalization of RAGE expression with CD31 (C) in the merged image (D). RAGE was also expressed in smooth muscle cells, based on colocalization with smooth muscle actin (G) in the merged image (H). Staining with nonimmune IgG revealed no specific immunoreactivity (E). Original magnification, ×400.
To delineate the sites of RAGE expression in the aorta, we performed immunohistochemistry on aortas of apoE–/– mice at age 10 weeks. RAGE expression was particularly evident in endothelial cells, as indicated by colocalization with CD31-expressing epitopes (Figure 1, B–D). Controls with nonimmune IgG revealed no specific immunoreactivity (Figure 1E). We performed immunohistochemistry to determine whether RAGE was expressed in smooth muscle cells at this early stage of vascular disease in apoE–/– mice. As illustrated in Figure 1, F–H, RAGE was expressed in medial smooth muscle cells, albeit to a lower degree than that observed in endothelial cells.
RAGE impacts atherosclerosis and endothelial function in apoE–/– mice. Based on these findings, we sought to dissect the role of the ligand-RAGE interaction in apoE–/– mice and used 2 distinct strategies. First, homozygous RAGE–/– mice, backcrossed more than 12 generations into C57BL/6, were bred into the apoE–/– background to obtain apoE–/–RAGE–/– mice. Second, to probe the role of endothelial RAGE in atherosclerosis, a Tg mouse was prepared to express human dominant-negative RAGE (DN-RAGE) specifically in endothelial cells by the preproendothelin-1 (PPET) promoter (18); these are referred to as Tg PPET DN-RAGE mice. Truncation of the cytoplasmic domain of RAGE mutes the impact of ligand-RAGE interaction in vitro and in vivo (1–3, 13). Multiple studies in RAGE-expressing cells have revealed the specificity of the DN-RAGE effect to RAGE ligands (2, 13). Specifically, when cells from DN-RAGE mice were incubated with RAGE ligands, abrogation of cellular stimulation was evident. However, when exposed to non-RAGE ligands, such as tumor necrosis factor–α or platelet-derived growth factor, cellular stimulation in DN-RAGE–expressing cells was intact and identical to that observed in WT RAGE-expressing cells (2, 13).
Based on these concepts, we introduced DN-RAGE into endothelial cells and prepared Tg mice. Southern blotting and PCR identified these DN-RAGE–expressing mice (Figure 2, A and B). Western blotting revealed a single band at approximately 55 kDa depicting RAGE antigen in WT mouse aortas; in contrast, aortas of RAGE–/– mice revealed no RAGE antigen (Figure 2C). Consistent with expression of WT and truncated RAGE, aortas retrieved from Tg PPET DN-RAGE mice revealed 2 bands; the upper band depicted WT RAGE and the lower band the truncated RAGE (Figure 2C).
RAGE impacts atherosclerosis and endothelial function in apoE–/– mice. Tg mice expressing DN-RAGE in endothelium. Founders were identified by Southern blotting (A) and by PCR (B). Lanes 1 and 6, lack of PPET DN-RAGE expression; lanes 2–5, expressing PPET DN-RAGE; C, control; M, base pair size marker. (C) Aortas were retrieved from the indicated mice and subjected to Western blotting using anti-RAGE IgG. (D) Thioglycollate-elicited macrophages were retrieved from the indicated mice and were incubated with S100b for 20 minutes. Western blotting was performed to detect phospho-JNK MAP kinase (P-JNK) followed by total JNK MAP kinase (T-JNK). Where indicated by the white line, lanes were run on the same gel but were noncontiguous. (E–J) Impact of RAGE on atherosclerosis at 14 weeks. Shown are representative images of aortic arches (E–G) and sections stained with oil red O (H–J). (K and L) Hearts were retrieved from apoE–/– (n = 12), apoE–/–RAGE–/– (n = 13), or apoE–/–Tg PPET DN-RAGE (n = 7) mice, and mean atherosclerotic lesion area (K) and lesion complexity profile (L) were determined. (M) Endothelium-dependent vasorelaxation was tested in isolated mouse aortic rings from apoE–/–, apoE–/–RAGE–/–, and apoE–/–Tg PPET DN-RAGE mice (n = 5 per group) sacrificed at 14 weeks of age. Relaxation is reported as percent of initial phenylephrine precontraction. Comparisons were conducted among groups for each agonist dose. *P < 0.05, _apoE–/–RAGE–/–_ versus _apoE–/–_ (doses >3 x 10–7 M); **P < 0.001, _apoE–/–Tg PPET DN-RAGE_ versus _apoE–/–_ (doses >10–8 M).
To further support that the effects observed in atherosclerosis would reflect the contribution of RAGE, we retrieved aorta from WT, RAGE–/–, and Tg PPET DN-RAGE mice and performed real-time quantitative PCR to detect mRNA transcripts for 3 distinct receptors implicated in RAGE ligand interactions: CD36, TLR2, and TLR4 (19, 20). Compared with WT mice, no differences in transcripts for CD36, TLR2, or TLR4 were evident in RAGE–/– or Tg PPET DN-RAGE mice (n = 4 per group; P > 0.05).
Furthermore, we performed additional experiments to ascertain that macrophages from Tg PPET DN-RAGE mice were not affected by introduction of DN-RAGE. Thioglycollate-elicited macrophages were retrieved from WT, RAGE–/–, and Tg PPET DN-RAGE mice. As illustrated in Figure 2D, macrophages from both WT and Tg PPET DN-RAGE mice displayed intact responses to RAGE ligand S100b as assessed by increased phosphorylation of JNK (Figure 2D). As expected, experiments in RAGE–/– macrophages revealed diminished phosphorylation of JNK MAP kinase in response to ligand S100b.
Following their characterization, we assessed the impact of RAGE on atherosclerosis in these 3 groups of apoE–/– mice. At age 14 weeks, compared with apoE–/– mice, in which mean atherosclerotic lesion area at the aortic sinus was 66,839 ± 6,940 μm2 (Figure 2, E, H, and K), apoE–/–RAGE–/– mice displayed significantly less atherosclerosis (32,084 ± 3,635 μm2; P < 0.0001; Figure 2, F, I, and K). apoE–/–Tg PPET DN-RAGE mice displayed reduced atherosclerosis (13,909 ± 3,222 μm2; P < 0.0001 versus apoE–/–; Figure 2, G, J, and K). The complexity of the lesions (21), as defined by the presence of cholesterol clefts, necrosis, calcifications, or fibrous caps, was significantly reduced in apoE–/–RAGE–/– and apoE–/–Tg PPET DN-RAGE compared with apoE–/– animals (P < 0.0001; Figure 2L). There were no differences in plasma cholesterol or triglyceride in these 3 groups of mice (Table 1).
Plasma cholesterol and triglyceride in 14-week-old _apoE_–/– mice
Based on the significant effects of RAGE on atherosclerosis, we prepared aortic rings from these mice to test whether RAGE affects endothelial-mediated vascular function. Upon exposure of aortic rings to increasing doses of acetylcholine, endothelium-dependent relaxation was significantly enhanced in rings retrieved from apoE–/–RAGE–/– and apoE–/–Tg PPET DN-RAGE mice compared with those of apoE–/– mice (Figure 2M).
RAGE impacts vascular inflammation in apoE–/– mice. We next focused on central mediators of inflammation and vascular tissue destruction and retrieved thoracic and abdominal aortas of mice at age 14 weeks. Levels of VCAM-1 antigen (5), one of the earliest molecular markers of vascular inflammation in atherogenesis, were significantly reduced in the aortas of apoE–/–RAGE–/– and apoE–/–Tg PPET DN-RAGE mice compared with apoE–/– mice (Figure 3A), as were levels of monocyte chemoattractant peptide–1 (MCP-1; Figure 3B) (8). Compared with apoE–/– mouse aortas, those of apoE–/–RAGE–/– and apoE–/–Tg PPET DN-RAGE mice revealed significant attenuation in MMP-2 antigen and activity (Figure 3, C and D) (22, 23). Furthermore, compared with apoE–/– mouse aortas, those of apoE–/–RAGE–/– or apoE–/–Tg PPET DN-RAGE mice revealed significantly decreased expression of IL-10 and CD40 (Figure 3, E and F) (24–26). To assess the effects of RAGE on expression of its inflammatory ligands, we examined levels of S100/calgranulins and HMGB1 and found that levels of both molecules were significantly attenuated in the aortas retrieved from apoE–/–RAGE–/– and apoE–/–Tg PPET DN-RAGE versus those of apoE–/– mice (Figure 3, G and H).
RAGE impacts vascular inflammation in the aortas of apoE–/– mice. (A–H) At age 14 weeks, apoE–/–, apoE–/–RAGE–/–, and apoE–/–Tg PPET DN-RAGE mice were sacrificed and aortas retrieved. Western blotting was performed to detect VCAM-1 (A), MCP-1 (B), MMP-2 (C), IL-10 (E), CD40 (F), S100b (G), and HMGB1 (H) followed by anti–β-actin IgG. In D, zymography was performed on aorta lysates to detect activity of MMP-2. *P < 0.001 versus apoE–/–. (I) Plasma was retrieved from 14-week-old mice and subjected to ELISA for determination of soluble VCAM-1 (sVCAM-1) levels. n ≥ 4 mice per group. *P < 0.05 versus apoE–/–.
To test that the effects of genetic RAGE modification impacted systemic evidence of vascular inflammation, we retrieved plasma from each of the 3 groups of mice at age 14 weeks. Compared with apoE–/– mice, plasma retrieved from apoE–/–RAGE–/– or apoE–/–Tg PPET DN-RAGE mice revealed significantly lower levels of soluble VCAM-1 (Figure 3I).
RAGE transduces the effects of S100/calgranulins on vascular inflammation. To specifically dissect the signal transduction mechanisms linking RAGE to endothelial dysfunction, we retrieved and characterized endothelial cells from the aortas of WT C57BL/6 and RAGE–/– and Tg PPET DN-RAGE mice. Uptake of diI-acetylated LDL and cord formation was observed in these primary murine aortic endothelial cells but not murine fibroblasts (data not shown). By Western blotting, although WT mouse aortas revealed a single band for RAGE antigen at approximately 55 kDa, endothelial cells retrieved from RAGE–/– aortas revealed no RAGE-immunoreactive bands (Figure 4A). Endothelial cells from Tg PPET DN-RAGE mice revealed 2 immunoreactive bands with anti-RAGE IgG; the higher band indicated full-length RAGE and the lower band the truncated DN-RAGE (Figure 4A).
RAGE-mediated upregulation of inflammatory molecules in murine aortic endothelial cells: S100b. (A) RAGE expression. Murine aortic endothelial cells from the indicated mice were subjected to Western blotting for detection of RAGE antigen. (B and C) Endothelial cells were isolated from the 3 genotypes and exposed to 10 μg/ml S100b for 4 hours. Western blotting was performed to detect VCAM-1 (B) and MMP-2 proteins (C) followed by anti–β-actin IgG. (D) Zymography for detection of MMP-2 activity was performed. (E and F) Signal transduction. Murine aortic endothelial cells from the indicated mice were incubated with 10 μg/ml S100b for 20 minutes. Western blotting for detection of phospho/total pERK and JNK MAP kinases was performed. (G and H) Endothelial cells were pretreated with the pERK MAP kinase inhibitor PD98059 (10 μM) or the JNK MAP kinase inhibitor SP600125 (10 μM) for 60 minutes prior to S100b for 20 minutes. Western blotting was performed for detection of VCAM-1 antigen. *P < 0.0001 versus unstimulated WT; **P < 0.0001 versus stimulated WT. (I) siRNA to knock down JNK MAP kinase was performed, and cells were exposed to S100b. *P < 0.0001 versus cells without S100b; **P < 0.0001 versus S100b without JNK knockdown.
Based on the observation that RAGE ligand S100/calgranulins was expressed in apoE–/– aortas (Figure 3G), we stimulated WT and DN-RAGE endothelial cells with S100b and probed the impact on vascular inflammation and signal transduction cascades. Stimulation of WT endothelial cells with S100b revealed an approximately 5-fold increase in VCAM-1 antigen compared with baseline (P < 0.0001; Figure 4B). When endothelial cells were retrieved from RAGE–/– or Tg PPET DN-RAGE mice, reduced upregulation of VCAM-1 antigen in response to S100b was noted versus stimulated WT endothelial cells (P < 0.0001; Figure 4B). When WT endothelial cells were stimulated with S100b, an approximately 8-fold increase in MMP-2 antigen and an approximately 11-fold increase in MMP-2 activity was noted versus baseline (P < 0.0001; Figure 4, C and D). However, when endothelial cells were retrieved from RAGE–/– and Tg PPET DN-RAGE aortas and incubated with S100b, upregulation of MMP-2 antigen and activity was markedly suppressed (P < 0.0001; Figure 4, C and D).
We determined the signal transduction mechanisms affected by S100b in aortic endothelial cells. Incubation of WT endothelial cells with S100b induced an approximately 7-fold increase in phosphorylated ERK MAP kinase versus baseline (P < 0.0001; Figure 4E). In contrast, when endothelial cells from RAGE–/– or Tg PPET DN-RAGE mice were incubated with S100b, a highly significant reduction in ERK phosphorylation was observed (P < 0.0001 versus S100b-stimulated WT; Figure 4E). When WT endothelial cells were incubated with S100b, an approximately 4.5-fold increase in phospho-JNK MAP kinase was observed versus baseline (P < 0.0001; Figure 4F). Key roles for RAGE were illustrated by a significant reduction in S100b-induced phosphorylation of JNK MAP kinase in RAGE–/– or Tg PPET DN-RAGE endothelial cells (P < 0.0001 versus stimulated WT). Of note, S100b failed to upregulate p38 MAP kinase signaling in WT endothelial cells (data not shown).
We next determined which signaling pathway(s) mediated S100b-induced upregulation of VCAM-1 in aortic endothelial cells. WT endothelial cells were incubated with S100b after pretreatment with either PD98059, a MAP kinase inhibitor, or SP600125, an inhibitor of JNK MAP kinases. Although PD98059 had no impact on S100b-induced upregulation of VCAM-1 antigen (Figure 4G), pretreatment of the endothelial cells with SP600125 resulted in highly significant reduction of S100b-mediated VCAM-1 upregulation (P < 0.0001; Figure 4H). In addition, introduction of siRNA to knock down JNK expression blunted the effect of S100b on upregulation of VCAM-1 (P < 0.0001; Figure 4I). Introduction of scrambled siRNA had no effect on S100b stimulation of mouse endothelial cells (data not shown).
RAGE transduces the effects of oxidized LDL–containing AGEs on vascular inflammation. Previous studies suggested that oxidized LDLs (oxLDLs) contained AGE epitopes (4); thus we probed this concept using affinity-purified anti-AGE IgG and oxLDL, the latter prepared by copper-induced oxidation. Compared with native LDL, Western blotting of oxLDL revealed markedly enhanced AGE immunoreactivity (Figure 5A).
RAGE-mediated upregulation of inflammatory molecules in murine aortic endothelial cells: oxLDL. (A) oxLDL contains AGE epitopes. Native LDL and oxLDL (5 μg/ml) were subjected to SDS-PAGE and Western blotting using affinity-purified rabbit anti-AGE IgG. (B) Endothelial cells were incubated with 5 μg/ml oxLDL for 4 hours. Western blotting was performed for detection of VCAM-1 antigen followed by anti–β-actin IgG. (C) Zymography for detection of MMP-2 activity was performed. *P < 0.0001 versus unstimulated WT; **P < 0.0001 versus stimulated WT. (D and E) Effect of anti-AGE IgG. Murine aortic endothelial cells were pretreated with rabbit anti-AGE IgG or nonimmune rabbit IgG (nI-IgG; 50 μg/ml) for 1 hour. OxLDL (5 μg/ml) was added for 4 hours; cells were harvested and Western blotting was performed to detect VCAM-1 (D) followed by anti–β-actin IgG, or MMP-2 activity by zymography (E). (F and G) Signal transduction. Endothelial cells were incubated with 5 μg/ml oxLDL for 20 minutes. Western blotting for detection of phospho/total pERK and JNK MAP kinases was performed. (H) Endothelial cells were pretreated with the pERK MAP kinase inhibitor PD98059 (10 μM) or the JNK MAP kinase inhibitor SP600125 (10 μM) for 1 hour prior to exposure to oxLDL for 20 minutes. Western blotting was performed for detection of VCAM-1 antigen. *P < 0.0001 versus unstimulated WT; **P < 0.0001 versus oxLDL-stimulated WT.
First, we determined whether oxLDL modulates expression of VCAM-1 antigen in WT aortic endothelial cells. Incubation of these cells with oxLDL resulted in an approximately 3.5-fold increase in VCAM-1 antigen by Western blotting (P < 0.0001 versus baseline; Figure 5B). When endothelial cells were retrieved from RAGE–/– or Tg PPET DN-RAGE aortas and incubated with oxLDL, a marked suppression in upregulation of VCAM-1 was noted versus that observed in WT cells (P < 0.0001; Figure 5B). Incubation of WT endothelial cells with oxLDL stimulated an approximately 11-fold increase in MMP-2 activity (P < 0.0001 versus baseline; Figure 5C). When endothelial cells were retrieved from RAGE–/– or Tg PPET DN-RAGE mice, a highly significant suppression in oxLDL-mediated upregulation of MMP2 activity was noted (P < 0.0001 versus WT; Figure 5C).
To test the premise that these oxLDL-mediated changes were evoked by AGE epitopes within oxLDL, we used antibodies to AGE. When WT endothelial cells were pretreated with anti-AGE IgG, significant suppression of oxLDL-induced upregulaton of VCAM-1 antigen and MMP-2 activity was observed compared with nonimmune IgG (P < 0.0001; Figure 5, D and E).
To establish the signal transduction pathways mediating the impact of oxLDL-RAGE on upregulation of inflammatory signaling in aortic endothelial cells, we tested the effects on MAP kinases. Incubation of WT endothelial cells with oxLDL induced approximately 4.5- and 2.5-fold increases in phospho-ERK MAP kinase and JNK MAP kinase, respectively, compared with baseline (P < 0.0001; Figure 5, F and G). Highly significant reductions in phospho-ERK and JNK MAP kinase were found in RAGE–/– and Tg PPET DN-RAGE endothelial cells in the presence of oxLDL compared with that observed in WT endothelial cells (P < 0.0001; Figure 5, F and G).
We then determined the specific signaling pathways stimulated by oxLDL that accounted for upregulation of VCAM-1 antigen. WT endothelial cells were pretreated with inhibitors of either phospho-ERK or JNK MAP kinases. Compared with PD98059 pretreatment, in which no significant suppression of oxLDL-mediated upregulation of VCAM-1 was noted, pretreatment with the JNK MAP kinase inhibitor SP600125 resulted in a highly significant reduction in oxLDL-induced upregulation of VCAM-1 antigen (P < 0.0001; Figure 5H). Introduction of siRNA to knock down JNK expression exerted similar suppressive effects on S100b-stimulated upregulation of VCAM-1 (data not shown).
Key roles for RAGE in mediating hyperpermeability and regulation of VCAM-1 in human aortic endothelial cells. In homeostasis, the endothelium maintains a competent barrier, thereby limiting infiltration of exogenous molecules or cells. We tested the role of RAGE in mediating barrier function in human aortic endothelial cells upon exposure to RAGE ligands. Lentiviral gene transduction was used to introduce full-length RAGE (to augment RAGE expression) or DN-RAGE (to block RAGE signaling) into human endothelial cells. In the presence of S100b, a significant increase in monolayer permeability of full-length RAGE–expressing endothelial cells was noted compared with unstimulated full-length RAGE–expressing cells (P < 0.0001; Figure 6A). When RAGE signaling was disrupted by introduction of DN-RAGE, S100b failed to increase monolayer permeability compared with unstimulated cells bearing DN-RAGE. Furthermore, a significant difference in permeability was observed between S100b-stimulated full-length RAGE–expressing cells and S100b-stimulated DN-RAGE–expressing cells (P < 0.0001; Figure 6A). We next tested the role of RAGE in vascular inflammation and used siRNA to suppress RAGE expression in human aortic endothelial cells. As illustrated in Figure 6, B and C, siRNA targeted against RAGE, but not control scramble siRNA, reduced RAGE transcripts and RAGE protein.
RAGE-mediated upregulation of inflammatory molecules in human aortic endothelial cells. (A) Human aortic endothelial cells were subjected to lentiviral infection introducing full-length or DN-RAGE. Cells were exposed to S100b and permeability assay performed. *P < 0.0001 versus unstimulated full-length RAGE; **P < 0.0001 versus S100b-stimulated full-length RAGE. (B and C) Human endothelial cells were subjected to introduction of siRNA to reduce RAGE transcripts (B) and RAGE protein (C) without effect on GAPDH transcripts or protein. (D) Endothelial cells were subjected to RAGE or scramble siRNA and incubated with 10 μg/ml S100b for 4 hours. Western blotting was performed to probe VCAM-1 antigen and β-actin. *P < 0.0001. (E) Human aortic endothelial cells were stimulated with 10 μg/ml S100b for 20 minutes. Western blotting was performed to detect phospho/total-JNK MAP kinases. *P < 0.0001. (F) Endothelial cells were treated with JNK MAP kinase inhibitor SP60025 (10 μM) for 60 minutes followed by S100b (10 μg/ml) for 4 hours and Western blotting performed with anti–VCAM-1 IgG and anti–β-actin IgG. **P < 0.0001 versus S100b-stimulated, non–SP600125-treated. (G) oxLDL (5 μg/ml) was incubated with human aortic endothelial cells and Western blotting performed for detection of phospho/total-JNK MAP kinase. (H) Cells were treated with 10 μM SP600125 for 1 hour followed by incubation with oxLDL for 4 hours. Western blotting was performed for detection of VCAM-1 antigen. *P < 0.0001. (I) Cells were treated with siRNA to knock down JNK MAP kinase followed by incubation with oxLDL for 4 hours. Western blotting was performed for detection of VCAM-1 antigen. Where indicated by the white line, lanes were run on the same gel but were noncontiguous. *P < 0.05; **P < 0.01.
We observed that RAGE siRNA suppressed S100b-stimulated upregulation of VCAM-1 compared with cells treated with scramble siRNA and S100b (Figure 6D). To test the possibility that JNK MAP kinase signaling mediated the effect of S100b in human endothelial cells, cells were treated with S100b. A significant time-dependent increase in phosphorylation of JNK MAP kinase was noted (about 2.5-fold that of baseline at 20 and 30 minutes of treatment; P < 0.0001; Figure 6E). The impact of S100b on upregulation of VCAM-1 was dependent on JNK MAP kinase signaling, as pretreatment with SP600125 significantly reduced VCAM-1 antigen (P < 0.0001; Figure 6F). Similarly, introduction of siRNA to knock down JNK expression in these cells suppressed S100b-stimulated upregulation of VCAM-1 (data not shown).
To extend these concepts and probe whether oxLDL-containing AGE epitopes impacted gene expression in these human endothelial cells, we first performed a time course to assess JNK MAP kinase signaling. As illustrated in Figure 6G, a time-dependent increase in phosphorylation of JNK MAP kinase was evident upon incubation with oxLDL. To examine if JNK signaling mediated upregulation of VCAM-1 by oxLDL in these cells, we used various means to block this pathway. Incubation of cells with SP600125 (Figure 6H) or introduction of siRNA to knock down JNK expression blunted oxLDL-stimulated upregulation of VCAM-1 (P < 0.05; Figure 6I).