A Deficiency of Herp, an Endoplasmic Reticulum Stress Protein, Suppresses Atherosclerosis in ApoE Knockout Mice by Attenuating Inflammatory Responses

<div><p>Herp was originally identified as an endoplasmic reticulum (ER) stress protein in vascular endothelial cells. ER stress is induced in atherosclerotic lesions, but it is not known whether Herp plays any role in the development of atherosclerosis. To address this question, we generated Herp- and apolipoprotein E (apoE)-deficient mice (Herp^−/−; apoE^−/− mice) by crossbreeding Herp^−/− mice and apoE^−/− mice. Herp was expressed in the endothelial cells and medial smooth muscle cells of the aorta, as well as in a subset of macrophages in the atherosclerotic lesions in apoE^−/− mice, while there was no expression of Herp in the Herp^−/−; apoE^−/− mice. The doubly deficient mice developed significantly fewer atherosclerotic lesions than the apoE^−/− mice at 36 and 72 weeks of age, whereas the plasma levels of cholesterol and triglycerides were not significantly different between the strains. The plasma levels of non-esterified fatty acids were significantly lower in the Herp^−/−; apoE^−/− mice when they were eight and 16 weeks old. The gene expression levels of ER stress response proteins (GRP78 and CHOP) and inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1) in the aorta were significantly lower in Herp^−/−; apoE^−/− mice than in apoE^−/− mice, suggesting that Herp mediated ER stress-induced inflammation. In fact, peritoneal macrophages isolated from Herp-deficient mice and RAW264.7 macrophages in which Herp was eliminated with a siRNA expressed lower levels of mRNA for inflammatory cytokines when they were treated with tunicamycin. Herp deficiency affected the major mediators of the unfolded protein response, including IRE1 and PERK, but not ATF6. These findings suggest that a deficiency of Herp suppressed the development of atherosclerosis by attenuating the ER stress-induced inflammatory reactions.</p></div

Herp expression in the artery.

<p>A: The amount for Herp mRNA was determined by a real-time PCR in the aortas of apoE^−/− mice (open columns) and Herp^−/−; apoE^−/−mice (closed columns). Each mRNA expression level was normalized to that of GAPDH. The mRNA levels are shown as relative ratios to the mRNA level of eight-week-old apoE^−/− mice. Each value represents the mean ± SEM of five mice. N.D. indicates non-detectable. ** p<0.01 vs. apoE^−/− mice at eight weeks of age. B: Immunostaining of Herp in the normal (upper panels) and atherosclerotic (lower panels) aortas of apoE^−/− and Herp^−/−; apoE^−/− mice at 72 weeks of age. Blue; hematoxylin, Red; Herp (diaminobenzidine; DAB). The bar shows 50 μm.</p

Herp-deficient RAW264.7 macrophages showed reduced expression of IL-1β and IL-6 in response to ER stress.

<p>RAW264.7 macrophages were treated with a siRNA against Herp or a control siRNA for 48 μM tunicamycin for 6 hrs. The mRNA expression levels of ER stress-related proteins and cytokines were analyzed by a real-time PCR analysis. Open columns show unstimulated cells (DMSO), closed columns show cells stimulated with tunicamycin (TM). The amounts of mRNA were normalized to that of GAPDH. The mRNA levels were shown as a relative ratio to the mRNA level of tunicamycin-treated macrophages, which were transfected with control siRNA. A: IL-1β, B: IL-6, C: Herp, D: GRP78, E: CHOP, F: MCP-1, G: TNF-α, H: SERP1, I: Sec61b, J: SEL1L and K: ATF4. Each column represents the mean ± SEM; n = 3 per group. * p<0.05, ** p<0.01, *** p<0.001, vs unstimulated control macrophages; ¶p<0.05, ¶¶p<0.01, ¶¶¶p<0.001 vs unstimulated siHerp macrophages. †p<0.05, ††p<0.01, †††p<0.001 vs stimulated siHerp macrophages. N.D.; not detected. n.d.; no difference.</p

The expression levels of IL-1β, MCP-1 and VCAM-1 were reduced in the aortas of Herp^−/−; apoE^−/− mice.

<p>Total RNA was prepared from the entire aortas of apoE^−/− and Herp^−/−; apoE^−/− mice. The mRNA expression levels were determined by real-time PCR. Each mRNA level was normalized to that of GAPDH mRNA. The mRNA levels are shown as the relative ratio to the mRNA level of eight or 20-week-old apoE^−/− mice. A: IL-1β, B: GRP78, C: IL-6, D: MCP-1, E: CHOP, F: VCAM-1, G: SERP1, H: Sec61b, I: SEL1L and J: ATF4. Open columns show apoE^−/−, and closed columns show Herp^−/−; apoE^−/− mice. Each column represents the mean ± SEM of five mice. N.D. indicates non-detectable. * p<0.05, ** p<0.01 vs. apoE^−/− mice at the same age. A, B: Immunostaining of IL-1β and GRP78 in the atherosclerotic aortas of apoE^−/− and Herp^−/−; apoE^−/− mice at 72 weeks of age. Blue; hematoxylin, Red; GRP78 or IL-1β (DAB). The bar shows 50 μm.</p

Effect of Herp deficiency on the plasma glucose and lipid levels.

<p>The body weight (A) and the plasma levels of glucose (B), cholesterol (C), triglycerides (D) and NEFA (E) following overnight fasting at eight, 16, 24, 36 and 72 weeks of age, were determined as described in the Methods. Open circles show apoE^−/−, and closed squares show Herp^−/−; apoE^−/− mice. Each value represents the mean ± SEM; n = 5 per group. * p<0.01 vs. Herp^−/−; apoE^−/− mice at the same age.</p

Herp-deficient peritoneal macrophages showed reduced expression of IL-1β and IL-6 in response to ER stress.

<p>Peritoneal macrophages were prepared from eight-week-old apoE^−/− and Herp^−/−; apoE^−/− mice, and were stimulated with 10 μM tunicamycin for 6 hrs. The mRNA levels of ER stress-related proteins and cytokines were analyzed by a real-time PCR analysis. Open columns show unstimulated cells (DMSO), and closed columns show cells stimulated with tunicamycin (TM). Each amount of mRNA was normalized to GAPDH. The mRNA levels are shown as the relative ratio to the mRNA level of tunicamycin-treated macrophages from apoE^−/− mice. A: IL-1β, B: IL-6, C: Herp, D: GRP78, E: CHOP, F: MCP-1, G: TNF-α, H: SERP1, I: Sec61b, J: SEL1L and K: ATF4. Each column represents the mean ± SEM; n = 5 per group. ** p<0.01, *** p<0.001, vs unstimulated apoE^−/− macrophages; ¶p<0.05, ¶¶p<0.01, ¶¶¶p<0.001 vs unstimulated Herp^−/−; apoE^−/− peritoneal macrophages. †p<0.05, ††p<0.01, †††p<0.001 vs stimulated Herp^−/−; apoE^−/− peritoneal macrophages. N.D.; not detected. n.d.; no difference.</p

Insulin-stimulated phosphorylation of Akt was significantly improved in liver of OLETF rats after voluntary exercise.

<p>At 15 weeks of age, insulin (0.5 U/kg BW) or saline was injected via the portal vein following overnight fasting. At 5 min after the injection, liver was taken under anesthesia. Insulin-stimulated phosphorylation of Akt at threonine 308 (C) and serine 473 (D) was significantly increased in the liver of OLETF rats after exercise, as compared with sedentary condition. Basal (exogenous insulin-naïve) Akt phosphorylation in the liver was not different between the voluntary exercise and sedentary OLETF rat groups. The protein expression of Akt did not differ between voluntary exercise and sedentary condition in the liver of OLETF rats (B). *,p<0.05; **,p<0.01 versus sedentary OLETF with saline, †,p<0.05 versus sedentary OLETF with insulin. N.S.: not significant.</p

Effects of voluntary exercise on the iNOS mRNA expression and S-nitrosylation of Akt and IRS-1 in the liver in the OLETF rats.

<p>The mRNA expression of iNOS was significantly increased in the liver in the sedentary OLETF rats (<b>A</b>). The S-nitrosylated Akt levels were significantly increased in the liver in the sedentary (SED) OLETF rats (<b>B</b>). Voluntary exercise decreased the S-nitrosylated Akt levels in the liver in the voluntary exercise (VE) OLETF rats compared with those observed in the OLETF-SED rats. Similarly, S-nitrosylation of IRS-1 was also increased in the liver of SED OLETF rats (<b>C</b>). The degree of S-nitrosylation was evaluated using a biotin switch analysis. All values are presented as the mean ± SEM. n = 9–11 per group, **,p<0.01 versus sedentary LETO, †,p<0.05; ††,p<0.01 versus voluntary exercise OLETF. N.S.: not significant.</p

Exercise suppressed the lipogenic gene expression and prevented the accumulation of TG and activation of JNK in the liver in the OLETF rats.

<p>The triglyceride content in the liver (<b>A</b>) was significantly lower in the voluntary exercise (VE) OLETF rats than in the sedentary (SED) OLETF rats. Consistent with the decreased triglyceride levels, the mRNA expression of <i>Srebp-1</i> (<b>B</b>) and <i>Scd-1</i> (<b>C</b>) was significantly decreased in the OLETF-VE rats. It is known that lipid accumulation in the liver increases the JNK activity. The total JNK amount was significantly increased in the OLETF rats compare with that observed in the LETO rats (<b>D</b> and <b>E</b>). The phosphorylation of JNK was significantly increased in the liver in the OLETF-SED rats compared with that observed in the LETO-SED rats (<b>D</b> and <b>F</b>). After 20 weeks of exercise, the activated JNK content in the liver decreased in the OLETF rats. The protein levels of JNK and p-JNK were normalized to that of actin. All values are presented as the mean ± SEM. n = 7–11 per group, *,p<0.05; **,p<0.01 versus sedentary LETO, †,p<0.05; ††,p<0.01 versus voluntary exercise OLETF. ‡,p<0.05; ‡‡,p<0.01 versus voluntary exercise LETO. N.S.: not significant.</p

Hyperglycemia and hyperinsulinemia in the OLETF rats were reversed by voluntary exercise.

<p>The blood glucose (<b>A</b>) and plasma insulin (<b>B</b>) levels were significantly greater in the OLETF rats under sedentary conditions (SED) than in the LETO rats on SED. There were no significant differences between the LETO and OLETF rats with respect to the effects of voluntary exercise (VE) on the plasma insulin levels. The OLETF-SED rats exhibited insulin resistance, as indicated by elevated HOMA insulin resistance index values (<b>C</b>). All values are presented as the mean ± SEM. n = 9–11 per group, *,p<0.05; **,p<0.01 versus sedentary LETO, †,p<0.05 versus voluntary exercise OLETF, ‡,p<0.05 versus voluntary exercise LETO. N.S.: not significant.</p