Human species-specific loss of CMP- N-acetylneuraminic acid hydroxylase enhances atherosclerosis via intrinsic and extrinsic mechanisms

Abstract

Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD-risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr^-/- mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr^-/- mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a "xeno-autoantigen" via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc "xeno-autoantibodies" potentiate chronic inflammation ("xenosialitis"). Cmah^-/-Ldlr^-/- mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah^-/-Ldlr^-/- mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.

Keywords: CMAH; N-glycolylneuraminic acid (Neu5Gc); atherosclerosis; cytidine-5′-monophosphate (CMP)-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH); human evolution.

Fig. 1.

Cmah^−/−Ldlr^−/− increases atherogenesis without changing lipoprotein profiles. (A) Six-week-old Cmah^−/−Ldlr^−/− and Cmah^+/+Ldlr^−/− mice were put on a 12-wk soy-based non-sialic acids (Sias) high-fat diet (HFD). (B) En face analysis of atherosclerosis (red dotted areas and yellow arrows indicate atheromatous lesions in female mice) and (C) quantification of Sudan IV-positive area. (Scale bars, 500 µm.) (D–G) Fast protein liquid chromatography analysis of lipoproteins after 12 wk of HFD feeding (pooled serum from n = 14 or 15). chylomicron, CR; very-low-density lipoprotein, VLDL; intermediate density lipoprotein, IDL; low-density lipoprotein, LDL; high-density lipoprotein, HDL. Mean values ± SEM, *P < 0.05 and **P < 0.01.

Fig. 2.

Cmah^−/−Ldlr^−/− increases atherogenesis without increased macrophage infiltration. Aortic sinus samples from Cmah^+/+Ldlr^−/− and Cmah^−/−Ldlr^−/− mice were analyzed after high-fat diet (HFD) feeding. (A and B) Quantification of total atherosclerotic lesion size (white dots) in the aortic sinus. (C) Area under the curve data. (D and E) Collagen fiber lesion (yellow dots) and analysis with Masson’s trichrome stain (female, n = 8 each, HFD for 12 wk). (F and G) F4/80-positive lesion area (yellow dots) and analysis. (H and I) CD68-positive lesion area (yellow dots) and analysis for measuring macrophage infiltration (female, n = 6 to 8, HFD for 12 wk). (J and K) Necrotic core (red dots) size analysis with Masson’s trichrome stain (female, n = 8 each, HFD for 12 wk). (L and M) Apoptotic cells (red color, yellow arrows) were detected with TUNEL per square millimeter lesion (white dots) (female, n = 4 each, HFD for 8 wk). (White scale bars, 300 µm; yellow scale bars, 100 µm.) Mean values ± SEM, *P < 0.05 and **P < 0.01.

Fig. 3.

Human-like Cmah^−/−Ldlr^−/− mice have a hyperglycemic phenotype. Cmah^+/+Ldlr^−/− and Cmah^−/−Ldlr^−/− male and female mice were fed either a control diet or a HFD for 11 wk. (A) Fasting glucose level (male, n = 5 to 13), serum insulin levels before and 15 min after an oral glucose gavage in control diet (B) and HFD (C), and (D) homeostasis model assessment for insulin resistance (HOMA-IR) (male, n = 5 each). (E) Fasting glucose level (female, n = 5 to 10), serum insulin levels before and 15 min after an oral glucose gavage in control diet (F) and HFD (G), and (H) HOMA-IR (female, n = 5 each). (I–L) Glucose tolerance tests and (M–P) insulin tolerance tests (relative to basal glucose level) (male and female, n = 10 each). Mean values ± SEM, *P < 0.05 and **P < 0.01.

Fig. 4.

Human-like Cmah^−/−Ldlr^−/− macrophages show elevated inflammatory cytokine expression. (A) Cytokine gene expression in bone marrow-derived macrophages (BMDM) obtained from female mice, 8 to 12 wk age, that were fed with control chow (n = 4 each). (B) Oil red O staining of peritoneal macrophages collected from female after 12 wk of HFD. (Scale bars, 100 µm.) (C) Quantification of Oil red O-positive peritoneal macrophages (female, n = 5 each) and (D) cholesterol ester levels (female, n = 10 each). (E) Expression of inflammatory cytokines in peritoneal macrophages collected after 12 wk of feeding with either control chow or (F) HFD (female, n = 8 each). (G) Expression of inflammatory cytokines in atheroma lesions of aortic arch collected after 12 wk of HFD feeding (female, n = 4 each). Mean values ± SEM, *P < 0.05 and **P < 0.01.

Fig. 5.

Combination of human-like anti-Neu5Gc antibodies and Neu5Gc-rich HFD increases atherogenesis. (A) Cmah^−/−Ldlr^−/− male mice were immunized with either a control antigen (Ctl Immunization) or a Neu5Gc antigen (Gc Immunization) and then fed with a Neu5Ac-rich, Neu5Gc-rich, or non-Sias high-fat diet (Ac-HFD, Gc-HFD, and HFD, respectively) for 9 wk (n = 14 to 16, each). (B and C) Graphs representing body weight changes over 12 wk (HFD started at 9 wk of age, indicated by an arrow) in each group. En face analysis of atherosclerosis lesion (red dots and yellow arrows) in (D) control immunization and (E) Neu5Gc immunization groups. (Scale bars, 500 µm.) (F) Quantification of Sudan IV-positive area in aorta. Mean values ± SEM, *P < 0.05 and **P < 0.01.

Fig. 6.

Human-like anti-Neu5Gc antibodies and Neu5Gc-rich HFD induce advanced atheroma. Cmah^−/−Ldlr^−/− male mice were immunized with either a control antigen (Ctl Immunization) or a Neu5Gc antigen (Gc Immunization) and then fed with a Neu5Ac-rich, Neu5Gc-rich, or non-Sias high-fat diet (Ac-HFD, Gc-HFD, and HFD, respectively) for 9 wk. (A and B) Atherosclerotic lesion in the aortic sinus. (C and D) Quantification of atherosclerotic lesion size in the aortic sinus. (E) Area under the curve. (F) Necrotic core size (male, n = 8 to 12). (G and H) Anti-Neu5Gc antibody titers measured by a bovine submaxillary mucin-coated ELISA (male, n = 14 to 16). (I) Representative images of expression of Neu5Gc in atheromas, using anti-Neu5Gc antibody immunohistochemistry: (a) secondary antibody (negative control) stain for Neu5Gc-immunized and Gc-HFD–fed mouse and Neu5Gc stains for (b) control immunized and Ac-HFD fed and (c and d) Neu5Gc-immunized and Gc-HFD–fed mice. (Scale bars, 100 µm.) Mean values ± SEM, *P < 0.05 and **P < 0.01.

Fig. 7.

Human species-specific loss of Neu5Gc increases atherosclerosis risk by multiple mechanisms. Inactivation of the CMP-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) occurred ∼2 to 3 Mya in the hominin lineage, which is now manifest as a human species-specific deficiency of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc). This Neu5Gc loss contributes to atherosclerosis risk via intrinsic mechanisms such as up-regulated inflammatory response and hyperglycemia as well as extrinsic mechanisms such as red meat-derived Neu5Gc-induced xenosialitis.