Significance Cardiovascular disease (CVD) events like heart attacks and strokes due to atherosclerotic narrowing of arteries are the commonest cause of worldwide deaths, but many first-time events occur in individuals without known risk factors. In contrast, such events are extremely rare in other animals despite some of the same risk factors. While environmental and behavioral factors likely contribute to the difference, we show here that a human-specific genetic mutation affecting cell-surface molecules called sialic acids may be one other factor. We also show that the same mutation can help explain the apparently human-specific increased risk of CVD events associated with red meat consumption. The humanized mouse systems we present could be explored as models for future studies of atherosclerosis.

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.

Atherosclerosis is the predominant cause of cardiovascular disease (CVD) events such as coronary thrombosis, myocardial infarction, and strokes, which are very common in humans and responsible for about one-third of deaths worldwide (1). Interestingly, these events rarely occur spontaneously in other mammals, in the absence of experimental or dietary manipulation. Human susceptibility is assumed to be due to a combination of physical inactivity and increased risk factors, such as dyslipidemia, hypertension, diabetes, obesity, red meat consumption, and smoking. However, ∼15% of initial CVD events occur in humans without obvious known risk factors (2, 3). Closely related chimpanzees also have many risk factors in captivity, including hypertension (4), high low-density lipoprotein (LDL) cholesterol (5), elevated lipoprotein(a) levels (6), and a sedentary lifestyle, yet rarely suffer atherosclerotic CVD events (5, 7⇓⇓–10), but instead often develop a different cardiac disease, interstitial myocardial fibrosis (5, 9, 11). While extrinsic factors related to transition from hunter-gatherer lifestyle/diet to current sedentary Western conditions likely contribute to the human propensity (12⇓–14); the prevalence of advanced atherosclerosis in well-preserved mummies from four diverse ancient cultures (15⇓–17) suggests a disease that is intrinsic to human aging. Taken together, the data suggest that one must also consider mechanisms intrinsic to the biology of our species, that is, genetic changes that occurred during the evolution of the human species after the common ancestor with the chimpanzee (>7 Mya).

The first reported clear-cut difference at the genetic and molecular level between humans and chimpanzees was a species-specific deficiency of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc). This loss is a result of pseudogenization of the cytidine monophosphate (CMP)-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) gene, which likely occurred in hominin ancestors about 2 to 3 Mya (18) and then fixed in the hominin lineage before the origin of modern humans (19). The loss of this common mammalian sialic acid is also associated with an excess of the sialic acid Neu5Ac, the precursor for Neu5Gc, in the glycocalyx of human cells. The intrinsic pathophysiological consequence of the loss of Neu5Gc has been extensively studied in human-like Cmah-deficient (Cmah−/−, thus Neu5Gc-deficient) mice. These mice present with many human-like phenotypes (20), are more susceptible to develop glucose intolerance (21), and have hyperreactive macrophages (22), T cells (23), and B cells (24).

In addition to intrinsic impacts of the loss of Neu5Gc production one also needs to consider its extrinsic impact, triggered by the dietary consumption of Neu5Gc (primarily derived from red meat) (25). Once Neu5Gc is absorbed by the gastrointestinal tract it can get metabolically incorporated into cellular glycoproteins and glycolipids following the same pathway utilized by the endogenous sialic acid Neu5Ac (26). In this way consumed exogenous Neu5Gc gets presented on the cell surface glycocalyx, where it can act as a foreign “xeno-autoantigen.” The actual amounts incorporated appear to be very small but tend to be enriched at sites such as epithelium and endothelium (27) and even within atherosclerotic plaques (28). Even though the incorporated amounts may be small, most humans have circulating anti-Neu5Gc “xeno-autoantibodies” and can thus develop local chronic inflammation or “xenosialitis” at sites of Neu5Gc accumulation. We have already shown that this novel form of antibody-mediated inflammation can potentiate cancer progression in the human-like Cmah-null mouse model (29). Based on these and previous observations we have suggested that xenosialitis may also help explain the increased risk of cancer and CVD epidemiologically associated with human red meat consumption (30).

Notably, dietary red meat-associated enhancement of cancer and CVD risk has not been reported in other carnivorous mammals. The human-specific red meat-related increase in CVD risk can be partially explained by increased choline intake (with increased conversion to trimethylamine N-oxide), cholesterol, and saturated fat content in red meat (25, 31⇓⇓–34). The suggestion of oxidant damage due to dietary heme iron is confounded by the high heme-binding capacity of plasma hemopexin, with complexes being efficiently cleared by the liver (25, 35, 36). Regardless, none of these mechanisms fully explains the increased human propensity for atherosclerosis, nor the red meat-specific and human-specific nature of the dietary component of risk.

Here we test the hypothesis that human CMAH deficiency contributes to CVD risk via both intrinsic and extrinsic mechanisms. To experimentally address the intrinsic CVD consequences Cmah-deficient (Cmah−/−) and congenic wild-type mice were bred into an Ldlr−/− background and fed a sialic acids (Sias)-free high-fat diet (HFD) to induce atherogenesis. We also probed the extrinsic CVD implications using Cmah−/−Ldlr−/− mice immunized with control antigens with Neu5Ac or with Neu5Gc-bearing antigens to generate human-like levels of anti-Neu5Gc antibodies. Subsequent feeding of a Neu5Gc-rich diet allowed evaluation of the contribution of diet-induced xenosialitis on atherogenesis. Our results suggest that human evolutionary loss of CMAH markedly promotes atherosclerosis development via both intrinsic and extrinsic mechanisms and may help explain the heightened predisposition of humans to develop CVD.

Discussion The underlying question that drove this study was the common observation that humans appear to be particularly prone to cardiovascular complications of atherosclerosis, in comparison with other mammals. The question is whether this difference is purely due to the commonly known risk factors or whether genetic factors unique to human evolution also contribute. Here we present evidence to implicate the homozygous fixed state of CMAH inactivation as such an intrinsic factor, complicated by an extrinsic dietary factor that is also contingent on this mutation. We show first that human-like Cmah inactivation in mice enhances atherosclerosis via multiple intrinsic mechanisms. An additional CMAH mutation-dependent extrinsic factor is “xenosialitis,” which arises from metabolic incorporation of the nonhuman sialic acid Neu5Gc (mimicking red meat intake) and polyclonal anti-Neu5Gc antibodies in the Cmah−/−Ldlr−/− mouse model. Given the plethora of cell types throughout the body affected by the loss of CMAH (20⇓⇓⇓–24), it is not surprising that there is not one dominant pathway responsible for the marked increase in atherosclerosis development. In addition to higher immune activation, we did observe a strong diabetic phenotype in Cmah−/−Ldlr−/− mice, which is a major driver of CVD in humans and to a lesser extent in mice. We have confirmed that glucose intolerance induced by HFD is more severe in Cmah−/−Ldlr−/−, without underlying insulin resistance, consistent with prior data (21). According to the previous study, the diabetic phenotype in Cmah−/− mice is explained by decreased islet size and numbers in pancreas (21) and the etiology is likely identical for our model. In our study, we measured inflammatory cytokine gene expression in pancreas; however, no significant difference was found between Cmah−/−Ldlr−/− and Cmah+/+Ldlr−/− (SI Appendix, Fig. S4J). One factor we can exclude is that loss of Cmah expression affects cholesterol levels in the absence (or presence) of LDL receptor (LDLR) expression (45). In fact, circulating triglyceride, cholesterol, and LDL levels indicated by the lipoprotein profiles were unchanged between the groups in Cmah−/−Ldlr−/− and Cmah+/+Ldlr−/− mice fed with HFD and among Cmah−/−Ldlr−/− mice, which were immunized with Neu5Gc or control antigen and a HFD containing either no Sias, Neu5Ac, or Neu5Gc. However, despite the lack of difference in circulating LDL-cholesterol levels, more foam cell conversion was observed in vivo in Cmah−/−Ldlr−/− mice. It is known that increased inflammation can promote foam cell conversion and consequently drive atherosclerosis development (37⇓–39). Indeed, this was observed in in Cmah−/−Ldlr−/− mice with increased expression of ACAT1 and ACAT2, which can convert macrophages more efficiently into foam cells in the context of hypercholesterolemia. Cmah−/− mouse macrophages showed stronger phagocytosis and cytokine production such as tumor necrosis factor-α and IL-6 than Cmah+/+ mouse macrophages, and similar differences were seen in direct comparisons between human macrophage and chimp macrophages (22). Furthermore, Neu5Gc feeding of Cmah−/− macrophages showed reduced phagocytosis in comparison Neu5Ac fed Cmah−/− macrophages (22). There are also likely to be adaptive immune factors we did not pursue in this study. For example, Cmah−/− mouse T cells showed a hyperactive phenotype following virus infection, and reintroduction of Neu5Gc to both human and Cmah−/− T cells blunted their activation and proliferation during in vitro stimulation (23). Cmah-deficient mice also show a moderate B cell hyperactivity: The immune response to thymus-independent antigens was increased, calcium signaling after anti-immunoglobulin M stimulation was enhanced, and the marginal zone and recirculating B cell populations were decreased (24). With regard to the extrinsic mechanism, the finding that only the combination of Neu5Gc antibodies and Neu5Gc-rich HFD feeding caused enhanced atherosclerosis indicates that the likely mechanism is antibody deposition with complement activation and/or recruitment of Fc receptor-positive leukocytes. However, these events probably occur early in the process, and we had difficulty showing clearly enhanced accumulation of antibody or complement in the extremely complex environment of the advanced atherosclerotic plaques, in which many other factors are operative. The obvious question arising from such experimental mouse studies is whether circulating anti-Neu5Gc antibodies correlate with CVD risk in human population studies. We are currently exploring this possibility (43) but are also aware of the numerous complicating variables in humans, such as the varying amount and unknown bioavailability of Neu5Gc from dietary red meat, the amount of Neu5Gc loading in tissues, the complex and variable polyclonal antibody profiles of individual humans, the skewed distribution of antibody levels within populations, and the variability of concurrent inflammatory conditions (30). Also, given that CMAH deletion results in variable loss of a major cell-surface molecule (Neu5Gc) and a corresponding increase in the precursor Neu5Ac throughout the body, it is not surprising that there is no single dominant mechanistic explanation for the striking phenotypic changes found in this study; rather, there are multiple interactive mechanisms (Fig. 7). 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. Overall our work may help explain why atherosclerosis and resulting CVD complications are very common in humans and why these events rarely occur spontaneously in other mammals, in the absence of experimental or dietary manipulation. Future studies of atherosclerosis in mice may also benefit from using the more human-like Cmah-deficient background, in which lesion progression is greater. With regard to the xenosialitis phenomenon, further work is also necessary to determine the exact pathways by which Neu5Gc is taken up into tissues, and potential approaches to prevent or eliminate such incorporation are being explored. The current findings indicate that this unique example of inflammation driven by a metabolically incorporated dietary glycan (30) is pathologically relevant and may be involved in other inflammation-driven diseases that are known to be associated with red meat consumption, including carcinomas (25), which also appear to be rare in chimpanzees (46).

Methods Ethics Statement. The proposed use of mice in this project was approved by the University of California San Diego (UCSD) Animal Subjects Committee (Evaluation of the Role of Glycans in Normal Physiology, Malignancies and Immune Responses, Protocol S01227). All procedures were approved by the Animal Care Program and Institutional Animal Care and Use Committee, UCSD. Human RBCs and LDL were obtained from venous blood provided by healthy volunteers with informed consent under a protocol approved by UCSD Human Research Protection Programs Institutional Review Board. Mice and Cell Culture. Cmah−/−Ldlr−/− mice were generated by crossing Cmah−/− mice (47) and Ldlr−/− mice (48) in a congenic C57BL/6 background and maintained in the UCSD vivarium according to Institutional Review Board guidelines for the care and use of laboratory animals. All animals were fully back-crossed and maintained on a 12-h light cycle and fed water and standard rodent chow for ad libitum consumption. Cmah−/−Ldlr−/− and Cmah+/+Ldlr−/− mice were also maintained on a control soy-based (Sias-free) chow after weaning at 3 wk. At 6 or 9 wk of age male and female mice were placed on a Sias-free soy-based HFD with 20% anhydrous milkfat and 0.2% cholesterol, a Neu5Gc-rich soy-based HFD containing 0.25 mg of Neu5Gc per gram of chow and made by adding purified porcine submaxillary mucin (PSM) as described previously (29), or a Neu5Ac-rich soy-based HFD containing 0.25 mg of Neu5Ac per gram of chow, made by adding edible bird’s nest (EBN) (Golden Nest, Inc.) (29). The amount of Neu5Gc in PSM and Neu5Ac in EBN was determined by HPLC with Neu5Ac (Nacalai) and Neu5Gc (Inalco) standards at UCSD and all chows were subsequently formulated and composed (Dyets, Inc.). Peritoneal macrophages were collected by peritoneal lavage after 12 wk of control diet or HFD feeding without any stimulants, and BMDMs were isolated from 8- to 12-wk-old female mice as described previously (37). BMDMs were maintained in M-CSF (Tonbo Biosciences) culture with lipoprotein-free bovine serum (Alfa Aesar) then fed with normal human LDL, ag LDL (40), or ox LDL (41). Macrophages or foam cell numbers were counted in a microscope and cholesterol ester level were measured with a kit (Invitrogen) and normalized with protein concentration (Thermo Fisher Scientific). Neu5Gc and Control Immunization. Chimpanzee and human RBC membrane ghosts were prepared as described previously (29). Six-week-old Cmah−/−Ldlr−/− male and female mice were immunized three times per week via intraperitoneal (i.p.) injection with pooled RBC membrane chimp ghosts (chimp, Neu5Gc 151.5 pmol/µg protein, Neu5Ac 24.1 pmol/µg protein, measured by HPLC) or control human ghosts (human, Neu5Gc 0.0 pmol/µg protein, Neu5Ac 97.7 pmol/µg protein, measured by HPLC), mixed with an equal volume of Freund’s adjuvant. Serum Lipoprotein and Lipid Analysis. Blood samples were obtained by mandibular plexus bleeding and cardiac puncture from mice fasted for 5 h. Plasma lipoproteins in 100-µL pooled samples were separated by size-exclusion chromatography using a polyethylene filter column (Sigma-Aldrich). Liver samples were homogenized in 250 mM sucrose buffer and the protein levels were measured by the bicinchoninic acid assay (Thermo Fisher Scientific). Cholesterol and triglyceride levels in separated lipoprotein fraction were measured by enzymatic kits (Sekisui) as well as total cholesterol and triglyceride levels in plasma and liver samples. Glucose Tolerance and Insulin Tolerance Test and HOMA-IR. Mice received an oral glucose gavage (2 mg/g body weight) for glucose tolerance test or an i.p. injection of recombinant human insulin (Novo Nordisk) for insulin tolerance test (0.5 U/kg body weight) after 5 h of fasting. Blood glucose levels were measured using blood collected via tail vein bleeding just before and at 15, 30, 60, 90, and 120 min after oral glucose or injection of insulin with the Accu-Chek Aviva glucose monitoring system (Roche Applied Science GmbH). Insulin levels were measured with the ultrasensitive mouse insulin ELISA kit (Chrystal Chem). HOMA-IR was calculated as follows: HOMA - IR = 26 × fasting insulin level ( ng / mL ) × fasting glucose level ( mg / dL ) / 405 . Quantification of Aortic Atherosclerosis. Mice were killed by isoflurane intoxication and perfused with a PBS–10% neutral buffered formalin solution. The heart and ascending aorta down to the iliac bifurcation were dissected using stereomicroscopy, the aortas were dissected open along the long axis, pinned flat and stained for lipids using Sudan IV. Serial 10-µm cryosections of the aortic sinus were stained with Masson’s trichrome to measure total atherosclerosis lesion, collagen fiber lesion, and necrotic core lesion area. Each parameter was calculated using ImageJ blindly by K.K. and R.D. Quantification of Inflammatory Cytokine Gene Expression. Cells or organs were collected after completion feeding regimen and mRNA was collected using a purification kit and converted into cDNA (Qiagen, Inc.). Expression of each cytokine gene (SI Appendix, Table S1) was measured using Cyber Green systems (Qiagen, Inc.). Immunohistochemistry. All aorta tissue samples were either flash frozen in OCT or fixed and processed for paraffin sections, and were stained with H&E, Masson-Trichrome, Oil Red O, Terminal TUNEL was done using an In Situ Cell Death Detection Kit (TMR red) as per kit manufacturer’s (Roche) instructions and counterstained with Hoechst (ThermoFisher), or with immunohistochemistry using anti-F4/80 (Bio-Rad/AbD Serotec), anti-CD68 (Abcam), and anti-Neu5Gc (BioLegend) counterstained with Mayer’s hematoxylin, on serial cross sections of the aortic sinus. TUNEL positive cells in aortic sinus and Oil Red O lipid positive areas in liver samples were photographed using Keyence BZ-X800 (KEYENCE, Cop.). Each parameter was measured using Image J and Keyence BZ-X800 blindly by K.K., C.D., and R.D. Blood Cell Analysis and Quantification of Serum Inflammatory Cytokine. Whole EDTA blood samples were analyzed in duplicate for complete blood count with differential leukocyte count were performed with a Hemavet 850FS Multi Species Hematology System (Drew Scientific) in mouse hematology settings. Serum haptoglobin (Life Diagnostics, Inc.) and serum amyloid protein A (Life Diagnostics, Inc.) were measured according to the manufacturers’ instructions. Multiplex inflammatory cytokine levels were measured with proinflammatory panel 1 (mouse) kits (Meso Scale Diagnostics, LLC). ELISA Detection of Anti-Neu5Gc Antibodies in Mice Serum. Microtiter plate wells were coated with Neu5Gc-rich bovine submaxillary mucin (Sigma) in sodium carbonate-bicarbonate buffer at 4 °C overnight. Wells were incubated with 1:100 dilutions of mouse serum at room temperature for 2 h. Thereafter, wells were incubated with HRP-conjugated goat anti-mouse IgG. Mild periodate treatment is used to selectively cleave sialic acid side-chains and eliminate specific reactivity, as described previously (29). Statistical Analysis. All data were analyzed by Student’s t test, Mann–Whitney U test, Kruskal–Wallis test, 1-way ANOVA, or 2-way ANOVA, Pearson correlation coefficient and presented as mean ± SEM. Statistical analyses were performed using Prism software (version 8; GraphPad Software). P values less than 0.05 were considered significant.

Acknowledgments We thank Sandra Diaz for helpful comments. This work was supported by NIH Grant R01GM32373 (to A.V.), American Heart Association Postdoctoral Fellowship 17POST33671176 (to K.K.), and Fondation Leducq Grant 16CVD01 (to P.L.S.M.G.).

Footnotes Author contributions: P.L.S.M.G. and A.V. designed research; K.K., C.D., R.D., and N.V. performed research; K.K., C.D., R.D., and P.L.S.M.G. analyzed data; and K.K., C.D., N.V., P.L.S.M.G., and A.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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