Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) has been implicated in physiological processes in the metabolic as well as in the vascular systems, as supported by the cardiometabolic abnormalities associated with insulin resistance that developed in global Ceacam1 null mutants. Given the role of CEACAM1 in maintaining vascular integrity, we aimed at identifying a role for CEACAM1-dependent pathways in endothelial cells in the pathogenesis of hepatic fibrosis. To this end, we generated endothelial cell-specific Ceacam1 knockout mice ( VECadCc1 -/- ) and propagated them on the C57BL/6J genetic background. The mice remain insulin sensitive even at 12-months of age, as determined by hyperinsulinemic-euglycemic clamp analysis at 7 months and by insulin tolerance test. The mice display normoinsulinemia associated with intact insulin clearance and without hepatic steatosis. However, these VECadCc1 -/- mutants display inflammatory foci in liver parenchyma, when stained by H&E staining. This was supported by ~2-fold increase in the mRNA levels of genes involved in inflammation (F4/80, CD68, IL-1β IL-6, and TNF­α) and by immunostaining for macrophage pool (F4/80) and their activation (CD68). Sirius Red stain detected a remarkable chicken-­wire deposition of collagen fibers interstitially and in the perivenular region in livers of VECadCc1 -/- but not controls. The mRNA levels of profibrogenic markers (Tgfβ, collagen 1A1, collagen 6a3, and α-SMA) are ~2-fold higher than controls. Moreover, TGFβ1 is a basally activated in null mice, as shown by increased Smad2/3 phosphorylation compared to their controls. Several mechanisms could contribute to hepatic fibrosis in VECadCc1 -/- mutants. These include: 1) increased endothelial-to-mesenchymal transition, supported by increased expression of mesenchymal markers (Snail, Slug, and [fibroblast-specific protein1 (FSP1)] with a reciprocal decrease in endothelial cell makers (CD31) in mouse liver endothelial cells (MLEC) isolated from VECadCc1 -/- mice; 2) increased capillarization, a liver injury event that precedes fibrogenesis, as shown by the ~2-fold increase in the mRNA levels of markers of capillarization (Gli1, Gli2, Gli3, iNOS, and osteopontin) in hepatic primary endothelial cells; 3) elevated levels of plasma endothelin-1 (ET-1) that promotes fibrogenesis. Mechanistically, the rise in plasma ET-1 results from increased coupling of the Ras/MAPKinase pathway to insulin receptor and VEGFR via Shc, a Src homology 2 (SH2)-containing cytoplasmic adaptor protein that upon its binding to phosphorylated CEACAM1 undergoes sequestration. Thus, endothelial cell CEACAM1 plays a key role in preventing liver injury and resulting fibrogenesis.

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Patients with nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) commonly develop atherosclerosis through a mechanism that is not well delineated. These diseases are associated with steatosis, inflammation, oxidative stress, and fibrosis. The role of insulin resistance in their pathogenesis remains controversial. Albumin ( Alb ) Cre + Cc1 flox ( fl ) /fl mice with the liver‐specific null deletion of the carcinoembryonic antigen‐related cell adhesion molecule 1 ( Ceacam1 ; alias Cc1 ) gene display hyperinsulinemia resulting from impaired insulin clearance followed by hepatic insulin resistance, elevated de novo lipogenesis, and ultimately visceral obesity and systemic insulin resistance. We therefore tested whether this mutation causes NAFLD/NASH and atherosclerosis. To this end, mice were propagated on a low‐density lipoprotein receptor ( Ldlr ) −/− background and at 4 months of age were fed a high‐cholesterol diet for 2 months. We then assessed the biochemical and histopathologic changes in liver and aortae. Ldlr −/− AlbCre + Cc1 fl/fl mice developed chronic hyperinsulinemia with proatherogenic hypercholesterolemia, a robust proinflammatory state associated with visceral obesity, elevated oxidative stress (reduced NO production), and an increase in plasma and tissue endothelin‐1 levels. In parallel, they developed NASH (steatohepatitis, apoptosis, and fibrosis) and atherosclerotic plaque lesions. Mechanistically, hyperinsulinemia caused down‐regulation of the insulin receptor followed by inactivation of the insulin receptor substrate 1–protein kinase B–endothelial NO synthase pathway in aortae, lowering the NO level. This also limited CEACAM1 phosphorylation and its sequestration of Shc‐transforming protein (Shc), activating the Shc–mitogen‐activated protein kinase–nuclear factor kappa B pathway and stimulating endothelin‐1 production. Thus, in the presence of proatherogenic dyslipidemia, hyperinsulinemia and hepatic insulin resistance driven by liver‐specific deletion of Ceacam1 caused metabolic and vascular alterations reminiscent of NASH and atherosclerosis. Conclusion: Altered CEACAM1‐dependent hepatic insulin clearance pathways constitute a molecular link between NASH and atherosclerosis.

(1) Background: Mice with global Ceacam1 deletion developed plaque-like aortic lesions even on C57BL/6J background in the presence of increased endothelial cell permeability and insulin resistance. Loss of endothelial Ceacam1 gene caused endothelial dysfunction and reduced vascular integrity without affecting systemic insulin sensitivity. Because endothelial cell injury precedes atherosclerosis, we herein investigated whether the loss of endothelial Ceacam1 initiates atheroma formation in the absence of insulin resistance. (2) Methods: Endothelial cell-specific Ceacam1 null mice on C57BL/6J.Ldlr−/− background (Ldlr−/−VECadCre+Cc1fl/fl) were fed an atherogenic diet for 3–5 months before metabolic, histopathological, and en-face analysis of aortae were compared to their control littermates. Sirius Red stain was also performed on liver sections to analyze hepatic fibrosis. (3) Results: These mice displayed insulin sensitivity without significant fat deposition on aortic walls despite hypercholesterolemia. They also displayed increased inflammation and fibrosis. Deleting Ceacam1 in endothelial cells caused hyperactivation of VEGFR2/Shc/NF-κB pathway with resultant transcriptional induction of NF-κB targets. These include IL-6 that activates STAT3 inflammatory pathways, in addition to endothelin-1 and PDGF-B profibrogenic effectors. It also induced the association between SHP2 phosphatase and VEGFR2, downregulating the Akt/eNOS pathway and reducing nitric oxide production, a characteristic feature of endothelial dysfunction. Similarly, hepatic inflammation and fibrosis developed in Ldlr−/−VECadCre+Cc1fl/fl mice without an increase in hepatic steatosis. (4) Conclusions: Deleting endothelial cell Ceacam1 caused hepatic and aortic inflammation and fibrosis with increased endothelial dysfunction and oxidative stress in the presence of hypercholesterolemia. However, this did not progress into frank atheroma formation. Because these mice remained insulin sensitive, the study provides an in vivo demonstration that insulin resistance plays a critical role in the pathogenesis of frank atherosclerosis.

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