SAT-174 Loss of CEACAM1 in Endothelial Cells Causes Hepatic Fibrogenesis

Abstract

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.

Conditional deletion of CEACAM1 causes hepatic stellate cell activation

“…We have previously shown that hepatic CEACAM1 expression is progressively reduced with advancing fibrosis stages in patients with MASH [15]. Moreover, single cell RNA-sequencing showed lower CEACAM1 expression in hepatocytes and LSECs of patients with fibrosis/cirrhosis [17]. S.M.N.…”

“…As detailed in Supplemental data, fixed liver sections were stained with hematoxylin-eosin (H&E) or with 0.1% Sirius Red stain to evaluate hepatic fibrosis. Images were taken using Nikon Eclipse 90i Microscope and 10 randomly selected high power fields (20X) per sample were imaged with ImageJ (v1.53t) to quantify Sirius Red stain as %area [17].…”

“…Microscope and 10 randomly selected high power fields (20X) per sample were imaged with ImageJ (v1.53t) to quantify Sirius Red stain as %area [17].…”

“…For immunohistochemical (IHC) analysis, liver sections underwent antigen-retrieval, blocking with rabbit or mouse serum, stained overnight at 4°C with specific antibodies, blotted with species-specific biotinylated secondary antibodies before being hematoxylin-counterstained [17]. Images were taken using Nikon Eclipse 90i Microscope and evaluated blindly to count positively stained cells in 5 fields/mouse at 40X magnification.…”

“…Reciprocally, liver-specific rescuing of CEACAM1 reverses metabolic dysregulation and hepatic fibrosis in global Cc1 −/− null mice [14]. In contrast, CEACAM1 loss in endothelial cells promotes hepatic fibrosis, driven by increased production of endothelin1, without insulin resistance or hepatic steatosis [17]. Consistent with these data in genetically-modified mice, patients with MASH exhibit a progressive loss of CEACAM1 in liver [15] and particularly in LSECs [17] as the disease advances.…”