Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Quantification of human MTTP activity at the plateau phase (170-210 min) non-steatosis (n=11) and steatosis samples (n=12). Each patient is represented by a group of approximately 5 data points (dots). (B-C) Representative western blot showing reduced CEPT1 and MTTP protein levels in human liver tissue from non-steatosis (n=11) and steatosis samples (n=12). GAPDH serves as a loading control. (D) Quantification of hepatic CEPT1 (C) and MTTP (D) protein levels. (E-F) Representative immunofluorescence images of human liver sections, comparing non-steatosis and steatosis conditions: (E) H&E staining with DAPI (blue) and MTTP (green); white arrows highlight regions of positive MTTP staining; (F) H&E staining alongside DAPI (blue), CD31 (red), and CEPT1 (green), white arrows indicate areas of CD31–CEPT1 colocalization, shown in yellow; (G) Quantification of MTTP integrated density, showing a significant decrease in steatosis (*p<0.05). (H) Quantification of CEPT1/CD31 co-localization, which is significantly reduced in steatotic samples (**p<0.01). Data represent mean ± SEM. Statistical significance was determined by Unpaired t-test.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Activity Assay, Western Blot, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Schematic illustrating the proposed impact of endothelial CEPT1 (EC CEPT1) on hepatic MTTP and PPARα regulation. EC CEPT1 influences hepatocyte function, potentially impacting VLDL assembly and fatty acid metabolism. Fenofibrate (FEN), a PPARα activator, is included to indicate pharmacological activation of PPARα in this pathway (B) Experimental setup illustrating co-culture of HUVECs with HepG2 cells ± FEN treatment (50 µM) for 48h. HUVECs were transfected with si CEPT1 +. (C-E) Relative mRNA expression of CEPT1 (C), PPARα (D), and MTTP (E) in HUVECs transfected with siRNA targeting Cept1 compared to untransfected HUVECs (control), normalized to controls (n=3). Relative mRNA expression levels of MTTP (F) and PPARα (G) in HepG2 cells co-cultured with HUVECs ± siRNA targeting CEPT1 and ±FEN treatment (50µM) for 48h, normalized to controls (n=3). (H) MTTP activity curve (pmol transferred/µg protein) over time for HepG2 cells from control and co-culture with HUVEC si CEPT1 + cells ±FEN treatment (50µM for 48h). Plateau indicates maximal MTTP transfer capacity, as determined by endpoint assay guidelines. (I) Quantification of MTTP activity at the plateau phase (195-240 min) for HepG2 cells from control and co-culture with HUVEC si CEPT1 + cells ±FEN treatment. Data were analyzed by Mann-Whitney U test; n=4. (J) Experimental setup illustrating co-culture of HepG2 with si PPARα + transfected HUVECs vs control (HUVECs si PPARα -) for 48h. (K) Relative mRNA expression of MTTP in HepG2 co-culture with HUVECs si PPARα + vs positive control (HUVECs si GFP + ) and negative control (un-transfected HUVECs) (n=9). Data were analyzed by t-test or one-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Activation Assay, Co-Culture Assay, Transfection, Expressing, Control, Cell Culture, Activity Assay, End Point Assay, MANN-WHITNEY, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Schematic representation of experimental design. Tamoxifen was administered intraperitoneally to induce Cre recombinase activity in Cept1 fl/fl Apoe +/+ and Cep1 fl/fl Apoe -/- mice. After tamoxifen treatment, mice were fed a 42% high-fat diet for 12 weeks. Serum samples were collected weekly for further analysis. (B) Body weight measurements (in grams) of mice over 12-week period. (C-E) Serum lipid profile after 12 weeks on a high-fat diet. Mean serum levels of total cholesterol (mg/dL) (C), triglycerides (mg/dL) (D), and free fatty acids (ng/dL) (E) in Cept1 fl/fl Apoe +/+ and Cep1 fl/fl Apoe -/- mice compared to controls. All data are presented as mean ± SEM. Statistical significance was determined by Unpaired t-test. *p<0.05, **p<0.01, ****p<0.0001, and n ≥ 6 for all groups.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Representative H&E-stained liver sections from Cept1 fl/fl Cre + Apoe -/- , Cept1 fl/fl Cre - Apoe -/- , Cept1 fl/fl Cre + Apoe +/+ and Cept1 fl/fl Cre - Apoe +/+ mice. Scale bar = 100 μm. (B) Quantification of hepatic lipid accumulation as measured by ORO intensity in liver sections across all experimental groups. Data analyzed by one-way ANOVA with Tukey’s post-hoc test; n=4-6 per group; **p<0.01. (C) Representative Western blots of MTTP, PPARα, CEPT1, and GAPDH in liver lysates of Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ . Molecular weights (kDa) are indicated. Quantification of hepatic Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ MTTP (D), PPARα (E), CEPT1 (F) protein levels normalized to GAPDH in all mice groups; n=3. Hepatic Mttp (G) and Cept1 (H) gene expression in Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ . (I) Representative Western blots of MTTP, PPARα, CEPT1, and GAPDH in liver lysates of Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- . Molecular weights (kDa) are indicated. Quantification of hepatic Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe -+/+ MTTP (J), PPARα (K), CEPT1 (L) protein levels normalized to GAPDH in all mice groups; n=3. Hepatic Mttp (M) and Cept1 (N) gene expression in Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- . Data were analyzed by t-test. (O) Liver MTTP activity curve (pmol transfered/μg protein) over time in Cept1 fl/fl Cre + Apoe -/- , and Cept1 fl/fl Cre + Apoe +/+ mice compared to controls. Plateau represents the maximal transfer capacity achieved under these experimental conditions, as per manufacturer’s guidelines for endpoint assays. (P-Q) MTTP activity at the plateau phase (180-240 min) in Cept1 fl/fl Cre + Apoe +/+ (P), and Cept1 fl/fl Cre + Apoe -/- (Q) mice compared to controls. Data were analyzed by Mann-Whitney U test; n=4. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Staining, Western Blot, Gene Expression, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: Representative images of ORO stained en face preparations of the aortas in Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ (A). Scale bar=5mm. Quantification of plaque area in the total aorta (B; n ≥ 5), aortic arch (C; n ≥ 4), thoracic aorta (D; n ≥ 5), and aortoiliac segment (E; n ≥ 4) of Cept1 fl/fl Cre - Apoe +/+ , and Cept1 fl/fl Cre + Apoe +/+ mice. Representative images of ORO stained en face preparations of the aortas in Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- (F). Scale bar=5mm. Quantification of plaque area in the total aorta (G; n ≥ 5), aortic arch (H; n ≥ 4), thoracic aorta (I; n ≥ 5), and aortoiliac segment (J; n ≥ 4) of Cept1 fl/fl Cre - Apoe -/- , and Cept1 fl/fl Cre + Apoe -/- mice. (K) Representative images of ORO-stained aortic root sections from Cept1 fl/fl Cre + Apoe +/+ and Cept1 fl/fl Cre + Apoe -/- mice compared to controls. Scale bar=100µm. (L-M) Quantification of lipid deposition in the aortic root sections; n ≥5 Cept1 fl/fl Cre + Apoe +/+ (L) and Cept1 fl/fl Cre + Apoe -/- (M) compared to their corresponding controls. (N) Representative immunofluorescence staining for CD68 (green) and DAPI (blue) in the aortic root of Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- mice. Scale bar=100μm. (O) Quantifying means CD68 intensity (arbitrary units, a.u.) (n=5). t-tests determined statistical significance. *p<0.05, **p<0.01.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Staining, Immunofluorescence