Fig 2: Schematic of the proposed role of CAR in endothelial mechanotransduction signaling under FSS.Endothelial CAR expression is regulated by FSS. Disturbed flow increases, whereas laminar shear stress decreases, CAR expression in ECs; these changes in CAR expression are regulated through the KLF2-AP-1 axis. Disturbed flow-induced CAR inhibits the phosphorylation of PECAM-1, leading to the suppression of endothelial mechanotransduction signaling through the phosphorylation of Src, PI3K, Akt, and eNOS. Therefore, CAR modulates mechanotransduction in ECs by blocking PECAM-1 phosphorylation, which influences endothelial function and vascular pathophysiology.

Fig 3: Fluid shear stress regulates CAR expression in endothelial cells.HUVECs were cultured under static, LSS, or OSS conditions for the indicated periods of time. a Levels of mRNAs encoding CAR and GAPDH (internal control) were determined by RT-PCR. b CAR protein levels were measured by western blotting. Representative images are shown (n = 5; compared with static conditions, *P < 0.05). c Mouse aortas were divided into two regions according to the distribution of vessel wall shear stress: the lesser curvature (LC; disturbed flow) and thoracic aorta (TA; normal laminar flow)20. The expression of CAR was evaluated by en face staining and confocal microscopy. Localization of CAR on the surface of the endothelium was verified by z-stack imaging (red, CAR; blue, nuclei; green, elastic lamina of vessel). d Frozen sections of human artery tissues20 were stained with anti-PECAM-1 and anti-CAR antibodies. PECAM-1 was used as an endothelial-specific marker. The expression of CAR was compared in two regions of the bifurcation site of the vessel with different wall shear stresses, the outer side with disturbed flow (D-Flow) and the inner side with normal laminar flow (NL-Flow), by confocal microscopy. Representative images from at least three experiments are shown (red, CAR; yellow, PECAM-1; blue, nuclei). Scale bars, 10 µm.

Fig 4: Characterization and comparison of manually seeded and bioprinted mono-cultures by LSM.Immunofluorescence labelling of F-actin cytoskeleton () and nuclei () in (a,b) EA.hy926 and (c,d) A549 mono-cultures cultivated for 3 days following (a,c) manual seeding and (b,d) bio-printing on Matrigel™ BM. Endothelial cells were labelled in addition with specific cell adhesion marker PECAM-1 (platelet endothelial adhesion molecule also referred to as CD31 ()). Single optical sections are presented in the x-y projection (central image) with respective side views in xz (bottom). Scale bars correspond to 20 µm.

Fig 5: CAR deletion protects the proatherogenic endothelium under disturbed flow.Wild-type (WT) C57BL/6 or EC-specific CAR KO mice were partially ligated in the LCA. a Twenty-four hours after LCA ligation, the mice were euthanized, and EC-enriched RNA was extracted from both carotid arteries. EC-enriched RNA was confirmed by assessing an endothelial-specific marker (PECAM-1), a smooth muscle cell-specific marker (a-smooth muscle actin [a-SMA]), and ß-actin (internal control). HUVECs were used as a positive control for PECAM-1 and a negative control for a-SMA. Human vascular smooth muscle cells (VSMCs) were used as a positive control for a-SMA and a negative control for PECAM-1. The knockout of endothelial CAR in EC-specific CAR KO mice was confirmed by RT-PCR. Representative images are shown (n = 5). b Levels of mRNAs encoding eNOS, VCAM-1, and ICAM-1 in EC-enriched RNA were determined by real-time PCR (n = 5; *P < 0.05, WT RCA vs. WT LCA, CAR KO RCA vs. CAR KO LCA; #P < 0.05, WT LCA vs. CAR KO LCA). c Immunofluorescence staining was performed in carotid arteries from WT or EC-specific CAR KO mice. Representative images are shown (n = 3; red, eNOS, VCAM-1, or ICAM-1; blue, nuclei; green, elastic lamina of vessel; scale bars, 10 µm). d Disturbed flow-induced atherosclerotic plaque formation was visualized in ligated LCA by Oil Red O staining. We measured the IMT by calculating the luminal area relative to the total vascular area. Representative images are shown (n = 11; *P < 0.05, WT vs. CAR KO). e Human arterial tissues were stained with hematoxylin and eosin (H&E) and anti-PECAM-1 and anti-CAR antibodies. PECAM-1 was used as an endothelial-specific marker. Expression of CAR was compared between normal and atherosclerotic regions of arterial tissue by confocal microscopy. Representative images from at least three experiments are shown (red, CAR; yellow, PECAM-1; blue, nuclei). Scale bars, 10 µm.