Fluid shear stress modulates endothelial inflammation by targeting LIMS2

Abstract

Mechanosensitive genes regulate multiple cardiovascular pathophysiological processes and disorders; however, the role of flow-sensitive genes in atherosclerosis is still unknown. In this study, we identify LIM Zinc Finger Domain Containing 2 (LIMS2) that acts as a mechanosensitive gene downregulated by disturbed flow (d-flow) both in human endothelial cells (ECs) in vitro and in mice in vivo. Mechanistically, d-flow suppresses LIMS2 expression, which leads to endothelial inflammation by upregulating typical inflammatory factors, VCAM-1, and ICAM-1 in human ECs. The findings indicate that LIMS2, the new flow-sensitive gene, may help us to find a new insight to explain how d-flow caused endothelial inflammation and provide a new therapeutic approach for atherosclerosis in the future.

Keywords: Atherosclerosis; biomarker; endothelial inflammation.

Figure 1.

D-flow induces atherosclerotic plaque formation in ApoE^−/− mice and causes inflammation in human and ECs. (a) Left carotid artery (LCA) showed atherosclerotic plaque formation compared with right carotid artery (RCA) after 28-day partial carotid ligation (PCL) in ApoE^−/− mice. (b) HE staining showed unbroken RCA and obvious atherosclerotic plaque accumulation in LCA after 28-day PCL surgery for ApoE^−/− mice (N = 4). (c) C57BL/6 mice were partially ligated for 48 h and then sacrificed. Endothelial-enriched total RNA (eRCA, eLCA) and total RNA of media and adventitia (RCA left over, RCA-L/O; LCA left over, LCA-L/O) were obtained from the LCA and RCA. VCAM-1 and ICAM-1 gene expression were measured and normalized to 18S (N = 4, *=p < 0.05, **=p < 0.01, ***=p < 0.001). (d) HAEC were exposed to LS or OS for 24 h. Following shear, RNA was collected for qPCR and VCAM-1 and ICAM-1 gene level were measured and normalized to 18S. (N = 4, **=p < 0.01, ***=p < 0.001). (A color version of this figure is available in the online journal.)

Figure 2.

LIMS2 is a novel mechanosensitive gene that is inhibited by d-flow in mice. (a) For building PCL animal model, C57BL/6 mice left carotid artery were partial ligated by suture, included internal carotid artery (ICA), external carotid artery (ECA), occipital artery (OA). (b) C57BL/6 mice were partially ligated for 48 h and then sacrificed. Endothelial-enriched total RNA (eRCA, eLCA) and total RNA of media and adventitia (RCA-L/O, LCA-L/O) were obtained from the LCA and RCA. KLF2 gene and TSP-1 gene level as positive control were measured and normalized to 18S (N = 4, **=p < 0.01, ***=p < 0.001). (c) C57BL/6 mice were partially ligated for 48 h (acute PCL group) or 2 week (chronic PCL group) and then sacrificed. Endothelial-enriched total RNA (eRCA, eLCA) and total RNA of media and adventitia (RCA-L/O, LCA-L/O) were obtained from the LCA and RCA. LIMS2 gene level was measured and normalized to 18S (N = 4, ***=p < 0.001). (d) C57BL/6 mice were partially ligated for 48 h (acute PCL group) and then sacrificed. RCA and LCA were separated and stained for LIMS2. (A color version of this figure is available in the online journal.)

Figure 3.

LIMS2 expression are suppressed by oscillatory shear (OS) in human ECs. (a) HAEC were subjected to laminar shear (LS, 20 dyn/cm²) or oscillatory shear (OS, ±5dyn/cm²) for 24 h. (b) HAEC were exposed to LS or OS for 24 h. Following shear, RNA was collected for qPCR and KLF2 gene and TSP-1 gene acted as positive control were measured and normalized to 18S. (N = 4, ***=p < 0.001). (C) HAEC were exposed to LS or OS for 24 h. Following shear, RNA was collected for qPCR and LIMS2 gene was measured and normalized to 18S. (N = 4, ***=p < 0.001). (d and e) HAEC were exposed to LS or OS for 24 h. Following shear, LIMS2 protein level was measured by western blot (N = 4, ***=p < 0.001). (A color version of this figure is available in the online journal.)

Figure 4.

Silencing LIMS2 expression modulates endothelial inflammation via upregulating VCAM-1 and ICAM-1 in human ECs. (a) HAECs were transfected with different dose of siLIMS2 for 48 h. Then, the cells were lysed and RNA was collected for determination of the LIMS2 gene level. (N = 4, **=p < 0.01, ***=p < 0.001). (b) HAECs were transfected with 150 nM of siLIMS2 and subjected to LS 24 h later. 24-h post shear, the cells were lysed and RNA was collected for determination of the LIMS2 gene level. (N = 4, ***=p < 0.001). (c) Alternatively, the cells were lysed at 24 h and RNA was collected for determination of the inflammatory markers VCAM-1 and ICAM-1. (N = 4, **=p < 0.01, ***=p < 0.001). (d and e) The protein was collected and measured by western blot for determination of the VCAM-1 and ICAM-1 protein expression. (N = 4, *=p < 0.05, **=p < 0.01). (A color version of this figure is available in the online journal.)