CLEC-2-dependent platelet subendothelial accumulation by flow disturbance contributes to atherogenesis in mice

Abstract

Rationale: Platelets play an essential role in atherosclerosis, but the underlying mechanisms remain to be addressed. This study is to investigate the role of platelets in d-flow induced vascular inflammation and the underlying mechanism. Methods: We established a disturbed blood flow (d-flow) model by partial carotid ligation (PCL) surgery using atherosclerosis-susceptible mice and wild-type mice to observe the d-flow induced platelet accumulation in the subendothelium or in the plaque by immunostaining or transmission electron microscopy. The mechanism of platelet subendothelial accumulation was further explored by specific gene knockout mice. Results: We observed presence of platelets in atherosclerotic plaques either in the atheroprone area of aortic arch or in carotid artery with d-flow using Ldlr -/- or ApoE -/- mice on high fat diet. Immunostaining showed the subendothelial accumulation of circulating platelets by d-flow in vivo . Transmission electron microscopy demonstrated the accumulation of platelets associated with monocytes in the subendothelial spaces. The subendothelial accumulation of platelet-monocyte/macrophage aggregates reached peak values at 2 days after PCL. In examining the molecules that may mediate the platelet entry, we found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) reduced the subendothelial accumulation of platelets and monocytes/macrophages by d-flow, and ameliorated plaque formation in Ldlr -/- mice on high fat diet. Supportively, CLEC-2 deficient platelets diminished their promoting effect on the migration of mouse monocyte/macrophage cell line RAW264.7. Moreover, monocyte podoplanin (PDPN), the only ligand of CLEC-2, was upregulated by d-flow, and the myeloid-specific PDPN deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages. Conclusions: Our results reveal a new CLEC-2-dependent platelet subendothelial accumulation in response to d-flow to regulate vascular inflammation.

Platelets at the Vessel Wall in Non-Thrombotic Disease

“…130 Under shear stress, PECAM-1 (platelet EC adhesion molecule 1) and CLEC-2 also mediate platelet adhesion to ECs. [131][132][133] In the study by Ilkan et al, 134 the authors showed the presence of Piezo1 mechanosensitive ion channel in human platelets and megakaryocytes. D-flow in the arteries stimulates platelets and Meg-01 (megakaryocytes cell line) Ca 2+ proving the presence of mechanosensitive cation channels.…”

“…141 Platelets in atherosclerosis also showed the expression of receptors like PECAM-1 and CLEC-2 mediating their accumulation in sub-endothelium and ameliorating plaque formation in mice models. 133,134…”

Podoplanin: A potential therapeutic target for thrombotic diseases

“…Pdpn was specifically associated with lymphatic endothelium number of adventitial lymphatics of human internal carotid artery, which demonstrated Pdpn may participate in atherosclerosis via regulating functions and regeneration of adventitial lymphatic vessels in atherosclerotic lesions ( 41 ). In a disturbed blood flow (d-flow) model, monocyte Pdpn was upregulated by d-flow, and the myeloid-specific Pdpn deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages, which ameliorated vascular inflammation ( 42 ) ( Table 1 ).…”

Podoplanin: Its roles and functions in neurological diseases and brain cancers

“…Interestingly, pro-inflammatory cytokines were found to enhance Pdpn expression in stromal and endothelial cells, further supporting a correlation between inflammation and podoplanin functions in atherosclerotic plaques ( Gröger et al, 2004 ; Suzuki et al, 2008 ). In a disturbed blood flow (d-flow) model, monocyte Pdpn was upregulated by d-flow, and myeloid-specific Pdpn deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages ( Tang et al, 2021 ). All these suggest that Pdpn may contribute to the atherosclerosis development in both CLEC-2-dependent and independent manners.…”