Background—: Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α ₁ β ₁ γ ₁ ) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α ₂ , α ₄ , or α ₅ chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. Methods and Results—: Using an in vitro flow assay, we show that laminin-411 (α ₄ β ₁ γ ₁ ), laminin-511 (α ₅ β ₁ γ ₁ ), and laminin-521 (α ₅ β ₂ γ ₁ ), but not laminin-211 (α ₂ β ₁ γ ₁ ), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α ₆ β ₁ and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α ₅ -containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α ₆ . Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α ₆ β ₁ -Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. Conclusions—: This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α ₆ β ₁ , could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Objective—: Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism. Approach and Results—: Using genetically engineered Sema7A ^−/− ApoE ^−/− mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE ^−/− mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 ( N -[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A ^+/+ ApoE ^−/− mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A ^−/− ApoE ^−/− mice. Further studies showed that blockage of β1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis. Conclusions—: Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a β1 integrin–dependent manner.

Besides their role in hemostasis and thrombosis, it has become increasingly clear that platelets are also involved in many other pathological processes of the vascular system, such as atherosclerotic plaque formation. Atherosclerosis is a chronic vascular inflammatory disease, which preferentially develops at sites under disturbed blood flow with low speeds and chaotic directions. Hyperglycemia, hyperlipidemia, and hypertension are all risk factors for atherosclerosis. When the vascular microenvironment changes, platelets can respond quickly to interact with endothelial cells and leukocytes, participating in atherosclerosis. This review discusses the important roles of platelets in the plaque formation under pro-atherogenic factors. Specifically, we discussed the platelet behaviors under disturbed flow, hyperglycemia, and hyperlipidemia conditions. We also summarized the molecular mechanisms involved in vascular inflammation during atherogenesis based on platelet receptors and secretion of inflammatory factors. Finally, we highlighted the studies of platelet migration in atherogenesis. In general, we elaborated an atherogenic role of platelets and the aspects that should be further studied in the future.

Glycoprotein VI (GPVI) has been proposed as a promising antiplatelet target, because its blockade prevents experimental thrombosis without impairing hemostasis. The objective of this study was to develop a preclinical tool to evaluate the role of human GPVI (hGPVI) in various models of thrombosis and to screen anti-GPVI compounds. A genetically modified mouse strain expressing hGPVI has been developed using a knockin strategy. The mice were viable and fertile and did not present any hematological defects. Approximately 3700 copies of human GPVI were detected at the platelet surface. Platelet aggregation, fibrinogen binding, and P-selectin exposure were normal in response to various agonists. The 9O12.2 Fab fragment directed against human GPVI bound to hGPVI platelets in vitro and ex vivo and markedly reduced collagen- and collagen-related peptide-induced responses. Injection of 9O12.2 into hGPVI animals did not prolong the tail bleeding time but provided protection against lethal thromboembolism induced by a collagen/adrenaline mixture. In addition, 9O12.2 reduced arterial thrombus growth by 44% after superficial laser injury, 43% after deep laser injury in mice pretreated with hirudin, and 48% after mechanical injury. In conclusion, we have developed a humanized mouse model that could be used in preclinical studies to evaluate the effects of anti-GPVI compounds.

Disturbed blood flow (d-flow) has been known to induce changes of the cells in the arterial wall, increasing the risk of atherosclerosis. However, the heterogeneity of the vascular cell populations under d-flow remains less understood. To generate d-flow in vivo, partial carotid artery ligation (PCL) was performed. Seven days after ligation, single-cell RNA sequencing of nine left carotid arteries (LCA) from the PCL group (10,262 cells) or control group (14,580 cells) was applied and a single-cell atlas of gene expression was constructed. The integrated analysis identified 15 distinct carotid cell clusters, including 10 d-flow-relevant subpopulations. Among endothelial cells, at least four subpopulations were identified, including Klk8^hi ECs, Lrp1^hi ECs, Dkk2^hi ECs, and Cd36^hi ECs. Analysis of GSVA and single-cell trajectories indicated that the previously undescribed Dkk2^hi ECs subpopulation was mechanosensitive and potentially transformed from Klk8^hi ECs under d-flow. D-flow-induced Spp1^hi VSMCs subpopulation that appeared to be endowed with osteoblast differentiation, suggesting a role in arterial stiffness. Among the infiltrating cell subpopulations, Trem2^hi Mφ, Birc5^hi Mφ, DCs, CD4⁺ T cells, CXCR6⁺ T cells, NK cells, and granulocytes were identified under d-flow. Of note, the novel Birc5^hi Mφ was identified as a potential contributor to the accumulation of macrophages in atherosclerosis. Finally, Dkk2^hi ECs, and Cd36^hi ECs were also found in the proatherosclerotic area of the aorta where the d-flow occurs. In conclusion, we presented a comprehensive single-cell atlas of all cells in the carotid artery under d-flow, identified previously unrecognized cell subpopulations and their gene expression signatures, and suggested their specialized functions.

Endothelial to mesenchymal transition (EndMT) is an important pathological change in many diseases. Semaphorin7A (Sema7A) has been reported to regulate nerve and vessel homeostasis, but its role in EndMT remains unclear. Here we investigate the effect of Sema7A on EndMT and the underlying mechanism. Sema7A-overexpressed human umbilical vein endothelial cells (Sema7A-HUVECs) were generated and showed lower levels of endothelial cell markers and higher levels of mesenchymal cell markers indicating the occurrence of EndMT. RNA-sequencing analysis showed a total of 1168 upregulated genes and 886 downregulated genes. Among them, most of the molecules associated with EndMT were upregulated in Sema7A-HUVECs. Mechanistically, Sema7A-HUVECs showed a higher TGF-β2 expression and activated TGF-β/Smad Signaling. Importantly, Sema7A overexpression upregulated activating transcription factor 3 (ATF3) that was found to selectively bind the promotor region of TGF-β2, but not TGF-β1, promoting TGF-β2 transcription, which was further confirmed by ATF3-siRNA knockdown approach. Blocking β1 integrin, a known Sema7A receptor, alleviated the expression of ATF3, TGF-β2, and EndMT in Sema7A-overexpressed HUVECs, implying a role of β1 integrin/ATF3/TGF-β2 axis in mediating Sema7A-induced EndMT. Using Sema7A-deficient mice and the partial carotid artery ligation (PCL) model, we showed that Sema7A deletion attenuated EndMT induced by blood flow disturbance in vivo. In conclusion, Sema7A promotes TGF-β2 secretion by upregulating transcription factor ATF3 in a β1 integrin-dependent manner, and thus facilitates EndMT through TGF/Smad signaling, implying Sema7A as a potential therapeutic target for EndMT-related vascular diseases.