During inflammation, leukocytes roll along the wall of postcapillary venules scanning the surface for immobilized CXCL1, a chemokine that triggers firm adhesion by activating CXCR2 on the neutrophil. PI-3K are signaling molecules important in cellular processes, ranging from cellular differentiation to leukocyte migration. PI-3K␥ can be activated directly by the ␤␥ dimer of heterotrimeric G proteins coupled to CXCR2. Here, we used in vivo and ex vivo intravital microscopy models to test the role of PI-3K␥ in leukocyte arrest. PI-3K␥ null mice showed an 80% decrease in CXCL1-induced leukocyte adhesion in venules of the exteriorized mouse cremaster muscle. In wildtype mice, rolling leukocytes showed rapid and sustained adhesion, but in PI-3K␥ ؊/؊ mice, adhesion was not triggered at all or was transient, suggesting that absence of PI-3K␥ interferes with integrin bond strengthening. Wild-type mice reconstituted with PI-3K␥ null bone marrow showed a 50% decrease in CXCL1-induced leukocyte adhesion. In a blood-perfused micro-flow chamber, leukocytes from PI-3K␥ ؊/؊ mice showed a defect in adhesion on a P-selectin/ICAM-1/CXCL1 substrate, indicating that leukocyte PI-3K␥ was required for adhesion. The adhesion defect in PI-3K␥ ؊/؊ mice was as severe as that in mice lacking LFA-1, the major integrin responsible for neutrophil adhesion. We conclude that the ␥ isoform of PI-3K must be functional in leukocytes to allow efficient adhesion from rolling in response to chemokine stimulation.

Lysophosphatidic acid (LPA) is a potent bioactive lysophospholipid. Accumulated evidence supports a role for LPA in inflammation. To profile LPA-induced cytokine production in vascular smooth muscle cells (SMCs), we used a cytokine antibody array system and found that LPA prominently induces the secretion of IL-6 and monocyte chemoattractant protein (MCP)-1 from human aortic SMCs (HASMCs). The mechanism by which LPA induces MCP-1 expression in SMCs has been previously reported. However, LPA induction of IL-6 secretion from vascular SMCs and its regulatory mechanism are unknown. The present study reveals that LPA induces the expression of IL-6 mRNA and protein in HASMCs as well as the secretion of IL-6 protein in a time-dependent manner. Our results demonstrate that LPA-specific receptor 1 (LPA(1)) mediates LPA-induced IL-6 secretion and that LPA induction of IL-6 is independent of the EGF receptor pathway. Our data further show that PKC-mediated p38 MAPK is responsible for the IL-6 secretion. Finally, small interfering RNA depletion experiments revealed that p38alpha is specifically responsible for the LPA-induced IL-6 secretion. The present study profiles the regulatory relationship between LPA and multiple cytokines in vascular SMCs for the first time, provides the first evidence that LPA upregulates IL-6 in vascular SMCs, and reveals the regulatory mechanism of LPA-induced IL-6 production in HASMCs. In light of the emerging roles of LPA and IL-6 in vascular inflammation, the understanding of the regulatory mechanism may contribute to the treatment and prevention of cardiovascular disorders.

Background: The molecular mechanism of smooth muscle cell (SMC) migration, a crucial event in atherosclerosis, is not well understood. Results: The de novo matricellular protein Cyr61 bridges lysophosphatidic acid (LPA) and integrin pathways, activating focal adhesion kinase (FAK) and leading to cell migration. Conclusion:The LPA-Cyr61-integrin-FAK axis controls SMC migration. Significance: This study provides new insights into mechanisms underlying cell migration-related disorders.

Vascular smooth muscle cell (SMC) migration is an essential step involved in neointimal formation in restenosis and atherosclerosis. Lysophosphatidic acid (LPA) is a bioactive component of oxidized low-density lipoprotein and is produced by activated platelets, implying that LPA influences vascular remodeling. Our previous study revealed that matricellular protein CCN1, a prominent extracellular matrix (ECM) protein, mediates LPA-induced SMC migration in vitro. Here we examined the role of CCN1 in LPA-induced neointimal formation. By using LPA infusion of carotid artery in a mouse model, we demonstrated that LPA highly induced CCN1 expression (approximately six- to sevenfold) in neointimal lesions. Downregulation of CCN1 expression with the specific CCN1 siRNA in carotid arteries blocked LPA-induced neointimal formation, indicating that CCN1 is essential in LPA-induced neointimal formation. We then used LPA receptor knockout (LPA-/-, LPA-/-, and LPA-/-) mice to examine LPA receptor function in CCN1 expression in vivo and in LPA-induced neointimal formation. Our data reveal that LPA deficiency, but not LPA or LPA deficiency, prevents LPA-induced CCN1 expression in vivo in mouse carotid arteries. We also observed that LPA deficiency blunted LPA infusion-induced neointimal formation, indicating that LPA is the major mediator for LPA-induced vascular remodeling. Our in vivo model of LPA-induced neointimal formation established a key role of the ECM protein CCN1 in mediating LPA-induced neointimal formation. Our data support the notion that the LPA-CCN1 axis may be the central control for SMC migration and vascular remodeling. CCN1 may serve as an important vascular disease marker and potential target for vascular therapeutic intervention.