Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca ²⁺ and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca ²⁺ response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brain's volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.

Aspirin prevents colorectal cancer metastasis in mice by splitting the crosstalk between platelets and tumor cells

Cyclooxygenase (COX)-2–derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E₂ (PGE₂) synthesis in HT29 cells was involved in the downregulation of p21^WAF1/CIP1 and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE₂. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in platelet-induced COX-2 overexpression. Inhibitors of galectin-3 function (β-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients.

Cyclooxygenase (COX)-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas. We have addressed whether the induction of COX-2–dependent prostanoids in endothelial cells by LSS plays a role in restraining endothelial tumor necrosis factor (TNF)-α generation, a proatherogenic cytokine, through the induction of heme oxygenase-1 (HO)-1, an antioxidant enzyme. In human umbilical vein endothelial cells (HUVECs) exposed to steady LSS of 10 dyn/cm²for 6 hours, COX-2 protein was significantly induced, whereas COX-1 and the downstream synthases were not significantly modulated. This was associated with significant (P<0.05) increase of 6-keto-prostaglandin (PG)F_1α(the hydrolysis product of prostacyclin), PGE₂, and PGD₂. In contrast, TNF-α released in the medium in 6 hours (3633±882 pg) or detected in cells lysates (1091±270 pg) was significantly (P<0.05) reduced versus static condition (9100±2158 and 2208±300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF-α generation and HO-1 induction were abrogated by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific prostacyclin receptor (IP) antagonist RO3244794 illuminates the central role played by LSS-induced COX-2–dependent prostacyclin in restraining endothelial inflammation. Carbacyclin, an agonist of IP, induced HO-1. Similarly to inhibition of prostacyclin biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC15 reversed TNF-α reduction by LSS. These findings suggest that inhibition of COX-2–dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking traditional nonsteroidal antiinflammatory drugs (NSAIDs) and NSAIDs selective for COX-2 through downregulation of HO-1, which halts TNF-α generation in human endothelial cells.

Background Even though the acetylation of platelet cyclooxygenase (COX)‐1 at serine‐529 is the direct mechanism of action of low‐dose aspirin, its antiplatelet effect has been characterized using indirect indexes of COX‐1 activity. Objectives We performed a clinical study with enteric‐coated low‐dose aspirin (EC‐aspirin), in healthy subjects, to evaluate the effects on the extent and duration of platelet COX‐1 acetylation, using a novel proteomic strategy for absolute protein quantification (termed AQUA), as compared with traditional pharmacokinetic and pharmacodynamic parameters. Subjects and methods In a phase I, single‐arm, open‐label study of EC aspirin (100 mg day⁻¹) administered to 24 healthy subjects, we compared, over a 24 h‐period on day 1 and 7, % platelet acetylated COX‐1 (AceCOX‐1) with traditional pharmacokinetic and pharmacodynamics [i.e. serum thromboxane (TX) B₂, platelet function by monitoring CEPI(collagen/epinephrine) closure time (CT) using whole‐blood PFA‐100 and urinary excretion of 11‐dehydro‐TXB₂] parameters. Results Acetylation of platelet COX‐1 was measurable before detection of aspirin levels in the systemic circulation and increased in a cumulative fashion upon repeated dosing. After the last dose of EC‐aspirin, %AceCOX‐1, serum TXB₂ and CEPI‐CT values were maximally and persistently modified throughout 24 h; they averaged 76 ± 2%, 99.0 ± 0.4% and 271 ± 5 s, respectively. EC‐aspirin caused 75% reduction in urinary 11‐dehydro‐TXB₂ excretion. After chronic dosing with aspirin, the pharmacokinetics of acetylsalicylic acid was completely dissociated from pharmacodynamics. Conclusions The demonstrated feasibility of quantifying the extent and duration of platelet COX‐1 acetylation will allow characterizing the genetic, pharmacokinetic and pharmacodynamic determinants of the inter‐individual variability in the antiplatelet response to low‐dose aspirin as well as identifying extra‐platelet sites of drug action.