CryoET reveals actin filaments within platelet microtubules.

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

FGL2/FcγRIIB Signalling Mediates Arterial Shear Stress-Mediated Endothelial Cell Apoptosis: Implications for Coronary Artery Bypass Vein Graft Pathogenesis.

The sudden exposure of venous endothelial cells (vECs) to arterial fluid shear stress (FSS) is thought to be a major contributor to coronary artery bypass vein graft failure (VGF). However, the effects of arterial FSS on the vEC secretome are poorly characterised. We propose that analysis of the vEC secretome may reveal potential therapeutic approaches to suppress VGF. Human umbilical vein endothelial cells (HUVECs) pre-conditioned to venous FSS (18 h; 1.5 dynes/cm2) were exposed to venous or arterial FSS (15 dynes/cm2) for 24 h. Tandem Mass Tagging proteomic analysis of the vEC secretome identified significantly increased fibroleukin (FGL2) in conditioned media from HUVECs exposed to arterial FSS. This increase was validated by Western blotting. Application of the NFκB inhibitor BAY 11-7085 (1 µM) following pre-conditioning reduced FGL2 release from vECs exposed to arterial FSS. Exposure of vECs to arterial FSS increased apoptosis, measured by active cleaved caspase-3 (CC3) immunocytochemistry, which was likewise elevated in HUVECs treated with recombinant FGL2 (20 ng/mL) for 24 h under static conditions. To determine the mechanism of FGL2-induced apoptosis, HUVECs were pre-treated with a blocking antibody to FcγRIIB, a receptor FGL2 is proposed to interact with, which reduced CC3 levels. In conclusion, our findings indicate that the exposure of vECs to arterial FSS results in increased release of FGL2 via NFκB signalling, which promotes endothelial apoptosis via FcγRIIB signalling. Therefore, the inhibition of FGL2/FcγRIIB signalling may provide a novel approach to reduce arterial FSS-induced vEC apoptosis in vein grafts and suppress VGF.

WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Mechanobiology of the endothelium in vascular health and disease: in vitro shear stress models.

In recent years, there has been growing evidence that vascular pathologies arise in sites experiencing an altered haemodynamic environment. Fluid shear stress (FSS) is an important contributor to vascular homeostasis and regulates endothelial cell (EC) gene expression, morphology, and behaviour through specialised mechanosensitive signalling pathways. The presence of an altered FSS profile is a pathological characteristic of many vascular diseases, with the most established example being the preferential localisation of atherosclerotic plaque development. However, the precise haemodynamic contributions to other vascular pathologies including coronary artery vein graft failure remains poorly defined. To evaluate potential novel therapeutics for the treatment of vascular diseases via targeting EC behaviour, it is important to undertake in vitro experiments using appropriate culture conditions, particularly FSS. There are a wide range of in vitro models used to study the effect of FSS on the cultured endothelium, each with the ability to generate FSS flow profiles through which the investigator can control haemodynamic parameters including flow magnitude and directionality. An important consideration for selection of an appropriate model of FSS exposure is the FSS profile that the model can generate, in comparison to the physiological and pathophysiological haemodynamic environment of the vessel of interest. A resource bringing together the haemodynamic environment characteristic of atherosclerosis pathology and the flow profiles generated by in vitro methods of applying FSS would be beneficial to researchers when selecting the appropriate model for their research. Consequently, here we summarise the widely used methods of exposing cultured endothelium to FSS, the flow profile they generate and their advantages and limitations in investigating the pathological contribution of altered FSS to vascular disease and evaluating novel therapeutic targets for the treatment and prevention of vascular disease.

Sphingosine-1-phosphate modulates PAR1-mediated human platelet activation in a concentration-dependent biphasic manner.

Sphingosine 1-phosphate (S1P) is a bioactive signalling sphingolipid that is increased in diseases such as obesity and diabetes. S1P can modulate platelet function, however the direction of effect and S1P receptors (S1PRs) involved are controversial. Here we describe the role of S1P in regulating human platelet function and identify the receptor subtypes responsible for S1P priming. Human platelets were treated with protease-activated receptor 1 (PAR-1)-activating peptide in the presence or absence of S1P, S1PR agonists or antagonists, and sphingosine kinases inhibitors. S1P alone did not induce platelet aggregation but at low concentrations S1P enhanced PAR1-mediated platelet responses, whereas PAR1 responses were inhibited by high concentrations of S1P. This biphasic effect was mimicked by pan-S1PR agonists. Specific agonists revealed that S1PR1 receptor activation has a positive priming effect, S1PR2 and S1PR3 have no effect on platelet function, whereas S1PR4 and S1PR5 receptor activation have an inhibitory effect on PAR-1 mediated platelet function. Although platelets express both sphingosine kinase 1/2, enzymes which phosphorylate sphingosine to produce S1P, only dual and SphK2 inhibition reduced platelet function. These results support a role for SphK2-mediated S1P generation in concentration-dependent positive and negative priming of platelet function, through S1PR1 and S1PR4/5 receptors, respectively.