The fibrinogen-derived peptide (RGDS) prevents proteolytic degradation of protein kinase C in platelets by inhibiting platelet aggregation

The effects of the fibrinogen-derived tetrapeptide, Arg-Gly-Asp-Ser (RGDS), on platelet activation processes was studied. At concentrations of 100-300 microM, RGDS completely prevented platelet aggregation induced by all the common platelet agonists, 'weak' and 'strong'. In agreement with earlier views on the aggregation-dependency of weak agonist-induced thromboxane synthesis and 5-hydroxytryptamine (5HT) secretion, RGDS (100-300 microM) inhibited these events induced by ADP, adrenaline and low concentrations of thrombin and collagen but not that induced by high concentrations of thrombin and collagen. 5HT secretion induced by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), was also not affected by RGDS, but proteolytic degradation of the translocated membrane-bound enzyme in PMA-treated platelets, due to the actions of the Ca2+-dependent protease (Ca-DP), was completely prevented such that in the presence of RGDS, sustained increases in membrane-bound PKC activity were observed. PMA alone caused only transient increases in membrane-bound PKC. This effect of RGDS was similar to the effect of E64-d, a recently described inhibitor of Ca-DP in platelets, or the effects seen with PMA in unstirred non-aggregating platelets. It is concluded that RGDS inhibits the actions of Ca-DP in platelets via inhibition of aggregation.     

Agonist-specific cross talk between ERKs and p38(mapk) regulates PGI(2) synthesis in endothelium

We have examined the mechanisms regulatingprostacyclin (PGI₂) synthesis after acute exposure of humanumbilical vein endothelial cells (HUVEC) to interleukin-1 (IL-1).IL-1 evoked an early (30 min) release of PGI₂ and[³H]arachidonate that was blocked by the cytosolicphospholipase A₂ (cPLA₂) inhibitorarachidonyl trifluoromethyl ketone. IL-1-mediated activationof extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44^mapk) coincided temporally with phosphorylation ofcPLA₂ and with the onset of PGI₂synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK)inhibitors, PD-98059 and U-0126, blocked IL-1-induced ERKactivation and partially attenuated cPLA₂phosphorylation and PGI₂ release, suggesting thatERK-dependent and -independent pathways regulate cPLA₂phosphorylation. SB-203580 treatment enhanced IL-1-induced MEK,p42/44^mapk, and cPLA₂ phosphorylation butreduced thrombin-stimulated MEK and p42/44^mapk activation.IL-1, but not thrombin, activated Raf-1 as assessed byimmune-complex kinase assay, as did SB-203580 alone. These results showthat IL-1 causes an acute upregulation of PGI₂generation in HUVEC, establish a role for theMEK/ERK/cPLA₂ pathway in this early release, and provideevidence for an agonist-specific cross talk between p38^mapkand p42/44^mapk that may reflect receptor-specificdifferences in the signaling elements proximal to MAPK activation.

     

Conventional myosins – unconventional functions

While the discovery of unconventional myosins raised expectations that their actions were responsible for most aspects of actin-based cell motility, few anticipated the wide range of cellular functions that would remain the purview of conventional two-headed myosins. The three nonsarcomeric, cellular myosins—M2A, M2B and M2C—participate in diverse roles including, but not limited to: neuronal dynamics, axon guidance and synaptic transmission; endothelial cell migration; cell adhesion, polarity, fusion and cytokinesis; vesicle trafficking and viral egress. These three conventional myosins each take on specific, differing functional roles during development and maturity, characteristic of each cell lineage; exact roles depend on the developmental stage of the cell, cellular location, upstream regulatory controls, relative isoform expression, orientation and associated state of the actin cytoscaffolds in which these myosins operate. Here, we discuss the separate yet related roles that characterise the actions of M2A, M2B and M2C in various cell types and show that these conventional myosins are responsible for functions as unconventional as any performed by unconventional myosins.     

Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to ‘remotely located’ ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE₂ on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE₂ increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF_1α release and EC proliferation. In contrast, PGE₂ attenuated VEGF₁₆₅-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE₂ restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH₂ (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.     

sPLA(2) cooperates with cPLA(2)alpha to regulate prostacyclin synthesis in human endothelial cells

The first step in prostacyclin (PGI(2)) synthesis involves the generation of arachidonic acid (AA) from membrane phospholipids mediated by the 85 kDa cytosolic phospholipase A(2) (cPLA(2)alpha). The current study examined the effects of secretory PLA(2)s (sPLA(2)s) on PGI(2) production by human umbilical vein endothelial cells (HUVEC). We demonstrate that exposure of HUVEC to sPLA(2) dose- and time-dependently enhances AA release and PGI(2) generation. sPLA(2)-stimulated AA mobilisation was blocked by AACOCF(3), an inhibitor of cPLA(2)alpha, suggesting cross-talk between the two classes of PLA(2). sPLA(2) induced the phosphorylation of cPLA(2)alpha and enhanced the phosphorylation states of p42/44(mapk), p38(mapk), and JNK, concomitant with elevated AA and PGI(2) release. The MEK inhibitor PD98059 attenuated sPLA(2)-stimulated cPLA(2)alpha phosphorylation and PGI(2) release. These data show that sPLA(2) cooperates with cPLA(2)alpha in a MAPK-dependent manner to regulate PGI(2) generation and suggests that cross-talk between sPLA(2) and cPLA(2)alpha is a physiologically important mechanism for enhancing prostanoid production in endothelial cells.     

Vascular endothelial growth factor receptor-2 couples cyclo-oxygenase-2 with pro-angiogenic actions of leptin on human endothelial cells

Background

The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs.

Methodology/Principal Findings

Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38^MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr¹¹⁷⁵. Phosphorylation of VEGFR2, p38^MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38^MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38^MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo.

Conclusions/Significance

We conclude that a functional endothelial p38^MAPK/Akt/COX-2 signalling axis is required for leptin''s pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin''s regulation of the vasculature in both non-obese and obese individuals.     

Postprandial lipoproteins and the molecular regulation of vascular homeostasis

Blood levels of triglyceride-rich lipoproteins (TRL) increase postprandially, and a delay in their clearance results in postprandial hyperlipidemia, an important risk factor in atherosclerosis development. Atherosclerosis is a multifactorial inflammatory disease, and its initiation involves endothelial dysfunction, invasion of the artery wall by leukocytes and subsequent formation of foam cells. TRL are implicated in several of these inflammatory processes, including the formation of damaging free radicals, leukocyte activation, endothelial dysfunction and foam cell formation. Recent studies have provided insights into the mechanisms of uptake and the signal transduction pathways mediating the interactions of TRL with leukocytes and vascular cells, and how they are modified by dietary lipids. Multiple receptor and non-receptor mediated pathways function in macrophage uptake of TRL. TRL also induce expression of adhesion molecules, cyclooxygenase-2 and heme-oxygenase-1 in endothelial cells, and activate intracellular signaling pathways involving mitogen-activated protein kinases, NF-κB and Nrf2. Many of these effects are strongly influenced by dietary components carried in TRL. There is extensive evidence indicating that raised postprandial TRL levels are a risk factor for atherosclerosis, but the molecular mechanisms involved are only now becoming appreciated. Here, we review current understanding of the mechanisms by which TRL influence vascular cell function.     

Reduced chondrogenic matrix accumulation by 4-methylumbelliferone reveals the potential for selective targeting of UDP-glucose dehydrogenase

4-Methylumbelliferone (4-MU) is described as a selective inhibitor of hyaluronan (HA) production. It is thought that 4-MU depletes UDP-glucuronic acid (UDP-GlcUA) substrate for HA synthesis and also suppresses HA-synthase expression. The possibility that 4-MU exerts at least some of its actions via regulation of UDP-glucose dehydrogenase (UGDH), a key enzyme required for both HA and sulphated-glycosaminoglycan (sGAG) production, remains unexplored. We therefore examined the effects of 4-MU on basal and retroviral UGDH-driven HA and sGAG release in cells derived from chick articular cartilage and its influence upon UGDH protein and mRNA expression and HA and sGAG production. We found that 4-MU: i) suppressed UGDH mRNA and protein expression and chondrogenic matrix accumulation in chick limb bud micromass culture, ii) significantly reduced both HA and sGAG production and iii) more selectively reversed the potentiating effects of UGDH overexpression on the production of HA than sGAG. Understanding how GAG synthesis is controlled and the mechanism of 4-MU action may inform its future clinical success.     

The expression of prostaglandin endoperoxide synthase 2 messenger RNA and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle

The use of transcervical artificial insemination in sheep is limited because of the anatomy of the cervix, which restricts the passage of an inseminating pipette into the uterine lumen. There is a degree of natural cervical relaxation at estrus that enables greater penetration with an inseminating pipette. We hypothesize that this relaxation may be regulated by cervical prostaglandin synthesis and remodeling of the cervical extracellular matrix. The present study investigated the changes in prostaglandin endoperoxide synthase 2 (PTGS2) mRNA expression and the proportion of smooth muscle and collagen in the sheep cervix during the estrous cycle. Sheep cervices were collected at four stages of the estrous cycle: prior to the LH surge, during the LH surge, after the LH surge, and during the luteal phase. The expression of cervical PTGS2 mRNA was determined by in situ hybridization, and the proportion of smooth muscle and collagen in the cervix was investigated by Masson trichrome staining. The expression of PTGS2 mRNA in the sheep cervix was greatest prior to the LH surge, when estradiol concentrations were also greatest. The increase in PTGS2 mRNA expression was associated with an increase in the proportion of collagen in the sheep cervix. We propose that prior to the LH surge, estradiol may stimulate PTGS2 mRNA expression and hence prostaglandin E2 synthesis in the sheep cervix to regulate cervical relaxation, most likely through the rearrangement of collagen bundles within the cervical extracellular matrix.