Mary E. W. Collier

Tissue factor-containing microparticles released from mesangial cells in response to high glucose and AGE induce tube formation in microvascular cells

Hyperglycaemia and the associated formation of advanced glycation end-products (AGE) have been implicated in the pathogenesis of diabetic vasculopathy. In addition to its role in coagulation, tissue factor (TF) is known to regulate vascular proliferation and angiogenesis. In this study, the influence of AGE and glucose on the expression of TF in human renal mesangial cells (HRMC) and the subsequent induction of capillary formation by human dermal microvascular endothelial cells (HDMEC) were measured. Furthermore, the activity of TF, incorporated into microparticles was investigated. Both AGE and elevated glucose were capable of upregulating the expression of TF expression in a concentration-dependent manner in HRMC but not in HDMEC. This TF antigen and activity in the conditioned media from HRMC was associated with microparticles. Moreover, the formation of capillaries was readily induced on supplementation of HDMEC with conditioned media, from AGE-treated or high glucose-treated HRMC but not on incubation of HDMEC with either AGE or hyperphysiological concentrations of glucose. Furthermore, the rate of capillary formation was suppressed on incubation of the conditioned media with a polyclonal antibody against TF but not against VEGF. This study indicates that TF-containing microparticles are an important pro-inflammatory mediator acting as a mediator between elevated glucose and the development of diabetic vasculopathy by altering the angiogenic properties of endothelial cells and offers one explanation for the correlation between diabetes and microvascular disease.

Low molecular weight heparin downregulates tissue factor expression and activity by modulating growth factor receptor-mediated induction of nuclear factor-κB

Treatment of cancer patients with low molecular weight heparin (LMWH) appears to have beneficial effects. In this study, the influence of low molecular weight heparin (LMWH) on tissue factor (TF) expression and activity in five cell lines from various tissues was analysed and explored. Incubation of cells with LMWH (0-2000μg/ml) resulted in the downregulation of TF mRNA expression which was both LMWH concentration-dependent and time-dependent. Downregulation of TF was also measured as decreased cellular TF antigen and activity. Consistently, incubation of cells with LMWH suppressed the nuclear localisation and the transcriptional activity of NFκB. Decreased TF mRNA was largely achievable by incubating the cells with an NFκB inhibitor alone whilst incubation with betulinic acid to activate NFκB reversed the inhibitory influence of LMWH. Cells were also incubated with a range of concentrations of EGF (0-10ng/ml), bFGF (0-20ng/ml) or VEGF (0-4ng/ml) in the presence or absence of LMWH (200μg/ml) for 24h and TF antigen measured. Inclusion of LMWH reduced TF expression in response to EGF, bFGF or VEGF but TF expression was partially restored by increasing concentrations of the growth factors. We conclude that LMWH downregulates TF expression in vitro through a mechanism that involves interference with the function of growth factors which in turn is mediated through the downregulation of the transcriptional activity of NFκB. This mechanism may also explain some of the beneficial influences attributed to LMWH therapy in the treatment of cancer patients.

Induction of Endothelial Cell Proliferation by Recombinant and Microparticle-Tissue Factor Involves β1-Integrin and Extracellular Signal Regulated Kinase Activation

Objective—Increased levels of circulating tissue factor (TF) in the form of microparticles increase the risk of thrombosis. However, any direct influence of microparticle-associated TF on vascular endothelial cell proliferation is not known. In this study, the influence of recombinant and microparticle-associated TF on endothelial cell proliferation and mitogen-activated protein kinase signaling mechanisms was examined. Methods and Results—Incubation of human coronary artery endothelial cells with lipidated recombinant full-length TF, or TF-containing microparticles (50 to 200 pmol/L TF), increased the rate of cell proliferation and induced phosphorylation of extracellular signal regulated kinase 1 in a TF-dependent manner. Inhibition of extracellular signal regulated kinase 1/2 using PD98059 or extracellular signal regulated kinase 1/2 antisense oligonucleotides or inhibition of c-Jun N-terminal kinase reduced recombinant TF-mediated cell proliferation. PD98059 also reduced cell proliferation in response to TF-containing microparticles. Inclusion of FVIIa (5 nmol/L) and FXa (10 nmol/L) or preincubation of cells with an inhibitory anti-FVIIa antibody had no additional influence on TF-mediated cell proliferation. However, preincubation of exogenous TF with a &bgr;1-integrin peptide (amino acids 579 to 799) reduced TF-mediated proliferation. Conclusion—High concentrations of recombinant or microparticle-associated TF stimulate endothelial cell proliferation through activation of the extracellular signal regulated kinase 1/2 pathway, mediated through a novel mechanism requiring the interaction of exogenous TF with cell surface &bgr;1-integrin and independent of FVIIa.

Microparticle-associated tissue factor is recycled by endothelial cells resulting in enhanced surface tissue factor activity

In this study the uptake of tissue factor (TF)-positive microparticles by endothelial cells and the recycling of the TF component were examined. Human dermal blood endothelial cells (HDBEC) were incubated with microparticles derived from cancer cell lines for up to 6 hours. Measurement of HDBEC cell surface TF antigen revealed two distinct peaks at 30 and 180-240 minutes post-incubation with TF-positive, but not TF-deficient microparticles. However, only the second peak was concurrent with high TF activity as determined by a chromogenic thrombin-generation assay. Annexin V-labelling of HDBEC showed phosphatidylserine exposure following 90 minutes incubation with microparticles, which explains the high TF activity associated with the second antigen peak. Analysis of TF mRNA levels revealed no de novo expression of TF mRNA in response to microparticles, and pre-incubation of cells with cycloheximide did not prevent the appearance of TF. However, blocking endocytosis with a dynamin inhibitor prolonged the disappearance and prevented the reappearance of TF antigen on the cell surface. Incubation of HDBEC with microparticles containing TF-GFP revealed the early co-localisation of TF with Rab4 and Rab5, followed by co-localisation with the late endosomal/trans-Golgi network marker Rab9, and the recycling endosome marker Rab11. siRNA-mediated suppression of Rab11 reduced the reappearance of TF on the cell surface. These data suggest a mechanism by which TF-containing microparticles are internalised by endothelial cells and the TF moiety recycled to the cell surface. Together with the exposure of phosphatidylserine, this is capable of inducing a substantial increase in the procoagulant potential of the surface of endothelial cells.

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF.

Characterization of physical properties of tissue factor containing microvesicles and a comparison of ultracentrifuge-based recovery procedures

Microvesicles were isolated from the conditioned media of 3 cell lines (MDA-MB-231, AsPC-1 and A375) by ultracentrifugation at a range of relative centrifugal forces, and the tissue factor (TF) protein and activity, microvesicle number, size distribution and relative density compared. Also, by expressing TF-tGFP in cells and isolating the microvesicles, the relative density of TF-containing microvesicles was established. Nanoparticle tracking analysis (NTA) indicated that the larger-diameter microvesicles (>200 nm) were primarily sedimented at 100,000g and possessed TF-dependent thrombin and factor Xa generation potential, while in the absence of factor VII, all microvesicles possessed some thrombin generation capacity. Immuno-precipitation of TF-containing microvesicles followed by NTA also indicated the range of these microvesicles to be 200–400 nm. Analysis of the microvesicles by gradient density centrifugation showed that lower-density (<1.1 g/ml) microvesicles were mainly present in the samples recovered at 100,000g and were associated with TF antigen and activity. Analysis of these fractions by NTA confirmed that these fractions were principally composed of the larger-diameter microvesicles. Similar analysis of microvesicles from healthy or patient plasma supported those obtained from conditioned media indicating that TF activity was mainly associated with lower-density microvesicles. Furthermore, centrifugation of healthy plasma, supplemented with TF-tGFP-containing microvesicles, resulted in 67% retrieval of the fluorescent microvesicles at 100,000g, but only 26% could be recovered at 20,000g. Pre-centrifugation of conditioned media or plasma at 10,000g improved the speed and yield of recovered TF-containing microvesicles by subsequent centrifugation at either 20,000g or 100,000g. In conclusion, TF appears to be associated with low-density (1.03–1.08 g/ml), larger-diameter (200–350 nm) microvesicles.

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles.

Influence of Exogenous Tissue Factor on Estrogen Receptorα Expression in Breast Cancer Cells: Involvement of β1-Integrin, PAR2, and Mitogen-Activated Protein Kinase Activation

Increased expression of tissue factor (TF) has been associated with invasive forms of breast cancer. Conversely, the loss of estrogen receptor α (ERα) is associated with increased cell invasiveness. We have examined the influence of exogenous truncated recombinant TF (rTF) on ERα expression and cell invasiveness and investigated the mechanism of rTF signaling. The influence of rTF on ERα expression in MCF-7 and T47D cell lines was investigated using reverse transcription-PCR and ELISA. Cell invasion was measured using Boyden chamber-based invasion assays. Additionally, the interaction of fluorescein-labeled rTF with the surface of MCF-7 cells and particularly with β1-integrin was examined. Treatment of cells with rTF resulted in the down-regulation of ERα mRNA and protein over 24 h, which required β1-integrin and involved the mitogen-activated protein kinase pathway but did not require PAR2 activation. The addition of rTF reduced estradiol-mediated cell proliferation as well as increased cell invasiveness requiring both PAR2 and β1-integrin activation. Fluorescein-labeled rTF was shown to bind to the surface of MCF-7 cells within 5 min and peaked at 15 min. The bound rTF colocalized with cellular β1-integrin and was disrupted in the presence of excess unlabeled rTF and an anti-β1 polyclonal antibody. Finally, affinity purification of β1-integrin using rTF-conjugated agarose showed a requirement for the presence of divalent cations but not factor VIIa. The results indicate that rTF is capable of down-regulating ERα expression in breast cancer cells, resulting in decreases in estrogen-mediated cell proliferation and increased invasiveness. Furthermore, the mechanisms by which rTF induces these changes involve both PAR2 and β1-integrin. (Mol Cancer Res 2008;6(12):1807–18)

Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90% suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation.

Enhanced binding of tissue factor-microparticles to collagen-IV and fibronectin leads to increased tissue factor activity in vitro.

The role of tissue factor (TF)-containing microparticles in clot propagation has been established, but the ability of circulating microparticles to initiate coagulation has been disputed. However, TF-bearing microparticles, particularly endothelial-microparticles generated during disease, may interact with extracellular matrices which in turn can localise circulating TF to sites of injury. In order to examine this hypothesis in vitro , microparticles were isolated from human coronary artery endothelial cells transfected to overexpress TF, tumour-necrosis factor (TNF) α-treated cells or non-transfected cells lacking TF. The ability of microparticles to bind collagen-IV, fibronectin and fibrin was examined under static conditions and arterial shear rates (650 s⁻¹), and also in the presence of inhibitory antibodies against β1-, β3-, α3- and αv-integrins or an anti-TF antibody. TF-microparticles showed increases of up to 43% and 24% in adherence to collagen-IV and fibronectin, respectively, compared to control microparticles under shear flow. Furthermore, TF-containing microparticles, but not the transfected parent cells had increased levels of β1-integrin compared to TF-deficient microparticles. Pre-incubation of microparticles with a β1-integrin-blocking antibody counteracted the additional adhesion of TF-microparticles compared to control microparticles. Finally, adherence of TF microparticles to collagen-IV or fibronectin resulted in increased TF activity by concentrating TF onto the surface. In conclusion, the presence of TF within microparticles enhances the interactions of endothelial cell-derived microparticles with extracellular matrices in an integrin-dependent manner. Accumulation and localisation of these microparticles in turn results in the enhancement of TF activity. This may be an innate mechanism by which TF-bearing microparticles induce coagulation upon vascular injury.