Thrombosis and Haemostasis: an interdisciplinary and translational endeavour.
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Animals↗
Biomedical subjects
Klaus T Preissner
Publications and source records attributed to Klaus T Preissner.
The G534E polymorphism of the gene encoding the factor VII-activating protease is associated with cardiovascular risk due to increased neointima formation.
The G534E polymorphism (Marburg I [MI]) of factor VII-activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.
Actins↗
The extracellular adherence protein from Staphylococcus aureus abrogates angiogenic responses of endothelial cells by blocking Ras activation.
The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF). Moreover, the induction of tissue factor and decay-accelerating factor were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the 16-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap's combined anti-inflammatory and antiangiogenic properties render this bacterial protein not only an important virulence factor during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.
Bacterial Proteins↗
Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.
Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth, Galleria mellonella, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report the activation of a serine proteinase cascade by thermolysin, as described for bacterial lipopolysaccharides (LPS), that results in subsequent prophenoloxidase activation leading to melanization, an elementary immune defense reaction of insects. Quantitative real-time reverse transcription-PCR analyses of the expression of immune-related genes encoding the inducible metalloproteinase inhibitor, gallerimycin, and lysozyme demonstrated increased transcriptional rates after challenge with purified protfrags similar to rates after challenge with LPS. Additionally, we determined the induction of a similar spectrum of immune-responsive proteins that were secreted into the hemolymph by using comparative proteomic analyses of hemolymph proteins from untreated larvae and from larvae that were challenged with either protfrags or LPS. Since G. mellonella was recently established as a valuable pathogenicity model for Cryptococcus neoformans infection, the present results add to our understanding of the mechanisms of immune responses in G. mellonella. The obtained results support the proposed danger model, which suggests that the immune system senses endogenous alarm signals during infection besides recognition of microbial pattern molecules.
Animals↗
Suppression of experimental autoimmune encephalomyelitis by extracellular adherence protein of Staphylococcus aureus.
Multiple sclerosis (MS) is a devastating inflammatory disorder of the central nervous system (CNS). A major hallmark of MS is the infiltration of T cells reactive against myelin components. T cell infiltration is mediated by the interaction of integrins of the beta1 and beta2 family expressed by lymphocytes with their endothelial counter-receptors, vascular cell adhesion molecule 1 and intercellular adhesion molecule (ICAM)-1, respectively. We have reported previously that extracellular adherence protein (Eap) of Staphylococcus aureus exerts antiinflammatory activities by interacting with ICAM-1 and blocking beta2-integrin-dependent neutrophil recruitment. Here, we report that Eap inhibits experimental autoimmune encephalomyelitis (EAE) in mice. In vitro, Eap reduced adhesion of peripheral blood T cells to immobilized ICAM-1 as well as their adhesion and transmigration of TNF-activated human endothelium under static and shear flow conditions. These inhibitory effects were corroborated in two mouse models of inflammation. In a delayed-type hypersensitivity model, both T cell infiltration and the corresponding tissue edema were significantly reduced by Eap. In addition, Eap administration prevented the development of EAE and markedly decreased infiltration of inflammatory cells into the CNS. Strikingly, intervention with Eap after the onset of EAE suppressed the disease. Collectively, our findings indicate that Eap represents an attractive treatment for autoimmune neuroinflammatory disorders such as MS.
Amino Acid Sequence↗
A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding.
FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.
Animals↗
Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.
The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.
Angiopoietin-1↗
Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac-1 integrin.
Lipoprotein(a) [Lp(a)], consisting of LDL and the unique constituent apolipoprotein(a) [apo(a)], which contains multiple repeats resembling plasminogen kringle 4, is considered a risk factor for the development of atherosclerotic disorders. However, the underlying mechanisms for the atherogenicity of Lp(a) are not completely understood. Here, we define a novel function of Lp(a) in promoting inflammatory cell recruitment that may contribute to its atherogenicity. Through its apo(a) moiety Lp(a) specifically interacts with the beta2-integrin Mac-1, thereby promoting the adhesion of monocytes and their transendothelial migration in a Mac-1-dependent manner. Interestingly, the interaction between Mac-1 and Lp(a) was strengthened in the presence of proatherogenic homocysteine and was blocked by plasminogen/angiostatin kringle 4. Through its interaction with Mac-1, Lp(a) induced activation of the proinflammatory transcription factor NFkappaB, as well as the NFkappaB-related expression of prothrombotic tissue factor. In atherosclerotic coronary arteries Lp(a) was found to be localized in close proximity to Mac-1 on infiltrating mononuclear cells. Taken together, our data demonstrate that Lp(a), via its apo(a) moiety, is a ligand for the beta2-integrin Mac-1, thereby facilitating inflammatory cell recruitment to atherosclerotic plaques. These observations suggest a novel mechanism for the atherogenic properties of Lp(a).
Aged↗
Analysis of thrombotic factors in severe acute respiratory syndrome (SARS) patients.
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Adult↗
Isolated pancreatic islets in three-dimensional matrices are responsive to stimulators and inhibitors of angiogenesis.
The formation of a new microvasculature is essential for the long-term survival and function of the islet graft. In this study we examined endothelium of isolated pancreatic islets by stimulation with growth factors, different culture conditions, and genetic modification. We also inspected the effect of immunosuppressives used in human transplantation on angiogenesis. Isolated islets were embedded in a three-dimensional fibrin or Matrigel matrix. The effect of hyperglycemia, hypoxia, and the addition of VEGF and bFGF was investigated. We exposed islets from transgenic mice expressing the VEGF gene (RIP1VEGF-A) to high glucose (16.7 mmol/L) medium and tested the immunosuppressive agents rapamycin (100 ng/ml) and FK506 (100 ng/ml). To quantify angiogenesis the percentage of sprouting islets was determined. New endothelial capillary-like structures protruded from isolated pancreatic islets. Addition of VEGF to the islets and transgenic RIP-VEGF islets showed a two- to threefold increase of sprouting islets compared to control. Hypoxic culture conditions stimulated angiogenesis, resulting in a twofold increase of capillary sprouting. Rapamycin and FK506 proved to be potent inhibitors of angiogenesis in this system, because a decrease of sprouting islets of more than 20% by both agents was observed. Isolated pancreatic islets are capable of forming new capillary structures and are susceptible to pro- and antiangiogenic stimuli.
Angiogenesis Inducing Agents↗
Tenilsetam prevents early diabetic retinopathy without correcting pericyte loss.
Hyperglycemia-induced mitochondrial overproduction of reactive oxygen species leads to the activation of different biochemical pathways involved in endothelial damage of the diabetic retina. Tenilsetam [(+/-)-3-(2-thienyl)-2-piperazinone] is a dicarbonyl scavenger in the millimolar range and a transition metal ion chelator in the micromolar range. We tested its effect on experimental diabetic retinopathy, and on endothelial cell characteristics in vitro. Streptozotocin diabetic male Wistar rats (60 mg/kg BW) received 50 mg/kg BW tenilsetam (D-T) for 36 weeks, or no treatment (D). The impact of tenilsetam (0-30 mM) on endothelial proliferation, apoptosis, sprouting, cytokine-induced leucocyte-endothelial interaction, and VEGF expression was tested in vitro. Tenilsetam did not affect glycemic control or body weight in diabetic animals. The 3.7 fold increase in acellular capillaries in diabetic rats [p < 0.001 vs. non-diabetic controls (N)] was reduced by 70% (p < 0.001) through treatment, but pericyte loss (D vs. N -33%; p < 0.001) remained unaffected. In vitro, tenilsetam inhibited endothelial proliferation at lower doses, while inducing apoptosis at high doses. Leucocyte adhesion was only inhibited at high doses. Sprouting angiogenesis of bovine retinal endothelial cells was promoted at lower doses (< or = 10 mM). At micromolar concentrations, endothelial VEGF expression was upregulated by 100%. Long-term treatment with the AGE-inhibitor and iron-chelating compound tenilsetam inhibits the formation of acellular capillaries without correcting pericyte loss. The compound has dose-dependent effects on endothelial cell function. These data suggest that, independent of known properties, tenilsetam shows important rescue functions on endothelial cells which could be useful for the treatment of early diabetic retinopathy.
Animals↗
Interleukin-4 differentially regulates osteoprotegerin expression and induces calcification in vascular smooth muscle cells.
Vascular calcification is characterized by cellular transdifferentiation and expression of bone-related matrix proteins that result in the presence of bone-like structures in the vascular wall. Interleukin (IL)-4, a pleiotropic cytokine, and osteoprotegerin (OPG), an essential regulator of osteoclast biology, have both been linked to vascular disease. Here, we assessed the role of IL-4 and OPG in vascular calcification in vitro. IL-4 induced OPG mRNA levels and protein secretion by 5-fold in a dose- and time-dependent fashion in human coronary artery smooth muscle cells (CASMC). Activation of the transcription factor STAT6 preceded IL-4-induced OPG expression, and blockade of IL-4-induced STAT6 activation by the phospholipase C inhibitor D609 decreased OPG expression. Long-term exposure of IL-4 for 4 weeks resulted in transformation of CASMC towards an osteoblastic phenotype, based on the expression of the transcription factor Cbfa1 and increased mineral deposition. Notably, calcification of CASMC was inhibited by gene silencing of Cbfa1. During osteogenic transformation, IL-4 down-regulated OPG production in CASMC. IL-4 has differential effects in CASMC: While short-term exposure enhances OPG production through a STAT6-dependent mechanism, long-term exposure causes Cbfa1-dependent osteogenic transformation and a decreased production of OPG, an inhibitor of bone resorption.
Bone Resorption↗
Fibronectin binding proteins contribute to the adherence of Staphylococcus aureus to intact endothelium in vivo.
Staphylococcal adhesins mediate attachment to matrix proteins and endothelial cells in vitro, yet, their role in primary adherence to the physiologic vessel wall has not been studied in vivo, and complex endocarditis models yielded ambiguous results. Recently, we developed a hamster model to study interaction kinetics of S. aureus with intact microvasculature using intravital fluorescence microscopy (Laschke et al. J Infect Dis 2005; 191: 435-443) providing the basis for this study. S. aureus Cowan 1 wild type (WT) log phase cells adhered to postcapillary venules to a significantly larger extent compared to stationary phase staphylococci, a finding in congruence with the fact that the staphylococcal adhesin repertoire largely depends on the growth phase. In comparison, the adherence rate of the fnbA deleted mutant (DU5895) to the vessel wall was significantly reduced to approximately 40% of WT. These DU5895 attachment rates were similar to those of an S. carnosus strain (TM300). In contrast, upon heterologous complementation of TM300 with either fnbA and fnbB, adherence of these transformants to the microvasculature increased, an increase found to be significant for fnbA transformant single cocci and clusters at 30 and 60 min when compared to S. carnosus TM300 WT. In conclusion, these results demonstrate that staphylococcal FnBPs significantly contribute to primary interaction with intact endothelium under physiologic conditions. Accordingly, this attribution of staphylococcal FnBPs provide a rationale for novel intervention strategies such as the use of anti-FnBP antibodies in endovascular S. aureus disease.
Adhesins, Bacterial↗
The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.
Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.
Animals↗
Functional characterisation of autoantibodies from patients with pediatric opsoclonus-myoclonus-syndrome.
Paraneoplastic opsoclonus-myoclonus-syndrome (OMS) both in children and adults is suspected to be the result of an autoimmune response directed against cross-reactive proteins of tumor and neuronal cells. We here characterised the binding and functional activities of anti-neuroblastoma antibodies in IgG fractions from 11 OMS children with and without neuroblastoma. IgG fractions from neuroblastoma without OMS (NB) and healthy children served as controls. Indirect immunofluorescence and Western blot revealed IgG binding to intracellular autoantigens in all OMS patients, but in only one of the controls (p<0.001). Using flow cytometry, we could demonstrate surface binding of IgG fractions in all OMS patients, but only in one of control (p<0.001). Moreover OMS IgG exhibited a significant anti-proliferative and a cytotoxic effect on neuroblastoma cells compared to control IgG (p<0.001 and p<0.01). TUNEL assay revealed increased apoptotic cell death of the neuroblastoma cells after exposure to OMS IgG, but not to NB or control IgG (p<0.01). Preabsorption of membrane binding abandoned the anti-proliferative effect of OMS IgG. These findings indicate that surface-binding autoantibodies are present in OMS patients and these autoantibodies cause inhibition of cell proliferation and induce apoptosis.
Apoptosis↗
The role of junctional adhesion molecule-C (JAM-C) in oxidized LDL-mediated leukocyte recruitment.
The junctional adhesion molecule-C (JAM-C) was recently shown to be a counter receptor for the leukocyte beta2-integrin Mac-1 (CD11b/CD18), thereby mediating interactions between vascular cells, particularly in inflammatory cell recruitment. Here, we investigated the role of JAM-C in oxidized low-density lipoprotein (LDL)-mediated leukocyte recruitment. As compared with normal arteries, immunostaining of atherosclerotic vessels revealed a high expression of JAM-C in association with neointimal smooth muscle cells and the endothelium. Moreover, JAM-C was strongly up-regulated in the spontaneous early lesions in ApoE -/- mice. In vitro, cultured human arterial smooth muscle cells (HASMC) were found to express JAM-C, and oxLDL, as well as enzymatically modified LDL (eLDL) significantly up-regulated JAM-C on both HASMC and endothelial cells in a time- and dose-dependent manner. Although under quiescent conditions, JAM-C predominantly localized to interendothelial cell-cell contacts in close proximity to zonula occludens-1 (ZO-1), oxLDL treatment induced a disorganization of JAM-C localization that was no more restricted to the interendothelial junctions. JAM-C thereby mediated both leukocyte adhesion and leukocyte transendothelial migration upon oxLDL treatment of endothelial cells, whereas JAM-C on quiescent endothelial cells only mediates leukocyte transmigration. Thus, upon oxLDL stimulation endothelial JAM-C functions as both an adhesion, as well as a transmigration receptor for leukocytes. Taken together, JAM-C is up-regulated by oxLDL and may thereby contribute to increased inflammatory cell recruitment during atherosclerosis. JAM-C may therefore provide a novel molecular target for antagonizing interactions between vascular cells in atherosclerosis.
Aged↗
Inhibition of pathologic retinal neovascularization by alpha-defensins.
Proliferative retinopathies, such as those complicating prematurity and diabetes, are major causes of blindness. A prominent feature of these retinopathies is excessive neovascularization, which is orchestrated by the hypoxia-induced vascular endothelial growth factor (VEGF) stimulating endothelial cells and the integrin-mediated adhesive interactions of endothelial cells with extracellular matrix components such as fibronectin (FN). Recently, we demonstrated that alpha-defensins interfere with alpha5beta1-FN interactions and dependent endothelial cell functions. Here, alpha-defensins were studied in hypoxia-induced proliferative retinopathy. In vitro, alpha-defensins specifically inhibited alpha5beta1-integrin-dependent migration of bovine retinal endothelial cells (BRECs) to FN, attenuated the VEGF-stimulated increase in endothelial permeability, and blocked BREC proliferation and capillary sprout formation in 3-dimensional fibrin-matrices. An up-regulation of beta1-integrin and FN was observed in the retinal vessels in the mouse model of hypoxia-induced retinal angiogenesis. Systemic and local administration of alpha-defensins reduced retinal neovascularization by 45% and 60%, respectively, and this effect was comparable to the inhibitory effect of alpha5beta1-blocking antibody. alpha-Defensins were detected in human diabetic retinas associated with normal retinal vessels but were absent from proliferative lesions. Together, these data show that alpha-defensins inhibit pathologic retinal neovascularization in vivo and may provide a clinically efficient strategy against proliferative retinopathies.
Animals↗
Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP).
FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K(D) estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.
Animals↗