Subendothelial Matrix Components Research Articles

Thrombotic Regulation From the Endothelial Cell Perspectives.

Thrombosis, the localized clotting of blood that affects arterial or venous circulation, is one of the leading causes of death worldwide.1 Currently used antithrombotic drugs have increased bleeding risk.2,3 Exploring new antithrombotic strategies that preserve physiological hemostasis is under intensive investigation. Classical triad of causative factors leading to thrombosis includes alterations in blood content, alterations in blood flow, and alterations in the vessel wall. Endothelium, the inner most single layer of cells lining the blood vessels, provides a surface for thrombosis formation and critically regulates blood fluidity and homeostasis. As barrier, endothelium separates blood clotting factors from exposure to subendothelial prothrombotic extracellular matrix components. Endothelium also secretes or expresses vasoactive factors that modulate platelet reactivity, coagulation, fibrinolysis, and vascular contractility, all of which contribute to thrombotic formation. Such factors include nitric oxide, prostacyclin, Von Willebrand factor (VWF), thrombomodulin, endothelin, etc. Accumulating evidences show that endothelial cells (ECs) play a pivotal role in modulating thrombosis, highlighting ECs as a potential target for thrombosis control. In this Recent Highlights, we reviewed recent publications in Arteriosclerosis, Thrombosis, and Vascular Biology and other leading journals that are focused on the mechanisms of endothelial regulation of thrombosis and discussed their merits in therapeutic discovery. Arterial thrombosis is commonly initiated by vascular endothelial erosions and ruptures of an atherosclerotic plaque3,4 while venous thrombosis mainly stems from blood stasis.5 Despite these differences, platelet adhesion/activation, and fibrin deposition as the result of coagulation constitute the fundamental processes of thrombus formation.6,7 Platelet activation occurs when they interact with activated ECs (with increased VWF release and selectin expression) via glycoprotein Ib-V-IX complex binding to VWF, or when they expose to subendothelial extracellular matrix components, such as collagen (via glycoprotein VI receptor), in the cases of endothelial injury or plaques …

Utility of the von Willebrand factor collagen binding assay in the diagnosis of von Willebrand disease.

von Willebrand Disease (VWD) is the most common inherited bleeding disorder and also arises as an acquired defect (AVWS). VWD and AVWS are due to quantitative deficiencies and/or qualitative defects in von Willebrand factor (VWF), an adhesive plasma protein with multiple activities. Diagnosis of VWD is problematic, being subject to overdiagnosis, underdiagnosis, and misdiagnosis. This is largely due to limitations in current test procedures and an over-reliance on these imperfect test systems for clinical diagnosis. VWF essentially acts to assist in the formation of a platelet thrombus to stop blood loss from sites of injury, achieving this by binding to platelets (primarily through the glycoprotein Ib receptor), binding to subendothelial matrix components (primarily collagen), and binding to factor VIII (FVIII), thus protecting FVIII from degradation and enabling its delivery to sites of vascular injury. VWD is classified into six separate types, which may each be differentially managed therapeutically, and this underscores the importance of a correct diagnosis. The current report concisely reviews the utility of a relatively underutilised assay, the VWF collagen binding assay (VWF:CB), in facilitating the correct diagnosis and typing of VWD. In particular, if laboratories do not utilise the VWF:CB, then (i) type 2M VWD will continue to be missed, and/or misdiagnosed as types 2A or 1 VWD, and (ii) types 2A, 2B and PT-VWD will continue to be missed, or else be misdiagnosed as type 1 VWD or ITP. Am. J. Hematol. 92:114-118, 2017. © 2016 Wiley Periodicals, Inc.

Towards personalised therapy for von Willebrand disease: a future role for recombinant products.

von Willebrand disease (VWD) is reportedly the most common bleeding disorder and is caused by deficiencies and/or defects in the adhesive plasma protein von Willebrand factor (VWF). Functionally, normal VWF prevents bleeding by promoting both primary and secondary haemostasis. In respect to primary haemostasis, VWF binds to both platelets and sub-endothelial matrix components, especially collagen, to anchor platelets to damaged vascular tissue and promote thrombus formation. VWF also stabilises and protects factor VIII in the circulation, delivering FVIII to the site of injury, which then facilitates secondary haemostasis and fibrin formation/thrombus stabilisation. As a result of this, patients with VWD suffer a bleeding diathesis reflective of a primary defect caused by defective/deficient VWF, which in some patients is compounded by a reduction in FVIII. Management of VWD, therefore, chiefly entails replacement of VWF, and sometimes also FVIII, to protect against bleeding. The current report principally focuses on the future potential for "personalised" management of VWD, given the emerging options in recombinant therapies. Recombinant VWF has been developed and is undergoing clinical trials, and this promising therapy may soon change the way in which VWD is managed. In particular, we can envisage a personalised treatment approach using recombinant VWF, with or without recombinant FVIII, depending on the type of VWD, the extent of deficiencies, and the period and duration of treatment.

Abstract SY28-04: Targeting the tumor extracellular matrix with armed antibodies

Abstract Antibodies can be used to deliver bioactive molecules (drugs, cytokines, photosensitizers, radionuclides, etc.) to the tumor environment, thus sparing normal tissues. The targeting of modified sub-endothelial extracellular matrix components using armed antibodies is particularly attractive, because of: (i) the abundance and stability of some of these antigens (e.g., splice isoforms of fibronectin and tenascin-C); (ii) the dependence of cancer on new blood vessels (iii) accessibility of these structures from the blood-stream (iv) the fact that some of these antigens are very aboundant in many different cancer types, while being virtually undetectable in most normal adult tissues. Advanced preclinical and clinical data on armed antibodies will be presented in this lecture.

Platelets guide lymphocytes to vascular injury sites

Insight on Spectre et al. Thromb Haemost 2012; 108: 328-337. In this issue of Thrombosis and Haemo stasis, Spectre et al. (1) demonstrate that (activated) platelets promote lymphocyte adhesion to extracellular matrix components under arterial flow conditions, thereby further connecting thrombotic processes to inflammation and immunity. At sites of vascular injury, platelets adhere and aggregate on exposed subendothelial extracellular matrix (ECM) components and thereby form a plug that seals the wound or eventually occludes a diseased vessel, e.g. at sites of atherosclerotic plaque rupture (2). Besides this essential role in haemostasis and thrombosis, platelets are increasingly recognised to also orchestrate inflammatory processes, a combination of events best described by the term “thrombo-inflammation” (3). Several studies have shown that platelets can directly interact with lymphocytes and that this interaction is important for the modulation of adaptive immunity (3). However, lymphocytes are also abundantly found in atherosclerotic plaques and it is not clear whether they are recruited there by direct interactions with exposed plaque ECM components or through a plateletdependent mechanism. Spectre and coworkers now show in a series of elegant experiments that platelets are essentially required to support lymphocyte adhesion to different ECM components under arterial flow conditions ex vivo (500 s-1). Interestingly, the nature of the adhesive substrate largely determined the ability of the attached platelets to recruit specific lymphocyte subsets. For example, while platelets attached to collagen or fibrinogen, but not von Willebrand factor (vWF) efficiently recruited CD4+ and CD8+ T cells, all three substrates primed platelets to recruit NK cells in this system. The authors further provide evidence that the differential expression of adhesion molecules, most notably PSGL1 (CD162) and Mac-1, on different lymphocyte subpopulations, which interact with P-selectin and GPIb on platelets, respectively, may contribute to this selectivity, but further studies will be required to address this in more detail. Together, these observations provide a possible mechanism how platelets promote chronic inflammatory diseases such as atherogenesis, and may also serve as a basis for a better understanding of acute “thrombo-inflammatory“ disease states, such as ischaemic stroke where platelets and T cells have been identified as essential pathogenic factors but their mechanistic link has not been identified (3).

Platelet glycoprotein Ibα is an important mediator of ischemic stroke in mice

BackgroundPlatelets play an important role in ischemic stroke. GPIbα is a major platelet receptor that is critical for platelet adhesion to exposed subendothelial matrix components at sites of vascular damage.MethodsIn this study, we used transgenic mice in which the extracellular part of GPIbα is replaced by human interleukin 4-receptor (GPIbα/IL4Rα). We observed normal brain vasculature in these mice. We compared infarct size in GPIbα/IL4Rα and wild-type (WT) mice 23 hours after 1-hour transient middle cerebral artery occlusion (tMCAO). In addition, the functional outcome was evaluated using a modified Bederson score.ResultsWe found a significantly smaller infarct size in GPIbα/IL4Rα mice compared to WT mice (38.0 ± 6.5 mm3 vs. 74.2 ± 8.6 mm3, p < 0.001). The decrease in infarct size was functionally relevant as indicated by a significantly better functional Bederson score in GPIbα/IL4Rα mice compared to WT animals (1.3 ± 0.4 vs. 2.7 ± 0.3, p < 0.05).ConclusionsOur data illustrate and further confirm the important role of platelet GPIbα in ischemic stroke, suggesting that targeted inhibition of this receptor may open new avenues in stroke treatment.

PWE-069 Colorectal cancer lymphatics display an altered phenotype associated with lymph node metastasis

<h3>Introduction</h3> Tumour cell metastasis to lymph nodes is a frequent clinical feature of colorectal cancer and often heralds a poor clinical outcome. Although tumoural lymphangiogenesis is assumed to contribute to this process there is little evidence to support any notion of a simple correlation between lymphatic vessel density and colorectal cancer metastasis. Growing evidence of complex functional and biochemical interactions between tumour cells and the tumour microvasculature imply that tumoural lymphatics may possess specific phenotypes capable of facilitating tumour cell entry and spread to draining lymph nodes. <h3>Methods</h3> We compared the RNA profile of primary lymphatic endothelial cells isolated from tumour and normal tissue using the lymph node metastasising murine T-241/VEGF-C fibrosarcoma model. These results were validated immunohistochemically in several human cancers, including colorectal cancer. The prognostic significance of two differentially expressed genes on tumoural lymphatics was assessed by immunofluorescence microscopy on a panel of mixed stage colorectal cancer tissues. <h3>Results</h3> We reveal significant differences in the expression of some 792 genes between normal and tumour lymphatic endothelial cells (ie, &gt;2× fold up/downregulation). These include genes that code for a variety of proteins including components of endothelial junctions, subendothelial matrix, and vessel growth/patterning. We investigated two of these differentially expressed genes in colorectal cancer; Endothelial Specific Adhesion Molecule (ESAM), a tight junction regulatory protein, and Endoglin (CD105), the transforming growth factor-β coreceptor. We reveal that ESAM and Endoglin are significantly upregulated on the lymphatics of colorectal cancer relative to normal tissue, with quantitative analysis of lymphatic vessel staining revealing a dramatic association between the incidence of ESAM positive tumour vessels and the presence of lymph node metastasis. <h3>Conclusion</h3> These findings reveal a remarkable degree of phenotypic plasticity in cancer lymphatics and provide new insight into the processes of lymphatic invasion and lymph node metastasis. The detailed characterisation of such qualitative changes may allow the development of better prognostic indicators in colorectal cancer and enlighten the search for improved anti-metastatic therapies.

The complexity of platelet metabolism and its contribution to atherothrombosis

Platelet functions are multiple, complex and not limited to haemostasis. In fact, platelets play a relevant role in vascular inflammation and atherosclerosis (ATS). In the presence of vascular lesions or inflammation, endothelial denudation or activation triggers mechanisms that render the circulating platelets adhesive for the vascular wall. Endothelial lesions expose subendothelial matrix components, such as collagen, von Willebrand factor, fibronectin and other adhesive proteins. Platelet adhesion depends on the interaction between these components and platelet receptors (mainly glycoprotein (GP) VI and GPIb-IX-V). Adhesion triggers the platelet release of inflammatory and mitogenic substances that alter the thromboresistant endothelial surface, enhance the chemoattraction of leukocytes, stimulate smooth muscle cell proliferation and contribute to matrix degradation. Finally, GPIIb- IIIa receptors are activated, leading to firm platelet aggregation and thrombus formation. Platelets participate in the formation of mural thrombi in the late stages of atherosclerotic disease, but also adhere to endothelial cells during the earlier stages of atherosclerotic plaque development. Moreover, platelets exert important functions in modulating inflammatory and immune processes. An improved comprehension of the complex platelet pathophysiology could suggest new therapeutic strategies to reduce the impact of atherosclerotic disease.

Platelets interact with bacterial pathogens

Thromb Haemost 2009; 102: 613–614 The predominant role of platelets is in haemostasis. Resting platelets patrol the vasculature and become activated in response to endothelial cell damage and exposure to subendothelial matrix components, such as collagen or von Willebrand factor. Adhesion and activation result in the release of the contents of platelet granules, and in the upregulation of surface receptors for fibrinogen/fibrin and other plasma proteins. This allows firm binding between platelets to occur, resulting in aggregation and formation of platelet thrombi. Platelets may also contribute to the immune response to endovascular bacterial pathogens by releasing pro-inflammatory factors including cytokines, chemokines, and antimicrobial peptides (1). Conflicting reports exist on the role of platelets during bacterial infection. On the one hand the production of antimicrobial peptides will kill bacteria, and the release of pro-inflammatory substances and the formation of platelet-leukocyte aggregates may prime the immune response. On the other hand, the ability to bind platelets may facilitate bacterial adherence to the endothelium, and bacteria contained within a platelet plug may be protected from immune defence. In order to initiate an infection, bacteria must adhere to and multiply at normally sterile sites within the host. Many bacterial pathogens bind to host matrix proteins such as fibrinogen/fibrin, collagen, or von Willebrand factor, and since many of these host proteins are also associated with activated platelets, the platelets may represent an abundant adhesive target for bacteria. Furthermore, a number of significant Gram-positive endovascular pathogens have been shown to directly stimulate platelet activation in vitro in the absence of other agonists (2). The most well characterised interactions occur between platelets and the bacteria that cause endocarditis: Staphylococcus aureus, Streptococcus sanguis and other streptococcal species. During endocarditis, bacteria adhere to the heart valves, and a vegetation of bacteria, fibrin and platelets is formed; thereby, the ability of bacteria to activate or adhere to platelets is considered to contribute to the formation of vegetations. A number of endocarditis pathogens have been reported to bind either directly to the platelet surface or indirectly via a plasma protein bridge. Binding in itself does not mediate activation, and plasma immunoglobulin G specific for the bacterium is also required to mediate platelet activation through the Fc-receptors on the platelet surface (2). As previously mentioned, activated platelets release antibacterial peptides, which could kill the bacteria. Interestingly, it has been shown that many of the S. aureus strains isolated from endocarditis are resistant to these antibacterial peptides (3, 4). To date, platelet interactions have been mainly studied for the causative agents of endocarditis, and the results obtained may be specific for this infection. In order to elucidate the role of platelets during infection, it is important that platelet interactions are investigated for other bacterial pathogens. In a study published in this edition of Thrombosis & Haemostasis, Niemann et al have investigated the interaction between platelets and the significant human pathogen, Streptococcus pneumoniae (5). Infection with S. pneumoniae is not a significant cause of endocarditis but is a leading cause of respiratory tract infections and may cause invasive infection and septicaemia. Niemann et al report that S. pneumoniae can bind to activated platelets, and this binding is mediated by platelet bound fibrin and further stimulated by thrombospondin released from activated platelets. Platelet activation in the absence of fibrin generation, for example collagen stimulation, does not stimulate bacteria-platelet associations. Niemann et al have previously described a similar interaction of S. aureus with activated platelets and this implies that the mechanism may be conserved for significant Gram-positive pathogens (6). As previously mentioned, S. aureus can also mediate platelet activation in the absence of other agonists. The authors did not investigate the ability of S. pneumoniae to stimulate platelet activation in the absence of other agonists, and it will be interesting to determine if this significant human pathogen has, like S. aureus, multiple mechanisms to interact with platelets. The ability to adhere to activated platelets on the damaged endothelium may provide an advantage for the bacteria by protecting against killing by immune cells, which may not recognise bacterial pathogens shielded by host proteins. Alternatively, bacteria attached to a platelet aggregate may also be able to disseminate via the bloodstream to other foci. Indeed, bacteria-platelet aggregates can become sequestered in the microvasculature, contributing to the organ damage observed in septicaemia and sepsis. The contribution of thrombospondin to these bacteria-platelet associations is interesting since thrombospondin has previously been reported to bind to bacterial peptidoglycan (7). Pep© 2009 Schattauer GmbH, Stuttgart

A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., >or=2-fold up- or down-regulated, P <or= 0.05) that code for a variety of proteins including components of endothelial junctions, subendothelial matrix, and vessel growth/patterning. The tumor LEC profile, validated by immunohistochemical staining, is distinct from that of normal, inflammatory cytokine, or mitogen-activated LEC, characterized by elevated expression of such functionally significant molecules as the tight junction regulatory protein endothelial specific adhesion molecule (ESAM), the transforming growth factor-beta coreceptor Endoglin (CD105), the angiogenesis-associated leptin receptor, and the immunoinhibitory receptor CD200, and reduced expression of subendothelial matrix proteins including collagens, fibrillin, and biglycan. Moreover, we show similar induction of ESAM, Endoglin, and leptin receptor within tumor lymphatics in a series of human head and neck and colorectal carcinomas, and uncover a dramatic correlation between ESAM expression and nodal metastasis that identifies this marker as a possible prognostic indicator. These findings reveal a remarkable degree of phenotypic plasticity in cancer lymphatics and provide new insight into the processes of lymphatic invasion and lymph node metastasis.

Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.

Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes

Phosphatidylserine (PS)-positive erythrocytes adhere to endothelium and subendothelial matrix components. While thrombospondin mediates these inter-actions, it is unknown whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control HbAA erythrocytes, we demonstrate that PS-positive erythrocytes adhered to human lung microendothelial cells in the absence of plasma ligands, that this adhesion was enhanced following endothelial activation with IL-1alpha, TNF-alpha, LPS, hypoxia, and heme, and that this adhesive interaction was selective to erythrocyte PS. We next explored whether microendothelial cells express an adhesion receptor that recognizes cell surface-expressed PS (PSR) similar to that expressed on activated macrophages. We demonstrate constitutive expression of both PSR mRNA and protein that were up-regulated in a time-dependent manner following endothelial activation. While minimal PSR expression was noted on unstimulated cells, endothelial activation up-regulated PSR surface expression. In antibody-blocking studies, using PS-positive erythrocytes generated either artificially via ionophore treatment of control erythrocytes or from patients with sickle cell disease, we demonstrate that PSR was functional, supporting PS-mediated erythrocyte adhesion to activated endothelium. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the microendothelium that is up-regulated by such pathologically relevant agonists as hypoxia, cytokines, and heme.

Microvascular Endothelial Cells Express Phosphatidylserine Receptor: A Functionally Active Receptor for Phosphatidylserine-Positive Erythrocytes.

Phosphatidylserine (PS)-positive erythrocytes adhere to both endothelial cells and the sub-endothelial matrix components. While thrombospondin mediates these interactions, it is not known whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control erythrocytes, we demonstrate that PS-positive erythrocytes adhered directly to human lung micro-endothelial cells in the absence of plasma ligands, that this adhesion was further enhanced following endothelial activation with IL-1α, TNF-α, LPS, and hypoxia (2.5- to 8-fold increase), and that this adhesive interaction was selective to erythrocyte-PS. We next explored whether micro-endothelial cells express an adhesion receptor that recognizes cell surface expressed PS (PSR) similar to that expressed on activated macrophages. Using RT-PCR and Western blotting, we demonstrate constitutive expression of both PSR mRNA and protein which were up-regulated in a time-dependent manner following endothelial activation (with maximal increases of 3-fold in mRNA and 2-fold in protein noted at 4 and 6 hour, respectively). While minimal PSR expression was noted on un-stimulated cell surface (8% positivity), endothelial activation up-regulated surface expression of this receptor (35–40% positivity in IL-1α and TNF-α activated cultures). In antibody blocking studies, using both artificially generated PS-positive erythrocytes and also using PS-positive erythrocytes from patient with sickle cell disease, we demonstrate that PSR was functionally active supporting PS-mediated erythrocyte adhesion to activated endothelial cells. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the micro-endothelium which is up-regulated by such pathologically relevant agonists as hypoxia and cytokines.

CXCR4 chemokine receptor engagement modifies integrin dependent adhesion of renal carcinoma cells

The mechanisms leading to renal cell carcinoma (RCC) metastasis are incompletely understood. Although evidence shows that the chemokine receptor CXCR4 and its ligand CXCL12 may regulate tumor dissemination, their role in RCC is not clearly defined. We examined CXCR4 expression and functionality on RCC cell lines, and explored CXCL12-triggered tumor adhesion to human endothelium (HUVEC) or extracellular matrix proteins. Functional CXCR4 was expressed on A498 tumor cells, enabling them to migrate towards a CXCL12 gradient. CXCR4 engagement by CXCL12 induced elevated cell adhesion to HUVEC, to immobilized fibronectin, laminin or collagen. Anti-CXCR4 antibodies or CXCR4 knock down by siRNA applied prior to CXCL12 stimulation impaired CXCL12-triggered tumor adhesion. However, blocking CXCR4 subsequent to CXCL12 stimulation did not. This pointed to an indirect control of tumor cell adhesion by CXCR4. In fact, CXCR4 engagement by CXCL12 also induced alterations of receptors of the integrin family, notably alpha3, alpha5, beta1 and beta3 subunits, and blocking beta1 integrins with a function-blocking antibody prevented CXCL12-induced A498 adhesion. Focal adhesion kinase (total and activated) and integrin-linked kinase significantly increased in CXCL12-treated A498 cells, accompanied by a distinct up-regulation of ERK1/2, JNK and p38 phosphorylation. Therefore, CXCR4 may be crucial in controlling adhesion of A498 cells via cross talking with integrin receptors. These data show that CXCR4 receptors contribute to RCC dissemination and may provide a novel link between CXCR4 chemokine receptor expression and integrin triggered RCC adhesion to the vascular wall and subendothelial matrix components.

Phosphatidylserine (PS)-Positive Erythrocytes Bind to Both Immobilized and Soluble Thrombospondin-1 (TSP-1) Via Its Heparin Binding Domain.

Phosphatidylserine (PS)-dependent erythrocyte adhesion to both cultured endothelial cells and the components of sub-endothelial matrix appears to be mediated in part via thrombospondin-1 (TSP). While TSP exhibits multiple cell-binding domains, the PS-binding site on TSP has not been identified. Since a cell-binding domain for anionic heparin is located at the amino-terminal domain of TSP, we hypothesized that anionic PS-positive (PS+ve) red cells bind to this domain. In a recent preliminary study, using a flow adhesion system and PS+ve erythrocytes (prepared by treating control AA red cells with A23187), we have demonstrated that heparin inhibited PS+ve erythrocyte adhesion to immobilized TSP in a concentration-dependent manner with 58 to 77% inhibition noted at concentrations between 1 and 50 U/ml (n=9, P<0.001). Other anionic polysaccharides including high molecular weight dextran sulfate and chondroitin sulfate A also inhibited PS+ve erythrocyte adhesion to immobilized TSP with the magnitude of the inhibitory effects comparable to heparin. These results suggested that the heparin-binding domain on TSP may be involved in PS-mediated red cell adhesion to immobilized TSP. We have extended these studies to characterize the PS-binding site on TSP using monoclonal antibodies directed against specific cell-binding domains on the molecule and also using specific TSP peptides. We demonstrate that pre-incubation of immobilized TSP with an antibody directed against the heparin-binding domain on TSP (TSP-Ab9, Lab Vision) blocked PS-mediated red cell adhesion to immobilized TSP (80% inhibition compared to an isotype-matched negative control antibody, n=7, P<0.001), whereas an antibody that recognizes the collagen-binding domain on TSP (TSP-Ab4) did not affect this process. In addition, incubation of PS+ve erythrocytes with a TSP peptide containing the specific heparin-binding motif, KKTRG, inhibited PS-mediated red cell adhesion by 55% (P<0.001, n=6), whereas a peptide lacking the binding motif had no effect. Since protein confirmation of immobilized TSP appears to be different from that of soluble TSP, we next investigated whether soluble TSP, like immobilized TSP, also interacted with PS+ve erythrocytes. Erythrocytes containing 8 to 10% PS+ve cells were incubated in the absence or the presence of increasing concentrations of soluble TSP (0.1 to 10 μg/ml), and then analyzed by flow cytometry for surface bound TSP using both adhesion blocking (TSP-Ab9) and non-blocking (TSP-Ab4) anti-TSP antibodies. We demonstrate that soluble TSP binds to PS+ve erythrocytes in a concentration-dependent manner with 3 to 11% TSP-positive (TSP+ve) red cells measured at soluble TSP concentrations between 1 to 10 μg/ml (n=4). In addition, TSP binding could be detected only with the non-adhesion blocking antibody TSP-Ab4, which recognizes the collagen-binding domain on TSP. The adhesion blocking antibody TSP-Ab9 that interacts with the heparin binding domain, failed to detect any TSP+ve red cells. No TSP+ve erythrocytes were detected when PS-negative control red cells were evaluated in binding assays. In parallel adhesion experiments, soluble TSP inhibited PS+ve erythrocyte adhesion to immobilized TSP at concentrations at which significant TSP binding to erythrocytes occurred (43% and 44% inhibition at 5 and 10 μg of soluble TSP per ml, n=4). These results conclusively demonstrate that PS-positive erythrocytes interact with both immobilized and fluid phase TSP through the heparin-binding domain, and that heparin blocks this interaction.

A novel hyaluronan-based biomaterial (Hyaff-11 ®) as a scaffold for endothelial cells in tissue engineered vascular grafts

Current prosthetic small diameter vascular grafts show poor long-term patency rates, leading to the pursuit of a biological alternative. Hyaff-11 is a hyaluronan-based biodegradable polymer developed for tissue-engineering applications. This study aimed to determine whether human vascular endothelial cells attach to Hyaff-11 scaffolds and produce a subendothelial matrix. Two forms of fibrous, non-woven Hyaff-11 scaffolds: unpressed and pressed felts, were analysed. Attachment of human venous endothelial cells was investigated after 1, 5, 10 and 20 days in culture using SEM and confocal microscopy. The deposition of subendothelial matrix components was investigated by immunofluorescent staining. We demonstrate that endothelial cells adhere to the individual fibres of both unpressed and pressed scaffolds: with a seeding density of 1×10 6 cells/cm 2, 94% of the cells attached to Hyaff-11 fibres after 24 h. The pressed material provided the best environment for cell growth, allowing the formation of a complete endothelial monolayer after 20 days. Furthermore, endothelial cells on Hyaff-11 pressed felts deposited an organised subendothelial matrix containing laminin, fibronectin, type IV and type VIII collagen. This work indicates Hyaff-11 based biopolymers as suitable scaffolds to promote endothelialisation within the next generation of vascular grafts.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Monocyte–macrophage differentiation in three dimensional collagen lattice

Human peripheral blood mononuclear cells (PBMC) upon transendothelial migration interact with subendothelial matrix components and differentiate into macrophages. In order to study whether the shape of the cells as dictated by the extracellular matrix can influence monocyte–macrophage (mo–mφ) differentiation, human PBMC were maintained in vitro on a three dimensional collagen I (COL I) lattice and studied for various macrophage specific functions, viz. endocytosis of [ 125I]acetyl bovine serum albumin (BSA), expression of specific cell surface antigens and expression of matrix metalloproteinases (MMPs). The cells maintained in three dimensional COL gel exhibited a higher rate of endocytosis of [ 125I]acetyl BSA than those on COL-coated plastic. FACS analysis showed that the mean fluorescence intensity (MFI) corresponding to monocyte specific LPS receptor CD14 was significantly decreased while MFI corresponding to macrophage specific transferrin receptor CD71 was significantly increased in cells maintained in vitro on three dimensional COL gel compared to two dimensional COL substrata. Expression of macrophage specific MMPs (gelatinase A and gelatinase B) was significantly high in cells maintained on COL gel than on COL I-coated plastic. Appearance of 67 kDa gelatinase in the COL gel suggested that induction as well as activation of MMPs occur when cells are maintained in a three dimensional environment. These results indicate that monocytes undergo a rapid rate of differentiation when maintained in vitro on three dimensional COL I lattice suggesting that apart from the chemical nature of the matrix, the shape of the cells as provided by the matrix also influences mo–mφ differentiation.

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix

Among potential virulence factors of Candida albicans, enzymes seem to play an important role. Many studies concern the secreted aspartic proteinases (saps), and the degradation of some components of the subendothelial extracellular matrix by the isoenzyme sap2 has been proved. Nevertheless, other proteolytic enzymes could be involved in the pathogenicity of the yeast. We studied the degradation of four constitutive proteins of the extracellular matrix: type I and IV collagens, laminin and fibronectin, by a 95-kDa metallopeptidase, localised in the cell wall of C. albicans. Each of these constituents was incubated with the purified enzyme and its degradation products analysed by an electrophoretic method. We observed that type I collagen and fibronectin were totally degraded by the enzyme whereas type IV collagen and laminin were only partially degraded. The C. albicans metallopeptidase may play a role in the degradation of the subendothelial extracellular matrix components. This enzyme could facilitate the migration of the yeast in the tissues after crossing the endothelial layer, allowing the fungal invasion of target organs.

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix.

Among potential virulence factors of Candida albicans, enzymes seem to play an important role. Many studies concern the secreted aspartic proteinases (saps), and the degradation of some components of the subendothelial extracellular matrix by the isoenzyme sap2 has been proved. Nevertheless, other proteolytic enzymes could be involved in the pathogenicity of the yeast. We studied the degradation of four constitutive proteins of the extracellular matrix: type I and IV collagens, laminin and fibronectin, by a 95-kDa metallopeptidase, localised in the cell wall of C. albicans. Each of these constituents was incubated with the purified enzyme and its degradation products analysed by an electrophoretic method. We observed that type I collagen and fibronectin were totally degraded by the enzyme whereas type IV collagen and laminin were only partially degraded. The C. albicans metallopeptidase may play a role in the degradation of the subendothelial extracellular matrix components. This enzyme could facilitate the migration of the yeast in the tissues after crossing the endothelial layer, allowing the fungal invasion of target organs.