Subendothelial matrix as the playground for “diabetic milieu” factors in development of angiopathy

Human monocytes have been shown to penetrate the endothelial layer of large blood vessels and to adhere to the subendothelial basement membrane. To determine the active components of this process, we have studied the ability of monocytes to adhere to isolated components of the subendothelial matrix. Using a quantitative dot-blot adhesion assay, we find that monocytes adhere preferentially to immobilized laminin and elastin. The monocytes adhere less well to fibronectin and bind poorly or not at all to collagen types I and IV, or to heparan sulfate. Monocyte binding to elastin requires an intact, crosslinked molecule as no binding was observed to soluble, acid-alcohol elastin extracts, to pepsin or elastase digests of elastin, to tropoelastin monomer, or to desmosine/isodesmosine crosslinks. Similar binding profiles to elastin, laminin, and fibronectin were seen with the established human leukocyte cell line U937. The promyelocytic cell line HL60 adhered equally well to laminin but showed slightly reduced adhesion to elastin when compared with the fresh monocytes or U937 cells. Freshly isolated human erythrocytes did not demonstrate significant adhesion to fibronectin, laminin, or elastin.

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Collagen-dependent platelet dysfunction and its relevance to either mitochondrial ROS or cytosolic superoxide generation: a question about the quality and functional competence of long-stored platelets

10.21203/rs.3.rs-29731/v2 ◽

2020 ◽

Author(s):

Ehteramolsadat Hosseini ◽

Saba Hojjati ◽

Safoora Afzalniaye gashti ◽

Mehran Ghasemzadeh

Keyword(s):

Platelet Adhesion ◽

Oxidant Stress ◽

Ros Generation ◽

Reverse Correlation ◽

Platelet Dysfunction ◽

Mitochondrial Ros ◽

Ros Accumulation ◽

The Matrix ◽

Subendothelial Matrix ◽

Platelet Spreading

Abstract Background: Upon vascular damage, the exposed subendothelial matrix recruits circulating platelets to site of injury while inducing their firm adhesion mainly via GPVI-collagen interaction. GPVI also supports aggregatory and pro-coagulant functions in arterial shear rate even on the matrix other than collagen. Reactive oxygen species (ROS) modulate these stages of thrombosis; however augmented oxidant stress also disturbs platelet functions. Stored-dependent platelet lesion is associated with the increasing levels of ROS. Whether ROS accumulation is also relevant to collagen-dependent platelet dysfunction is the main interest of this study. Methods: Fresh PRP-PCs (platelet concentrates) were either stimulated with potent ROS-inducers PMA and CCCP or stored for 5 days. Intra-platelet superoxide (O2--) or mitochondrial-ROS and GPVI expression were detected by flowcytometery. GPVI shedding, platelet aggregation and spreading/adhesion to collagen were analyzed by western blot, aggregometry and fluorescence-microscopy respectively. Results: Mitochondrial-ROS levels in 5 days-stored PCs were comparable to those induced by mitochondrial uncoupler, CCCP while O2-- generations were higher than those achieved by PMA. Shedding levels in 5 days-stored PCs were higher than those induced by these potent stimuli. GPVI expressions were reduced comparably in CCCP treated and 5 days-stored PCs. Platelet adhesion was also diminished during storage while demonstrating significant reverse correlation with GPVI shedding. However, only firm adhesion (indicated by spreading or platelet adhesion surface area) was relevant to GPVI expression. Platelet adhesion and aggregation also showed reverse correlations with both O2-- and mitochondrial-ROS formations; nonetheless mitochondrial-ROS was only relevant to firm adhesion. Conclusion: As a sensitive indicator of platelet activation, GPVI shedding correlated with either simple adhesion or spreading to collagen, while GPVI expression was only relevant to platelet spreading. Thereby, if the aim of GPVI evaluation is to examine platelet firm adhesion, expression seems to be a more specific choice. Furthermore, the comparable levels of ROS generation in 5 days-stored PCs and CCCP treated platelets, indicated that these products are significantly affected by oxidative stress. Reverse correlation of accumulating ROS with collagen-dependent platelet dysfunction is also a striking sign of an oxidant-induced lesion that may raise serious question about the post-transfusion quality and competence of longer stored products.

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Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-2002-23488 ◽

2002 ◽

Vol 110 (02) ◽

pp. 67-73 ◽

Cited By ~ 9

Author(s):

J. Grulich-Henn ◽

J. Ritter ◽

S. Mesewinkel ◽

U. Heinrich ◽

M. Bettendorf ◽

...

Keyword(s):

Endothelial Cell ◽

Growth Factor ◽

Insulin Like Growth Factor ◽

Cell Monolayers ◽

Factor I ◽

Subendothelial Matrix ◽

Growth Factor I

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CD11/CD18-independent neutrophil adherence to laminin is mediated by the integrin VLA-6

Blood ◽

10.1182/blood.v79.6.1545.1545 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1545-1552 ◽

Cited By ~ 55

Author(s):

JF Bohnsack

Keyword(s):

Basement Membrane ◽

Critical Event ◽

Vascular Walls ◽

Subendothelial Matrix ◽

Neutrophil Adherence ◽

Human Pmns ◽

And Migration ◽

Coated Surfaces ◽

Reduced Adherence

Abstract Regulated adherence of polymorphonuclear leukocytes (PMNs) to endothelium and subendothelial matrix is a critical event for PMN localization at and migration into inflammatory sites. We previously reported that human PMNs stimulated in vitro adhere to laminin, the major glycoprotein of mammalian basement membrane, by both CD11/CD18 (beta 2 integrin)-dependent and CD11/CD18-independent mechanisms. This CD11/CD18-independent adherence is inhibited by monoclonal antibodies (MoAbs) directed against the beta 1 subunit of integrins (very late antigens [VLA]). The specific PMN VLA receptor responsible for stimulated CD11/CD18-independent PMN adherence to laminin was not elucidated. We show here that this CD11/CD18-independent adherence is mediated by a member of the beta 1 integrins, VLA-6. MoAbs GoH3 and 450– 30, which bind the alpha 6 subunit of VLA-6, significantly reduced adherence of phorbol myristate acetate-stimulated PMNs to laminin- coated surfaces when CD11/CD18-independent adherence was blocked with anti-CD11/CD18 MoAbs. Furthermore, GoH3 completely inhibited stimulated adherence of CD11/CD18-deficient PMNs to laminin. Analysis by flow cytometry showed that human PMNs express VLA-6. The PMN alpha 6 is identical in size and pl to the platelet alpha 6, but the PMN beta 1 exhibits considerable heterogeneity in molecular weight compared with the platelet beta 1. This activation-dependent adherence receptor for laminin may play a role in PMN interaction with basement membrane laminin during PMN movement through vascular walls.

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Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation

Blood ◽

10.1182/blood.v75.12.2335.2335 ◽

1990 ◽

Vol 75 (12) ◽

pp. 2335-2342 ◽

Cited By ~ 35

Author(s):

AP Kowalczyk ◽

RH Tulloh ◽

PJ McKeown-Longo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Binding Sites ◽

Affinity Binding ◽

Apical Surface ◽

Matrix Assembly ◽

High Affinity Binding ◽

High Affinity ◽

Fibronectin Matrix ◽

Subendothelial Matrix

Abstract Endothelial cells in vivo form the interface between the vascular and interstitial compartments and are strategically located to mediate vascular permeability and hemostasis. One mechanism endothelial cells use to maintain a nonthrombogenic surface is to polarize basement membrane constituents to the basolateral cell surface. In the present study, we began characterization of the mechanisms used by endothelial cells in the assembly of a subcellular fibronectin matrix. Immunofluorescence microscopy was used to localize extracellular matrix fibronectin in endothelial cell cultures. In contrast to preconfluent and newly confluent cultures, post-confluent cultures assembled a fibronectin matrix that was restricted to the basolateral cell surface. To determine if endothelial cells polarize fibronectin secretion, Millicell culture inserts were used to distinguish proteins secreted from apical and basal surfaces. Preconfluent and newly confluent cultures secreted fibronectin equally into apical and basal media. In contrast, post-confluent endothelial cells secreted fibronectin preferentially into the basal chamber. The degree to which fibronectin secretion was polarized varied with time in culture and with the ability of the monolayers to act as a barrier to the movement of 125I- fibronectin from the apical to basal chamber. In addition, high affinity binding sites for exogenous 125I-fibronectin were found to be present on the basolateral, but not apical, surface of post-confluent endothelial monolayers. These results indicate that subendothelial matrix assembly correlates with polarized fibronectin secretion, culture confluence, and expression of high affinity binding sites for fibronectin on the basolateral cell surface.

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Fibrinogen induces endothelial cell adhesion and spreading via the release of endogenous matrix proteins and the recruitment of more than one integrin receptor

Blood ◽

10.1182/blood.v75.7.1509.1509 ◽

1990 ◽

Vol 75 (7) ◽

pp. 1509-1517 ◽

Cited By ~ 55

Author(s):

E Dejana ◽

MG Lampugnani ◽

M Giorgi ◽

M Gaboli ◽

PC Marchisio

Keyword(s):

Endothelial Cell ◽

Matrix Protein ◽

Matrix Proteins ◽

Extracellular Matrix Component ◽

Integrin Receptor ◽

Complex Type ◽

Matrix Component ◽

Cytoskeletal Organization ◽

Subendothelial Matrix ◽

Alpha V Beta 3

Abstract We have previously shown that fibrinogen (fg) acts as a subendothelial matrix protein in promoting human endothelial cell (EC) adhesion and cytoskeletal organization. In this study we report that EC spreading on fg, at variance with other matrix proteins, requires endogenous matrix protein synthesis and secretion. ECs, upon seeding on fg, promptly released and organized a fibronectin (fn) matrix. Fg was more effective than vitronectin (vn) in promoting the deposition of this protein. ECs treated with monensin to block matrix protein secretion still adhered to fg but did not properly organize their cytoskeleton and adhesion structures. In contrast, monensin did not affect EC spreading either on vn or on fn. Using antibodies to the alpha and beta chains of fn (alpha 5 beta 1) and vn (alpha v beta 3) receptors, it was found that ECs adherent to fg show clustering and organization in adhesion structures of both type of receptors. A faint staining of adhesion structures with alpha 2 but not alpha 3 and alpha 6 antibodies was also observed. Antibodies either to vn or fn receptors were able to disrupt the EC monolayer and to induce EC retraction and detachment, thus indicating that both receptors are important in maintaining a sustained EC adhesion to fg. However, when ECs were treated with monensin only the vn receptor was organized in adhesion structures while the fn receptor was diffusely distributed. This suggests that clustering of the fn receptor is mediated by the release of endogenous matrix proteins induced by the exposure to fg. In conclusion, fg has a peculiar and complex type of interaction with ECs since it requires endogenous matrix protein release and the recruitment of more than one adhesive receptor. This suggests a specific way of response of ECs to each extracellular matrix component.

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Platelets selectively enhance lymphocyte adhesion on subendothelial matrix under arterial flow conditions

Thrombosis and Haemostasis ◽

10.1160/th12-02-0064 ◽

2012 ◽

Vol 108 (08) ◽

pp. 328-337 ◽

Cited By ~ 16

Author(s):

Maria Ersoy ◽

Paul Hjemdahl ◽

Naphtali Savion ◽

David Varon ◽

Galia Spectre ◽

...

Keyword(s):

Cell Adhesion ◽

B Cells ◽

Nk Cells ◽

Platelet Activation ◽

Platelet Adhesion ◽

Nk Cell ◽

Lymphocyte Adhesion ◽

Subendothelial Matrix ◽

Coated Surface ◽

Coated Surfaces

SummaryPlatelet adhesion at sites of cardiovascular injury may facilitate leukocyte deposition. We asked if and how platelets enhance lymphocyte adhesion on different subendothelial matrix protein (SEMP)-coated surface at arterial shear stress. Hirudinised whole blood was subjected to an arterial shear rate (500 s−1) in a Cone and Plate(let) analyser (CPA) for 5 minutes using plates coated with bovine serum albumin (BSA), collagen, fibrinogen, von Willebrand factor (vWF), or fibronectin. Platelet and lymphocyte adhesion were monitored by CPA and flow cytometry. Exposure of blood to collagen, fibrinogen, and vWF-coated surfaces induced platelet activation. The most marked effect was seen with collagen-coating, which markedly enhanced the adhesion of all lymphocyte subpopulations compared to BSA-coating. Fibrinogen-coating supported both T and NK cell adhesion, while vWF-coated surface only enhanced NK cell deposition. In contrast, fibronectin enhanced neither platelet activation nor lymphocyte adhesion. Moreover, platelets preferentially facilitated adhesion of large CD4+ and CD8+ T cells and NK cells, and of small B cells. Enhanced cell adhesion of larger lymphocytes was associated with elevated platelet conjugation and higher lymphocyte expression of PSGL-1, Mac-1, and CD40L. The enhancement of lymphocyte adhesion was totally platelet-dependent, and was abolished in platelet-depleted blood. Moreover, blockade of the platelet adhesion molecules P-selectin, GPIIb/IIIa, and CD40L attenuated platelet-dependent lymphocyte deposition. In conclusion, platelets support lymphocyte adhesion on SEMP-coated surfaces under arterial shear. The enhancement is selective for large T and NK cells and small B cells.

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Interaction of Thrombin-Stimulated Platelets with Vitronectin (S-Protein of Complement) Substrate: Inhibition by a Monoclonal Antibody to Glycoprotein IIb-IIIa Complex

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647002 ◽

1988 ◽

Vol 60 (03) ◽

pp. 514-517 ◽

Cited By ~ 11

Author(s):

Perumal Thiagarajan ◽

Kathleen Kelly

Keyword(s):

Synthetic Peptides ◽

Vessel Wall ◽

Divalent Cations ◽

Substrate Inhibition ◽

Adhesion Assay ◽

Dependent Manner ◽

Activated Platelets ◽

Subendothelial Matrix ◽

Dose Dependent ◽

Adhesion Of Platelets

SummaryPlatelets adhere to vitronectin substrate following activation with physiological concentrations of thrombin. Adhesion of activated platelets to vitronectin substrate is dependent upon the presence of divalent cations, the amount of vitronectin, and the duration of adhesion assay. The adhesion of platelets is inhibited by synthetic peptides containing the sequence of Arg-Gly-Asp. In addition, monoclonal antibodies to glycoprotein IIb-IIIa complex inhibit the adhesion of activated platelets to vitronectin substrate in a dose-dependent manner. These studies suggest that the glycoprotein IIb-IIIa complex on activated platelets may interact with vitronectin substrate through the Arg-Gly-Asp mechanism. Since vitronectin is present in the subendothelial matrix, it might be involved in platelet-vessel wall interactions

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