scholarly journals MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells

2019 ◽  
Vol 20 (12) ◽  
pp. 3107 ◽  
Author(s):  
Mikel Aristorena ◽  
Eunate Gallardo-Vara ◽  
Matej Vicen ◽  
Mateo de Las Casas-Engel ◽  
Luisa Ojeda-Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Macrophage Polarization ◽  
Cell Surface Receptor ◽  
Soluble Form ◽  
Specific Marker ◽  
Good Resolution ◽  
Angiogenic Activity ◽  
Soluble Endoglin

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.

Inhibition of bovine retinal microvascular pericyte proliferation in vitro by adenosine

1992 ◽  
Vol 263 (2) ◽  
pp. H634-H640 ◽  
Author(s):  
J. A. Jackson ◽  
E. C. Carlson
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Adenosine Receptor ◽  
Inhibitory Effect ◽  
Cell Surface Receptor ◽  
Dual Function ◽  
Receptor Subtypes ◽  
Retinal Pericytes ◽  
Surface Receptor

Adenosine acts on bovine retinal microvascular pericytes through one or more adenosine receptor subtypes present on the cell surface. Retinal pericytes cultured in medium containing adenosine at concentrations from 10(-6) to 10(-4) M showed significant reduction in proliferation following several days in vitro compared with control cultures. The effects of adenosine were mimicked by polyadenylic acid and inhibited by 8-phenyltheophylline, indicating involvement of a cell surface receptor. Metabolites of adenosine had no effect on pericyte proliferation. An A2 adenosine receptor-specific analogue also inhibited pericyte growth, suggesting that inhibition by adenosine is mediated by A2-receptors and might involve a transient increase in adenosine 3',5'-cyclic monophosphate levels. The results of the present study demonstrate that in addition to demonstrated stimulatory effects on capillary endothelial cells, adenosine also has a direct inhibitory effect on retinal pericytes. We hypothesize a dual function of adenosine within the capillary wall resulting in loss of inhibition of endothelial cells and suggest a role for this nucleoside in pathological neovascularization processes such as proliferative diabetic retinopathy.

scholarly journals Regulation of Tie Receptor Expression on Human Endothelial Cells by Protein Kinase C-Mediated Release of Soluble Tie

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 706-715 ◽  
Author(s):  
Rachel Yabkowitz ◽  
Susanne Meyer ◽  
Donna Yanagihara ◽  
David Brankow ◽  
Tabitha Staley ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Extracellular Domain ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Soluble Form ◽  
Human Endothelial Cells ◽  
Kinase C

Abstract The expression and activity of receptor tyrosine kinases (RTK) at the cell surface can be modulated by several different pathways including the proteolytic release of the extracellular domain as a soluble receptor. We investigated the regulation of tie receptor expression, an orphan RTK restricted to cells of hematopoietic and endothelial lineages, on primary human endothelial cells and a stably transfected Chinese hamster ovary (CHO) cell line. Tie was expressed in cells as a doublet of 135 and 125 kD; the 135-kD band represented mature cell surface receptor containing sialic acid and N-linked oligosaccharide residues, whereas the 125-kD band represented an intracellular pool of immature receptor. Phorbol 12-myristate 13-acetate (PMA) had dramatic effects on tie expression at the cell surface. Within 15 minutes of PMA treatment, the 135-kD band disappeared from the cell surface and was accompanied by the appearance of a 100-kD band in cell supernatants. The 100-kD band continued to accumulate in the media throughout the duration of PMA treatment during which mature tie receptor was undetectable on the cell surface by fluorescence-activated cell sorting (FACS) or in cell lysates by immunoblot analysis. Using specific antibodies, this 100-kD species was shown to be a soluble form of the tie receptor containing the extracellular domain. PMA-dependent release of soluble tie was mediated through the activation of protein kinase C (PKC); soluble tie was not released in the presence of PKC inhibitors, an inactive PMA analog, or following the downregulation of PKC through chronic PMA treatment. These results indicate that tie receptor expression on endothelial cells is regulated by the release of a soluble extracellular fragment following activation of PKC. Parallel pathways regulating c-kit, tumor necrosis factor (TNF), and colony-stimulating factor (CSF) receptor expression suggest that the release of extracellular receptor fragments represents an alternative mechanism through which cells modulate responses to growth factors and cytokines.

scholarly journals Regulation of Tie Receptor Expression on Human Endothelial Cells by Protein Kinase C-Mediated Release of Soluble Tie

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 706-715 ◽  
Author(s):  
Rachel Yabkowitz ◽  
Susanne Meyer ◽  
Donna Yanagihara ◽  
David Brankow ◽  
Tabitha Staley ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Extracellular Domain ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Soluble Form ◽  
Human Endothelial Cells ◽  
Kinase C

The expression and activity of receptor tyrosine kinases (RTK) at the cell surface can be modulated by several different pathways including the proteolytic release of the extracellular domain as a soluble receptor. We investigated the regulation of tie receptor expression, an orphan RTK restricted to cells of hematopoietic and endothelial lineages, on primary human endothelial cells and a stably transfected Chinese hamster ovary (CHO) cell line. Tie was expressed in cells as a doublet of 135 and 125 kD; the 135-kD band represented mature cell surface receptor containing sialic acid and N-linked oligosaccharide residues, whereas the 125-kD band represented an intracellular pool of immature receptor. Phorbol 12-myristate 13-acetate (PMA) had dramatic effects on tie expression at the cell surface. Within 15 minutes of PMA treatment, the 135-kD band disappeared from the cell surface and was accompanied by the appearance of a 100-kD band in cell supernatants. The 100-kD band continued to accumulate in the media throughout the duration of PMA treatment during which mature tie receptor was undetectable on the cell surface by fluorescence-activated cell sorting (FACS) or in cell lysates by immunoblot analysis. Using specific antibodies, this 100-kD species was shown to be a soluble form of the tie receptor containing the extracellular domain. PMA-dependent release of soluble tie was mediated through the activation of protein kinase C (PKC); soluble tie was not released in the presence of PKC inhibitors, an inactive PMA analog, or following the downregulation of PKC through chronic PMA treatment. These results indicate that tie receptor expression on endothelial cells is regulated by the release of a soluble extracellular fragment following activation of PKC. Parallel pathways regulating c-kit, tumor necrosis factor (TNF), and colony-stimulating factor (CSF) receptor expression suggest that the release of extracellular receptor fragments represents an alternative mechanism through which cells modulate responses to growth factors and cytokines.

scholarly journals Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 3099-3107 ◽  
Author(s):  
Masashi Narazaki ◽  
Marta Segarra ◽  
Xu Hou ◽  
Toshio Tanaka ◽  
Xuri Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Ligand Interaction ◽  
Surface Receptors ◽  
Neuropilin 1 ◽  
And Function

Abstract Ligand interaction with cognate cell-surface receptor often promotes receptor internalization, protecting cells from prolonged or excessive signaling from extracellular ligands. Compounds that induce internalization of surface receptors prevent ligand binding to cognate cell-surface receptors serving as inhibitors. Here, we show that synthetic polyriboguanosine (poly G) and oligo-deoxyriboguanosine (oligo G) reduce endothelial levels of surface neuropilin-1 (NRP1), a receptor shared by semaphorin 3A and vascular endothelial growth factor (VEGF), which plays critical roles in angiogenesis. Oligo G also reduces levels of cell-surface scavenger receptor expressed by endothelial cells I (SREC-I), but not levels of NRP2, gp130, CD31, VEGFR-1, or VEGFR-2. Poly or oligo A, T, and C do not promote NRP1 or SREC-I internalization. We find that oligo G binds to NRP1 with high affinity (Kd:1.3 ± 0.16nM), bridges the extracellular domain of NRP1 to that of SREC-I, and induces coordinate internalization of NRP1 and SREC-I. In vitro, oligo G blocks the binding and function of VEGF165 in endothelial cells. In vivo, intravitreal administration of oligo G reduces choroidal neovascularization in mice. These results demonstrate that synthetic oligo G is an inhibitor of pathologic angiogenenesis that reduces cell-surface levels and function of NRP1 acting as an internalization inducer.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

scholarly journals Autoantibodies against the fibrinolytic receptor, annexin 2, in antiphospholipid syndrome

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4375-4382 ◽  
Author(s):  
Gabriela Cesarman-Maus ◽  
Nina P. Ríos-Luna ◽  
Arunkumar B. Deora ◽  
Bihui Huang ◽  
Rosario Villa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Antiphospholipid Syndrome ◽  
Lupus Erythematosus ◽  
Antiphospholipid Antibodies ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Antigen ◽  
Healthy Individuals ◽  
Annexin 2

AbstractThe association of thrombosis and gestational morbidity with antiphospholipid antibodies is termed antiphospholipid syndrome (APS). Annexin 2 (A2) is a profibrinolytic endothelial cell surface receptor that binds plasminogen, its tissue activator (tPA), and β2-glycoprotein I (β2GPI), the main antigen for antiphospholipid antibodies. Here, we evaluate A2 as a target antigen in APS. Serum samples from 434 individuals (206 patients with systemic lupus erythematosus without thrombosis, 62 with APS, 21 with nonautoimmune thrombosis, and 145 healthy individuals) were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblot for antiphospholipid and A2 antibodies. Anti-A2 antibodies (titer > 3 SDs) were significantly more prevalent in patients with APS (22.6%; venous, 17.5%; arterial, 34.3%; and mixed thrombosis, 40.4%) than in healthy individuals (2.1%, P < .001), patients with nonautoimmune thrombosis (0%, P = .017), or patients with lupus without thrombosis (6.3%, P < .001). Anti–A2 IgG enhanced the expression of tissue factor on endothelial cells (6.4-fold ± 0.13-fold SE), blocked A2-supported plasmin generation in a tPAdependent generation assay (19%-71%) independently of β2GPI, and inhibited cell surface plasmin generation on human umbilical vein endothelial cells (HUVECs) by 34% to 83%. We propose that anti-A2 antibodies contribute to the prothrombotic diathesis in antiphospholipid syndrome.

scholarly journals ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

2018 ◽  
Vol 19 (10) ◽  
pp. 3089 ◽  
Author(s):  
Marie Hlavničková ◽  
Milan Kuchař ◽  
Radim Osička ◽  
Lucie Vaňková ◽  
Hana Petroková ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Manifestations ◽  
Cell Surface Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interleukin 17 ◽  
Normal Human Skin ◽  
High Affinity ◽  
Th 17 ◽  
Surface Receptor

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.

Abstract 34: Human Cardiac Mesenchymal Cell Derived Extracellular Vesicles Promote Angiogenesis Through Angiopoietin-TIE2 Signaling

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Asif Pathan ◽  
Talha Farid ◽  
Abdur Rahman Khan ◽  
Marjan Nasr ◽  
Marcin Wysoczynski ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Embryonic Stem ◽  
Tube Formation ◽  
Right Atrial ◽  
Boyden Chamber ◽  
Human Umbilical Cord ◽  
Angiogenic Activity ◽  
Cell Based Therapy

Cell-based therapy is considered a promising approach to treat the damaged heart due to myocardial infarction. Although the mechanisms for their beneficial action are not yet clear, exosome/extracellular vesicles (EVs) secreted by these cells may be involved in their reparative paracrine signaling. Previous studies have suggested that EVs isolated from several cell types (e.g. cardiosphere-derived cells, embryonic stem cell, CD34+ stem cells) induce angiogenic activity both in vitro and in vivo . Here, we investigated whether EVs secreted by adult human cardiac mesenchymal cells (hCMCs) exhibit pro-angiogenic activity, and if so, what signaling molecules are involved in this process. hCMCs were isolated from right atrial appendage of patients undergoing cardiac procedures and were characterized by the expression of classical mesenchymal markers- CD29 (99.1%), CD73 (99.0%), CD90 (20.4%), CD105 (99.3%), CD 31 (16.8%), CD34 (0.9%) and CD45 (0.1%). EVs isolated from serum-free 24-hour hCMC conditioned media using PEG4000-based precipitation technique exhibited two distinct population of particles with size range of 10-60nm and 100-500nm in diameter; expressed characteristic exosomal markers- CD63, HSP70, Flotillin-1 and were negative for cellular organelle markers- calreticulin (ER and apoptotic bodies), prohibitin (mitochondria), GM130 (Golgi), Lamin B (nuclear protein), β-actin (cytoskeleton) and PMP70 (peroxisomes) as determined by immunoblotting. In vitro assays revealed that hCMC EVs promote human umbilical cord endothelial cells (HUVECs) proliferation, transwell migration in Boyden chamber and tube formation on Matrigel, indicative of enhanced angiogenesis. Angiogenic proteomic array identified that angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) proteins are highly enriched in EVs secreted by hCMCs. Furthermore, hCMC EV mediated HUVEC migration and tube formation was inhibited by TIE2 kinase inhibitor. Overall, these findings suggest that ANG-1 and ANG-2 are the key component of hCMC secreted EVs and they promote angiogenesis by activating TIE2 receptor in endothelial cells.

scholarly journals Evaluation of Angiogenesis Assays

Biomedicines ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 37 ◽  
Author(s):  
Zachary I. Stryker ◽  
Mehdi Rajabi ◽  
Paul J. Davis ◽  
Shaker A. Mousa
Keyword(s):  
Endothelial Cells ◽  
Tube Formation ◽  
Angiogenic Activity ◽  
In Vitro Methods ◽  
Current State ◽  
Angiogenesis Process ◽  
Pertinent Information ◽  
Exogenous Factors

Angiogenesis assays allow for the evaluation of pro- or anti-angiogenic activity of endogenous or exogenous factors (stimulus or inhibitors) through investigation of their pro-or anti- proliferative, migratory, and tube formation effects on endothelial cells. To model the process of angiogenesis and the effects of biomolecules on that process, both in vitro and in vivo methods are currently used. In general, in vitro methods monitor specific stages in the angiogenesis process and are used for early evaluations, while in vivo methods more accurately simulate the living microenvironment to provide more pertinent information. We review here the current state of angiogenesis assays as well as their mechanisms, advantages, and limitations.

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