scholarly journals C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1925
Author(s):  
Laura T. Fee ◽  
Debananda Gogoi ◽  
Michael E. O’Brien ◽  
Emer McHugh ◽  
Michelle Casey ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Plasma Levels ◽  
Plasma Membranes ◽  
Ex Vivo ◽  
Endothelial Cell Migration ◽  
Increased Risk ◽  
Alpha 1 Antitrypsin ◽  
The Impact

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

scholarly journals Zinc deficiency promotes endothelin secretion and endothelial cell migration through nuclear hypoxia-inducible factor-1 translocation

2019 ◽  
Vol 317 (2) ◽  
pp. C270-C276 ◽  
Author(s):  
Jessica Morand ◽  
Anne Briançon-Marjollet ◽  
Emeline Lemarie ◽  
Brigitte Gonthier ◽  
Josiane Arnaud ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Zinc Deficiency ◽  
Hypoxia Inducible Factor ◽  
Endothelial Cell Migration ◽  
Hypoxia Inducible Factor 1 ◽  
Obstructive Sleep ◽  
And Function

Zinc is involved in the expression and function of various transcription factors, including the hypoxia-inducible factor-1 (HIF-1). HIF-1 and its target gene endothelin-1 (ET-1) are activated by intermittent hypoxia (IH), one of the main consequences of obstructive sleep apnea (OSA), and both play a key role in the cardiovascular consequences of IH. Because OSA and IH are associated with zinc deficiency, we investigated the effect of zinc deficiency caused by chelation on the HIF-1/ET-1 pathway and its functional consequences in endothelial cells. Primary human microvascular endothelial cells (HMVEC) were incubated with submicromolar doses of the zinc-specific membrane-permeable chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN, 0.5 µM) or ET-1 (0.01 µM) with or without bosentan, a dual ET-1-receptor antagonist. HIF-1α expression was silenced by transfection with specific siRNA. Nuclear HIF-1 content was assessed by immunofluorescence microscopy and Western blot. Migratory capacity of HMVEC was evaluated with a wound-healing scratch assay. Zinc chelation by TPEN exposure induced the translocation of the cytosolic HIF-1α subunit of HIF-1 to the nucleus as well as an HIF-1-mediated ET-1 secretion by HMVEC. Incubation with either TPEN or ET-1 increased endothelial wound-healing capacity. Both HIF-1α silencing or bosentan abolished this effect. Altogether, these results suggest that zinc deficiency upregulates ET-1 signaling through HIF-1 activation and stimulates endothelial cell migration, suggesting an important role of zinc in the vascular consequences of IH and OSA mediated by HIF-1-ET- signaling.

scholarly journals Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

2021 ◽  
Vol 23 (1) ◽  
pp. 294
Author(s):  
Hanna Mannell ◽  
Petra Kameritsch ◽  
Heike Beck ◽  
Alexander Pfeifer ◽  
Ulrich Pohl ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Connexin 43 ◽  
Ex Vivo ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Endothelial Cell Migration ◽  
Junction Protein ◽  
Endothelial Migration

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

scholarly journals A microfluidic wound-healing assay for quantifying endothelial cell migration

2010 ◽  
Vol 298 (2) ◽  
pp. H719-H725 ◽  
Author(s):  
Andries D. van der Meer ◽  
Kim Vermeul ◽  
André A. Poot ◽  
Jan Feijen ◽  
István Vermes
Keyword(s):  
Wound Healing ◽  
Shear Stress ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Migration Rate ◽  
Microfluidic Channel ◽  
Endothelial Cell Migration ◽  
Wound Healing Assay ◽  
Gradient Generation ◽  
Endothelial Growth Factor

Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-μm-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF165) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF165 could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.

scholarly journals Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation

2014 ◽  
Vol 112 (12) ◽  
pp. 1244-1251 ◽  
Author(s):  
Sergiy Yakovlev ◽  
Irina Mikhailenko ◽  
Galina Tsurupa ◽  
Alexey Belkin ◽  
Leonid Medved
Keyword(s):  
Cell Proliferation ◽  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Wound Closure ◽  
Signalling Pathway ◽  
Endothelial Cell Migration ◽  
Specific Antagonist ◽  
Cell Migration And Proliferation

SummaryUpon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by timelapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing

2000 ◽  
Vol 278 (3) ◽  
pp. C612-C618 ◽  
Author(s):  
Hsinyu Lee ◽  
Edward J. Goetzl ◽  
Songzhu An
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Lysophosphatidic Acid ◽  
Mitogen Activated Protein Kinase ◽  
Endothelial Cell Migration ◽  
Mapk Phosphorylation ◽  
Cell Functions ◽  
Sphingosine 1 Phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent lipid growth factors with similar abilities to stimulate cytoskeleton-based cellular functions. Their effects are mediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes ( edgs). We hypothesize that large quantities of LPA and S1P generated by activated platelets may influence endothelial cell functions. Using an in vitro wound healing assay, we observed that LPA and S1P stimulated closure of wounded monolayers of human umbilical vein endothelial cells and adult bovine aortic endothelial cells, which express LPA receptor Edg2, and S1P receptors Edg1 and Edg3. The two major components of wound healing, cell migration and proliferation, were stimulated individually by both lipids. LPA and S1P also stimulated intracellular Ca2+mobilization and mitogen-activated protein kinase (MAPK) phosphorylation. Pertussis toxin partially blocked the effects of both lipids on endothelial cell migration, MAPK phosphorylation, and Ca2+ mobilization, implicating Gi/o-coupled Edg receptor signaling in endothelial cells. LPA and S1P did not cross-desensitize each other in Ca2+ responses, suggesting involvement of distinct receptors. Thus LPA and S1P affect endothelial cell functions through signaling pathways activated by distinct GPCRs and may contribute to the healing of wounded vasculatures.

scholarly journals Role of heparan sulfate in mediating CXCL8-induced endothelial cell migration

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1669 ◽  
Author(s):  
Zhiping Yan ◽  
Jingxia Liu ◽  
Linshen Xie ◽  
Xiaoheng Liu ◽  
Ye Zeng
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Actin Cytoskeleton ◽  
Heparan Sulfate ◽  
Rho Gtpases ◽  
Umbilical Vein ◽  
Endothelial Cell Migration

CXCL8 (Interleukin-8, IL-8) plays an important role in angiogenesis and wound healing by prompting endothelial cell migration. It has been suggested that heparan sulfate (HS) could provide binding sites on endothelial cells to retain and activate highly diffusible cytokines and inflammatory chemokines. In the present study, we aimed to test the hypothesis that HS is essential for enhancement of endothelial cell migration by CXCL8, and to explore the underlying mechanism by detecting the changes in expression and activity of Rho GTPases and in the organization of actin cytoskeleton after enzymatic removal of HS on human umbilical vein endothelial cells (HUVECs) by using heparinase III. Our results revealed that the wound healing induced by CXCL8 was greatly attenuated by removal of HS. The CXCL8-upregulated Rho GTPases including Cdc42, Rac1, and RhoA, and CXCL8-increased Rac1/Rho activity were suppressed by removal of HS. The polymerization and polarization of actin cytoskeleton, and the increasing of stress fibers induced by CXCL8 were also abolished by heparinase III. Taken together, our results demonstrated an essential role of HS in mediating CXCL8-induced endothelial cell migration, and highlighted the biological importance of the interaction between CXCL8 and heparan sulfate in wound healing.

An in vitro model for investigation of vitamin A effects on wound healing

2021 ◽  
pp. 1-6
Author(s):  
Hoda Keshmiri Neghab ◽  
Mohammad Hasan Soheilifar ◽  
Gholamreza Esmaeeli Djavid
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Vitamin A ◽  
In Vitro Model ◽  
Cellular Proliferation ◽  
Endothelial Cell Migration ◽  
Normal Fibroblast ◽  
Anti Inflammatory ◽  
Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inflammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inflammation responses.

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