scholarly journals Distribution of Cytoskeletal Components in Endothelial Cells in the Guinea Pig Renal Artery

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kazuo Katoh ◽  
Yasuko Noda
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Renal Artery ◽  
Laser Scanning ◽  
Fluid Shear Stress ◽  
Focal Adhesions ◽  
Laser Scanning Microscopy ◽  
Stress Fibers ◽  
Confocal Laser ◽  
Pulsatile Pressure

The cytoskeletal components of endothelial cells in the renal artery were examined by analysis of en face preparations under confocal laser scanning microscopy. Renal arterial endothelial cells were shown to be elongated along the direction of blood flow, while stress fibers ran perpendicular to the flow in the basal portion. Focal adhesions were observed along the stress fibers in dot-like configurations. On the other hand, stress fibers in the apical portion of cells ran along the direction of flow. The localizations of stress fibers and focal adhesions in endothelial cells in the renal artery differed from those of unperturbed aortic and venous endothelial cells. Tyrosine-phosphorylated proteins were mainly detected at the sites of cell-to-cell apposition, but not in focal adhesions. Pulsatile pressure and fluid shear stress applied over endothelial cells in the renal artery induce stress fiber organization and localization of focal adhesions. These observations suggest that the morphological alignment of endothelial cells along the direction of blood flow and the organization of cytoskeletal components are independently regulated.

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

2004 ◽  
Vol 287 (5) ◽  
pp. H2287-H2294 ◽  
Author(s):  
Akinori Ueda ◽  
Manabu Shimomura ◽  
Mariko Ikeda ◽  
Ryuhei Yamaguchi ◽  
Kazuo Tanishita
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Laser Scanning ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Interface Barrier ◽  
Uptake Process ◽  
Static Conditions

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.

Platelet Binding to Monocytes Induces Changes in Integrin Functionality Promoting Monocyte Adhesion and Transendothelial Migration. Do Platelets Migrate Along?.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2214-2214
Author(s):  
Paula da Costa Martins ◽  
Janine M. van Gils ◽  
Anita Mol ◽  
Peter L. Hordijk ◽  
Jaap-Jan Zwaginga
Keyword(s):  
Endothelial Cells ◽  
Laser Scanning ◽  
Transendothelial Migration ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Monocyte Adhesion ◽  
Fibrin Gel ◽  
Endothelial Layer ◽  
Monocyte Migration ◽  
Platelet Binding

Abstract Monocyte adhesion to and transmigration across the endothelium are essential steps in atherogenesis. We have shown that the adhesive interactions between monocytes and the activated endothelium are increased when platelets bind to the monocyte surface and form platelet-monocyte complexes (PMC). PMC formation is dependent on interactions between platelet-displayed P-selectin and PSGL-1 on the monocyte surface. To better understand the effect of platelet binding on the capacity of monocytes to adhere to activated endothelium the P-selectin-PSGL-1 interaction - induced changes in integrin functionality were studied. The binding of platelets to monocytes via P-selectin-PSGL-1 interactions was shown to increase expression and activity of α4 beta;1 - and αM β2 - integrins which, resulted in increased monocyte adhesion to ICAM-1, VCAM-1, fibronectin and subsequently to stimulated endothelial cells. Platelet binding also induced monocyte migration (up to a 3-fold increase), compared to monocytes without platelets on their surface. To investigate the role of platelets in this process we determined the fate of platelets (within the PMC) during monocyte transendothelial migration. After forming PMC by mixing freshly isolated and fluorescently labeled platelets and monocytes, PMC were seeded on endothelial cells cultured on top of a fibrin gel. The cells were allowed to migrate across the endothelial layer into the gel where afterwards the position of platelets and monocytes was analyzed by confocal laser scanning microscopy. We found that the platelets were retained at the endothelium, suggesting that they detach from the PMC and are left behind on the endothelial layer upon monocyte transendothelial migration. In line with this we observed that the monocytes that were in the fibrin gel, underneath the endothelial layer, did not carry any platelets. This was confirmed by PMC migration over endothelial layered transwell filters since almost only monocytes were found in the lower compartment after migration. After testing different migration barriers, platelets seem to be shed from the monocyte surface upon monocyte migration by mechanical stress rather than only endothelial interaction. Our data suggest that monocytes bound to platelets are in a higher state of activation and have an increased atherogenic capacity. Furthermore, platelets seem to mainly play a role in the monocyte recruitment to the endothelium because once the monocytes cross the endothelial layer, platelets detach from their surface. Altogether, our findings, by showing that PMC have a strong atherogenic capacity, might be helpful in finding new therapeutic ways to prevent atherosclerosis and inflammation.

scholarly journals OP0141 HIGH DIMENSIONAL ANALYSIS REVEAL A NETWORK OF CERTAIN TRANSCRIPTION FACTORS THAT LINK VASCULOPATHY AND ORGAN FIBROSIS IN SYSTEMIC SCLEROSIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 90.2-91
Author(s):  
C. G. Anchang ◽  
B. Matalobos Lawaree ◽  
S. Weber ◽  
S. Rauber ◽  
T. Wohlfahrt ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Muscle Cells ◽  
Laser Scanning Microscopy ◽  
Tube Length ◽  
Confocal Laser ◽  
Research Support ◽  
Organ Fibrosis

Background:Since vascular manifestations such as Raynaud’s phenomenon often precede the onset of other clinical manifestations of systemic sclerosis (SSc), the identification of pathways linking vasculopathy to organ fibrosis might thus provide important insights into early disease mechanisms and allow early targeted intervention for both fibrotic and vascular events.Objectives:In this study we performed high dimensional (HD) analyses to identify mediators that link vasculopathy to organ fibrosis.Methods:HD techniques including RNA-seq, ChIP-seq, ATAC-seq and FISH-seq have been performed to identify mediators in vessels and fibrotic lesions of human skin samples of SSc patients and healthy volunteers. In addition, murine skin and lung tissue samples were analyzed by multi-channel immunofluorescence (IF) and confocal laser scanning microscopy. Microvascular endothelial cells, smooth muscle cells and fibroblasts have been further processed to address their functional attributes with regard to their proliferative, migratory and chemotactic capacity. In vivo models and ex vivomouse fetal metatarsal assays were performed to study fibrotic and angiogenic processes.Results:Bioinformatic HD analyses revealed the ETS transcription factor PU.1 as molecular checkpoint of a network of factors that drive matrix production and fibrotic imprinting in SSc. Within this network ATF3 was significantly upregulated in fibroblasts of skin biopsies of SSc patients and of various organs of fibrosis models. ATF3 deficiency ameliorated fibrosis in various mouse models. Notably, ATF3 was significantly upregulated in vascular cells of fibrotic tissues of SSc patients. Multi-channel IF and confocal laser scanning microscopy of skin and lung biopsies of SSc patients revealed an increased expression of ATF3 especially in microvascular endothelial cells and smooth muscle cells. ATF3 overexpression in smooth muscle cells led to an extensively enhanced proliferation and increased migratory capacity whereas endothelial cells showed a SSc-like phenotype with reduced proliferation and migration. After ATF3 overexpression, tube formation capacity was completely altered as assessed by cumulative tube length, tube numbers and capillary sprouting. To investigate vessel outgrowth from a different perspective, we used theex vivofetal mouse metatarsal assay. ATF3 knockout mice showed a completely altered angiogenic response as assessed by tube length, number of branches and number junctions compared to wildtype controls.Conclusion:We identified PU.1 and ATF3 as key factors in disturbed vasculature and endogenous activated fibroblasts suggesting this axis as a potential therapeutic target intervening both fibrotic and vascular manifestations.Disclosure of Interests:Charles Gwellem Anchang: None declared, Bettina Matalobos Lawaree: None declared, Stefanie Weber: None declared, Simon Rauber: None declared, Thomas Wohlfahrt: None declared, Markus Luber: None declared, Alexander Kreuter: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim, Andreas Ramming Grant/research support from: Pfizer, Novartis, Consultant of: Boehringer Ingelheim, Novartis, Gilead, Pfizer, Speakers bureau: Boehringer Ingelheim, Roche, Janssen

scholarly journals Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1221-1229
Author(s):  
WJ Mayet ◽  
E Csernok ◽  
C Szymkowiak ◽  
WL Gross ◽  
KH Meyer zum Buschenfelde
Keyword(s):  
Endothelial Cells ◽  
Wegener’S Granulomatosis ◽  
Laser Scanning ◽  
Interleukin 1 ◽  
Wegener's Granulomatosis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Target Antigen ◽  
Proteinase 3 ◽  
Human Endothelial Cells

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool confirming the diagnosis or monitoring disease activity of Wegener's granulomatosis (WG). Although several concepts concerning the pathophysiologic potentials of ANCA have been discussed, only sparse data about ANCA-endothelium interactions have been available. In this study, we have investigated the expression of PR-3 in cytokine- treated human endothelial cells using purified anti-PR-3 antibodies of patients with WG, murine and human monoclonal anti-PR-3 antibodies as probes. We were able to show that tumor necrosis factor-alpha, interleukin-1 alpha/beta, and interferon-gamma led to an increased PR-3 expression in the cytoplasm of endothelial cells by performing polymerase chain reaction analysis, Western blot, cyto-enzyme-linked immunosorbent assays, and confocal laser scanning microscopy. Moreover, PR-3 was also translocated into the cell membrane, becoming accessible to ANCA. Our data suggest a possible direct pathogenic effect of anti- PR-3 antibodies in WG and other vasculitides. Anti-PR-3 antibodies represent an important missing link in ANCA-endothelial interactions.

scholarly journals A new low-nephron CKD model with hypertension, progressive decline of renal function, and enhanced inflammation in C57BL/6 mice

2018 ◽  
Vol 314 (5) ◽  
pp. F1008-F1019 ◽  
Author(s):  
Jin Wei ◽  
Jie Zhang ◽  
Lei Wang ◽  
Byeong Jake Cha ◽  
Shan Jiang ◽  
...  
Keyword(s):  
Renal Function ◽  
Renal Artery ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Renal Infarction ◽  
Contralateral Kidney ◽  
Inflammatory Status ◽  
Transmission Electron ◽  
Artery Branch

Chronic kidney disease (CKD) is a major health issue in the US. The typical five-sixths nephrectomy (typical 5/6 NX) is a widely used experimental CKD model. However, the typical 5/6 NX model is hypertensive in rats but strain dependent in mice. In particular, C57BL/6 mice with the typical 5/6 NX exhibits normal blood pressure and well-preserved renal function. The goal of the present study was to create a new hypertensive CKD model in C57BL/6 mice. We first characterized the vascular architecture originated from each renal artery branch by confocal laser-scanning microscopy with fluorescent lectin. Then, a novel 5/6 NX-BL model was generated by uninephrectomy combined with 2/3 renal infarction via a ligation of upper renal artery branch on the contralateral kidney. Compared with 5/6 NX-C, the 5/6 NX-BL model exhibited elevated mean arterial pressure (137.6 ± 13.9 vs. 104.7 ± 8.2 mmHg), decreased glomerular filtration rate (82.9 ± 19.2 vs. 125.0 ± 13.9 µl/min) with a reciprocal increase in plasma creatinine (0.31 ± 0.03 vs. 0.19 ± 0.04 mg/dl), and significant renal injury as assessed by proteinuria, histology with light, and transmission electron microscopy. In addition, inflammatory status, as indicated by the level of proinflammatory cytokine TNFα and the leukocyte counts, was significantly upregulated in 5/6 NX-BL compared with the 5/6 NX-C. In summary, we developed a new hypertensive CKD model in C57BL/6 mice with 5/6 renal mass reduction by uninephrectomy and upper renal artery branch ligation on the contralateral kidney. This 5/6 NX-BL model exhibits an infarction zone-dependent hypertension and progressive deterioration of the renal function accompanied by enhanced inflammatory response.

scholarly journals Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1221-1229 ◽  
Author(s):  
WJ Mayet ◽  
E Csernok ◽  
C Szymkowiak ◽  
WL Gross ◽  
KH Meyer zum Buschenfelde
Keyword(s):  
Endothelial Cells ◽  
Wegener’S Granulomatosis ◽  
Laser Scanning ◽  
Interleukin 1 ◽  
Wegener's Granulomatosis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Target Antigen ◽  
Proteinase 3 ◽  
Human Endothelial Cells

Abstract Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (PR-3), have proved to be a useful clinical tool confirming the diagnosis or monitoring disease activity of Wegener's granulomatosis (WG). Although several concepts concerning the pathophysiologic potentials of ANCA have been discussed, only sparse data about ANCA-endothelium interactions have been available. In this study, we have investigated the expression of PR-3 in cytokine- treated human endothelial cells using purified anti-PR-3 antibodies of patients with WG, murine and human monoclonal anti-PR-3 antibodies as probes. We were able to show that tumor necrosis factor-alpha, interleukin-1 alpha/beta, and interferon-gamma led to an increased PR-3 expression in the cytoplasm of endothelial cells by performing polymerase chain reaction analysis, Western blot, cyto-enzyme-linked immunosorbent assays, and confocal laser scanning microscopy. Moreover, PR-3 was also translocated into the cell membrane, becoming accessible to ANCA. Our data suggest a possible direct pathogenic effect of anti- PR-3 antibodies in WG and other vasculitides. Anti-PR-3 antibodies represent an important missing link in ANCA-endothelial interactions.

scholarly journals In Vitro Evidences of Different Fibroblast Morpho-Functional Responses to Red, Near-Infrared and Violet-Blue Photobiomodulation: Clues for Addressing Wound Healing

2020 ◽  
Vol 10 (21) ◽  
pp. 7878
Author(s):  
Flaminia Chellini ◽  
Alessia Tani ◽  
Sandra Zecchi-Orlandini ◽  
Marco Giannelli ◽  
Chiara Sassoli
Keyword(s):  
Wound Healing ◽  
Laser Scanning ◽  
Near Infrared ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Laser Scanning Microscopy ◽  
Wound Management ◽  
Matrix Metalloproteinase 2 ◽  
Confocal Laser ◽  
Functional Responses

Although photobiomodulation (PBM) has proven promising to treat wounds, the lack of univocal guidelines and of a thorough understanding of light–tissue interactions hampers its mainstream adoption for wound healing promotion. This study compared murine and human fibroblast responses to PBM by red (635 ± 5 nm), near-infrared (NIR, 808 ± 1 nm), and violet-blue (405 ± 5 nm) light (0.4 J/cm2 energy density, 13 mW/cm2 power density). Cell viability was not altered by PBM treatments. Light and confocal laser scanning microscopy and biochemical analyses showed, in red PBM irradiated cells: F-actin assembly reduction, up-regulated expression of Ki67 proliferation marker and of vinculin in focal adhesions, type-1 collagen down-regulation, matrix metalloproteinase-2 and metalloproteinase-9 expression/functionality increase concomitant to their inhibitors (TIMP-1 and TIMP-2) decrease. Violet-blue and even more NIR PBM stimulated collagen expression/deposition and, likely, cell differentiation towards (proto)myofibroblast phenotype. Indeed, these cells exhibited a higher polygonal surface area, stress fiber-like structures, increased vinculin- and phospho-focal adhesion kinase-rich clusters and α-smooth muscle actin. This study may provide the experimental groundwork to support red, NIR, and violet-blue PBM as potential options to promote proliferative and matrix remodeling/maturation phases of wound healing, targeting fibroblasts, and to suggest the use of combined PBM treatments in the wound management setting.

Abstract 201: A New Multimodal Contrast Agent for Molecular Imaging of Adhesion Molecules Expressed in Atherosclerosis

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Mona Ahmed ◽  
Björn Gustafsson ◽  
Kenneth Caidahl
Keyword(s):  
Endothelial Cells ◽  
Contrast Agent ◽  
Adhesion Molecules ◽  
Laser Scanning ◽  
Specific Interaction ◽  
Negative Control ◽  
Laser Scanning Microscopy ◽  
Confocal Laser

Background: Inflammation is involved in many chronic diseases, one of which is atherosclerosis. Early detection of inflammation could improve clinical management. Most existing contrast agents on the market are non-specific and single-modal. Our aim was to apply an innovative new multimodal contrast agent, a polyvinyl alcohol based microbubble (MB), to target adhesion molecules expressed in atherosclerosis, opening a new scenario for biomedicine. Objective: To evaluate a contrast agent with different surface modifications and their adhesion to inflammatory in vitro models as a proof of concept, by imaging inflamed endothelium, that is involved in the pathogenesis of atherosclerosis. Methods: Microbubbles were antibody (Ab) or peptide conjugated to the adhesion molecule of interest. To evaluate attachment between MBs and an inflamed murine cell line, endothelial cells (EC), confocal laser scanning microscopy (CLSM) and flow cytometry (FACS) were used. To verify expression of cell adhesion molecules we used FACS and western blot. Stimulated and non-stimulated cells were incubated with MBs conjugated with active ligands, or a negative control. Results: In endothelial cells CLSM showed an increased uptake, or adherence, of peptide-conjugated microbubbles compared to non-stimulated control. MBs tagged with Abs against; ICAM-1, VCAM-1, E-Selectin, or with Cyclic Arginine-Glycine-Aspartate (cRGD) peptide showed stronger adherence to EC compared to non-targeted control. Microbubbles labeled with cRGD-peptide was shown to be the most prominent, indicating a specific interaction. Conclusions: Adhesion molecules expressed in atherosclerosis may be targeted by our labeled multimodal contrast agent in vitro , with potential for disease specific diagnostics in vivo.

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