Two-Photon Microscopy of Vital Murine Elastic and Muscular Arteries

Megens, Remco T. A.; Reitsma, Sietze; Schiffers, Paul H M; Hilgers, Rob H. P.; Mey, Jo G. R. De; Slaaf, Dick W.; Egbrink, Mirjam G.A. oude; Zandvoort, Marc A. M. J. van

doi:10.1159/000098259

Cited by 162 publications

(107 citation statements)

References 71 publications

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“…Previous studies have demonstrated that the vessel, including the endothelial and smooth muscle cells, remains intact. 17,30 Whole blood was diluted with isotonic citrate buffer to adjust the platelet count to 1×10 8 platelets/mL blood. Before perfusion, the endothelium was labeled with anti-CD31-eFluor 450 antibody for 30 minutes.…”

Section: Ex Vivo Adhesion Assaymentioning

confidence: 99%

Hyperreactivity of Junctional Adhesion Molecule A-Deficient Platelets Accelerates Atherosclerosis in Hyperlipidemic Mice

Karshovska

Zhang

Blanchet

et al. 2015

Circulation Research

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Rationale: Besides their essential role in hemostasis, platelets also have functions in inflammation. In platelets, junctional adhesion molecule (JAM)-A was previously identified as an inhibitor of integrin α IIb β 3 -mediated outside-in signaling and its genetic knockdown resulted in hyperreactivity. Objective: This gain-of-function was specifically exploited to investigate the role of platelet hyperreactivity in plaque development. Methods and Results: JAM-A–deficient platelets showed increased aggregation and cellular and sarcoma tyrosine-protein kinase activation. On α IIb β 3 ligation, JAM-A was shown to be dephosphorylated, which could be prevented by protein tyrosine phosphatase nonreceptor type 1 inhibition. Mice with or without platelet-specific (tr)JAM-A-deficiency in an apolipoprotein e ( apoe –/– ) background were fed a high-fat diet. After ≤12 weeks of diet, trJAM-A –/– apoe–/– mice showed increased aortic plaque formation when compared with trJAM-A +/+ apoe –/– controls, and these differences were most evident at early time points. At 2 weeks, the plaques of the trJAM-A –/– apoe –/– animals revealed increased macrophage, T cell, and smooth muscle cell content. Interestingly, plasma levels of chemokines CC chemokine ligand 5 and CXC-chemokine ligand 4 were increased in the trJAM-A –/– apoe –/– mice, and JAM-A–deficient platelets showed increased binding to monocytes and neutrophils. Whole-blood perfusion experiments and intravital microscopy revealed increased recruitment of platelets and monocytes to the inflamed endothelium in blood of trJAM-A –/– apoe –/– mice. Notably, these proinflammatory effects of JAM-A–deficient platelets could be abolished by the inhibition of α IIb β 3 signaling in vitro. Conclusions: Deletion of JAM-A causes a gain-of-function in platelets, with lower activation thresholds and increased inflammatory activities. This leads to an increase of plaque formation, particularly in early stages of the disease.

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Section: Ex Vivo Adhesion Assaymentioning

confidence: 99%

Hyperreactivity of Junctional Adhesion Molecule A-Deficient Platelets Accelerates Atherosclerosis in Hyperlipidemic Mice

Karshovska

Zhang

Blanchet

et al. 2015

Circulation Research

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“…DAPI was used to stain and image nuclei and was excited with a multi-photon laser at 720 nm (43). Collagen was imaged using a multi-photon laser at 850 nM via second-harmonic image, a scattering nonlinear process that requires no staining (30). Images were processed, and all channels were quantified to determine the total volume (number of expressed voxels) occupied by vascular smooth muscle cell (VSMC) nuclei, elastin, actin (within the media), and collagen with an in-house MATLAB algorithm available upon request.…”

Section: Animalsmentioning

confidence: 99%

Regional variation in arterial stiffening and dysfunction in Western diet-induced obesity

Bender

Castorena‐Gonzalez

Garro

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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, is an independent predictor of cardiovascular event risk. Recent evidence demonstrates that accelerated aortic stiffening occurs in obesity; however, little is known regarding stiffening of other disease-relevant arteries or whether regional variation in arterial stiffening occurs in this setting. We addressed this gap in knowledge by assessing femoral PWV in vivo in conjunction with ex vivo analyses of femoral and coronary structure and function in a mouse model of Western diet (WD; high-fat/high-sugar)-induced obesity and insulin resistance. WD feeding resulted in increased femoral PWV in vivo. Ex vivo analysis of femoral arteries revealed a leftward shift in the strain-stress relationship, increased modulus of elasticity, and decreased compliance indicative of increased stiffness following WD feeding. Confocal and multiphoton fluorescence microscopy revealed increased femoral stiffness involving decreased elastin/collagen ratio in conjunction with increased femoral transforming growth factor-␤ (TGF-␤) content in WD-fed mice. Further analysis of the femoral internal elastic lamina (IEL) revealed a significant reduction in the number and size of fenestrae with WD feeding. Coronary artery stiffness and structure was unchanged by WD feeding. Functionally, femoral, but not coronary, arteries exhibited endothelial dysfunction, whereas coronary arteries exhibited increased vasoconstrictor responsiveness not present in femoral arteries. Taken together, our data highlight important regional variations in the development of arterial stiffness and dysfunction associated with WD feeding. Furthermore, our results suggest TGF-␤ signaling and IEL fenestrae remodeling as potential contributors to femoral artery stiffening in obesity.

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“…There have been many different approaches used in preparing vessels for ex vivo microscopy reported in the literature. Some have included mounting variably sized vessel segments into a perfusion chamber (Megens et al 2007a;Megens et al 2007b), fresh and/or formalinfixed tissue preparations mounted between a glass slide and a coverslip (Kwon et al 2008;Lee et al 2009;Kim et al 2010, Lim et al 2010, Lim et al 2011Suhalim et al 2012), as well as agar gel-infused vascular casts that are segmented into thinly sliced molds and mounted on a glass slide (van Zandvoort et al 2004;Megens et al 2007a;Megens et al 2008;Le et al 2010). In vivo approaches have also been described but these are with the addition of mechanical techniques to help minimize motion artifact.…”

Section: Discussionmentioning

confidence: 99%

Study of the Development of the Mouse Thoracic Aorta Three-Dimensional Macromolecular Structure using Two-Photon Microscopy

Zadrozny

Neufeld

Lucotte

et al. 2014

J Histochem Cytochem.

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SummaryUsing the intrinsic optical properties of collagen and elastin, two-photon microscopy was applied to evaluate the threedimensional (3D) macromolecular structural development of the mouse thoracic aorta from birth to 60 days old. Baseline development was established in the Scavenger Receptor Class B Type I-Deficient, Hypomorphic Apolipoprotein ER61 (SR-BI KO/ApoeR61 h/h ) mouse in preparation for modeling atherosclerosis. Precise dissection enabled direct observation of the artery wall in situ. En-face, optical sectioning of the aorta provided a novel assessment of the macromolecular structural development. During aortic development, the undulating lamellar elastin layers compressed consistent with the increases in mean aortic pressure with age. In parallel, a net increase in overall wall thickness (p<0.05, in day 60 compared with day 1 mice) occurred with age whereas the ratio of the tunicas adventitia and media to full aortic thickness remained nearly constant across age groups (~1:2.6, respectively). Histochemical analyses by brightfield microscopy and ultrastructure validated structural proteins and lipid deposition findings derived from two-photon microscopy. Development was associated with decreased decorin but not biglycan proteoglycan expression. This non-destructive 3D in situ approach revealed the aortic wall microstructure development. Coupling this approach with the intrinsic optical properties of the macromolecules may provide unique vascular wall 3D structure in many pathological conditions, including aortic atherosclerosis, dissections and aneurysms. (J Histochem Cytochem 63:8-21, 2015)

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Two-Photon Microscopy of Vital Murine Elastic and Muscular Arteries

Cited by 162 publications

References 71 publications

Hyperreactivity of Junctional Adhesion Molecule A-Deficient Platelets Accelerates Atherosclerosis in Hyperlipidemic Mice

Hyperreactivity of Junctional Adhesion Molecule A-Deficient Platelets Accelerates Atherosclerosis in Hyperlipidemic Mice

Regional variation in arterial stiffening and dysfunction in Western diet-induced obesity

Study of the Development of the Mouse Thoracic Aorta Three-Dimensional Macromolecular Structure using Two-Photon Microscopy

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