Liesbeth H. P. Hekking

Focused ion beam‐scanning electron microscope: exploring large volumes of atherosclerotic tissue

Atherogenesis is a pathological condition in which changes in the ultrastructure and in the localization of proteins occur within the vasculature during all stages of the disease. To gain insight in those changes, high‐resolution imaging is necessary. Some of these changes will only be present in a small number of cells, positioned in a ‘sea’ of non‐affected cells. To localize this relatively small number of cells, there is a need to first navigate through a large area of the sample and subsequently zoom in onto the area of interest. This approach enables the study of specific cells within their in vivo environment and enables the study of (possible) interactions of these cells with their surrounding cells/environment. The study of a sample in a correlative way using light and electron microscopy is a promising approach to achieve this; however, it is very laborious and additional ultrastructural techniques might be very valuable to find the places of interest.

Zebrafish Tie-2 shares a redundant role with Tie-1 in heart development and regulates vessel integrity.

SUMMARY Tie-2 is a member of the receptor tyrosine kinase family and is required for vascular remodeling and maintenance of mammalian vessel integrity. A number of mutations in the human TIE2 gene have been identified in patients suffering from cutaneomucosal venous malformations and ventricular septal defects. How exactly Tie-2 signaling pathways play different roles in both vascular development and vascular stability is unknown. We have generated a zebrafish line carrying a stop mutation in the kinase domain of the Tie-2 receptor. Mutant embryos lack Tie-2 protein, but do not display any defect in heart and vessel development. Simultaneous loss of Tie-1 and Tie-2, however, leads to a cardiac phenotype. Our study shows that Tie-1 and Tie-2 are not required for early heart development, yet they have redundant roles for the maintenance of endocardial-myocardial connection in later stages. Tie-2 and its ligand Angiopoietin-1 have also been reported to play an important role in vessel stability. We used atorvastatin and simvastatin, drugs that cause bleeding in wild-type zebrafish larvae, to challenge vessel stability in tie-2 mutants. Interestingly, recent clinical studies have reported hemorrhagic stroke as a side effect of atorvastatin treatment. Exposure of embryos to statins revealed that tie-2 mutants are significantly protected from statin-induced bleeding. Furthermore, tie-2 mutants became less resistant to bleeding after VE-cadherin knockdown. Taken together, these data show that atorvastatin affects vessel stability through Tie-2, and that VE-cadherin and Tie-2 act in concert to allow vessel remodeling while playing a role in vessel stability. Our study introduces an additional vertebrate model to study in vivo the function of Tie-2 in development and disease.

Endothelial cell senescence is associated with disrupted cell-cell junctions and increased monolayer permeability

BackgroundCellular senescence is associated with cellular dysfunction and has been shown to occur in vivo in age-related cardiovascular diseases such as atherosclerosis. Atherogenesis is accompanied by intimal accumulation of LDL and increased extravasation of monocytes towards accumulated and oxidized LDL, suggesting an affected barrier function of vascular endothelial cells. Our objective was to study the effect of cellular senescence on the barrier function of non-senescent endothelial cells.MethodsHuman umbilical vein endothelial cells were cultured until senescence. Senescent cells were compared with non-senescent cells and with co-cultures of non-senescent and senescent cells. Adherens junctions and tight junctions were studied. To assess the barrier function of various monolayers, assays to measure permeability for Lucifer Yellow (LY) and horseradish peroxidase (PO) were performed.ResultsThe barrier function of monolayers comprising of senescent cells was compromised and coincided with a change in the distribution of junction proteins and a down-regulation of occludin and claudin-5 expression. Furthermore, a decreased expression of occludin and claudin-5 was observed in co-cultures of non-senescent and senescent cells, not only between senescent cells but also along the entire periphery of non-senescent cells lining a senescent cell.ConclusionsOur findings show that the presence of senescent endothelial cells in a non-senescent monolayer disrupts tight junction morphology of surrounding young cells and increases the permeability of the monolayer for LY and PO.

Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion.

AIMS In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation. METHODS AND RESULTS Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor alpha stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes. CONCLUSION Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response.

Novel Role for Mast Cells in Omental Tissue Remodeling and Cell Recruitment in Experimental Peritoneal Dialysis

Because of its dynamic structure, the omentum plays a key role in the immunity of the peritoneal cavity by orchestrating peritoneal cell recruitment. Because mast cells accumulate in the omentum upon experimental peritoneal dialysis (PD) and may produce angiogenic/profibrotic factors, it was hypothesized that mast cells mediate omental tissue remodeling during PD. Daily treatment with conventional PD fluid (PDF) for 5 wk resulted in a strong omental remodeling response, characterized by an approximately 10-fold increase in mast cell density (P < 0.01), an approximately 20-fold increase in vessel density (P < 0.02), an approximately 20-fold increase in the number of milky spots (P < 0.01), and a four-fold increase in submesothelial matrix thickness (P < 0.0003) in wild-type rats. In contrast, all PDF-induced omental changes were significantly reduced in mast cell-deficient Ws/Ws rats or in wild-type rats that were treated orally with a mast cell stabilizer cromoglycate. A time-course experiment showed mast cell accumulation immediately before the formation of blood vessels and milky spots. Functionally, PDF evoked a peritoneal cell influx, which was significantly reduced in Ws/Ws rats (P < 0.04) and in wild-type rats that were treated with cromoglycate (P < 0.03). Cromoglycate treatment also completely prevented PDF-induced omental adhesions to the catheter tip (P = 0.0002). Mesothelial damage, angiogenesis, and fibrosis of mesentery and parietal peritoneum as well as glucose absorption rate and ultrafiltration capacity proved to be mast cell independent. Data strongly support the hypothesis that mast cells mediate PDF-induced omental tissue remodeling and, subsequently, peritoneal cell influx and adhesion formation, providing therapeutic possibilities of modulating omental function.

Spiral Coating of the Endothelial Caveolar Membranes as Revealed by Electron Tomography and Template Matching

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3‐D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high‐pressure frozen, freeze‐substituted and epon‐embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises – as confirmed by 3‐D immunolabeling for caveolin of ‘intact’ cells –bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi‐caveolar bodies. Application of template matching to tomograms allowed the 3‐D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze‐etched caveolae from fibroblasts. Meticulous 3‐D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the ‘intact’ caveolar system, setting a detailed morphological basis for its functional diversity.

Endothelial glycocalyx dimensions are reduced in growing collateral arteries and modulate leucocyte adhesion in arteriogenesis

During collateral artery growth, monocytes adhere to the endothelium and secrete cytokines from the perivascular space promoting arteriogenesis. Recently, the endothelial glycocalyx has been shown to modulate leucocyte infiltration in atherogenic regions. The role of this endothelial surface coating in arteriogenesis, however, has not been investigated so far. We now report that local plasma levels of hyaluronic acid are specifically increased in collateral arterial blood of coronary artery disease patients and hypothesized that components of the endothelial glycocalyx are shed during arteriogenesis, resulting in decreased glycocalyx dimensions and an increased leucocyte extravasation. In a rabbit model of femoral artery ligation, electron microscopy revealed a decrease in glycocalyx dimensions in collateral arteries compared with quiescent anastomoses (67.5 ± 47.2 nm versus 101.0 ± 11.3 nm; P < 0.001). This decrease was correlated with a higher number of perivascular macrophages around collateral arteries. The additional glycocalyx perturbation by local hyaluronidase infusion almost completely removed the endothelial surface layer and temporarily stimulated leucocyte accumulation in the perivascular space. However, complete perturbation of the glycocalyx by hyaluronidase infusion resulted in a significant attenuation of collateral artery growth assessed by microsphere‐based perfusion measurements (ml/min/100 mmHg: hyaluronidase: 27.5 ± 3.5; Controls: 47.1 ± 3.83; P < 0.001) and a lower percentage of actively proliferating vascular smooth muscle cells. A decreased expression of the shear‐stress regulated pro‐arteriogenic genes eNOS and TGF‐β1 suggests an impaired mechanotransduction as the underlying mechanisms. For the first time, we describe the role of the endothelial glycocalyx in collateral artery growth. Although complete abrogation led to attenuated arteriogenesis, shedding of glycocalyx components is observed during collateral artery growth.

P-glycoprotein regulates trafficking of CD8+ T cells to the brain parenchyma

The trafficking of cytotoxic CD8+ T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8+ T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8+ T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell–endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C–C motif) ligand 2 (CCL2), a chemokine that mediates CD8+ T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8+ T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8+ T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

Comparative Ultrastructural and Stereological Analyses of Unruptured and Ruptured Saccular Intracranial Aneurysms

Insight into processes leading to rupture of intracranial aneurysms (IAs) may identify biomarkers for rupture or lead to management strategies reducing the risk of rupture. We characterized and quantified (ultra)structural differences between unruptured and ruptured aneurysmal walls. Six unruptured and 6 ruptured IA fundi were resected after microsurgical clipping and analyzed by correlative light microscopy for quantitative analysis (proportion of the vessel wall area) and transmission electron microscopy for qualitative ultrastructural analysis. Quantitative analysis revealed extensive internal elastic lamina (IEL) thickening in ruptured IA (36.3% ± 15%), while thin and fragmented IEL were common in unruptured IA (5.6% ± 7.1%). Macrophages were increased in ruptured IA (28.3 ± 24%) versus unruptured IA (2.7% ± 5.5%), as were leukocytes (12.85% ± 10% vs 0%). Vasa vasorum in ruptured but not in unruptured IA contained vast numbers of inflammatory cells and extravasation of these cells into the vessel wall. In conclusion, detection of thickened IEL, leaky vasa vasorum, and heavy inflammation as seen in ruptured IA in comparison to unruptured IA may identify aneurysms at risk of rupture, and management strategies preventing development of vasa vasorum or inflammation may reduce the risk of aneurysmal rupture.

D-CAT: Density and Clustering Annotation Tool for three dimensional electron microscopic volumes.

Three dimensional (3D) electron microscopy techniques have become valuable tools for investigating cellular architecture and the processes that govern it. A vast amount of information is available in every 3D tomogram but the options for presenting this information in a clear and visually appealing way are limited. To address this, we developed D-CAT; a MatLab-application to accurately visualize the distribution of membrane proteins and/or membrane-bound structures. Presence (density) and distribution (clustering, depletion) are presented as color-coded areas on membranes. By using IMOD models both as input and output format, we ensure that the application fits within workflows common in the field of 3D electron microscopy.