Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study

Holte, Christopher Florian; Kruse, Larissa; Mao, Hong; Øie, Cristina Ionica; Szymoński, Marek; Zapotoczny, Bartłomiej; McCourt, Peter

doi:10.1016/j.micron.2021.103121

Cited by 11 publications

(16 citation statements)

References 19 publications

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“…We observed broadening of the distribution of the fenestrations’ sizes towards smaller values. The detection of fenestrations in the range of 50–90 nm was reported to be overburdened with the error arising from the quality of SEM images [ 28 ]. Here, however, we were able to clearly distinguish the second group of fenestrations < 100 nm.…”

Section: Resultsmentioning

confidence: 99%

“…We also provide data acquired with structured illumination microscopy (SIM), a method with resolution better than those imposed by the diffraction limit of light, that would allow us to colocalize pMLC and ppMLC to sieve plates and fenestrations. The selected methodology and data analysis methods were carefully selected based on our recent reports [ 28 , 29 ]. We supported our data with the assessment of LSEC functions, by performing the following: a viability test (lactate dehydrogenase (LDH) assay), a mitochondrial activity test (resazurin assay), and an assessment of endocytic activity (FSA scavenging assay).…”

Section: Introductionmentioning

confidence: 99%

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Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Zapotoczny

Lekka

Ahluwalia

et al. 2022

IJMS

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Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Zapotoczny

Lekka

Ahluwalia

et al. 2022

IJMS

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“…The comparison between the groups in a single report provides information about the alterations as the same microscopy method is applied. The methodological details enabling avoiding errors in imaging and data analysis were described: SEM (Wisse et al, 2010;Cogger et al, 2015;Szafranska et al, 2021), AFM (Zapotoczny et al, 2017a(Zapotoczny et al, , 2020Szafranska et al, 2021), SIM (Kong et al, 2021;Szafranska et al, 2021). Moreover, the comparative measurements using different microscopies were reported in the past showing good correlation between the methods.…”

Section: Introductionmentioning

confidence: 99%

The wHole Story About Fenestrations in LSEC

et al. 2021

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The porosity of liver sinusoidal endothelial cells (LSEC) ensures bidirectional passive transport of lipoproteins, drugs and solutes between the liver capillaries and the liver parenchyma. This porosity is realized via fenestrations – transcellular pores with diameters in the range of 50–300 nm – typically grouped together in sieve plates. Aging and several liver disorders severely reduce LSEC porosity, decreasing their filtration properties. Over the years, a variety of drugs, stimulants, and toxins have been investigated in the context of altered diameter or frequency of fenestrations. In fact, any change in the porosity, connected with the change in number and/or size of fenestrations is reflected in the overall liver-vascular system crosstalk. Recently, several commonly used medicines have been proposed to have a beneficial effect on LSEC re-fenestration in aging. These findings may be important for the aging populations of the world. In this review we collate the literature on medicines, recreational drugs, hormones and laboratory tools (including toxins) where the effect LSEC morphology was quantitatively analyzed. Moreover, different experimental models of liver pathology are discussed in the context of fenestrations. The second part of this review covers the cellular mechanisms of action to enable physicians and researchers to predict the effect of newly developed drugs on LSEC porosity. To achieve this, we discuss four existing hypotheses of regulation of fenestrations. Finally, we provide a summary of the cellular mechanisms which are demonstrated to tune the porosity of LSEC.

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“…Fenestrations were quantified using an intensity-based threshold method similar to the semiautomatic method described in ref. 46 .…”

Section: Methodsmentioning

confidence: 99%

Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

Butola

Coucheron

Ahmad

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.

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Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study

Cited by 11 publications

References 19 publications

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

The wHole Story About Fenestrations in LSEC

Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

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