The Computed Sinusoid

Hepatic sinusoids are lined with thin endothelial cells with transcellular pores, termed fenestrations. These fenestrations are open channels that connect the sinusoidal lumen to the underlying Space of Disse (SoD) and the hepatocytes of the liver parenchyma. Fenestrations range from 0.05 to 0.35 µm in diameter and cover 5–15% of the sinusoidal endothelial surface area, depending on their location along the sinusoids. The direct measurement of hemodynamic parameters, such as pressure and flow velocity, remains challenging within the narrow sinusoids. Such knowledge would increase our understanding of the physiology of the hepatic niche and possible implications in aging or diseases in which fenestrations are reduced or lost. Few simulations of liver blood flow focus on the level of the individual sinusoid, and fewer still include the transcellular pores (fenestrations) of the sinusoidal endothelium. Furthermore, none have included (i) a porosity gradient along the sinusoid wall, modeled using through-all pores rather than a porous medium, (ii) the presence of the SoD, or (iii) lymphatic drainage. Herein, computed fluid dynamics (CFD) simulations were performed using a numerical model with relevant anatomical characteristics (length, diameter, porosity, inlet/outlet pressure, and lymphatic outflow from the portal region of the SoD). The greatest contribution to luminal velocity magnitude and pressure was the overall shape of the vessel. Divergent-radius models yielded velocity magnitudes 1.5–2 times higher than constant-radius models, and pressures were 5–8% lower in the divergent-radius models compared to the constant-radius models. Porosity only modestly contributed to luminal pressure. The luminal velocity magnitude was largely unaffected by the presence or absence of lymphatic drainage. Velocity magnitudes through fenestrations were lower in higher-porosity models (20%) vs. lower-porosity models (5%) across all models (0.4–0.55-fold lower). Velocity magnitudes through the space of Disse were increased 3–4 times via the addition of lymphatic drainage to the models, while pressures were decreased by 6–12%. The flow velocity in the SoD was modified via differences in porosity, while the flow velocity in the lumens of the sinusoids was largely unaffected. The overall shape of the vessel is the single most important factor in the pressure flow behavior of the sinusoidal lumen. The flow rate over hepatocytes and the SoD is modestly affected by the distribution of porosity along the sinusoid and greatly affected by the lymphatic drainage, parameters that would be of interest for modeling the exchange of blood with the hepatic parenchyma

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

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

The scavenger function of liver sinusoidal endothelial cells in health and disease

The aim of this review is to give an outline of the blood clearance function of the liver sinusoidal endothelial cells (LSECs) in health and disease. Lining the hundreds of millions of hepatic sinusoids in the human liver the LSECs are perfectly located to survey the constituents of the blood. These cells are equipped with high-affinity receptors and an intracellular vesicle transport apparatus, enabling a remarkably efficient machinery for removal of large molecules and nanoparticles from the blood, thus contributing importantly to maintain blood and tissue homeostasis. We describe here central aspects of LSEC signature receptors that enable the cells to recognize and internalize blood-borne waste macromolecules at great speed and high capacity. Notably, this blood clearance system is a silent process, in the sense that it usually neither requires or elicits cell activation or immune responses. Most of our knowledge about LSECs arises from studies in animals, of which mouse and rat make up the great majority, and some species differences relevant for extrapolating from animal models to human are discussed. In the last part of the review, we discuss comparative aspects of the LSEC scavenger functions and specialized scavenger endothelial cells (SECs) in other vascular beds and in different vertebrate classes. In conclusion, the activity of LSECs and other SECs prevent exposure of a great number of waste products to the immune system, and molecules with noxious biological activities are effectively “silenced” by the rapid clearance in LSECs. An undesired consequence of this avid scavenging system is unwanted uptake of nanomedicines and biologics in the cells. As the development of this new generation of therapeutics evolves, there will be a sharp increase in the need to understand the clearance function of LSECs in health and disease. There is still a significant knowledge gap in how the LSEC clearance function is affected in liver disease

High-speed TIRF and 2D super-resolution structured illumination microscopy with a large field of view based on fiber optic components

Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fast, and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high, and the physical size of the instruments based on the implementation of their optics is still rather large. Here, we present our latest results towards realizing a new generation of compact, cost-efficient, and high-speed SR-SIM instruments. Tight integration of the fiber-based structured illumination microscope capable of multi-color 2D- and TIRF-SIM imaging, allows us to demonstrate SR-SIM with a field of view of up to 150 × 150 µm2 and imaging rates of up to 44 Hz while maintaining highest spatiotemporal resolution of less than 100 nm. We discuss the overall integration of optics, electronics, and software that allowed us to achieve this, and then present the fiberSIM imaging capabilities by visualizing the intracellular structure of rat liver sinusoidal endothelial cells, in particular by resolving the structure of their trans-cellular nanopores called fenestrations

Grazing incidence to total internal reflection fluorescence structured illumination microscopy enabled by a prism telescope

In super-resolution structured illumination microscopy (SR-SIM) the separation between opposing laser spots in the back focal plane of the objective lens affects the pattern periodicity, and, thus, the resulting spatial resolution. Here, we introduce a novel hexagonal prism telescope which allows us to seamlessly change the separation between parallel laser beams for 3 pairs of beams, simultaneously. Each end of the prism telescope is composed of 6 Littrow prisms, which are custom-ground so they can be grouped together in the form of a tight hexagon. By changing the distance between the hexagons, the beam separation can be adjusted. This allows us to easily control the position of opposing laser spots in the back focal plane and seamlessly adjust the spatial frequency of the resulting interference pattern. This also enables the seamless transition from 2D-SIM to total internal reflection fluorescence (TIRF) excitation using objective lenses with a high numerical aperture. In linear SR-SIM the highest spatial resolution can be achieved for extreme TIRF angles. The prism telescope allows us to investigate how the spatial resolution and contrast depend on the angle of incidence near, at, and beyond the critical angle. We demonstrate this by imaging the cytoskeleton and plasma membrane of liver sinusoidal endothelial cells, which have a characteristic morphology consisting of thousands of small, transcellular pores that can only be observed by super-resolution microscopy

Uptake and Degradation of Bacteriophages by Liver Sinusoidal Endothelial Cells

Bacteriophages (briefly, “phages”) are viruses which target bacteria, and are non-infectious to eukaryotic cells. It is estimated that more than 30 billion phages cross into the human body from the gut each day1, and eventually need to be cleared from the blood circulation. The liver plays a central role in pathogen clearance, and liver sinusoidal endothelial cells (LSECs), which form the lining of the numerous capillaries in the liver, are therefore on the front lines for this removal process. However, despite their strategic location and efficiency in removing small (<200 nm) particles2, LSECs have historically been poorly studied in terms of removal of phages. We hypothesized that LSECs play a critical role in the removal of phages from the bloodstream through endocytic uptake and lysosomal degradation, and used GFP-labeled T4 bacteriophages as a model system to study this clearance process. Uptake and trafficking of phages in primary cultured LSECs was monitored by deconvolution microscopy on both short (1 hour) and long (24 hours) term timescales, and structured illumination microscopy was used to confirm the identity of the LSECs using their unique, sub-diffraction scale morphological features: tiny holes called fenestrations. After being taken up by the cells, the phages were rapidly transported to late endosomes/lysosomes, as confirmed by colocalization studies with an LSEC-specific lysosomal vital marker. Challenging the LSEC cultures with radiolabeled phages for up to 24 hours showed that the phages were degraded about 4h after being taken up by the cells, with degradation products being increasingly released to the spent medium up to about 18h after uptake. In conclusion, our novel finding that LSECs internalize and degrade bacteriophages lends support to the hypothesis that LSECs play an important role in the clearance of blood borne phages

Cost-efficient nanoscopy reveals nanoscale architecture of liver cells and platelets

Single-molecule localization microscopy (SMLM) provides a powerful toolkit to specifically resolve intracellular structures on the nanometer scale, even approaching resolution classically reserved for electron microscopy (EM). Although instruments for SMLM are technically simple to implement, researchers tend to stick to commercial microscopes for SMLM implementations. Here we report the construction and use of a “custom-built” multi-color channel SMLM system to study liver sinusoidal endothelial cells (LSECs) and platelets, which costs significantly less than a commercial system. This microscope allows the introduction of highly affordable and low-maintenance SMLM hardware and methods to laboratories that, for example, lack access to core facilities housing high-end commercial microscopes for SMLM and EM. Using our custom-built microscope and freely available software from image acquisition to analysis, we image LSECs and platelets with lateral resolution down to about 50 nm. Furthermore, we use this microscope to examine the effect of drugs and toxins on cellular morphology

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Physical, cognitive, and mental health impacts of COVID-19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research