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

The wHole Story About Fenestrations in LSEC

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

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

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50−300 nm in diameter, which is below the resolution limit of a conventional light microscopy. To date, there is no standardized method of fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, a semi-automatic (threshold-based) method, and an automatic method based on user-friendly open source machine learning software. Images were obtained using three super resolution techniques – atomic force microscopy (AFM), scanning electron microscopy (SEM), and structured illumination microscopy (SIM). Parameters describing fenestrations such as diameter, area, roundness, frequency, and porosity were measured. Finally, we studied the user bias by comparison of the data obtained by five different users applying provided analysis methods

Novel insights into the fenestrated scavenger endothelium of the liver sinusoid

The sinusoids (specialized small blood vessels) of the liver are covered by endothelium (blood vessel wall cells) with open transcellular pores (holes that go from one side to the other) called fenestrations. This allows for efficient bidirectional transfer of solutes between the blood and the hepatocytes (main metabolic liver cell). These fenestrations can disappear or reduce in number and size in disease states or in ageing. We therefore sought to map the literature on compounds, that affect these fenestrations, and to hypothesize how the mechanism regulating them operates. The fenestrations are unevenly distributed along the sinusoid, with there being a greater fraction of the cell surfaces covered by these pores towards the end (the pericentral area) compared with the start of the vessel (the periportal area). There are also lymphatic vessels in the periportal area, in a space behind the portal vein and hepatic artery, which is often omitted from consideration in anatomical illustrations and flow models of the liver. We therefore sought to make a digital model at the single sinusoid level, including these ultrastructural details, to assess their influence on fluid flow parameters. The liver endothelium is a scavenging endothelium, that is to say high capacity waste removal cells specialized in macromolecular and nanoparticle sized waste from the blood stream. Albumin is the single most abundant protein in blood, with chemically modified forms of it being found in several pathologies, especially diabetes or liver disease. It was found by a Japanese research group, Iwao et al., that when albumin is highly oxidized, it is rapidly removed from the blood stream, mainly by the liver. The properties of the liver sinusoidal endothelium as a scavenger endothelium, and the clearance kinetics led us to believe this was done by the liver sinusoidal endothelium and its stabilin receptors, because of the functions of these in respect to other modified albumins. We indeed found that this was the case. The analysis of fenestrations from microscopy images is a laborious process, and contains the possibility of introducing user bias into quantifications. We assessed three different methods of image analysis for the purpose of quantifying fenestration parameters. These were manual, semi-automated/thresholding based, and fully automated/neural network based approaches. The manual classification method had little bias with regards to number, whilst showing significant user bias for diameter/size of fenestration. The semi-automated was the least biased with regard to diameter/size, but significantly biased with regards to number. The fully automated also showed considerable user bias for all parameters, however it can be used for batch processing. The methods are roughly ordered by speed (manual, semi-automated, fully automated), with regards to larger data sets

In vitro and in vivo studies of a novel blood brain barrier permeabilizing peptide K16ApoE

Brain metastasis is a common and severe complication arising in up to 40 % of cancer patients. The prognoses for patients with brain metastases are dismal, with a median treated survival of around 10 months. Treatment of brain metastases is mostly limited to surgery and radiotherapy, as an intact blood brain barrier (BBB) effectively protects smaller tumors from chemotherapeutic agents. This means that even if there would be effective chemotherapy treatment regimens for metastases elsewhere in the body, brain metastases would not be affected by such strategies until late stages of cancer development, when the BBB is disrupted. The prevalence of metastatic melanoma has been increasing steadily the last years accounting for 10 % of all brain metastatic cancers. Advanced stage melanoma patients are commonly treated with targeted inhibitors, such as vemurafenib (a small molecule inhibitor, tailored to have a higher affinity to the mutated BRAF kinase). However an intact BBB then prevents the brain metastases from being affected by vemurafenib as well as most chemotherapeutic agents. Based on this a novel BBB permeabilizing peptide was of great interest for the work done in this thesis. The peptide is a combination of the low density lipoprotein receptor binding domain of ApolipoproteinE, and a 16 Lysine tail, which was named K16ApoE. In this study the in vitro mechanisms of K16ApoE was studied. Thereafter the ability of opening the BBB by the peptide was investigated, followed by a pilot study combining vemurafenib and K16ApoE in a small animal tumor model. Cell lines exposed to K16ApoE showed a high degree of cytotoxicity at prolonged exposures of high doses, by affecting cell viability and causing lytic cell death. However lower doses were in general tolerated by the cells. Scanning electron microscopy showed that the peptide caused damages to confluent endothelial cell monolayers likely by inducing lytic cell death. In contrast transmission electron microscopy showed the peptide did not affect cell-cell junctions. DCE-MRI on mice after intravenous administration of 200 μg of K16ApoE showed that the BBB was opened at already 10 minutes after injection, and was permeable at least 2 hours after injection. In animals given 100 μg of peptide the BBB was also permeabilized 10 minutes after injection of the peptide. 10 The pilot treatment study using the xenograft mouse model for brain-metastatic melanoma, indicated a treatment effect when co-administering the peptide and a chemotherapeutic agent. The promising results of this study indicate the potential value of K16ApoE as a therapeutic strategy in brain metastatic cancers. Following this study full scale pre-clinical studies will follow, and based upon those the peptide may move forward towards clinical safety studies, and clinical studies in turn

From fixed-dried to wet-fixed to live-comparative super-resolution microscopy of liver sinusoidal endothelial cell fenestrations

Fenestrations in liver sinusoidal endothelial cells (LSEC) are transcellular nanopores of 50–350 nm diameter that facilitate bidirectional transport of solutes and macromolecules between the bloodstream and the parenchyma of the liver. Liver diseases, ageing, and various substances such as nicotine or ethanol can negatively influence LSECs fenestrations and lead to defenestration. Over the years, the diameter of fenestrations remained the main challenge for imaging of LSEC in vitro. Several microscopy, or rather nanoscopy, approaches have been used to quantify fenestrations in LSEC to assess the effect of drugs and, and toxins in different biological models. All techniques have their limitations, and measurements of the “true” size of fenestrations are hampered because of this. In this study, we approach the comparison of different types of microscopy in a correlative manner. We combine scanning electron microscopy (SEM) with optical nanoscopy methods such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy. In addition, we combined atomic force microscopy (AFM) with SEM and STED, all to better understand the previously reported differences between the reports of fenestration dimensions. We conclude that sample dehydration alters fenestration diameters. Finally, we propose the combination of AFM with conventional microscopy that allows for easy super-resolution observation of the cell dynamics with additional chemical information that can be traced back for the whole experiment. Overall, by pairing the various types of imaging techniques that provide topological 2D/3D/label-free/chemical information we get a deeper insight into both limitations and strengths of each type microscopy when applied to fenestration analysis

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially