Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors

Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure

Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterise the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs).Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterised by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (GSH/GSSH ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, co-incubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor (ABH, 2(S)-amino-6-boronohexanoic acid).In the Tgαq*44 murine model of HF, systemic endothelial dysfunction accelerates erythropathy and, conversely, erythropathy may contribute to endothelial dysfunction. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic endothelial dysfunction in HF. In particular, targeting RBC arginase may represent a novel treatment strategy for systemic endothelial dysfunction in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic endothelial dysfunction in HF.Endothelial dysfunction (ED) and red blood cell distribution width (RDW) both have prognostic value for heart failure (HF), but it is not known whether these pathologies are related. We comprehensively characterized endothelial and RBC functional status in a unique murine model of chronic heart failure with a prolonged time course of HF progression. Our results suggest that ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. Accordingly, erythropathy in HF reflects ED and involves various changes (in functional, structural, nanomechanical, and biochemical levels) that could have diagnostic and therapeutic significance for HF

Assassment of nanomechanical changes of cells including the surface layer of glycocalyx.

W niniejszej pracy przedstawiono analizę danych eksperymentalnych uzyskanych dla komórek śródbłonka i komórek nowotworowych, zmierzonych metodą nanoindentacji próbnikiem kulowym. Korzystając z modelu Hertza oraz teorii Aleksander-de Gennes wykonano analizę krzywych indentacji. Otrzymane wartości przedstawiono w postaci przestrzennych map analizowanych parametrów nanomechanicznych, tj. modułu elastyczności oraz długości i gęstości szczepienia glikokaliksu, jak również odpowiadającym im histogramom obrazującym rozkład Logarytmiczny Normalny otrzymanych wartości. Następnie na podstawie przeprowadzonej analizy statystycznej oraz analizie wariancji ANOVA wyznaczono wartości średnie modułu elastyczności badanych komórek jak również wartości średnie długości i gęstości szczepienia włókien glikokaliksu. Otrzymane wyniki wskazują, iż pod wpływem hiperglikemii następuje wzrost sztywności (wzrost modułu elastyczności) oraz redukcja długości glikokaliksu w komórkach śródbłonka (linia HUVEC) . Natomiast dla komórek nowotworowych płaskonabłonkowego raka płuc (linia A549) w warunkach hiperglikemii moduł elastyczności jest znacząco mniejszy niż dla komórek śródbłonka. Dodatkowo dla linii nowotworowej otrzymane parametry glikokaliksu wskazują na brak redukcji glikokaliksu w warunkach hiperglikemii.This work presents the analysis of experimental data obtained for endothelial cells (HUVEC line) and tumor cells (A549 line), measured by using the nanoindentation method with a spherical AFM probe. By using the Hertz model and Alexander-de Gennes theory of polymer brushes an analysis of the indentation curves was performed. The obtained values are presented in the form of spatial maps of the analyzed nanomechanical parameters, i.e. the elastic modulus and the length and the grafting density of glycocalyx layer. The all data was presented in the form of histograms illustrating the Logarithmic Normal distribution of the obtained values. Next, on the basis of statistical analysis and ANOVA test, the mean values of the elastic modulus of the studied cell were determined as well as the mean values of the length and grafting density of glycocalyx brush.The obtained results indicate that under the influence of hyperglycemia, there is an increase in stiffness (increase of the elastic modulus) and reduction of the length of glycocalyx in endothelial cells (HUVEC line). However, for tumor cells (line A549) incubated in hyperglycemic conditions, the elastic modulus is significantly smaller than for endothelial cells. In addition, for the tumor cells, the obtained parameters of glycocalyx indicate an absence of reduction of glycocalyx under hyperglycemia condition

The connection between the membrane lipid composition and cell elasticity

Celem niniejszej pracy było wykazanie, że modyfikacja składu lipidów błonowych na skutek działania oksysteroli wpływa na elastyczność komórek, co ma bezpośrednie odzwierciedlenie w upośledzeniu ich funkcji biologicznych. W badaniach zastosowano wybrane oksysterole (pochodne cholesterolu): 7-ketocholesterol (7-K), 7α-hydroksycholesterol (7α-OH) oraz 7β-hydroksycholesterol (7β-OH), którymi traktowano czerwone krwinki (erytrocyty) oraz komórki glejaka (linia komórkowa U-251). Stężenia wszystkich oksysteroli, z którymi inkubowano erytrocyty wynosiły 20 μM (oraz dodatkowe 50 μM dla 7-K). Komórki glejaka inkubowano z 25 μM i 50 μM 7-ketocholesterolem. Jednoczesny pomiar morfologii i elastyczności czerwonych krwinek oraz komórek nowotworowych wykonano metodami bazującymi na mikroskopii sił atomowych AFM. W tym celu zastosowano następujące tryby pracy mikroskopu: Imaging, Force Mapping oraz Quantitative Imaging. Otrzymano obrazy topografii pozwalające na monitorowanie zmian kształtu komórek pod wpływem oksysteroli, jak również mapy elastyczności na podstawie których przeprowadzono ilościową analizę zmian modułu elastyczności komórek. Utworzone histogramy modułu elastyczności przedstawiały rozkład normalny z pojedynczym lub wielokrotnymi pikami, świadczącymi o złożoności zmian elastyczności komórek wywołanych oksysterolami. Istotność statystyczną zbadano przy użyciu jednoczynnikowego testu ANOVA, a następnie testami Bonferroniego. Ponadto dla badanych erytrocytów wykonano także analizę współczynnika kształtu. Otrzymane wyniki wskazują, iż zmiana struktury lipidowej na skutek działania oksysteroli prowadzi do znacznych zmian w elastyczności zarówno erytrocytów jak i komórek glejaka. Substancjami powodującymi wzrost sztywności w obu typach komórek, zarówno żywych jak i utrwalonych, były 7-ketocholesterol oraz 7β-hydroksycholesterol. Natomiast spadek sztywności erytrocytów zaobserwowano po zastosowaniu 7α-hydroksycholesterolu. Przedstawione w pracy zmiany elastyczności erytrocytów jak i komórek glejaka pogłębiają wiedzę na temat wpływu procesów utleniania lipidów w przebiegu procesów chorobowych.The aim of this study was to show that the modification of membrane lipid composition caused by the action of oxysterols affects cell elasticity, which is directly reflected in the impairment of biological functions of cells. Red blood cells (erythrocytes) and glioblastoma cells (line cellular U-251) were treated with selected oxysterols (cholesterol derivatives): 7-ketocholesterol (7-K), 7α-hydroxycholesterol (7α-OH) and 7β-hydroxycholesterol (7β-OH). For erythrocytes, the concentrations of all oxysterols was 20 μM. For 7K, an additional concentration of 50 μM was tested. Glioblastoma cells were incubated with 25 μM and 50 μM of 7-K. Simultaneous measurement of morphology and elasticity of red blood cells and cancer cells was performed using methods based on atomic force microscopy AFM. For this purpose, the following microscope operating modes were used: imaging, force mapping and quantitative imaging. Applied operating modes enabled to obtain topographic images, which evidenced the shape changes of cell under the influence of oxysterols as well as elasticity maps for quantitative monitoring of cell elastic modulus. The derived elasticity modulus histograms presented normal distribution with single or multiple peaks what indicates a complex character of changes in cell elasticity caused by oxysterols. In addition, the aspect ratio analysis was performed for the erythrocytes. The obtained results indicate that the change in lipid structure due to the action of oxysterols leads to significant modification in the elasticity of both erythrocytes and glioblastoma cells. 7-ketocholesterol and 7β-hydroxycholesterol were the substances that caused an statistically significant increase in stiffness in both living and fixed cells. In all cases statistical significance was tested using a one-way ANOVA test followed by Bonferroni tests. In contrast, the decrease of cell stiffness was observed for 7α-hydroxycholesterol. The changes in the elasticity of erythrocytes and glioblastoma cells presented in the Thesis give insights on the impact of lipid oxidation processes in the course of disease processes

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