Protein disulfide isomerase A1 regulates fenestration dynamics in primary mouse liver sinusoidal endothelial cells (LSECs).

Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.

Effects of Polarizer-Analyzer Configurations for FTIR Spectroscopy: Implications for Multiple Polarization Algorithms.

Polarized Fourier transform infrared (p-FTIR) spectroscopy is a widely used technique for determining orientational information in thin organic materials. Conventionally, a single polarizer is placed in the path of the incident light (termed the polarizer). Occasionally, a second polarizer is also placed after the sample (referred to as the analyzer). However, this polarizer-analyzer configuration has the potential to induce polarization-dependent variances in the final spectra beyond those that are expected, i.e., the squared-cosine relationship of absorptance with respect to polarization angle is no longer accurate. These variances are due to changes in the polarization state of the transmitted light induced by the sample and have yet to be explored in the context of p-FTIR. Consequently, this study employs both theoretical and experimental approaches to identify the effects of including a second polarizer in p-FTIR analyses of anisotropic organic samples. For thin samples, the most significant spectral variance arising from only birefringence is observed on the shoulders of the dichroic peaks. By adopting a crossed polarizer configuration, it is shown that there is potential to identify anisotropy of samples that are generally considered too thick for p-FTIR analysis by exploiting this feature. Furthermore, the squared-cosine relationship of absorptance with respect to the polarization angle is also shown to be inapplicable when a second parallel-oriented polarizer is included. Accordingly, a function that accounts for the second polarizer is proposed for multiple polarization techniques.

Chronic heart failure induces early defenestration of liver sinusoidal endothelial cells (LSECs) in mice.

AIM: Chronic heart failure (CHF) is often linked to liver malfunction and systemic endothelial dysfunction. However, whether cardio-hepatic interactions in heart failure involve dysfunction of liver sinusoidal endothelial cells (LSECs) is not known. Here we characterize LSECs phenotype in early and end stages of chronic heart failure in a murine model. METHODS: Right ventricle (RV) function, features of congestive hepatopathy, and the phenotype of primary LSECs were characterized in Tgαq*44 mice, with cardiomyocyte-specific overexpression of the Gαq protein, at the age of 4- and 12-month representative for early and end-stage phases of CHF, respectively. RESULTS: 4- and 12-month-old Tgαq*44 mice displayed progressive impairment of RV function and alterations in hepatic blood flow velocity resulting in hepatic congestion with elevated GGT and bilirubin plasma levels and decreased albumin concentration without gross liver pathology. LSECs isolated from 4- and 12-month-old Tgαq*44 mice displayed significant loss of fenestrae with impaired functional response to cytochalasin B, significant changes in proteome related to cytoskeleton remodeling, and altered vasoprotective function. However, LSECs barrier function and bioenergetics were largely preserved. In 4- and 12-month-old Tgαq*44 mice, LSECs defenestration was associated with prolonged postprandial hypertriglyceridemia and in 12-month-old Tgαq*44 mice with proteomic changes of hepatocytes indicative of altered lipid metabolism. CONCLUSION: Tgαq*44 mice displayed right-sided HF and altered hepatic blood flow leading to LSECs dysfunction involving defenestration, shift in eicosanoid profile, and proteomic changes. LSECs dysfunction appears as an early and persistent event in CHF, preceding congestive hepatopathy and contributing to alterations in lipoprotein transport and CHF pathophysiology.

The role of the cortex in indentation experiments of animal cells.

We present a model useful for interpretation of indentation experiments on animal cells. We use finite element modeling for a thorough representation of the complex structure of an animal cell. In our model, the crucial constituent is the cell cortex-a rigid layer of cytoplasmic proteins present on the inner side of the cell membrane. It plays a vital role in the mechanical interactions between cells. The cell cortex is modeled by a three-dimensional solid to reflect its bending stiffness. This approach allows us to interpret the results of the indentation measurements and extract the mechanical properties of the individual elements of the cell structure. During the simulations, we scan a broad range of parameters such as cortex thickness and Young's modulus, cytoplasm Young's modulus, and indenter radius, which define cell properties and experimental conditions. Finally, we propose a simple closed-form formula that approximates the simulated results with satisfactory accuracy. Our formula is as easy to use as Hertz's function to extract cell properties from the measurement, yet it considers the cell's inner structure, including cell cortex, cytoplasm, and nucleus.

Endothelial glycocalyx detection and characterization by means of atomic force spectroscopy: Comparison of various data analysis approaches.

In recent years, atomic force spectroscopy (AFS) has been used to detect and characterize the endothelial glycocalyx (eGlx) in in vitro and ex vivo experiments. Several analysis methods were proposed, which differ not only in the numerical implementations, but also in physical models of glycocalyx description. Therefore, it is difficult to directly relate the experiments performed by different groups. In this work, we compared different models used for quantitative analysis of atomic force spectroscopy datasets recorded for eGlx. To capture glycocalyx at various structural conditions, we used basic enzymatic protocols for glycocalyx removal and restoration in human aortal endothelial cells (HAEC). Nanoindentation experiments for this model system were performed for (i) untreated cells, (ii) for cells after heparinase incubation, which enzymatically removes glycocalyx, (iii) for cells with successive heparin treatment, which partially restores the glycocalyx layer. Analysis of nanoindentation data was performed using different models: (a) a single-layer contact mechanics, (b) a double-layer model contact mechanics, (c) a polymer "brush" two-layer model based on the Alexander - de Gennes theory and (d) a simple single-layer "mechanical spring" model. Although different physical parameters are evaluated in methods (a-d), we show that all approaches revealed similar qualitative changes of the glycocalyx layer, which reflected the processes of glycocalyx degradation and its partial restoration. This paper may facilitate a direct comparison of past and future glycocalyx oriented AFS experiments that are analysed with different approaches.