Intracellular organelles remodeling in myocardial endotheliocytes in COVID-19: an autopsy-based study.

It is known that the unfavorable outcome in patients infected with SARS-CoV-2 may be associated with the development of complications caused by heart damage due to the direct virus action. The mechanism of these cardiovascular injuries caused by SARS-CoV-2 infection has not been fully understood; however, the study of COVID-19-associated myocardial microcirculatory dysfunction can represent the useful strategy to solving this challenge. Thus, here we aimed to study the ultrastructural organization of endothelial cells of myocardial capillaries in patients with COVID-19. The morphology of endotheliocytes of the myocardial blood capillaries in patients with COVID-19 was studied on cardiac autopsy material using transmission electron microscopy. The endotheliocytes of myocardial capillaries in patients with COVID-19 were characterized by the abundant rough endoplasmic reticulum (ER) membranes, the Golgi complex, and free polysomal complexes of ribosomes and lipids. The presence of double membrane vesicles with virions and zippered ER was detected in the cytoplasm of endotheliocytes. The revealed endothelial ultrastructural changes indicate the remodeling of intracellular membranes during SARS-CoV-2 infection. Our findings confirm the formation of virus-induced structures in myocardial endothelial cells considered critical for viral replication and assembly. The data may elucidate the mechanisms of endothelial dysfunction development in patients with COVID-19 to provide potential targets for drug therapy.

Lithium salts cytotoxicity and accumulation in melanoma cells in vitro.

Boron neutron capture therapy is a perspective selective technology for the destruction of cancer cells, while the use of lithium instead of boron may represent a new and promising vector for the development of neutron capture therapy (NCT). The aim of the study was a comparative assessment of the cytotoxicity of various lithium salts, as well as an analysis of the accumulation of lithium in tumor cells in vitro to determine the possibility of using lithium in NCT. The cytotoxicity of lithium salts was determined using MTT-test and colony forming assay on human fibroblasts BJ-5ta, human skin melanoma SK-Mel-28, and mouse skin melanoma B16 cell lines. An assessment of lithium concentration in cells was performed using inductively coupled plasma atomic emission spectrometry. Our results showed that three different lithium salts at a concentration of 40 µg/ml are not toxic for both tumor and normal cells. The highest uptake values were obtained on murine melanoma B16 cells when exposed to lithium carbonate (0.8 µg/106 cells); however, human melanoma SK-Mel-28 cells effectively accumulated both lithium carbonate and lithium citrate (about 0.46 µg/106 cells for two salts). Thus, our results demonstrate a range of non-toxic doses of lithium salts and a high uptake of lithium by tumor cells, which indicates the possibility to use the lithium in NCT.

Autophagy and vesicular trafficking in human uveal melanoma: A histopathological study.

Uveal melanoma is an ocular tumor with a high risk of developing metastases. The endo-lysosomal system can affect the melanoma progression by accelerating and facilitating invasion or metastasis. This study aims to conduct comparative analysis of normal choroidal melanocytes and uveal melanoma cells ultrastructure with a focus on intracellular transport system, and to examine the patterns of autophagy- and vesicular trafficking-related proteins expression in a case series of uveal melanomas. Transmission electron microscopy was used to assess the ultrastructure of normal choroidal melanocytes and uveal melanoma cells. The expression levels of autophagy- and vesicular trafficking-related proteins in three histological types of uveal melanoma were analyzed by immunofluorescence staining. Electron microscopy results showed that the autophagic vacuoles were more abundant in normal choroidal melanocytes, than in uveal melanoma cells. The normal choroidal melanocytes were characterized by active intracellular vesicular trafficking; however, the proportion of caveolae was higher in uveal melanoma cells. The spindle type of tumor was characterized by a high expression levels of LC3 beta, while Rab7 and Rab11 proteins expression was significantly up-regulated in the mixed-type tumor cells. The results indicate that uveal melanoma cells probably have lower basal levels of autophagy and higher receptor-mediated endocytic trafficking-associated with caveolae than normal choroidal melanocytes. RESEARCH HIGHLIGHTS: The autophagic vacuoles are abundant in normal choroidal melanocytes. Uveal melanoma cells are characterized by a high proportion of caveolae. The high expression levels of LC3 beta were revealed in a spindle type of tumor, while Rab7 and Rab11 proteins expression was up-regulated in the mixed-type tumor cells.

An Experimental Model of Proton-Beam-Induced Radiation Dermatitis In Vivo.

Radiation dermatitis (RD) is one of the most common side effects of radiation therapy. However, to date, there is a lack of both specific treatments for RD and validated experimental animal models with the use of various sources of ionizing radiation (IR) applied in clinical practice. The aim of this study was to develop and validate a model of acute RD induced using proton radiation in mice. Acute RD (Grade 2-4) was obtained with doses of 30, 40, and 50 Gy, either with or without depilation. The developed model of RD was characterized by typical histological changes in the skin after irradiation. Moreover, the depilation contributed to a skin histology alteration of the irradiated mice. The assessment of animal vital signs indicated that there was no effect of proton irradiation on the well-being or general condition of the animals. This model can be used to develop effective therapeutic agents and study the pathogenesis of radiation-induced skin toxicity, including that caused by proton irradiation.

Study of Lithium Biodistribution and Nephrotoxicity in Skin Melanoma Mice Model: The First Step towards Implementing Lithium Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is one of the promising treatment methods for malignant melanoma. The main issue of this technology is the insufficient selectivity of 10B accumulation in tumor cells. As a result of the neutron absorption by boron, an 84% energy release occurred within the cell by the nuclear reaction 10B (n, α)7Li, which lead to tumor cell death. The use of lithium instead of boron brings a new unique opportunity-local 100% energy release-since all products of the 6Li (n, α)3H reaction have high linear energy transfer characteristics. The aim of this study was to determine the concentrations of Li in the tumor, skin, blood, brain and kidney in experimental animals with B16 melanoma and to analyze the potential Li toxicity after lithium carbonate administration at single doses of 300 and 400 mg/kg. Lithium carbonate was chosen since there is a long-term experience of its use in clinical practice for the treatment of psychiatric disorders. The inductively coupled plasma atomic emission spectrometry was used to evaluate Li concentrations in tissue samples. The accumulation efficiency of Li in the tumor was the highest at a time point of 30 min (22.4 µg/g; at a dose of 400 mg/kg). Despite the high lithium accumulation in the kidneys, the pathological changes in kidney tissues were not found. Thus, lithium may actually be used for the Li-NCT development and future studies can be conducted using 6Li and following irradiation of tumor cells using the schemes of lithium administration tested in this work.

Lithium Enhances Autophagy and Cell Death in Skin Melanoma: An Ultrastructural and Immunohistochemical Study.

Lithium is an inhibitor of glycogen synthase kinase 3 beta, which is traditionally used in the treatment of bipolar disorders and has antitumor effects. The aim of the current study was to determine if lithium salt causes autophagy and apoptosis in skin melanoma cells to enhance cell death. Light microscopy, transmission electron microscopy, immunohistochemistry, and immunofluorescence were used to study the mechanism of action of lithium carbonate in B16 melanoma cells in vivo. Proliferating cell nuclear antigen immunofluorescence assay revealed that the proliferation of B16 melanoma cells was suppressed by lithium treatment for 7 days. Electron microscopy demonstrated a significant increase in the number of autophagic vacuoles in lithium-treated cells relative to control. In addition, levels of autophagy markers LC3 beta and LAMP1 found in lithium-treated tumor xenografts were higher than levels of these markers in the control tumors. Lithium induced caspase-3 expression and apoptotic cell death in tumor cells. Thus, lithium carbonate is the compound that inhibits cell proliferation and stimulates cell death in melanoma cells through induction of autophagy and apoptosis. Stimulation of autophagy by lithium could contribute to the development of autophagic cell death in tumor cells.

Influence of distant tumor growth and lithium treatment on ultrastructural organization of kidney proximal tubules.

Tumor growth causes significant metabolic disturbances, tissue damage and the accumulation of toxic metabolites in the blood. The kidney is an organ with highly developed capillary network and therefore can be exposed to toxic metabolites. Here, the proximal renal tubule cells were studied by immunohistochemistry and electron microscopy, on a model of hepatocellular carcinoma-29 growth in the thigh of CBA mice and lithium carbonate treatment. An increase of autophagy markers (LC3 and LAMP-1) expression was revealed under conditions of distant tumor growth and especially after lithium carbonate treatment. Under conditions of distant tumor we found decrease of numerical density of endosomes and dense apical tubules in the apical part of the cells. In the perinuclear cell compartment, there were swelling of mitochondria and a decrease in their cristae, a decrease of volume density of rough endoplasmic reticulum and the presence of autophagosomes and autolysosomes. The use of lithium carbonate led to an increase of autophagic structures volume density and of dense apical tubules numerical density in the proximal tubule cells. It is possible that the activation of autophagy by lithium can promote an increase in protein recycling in the proximal tubule cells.

Method of Measuring High-LET Particles Dose.

In boron neutron capture therapy, the total absorbed dose is the sum of four dose components with different relative biological effectiveness (RBE): boron dose, "nitrogen" dose, fast neutron dose and γ-ray dose. We present a new approach for measuring the first three doses. In this work, we provide the details of this method of dose measurement and results when this proposed method is employed.

Microvasculature in hepatocellular carcinoma: An ultrastructural study.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most vascularized tumor types, and is characterized by development of heterogeneous immature vessels with increased permeability. Here, we analyzed morphology and vascular permeability-related structures in endothelial cells of HCC microvessels. METHODS: Small (Type I) and large (Type II) peritumoral blood microvessels were assessed in HCC-bearing mice. By transmission electron microscopy, endothelial cell cytoplasm area, free transport vesicles, vesiculo-vacuolar organelles and clathrin-coated vesicles were measured. RESULTS: The phenotypic changes in the HCC microvessels included presence of sinusoidal capillarization, numerous luminal microprocesses and abnormal luminal channels, irregular dilatations of interendothelial junctions, local detachment of basement membranes and widened extracellular space. Endothelial cells Type I microvessels showed increased vesicular trafficking-related structures. CONCLUSION: Ultrastructural characteristics of microvessels Type I can associate with HCC new-formed microvessels. The morphological changes observed in HCC microvessels might explain the increased transcellular and paracellular permeability in HCC endothelial cells.

In Vitro and In Vivo Evaluation of Fluorescently Labeled Borocaptate-Containing Liposomes.

Boron neutron capture therapy (BNCT), a binary cancer therapeutic modality, has moved to a new phase since development of accelerator-based neutron sources and establishment of BNCT centers in Finland and Japan. That stimulated efforts for better boron delivery agent development. As liposomes have shown effective boron delivery properties and sufficient tumor retention, fluorescent liposome labelling may serve as a rapid method to study initial ability of newly synthesized liposomes to be captured by tumor cells prior to experiments on boron accumulation and neutron irradiation. In this work, we studied the accumulation and biodistribution of pegylated liposomes with encapsulated borocaptate (BSH) and a fluorescent label (Nile Red) in U87 (human glioblastoma), SW-620 (human colon carcinoma), SK-MEL-28 (human melanoma), FetMSC (mesenchymal human embryo stem cells), and EMBR (primary embryocytes) cell lines as well as an orthotopic xenograft model of U87 glioma in SCID mice. Results indicate that fluorescent microscopy is effective at determining the intracellular localization of the liposomes using a fluorescent label. The synthesized, pegylated liposomes showed higher accumulation in tumors compared to normal cells, with characteristic concentration peaks in SW-620 and U87 cell lines, and provided in vivo tumor selectivity with several-fold higher tumor tissue fluorescence at the 6-h timepoint. Graphical abstract Fluorescent images of U-87 glioma cells after 24 hours of incubation with BSH-containing liposomes labeled with lipophilic Nile Red (red color)and water-soluble FITC-Dextran (green color); cell nuclei in blue color (DAPI-staining) (×400). Scale bar is 50 µm. Fluorescent labelling serves as anexpress method to study liposome delivery efficiency prior to boron accumulation evaluation and BNCT irradiation experiments.

Ultrastructure of the kidney filtration barrier in conditions of distant tumor growth and lithium treatment.

Toxic products generated in the process of tumor growth and as a result of treatment may cause damage to organs distant from the tumor growth, including kidneys. The aim of the work was to identify ultrastructural changes in the components of kidney filtration barrier in conditions of distant tumor growth and lithium carbonate treatment. Tumor growth was induced by the inoculation of hepatocellular carcinoma-29 cells into the right thigh muscle of CBA mice. Lithium carbonate was injected along the periphery of the tumor. Ultrastructural analysis of podocytes and endotheliocytes of glomerular capillaries in the dynamics of tumor growth and lithium carbonate treatment was carried out. Under conditions of distant tumor growth, ultrastructural changes of the kidney filtration barrier were noted. Podocyte hypertrophy was detected, the width of foot process and glomerular membrane were increased. The number of fenestrae was decreased and cell hypertrophy was developed in the structure endothelium of glomerular capillary. Lithium carbonate had some protective effect on podocytes, but caused a significant hypertrophy of endotheliocytes leading to glomerular capillary occlusion.

SGLT2 Inhibitor Empagliflozin and DPP4 Inhibitor Linagliptin Reactivate Glomerular Autophagy in db/db Mice, a Model of Type 2 Diabetes.

Recent data have indicated the emerging role of glomerular autophagy in diabetic kidney disease. We aimed to assess the effect of the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and their combination, on glomerular autophagy in a model of type 2 diabetes. Eight-week-old male db/db mice were randomly assigned to treatment with empagliflozin, linagliptin, empagliflozin-linagliptin or vehicle for 8 weeks. Age-matched non-diabetic db/+ mice acted as controls. To estimate glomerular autophagy, immunohistochemistry for beclin-1 and LAMP-1 was performed. Podocyte autophagy was assessed by counting the volume density (Vv) of autophagosomes, lysosomes and autolysosomes by transmission electron microscopy. LC3B and LAMP-1, autophagy markers, and caspase-3 and Bcl-2, apoptotic markers, were evaluated in renal cortex by western blot. Vehicle-treated db/db mice had weak glomerular staining for beclin-1 and LAMP-1 and reduced Vv of autophagosomes, autolysosomes and lysosomes in podocytes. Empagliflozin and linagliptin, both as monotherapy and in combination, enhanced the areas of glomerular staining for beclin-1 and LAMP-1 and increased Vv of autophagosomes and autolysosomes in podocytes. Renal LC3B and Bcl-2 were restored in actively treated animals. LAMP-1 expression was enhanced in the empagliflozin group; caspase-3 expression decreased in the empagliflozin-linagliptin group only. Mesangial expansion, podocyte foot process effacement and urinary albumin excretion were mitigated by both agents. The data provide further explanation for the mechanism of the renoprotective effect of SGLT2 inhibitors and DPP4 inhibitors in diabetes.

Empagliflozin alleviates podocytopathy and enhances glomerular nephrin expression in db/db diabetic mice.

BACKGROUND: Modern guidelines recommend sodium-glucose cotransporter-2 (SGLT2) inhibitors as the preferred antihyperglycemic agents for patients with type 2 diabetes and chronic kidney disease. However, the mechanisms underlying the renal protective effect of SGLT2 inhibitors are not fully understood. AIM: To estimate the effect of the SGLT2 inhibitor, empagliflozin (EMPA), on the structure of podocytes and nephrin expression in glomeruli in db/db diabetic mice. METHODS: We treated 8-wk-old male db/db mice with EMPA (10 mg/kg/d) or vehicle for 8 wk. Age-matched male db/+ mice were included as non-diabetic controls. Parameters of body composition, glycemic and lipid control, and plasma concentrations of leptin, insulin and glucagon were assessed. We evaluated renal hypertrophy as kidney weight adjusted to lean mass, renal function as plasma levels of creatinine, and albuminuria as the urinary albumin-to-creatinine ratio (UACR). Renal structures were studied by light and transmission electron microscopy with a focus on mesangial volume and podocyte structure, respectively. Glomerular nephrin and transforming growth factor beta (TGF-ß) were assessed by immunohistochemistry. RESULTS: Severe obesity and hyperglycemia developed in db/db mice prior to the start of the experiment; increased plasma concentrations of fructosamine, glycated albumin, cholesterol, leptin, and insulin, and elevated UACR were detected. Mesangial expansion, glomerular basement membrane thickening, and increased area of TGF-ß staining in glomeruli were revealed in vehicle-treated mice. Podocytopathy was manifested by effacement of foot processes; nephrin-positive areas in glomeruli were reduced. EMPA decreased the levels of glucose, fructosamine and glycated albumin, UACR, kidney hypertrophy, mesangial expansion, glomerular basement membrane thickening, and glomerular TGF-ß staining, alleviated podocytopathy and restored glomerular staining of nephrin. CONCLUSION: These data indicate that EMPA attenuates podocytopathy in experimental diabetic kidney disease. The anti-albuminuric effect of EMPA could be attributed to mitigation of podocyte injury and enhancement of nephrin expression.

Lithium effects on vesicular trafficking in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most commonly malignant tumors worldwide, characterized by the presence of many heterogeneous molecular cell events that contribute to tumor growth and progression. Endocytic processes are intimately involved in various pathological conditions, including cancer, since they interface with various cellular signaling programs. The ability of lithium to induce cell death and autophagy and affect cell proliferation and intracellular signaling has been shown in various experimental tumor models. The aim of this study was to evaluate the effects of lithium on vesicular transport in hepatocellular carcinoma cells. Using transmission electron microscopy we have characterized the endocytic apparatus in hepatocellular carcinoma-29 (HCC-29) cells in vivo and detailed changes in endocytotic vesicles after 20 mM lithium carbonate administration. Immunofluorescent analysis was used to quantify cells positive for EEA1-positive early endosomes, Rab11-positive recycling endosomes and Rab7-positive late endosomes. Lithium treatment caused an increase in EEA1- and Rab11-positive structures and a decrease in Rab7-positive vesicles. Thus, lithium affects diverse endocytic pathways in HCC-29 cells which may modulate growth and development of hepatocellular carcinoma.