Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.

Our previous findings performed in rat tissues demonstrated that intermediate filament nestin is expressed in endothelial cells of newly formed blood vessels of developing organs and neural transplants. The aim of the present study was to identify other cellular markers expressed in nestin-positive (nestin+) blood vessels. To reach this goal we performed double immunofluorescent study to co-localize nestin with endothelium-specific markers (CD31, CD34 II, vimentin) or markers of perivascular cells (GFAP, SMA) in paraffin-embedded sections of normal human brain tissue, low- and high-grade gliomas, postinfarcted heart and samples of non-neural tumours. Our findings documented that all the samples examined contained blood vessels with different ratio of nestin+ endothelial cells. Double immunostaining provided unambiguous evidence that endothelial cells expressed nestin and allowed them to distinguish from other nestin+ elements (perivascular astrocytic endfeet, undifferentiated tumour cells, smooth muscle cells and pericytes). Nestin+ endothelium was not confined only to newly formed capillaries but was also observed in blood vessels of larger calibres, frequently in arterioles and venules. We conclude that nestin represents a reliable vascular marker that is expressed in endothelial cells. Elevation of nestin expression likely corresponds to reorganization of intermediate filament network in the cytoskeleton of endothelial cells in the course of their maturation or adaptation to changes in growing tissues.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor—Lys05—with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

Purpose: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO₂ P25), are among the most produced nanomaterials worldwide. The broad use of TiO₂ P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO₂ P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO₂ P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO₂ P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO₂ P25. We used four techniques for TiO₂ P25 dispersion. We estimated the colloid stability of TiO₂ P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO₂ P25 (0– 100 μg.mL^{− 1}) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO₂ P25. Under these conditions, TiO₂ P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO₂ P25 in A549 cells were met, similar results on the biological effects of TiO₂ P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO₂ P25 preparation for toxicity testing in A549 cells. The results presented here on TiO₂ P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO₂ P25 as a reference material.