Microglia accumulate in malignant gliomas and play a pivotal role in tumor progression. Using single‑cell RNA sequencing studies researchers have probed gene expression in the myeloid cells in experimental gliomas at relatively late stages of the tumor development. Therefore, the early changes in gene expression in microglia in response to glioma are not fully characterized. We have previously reported distinct profiles of gene expression in the rat primary microglia cultures treated for 6 hours with either rat C6 glioma‑conditioned medium (GCM) or lipopolysaccharide. In the current study, using RNA‑seq, we characterized the transcriptional response of rat primary microglia to GCM in vitro at different time‑points: 6 h, 24 h, and 48 h, as compared to the control treated for 6 h with its own medium. We observed that during the GCM treatment gene expression changes in a biphasic, swing‑like pattern. This includes the genes involved in innate immune response, which are mostly down‑regulated at 6 h by the GCM treatment, as compared to the time‑matched control, and subsequently up‑regulated at 48 h, as compared to the earlier time‑points of the GCM treatment. Conversely, the genes involved in the cell cycle are up‑regulated at 6 h and down‑regulated at 48 h, which coincides with the induction of Tgfb1. Notable exceptions to this biphasic pattern include key genes activating immune response, such as Tlr9 and Myd88, which are down‑regulated early and persistently, while genes inhibiting immune activation, such as Trem1, and genes involved in a metabolic switch, such as Pfkl, are persistently up‑regulated. Most notably, the up‑regulated genes include Ptgs1 (alias Cox1) and Tbxas1, which encode the enzymes catalyzing the synthesis of thromboxane A2, a known inducer of T cell suppression. Further studies are needed to test the functional consequences of their up‑regulation.

Continuous-wave parallel interferometric near-infrared spectroscopy (CW-pi NIRS) has been recently introduced to noninvasively monitor cerebral blood flow in humans in vivo. With almost 10,000 parallel detection channels, CW-pi NIRS enabled the monitoring of prefrontal cortex activation. However, CW-pi NIRS lacked time-of-flight (TOF) resolution, requiring operation at long source-collector separations (SCS), which led to poor spatial resolution. Here, we demonstrate TOF-resolved parallel iNIRS, which enables us to shorten the SCS and overcome the limitations of CW-pi NIRS. (c) 2025 The Authors

We developed an ordinary differential equations (ODEs) model of hypoxia signaling that, in addition to HIF-1α, takes into account also HIF-2α. Our model can be separated into two parts, the first, describing the production and degradation of the α subunits of HIF-1 and HIF-2, and their accumulation in response to hypoxia; and the second, describing how the α subunits cooperate with the β subunit in binding to cis-regulatory regions and activation of HIF-target genes in response to hypoxia. In our previous work [1], using the first part of our model trained on time-series data from 0.9 % hypoxia, we successfully predicted the response of the system to a further drop of the oxygen to 0.3 % hypoxia. This modeling result contributed to explaining the mechanism of the switch of the control from HIF-1 to HIF-2 during the response of human primary endothelial cells to hypoxia. In another work [2], we experimentally demonstrated a linear proportionality between the counts of motifs assigned to HIF-1 in promoter open chromatin regions of genes and the effects of HIF-1 on the induction of these genes under hypoxia. We furthermore showed that such a proportionality is predicted by the subset of the ODE model of Nguyen et al. (2013) [3] common with the second part of our ODE model. In the current work, we provide the details of our full ODE model and show that it leads to a prediction that HIF-1β can be a limiting factor of the response to hypoxia.

[This corrects the article DOI: 10.3389/fmed.2023.1293640.].

Two-photon excitation fluorescence (TPEF) is emerging as a powerful imaging technique with superior penetration power in scattering media, allowing for functional imaging of biological tissues at a subcellular level. TPEF is commonly used in cancer diagnostics, as it enables the direct observation of metabolism within living cells. The technique is now widely used in various medical fields, including ophthalmology. The eye is a complex and delicate organ with multiple layers of different cell types and tissues. Although this structure is ideal for visual perception, it generates aberrations in TPEF eye imaging. However, adaptive optics can now compensate for these aberrations, allowing for improved imaging of the eyes of animal models for human diseases. The eye is naturally built to filter out harmful wavelengths, but these wavelengths can be mimicked and thereby utilized in diagnostics via two-photon (2Ph) excitation. Recent advances in laser-source manufacturing have made it possible to minimize the exposure of in vivo measurements within safety, while achieving sufficient signals to detect for functional images, making TPEF a viable option for human application. This review explores recent advances in wavefront-distortion correction in animal models and the safety of use of TPEF on human subjects, both of which make TPEF a potentially powerful tool for ophthalmological diagnostics.

Chronic myeloid leukemia (CML) cells circulate between blood and bone marrow niche, representing different microenvironments. We studied the role of the two RNA-binding proteins, T-cell-restricted intracellular antigen (TIAR), and the fragile X mental retardation protein (FMRP) in the regulation of protein translation in CML cells residing in settings mimicking peripheral blood microenvironment (PBM) and bone marrow microenvironment (BMM). The outcomes showed how conditions shaped the translation process through TIAR and FMRP activity, considering its relevance in therapy resistance. The QuaNCAT mass-spectrometric approach revealed that TIAR and FMRP have a discrete modulatory effect on protein synthesis and thus affect distinct aspects of leukemic cells functioning in the hypoxic niche. In the BMM setup, FMRP impacted metabolic adaptation of cells and TIAR substantially supported the resistance of CML cells to translation inhibition by homoharringtonine. Overall, our results demonstrated that targeting post-transcriptional control should be considered when designing anti-leukemia therapeutic solutions.

Noninvasive imaging of endogenous retinal fluorophores, including vitamin A derivatives, is vital to developing new treatments for retinal diseases. Here, we present a protocol for obtaining in vivo two-photon excited fluorescence images of the fundus in the human eye. We describe steps for laser characterization, system alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays safety concerns by allowing for the acquisition of informative images at low laser exposure. For complete details on the use and execution of this protocol, please refer to Bogusławski et al. (2022).1.

Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.

The cellular adaptive response to hypoxia relies on the expression of hypoxia-inducible factors (HIFs), HIF-1 and HIF-2. HIFs regulate global gene expression changes during hypoxia that are necessary for restoring oxygen homeostasis and promoting cell survival. In the early stages of hypoxia, HIF-1 is elevated, whereas at the later stages, HIF-2 becomes the predominant form. What governs the transition between the two HIFs (the HIF switch) and the role of miRNAs in this regulation are not completely clear. Genome-wide expression studies on the miRNA content of RNA-induced silencing complexes (RISC) in HUVECs exposed to hypoxia compared to the global miRNA-Seq analysis revealed very specific differences between these two populations. We analyzed the miRNA and mRNA composition of RISC at 2 h (mainly HIF-1 driven), 8 h (HIF-1 and HIF-2 elevated), and 16 h (mainly HIF-2 driven) in a gene ontology context. This allowed for determining the direct impact of the miRNAs in modulating the cellular signaling pathways involved in the hypoxic adaptive response. Our results indicate that the miRNA-mRNA RISC components control the adaptive responses, and this does not always rely on the miRNA transcriptional elevations during hypoxia. Furthermore, we demonstrate that the hypoxic levels of the vast majority of HIF-1-dependent miRNAs (including miR-210-3p) are also HIF-2 dependent and that HIF-2 governs the expression of 11 specific miRNAs. In summary, the switch from HIF-1 to HIF-2 during hypoxia provides an important level of miRNA-driven control in the adaptive pathways in endothelial cells.

We analyzed the effects of selective knockdown of either HIF-1 alpha or HIF-2 alpha on the transcriptional response to hypoxia of human umbilical endothelial cells at two time-points (2 h and 8 h) of hypoxia. We focused on 13 previously identified hypoxia-responsive genes, pre-selected to have different activation kinetics and different proportions of HRE motifs annotated to either HIF-1 or HIF-2 in open promoters - open chromatin DNase-hypersensitive sites (DHS) regions within +/- 1 kb of the gene start. We report that genes activated by both HIF-1 and 2 tend to be activated earlier than genes activated by HIF-1 only, which, in turn, tend to be activated earlier than genes activated by HIF-2 only. Moreover, for the 13 analyzed genes, we found that the effect of silencing HIF1A on the gene induction by hypoxia is greater for the genes with more HRE motifs annotated to HIF-1 in their promoter open chromatin DHS regions within & PLUSMN;1 kb and also within +/- 10 kb of the gene start. We corroborated and extended this finding by showing that among 232 genes previously identified as activated by hypoxia, the genes with ChIP-seq peak(s) for HIF-1 alpha within a +/- 10 kb flank of the gene start contain more HRE motifs annotated to HIF-1 in the DHS regions within this flank than the genes with no ChIP-seq peaks. Also in the whole genome, the DHS regions intersecting ChIP-seq peaks for HIF-1 alpha contain more HRE motifs annotated to HIF-1 than the DHS regions not intersecting the ChIP-seq peaks. This suggests a mechanism, by which higher promoter content of HRE motifs in DHS regions increases HIF-1 binding, which in turn increases gene induction by hypoxia.

The hypoxia-inducible factors (HIF) are transcription factors that activate the adaptive hypoxic response when oxygen levels are low. The HIF transcriptional program increases oxygen delivery by inducing angiogenesis and by promoting metabolic reprograming that favors glycolysis. The two major HIFs, HIF-1 and HIF-2, mediate this response during prolonged hypoxia in an overlapping and sequential fashion that is referred to as the HIF switch. Both HIF proteins consist of an unstable alpha chain and a stable beta chain. The instability of the alpha chains is mediated by prolyl hydroxylase (PHD) activity during normoxic conditions, which leads to ubiquitination and proteasomal degradation of the alpha chains. During normoxic conditions, very little HIF-1 or HIF-2 alpha–beta dimers are present because of PHD activity. During hypoxia, however, PHD activity is suppressed, and HIF dimers are stable. Here we demonstrate that HIF-1 expression is maximal after 4 h of hypoxia in primary endothelial cells and then is dramatically reduced by 8 h. In contrast, HIF-2 is maximal at 8 h and remains elevated up to 24 h. There are differences in the HIF-1 and HIF-2 transcriptional profiles, and therefore understanding how the transition between them occurs is important and not clearly understood. Here we demonstrate that the HIF-1 to HIF-2 transition during prolonged hypoxia is mediated by two mechanisms: (1) the HIF-1 driven increase in the glycolytic pathways that reactivates PHD activity and (2) the much less stable mRNA levels of HIF-1α (HIF1A) compared to HIF-2α (EPAS1) mRNA. We also demonstrate that the alpha mRNA levels directly correlate to the relative alpha protein levels, and therefore to the more stable HIF-2 expression during prolonged hypoxia.

Non-alcoholic fatty liver disease (NAFLD) is a health concern affecting 24% of the population worldwide. Although the pathophysiologic mechanisms underlying disease are not fully clarified, mitochondrial dysfunction and oxidative stress are key players in disease progression. Consequently, efforts to develop more efficient pharmacologic strategies targeting mitochondria for NAFLD prevention/treatment are underway. The conjugation of caffeic acid anti-oxidant moiety with an alkyl linker and a triphenylphosphonium cation (TPP+), guided by structure-activity relationships, led to the development of a mitochondria-targeted anti-oxidant (AntiOxCIN4) with remarkable anti-oxidant properties. Recently, we described that AntiOxCIN4 improved mitochondrial function, upregulated anti-oxidant defense systems, and cellular quality control mechanisms (mitophagy/autophagy) via activation of the Nrf2/Keap1 pathway, preventing fatty acid-induced cell damage. Despite the data obtained, AntiOxCIN4 effects on cellular and mitochondrial energy metabolism in vivo were not studied.In the present work, we proposed that AntiOxCIN4 (2.5 mg/day/animal) may prevent non-alcoholic fatty liver (NAFL) phenotype development in a C57BL/6J mice fed with 30% high-fat, 30% high-sucrose diet for 16 weeks. HepG2 cells treated with AntiOxCIN4 (100 μM, 48 h) before the exposure to supraphysiologic free fatty acids (FFAs) (250 μM, 24 h) were used for complementary studies. AntiOxCIN4 decreased body (by 43%), liver weight (by 39%), and plasma hepatocyte damage markers in WD-fed mice. Hepatic-related parameters associated with a reduction of fat liver accumulation (by 600%) and the remodeling of fatty acyl chain composition compared with the WD-fed group were improved. Data from human HepG2 cells confirmed that a reduction of lipid droplets size and number can be a result from AntiOxCIN4-induced stimulation of fatty acid oxidation and mitochondrial OXPHOS remodeling. In WD-fed mice, AntiOxCIN4 also induced a hepatic metabolism remodeling by upregulating mitochondrial OXPHOS, anti-oxidant defense system and phospholipid membrane composition, which is mediated by the PGC-1α-SIRT3 axis. AntiOxCIN4 prevented lipid accumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity.AntiOxCIN4 improved NAFL phenotype of WD-fed mice, via three main mechanisms: a) increase mitochondrial function (fatty acid oxidation); b) stimulation anti-oxidant defense system (enzymatic and non-enzymatic) and; c) prevent the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/treatment of NAFLD.

Get PDF Email Share Share with Facebook Tweet This Post on reddit Share with LinkedIn Add to CiteULike Add to Mendeley Add to BibSonomy Get Citation Copy Citation Text J. Bogusławski, G. Palczewska, M. Dąbrowski, S. Tomczewski, J. Milkiewicz, D. Stachowiak, K. Komar, M. Marzejon, B. L. Sikorski, A. Hudzikowski, A. Głuszek, Z. Łaszczych, G. Soboń, K. Palczewski, and M. Wojtkowski, "Two-photon excited fluorescence scanning laser ophthalmoscope for in vivo imaging of the human eye," in Biophotonics Congress: Biomedical Optics 2022 (Translational, Microscopy, OCT, OTS, BRAIN), Technical Digest Series (Optica Publishing Group, 2022), paper TTu2B.4. Export Citation BibTex Endnote (RIS) HTML Plain Text Citation alert Save article