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Heme released from damaged and senescent red blood cells (RBCs) may contribute to oxidant-mediated cell injury. One of the recently investigated physiological processes, essential in preventing the inflammatory impact of labile heme, is its uptake from the bloodstream by endothelial cells (ECs). In this study, we investigated heme uptake by ECs starting from the model studies on the in vitro cellular level, through the endothelium layer on the ex vivo murine aortic tissues. As the cellular model, Human Aortic Endothelial Cells (HAECs) were chosen, and the concentration of labile heme was adjusted so to avoid the excessive toxic effect of the labile heme. We utilized label-free Raman imaging with two different excitation wavelengths to capture the uptake process in situ and characterize the oxidation state of the iron ion in the intercalated heme. The phenomenon of heme uptake was demonstrated in both, the healthy control C57Bl/6J and FVB animals, as well as in mice with developed atherosclerosis (ApoE/LDLR -/- mice). In the presented work, we presented for the first time Raman-based evidence on the heme uptake process by endothelial cells in both, in vitro and ex vivo systems., (© 2024. The Author(s).)

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing., Competing Interests: P.R. has licensed the PGK:FANCAWpre∗ LV medicinal product and receives funding and equity from Rocket Pharmaceuticals, Inc., patents and royalties, research & consulting funding. D.L. is an inventor on a patent National Institute of Chemistry filed (WO/2021/032759 patent application, European patent application EP 3783104, China patent application CN 114269930 with National Phase entry EP2020756868). R.O.B. holds patents related to CRISPR-Cas genome editing and has equity in Graphite Bio and is consultant for UNIKUM Tx. G.G. holds patents related to CRISPR-Cas genome editing, is an employee of CasZyme, and has equity in CasZyme. S.Q.T. is a co-inventor on patents for GUIDE-seq, CHANGE-seq, and other genome editing technologies and a member of the scientific advisory boards of Prime Medicine and Ensoma. T.C. is a co-inventor on patents for CAST-seq, Abnoba-Seq, and other genome editing technologies, and a member of the scientific advisory boards of Cimeo Therapeutics, Excision BioTherapeutics, and GenCC. A.C. and G.T. are inventors on a patent for MEGA (WO/2023/079285), G.T. is also co-inventor on a patent for CAST-seq., (© 2023 The Author(s).)

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDH high ) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous group of monogenic endocrine disorders that is characterised by autosomal dominant inheritance and pancreatic β-cell dysfunction. These patients are commonly misdiagnosed with type 1 or type 2 diabetes, as the clinical symptoms largely overlap. Even though several biomarkers have been tested none of which could be used as single clinical discriminator. The correct diagnosis for individuals with MODY is of utmost importance, as the applied treatment depends on the gene mutation or is subtype-specific. Moreover, in patients with HNF1A-MODY, additional clinical monitoring can be included due to the high incidence of vascular complications observed in these patients. Finally, stratification of MODY patients will enable better and newer treatment options for MODY patients, once the disease pathology for each patient group is better understood. In the current review the clinical characteristics and the known disease-related abnormalities of the most common MODY subtypes are discussed, together with the up-to-date applied diagnostic criteria and treatment options. Additionally, the usage of pluripotent stem cells together with CRISPR/Cas9 gene editing for disease modelling with the possibility to reveal new pathophysiological mechanisms in MODY is discussed.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as a master cytoprotective factor regulating the expression of genes encoding anti-oxidant, anti-inflammatory, and detoxifying proteins. The role of Nrf2 in the pathophysiology of skeletal muscles has been evaluated in different experimental models, however, due to inconsistent data, we aimed to investigate how Nrf2 transcriptional deficiency (Nrf2 tKO ) affects muscle functions both in an acute and chronic injury. The acute muscle damage was induced in mice of two genotypes-WT and Nrf2 tKO mice by cardiotoxin (CTX) injection. To investigate the role of Nrf2 in chronic muscle pathology, mdx mice that share genetic, biochemical, and histopathological features with Duchenne muscular dystrophy (DMD) were crossed with mice lacking transcriptionally active Nrf2 and double knockouts (mdx/Nrf2 tKO ) were generated. To worsen the dystrophic phenotype, the analysis of disease pathology was also performed in aggravated conditions, by applying a long-term treadmill test. We have observed slightly increased muscle damage in Nrf2 tKO mice after CTX injection. Nevertheless, transcriptional ablation of Nrf2 in mdx mice did not significantly aggravate the most deleterious, pathological hallmarks of DMD related to degeneration, inflammation, fibrotic scar formation, angiogenesis, and the number and proliferation of satellite cells in non-exercised conditions. On the other hand, upon chronic exercises, the degeneration and inflammatory infiltration of the gastrocnemius muscle, but not the diaphragm, turned to be increased in Nrf2 tKO mdx in comparison to mdx mice. In conclusion, the lack of transcriptionally active Nrf2 influences moderately muscle pathology in acute CTX-induced muscle injury and chronic DMD mouse model, without affecting muscle functionality. Hence, in general, we demonstrated that the deficiency of Nrf2 transcriptional activity has no profound impact on muscle pathology in various models of muscle injury.

Cell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI. ADSCs and hiPSCs were transduced with lentiviral vectors encoding luciferase (Luc), GFP and either HO-1 or SDF-1α. hiPSCs were then differentiated to hiPSC-CMs using small molecules modulating the WNT pathway. Genetically modified ADSCs were firstly administered via intracardiac injection after MI induction in Nude mice. Next, ADSCs-Luc-GFP and genetically modified hiPSC-CMs were injected into the hearts of the more receptive NOD/SCID strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated Nude and NOD/SCID mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration.

Inadequate supply of oxygen (O 2 ) is a hallmark of many diseases, in particular those related to the cardiovascular system. On the other hand, tissue hypoxia is an important factor regulating (normal) embryogenesis and differentiation of stem cells at the early stages of embryonic development. In culture, hypoxic conditions may facilitate the derivation of embryonic stem cells (ESCs) and the generation of induced pluripotent stem cells (iPSCs), which may serve as a valuable tool for disease modeling. Endothelial cells (ECs), multifunctional components of vascular structures, may be obtained from iPSCs and subsequently used in various (hypoxia-related) disease models to investigate vascular dysfunctions. Although iPSC-ECs demonstrated functionality in vitro and in vivo, ongoing studies are conducted to increase the efficiency of differentiation and to establish the most productive protocols for the application of patient-derived cells in clinics. In this review, we highlight recent discoveries on the role of hypoxia in the derivation of ESCs and the generation of iPSCs. We also summarize the existing protocols of hypoxia-driven differentiation of iPSCs toward ECs and discuss their possible applications in disease modeling and treatment of hypoxia-related disorders.

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies., (© 2019 UICC.)

Tumor hypoxia and high activity of hypoxia-inducible factor-1 (HIF-1) correlate with adverse disease outcomes, malignancy, resistance to therapy and metastasis. Nonetheless, recent studies indicate that under certain circumstances, HIF-1 stabilization may exert protective effects and even decrease tumor cell aggressiveness. This study aimed to characterize the potential anticancer effect of molidustat (BAY 85-3934), the prolyl hydroxylase (PHD) inhibitor and HIF-1 stabilizator. We confirmed that molidustat stabilizes HIF-1α and induces the expression of vascular endothelial growth factor (VEGF) in MDA-MB-231 breast cancer cells, to a similar or even greater extent than hypoxia. Interestingly, decreased cell survival and colony formation capabilities, together with S/G2 cell cycle arrest, were observed after treatment with PHD inhibitor. Importantly, molidustat enhanced the effectiveness of the chemotherapeutic drug, gemcitabine, on cancer cells. Finally, the xenograft model revealed decreased tumor growth in vivo after molidustat treatment. Both in vitro and in vivo analysis showed no differences in the angiogenic potential of endothelial cells treated with tumor-conditioned media or vascularization of the MDA-MB-231 xenografts, respectively. In summary, molidustat treatment exhibits an inhibitory effect on breast cancer cell survival, self-renewal capacity and potentiates the efficacy of chemotherapeutic gemcitabine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Burkitt lymphoma (BL) is a rapidly growing tumor, characterized by high anabolic requirements. The MYC oncogene plays a central role in the pathogenesis of this malignancy, controlling genes involved in apoptosis, proliferation, and cellular metabolism. Serine biosynthesis pathway (SBP) couples glycolysis to folate and methionine cycles, supporting biosynthesis of certain amino acids, nucleotides, glutathione, and a methyl group donor, S-adenosylmethionine (SAM). We report that BLs overexpress SBP enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1). Both genes are controlled by the MYC-dependent ATF4 transcription factor. Genetic ablation of PHGDH/PSAT1 or chemical PHGDH inhibition with NCT-503 decreased BL cell lines proliferation and clonogenicity. NCT-503 reduced glutathione level, increased reactive oxygen species abundance, and induced apoptosis. Consistent with the role of SAM as a methyl donor, NCT-503 decreased DNA and histone methylation, and led to the re-expression of ID4 , KLF4 , CDKN2B and TXNIP tumor suppressors. High H3K27me3 level is known to repress the MYC negative regulator miR-494. NCT-503 decreased H3K27me3 abundance, increased the miR-494 level, and reduced the expression of MYC and MYC-dependent histone methyltransferase, EZH2. Surprisingly, chemical/genetic disruption of SBP did not delay BL and breast cancer xenografts growth, suggesting the existence of mechanisms compensating the PHGDH/PSAT1 absence in vivo., Competing Interests: P. Juszczynski is a member of the Scientific Advisory Board at Selvita S.A. and served as a consultant for Selvita S.A. Dr. T. Rzymski, Dr. K. Brzozka and P. Kowalczyk are Selvita S.A. employees.

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche-endothelial cells (ECs) and CXCL12-abundant reticular cells (CARs)-highly express the heme-degrading enzyme, heme oxygenase 1 (HO-1), but then decrease its expression with age. HO-1-deficient animals (HO-1 -/- ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co-cultured in vitro with HO-1 -/- mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO-1 -/- animals have reduced quiescence and regenerative potential. Young HO-1 -/- HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO-1 +/+ HSCs transplanted into HO-1 -/- recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO-1 -/- HSCs to the HO-1 +/+ recipients recovers the regenerative potential of HO-1 -/- HSCs and reverses their transcriptional alterations. Thus, HSC-extrinsic activity of HO-1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Granulocyte colony-stimulating factor (G-CSF) is used in clinical practice to mobilize cells from the bone marrow to the blood; however, it is not always effective. We show that cobalt protoporphyrin IX (CoPP) increases plasma concentrations of G-CSF, IL-6, and MCP-1 in mice, triggering the mobilization of granulocytes and hematopoietic stem and progenitor cells (HSPC). Compared with recombinant G-CSF, CoPP mobilizes higher number of HSPC and mature granulocytes. In contrast to G-CSF, CoPP does not increase the number of circulating T cells. Transplantation of CoPP-mobilized peripheral blood mononuclear cells (PBMC) results in higher chimerism and faster hematopoietic reconstitution than transplantation of PBMC mobilized by G-CSF. Although CoPP is used to activate Nrf2/HO-1 axis, the observed effects are Nrf2/HO-1 independent. Concluding, CoPP increases expression of mobilization-related cytokines and has superior mobilizing efficiency compared with recombinant G-CSF. This observation could lead to the development of new strategies for the treatment of neutropenia and HSPC transplantation., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Patients with HNF1A -maturity-onset diabetes of the young (MODY) often develop endothelial dysfunction and related microvascular complications, like retinopathy. As the clinical phenotype of HNF1A -MODY diabetes varies considerably, we used human induced pluripotent stem cells (hiPSCs) from two healthy individuals (control) to generate isogenic lines with mutation in HNF1A gene. Subsequently, control hiPSCs and their respective HNF1A clones were differentiated toward endothelial cells (hiPSC-ECs) and different markers/functions were compared. Human iPSC-ECs from all cell lines showed similar expression of CD31 and Tie-2. VE-cadherin expression was lower in HNF1A -mutated isogenic lines, but only in clones derived from one control hiPSCs. In the other isogenic set and cells derived from HNF1A -MODY patients, no difference in VE-cadherin expression was observed, suggesting the impact of the genetic background on this endothelial marker. All tested hiPSC-ECs showed an expected angiogenic response regardless of the mutation introduced. Isogenic hiPSC-ECs responded similarly to stimulation with pro-inflammatory cytokine TNF- with the increase in ICAM-1 and permeability, however, HNF1A mutated hiPSC-ECs showed higher permeability in comparison to the control cells. Summarizing, both mono- and biallelic mutations of HNF1A in hiPSC-ECs lead to increased permeability in response to TNF- in normal glycemic conditions, which may have relevance to HNF1A- MODY microvascular complications., Competing Interests: The authors declare no conflict of interest.

Heme oxygenase-1 (HO-1, HMOX1) degrades pro-oxidant heme into carbon monoxide (CO), ferrous ions (Fe 2+ ) and biliverdin. The enzyme exerts multiple cytoprotective functions associated with the promotion of angiogenesis and counteraction of the detrimental effects of cellular stress which are crucial for the survival of both normal and tumor cells. Accordingly, in many tumor types, high expression of HO-1 correlates with poor prognosis and resistance to treatment, i.e. chemotherapy, suggesting inhibition of HO-1 as a possible antitumor approach. At the same time, the lack of selective and well-profiled inhibitors of HO-1 determines the unmet need for new modulators of this enzyme, with the potential to be used in either adjuvant therapy or as the stand-alone targeted therapeutics. In the current study, we provided novel inhibitors of HO-1 and validated the effect of pharmacological inhibition of HO activity by the imidazole-based inhibitor (SLV-11199) in human pancreatic (PANC-1) and prostate (DU-145) cancer cell lines. We demonstrated potent inhibition of HO activity in vitro and showed associated anticancer effectiveness of SLV-11199. Treatment with the tested compound led to decreased cancer cell viability and clonogenic potential. It has also sensitized the cancer cells to chemotherapy. In PANC-1 cells, diminished HO activity resulted in down-regulation of pro-angiogenic factors like IL-8. Mechanistic investigations revealed that the treatment with SLV-11199 decreased cell migration and inhibited MMP-1 and MMP-9 expression. Moreover, it affected mesenchymal phenotype by regulating key modulators of the epithelial to mesenchymal transition (EMT) signalling axis. Finally, F-actin cytoskeleton and focal contacts were destabilized by the reported compound. Overall, the current study suggests a possible relevance of the tested novel inhibitor of HO activity as a potential anticancer compound. To support such utility, further investigation is still needed, especially in in vivo conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Myeloid angiogenic cells (MAC) derive from hematopoietic stem/progenitor cells (HSPCs) that are mobilized from the bone marrow. They home to sites of neovascularization and contribute to angiogenesis by production of paracrine factors. The number and function of proangiogenic cells are impaired in patients with diabetes or cardiovascular diseases. Both conditions can be accompanied by decreased levels of heme oxygenase-1 (HMOX1), cytoprotective, heme-degrading enzyme. Our study is aimed at investigating whether precursors of myeloid angiogenic cells (PACs) treated with known pharmaceuticals would produce media with better proangiogenic activity in vitro and if such media can be used to stimulate blood vessel growth in vivo . We used G-CSF-mobilized CD34 + HSPCs, FACS-sorted from healthy donor peripheral blood mononuclear cells (PBMCs). Sorted cells were predominantly CD133 + . CD34 + cells after six days in culture were stimulated with atorvastatin (AT), acetylsalicylic acid (ASA), sulforaphane (SR), resveratrol (RV), or metformin (Met) for 48 h. Conditioned media from such cells were then used to stimulate human aortic endothelial cells (HAoECs) to enhance tube-like structure formation in a Matrigel assay. The only stimulant that enhanced PAC paracrine angiogenic activity was atorvastatin, which also had ability to stabilize endothelial tubes in vitro . On the other hand, the only one that induced heme oxygenase-1 expression was sulforaphane, a known activator of a HMOX1 inducer-NRF2. None of the stimulants changed significantly the levels of 30 cytokines and growth factors tested with the multiplex test. Then, we used atorvastatin-stimulated cells or conditioned media from them in the Matrigel plug in vivo angiogenic assay. Neither AT alone in control media nor conditioned media nor AT-stimulated cells affected numbers of endothelial cells in the plug or plug's vascularization. Concluding, high concentrations of atorvastatin stabilize tubes and enhance the paracrine angiogenic activity of human PAC cells in vitro . However, the effect was not observed in vivo . Therefore, the use of conditioned media from atorvastatin-treated PAC is not a promising therapeutic strategy to enhance angiogenesis., Competing Interests: The authors declare no existing conflict of interest.

Background: Sarcoidosis is characterized by exaggerated immune response to unknown agent and can affect different organs. One of the main players in the pathology of the disease are regulatory T cells (Tregs), however, up to date the mechanisms of the possible molecular alterations of this particular cell subset are not known., Methods: In the current study we looked for the global transcriptomic changes of miRNAs, using predefined array, and mRNAs (RNA seq analysis) of Tregs of patients with the most predominant form of the disease - acute pulmonary sarcoidosis (PS). For this purpose sorted CD4+/CD25+/CD127- Tregs from peripheral blood (PB) and CD4+/CD25 + Tregs from bronchoalveolar lavage (BAL) were used., Results: MiRNA analysis revealed that Tregs isolated from PB and BAL display significantly different miRNA profile, suggesting an important role of the pulmonary microenvironment in creating these changes. Among disease-related miRNAs of PB Tregs we identified miR-155 and miR-223. Moreover, looking at the global transcriptome of PB Tregs, we recognized alterations in TLR-2 signaling pathway and in the downstream of NF-κB apoptosis and proliferation signals. However, induction of TLR-2 expression was found not only in Tregs, but also in the heterogeneous population of peripheral blood mononuclear cells (PBMC) as well as two PBMC subpopulations (CD4+/CD25-and CD4-/CD25-) of patients with PS. This indicates that activation of TLR signaling pathway in sarcoidosis does not occur only in Tregs., Conclusion: Our findings offer a deeper insight into the molecular mechanisms of Tregs reduced suppression and increased apoptosis in patients with PS. Based on the current results, future studies should focus on possible therapeutic effect of TLR-2 signaling inhibition., (Copyright © 2018. Published by Elsevier Ltd.)

Heme oxygenase 1 (Hmox1), a ubiquitous enzyme degrading heme to carbon monoxide, iron, and biliverdin, is one of the cytoprotective enzymes induced in response to a variety of stimuli, including cellular oxidative stress. Gangliosides, sialic acid-containing glycosphingolipids expressed in all cells, are involved in cell recognition, signalling, and membrane stabilization. Their expression is often altered under many pathological and physiological conditions including cell death, proliferation, and differentiation. The aim of this study was to assess the possible role of Hmox1 in ganglioside metabolism in relation to oxidative stress. The content of liver and brain gangliosides, their cellular distribution, and mRNA as well as protein expression of key glycosyltransferases were determined in Hmox1 knockout mice as well as their wild-type littermates. To elucidate the possible underlying mechanisms between Hmox1 and ganglioside metabolism, hepatoblastoma HepG2 and neuroblastoma SH-SY5Y cell lines were used for in vitro experiments. Mice lacking Hmox1 exhibited a significant increase in concentrations of liver and brain gangliosides and in mRNA expression of the key enzymes of ganglioside metabolism. A marked shift of GM1 ganglioside from the subsinusoidal part of the intracellular compartment into sinusoidal membranes of hepatocytes was shown in Hmox1 knockout mice. Induction of oxidative stress by chenodeoxycholic acid in vitro resulted in a significant increase in GM3, GM2, and GD1a gangliosides in SH-SY5Y cells and GM3 and GM2 in the HepG2 cell line. These changes were abolished with administration of bilirubin, a potent antioxidant agent. These observations were closely related to oxidative stress-mediated changes in sialyltransferase expression regulated at least partially through the protein kinase C pathway. We conclude that oxidative stress is an important factor modulating synthesis and distribution of gangliosides in vivo and in vitro which might affect ganglioside signalling in higher organisms.

Aims: Mesenchymal stromal cells (MSCs) are heterogeneous cells from adult tissues that are able to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes. Such cells are widely studied in regenerative medicine. However, the success of cellular therapy depends on the cell survival. Heme oxygenase-1 (HO-1, encoded by the Hmox1 gene), an enzyme converting heme to biliverdin, carbon monoxide, and Fe 2+ , is cytoprotective and can affect stem cell performance. Therefore, our study aimed at assessing whether Hmox1 is critical for survival and functions of murine bone marrow MSCs., Results: Both MSC Hmox1 +/+ and Hmox1 -/- showed similar phenotype, differentiation capacities, and production of cytokines or growth factors. Hmox1 +/+ and Hmox1 -/- cells showed similar survival in response to 50 μmol/L hemin even in increased glucose concentration, conditions that were unfavorable for Hmox1 -/- bone marrow-derived proangiogenic cells (BDMC). Hmox1 +/+ MSCs but not fibroblasts retained low ROS levels even after prolonged incubation with 50 μmol/L hemin, although both cell types have a comparable Hmox1 expression and similarly increase its levels in response to hemin. MSCs Hmox1 -/- treated with hemin efficiently induced expression of a vast panel of antioxidant genes, especially enzymes of the glutathione pathway. Innovation and Conclusion: Hmox1 overexpression is a popular strategy to enhance viability and performance of MSCs after the transplantation. However, murine MSCs Hmox1 -/- do not differ from wild-type MSCs in phenotype and functions. MSC Hmox1 -/- show better resistance to hemin than fibroblasts and BDMCs and rapidly react to the stress by upregulation of quintessential genes in antioxidant response. Antioxid. Redox Signal. 00, 000-000.

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1 -/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1 +/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1 +/+ and Hmox1 -/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2 +/+ and Nfe2l2 -/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may serve as a valuable therapy in various pathophysiological processes, including tumorigenesis. We compared the effect of chemical inhibitors - metalloporphyrins, with genetic tools - shRNA and CRISPR/Cas9 systems, to knock-down (KD)/knock-out (KO) HO-1 expression/activity. 293T cells were incubated with metalloporphyrins, tin and zinc protoporphyrins (SnPPIX and ZnPPIX, respectively) or were either transduced with lentiviral vectors encoding different shRNA sequences against HO-1 or were modified by CRISPR/Cas9 system targeting HMOX1. Metalloporphyrins decreased HO activity but concomitantly strongly induced HO-1 mRNA and protein in 293T cells. On the other hand, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. Nevertheless, silencing effect was much stronger when CRISPR/Cas9-mediated knock-out was performed. Most of the clones harboring mutations within HMOX1 locus did not express HO-1 protein and failed to increase bilirubin concentration after hemin stimulation. Furthermore, CRISPR/Cas9-mediated HO-1 depletion decreased 293T viability, growth, clonogenic potential and increased sensitivity to H 2 O 2 treatment. In summary, we have shown that not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach.

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Induced pluripotent stem cells (iPSCs) have shown great potential in regenerative medicine and research applications like disease modeling or drug discovery. Endothelium is indispensable for vascular homeostasis, whereas endothelial dysfunction could lead to different diseases. Therefore, generating autologous cells, able to restore the endothelial lining, can be crucial for slowing or reversing certain pathological processes. In the current study we show efficient differentiation of murine iPSCs into endothelial cells (ECs) with stable CD34+/Tie-2+/Sca-1+/CD45- phenotype and proven functionality. iPS-derived ECs (iPS-ECs) were positive for phospho-eNOS and von Willebrand factor, and responded to shear stress with up-regulation of KLF-2, KDR, HO-1, and increased nitric oxide and VEGF production. These cells reacted to cytokine stimulation through increase in VCAM-1 and inflammatory cytokine secretion. iPS-ECs showed also certain progenitor features, like expression of progenitor markers (CD34, Sca-1, c-kit) and high clonogenic potential. The angiogenic capacity of iPS-ECs in spheroid sprouting assay was similar to primary ECs, whereas on Matrigel, tube structures could be formed only in the presence of other support cells. Angiogenic potential of iPS-ECs in vivo, was similar to murine endothelial cell line MS-1. Summarizing, our approach enabled generation of functional progenitor-like ECs, which can be used as a research model., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sarcoidosis is a multi-organ disease in which affected tissues are invaded with non-necrotizing granulomatous structures, mostly consisted of T helper 1 (Th1) cells and multinucleate giant cells. However, the etiology and pathogenesis of sarcoidosis is not known and the diagnosis is usually based on clinical examination involving radiography and histopathological analysis of biopsies of affected organs. Although the knowledge on the molecular background of sarcoidosis is limited, it seems that the important pathways involve transforming growth factor-β (TGF-β) and JAK/STAT, which may influence the interferon-γ (IFN-γ)-mediated signaling. Additionally, recently the role of microRNAs (miRNAs), the small non-coding RNA molecules, has been emphasized in different pathological conditions including autoimmune diseases. This review summarizes the current knowledge on the molecular pathways in the pathogenesis of sarcoidosis with a special emphasis on cytokines and miRNAs controlling immune cells proliferation and differentiation. Moreover, the possible role of T regulatory cells (CD4(+) CD25(+) FoxP3(+)) in this disease has been discussed.

Endothelial progenitor cells (EPCs) have been extensively studied for almost 19 years now and were considered as a potential marker for endothelial regeneration ability. On the other hand, circulating endothelial cells (CEC) were studied as biomarker for endothelial injury. Yet, in the literature, there is also huge incoherency in regards to terminology and protocols used. This results in misleading conclusions on the role of so called "EPCs", especially in the clinical field. The discrepancies are mainly due to strong phenotypic overlap between EPCs and circulating angiogenic cells (CAC), therefore changes in "EPC" terminology have been suggested. Other factors leading to inconsistent results are varied definitions of the studied populations and the lack of universal data reporting, which could strongly affect data interpretation. The current review is focused on controversies concerning the use of "EPCs"/CAC and CEC as putative endothelial diagnostic markers., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

In this work, 3D linear Raman spectroscopy was used to study lipid droplets (LDs) ex vivo in liver tissue and also in vitro in a single endothelial cell. Spectroscopic measurements combined with fluorescence microscopy and/or histochemical staining gave complex chemical information about LD composition and enabled detailed investigations of the changes occurring in various pathological states. Lipid analysis in fatty liver tissue was performed using a dietary mouse model of liver steatosis, induced by a high fat diet (HFD). HFD is characterized by a high percentage of calories from saturated fat (60%) and reflects closely the detrimental effects of dietary habits responsible for increased morbidity due to obesity and its complications in well-developed Western societies. Such diets lead to obesity, hyperlipidemia, insulin resistance, and steatosis that may also be linked to endothelial dysfunction. In the present work, Raman spectroscopy was applied to characterized chemical composition of lipid droplets in hepatocytes from mice fed HFD and in the endothelium treated with exogenous unsaturated free fatty acid (arachidonic acid). The results demonstrate the usefulness of Raman spectroscopy to characterize intracellular lipid distribution in 2D and 3D images and can be used to determine the degree of saturation. Raman spectroscopy shows the potential to be a valuable tool for studying the role of LDs in physiology and pathology. The method is generally applicable for the determination of LDs of different size, origin, and composition. Moreover, for the first time, the process of LD formation in the endothelium was detected and visualized in 3D.

Supravital species identification of morphologically similar syntopic earthworms inhabiting dung and compost heaps or those from commercial cultures is difficult. The aim of the studies was to find out non-invasive species-specific markers for proper segregation of earthworm species from a dense mixed colony of waste decomposers. Worms were segregated according to external characteristics into Eisenia andrei, Eisenia fetida, and Dendrobaena veneta, and left for reproduction and analysis of non-invasively retrieved coelomocyte-containing coelomic fluid and/or species-specific partial sequences of cytochrome c oxidase subunit I (COI) gene in DNA extracted from amputated tail tips of adults and their offspring. Flow cytometric analysis of coelomocyte samples revealed that amount of nuclear DNA increases in order D. veneta ≪ E. andrei < E. fetida, and intensity of eleocyte-derived fluorescence is lower in D. veneta than in Eisenia spp. Spectrofluorimetry of coelomocyte lysates revealed that the amount of eleocyte-stored riboflavin is significantly lower in coelomocyte lysates from D. veneta than from Eisenia spp., and the emission peak of X-fluorophore is much more distinct in D. veneta than in Eisenia spp. Coelomic fluid of E. andrei exhibits a very distinct spectra of MUG fluorophore which are absent in D. veneta and in the majority of E. fetida, while some E. fetida possess MUG-like fluorophore. Sequences of the COI gene in the DNA of the worms from the mixed colony and their offspring confirmed species identity. In conclusion, species-specific coelomocyte-derived markers may be a useful complement to morphological and DNA-based taxonomy during studies on syntopic earthworms., (© 2013 Wiley Periodicals, Inc.)

Murine very small embryonic-like (VSEL) cells, defined by the Lin(-)Sca-1(+)CD45(-) phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin(-)Sca(+)CD45(-) population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin(-)Sca-1(+)CD45(-) population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit(+)KDR(-), c-Kit(-)KDR(+), and c-Kit(-)KDR(-) subpopulations could be distinguished, while c-Kit(+)KDR(+) events were very rare. The c-Kit(+)KDR(-) subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit(-)KDR(+) cells. The c-Kit(-)KDR(-)FSC(low) subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin(-)Sca-1(+)CD45(-)FSC(low) population, even by single-cell qRT-PCR. We also found that the Lin(-)Sca-1(+)CD45(-)c-Kit(+) subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1(+)c-Kit(+)Lin(-) (SKL) CD34(-)CD45(-)CD105(+) cells, expressing particular HSC markers. Co-culture of Lin(-)Sca-1(+)CD45(-)FSC(low) with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45(-)CD105(+) subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin(-)Sca-1(+)CD45(-)FSC(low) cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin(-)Sca-1(+)CD45(-)c-Kit(+)KDR(-) subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.

Raman imaging was used to illustrate heterogeneity of a single endothelial cell and the vascular wall sample. The spectral analysis allowed for exploring the complexity of the studied systems in three dimensions and defining the size, volume, shape and biochemical composition of cellular organelles. The ability to construct the 3D maps by a method that does not disrupt the spatial integrity of the cell provided a unique insight into biochemical architecture and cellular processes of endothelium and vascular wall. 3D Raman imaging may be considered as a new trend in analytical spectroscopy to be applied in biomedical research. This method has a potential to be used for medical imaging and diagnostic purposes together with other, already established, imaging techniques.

Cell cycle (CC) reactivation in neurons seems to underlie the development of Alzheimer's disease (AD). We analyzed whether CC alterations can be detected in immortalized lymphocytes from patients with the sporadic and the familial form of AD (SAD and FAD). Real-time polymerase chain reaction (PCR)-arrays, immunoblotting, and flow cytometry demonstrated differences in the regulation of G1/S phases between SAD lymphocytes and cells from nondemented subjects, as well as between SAD and FAD cells. SAD compared to FAD lymphocytes showed differences in expression profiles of the 90 CC genes, and a marked increase in the level of the p21 protein, which promotes G1-arrest. Accordingly, SAD but not FAD cells had a prolonged G1-phase. γ-secretase inhibition did not change the CC profiles of the cell lines. These data show that SAD involves a prolongation of the G1 phase driven by p21 pathway, which is not activated in FAD cells. Thus, the mechanism in SAD differs from FAD. Moreover, disturbances of the CC in lymphocytes have a potential diagnostic value., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners., Results: By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway., Conclusion: Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3/7, suggesting that this protein may indirectly participate in apoptosis.