Publications

Single-cell transcriptomics reveals subtype-specific molecular profiles in Nrf2-deficient macrophages from murine atherosclerotic aortas

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator of antioxidant and anti-inflammatory response in all cell types. It also activates the transcription of genes important for macrophage function. Nrf2 activity declines with age and has been closely linked to atherosclerosis, but its specific role in this vascular pathology is not clear. Atherosclerotic plaques contain several macrophage subsets with distinct, yet not completely understood, functions in the lesion development. The aim of this study was to analyze the transcriptome of diverse Nrf2-deficient macrophage subpopulations from murine atherosclerotic aortas. Mice with transcriptionally inactive Nrf2 in Cdh5-expressing cells ( Nrf2 Cdh5tKO ) were used in the experiments. These mice lack transcriptional Nrf2 activity in endothelial cells, but also in a proportion of leukocytes. We confirmed that the bone marrow-derived and tissue-resident macrophages isolated from Nrf2 Cdh5tKO mice exhibit a significant decline in Nrf2 activity. Atherosclerosis was induced in Nrf2 Cdh5tKO and appropriate control mice via adeno-associated viral vector (AAV)-mediated overexpression of murine proprotein convertase subtilisin/kexin type 9 (Pcsk9) in the liver and high-fat diet feeding. After 21 weeks, live aortic cells were sorted on FACS and single-cell RNA sequencing (scRNA-seq) was performed. Unsupervised clustering singled out 13 distinct aortic cell types. Among macrophages, 9 subclusters were identified. Differential gene expression analysis revealed cell subtype-specific expression patterns. A subset of inflammatory macrophages from atherosclerotic Nrf2 Cdh5tKO mice demonstrated downregulation of DNA replication genes (e.g. Mcm7 , Lig1 , Pola1 ) concomitant with upregulation of DNA damage sensor Atr gene. Atherosclerotic Nrf2 Cdh5tKO Lyve1+ resident macrophages showed strong upregulation of IFN-stimulated genes, as well as changes in the expression of death pathways-associated genes ( Slc40a1 , Bcl2a1 ). Furthermore, we observed subtype-specific expression of core ferroptosis genes (e.g. Cp, Hells , Slc40a1 ) in inflammatory versus tissue resident macrophages. This observation suggested a link between ferroptosis and inflammatory microenvironment appearing at a very early stage of atherogenesis. Our findings indicate that Nrf2 deficiency in aortic macrophages leads to subtype-specific transcriptomic changes associated with inflammation, iron homeostasis, cell injury or death pathways. This may help understanding the role of aging-associated decline of Nrf2 activity and the function of specific macrophage subtypes in atherosclerotic lesion development.

Splenic Ly6Chi monocytes contribute to adverse late post-ischemic left ventricular remodeling in heme oxygenase-1 deficient mice

Heme oxygenase-1 (Hmox1) is a stress-inducible protein crucial in heme catabolism. The end products of its enzymatic activity possess anti-oxidative, anti-apoptotic and anti-inflammatory properties. Cardioprotective effects of Hmox1 were demonstrated in experimental models of myocardial infarction (MI). Nevertheless, its importance in timely resolution of post-ischemic inflammation remains incompletely understood. The aim of this study was to determine the role of Hmox1 in the monocyte/macrophage-mediated cardiac remodeling in a mouse model of MI. Hmox1 knockout (Hmox1^-/-) and wild-type (WT, Hmox1^+/+) mice were subjected to a permanent ligation of the left anterior descending coronary artery. Significantly lower incidence of left ventricle (LV) free wall rupture was noted between 3rd and 5th day after MI in Hmox1^-/- mice resulting in their better overall survival. Then, starting from 7th until 21st day post-MI a more potent deterioration of LV function was observed in Hmox1^-/- than in the surviving Hmox1^+/+ mice. This was accompanied by higher numbers of Ly6C^hi monocytes in peripheral blood, as well as higher expression of monocyte chemoattractant protein-1 and adhesion molecules in the hearts of MI-operated Hmox1^-/- mice. Consequently, a greater post-MI monocyte-derived myocardial macrophage infiltration was noted in Hmox1-deficient individuals. Splenectomy decreased the numbers of circulating inflammatory Ly6C^hi monocytes in blood, reduced the numbers of proinflammatory cardiac macrophages and significantly improved the post-MI LV function in Hmox1^-/- mice. In conclusion, Hmox1 deficiency has divergent consequences in MI. On the one hand, it improves early post-MI survival by decreasing the occurrence of cardiac rupture. Afterwards, however, the hearts of Hmox1-deficient mice undergo adverse late LV remodeling due to overactive and prolonged post-ischemic inflammatory response. We identified spleen as an important source of these cardiovascular complications in Hmox1^-/- mice.

Carbon monoxide and nitric oxide-mediated induction of VEGF synthesis in vascular smooth muscle cells

Global miRNA expression in Treg cells from patients with pulmonary sarcoidosis indicates for targeting of MAPK and FOXO signaling pathways

Sarcoidosis is a multisystem granulomatous disorder predominantly involving the lungs. The disequilibrium between effector and regulatory lymphocytes (Treg cells) in sarcoidosis has been suggested. MicroRNAs (miRNAs) are small non-coding molecules necessary for proper T cell functioning. Certain miRNAs can lead to polarization of the inflammatory response via IL-2 and IFN-g signaling pathways, implicated in sarcoidosis. In the current study, increase in the plasma levels of IFN-g induced protein 10, MIG, IL-12, IL-2R and VEGF was found in patients with pulmonary sarcoidosis (PS). These results point at persistence of the Th1 immune response with putative defective Treg functions. Therefore, the global miRNA expression of Treg (CD4+/CD25+) subpopulation, from peripheral blood (PB) and bronchoalveolar lavage (BAL) of PS patients, was assessed. As a control Treg cells from PB of healthy individuals (HI) were used. In the array 743 miRNAs were tested, but only 139 miRNA were found to be expressed in Tregs. Among them 15 were significantly different from the Treg cells of HI. Most of those miRNAs were up-regulated in BAL Treg cells and only two of them were down-regulated (miR-130b and miR-150). According to DIANA miR-Path the miRNAs differentially expressed (DE) in PS Treg cells target genes from MAPK signaling pathway. Interestingly the same DE-miRNAs also target genes in FOXO signaling pathway. Several DE-miRNAs (miR-223, -155 and -132) were predicted to target FOXO directly, suggesting impairment in PS Treg cell function and differentiation. <b>Acknowledgments:</b> Supported by OPUS grant No. 2012/07/B/NZ5/02506 (NCN) and KNOW grants.

Prostaglandin-J2 induces the endothelial synthesis of IL-8 independently of PPARgamma activation

Cross‐talk between micro<scp>RNA</scp>s, nuclear factor <scp>E</scp>2‐related factor 2, and heme oxygenase‐1 in ochratoxin <scp>A</scp>‐induced toxic effects in renal proximal tubular epithelial cells

Scope Ochratoxin A ( OTA ) is a mycotoxin exhibiting nephrotoxic and potential carcinogenic activity. We investigated the cross‐talk between micro RNA s, nuclear factor E 2‐related factor 2 ( N rf2), and heme oxygenase‐1 ( HO ‐1) in ochratoxin A ‐mediated effects. Methods and results In porcine renal proximal tubular cells, OTA increased expression of profibrotic transforming growth factors β ( TGF β) while concomitantly decreasing expression of N rf2, HO ‐1, and erythropoietin. Adenoviral overexpression of N rf2 counteracted OTA ‐mediated reduction in HO ‐1 and erythropoietin expression and cell proliferation as well as increase in reactive oxygen species ( ROS ) generation and TGF β expression. Additionally, inhibition of HO activity enhanced whereas adenoviral overexpression of HO ‐1 reduced expression of TGF β. Moreover, antioxidants, N ‐acetyl‐cysteine and desferioxamine, prevented OTA ‐mediated enhancement of ROS generation, and TGF β expression. Finally, OTA modulated micro RNA processing by upregulating LIN eage protein 28 and D i G eorge syndrome critical region‐8, increasing the total pool of cellular micro RNA s and elevating the expression of mi R ‐132 and mi R ‐200c. Inhibition of mi R ‐132 by specific antagomir restored the OTA ‐driven reduction in N rf2 expression. Moreover, anti‐mi R ‐132 and anti‐mi R ‐200c counteracted OTA ‐mediated decrease in HO ‐1 levels as well as increase in ROS production and TGF β expression. Conclusion We showed that attenuation of N rf2 and HO ‐1 expression through induction of mi R ‐132 and mi R ‐200c by OTA elevates ROS levels and profibrotic TGF β expression.

Valproic acid downregulates heme oxygenase-1 independently of Nrf2 by increasing ubiquitination and proteasomal degradation

The role of oxidative stress in skeletal muscle injury and regeneration: focus on antioxidant enzymes

Carbon Monoxide and Iron, by-Products of Heme Oxygenase, Modulate Vascular Endothelial Growth Factor Synthesis in Vascular Smooth Muscle Cells

Serine Biosynthesis Pathway Supports MYC–miR-494–EZH2 Feed-Forward Circuit Necessary to Maintain Metabolic and Epigenetic Reprogramming of Burkitt Lymphoma Cells

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.

Activation of Tyk2 and Stat3 Is Required for the Apoptotic Actions of Interferon-β in Primary Pro-B Cells

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.

Long-Term Neuroprotective Effects of NT-4–Engineered Mesenchymal Stem Cells Injected Intravitreally in a Mouse Model of Acute Retinal Injury

Transplantation of genetically modified MSCs that produce neurotrophic growth factors may represent a useful strategy for treatment of different forms of retinopathies in the future.

Human-induced pluripotent stem cell-derived cardiomyocytes, 3D cardiac structures, and heart-on-a-chip as tools for drug research

Stimulation of haematopoiesis in murine model of myocardial infarction. Role of heme oxygenase 1

Supplementary Figure 4 from Hypoxia-Regulated Overexpression of Soluble VEGFR2 Controls Angiogenesis and Inhibits Tumor Growth

&lt;p&gt;PDF - 2705KB, Expression of VEGFRs on the surface of B16F10-msVEGFR2 melanoma clone 13.3 and 16.4.&lt;/p&gt;

Cobalt protoporphyrin <scp>IX</scp> increases endogenous G‐ <scp>CSF</scp> and mobilizes <scp>HSC</scp> and granulocytes to the blood

Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells

Nrf2 deficiency exacerbates ochratoxin A-induced toxicity in vitro and in vivo