Publications

Supplementary Figure 5 from Hypoxia-Regulated Overexpression of Soluble VEGFR2 Controls Angiogenesis and Inhibits Tumor Growth

<p>PDF - 3176KB, Characterization of B16F10 wt and msVEGFR2 expressing clones 13.3 and 16.4 tumors in vivo.</p>

Hypoxic angiogenic transcriptome in human keratinocytes and microvascular endothelial cells: Macroarray and real-time PCR analysis.

Interplay Between Heme Oxygenase-1 and miR-378 Affects Non-Small Cell Lung Carcinoma Growth, Vascularization, and Metastasis

In vitro and in vivo data indicate that the interplay between HMOX1 and miR-378 significantly modulates NSCLC progression and angiogenesis, suggesting miR-378 as a new therapeutic target. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16, 293-296, 2012) with the following serving as open reviewers: James F. George, Mahin D. Maines, Justin C. Mason, and Yasufumi Sato.

Generation of human induced pluripotent stem cell line derived from Becker muscular dystrophy patient with CRISPR/Cas9-mediated correction of DMD gene mutation

Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by in-frame deletions in the dystrophin gene (DMD), leading to progressive muscle degeneration and weakness. We generated a human induced pluripotent stem cell (hiPSC) line from a BMD patient. BMD hiPSCs were then engineered by CRISPR/Cas9-mediated knock-in of missing exons 3-9 of DMD gene. Obtained hiPSC line may be a valuable tool for investigating the mechanisms underlying BMD pathogenesis.

Mięśniowe komórki progenitorowe - charakterystyka i funkcja

P1732 Augmentation of vascular endothelial growth factor synthesis by gene transfer of copper-zinc superoxide dismutase: the role of hydrogen proxide and involvement of heme oxygenase-1

The role of nitric oxide synthase, superoxide dismutase and heme oxygenase-1 in the synthesis of vascular endothelial growth factor

Tyk2 Tyrosine Kinase Expression Is Required for the Maintenance of Mitochondrial Respiration in Primary Pro-B Lymphocytes

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-beta. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-beta. Finally, Tyk2(-/-) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.

Heme Oxygenase-1 Influences Satellite Cells and Progression of Duchenne Muscular Dystrophy in Mice

In dystrophin-deficient mdx mice, expression of Hmox1 in limb skeletal muscles and diaphragm is higher than in wild-type animals, being consistently elevated from 8 up to 52 weeks, both in myofibers and inflammatory leukocytes. Accordingly, HO-1 expression is induced in muscles of DMD patients. Pharmacological inhibition of HO-1 activity or genetic ablation of Hmox1 aggravates muscle damage and inflammation in mdx mice. Double knockout animals (Hmox1^-/-mdx) demonstrate impaired exercise capacity in comparison with mdx mice. Interestingly, in contrast to the effect observed in muscle fibers, in dystrophin-deficient muscle satellite cells (SCs) expression of Hmox1 is decreased, while MyoD, myogenin, and miR-206 are upregulated compared with wild-type counterparts. Mdx SCs demonstrate disturbed and enhanced differentiation, which is further intensified by Hmox1 deficiency. RNA sequencing revealed downregulation of Atf3, MafK, Foxo1, and Klf2 transcription factors, known to activate Hmox1 expression, as well as attenuation of nitric oxide-mediated cGMP-dependent signaling in mdx SCs. Accordingly, treatment with NO-donor induces Hmox1 expression and inhibits differentiation. Finally, differentiation of mdx SCs was normalized by CO, a product of HO-1 activity. Innovation and Conclusions: HO-1 is induced in DMD, and HO-1 inhibition aggravates DMD pathology. Therefore, HO-1 can be considered a therapeutic target to alleviate this disease. Antioxid. Redox Signal. 00, 000-000.

Atorvastatin and Conditioned Media from Atorvastatin-Treated Human Hematopoietic Stem/Progenitor-Derived Cells Show Proangiogenic Activity <i>In Vitro</i> but Not <i>In Vivo</i>

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.

The multifaceted view of heart problem in Duchenne muscular dystrophy

Proangiogenic enzymes in non-small cell lung carcinoma — An interplay between thymidine phosphorylase and heme oxygenase-1?

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells.

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.

Influence of osteoclasts and osteoprotegerin on the mode of calcific degeneration of aortic valves

The presence of osteoclasts in stenotic valves associated with low circulating OPG levels and an enhanced proportion of M2 macrophages can represent a variant of calcific aortic valve disease with a specifically regulated calcification process.

585 TISSUE INHIBITOR OF METALLOPROTEINASES (TIMP1, TIMP2) ARE RELATED TO HYPERTENSION AND AORTIC STENOSIS?

Background: Aortic stenosis (AS) and hypertension (HT) are both associated with chronic pressure overload and left vetricular hyperthrophy. Matrix metalloproteinases (MMPs), specific tissue inhibitors of metalloproteinases (TIMPs) and inflammatory cytokines are involved in left ventricular remodeling. Local expression of TIMP-1 and TIMP-2 was found to be related to cardiac interstitial fibrosis and left ventricular overload. Objectives: The aim of study was to compare the serum level of metalloproteinase inhibitors (TIMP1, TIMP2) and selected mediators of inflammation and tissue remodeling (IL-1b, IL-6, monocyte chemoattractant protein 1 (MCP-1), TNFα, osteoprotegerin (OPG), osteopontin (OPN), NT-proBNP) in hypertensive and non-hypertensive stenotic patients and to evaluate the relationship of examined clinical and biochemical parameters to hypertension. Patients and Methods: Sixty six patients accepted for surgical aortic valve replacement with moderate (n=27) and severe (n=39) aortic stenosis were included. There were fifty two (78,8%) cases with concomitant arterial hypertension. In all patients we performed transthoracal echo with thypical projections, severe AS in 33 (50,0%) patients wtih mean gradient ≥ 50mmHg with left ventricular ejection fraction LVEF ≥50%, moderate AS 27 (40,9%) patients with mean gradient <50mmHg with LVEF < 50%. The panel of serum biomarkers: IL-1b, IL-6, TIMP1, 2 TIMP2, MCP-1, TNFα, OPG, OPN, NT-proBNP was measured using multiplex method based on Luminex technology (Flexmap 3D analyser). The data were analyzed with Mann-Whitney test, chi-squared/Fisher's exact test. Variables were also tested in multivariate logistic regression models. Results: The comparison of hypertensive and non-hypertensive patients disclosed the following significant differences: age (years) 67 (61-73) [median (inter-quartile range)] vs 57 (53-72); body mass index (BMI) 29,1 (26,9-32,1) vs 25,3 (22,8-29,6); IL-6 (pg/ml) 1,291 (0,82-2,872) vs 0,802 (0,563-1,17); TIMP2 (ng/ml) 113,6 (98,95-128,35) vs 95,73 (83,34-105,85) respectively. There were no significant differences in severity (moderate/severe) of aortic stenosis between hypertensive and non-hypertensive patients. In multivariate logistic regression analysis TIMP2 was the only factor significantly contributed to hypertension (OR=1.05, 95% CI=1.001-1.09, P=0.046). Conclusions: In our study the level of TIMP2 is related with arterial hypertension in patients with moderate to severe aortic stenosis influencing on pressure overload of the left ventricle.

Lack of miR-378 attenuates muscular dystrophy in mdx mice

The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 (Fgf1) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology.

Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3' untranslated region (3' UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J × FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J × FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability.

274P Generation of cardiac organoids from DuchenneMuscularDystrophy patient-derived induced pluripotent stem cells: a novel approach to understanding cardiomyopathy

Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells