Publications

Single-Cell RNA-Sequencing and Metabolomics Analyses Reveal the Contribution of Perivascular Adipose Tissue Stem Cells to Vascular Remodeling

Objective: Perivascular adipose tissue (PVAT) plays a vital role in maintaining vascular homeostasis. However, most studies ascribed the function of PVAT in vascular remodeling to adipokines secreted by the perivascular adipocytes. Whether mesenchymal stem cells exist in PVAT and play a role in vascular regeneration remain unknown. Approach and Results: Single-cell RNA-sequencing allowed direct visualization of the heterogeneous PVAT-derived mesenchymal stem cells (PV-ADSCs) at a high resolution and revealed 2 distinct subpopulations, among which one featured signaling pathways crucial for smooth muscle differentiation. Pseudotime analysis of cultured PV-ADSCs unraveled their smooth muscle differentiation trajectory. Transplantation of cultured PV-ADSCs in mouse vein graft model suggested the contribution of PV-ADSCs to vascular remodeling through smooth muscle differentiation. Mechanistically, treatment with TGF-β1 (transforming growth factor β1) and transfection of microRNA (miR)-378a-3p mimics induced a similar metabolic reprogramming of PV-ADSCs, including upregulated mitochondrial potential and altered lipid levels, such as increased cholesterol and promoted smooth muscle differentiation. Conclusions: Single-cell RNA-sequencing allows direct visualization of PV-ADSC heterogeneity at a single-cell level and uncovers 2 subpopulations with distinct signature genes and signaling pathways. The function of PVAT in vascular regeneration is partly attributed to PV-ADSCs and their differentiation towards smooth muscle lineage. Mechanistic study presents miR-378a-3p which is a potent regulator of metabolic reprogramming as a potential therapeutic target for vascular regeneration.

Stem cells: applications, perspectives, misunderstandings

Stem cells exist and can do a lot. For several decades, bone marrow and umbilical cord blood transplants containing haematopoietic stem cells have been used in the treatment of blood diseases. Genetic modifications (gene therapy) of such cells help to cure complex immunodeficiencies and severe anaemias. The limbal stem cells taken from the eye and properly multiplied can regenerate the damaged cornea, and the epidermal stem cells help in the treatment of severe burns and some hereditary, severe skin diseases. Promising experimental research is under way on other uses of stem cells. However, these cells are appropriately selected, having real ability to differentiate into specialized cells whose malfunction is the cause of the disease. Therapeutic applications of stem cells are apparently limited to date. Meanwhile, the Internet is full of advertisements for supposedly miraculous treatments for almost any disease. Stem cells have become a modern synonym of the Holy Grail. A wonderful dish, transforming every drink into elixir of health, youth and long life. Stem cells from a single source, e.g., umbilical cord blood, or so-called cells, although without proven properties of stem cells, are offered in commercial private clinics as a panacea for autism, cerebral palsy, spina bifida, eye diseases, amyotrophic lateral sclerosis and dozens other disorders. Without justification for their action in these diseases, without convincing evidence of safety, but for a high fee. This article discusses stem cells and misunderstandings about including any cells among them. It draws attention to the real possibilities and confirmed uses of stem cells and presents the problems, doubts and dangers for patients associated with commercial offers of treatments using “stem” cells. The author cites the positions of scientific institutions and societies warning against premature commercialization of unjustified and potentially dangerous therapies

Lymphoid organs of <i>Gasterosteus aculeatus</i>

15d-PGJ ² Upregulates Synthesis of IL-8 in Endothelial Cells Through Induction of Oxidative Stress

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a cyclopentenone prostaglandin regarded as antiinflammatory mediator, which can act through peroxisome proliferator-activated receptor-gamma (PPARgamma) or through G protein-coupled surface receptors. It has been demonstrated that 15d-PGJ(2) potently increases the generation of interleukin-8 (IL-8) in human microvascular endothelial cells (HMEC-1s); however, the mechanism of this induction is not known. The aim of the study was to find the pathway involved in 15d-PGJ(2)-mediated IL-8 stimulation. Our data confirmed that the effect of 15d-PGJ(2) is independent of PPARgamma. For the first time, we excluded the activation of G proteins and the contribution of G protein-coupled surface receptors in endothelial cells treated with 15d-PGJ(2). Instead, we demonstrated that stimulation of IL-8 involved induction of oxidative stress, activation of p38 kinases, and increase in stability of IL-8 mRNA. Upregulation of IL-8 promoter, although measurable, seemed to play a less-pronounced role. Additionally, our results indicate the involvement of cAMP elevation and may suggest a role for ATF2 transcription factor. Concomitant induction of heme oxygenase-1 in HMEC-1s did not influence the synthesis of IL-8. In summary, we showed that 15d-PGJ(2), acting through oxidative stress, may exert proinflammatory effects. The upregulation of IL-8 is mostly associated with p38-mediated stabilization of mRNA.

HO-1 and miR-378 regulate tumor microenvironment and angiogenic potential of human lung cancer

Senescence of endothelial cells promotes phenotypic changes in adventitial fibroblasts: possible implications for vascular aging

A novel 3D cardiac microtissue model for investigation of cardiovascular complications in rheumatoid arthritis

Pharmacological versus genetic inhibition of heme oxygenase-1 – the comparison of metalloporphyrins, shRNA and CRISPR/Cas9 system

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may be 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, basal and hemin-induced HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA, protein and activity level. 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 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 H2O2 treatment. In summary, we have shown that all technologies can be used for inhibition of HO activity in vitro; however, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach.

Selectin‐P (PADGEM, GMP‐140)‐ mediated Adhesion of Human Platelets to Neutrophils <i>in Vitro</i> and Immune Complex‐induced Peritonitis in Rats Is Influenced by Interleukin‐8

Comparison of shRNA and CRISPR/Cas9 in heme oxygenase-1 targeting efficiency

HIF-1 induction attenuates inter-leukine-8-synthesis in human endothelial cells

Clock and clock-controlled genes are differently expressed in the retina, lamina and in selected cells of the visual system of Drosophila melanogaster

The retina and the first optic neuropil (lamina) of Drosophila show circadian rhythms in various processes. To learn about the regulation of circadian rhythms in the retina and lamina and in two cell types, glial and the lamina L2 interneurons, we examined expression of the following clock genes; per, tim, clk, and cry and clock-controlled genes (ccgs); Atpα, nrv2, brp, Pdfr. We found that the expression of gene studied is specific for the retina and lamina. The rhythms of per and tim expression in the retina and glial cells are similar to that observed in the whole head and in clock neurons, while they differ in the lamina and L2 cells. In both the retina and lamina, CRY seems to be a repressor of clk expression. In L2 interneurons per expression is not cyclic indicating the other function of PER in those cells than in the circadian molecular clock. In contrast to per and tim, the pattern of clk and cry expression is similar in both the retina and lamina. The retina holds the autonomous oscillators but the expression of cry and ccgs, Atpα and nrv2, is also regulated by inputs from the pacemaker transmitted by PDF and ITP neuropeptides.

Supplementary Figure 6 from Hypoxia-Regulated Overexpression of Soluble VEGFR2 Controls Angiogenesis and Inhibits Tumor Growth

&lt;p&gt;PDF - 1037KB, Effect of the IFP1.4 and mCherry expression on the B16F10 cells proliferation.&lt;/p&gt;

Supplementary Figure 2 from Hypoxia-Regulated Overexpression of Soluble VEGFR2 Controls Angiogenesis and Inhibits Tumor Growth

&lt;p&gt;PDF - 55KB, Shortening of the minCMV promoter.&lt;/p&gt;

A novel spheroid–plug model to study tumor angiogenesis and development

Ciglitazone, Ligand of Peroxisome Proliferator-Activated Receptor-gamma, Inhibits Vascular Endothelial Growth Factor Activity

Summary: Background: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands were shown to induce the vascular endothelial growth factor (VEGF) synthesis in several cell types. We tested the effect of ciglitazone, one of the most specific PPARγ agonists, on the angiogenic activity of VEGF. Methods: Experiments were performed on human umbilical vein endothelial cell (HUVEC), incubated for 12 to 48 h with human VEGF165 (30 ng/ml) in the presence or absence of ciglitazone (1 – 10 µM). Cell proliferation was tested by BrdU incorporation. Migration was measured in modified Boyden chambers. Morphogenesis and capillary outgrowth were assessed by tube formation assay and spheroid cell culture, respectively. Results: Treatment with ciglitazone inhibited by about 50 % VEGF-induced proliferation, migration, morphogenesis, and sprouting of capillaries. Conclusions: Thus we conclude that, despite augmentation of VEGF generation, ciglitazone may act as an antiangiogenic agent attenuating VEGF activity by directly influencing endothelial cells. Zusammenfassung: Grundlagen: Peroxisome proliferator-activated receptor-γ (PPARγ)-Liganden induzieren die Synthese von Vascular Endothelial Growth Factor (VEGF) in verschiedenen Zellen. Methodik: Es wurde die Wirkung von Ciglitazon, einer der potentesten PPARγ-Agonisten, auf die angiogenetische Wirkung von VEGF untersucht. HUVEC (human umblical endothelial cells) wurden für 12 bis 48 Stunden mit humanem VEGF165 (30 ng/ml) sowohl mit, als auch ohne Ciglitazon (1 – 10 µM) inkubiert. Ergebnisse: Ciglitazon hat die angiogenetischen Aktivitäten von Endothelzellen gehemmt. Die Behandlung mit Ciglitazon hat die VEGF-induzierte Proliferation und Migration von HUVEC unterdrückt. Weiteres konnte mit der Ciglitazon-Behandlung eine Reduktion der morphogenetischen Kapazität von Endothelzellen („tube formation assay”) sowie ein vermindertes Aussprossen der Kapillaren (Sphärenzellkultur) nachgewiesen werden. Schlußfolgerungen: Obwohl Ciglitazon die Synthese von VEGF erhöht, hat Ciglitazon eine anti-angiogenetische Wirkung, welche durch Hemmung der VEGF-Aktivität und direkten Einfluß auf Endothelzellen bedingt ist.

Angiogenic Gene Therapy With Vascular Endothelial Growth Factor - Hope or Hype?

Summary: Background: Angiogenesis is a promising novel therapeutic strategy to provide new venues for blood flow in patients with severe ischaemic heart and peripheral vascular diseases. Among several stimulators, the vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2, basic FGF) have been most widely studied. Methods: Preclinical animal studies suggested the strong therapeutic potentials of VEGF, and such positive effects have also been confirmed in a limited number of phase I clinical trials. However, some animal studies suggest the existence of a considerable risk associated with angiogenic therapy. Results: VEGF is strongly expressed in human atherosclerotic plaques, it promotes atherosclerosis in animal models, and there is evidence indicating that the plaque growth is dependent on the formation of new blood vessels. Therefore, for a convincing demonstration of the safety and efficacy of VEGF gene therapy, the clinical effects need to be evaluated in larger randomized, double-blinded clinical trials. Conclusions: Further understanding of the mechanisms of activity of angiogenic growth factors and the formation of blood vessels is required, and improved gene transfer methods are necessary to introduce therapeutic angiogenesis in clinical practice. Zusammenfassung: Grundlagen: Die Angiogenese ist eine vielversprechende neue therapeutische Strategie bei Patienten mit Myokardischämie und peripheren Gefäßerkrankungen. Unter den verschiedensten Stimulatoren sind der Vascular Endothelial Growth Factor (VEGF) und der Fibroblast Growth Factor-2 (FGF-2, basic FGF) am besten erforscht. Methodik: Vorklinische Tierversuche lassen ein großes therapeutisches Potential erwarten, und diese positiven Effekte werden von einer begrenzten Zahl von Phase-I-Studien bestätigt. Einige der experimentellen Studien lassen erhebliche Risiken in Verbindung mit einer angiogenetischen Therapie vermuten. Ergebnisse: VEGF wird von humanen atherosklerotischen Plaques verstärkt gebildet, fördert Atherosklerose am Tiermodell, und es gibt Anzeichen, daß das Plaquewachstum von der Bildung neuer Blutgefäße abhängig ist. Daher sollten die klinischen Effekte, um die Sicherheit und Effizienz einer VEGF-Gentherapie zu zeigen, in großen, randomisierten, doppelblinden, klinischen Studien evaluiert werden. Schlußfolgerungen: Um eine therapeutische Angiogenese in der klinischen Praxis anwenden zu können, ist ein genaueres Verständnis der Mechanismen der Aktivität von angiogenetischen Wachstumsfaktoren und der Bildung von Blutgefäßen und die Verbesserung der Gentransfermethoden nötig.

The role of heme oxygenase-1 in the inflammatory bowel diseases