Publications

Abstract Aims Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD. Methods and results For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM. Conclusion To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy.

Abstract Blood vessel formation is a fundamental process for the development of organism and tissue regeneration. Of importance, angiogenesis occurring during postnatal development is usually connected with inflammation. Here, we review how molecular and cellular mechanisms underlying inflammatory reactions regulate angiogenesis. Inflamed tissues are characterized by hypoxic conditions and immune cell infiltration. In this review, we describe an interplay of hypoxia‐inducible factors (HIFs), HIF1 and HIF2, as well as NF‐κB and nitric oxide in the regulation of angiogenesis. The mobilization of macrophages and the differential role of M1 and M2 macrophage subsets in angiogenesis are also discussed. Next, we present the current knowledge about microRNA regulation of inflammation in the context of new blood vessel formation. Finally, we describe how the mechanisms involved in inflammation influence tumor angiogenesis. We underlay and discuss the role of NF‐E2‐related factor 2/heme oxygenase‐1 pathway as crucial in the regulation of inflammation‐induced angiogenesis. © 2015 IUBMB Life, 67(3):145–159, 2015

Numerous bioactive chemical compounds of plant origin may influence the angiogenic activity of various cell types and may thus affect the formation of blood vessels. Here we present the angiogenic effects of extracts of edible plants collected in Crete, Southern Italy and Southern Spain. Extracts have been applied to cultured human microvascular endothelial cells (HMEC-1), human umbilical vein endothelial cells (HUVEC) and human keratinocytes (HaCaT). About half out of 96 extracts exerted an inhibitory effect on HMEC-1 proliferation. Additionally, we have noted the inhibitory effects of extracts on HUVEC differentiation on a Matrigel layer. None of the extracts showed a stimulatory activity. The extract of Thymus piperella exerted moderate inhibitory effect on cobalt-chloride induced VEGF synthesis, however, CoCl(2)-induced activation of hypoxia responsive element of VEGF promoter was significantly attenuated only by extract of Origanum heracleoticum. Our study indicates that extracts of local food plants, of potential value as nutraceuticals, contain chemical compounds which may inhibit angiogenesis. Demonstration of their real influence on human health requires, however, extensive animal studies and controlled clinical investigations.

Abstract The nuclear factor erythroid 2-related factor (Nrf2) is considered as a master cytoprotective factor regulating the expression of genes coding for 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 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. We have observed slightly increased muscle degeneration and delayed regeneration in Nrf2 tKO mice after CTX treatment. Nevertheless, transcriptional ablation of Nrf2 in mdx mice did not significantly aggravate the most deleterious pathological hallmarks of DMD such as degeneration, inflammation, fibrotic scar formation, and decreased angiogenesis, as well as the number and proliferation of satellite cells. In conclusion, our analyses in both acute and chronic injury mouse models have revealed no significant influence of Nrf2 transcriptional deficiency on skeletal muscle regeneration and function.

Psoriasis (Ps) is a chronic, inflammatory skin disease characterized by thickened, red and scaly plaques. Systemic inflammation associated with psoriasis results in an increased risk of death due to the development of psoriasis-associated comorbidities such as cardiovascular disease (CVD) and metabolic syndrome. Although the cardiometabolic features in psoriasis are clinically well described, the underlying molecular mechanisms linking these comorbidities remain poorly understood. Generation of induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMCs) and skin fibroblasts (SFs) of psoriatic patients provides a novel approach to investigate the pathway by which cutaneous inflammation promotes CV complications in this disorder.

Numerous bioactive chemical compounds of plant origin may influence the angiogenic activity of various cell types and may thus affect the formation of blood vessels. Here we present the angiogenic effects of extracts of edible plants collected in Crete, Southern Italy and Southern Spain. Extracts have been applied to cultured human microvascular endothelial cells (HMEC-1), human umbilical vein endothelial cells (HUVEC) and human keratinocytes (HaCaT). About half out of 96 extracts exerted an inhibitory effect on HMEC-1 proliferation. Additionally, we have noted the inhibitory effects of extracts on HUVEC differentiation on a Matrigel layer. None of the extracts showed a stimulatory activity. The extract of Thymus piperella exerted moderate inhibitory effect on cobalt-chloride induced VEGF synthesis, however, CoCl(2)-induced activation of hypoxia responsive element of VEGF promoter was significantly attenuated only by extract of Origanum heracleoticum. Our study indicates that extracts of local food plants, of potential value as nutraceuticals, contain chemical compounds which may inhibit angiogenesis. Demonstration of their real influence on human health requires, however, extensive animal studies and controlled clinical investigations.

Heme oxygenase-1 (HO-1, encoded by HMOX1) is a cytoprotective enzyme degrading heme into CO, Fe2+, and biliverdin. HO-1 was demonstrated to affect cardiac differentiation of murine pluripotent stem cells (PSCs), regulate the metabolism of murine adult cardiomyocytes, and influence regeneration of infarcted myocardium in mice. However, the enzyme’s effect on human cardiogenesis and human cardiomyocytes’ electromechanical properties has not been described so far. Thus, this study aimed to investigate the role of HO-1 in the differentiation of human induced pluripotent stem cells (hiPSCs) into hiPSC-derived cardiomyocytes (hiPSC-CMs). hiPSCs were generated from human fibroblasts and peripheral blood mononuclear cells using Sendai vectors and subjected to CRISPR/Cas9-mediated HMOX1 knock-out. After confirming lack of HO-1 expression on the protein level, isogenic control and HO-1-deficient hiPSCs were differentiated into hiPSC-CMs. No differences in differentiation efficiency and hiPSC-CMs metabolism were observed in both cell types. The global transcriptomic analysis revealed, on the other hand, alterations in electrophysiological pathways in hiPSC-CMs devoid of HO-1, which also demonstrated increased size. Functional consequences in changes in expression of ion channels genes were then confirmed by patch-clamp analysis. To the best of our knowledge, this is the first report demonstrating the link between HO-1 and electrophysiology in human cardiomyocytes.

<p>Effect of SnPP (10 μM, 24 h) on RMS cell lines. A - Expression of cMET, HGF, CXCR4 and SDF1 mRNAs in CW9019 aRMS cell line.</p>

We present a case of widespread cutaneous telangiectasias in a patient with a B-cell intravascular lymphoma most likely representing tumor-induced angiogenesis. The patient presented with a rapid onset of large cutaneous telangiectasias and skin edema, followed by the development of multiorgan failure. We describe difficulties with the ante-mortem diagnosis in the patient with predominant, clinically observed, skin lesions. The patient had disseminated disease involving many organs with a rapidly fatal outcome. The final diagnosis of intravascular malignant lymphoma (IVL) was established post-mortem after morphological and immunohistochemical studies of the autopsy material.