MCPIP1 Inhibits Hepatic Stellate Cell Activation in Autocrine and Paracrine Manners, Preventing Liver Fibrosis.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by enhanced deposition of extracellular matrix (ECM), which results from the wound healing response to chronic, repeated injury of any etiology. Upon injury, hepatic stellate cells (HSCs) activate and secrete ECM proteins, forming scar tissue, which leads to liver dysfunction. Monocyte-chemoattractant protein-induced protein 1 (MCPIP1) possesses anti-inflammatory activity, and its overexpression reduces liver injury in septic mice. In addition, mice with liver-specific deletion of Zc3h12a develop features of primary biliary cholangitis. In this study, we investigated the role of MCPIP1 in liver fibrosis and HSC activation. METHODS: We analyzed MCPIP1 levels in patients' fibrotic livers and hepatic cells isolated from fibrotic murine livers. In vitro experiments were conducted on primary HSCs, cholangiocytes, hepatocytes, and LX-2 cells with MCPIP1 overexpression or silencing. RESULTS: MCPIP1 levels are induced in patients' fibrotic livers compared with their nonfibrotic counterparts. Murine models of fibrosis revealed that its level is increased in HSCs and hepatocytes. Moreover, hepatocytes with Mcpip1 deletion trigger HSC activation via the release of connective tissue growth factor. Overexpression of MCPIP1 in LX-2 cells inhibits their activation through the regulation of TGFB1 expression, and this phenotype is reversed upon MCPIP1 silencing. CONCLUSIONS: We demonstrated that MCPIP1 is induced in human fibrotic livers and regulates the activation of HSCs in both autocrine and paracrine manners. Our results indicate that MCPIP1 could have a potential role in the development of liver fibrosis.

Proteome Profiling of the Dystrophic mdx Mice Diaphragm.

Mdx mice with a spontaneous mutation in exon 23 of the Dmd gene represent the most common model to investigate the pathophysiology of Duchenne muscular dystrophy (DMD). The disease, caused by the lack of functional dystrophin, is characterized by irreversible impairment of muscle functions, with the diaphragm affected earlier and more severely than other skeletal muscles. We applied a label-free (LF) method and the more thorough tandem mass tag (TMT)-based method to analyze differentially expressed proteins in the diaphragm of 6-week-old mdx mice. The comparison of both methods revealed 88 commonly changed proteins. A more in-depth analysis of the TMT-based method showed 953 significantly changed proteins, with 867 increased and 86 decreased in dystrophic animals (q-value < 0.05, fold-change threshold: 1.5). Consequently, several dysregulated processes were demonstrated, including the immune response, fibrosis, translation, and programmed cell death. Interestingly, in the dystrophic diaphragm, we found a significant decrease in the expression of enzymes generating hydrogen sulfide (H2S), suggesting that alterations in the metabolism of this gaseous mediator could modulate DMD progression, which could be a potential target for pharmacological intervention.

Hepatic Mcpip1 regulates adaptation to food restriction in mice.

Monocyte-chemoattractant protein-induced protein 1 (MCPIP1, or Regnase-1) is an endoribonuclease that degrades translationally active mRNA molecules. MCPIP1 is mostly known for its anti-inflammatory actions, but it is also an important regulator of adipogenesis and lipid metabolism. Its overexpression impairs adipogenesis by reducing mRNA levels of C/EBPß and PPARγ, key transcription factors regulating this process. Although adipocytes overexpressing MCPIP1 are characterised by impaired glucose uptake, the function of MCPIP1 in hepatocyte metabolism remains unknown. In this study, conditional deletion of Zc3h12a in murine liver epithelial cells was used to characterise the role of Mcpip1 in adaptation to 24-hour food restriction. We found that Mcpip1 deficiency in liver epithelial cells (Mcpip1fl/flAlbCre mice) resulted in higher blood glucose levels in response to fasting in comparison to Mcpip1fl/fl counterparts. Hepatic proteome analysis showed 26 down-regulated and 117 up-regulated proteins in Mcpip1fl/flAlbCre animals that were involved in cellular adhesion, extracellular matrix and metabolic processes. In conclusion, our studies provide new insight into the hepatic function of Mcpip1 and its involvement in metabolic control.

miR-378 influences muscle satellite cells and enhances adipogenic potential of fibro-adipogenic progenitors but does not affect muscle regeneration in the glycerol-induced injury model.

Skeletal muscle regeneration relies on the reciprocal interaction between many types of cells. Regenerative capacity may be altered in different disorders. In our study, we investigated whether the deletion of miR-378a (miR-378) affects muscle regeneration. We subjected 6-week-old wild-type (WT) and miR-378 knockout (miR-378-/-) animals to the glycerol-induced muscle injury and performed analyses in various time-points. In miR-378-/- animals, an elevated abundance of muscle satellite cells (mSCs) on day 3 was found. Furthermore, fibro-adipogenic progenitors (FAPs) isolated from the muscle of miR-378-/- mice exhibited enhanced adipogenic potential. At the same time, lack of miR-378 did not affect inflammation, fibrosis, adipose tissue deposition, centrally nucleated fiber count, muscle fiber size, FAP abundance, and muscle contractility at any time point analyzed. To conclude, our study revealed that miR-378 deletion influences the abundance of mSCs and the adipogenic potential of FAPs, but does not affect overall regeneration upon acute, glycerol-induced muscle injury.

Sodium hydrosulfide moderately alleviates the hallmark symptoms of Duchenne muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is an incurable disease caused by mutations in the X-linked DMD gene that encodes a structural muscle protein, dystrophin. This, in turn, leads to progressive degeneration of the skeletal muscles and the heart. Hydrogen sulfide (H2S), the pleiotropic agent with antioxidant, anti-inflammatory, and pro-angiogenic activities, could be considered a promising therapeutic factor for DMD. In this work, we studied the effect of daily intraperitoneal administration of the H2S donor, sodium hydrosulfide (NaHS, 100 µmol/kg/day for 5 weeks) on skeletal muscle (gastrocnemius, diaphragm and tibialis anterior) pathology in dystrophin-deficient mdx mice, characterized by decreased expression of H2S-generating enzymes. NaHS reduced the level of muscle damage markers in plasma (creatine kinase, lactate dehydrogenase and osteopontin). It lowered oxidative stress by affecting the GSH/GSSG ratio, up-regulating the level of cytoprotective heme oxygenase-1 (HO-1) and down-regulating the NF-κB pathway. In the gastrocnemius muscle, it also increased angiogenic vascular endothelial growth factor (Vegf) and its receptor (Kdr) expression, accompanied by the elevated number of α-SMA/CD31/lectin-positive blood vessels. The expression of fibrotic regulators, like Tgfß, Col1a1 and Fn1 was decreased by NaHS in the tibialis anterior, while the level of autophagy markers (AMPKα signalling and Atg genes), was mostly affected in the gastrocnemius. Histological and molecular analysis showed no effect of H2S donor on regeneration and the muscle fiber type composition. Overall, the H2S donor modified the gene expression and protein level of molecules associated with the pathophysiology of DMD, contributing to the regulation of oxidative stress, inflammation, autophagy, and angiogenesis.

miR-378 affects metabolic disturbances in the mdx model of Duchenne muscular dystrophy.

Although Duchenne muscular dystrophy (DMD) primarily affects muscle tissues, the alterations to systemic metabolism manifested in DMD patients contribute to the severe phenotype of this fatal disorder. We propose that microRNA-378a (miR-378) alters carbohydrate and lipid metabolism in dystrophic mdx mice. In our study, we utilized double knockout animals which lacked both dystrophin and miR-378 (mdx/miR-378-/-). RNA sequencing of the liver identified 561 and 194 differentially expressed genes that distinguished mdx versus wild-type (WT) and mdx/miR-378-/- versus mdx counterparts, respectively. Bioinformatics analysis predicted, among others, carbohydrate metabolism disorder in dystrophic mice, as functionally proven by impaired glucose tolerance and insulin sensitivity. The lack of miR-378 in mdx animals mitigated those effects with a faster glucose clearance in a glucose tolerance test (GTT) and normalization of liver glycogen levels. The absence of miR-378 also restored the expression of genes regulating lipid homeostasis, such as Acly, Fasn, Gpam, Pnpla3, and Scd1. In conclusion, we report for the first time that miR-378 loss results in increased systemic metabolism of mdx mice. Together with our previous finding, demonstrating alleviation of the muscle-related symptoms of DMD, we propose that the inhibition of miR-378 may represent a new strategy to attenuate the multifaceted symptoms of DMD.

Simvastatin does not alleviate muscle pathology in a mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an incurable disease, caused by the mutations in the DMD gene, encoding dystrophin, an actin-binding cytoskeletal protein. Lack of functional dystrophin results in muscle weakness, degeneration, and as an outcome cardiac and respiratory failure. As there is still no cure for affected individuals, the pharmacological compounds with the potential to treat or at least attenuate the symptoms of the disease are under constant evaluation. The pleiotropic agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, known as statins, have been suggested to exert beneficial effects in the mouse model of DMD. On the other hand, they were also reported to induce skeletal-muscle myopathy. Therefore, we decided to verify the hypothesis that simvastatin may be considered a potential therapeutic agent in DMD. METHODS: Several methods including functional assessment of muscle function via grip strength measurement, treadmill test, and single-muscle force estimation, enzymatic assays, histological analysis of muscle damage, gene expression evaluation, and immunofluorescence staining were conducted to study simvastatin-related alterations in the mdx mouse model of DMD. RESULTS: In our study, simvastatin treatment of mdx mice did not result in improved running performance, grip strength, or specific force of the single muscle. Creatine kinase and lactate dehydrogenase activity, markers of muscle injury, were also unaffected by simvastatin delivery in mdx mice. Furthermore, no significant changes in inflammation, fibrosis, and angiogenesis were noted. Despite the decreased percentage of centrally nucleated myofibers in gastrocnemius muscle after simvastatin delivery, no changes were noticed in other regeneration-related parameters. Of note, even an increased rate of necrosis was found in simvastatin-treated mdx mice. CONCLUSION: In conclusion, our study revealed that simvastatin does not ameliorate DMD pathology.

Age-Dependent Dysregulation of Muscle Vasculature and Blood Flow Recovery after Hindlimb Ischemia in the mdx Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by a lack of functional dystrophin, is characterized by progressive muscle degeneration. Interestingly, dystrophin is also expressed in endothelial cells (ECs), and insufficient angiogenesis has already been hypothesized to contribute to DMD pathology, however, its status in mdx mice, a model of DMD, is still not fully clear. Our study aimed to reveal angiogenesis-related alterations in skeletal muscles of mdx mice compared to wild-type (WT) counterparts. By investigating 6- and 12-week-old mice, we sought to verify if those changes are age-dependent. We utilized a broad spectrum of methods ranging from gene expression analysis, flow cytometry, and immunofluorescence imaging to determine the level of angiogenic markers and to assess muscle blood vessel abundance. Finally, we implemented the hindlimb ischemia (HLI) model, more biologically relevant in the context of functional studies evaluating angiogenesis/arteriogenesis processes. We demonstrated that both 6- and 12-week-old dystrophic mice exhibited dysregulation of several angiogenic factors, including decreased vascular endothelial growth factor A (VEGF) in different muscle types. Nonetheless, in younger, 6-week-old mdx animals, neither the abundance of CD31+α-SMA+ double-positive blood vessels nor basal blood flow and its restoration after HLI was affected. In 12-week-old mdx mice, although a higher number of CD31+α-SMA+ double-positive blood vessels and an increased percentage of skeletal muscle ECs were found, the abundance of pericytes was diminished, and blood flow was reduced. Moreover, impeded perfusion recovery after HLI associated with a blunted inflammatory and regenerative response was evident in 12-week-old dystrophic mice. Hence, our results reinforce the hypothesis of age-dependent angiogenic dysfunction in dystrophic mice. In conclusion, we suggest that older mdx mice constitute an appropriate model for preclinical studies evaluating the effectiveness of vascular-based therapies aimed at the restoration of functional angiogenesis to mitigate DMD severity.

Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

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.

Dysregulated Autophagy and Mitophagy in a Mouse Model of Duchenne Muscular Dystrophy Remain Unchanged Following Heme Oxygenase-1 Knockout.

Dysregulation of autophagy may contribute to the progression of various muscle diseases, including Duchenne muscular dystrophy (DMD). Heme oxygenase-1 (HO-1, encoded by Hmox1), a heme-degrading enzyme, may alleviate symptoms of DMD, inter alia, through anti-inflammatory properties. In the present study, we determined the role of HO-1 in the regulation of autophagy and mitophagy in mdx animals, a commonly used mouse model of the disease. In the gastrocnemius of 6-week-old DMD mice, the mRNA level of mitophagy markers: Bnip3 and Pink1, as well as autophagy regulators, e.g., Becn1, Map1lc3b, Sqstm1, and Atg7, was decreased. In the dystrophic diaphragm, changes in the latter were less prominent. In older, 12-week-old dystrophic mice, diminished expressions of Pink1 and Sqstm1 with upregulation of Atg5, Atg7, and Lamp1 was depicted. Interestingly, we demonstrated higher protein levels of autophagy regulator, LC3, in dystrophic muscles. Although the lack of Hmox1 in mdx mice influenced blood cell count and the abundance of profibrotic proteins, no striking differences in mRNA and protein levels of autophagy and mitophagy markers were found. In conclusion, we demonstrated complex, tissue, and age-dependent dysregulation of mitophagic and autophagic markers in DMD mice, which are not affected by the additional lack of Hmox1.

Hypoxia as a Driving Force of Pluripotent Stem Cell Reprogramming and Differentiation to Endothelial Cells.

Inadequate supply of oxygen (O2) 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.

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.

Synthetically Lethal Interactions of Heme Oxygenase-1 and Fumarate Hydratase Genes.

Elevated expression of heme oxygenase-1 (HO-1, encoded by HMOX1) is observed in various types of tumors. Hence, it is suggested that HO-1 may serve as a potential target in anticancer therapies. A novel approach to inhibit HO-1 is related to the synthetic lethality of this enzyme and fumarate hydratase (FH). In the current study, we aimed to validate the effect of genetic and pharmacological inhibition of HO-1 in cells isolated from patients suffering from hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-an inherited cancer syndrome, caused by FH deficiency. Initially, we confirmed that UOK 262, UOK 268, and NCCFH1 cell lines are characterized by non-active FH enzyme, high expression of Nrf2 transcription factor-regulated genes, including HMOX1 and attenuated oxidative phosphorylation. Later, we demonstrated that shRNA-mediated genetic inhibition of HMOX1 resulted in diminished viability and proliferation of cancer cells. Chemical inhibition of HO activity using commercially available inhibitors, zinc and tin metalloporphyrins as well as recently described new imidazole-based compounds, especially SLV-11199, led to decreased cancer cell viability and clonogenic potential. In conclusion, the current study points out the possible relevance of HO-1 inhibition as a potential anti-cancer treatment in HLRCC. However, further studies revealing the molecular mechanisms are still needed.

miR-378a influences vascularization in skeletal muscles.

AIMS: MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. METHODS AND RESULTS: Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a-/-) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a-/- aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a-/- muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a-/- and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G-) was higher in the ischaemic muscles of miR-378a-/- mice, suggesting an anti-inflammatory action of miR-378a. CONCLUSIONS: Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.

miR-146a deficiency does not aggravate muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is a genetic disease evoked by a mutation in the dystrophin gene. It is associated with progressive muscle degeneration and increased inflammation. Up to this date, mainly anti-inflammatory treatment is available for patients suffering from DMD. miR-146a is known to diminish inflammation and fibrosis in different tissues by downregulating the expression of proinflammatory cytokines. However, its role in DMD has not been studied so far.In our work, we have generated mice globally lacking both dystrophin and miR-146a (miR-146a-/-mdx) and examined them together with wild-type, single miR-146a knockout and dystrophic (mdx-lacking dystrophin) mice in a variety of aspects associated with DMD pathophysiology (muscle degeneration, inflammatory reaction, muscle satellite cells, muscle regeneration, and fibrosis).We have shown that miR-146a level is increased in dystrophic muscles in comparison to wild-type mice. Its deficiency augments the expression of proinflammatory cytokines (IL-1ß, CCL2, TNFα). However, muscle degeneration was not significantly worsened in mdx mice lacking miR-146a up to 24 weeks of age, although some aggravation of muscle damage and inflammation was evident in 12-week-old animals, though no effect of miR-146a deficiency was visible on quantity, proliferation, and in vitro differentiation of muscle satellite cells isolated from miR-146a-/-mdx mice vs. mdx. Similarly, muscle regeneration and collagen deposition were not changed by miR-146a deficiency. Nevertheless, the lack of miR-146a is associated with decreased Vegfa and increased Tgfb1.Overall, the lack of miR-146a did not aggravate significantly the dystrophic conditions in mdx mice, but its effect on DMD in more severe conditions warrants further investigation.

Role of the kidneys in the redistribution of heme-derived iron during neonatal hemolysis in mice.

Moderate intravascular hemolysis is a common condition in newborns. It is followed by the accumulation of bilirubin, which is a secondary product of the activity of heme oxygenase-1, an enzyme that catalyzes the breakdown of heme released from disrupted erythrocytes and taken up by hepatic macrophages. Although these cells are a major site of enzymatic heme breakdown in adults, we show here that epithelial cells of proximal tubules in the kidneys perform the functions of both heme uptake and catabolism in mouse neonates. A time-course study examining mouse pups during the neonatal period showed a gradual recovery from hemolysis, and concomitant decreases in the expression of heme-related genes and non-heme iron transporters in the proximal tubules. By adjusting the expression of iron-handling proteins in response to the disappearance of hemolysis in mouse neonates, the kidneys may play a role in the detoxification of iron and contribute to its recirculation from the primary urine to the blood.

Development and characterization of a new inhibitor of heme oxygenase activity for cancer treatment.

Heme oxygenase-1 (HO-1, HMOX1) degrades pro-oxidant heme into carbon monoxide (CO), ferrous ions (Fe2+) 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.

Targeting angiogenesis in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) represents one of the most devastating types of muscular dystrophies which affect boys already at early childhood. Despite the fact that the primary cause of the disease, namely the lack of functional dystrophin is known already for more than 30 years, DMD still remains an incurable disease. Thus, an enormous effort has been made during recent years to reveal novel mechanisms that could provide therapeutic targets for DMD, especially because glucocorticoids treatment acts mostly symptomatic and exerts many side effects, whereas the effectiveness of genetic approaches aiming at the restoration of functional dystrophin is under the constant debate. Taking into account that dystrophin expression is not restricted to muscle cells, but is present also in, e.g., endothelial cells, alterations in angiogenesis process have been proposed to have a significant impact on DMD progression. Indeed, already before the discovery of dystrophin, several abnormalities in blood vessels structure and function have been revealed, suggesting that targeting angiogenesis could be beneficial in DMD. In this review, we will summarize current knowledge about the angiogenesis status both in animal models of DMD as well as in DMD patients, focusing on different organs as well as age- and sex-dependent effects. Moreover, we will critically discuss some approaches such as modulation of vascular endothelial growth factor or nitric oxide related pathways, to enhance angiogenesis and attenuate the dystrophic phenotype. Additionally, we will suggest the potential role of other mediators, such as heme oxygenase-1 or statins in those processes.

Kidney injury by cyclosporine A is aggravated in heme oxygenase-1 deficient mice and involves regulation of microRNAs.

Cyclosporine A (CsA), a widely used immunosuppressive drug, exerts nephrotoxic activities, as demonstrated by increased tubulointerstitial fibrosis, inflammation and podocyte damage. Recently, a number of microRNAs expressed in the kidney have been reported to be elevated during renal damage. Our aim was to investigate the effect of CsA on selected microRNAs in the mouse kidney after CsA treatment. Moreover, as heme oxygenase-1 (HO-1, encoded by the Hmox1 gene) was shown to play a protective role during kidney disorders, we assessed whether HO-1 deficiency in vivo influences the CsA-regulated microRNAs' expression. We have observed that the pro-fibrotic miR-21 and pro-apoptotic miR-34a expression was upregulated in kidneys of HO-1 deficient mice and it was further enhanced by CsA. Concomitantly, the level of anti-fibrotic microRNAs, belonging to miR-29 and miR-200 families, was down-regulated after CsA treatment. Generally, Hmox1 knock-out (Hmox1-/-) animals were more susceptible to CsA treatment, as the mortality rate was 4 out of 9 Hmox1-/- mice, and increased fibrosis (Tgfb2, Pai1), inflammation (Il6) and apoptosis (Cdkn1a-p21) were noticed in the HO-1 deficient kidneys. In summary, our data demonstrate that CsA induces significant changes in the expression of renal microRNAs and emphasize HO-1 deficiency as an important factor contributing to the CsA-mediated renal toxicity.

Heme oxygenase inhibition in cancers: possible tools and targets.

Heme oxygenase-1 (HO-1, encoded by HMOX1) through degradation of pro-oxidant heme into carbon monoxide (CO), ferrous ions (Fe2+) and biliverdin, exhibits cytoprotective, anti-apoptotic and anti-inflammatory properties. All of these potentially beneficial functions of HO-1 may play an important role in tumors' development and progression. Moreover, HO-1 is very often upregulated in tumors in comparison to healthy tissues, and its expression is further induced upon chemo-, radio- and photodynamic therapy, what results in decreased effectiveness of the treatment. Consequently, HO-1 can be proposed as a therapeutic target for anticancer treatment in many types of tumors. Nonetheless, possibilities of specific inhibition of HO-1 are strongly limited. Metalloporphyrins are widely used in in vitro studies, however, they are unselective and may exert serious side effects including an increase in HMOX1 mRNA level. On the other hand, detailed information about pharmacokinetics and biodistribution of imidazole-dioxolane derivatives, other potential inhibitors, is lacking. The genetic inhibition of HO-1 by RNA interference (RNAi) or CRISPR/Cas9 approaches provides the possibility to specifically target HO-1; however, the potential therapeutic application of those methods are distant at best. In summary, HO-1 inhibition might be the valuable anticancer approach, however, the ideal strategy for HO-1 targeting requires further studies.