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 H$_{2}$S-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 ($\textit{Vegf}$) and its receptor ($\textit{Kdr}$) expression, accompanied by the elevated number of α-SMA/CD31/lectin-positive blood vessels. The expression of fibrotic regulators, like Tgfβ, 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 H$_{2}$S donor on regeneration and the muscle fiber type composition. Overall, the H$_{2}$S 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

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 serve as 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, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. 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 protein 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 not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach

Human induced pluripotent stem cell-derived cardiomyocytes, in contrast to adipose tissue-derived stromal cells, efficiently improve heart function in murine model of myocardial infarction

Cell therapies are extensively tested to restore heart function after myocardial infarction (MI). Survival of any cell type after intracardiac administration, however, may be limited due to unfavorable conditions of damaged tissue. Therefore, the aim of this study was to evaluate the therapeutic effect of adipose-derived stromal cells (ADSCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing either the proangiogenic SDF-1α or anti-inflammatory heme oxygenase-1 (HO-1) in a murine model of MI. ADSCs and hiPSCs were transduced with lentiviral vectors encoding luciferase (Luc), GFP and either HO-1 or SDF-1α. hiPSCs were then differentiated to hiPSC-CMs using small molecules modulating the WNT pathway. Genetically modified ADSCs were firstly administered via intracardiac injection after MI induction in Nude mice. Next, ADSCs-Luc-GFP and genetically modified hiPSC-CMs were injected into the hearts of the more receptive NOD/SCID strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated Nude and NOD/SCID mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration

Badanie antynowotworowego efektu genetycznego i farmakologicznego zahamowania oksygenazy hemowej-1 in vitro

Oksygenaza hemowa-1 (HO-1, HMOX1) poprzez rozkład pro-oksydacyjnego hemu do dwutlenku węgla, jonów żelaza oraz biliwerdyny, wykazuje silne działanie cytoprotekcyjne, które może mieć również znaczenie w procesach patofizjologicznych, takich jak nowotworzenie. Szereg badań wykazało podwyższony poziom HO-1 w wielu typach nowotworów, który korelował z progresją choroby i opornością na stosowaną terapię. W związku z tym sugeruje się, że HO-1 może stanowić potencjalny cel terapii przeciwnowotworowej. Obecnie stosowane strategie hamowania HO-1, w tym genetyczna modyfikacja komórek i znane inhibitory aktywności oksygenazy hemowej (HO), metaloporfiryny, posiadają jednak wiele wad. Alternatywę może stanowić inna klasa inhibitorów aktywności HO, pochodnych imidazolowych, nie mniej jednak wciąż niewiele wiadomo na temat ich biodystrybucji i farmakokinetyki. W związku z tym opracowanie i zbadanie nowych, chemicznych inhibitorów jest wciąż potrzebne. Celem poniższej pracy było zbadanie genetycznej oraz farmakologicznej inhibicji HO-1 in vitro w nowotworze trzustki oraz nowotworze nerek związanym z wrodzoną mięśniakowatością gładkokomórkową i rakiem nerkowokomórkowym (HLRCC) stanowiącym model syntetycznej letalności defektu genów HMOX1 oraz hydratazy fumaranu (FH). Wykazano, że wysoki poziom HO-1 koreluje z niższą wrażliwością komórek nowotworu trzustki, PANC-1, na stosowaną chemioterapię. Genetyczne zahamowanie HMOX1 przy użyciu shRNA skutkowało niewielkim spadkiem żywotności i proliferacji komórek PANC-1 oraz uwrażliwieniem komórek na gemcytabinę. W przypadku linii komórkowej z defektem genu FH, UOK 262, genetyczne zahamowanie HMOX1 drastycznie obniżyło żywotność oraz proliferację tych komórek. Farmakologiczne zahamowanie aktywności HO przy użyciu znanego inhibitora HO, ZnPPIX, prowadziło do znacznego spadku żywotności oraz potencjału klonogennego komórek PANC-1 oraz UOK 262. Jednocześnie jednak, ZnPPIX silnie indukowało HMOX1 na poziomie mRNA. Niektóre z nowych inhibitorów HO spowodowały spadek żywotności oraz potencjału klonogennego komórek PANC-1 i UOK 262. Podsumowując, powyższe wyniki wskazują na potencjalny, antynowotworowy efekt zahamowania HO-1 in vitro w nowotworze trzustki oraz związanym z zespołem HLRCC nowotworze nerek. Nie mniej jednak, dalsze badania, w szczególności związanie z działaniem nowych inhibitorów HO, wydają się być niezbędne.Heme oxygenase-1 (HO-1, HMOX1) through degradation of pro-oxidant heme into carbon monoxide, ferrous ions and biliverdin exhibits cytoprotective properties which may be implicated in pathophysiological processes, such as tumorigenesis. Substantial number of studies reported that elevated level of HO-1, found in many types of cancer, contributed to progression of the disease and correlated with impaired susceptibility to anticancer treatment. Therefore, inhibition of HO-1 can be proposed as novel, anti-tumor treatment. Nonetheless, possibilities of specific silencing of HO-1, including genetic modifications and known inhibitors of HO activity, metalloporphyrins (MPs), possess many disadvantages. On the other hand detailed information about pharmacokinetics and biodistribution of another class of HO activity inhibitors, imidazole-derivatives are still lacking. Thus, there is still a need to develop and evaluate new, chemical inhibitors of HO activity. In the current study we examined genetic and pharmacological inhibition of HO-1 in vitro in pancreatic cancer and hereditary leiomyomatosis and renal cell cancer (HLRCC) associated kidney cancer, as a model of synthetic lethality of HMOX1 and fumarate hydratase (FH) genes. High HO-1 level in human pancreatic cancer cell line, PANC-1, correlated with lower susceptibility to anticancer treatment. Genetic inhibition of HMOX1 by means of shRNA approach resulted in slight decrease in viability and proliferation of cancer cells and increased susceptibility to gemcitabine. In case of FH-deficient cell line, UOK 262, genetic inhibition of HMOX1 caused drastically diminished viability and proliferation of cancer cells. Pharmacological inhibition of HO activity using well-known inhibitor, ZnPPIX, decreased viability and clonogenic potential of PANC-1 and UOK 262 cell lines, but strongly induced HMOX1 mRNA level. Some of novel inhibitors of HO activity affected viability and clonogenic potential of UOK 262 and PANC-2 cell lines. In conclusion, current study points out the possible anticancer effectiveness of HO-1 inhibition in vitro in two analysed cancer types. However, further validation of obtained results, especially in terms of novel HO inhibitors, is still needed

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