Effect of heme oxygenase-1 on the differentiation of human myoblasts and the regeneration of murine skeletal muscles after acute and chronic injury.

BACKGROUND: Impaired muscle regeneration is a hallmark of Duchenne muscular dystrophy (DMD), a neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. The lack of heme oxygenase-1 (HO-1, Hmox1), a known anti-inflammatory and cytoprotective enzyme, was shown to aggravate DMD pathology. METHODS: We evaluated the role of HO-1 overexpression in human induced pluripotent stem cell (hiPSC)-derived skeletal muscle cells (hiPSC-SkM) in vitro and in the regeneration process in vivo in wild-type mice. Furthermore, the effect of cobalt protoporphyrin IX (CoPP), a pharmacological inducer of HO-1 expression, on regeneration markers during myogenic hiPSC differentiation and progression of the dystrophic phenotype was analysed in the mdx mouse DMD model. RESULTS: HO-1 has an impact on hiPSC-SkM generation by decreasing cell fusion capacity and the expression of myogenic regulatory factors and muscle-specific microRNAs (myomiRs). Also, strong induction of HO-1 by CoPP totally abolished hiPSC-SkM differentiation. Injection of HO-1-overexpressing hiPSC-SkM into the cardiotoxin (CTX)-injured muscle of immunodeficient wild-type mice was associated with decreased expression of miR-206 and Myh3 and lower number of regenerating fibers, suggesting some advanced regeneration. However, the very potent induction of HO-1 by CoPP did not exert any protective effect on necrosis, leukocyte infiltration, fibrosis, myofiber regeneration biomarkers, and exercise capacity of mdx mice. CONCLUSIONS: In summary, HO-1 inhibits the expression of differentiation markers in human iPSC-derived myoblasts. Although moderate overexpression of HO-1 in the injected myoblast was associated with partially advanced muscle regeneration, the high systemic induction of HO-1 did not improve muscle regeneration. The appropriate threshold of HO-1 expression must be established for the therapeutic effect of HO-1 on muscle regeneration.

NRF2 Regulates Viability, Proliferation, Resistance to Oxidative Stress, and Differentiation of Murine Myoblasts and Muscle Satellite Cells.

Increased oxidative stress can slow down the regeneration of skeletal muscle and affect the activity of muscle satellite cells (mSCs). Therefore, we evaluated the role of the NRF2 transcription factor (encoded by the Nfe2l2 gene), the main regulator of the antioxidant response, in muscle cell biology. We used (i) an immortalized murine myoblast cell line (C2C12) with stable overexpression of NRF2 and (ii) primary mSCs isolated from wild-type and Nfe2l2 (transcriptionally)-deficient mice (Nfe2l2tKO). NRF2 promoted myoblast proliferation and viability under oxidative stress conditions and decreased the production of reactive oxygen species. Furthermore, NRF2 overexpression inhibited C2C12 cell differentiation by down-regulating the expression of myogenic regulatory factors (MRFs) and muscle-specific microRNAs. We also showed that NRF2 is indispensable for the viability of mSCs since the lack of its transcriptional activity caused high mortality of cells cultured in vitro under normoxic conditions. Concomitantly, Nfe2l2tKO mSCs grown and differentiated under hypoxic conditions were viable and much more differentiated compared to cells isolated from wild-type mice. Taken together, NRF2 significantly influences the properties of myoblasts and muscle satellite cells. This effect might be modulated by the muscle microenvironment.

Role of HMOX1 Promoter Genetic Variants in Chemoresistance and Chemotherapy Induced Neutropenia in Children with Acute Lymphoblastic Leukemia.

Whilst the survival rates of childhood acute lymphoblastic leukemia (ALL) have increased remarkably over the last decades, the therapy resistance and toxicity are still the major causes of treatment failure. It was shown that overexpression of heme oxygenase-1 (HO-1) promotes proliferation and chemoresistance of cancer cells. In humans, the HO-1 gene (HMOX1) expression is modulated by two polymorphisms in the promoter region: (GT)n-length polymorphism and single-nucleotide polymorphism (SNP) A(-413)T, with short GT repeat sequences and 413-A variants linked to an increased HO-1 inducibility. We found that the short alleles are significantly more frequent in ALL patients in comparison to the control group, and that their presence may be associated with a higher risk of treatment failure, reflecting the role of HO-1 in chemoresistance. We also observed that the presence of short alleles may predispose to develop chemotherapy-induced neutropenia. In case of SNP, the 413-T variant co-segregated with short or long alleles, while 413-A almost selectively co-segregated with long alleles, hence it is not possible to determine if SNPs are actually of phenotypic significance. Our results suggest that HO-1 can be a potential target to overcome the treatment failure in ALL patients.

The role of Nrf2 in acute and chronic muscle injury.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as a master cytoprotective factor regulating the expression of genes encoding 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 (Nrf2tKO) affects muscle functions both in an acute and chronic injury. The acute muscle damage was induced in mice of two genotypes-WT and Nrf2tKO 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/Nrf2tKO) were generated. To worsen the dystrophic phenotype, the analysis of disease pathology was also performed in aggravated conditions, by applying a long-term treadmill test. We have observed slightly increased muscle damage in Nrf2tKO mice after CTX injection. Nevertheless, transcriptional ablation of Nrf2 in mdx mice did not significantly aggravate the most deleterious, pathological hallmarks of DMD related to degeneration, inflammation, fibrotic scar formation, angiogenesis, and the number and proliferation of satellite cells in non-exercised conditions. On the other hand, upon chronic exercises, the degeneration and inflammatory infiltration of the gastrocnemius muscle, but not the diaphragm, turned to be increased in Nrf2tKOmdx in comparison to mdx mice. In conclusion, the lack of transcriptionally active Nrf2 influences moderately muscle pathology in acute CTX-induced muscle injury and chronic DMD mouse model, without affecting muscle functionality. Hence, in general, we demonstrated that the deficiency of Nrf2 transcriptional activity has no profound impact on muscle pathology in various models of muscle injury.

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.

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.

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

AIMS: Muscle damage in Duchenne muscular dystrophy (DMD) caused by the lack of dystrophin is strongly linked to inflammation. Heme oxygenase-1 (HO-1; Hmox1) is an anti-inflammatory and cytoprotective enzyme affecting myoblast differentiation by inhibiting myomiRs. The role of HO-1 has not been so far well addressed in DMD. RESULTS: 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.

Lack of Heme Oxygenase-1 Induces Inflammatory Reaction and Proliferation of Muscle Satellite Cells after Cardiotoxin-Induced Skeletal Muscle Injury.

Heme oxygenase-1 (HO-1, Hmox1) regulates viability, proliferation, and differentiation of many cell types; hence, it may affect regeneration of injured skeletal muscle. Here, we injected cardiotoxin into gastrocnemius muscle of Hmox1+/+ and Hmox1-/- animals and analyzed cellular response after muscle injury, focusing on muscle satellite cells (SCs), inflammatory reaction, fibrosis, and formation of new blood vessels. HO-1 is strongly induced after muscle injury, being expressed mostly in the infiltrating leukocytes (CD45+ cells), including macrophages (F4/80+ cells). Lack of HO-1 augments skeletal muscle injury, evidenced by increased creatinine kinase and lactate dehydrogenase, as well as expression of monocyte chemoattractant protein-1, IL-6, IL-1ß, and insulin-like growth factor-1. This, together with disturbed proportion of M1/M2 macrophages, accompanied by enhanced formation of arterioles, may be responsible for shift of Hmox1-/- myofiber size distribution toward larger one. Importantly, HO-1-deficient SCs are prone to activation and have higher proliferation on injury. This effect can be partially mimicked by stimulation of Hmox1+/+ SCs with monocyte chemoattractant protein-1, IL-6, IL-1ß, and is associated with increased MyoD expression, suggesting that Hmox1-/- SCs are shifted toward more differentiated myogenic population. However, multiple rounds of degeneration/regeneration in conditions of HO-1 deficiency may lead to exhaustion of SC pool, and the number of SCs is decreased in old Hmox1-/- mice. In summary, HO-1 modulates muscle repair mechanisms preventing its uncontrolled acceleration.

Heme Oxygenase-1 Controls an HDAC4-miR-206 Pathway of Oxidative Stress in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue cancer characterized by disturbed myogenic differentiation. Here we report a role for the oxidative stress response factor HO-1 in progression of RMS. We found that HO-1 was elevated and its effector target miR-206 decreased in RMS cell lines and clinical primary tumors of the more aggressive alveolar phenotype (aRMS). In embryonal RMS (eRMS), HO-1 expression was induced by Pax3/7-FoxO1, an aRMS hallmark oncogene, followed by a drop in miR-206 levels. Inhibition of HO-1 by tin protoporphyrin (SnPP) or siRNA downregulated Pax3/7-FoxO1 target genes and induced a myogenic program in RMS. These effects were not mediated by altered myoD expression; instead, cells with elevated HO-1 produced less reactive oxygen species, resulting in nuclear localization of HDAC4 and miR-206 repression. HO-1 inhibition by SnPP reduced growth and vascularization of RMS tumors in vivo accompanied by induction of miR-206. Effects of SnPP on miR-206 expression and RMS tumor growth were mimicked by pharmacologic inhibition of HDAC. Thus, HO-1 inhibition activates an miR-206-dependent myogenic program in RMS, offering a novel therapeutic strategy for treatment of this malignancy. Cancer Res; 76(19); 5707-18. ©2016 AACR.

Preliminary Analysis of the Expression of Selected Proangiogenic and Antioxidant Genes and MicroRNAs in Patients with Non-Muscle-Invasive Bladder Cancer.

Heme oxygenase-1 (HO-1) is an enzyme contributing to the development and progression of different cancer types. HO-1 plays a role in pathological angiogenesis in bladder cancer and contributes to the resistance of this cancer to therapy. It also regulates the expression of microRNAs in rhabdomyosarcoma and non-small cell lung cancer. The expression of HO-1 may be regulated by hypoxia inducible factors (HIFs) and Nrf2 transcription factor. The expression of HO-1 has not so far been examined in relation to Nrf2, HIF-1α, and potential mediators of angiogenesis in human bladder cancer. We measured the concentration of proinflammatory and proangiogenic cytokines and the expression of cytoprotective and proangiogenic mRNAs and miRNAs in healthy subjects and patients with bladder cancer. HO-1 expression was upregulated together with HIF-1α, HIF-2α, and Nrf2 in bladder cancer in comparison to healthy tissue. VEGF was elevated both at mRNA and protein level in the tumor and in sera, respectively. Additionally, IL-6 and IL-8 were increased in sera of patients affected with urothelial bladder cancer. Moreover, miR-155 was downregulated whereas miR-200c was elevated in cancer biopsies in comparison to healthy tissue. The results indicate that the increased expression of HO-1 in bladder cancer is paralleled by changes in the expression of other potentially interacting genes, like Nrf2, HIF-1α, HIF-2α, IL-6, IL-8, and VEGF. Further studies are necessary to also elucidate the potential links with miR-155 and miR-200c.

Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species.

Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34(+) cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34(+) cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P<0.05) and induced apoptosis of BNML cells more efficiently than etoposide alone (1.54-fold change versus etoposide alone; P<0.05), but this treatment protected nonleukemic B-cells from apoptosis. Thus, curcumin can increase the antileukemic effect of etoposide through reactive oxygen species in sensitive myeloid leukemia cells, and it is harmless to normal human cells.

[Heme oxygenase-1 - more than the cytoprotection]. / Oksygenaza hemowa-4 - wiecej niz cytoprotekcja.

Heme oxygenase-1 (HO-1) is an enzyme degrading heme to three products - ferrous ions, carbon monoxide and biliverdin. Its function extends, however, far beyond removal of pro-oxidative heme from microenvironment. During the last few decades it was proven that apart from cytoprotective and antioxidative properties HO-1 regulates also a variety of cellular processes. It exerts an impact on both innate and adaptive immune response. HO-1 accelerates development of new blood vessels in a process called angiogenesis. Moreover, it controls cell cycle and depending on a cell type increases or decreases the rate of cell division. Finally, the most recent data indicate, that HO-1 regulates also differentiation of various stem and progenitor cells. Interestingly, that aspect of HO-1 function seems also to depend on cell type. In this review, both effects and mechanisms of above-mentioned processes in different cell types are discussed.

Myoblast-conditioned media improve regeneration and revascularization of ischemic muscles in diabetic mice.

INTRODUCTION: Diabetes is associated with reduced expression of heme oxygenase-1 (HO-1), a heme-degrading enzyme with cytoprotective and proangiogenic properties. In myoblasts and muscle satellite cells HO-1 improves survival, proliferation and production of proangiogenic growth factors. Induction of HO-1 in injured tissues facilitates neovascularization, the process impaired in diabetes. We aimed to examine whether conditioned media from the HO-1 overexpressing myoblast cell line can improve a blood-flow recovery in ischemic muscles of diabetic mice. METHODS: Analysis of myogenic markers was performed at the mRNA level in primary muscle satellite cells, isolated by a pre-plate technique from diabetic db/db and normoglycemic wild-type mice, and then cultured under growth or differentiation conditions. Hind limb ischemia was performed by femoral artery ligation in db/db mice and blood recovery was monitored by laser Doppler measurements. Mice were treated with a single intramuscular injection of conditioned media harvested from wild-type C2C12 myoblast cell line, C2C12 cells stably transduced with HO-1 cDNA, or with unconditioned media. RESULTS: Expression of HO-1 was lower in muscle satellite cells isolated from muscles of diabetic db/db mice when compared to their wild-type counterparts, what was accompanied by increased levels of Myf5 or CXCR4, and decreased Mef2 or Pax7. Such cells also displayed diminished differentiation potential when cultured in vitro, as shown by less effective formation of myotubes and reduced expression of myogenic markers (myogenic differentiation antigen - myoD, myogenin and myosin). Blood flow recovery after induction of severe hind limb ischemia was delayed in db/db mice compared to that in normoglycemic individuals. To improve muscle regeneration after ischemia, conditioned media collected from differentiating C2C12 cells (control and HO-1 overexpressing) were injected into hind limbs of diabetic mice. Analysis of blood flow revealed that media from HO-1 overexpressing cells accelerated blood-flow recovery, while immunohistochemical staining assessment of vessel density in injected muscle confirmed increased angiogenesis. The effect might be mediated by stromal-cell derived factor-1α proangiogenic factor, as its secretion is elevated in HO-1 overexpressing cells. CONCLUSIONS: In conclusion, paracrine stimulation of angiogenesis in ischemic skeletal muscle using conditioned media may be a safe approach exploiting protective and proangiogenic properties of HO-1 in diabetes.

The role of oxidative stress in skeletal muscle injury and regeneration: focus on antioxidant enzymes.

Reactive oxygen species (ROS) are generated in skeletal muscle both during the rest and contractile activity. Myogenic cells are equipped with antioxidant enzymes, like superoxide dismutase, catalase, glutathione peroxidase, γ-glutamylcysteine synthetase and heme oxygenase-1. These enzymes not only neutralise excessive ROS, but also affect myogenic regeneration at several stages: influence post-injury inflammatory reaction, enhance viability and proliferation of muscle satellite cells and myoblasts and affect their differentiation. Finally, antioxidant enzymes regulate also processes accompanying muscle regeneration-induce angiogenesis and reduce fibrosis. Elevated ROS production was also observed in Duchenne muscular dystrophy (DMD), a disease characterised by degeneration of muscle tissue and therefore-increased rate of myogenic regeneration. Antioxidant enzymes are consequently considered as target for therapies counteracting dystrophic symptoms. In this review we present current knowledge regarding the role of oxidative stress and systems of enzymatic antioxidant defence in muscular regeneration after both acute injury and persistent muscular degeneration.

PPARγ activation but not PPARγ haplodeficiency affects proangiogenic potential of endothelial cells and bone marrow-derived progenitors.

BACKGROUND: Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs). METHODS: PACs were isolated from bone marrow of 10-12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 µmol/L) or GW9662 (10 µmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model. RESULTS: ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells. CONCLUSIONS: In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

Heme oxygenase-1 is required for angiogenic function of bone marrow-derived progenitor cells: role in therapeutic revascularization.

AIMS: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. RESULTS: Lack of HO-1 decreased the number of endothelial progenitor cells (Lin(-)CD45(-)cKit(-)Sca-1(+)VEGFR-2(+)) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1(-/-) BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1(+/-) diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1(+)CXCR-4(+)) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. INNOVATION: This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. CONCLUSIONS: HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs.

Nrf2 regulates angiogenesis: effect on endothelial cells, bone marrow-derived proangiogenic cells and hind limb ischemia.

AIMS: Nuclear factor E2-related factor 2 (Nrf2), a key cytoprotective transcription factor, regulates also proangiogenic mediators, interleukin-8 and heme oxygenase-1 (HO-1). However, hitherto its role in blood vessel formation was modestly examined. Particularly, although Nrf2 was shown to affect hematopoietic stem cells, it was not tested in bone marrow-derived proangiogenic cells (PACs). Here we investigated angiogenic properties of Nrf2 in PACs, endothelial cells, and inflammation-related revascularization. RESULTS: Treatment of endothelial cells with angiogenic cytokines increased nuclear localization of Nrf2 and induced expression of HO-1. Nrf2 activation stimulated a tube network formation, while its inhibition decreased angiogenic response of human endothelial cells, the latter effect reversed by overexpression of HO-1. Moreover, lack of Nrf2 attenuated survival, proliferation, migration, and angiogenic potential of murine PACs and affected angiogenic transcriptome in vitro. Additionally, angiogenic capacity of PAC Nrf2(-/-) in in vivo Matrigel assay and PAC mobilization in response to hind limb ischemia of Nrf2(-/-) mice were impaired. Despite that, restoration of blood flow in Nrf2-deficient ischemic muscles was better and accompanied by increased oxidative stress and inflammatory response. Accordingly, the anti-inflammatory agent etodolac tended to diminish blood flow in the Nrf2(-/-) mice. INNOVATION: Identification of a novel role of Nrf2 in angiogenic signaling of endothelial cells and PACs. CONCLUSION: Nrf2 contributes to angiogenic potential of both endothelial cells and PACs; however, its deficiency increases muscle blood flow under tissue ischemia. This might suggest a proangiogenic role of inflammation in the absence of Nrf2 in vivo, concomitantly undermining the role of PACs in such conditions.

Role of heme oxygenase-1 in postnatal differentiation of stem cells: a possible cross-talk with microRNAs.

SIGNIFICANCE: Heme oxygenase-1 (HO-1) converts heme to biliverdin, carbon monoxide, and ferrous ions, but its cellular functions are far beyond heme metabolism. HO-1 via heme removal and degradation products acts as a cytoprotective, anti-inflammatory, immunomodulatory, and proangiogenic protein, regulating also a cell cycle. Additionally, HO-1 can translocate to nucleus and regulate transcription factors, so it can also act independently of enzymatic function. RECENT ADVANCES: Recently, a body of evidence has emerged indicating a role for HO-1 in postnatal differentiation of stem and progenitor cells. Maturation of satellite cells, skeletal myoblasts, adipocytes, and osteoclasts is inhibited by HO-1, whereas neurogenic differentiation and formation of cardiomyocytes perhaps can be enhanced. Moreover, HO-1 influences a lineage commitment in pluripotent stem cells and maturation of hematopoietic cells. It may play a role in development of osteoblasts, but descriptions of its exact effects are inconsistent. CRITICAL ISSUES: In this review we discuss a role of HO-1 in cell differentiation, and possible HO-1-dependent signal transduction pathways. Among the potential mediators, we focused on microRNA (miRNA). These small, noncoding RNAs are critical for cell differentiation. Recently we have found that HO-1 not only influences expression of specific miRNAs but also regulates miRNA processing enzymes. FUTURE DIRECTIONS: It seems that interplay between HO-1 and miRNAs may be important in regulating fates of stem and progenitor cells and needs further intensive studies.

Interplay between heme oxygenase-1 and miR-378 affects non-small cell lung carcinoma growth, vascularization, and metastasis.

AIMS: Heme oxygenase-1 (HO-1, HMOX1) can prevent tumor initiation; while in various tumors, it has been demonstrated to promote growth, angiogenesis, and metastasis. Here, we investigated whether HMOX1 can modulate microRNAs (miRNAs) and regulate human non-small cell lung carcinoma (NSCLC) development. RESULTS: Stable HMOX1 overexpression in NSCLC NCI-H292 cells up-regulated tumor-suppressive miRNAs, whereas it significantly diminished the expression of oncomirs and angiomirs. The most potently down-regulated was miR-378. HMOX1 also up-regulated p53, down-regulated angiopoietin-1 (Ang-1) and mucin-5AC (MUC5AC), reduced proliferation, migration, and diminished angiogenic potential. Carbon monoxide was a mediator of HMOX1 effects on proliferation, migration, and miR-378 expression. In contrast, stable miR-378 overexpression decreased HMOX1 and p53; while enhanced expression of MUC5AC, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and Ang-1, and consequently increased proliferation, migration, and stimulation of endothelial cells. Adenoviral delivery of HMOX1 reversed miR-378 effect on the proliferation and migration of cancer cells. In vivo, HMOX1 overexpressing tumors were smaller, less vascularized and oxygenated, and less metastatic. Overexpression of miR-378 exerted opposite effects. Accordingly, in patients with NSCLC, HMOX1 expression was lower in metastases to lymph nodes than in primary tumors. INNOVATION AND CONCLUSION: 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.