Objective-Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. Methods and Results-On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT Յ23), medium (moderately active, GTϭ24 to 28), and long (least active, GT Ն29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J 2 , hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor-induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1, interleukin-6, and soluble intercellular adhesion molecule-1. Conclusion-The
BackgroundImpaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.MethodsLeptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.ResultsComplete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.ConclusionCombined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.
Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1+/+, HO-1+/−, and HO-1−/− mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1−/− and HO-1+/− than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1−/− lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.
BackgroundGene therapy stimulating the growth of blood vessels is considered for the treatment of peripheral and myocardial ischemia. Here we aimed to achieve angiogenic synergism between vascular endothelial growth factor-A (VEGF-A, VEGF) and fibroblast growth factor 4 (FGF4) in murine normoperfused and ischemic limb muscles.MethodsAdeno-associated viral vectors (AAVs) carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A) or two angiogenic genes (AAV-FGF4-IRES-VEGF-A) were injected into the normo-perfused adductor muscles of C57Bl/6 mice. Moreover, in a different experiment, mice were subjected to unilateral hindlimb ischemia by femoral artery ligation followed by intramuscular injections of AAV-LacZ, AAV-VEGF-A or AAV-FGF4-IRES-VEGF-A below the site of ligation. Post-ischemic blood flow recovery was assessed sequentially by color laser Doppler. Mice were monitored for 28 days.ResultsVEGF-A delivered alone (AAV-VEGF-A) or in combination with FGF4 (AAV-FGF4-IRES-VEGF-A) increased the number of capillaries in normo-perfused hindlimbs when compared to AAV-LacZ. Simultaneous overexpression of both agents (VEGF-A and FGF4) stimulated the capillary wall remodeling in the non-ischemic model. Moreover, AAV-FGF4-IRES-VEGF-A faster restored the post-ischemic foot blood flow and decreased the incidence of toe necrosis in comparison to AAV-LacZ.ConclusionsSynergy between VEGF-A and FGF4 to produce stable and functional blood vessels may be considered a promising option in cardiovascular gene therapy.
BackgroundPeroxisome 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).MethodsPACs 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.ResultsECs 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.Transcriptome analysis showed an upregulation of prooxidative and proinflammatory pathways, and downregulation of several proangiogenic genes in db/db PACs. Interestingly, db/db PACs had also a decreased level of PPARγ and changed expression of PPARγ-regulated genes. Using normoglycemic PPARγ+/− mice we demonstrated that reduced expression of PPARγ does not influence neovascularization either in wound healing or in hind limb ischemia models.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-014-0150-7) contains supplementary material, which is available to authorized users.
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.
Both MSC Hmox1 and Hmox1 showed similar phenotype, differentiation capacities, and production of cytokines or growth factors. Hmox1 and Hmox1 cells showed similar survival in response to 50 μmol/L hemin even in increased glucose concentration, conditions that were unfavorable for Hmox1 bone marrow-derived proangiogenic cells (BDMC). Hmox1 MSCs but not fibroblasts retained low ROS levels even after prolonged incubation with 50 μmol/L hemin, although both cell types have a comparable Hmox1 expression and similarly increase its levels in response to hemin. MSCs Hmox1 treated with hemin efficiently induced expression of a vast panel of antioxidant genes, especially enzymes of the glutathione pathway. Innovation and Conclusion: Hmox1 overexpression is a popular strategy to enhance viability and performance of MSCs after the transplantation. However, murine MSCs Hmox1 do not differ from wild-type MSCs in phenotype and functions. MSC Hmox1 show better resistance to hemin than fibroblasts and BDMCs and rapidly react to the stress by upregulation of quintessential genes in antioxidant response. Antioxid. Redox Signal. 00, 000-000.
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