title: Pre-emptive hypoxia-regulated HO-1 gene therapy improves post-ischaemic limb perfusion and tissue regeneration in mice.
abstract: OBJECTIVE
  Haem oxygenase-1 (HO-1) is a haem-degrading enzyme that generates carbon monoxide, bilirubin, and iron ions. Through these compounds, HO-1 mitigates cellular injury by exerting antioxidant, anti-apoptotic, and anti-inflammatory effects. Here, we examined the influence of HO-1 deficiency and transient hypoxia/ischaemia-induced HO-1 overexpression on post-injury hindlimb recovery.
  
  
  RESULTS
  Mice lacking functional HO-1 (HO-1(-/-)) showed reduced reparative neovascularization in ischaemic skeletal muscles, impaired blood flow (BF) recovery, and increased muscle cell death compared with their wild-type littermates. Human microvascular endothelial cells (HMEC-1) transfected with plasmid vector (pHRE-HO-1) carrying human HO-1 driven by three hypoxia response elements (HREs) and cultured in 0.5% oxygen demonstrated markedly increased expression of HO-1. Such upregulated HO-1 levels were effective in conferring protection against H(2)O(2)-induced cell death and in promoting the proangiogenic phenotype of HMEC-1 cells. More importantly, when delivered in vivo, pHRE-HO-1 significantly improved the post-ischaemic foot BF in mice subjected to femoral artery ligation. These effects were associated with reduced levels of pro-inflammatory cytokines (IL-6 and CXCL1) and lower numbers of transferase-mediated dUTP nick-end labelling-positive cells. Moreover, HO-1 delivered into mouse skeletal muscles seems to influence the regenerative potential of myocytes as it significantly changed the expression of transcriptional (Pax7, MyoD, myogenin) and post-transcriptional (miR-146a, miR-206) regulators of skeletal muscle regeneration.
  
  
  CONCLUSIONS
  Our results suggest the therapeutic potential of HO-1 for prevention of adverse effects in critical limb ischaemia.
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. agnieszka.jazwa@uj.edu.pl
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Jazwa
authors.forename: Jacek
authors.initials: J
authors.lastname: Stepniewski
authors.forename: Martin
authors.initials: M
authors.lastname: Zamykal
authors.forename: Jolanta
authors.initials: J
authors.lastname: Jagodzinska
authors.forename: Marco
authors.initials: M
authors.lastname: Meloni
authors.forename: Costanza
authors.initials: C
authors.lastname: Emanueli
authors.forename: Alicja
authors.initials: A
authors.lastname: Jozkowicz
authors.forename: Jozef
authors.initials: J
authors.lastname: Dulak
mesh_terms: D000818:Animals; D017209:Apoptosis; D015687:Cell Hypoxia; D002478:Cells, Cultured; D054360:Chemokine CXCL1 / Q000097:blood; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D015316:Genetic Therapy; D051547:Heme Oxygenase-1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006614:Hindlimb; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018836:Inflammation Mediators / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D007511:Ischemia / Q000097:blood / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000503:physiopathology / Q000628:therapy*; D008297:Male; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D035683:MicroRNAs / Q000378:metabolism; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000473:pathology; D017570:MyoD Protein / Q000378:metabolism; D018008:Myogenin / Q000378:metabolism; D009336:Necrosis; D018919:Neovascularization, Physiologic; D016877:Oxidants / Q000494:pharmacology; D051763:PAX7 Transcription Factor / Q000378:metabolism; D020127:Recovery of Function; D012038:Regeneration; D012039:Regional Blood Flow; D020218:Response Elements; D013997:Time Factors
chemical_list: D054360:Chemokine CXCL1; C475895:Cxcl1 protein, mouse; D018836:Inflammation Mediators; D015850:Interleukin-6; D008565:Membrane Proteins; D035683:MicroRNAs; C534190:Mirn146 microRNA, mouse; C540669:Mirn206 microRNA, mouse; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; C503801:Myog protein, mouse; D018008:Myogenin; D016877:Oxidants; D051763:PAX7 Transcription Factor; C497322:Pax7 protein, mouse; D006861:Hydrogen Peroxide; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse