Inhibition of prolyl hydroxylase domain proteins promotes therapeutic revascularization.
BACKGROUND
The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O(2)-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization.


RESULTS
Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1alpha (shHIF-1alpha), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1alpha. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1alpha-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2- and 2.6-fold, capillary density by 1.8- and 2.1-fold, and foot perfusion by 1.2- and 1.4-fold, respectively, compared with shCON (P<0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1alpha with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing.


CONCLUSIONS
We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization.
Loinard
Céline
C
Paris Cardiovascular Research Center, INSERM U970, Hôpital Européen Georges Pompidou, Université Paris 5, Paris, France.
Ginouvès
Amandine
A
Vilar
José
J
Cochain
Clément
C
Zouggari
Yasmine
Y
Recalde
Alice
A
Duriez
Micheline
M
Lévy
Bernard I
BI
Pouysségur
Jacques
J
Berra
Edurne
E
Silvestre
Jean-Sébastien
JS
D000818:Animals; D018925:Chemokines / Q000378:metabolism; D005263:Femoral Artery; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D006614:Hindlimb / Q000098:blood supply; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007249:Inflammation / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010957:Plasmids / Q000494:pharmacology; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D018925:Chemokines; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C522721:PHD1 protein, mouse; C522722:PHD3 protein, mouse; D011393:Procollagen-Proline Dioxygenase; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse