Inhibition of Prolyl Hydroxylase Domain Proteins Promotes Therapeutic Revascularization

Abstract

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. Methods and 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-1␣ (shHIF-1␣), 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-1␣. 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-1␣-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 (PϽ0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1␣ 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. (Circulation. 2009;120:50-59.)

Short Hairpin RNA Gene Silencing of Prolyl Hydroxylase-2 with a Minicircle Vector Improves Neovascularization of Hindlimb Ischemia

“…In line with our results, a recent report showed that downregulation of PHD2 by shRNA enhanced neoangiogenesis in a mouse model of myocardial infarction (Huang et al, 2008). A different study evaluated the effects of shPHD1, shPHD2, and shPHD3 on neovascularization in a mouse hindlimb ischemia model (Loinard et al, 2009). Electroporation of plasmid vectors was used and silencing of PHDs triggered postischemic neovascularization, with shPHD2 and shPHD3 having the most robust effects.…”

“…Inhibition of HIF-1-alpha degradation through short hairpin RNA (shRNA) knockdown of PHD2 has been shown to significantly improve neovascularization, which in turn improved cardiac function in a mouse model of myocardial infarction (Huang et al, 2008). Moreover, inhibition of PHD by shRNA provides a promising proangiogenic therapeutic approach, as in vivo electrotransfer of plasmids with either shPHD2 or shPHD3 has been shown to significantly improve postischemic neovascularization in a mouse model of PAD (Loinard et al, 2009).…”

EGLN1 involvement in high-altitude adaptation revealed through genetic analysis of extreme constitution types defined in Ayurveda

“…We hypothesized that these variations, which are linked to Prakriti groups that are differently predisposed to diseases, may lead us to identification of predictive markers for differential responsiveness to disease and environment. As a proof of concept, we studied EGLN1 because it plays a key role in oxygen homeostasis and is also believed to be a target of many pharmacological interventions that aim to stabilize HIF or lower HIF activity (9)(10)(11). Although variations in genes of pathways related to hypoxia, such as HIF-1, endothelial function, and vascular remodeling, have been studied (12)(13)(14)(15)(16)(17), none of the studies so far have reported polymorphisms linked to EGLN1 in high-altitude adaptation.…”

PHD3-SUMO conjugation optimizes HIF1 repression independently of PHD3 catalytic activity

“…This complexity reflects not only the crucial role of HIF1 in maintaining oxygen homeostasis but also the need to safeguard against its inappropriate activation. Previous reports have linked the PHD3-HIF and SUMOylation pathways with pathologies such as cancer and ischemia (Cheng et al, 2006;Chen et al, 2011;Flotho and Melchior, 2013;Fox et al, 2011;Loinard et al, 2009;Mo et al, 2005;Silveirinha et al, 2013;Su et al, 2010;Wang and Banerjee, 2004;Xue et al, 2010). In light of these findings, there is a great interest in the development of drugs targeting these pathways.…”