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. 2016 Mar 11;11(3):e0150927.
doi: 10.1371/journal.pone.0150927. eCollection 2016.

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs

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Free PMC article

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs

Kevin J Paik et al. PLoS One. .
Free PMC article

Abstract

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. shPHD-2 Suppresses PHD-2 and Upregulates HIF-1α and Downstream Angiogenic Genes In Vitro.
Cells for all experiments were cultured under hypoxic conditions of 5% oxygen. (a) Schematics of shPHD-2 and shScr constructs. (b) qRT-PCR showed decreased PHD-2 expression in vitro from diabetic mouse fibroblasts transfected with shPHD-2 compared to fibroblasts transfected with shScr (****p<0.0001). (c) Western blot revealed higher levels of HIF-1α protein from shPHD-2 fibroblasts compared to shScr fibroblasts. (d) qRT-PCR additionally showed that SDF-1, an angiogenic chemokine downstream of HIF-1α, was more highly expressed in the shPHD-2 group than in shScr in vitro (****p<0.0001), as was (e) VEGFα (*p<0.05).
Fig 2
Fig 2. shPHD-2 Promotes Angiogenesis In Vivo.
(a) BLI confirmed uptake of shPHD-2 plasmid in vivo as indicated by expression of firefly luciferase (right) compared to a non-injected mouse (left). (b) RNA derived from the tissue surrounding the wound beds five days after injection showed decreased PHD-2 transcript as a result of shPHD-2 plasmid treatment compared to shScr (**p<0.01). (c) Protein derived from the same tissue samples congruously showed higher levels of HIFα protein in the shPHD-2 group. (d) qRT-PCR further demonstrated the upregulation of angiogenic gene PDGFα in the shPHD-2 treatment group (*p<0.05).
Fig 3
Fig 3. shPHD-2 Promotes Accelerated Wound Healing and Increased Vascular Density In Vivo.
(a,b) Diabetic wounds treated with shPHD-2 healed significantly faster than wounds treated with shScr, (c) closing in an average of 14.33 ± 0.45 days compared to 19 ± 0.33 days (*****p<0.00001). (d,e) CD31 staining revealed enhanced vascular density in wound beds treated with shPHD-2 versus shScr (***p<0.001). Scale bar = 100μm.
Fig 4
Fig 4. Local Injection of shPHD-2 Accelerates Revascularization Following Ischemic Injury and Improves Tissue Survival.
(a) Representative laser doppler images of ischemic and control limbs for shPHD-2 and shScr-treated animals demonstrating accelerated neovascularization with shPHD-2 application (arrows indicate ischemic limbs). (b) Quantification of mean perfusion of ischemic to control limbs, with the greatest difference between shPHD-2 (red) and shScr (blue) occurring at Day 10 (***p<0.001). (c) Distal toe necrosis was observed in the shScr group, but not in shPHD-2 treated limbs (arrows indicate ischemic limbs). (d) Representative high-power H&E stained images (200x) and (e) quantification revealing enhanced muscle survival at Day 14 in ischemic hind limbs treated with shPHD-2 as compared to shScr (*p<0.05).

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