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

Kevin J Paik,Alexander J Whittam,Shane D Morrison,Geoffrey C Gurtner,Derrick C Wan,Michael S Hu,Elizabeth R Zielins,Elizabeth A Brett,Dominik Duscher,Michael T Longaker,Zeshaan N Maan,Joseph C Wu,Ryan C Ransom,Masuko Ushio-Fukai

doi:10.1371/journal.pone.0150927

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.

Highlights

Chronic wounds, often associated with peripheral limb ischemia in diabetic and aged individuals [1], severely impair quality of life and exert a substantial burden on health care systems worldwide, with an estimated 6.5 million patients with chronic wounds managed each year in the United States alone [2]
We first examined the effects of shPHD-2 therapy by transfecting diabetic mouse fibroblasts in vitro with either shPHD-2 or shScr plasmids. shScr plasmids were used as controls to ensure that any phenotype observed was not the result of off-target sh plasmid effects
Total RNA was collected from transfected fibroblasts after allowing 24 hours for recovery. quantitative real-time PCR (qRT-PCR) analysis demonstrated that diabetic mouse fibroblasts transfected with shPHD-2 resulted in significantly decreased levels of prolyl hydroxylases (PHDs)-2 transcript, compared to fibroblasts transfected with the shScr control (ÃÃÃÃp

Summary

Introduction

Often associated with peripheral limb ischemia in diabetic and aged individuals [1], severely impair quality of life and exert a substantial burden on health care systems worldwide, with an estimated 6.5 million patients with chronic wounds managed each year in the United States alone [2]. An impaired response to hypoxia is a major factor contributing to compromised wound healing [3]. Hypoxia inducible factor-1 (HIF-1) regulates the majority of adaptive cellular responses to hypoxia, and consists of a highly regulated α-subunit and a constitutively expressed β-subunit [3]. Under hypoxic conditions the hydroxylation of proline residues on the HIF-1α subunit by prolyl hydroxylases (PHDs) is inhibited, which blocks the degradation of HIF-1α [4]. HIF-1α is hydroxylated by Prolyl Hydroxylase Domain-2 (PHD-2), ubiquitinated, and eventually degraded [5, 6]

Methods

Results

Conclusion