Plasmid-based gene therapy with hepatocyte growth factor stimulates peripheral nerve regeneration after traumatic injury

https://doi.org/10.1016/j.biopha.2018.02.138Get rights and content

Abstract

Peripheral nerve injury remains a common clinical problem with no satisfactory treatment options. Numerous studies have shown that hepatocyte growth factor (HGF) exerts neurotrophic effect in motor, sensory, and parasympathetic neurons in addition to mitogenic, morphogenic, angiogenic, antiapoptotic, antifibrotic, and anti-inflammatory effect on various tissues and cells. In our study we examined efficacy of gene therapy with HGF-bearing plasmid (pC4W-hHGF) to improve consequences of traumatic nerve injury in mice.

Treatment by pC4W-hHGF led to restoration of nerve structure and functional recovery compared to similar parameters in control animals. Compound action potentials (CAP) in experimental groups treated with 100 or 200 μg of pC4W-hHGF demonstrated increased amplitude and latency decrease compared to spontaneous recovery control group. In HGF-treated mice histological analysis showed a three-fold increase in axon number in nerve portion located distal to the lesion site compared to control. Moreover, significant functional recovery of n. peroneus communis triggered by pC4W-hHGF gene therapy was observed using the footprints analysis. Obtained results provide evidence for plasmid-based HGF gene therapy as a potential treatment for traumatic injury of peripheral nerve.

Introduction

Peripheral nerve injury (PNI) has a prevalence of 3%–5% in patients with polytrauma [1,2] and may lead to long-term functional deficiencies significantly affecting quality of life and causing significant financial burden due to patient’s disability [3]. Peripheral nervous system has certain potential for full regeneration after trauma in contrast to central nervous system known to have a very limited regenerative capacity [4,5]. However, in case of severe peripheral nerve injury functional recovery is often unsatisfactory and clinical intervention is necessary.

Hepatocyte growth factor (HGF) exhibits unique features that make it a promising agent for PNI treatment. Several studies showed that HGF and its receptor c-Met are expressed in peripheral nervous system cells [6] and in adult brain [7,8]. HGF/c-Met axis is involved in mitogenic, morphogenic, angiogenic and antiapoptotic effects in various kinds of cells and tissues [9,10]. Early studies have demonstrated that HGF functions as a guidance and survival factor in the developing nervous system being an essential component of muscle-derived support for motoneurons in development [11,12]. HGF receptor c-Met is expressed by Schwann cells as well as by peripheral sensory and motor neurons [13,14]. Both in vitro and in vivo there is substantial evidence that HGF is essential for peripheral sensory, sympathetic and motor neurons and enhances neuronal survival and axonal outgrowth [6,[15], [16], [17]].

Neuroprotective effects of HGF in diseases affecting central and peripheral nervous systems were demonstrated in several studies. Treatment by HGF in acute phase induces long-term neuroprotection and recovery from stroke via induction of proliferation and differentiation of neural precursor cell [18]. After optic nerve injury HGF promotes long-term survival and axonal regeneration of retinal ganglion cells [19]. Adenoviral transfer of HGF gene prevents death of injured adult motoneurons in a rat model of peripheral nerve avulsion [20]. Furthermore, non-viral HGF gene therapy by intramuscular injections in patients with painful diabetic neuropathy provided symptomatic relief with improvement in quality of life [21]. In addition, plasmid-based HGF gene therapy by intrathecal injection significantly attenuated pain induced by nerve injury in mice through direct inhibition of spinal cord microglia and astrocyte activation due to anti-inflammatory action of HGF [22].

Besides neuroprotective activity HGF is considered as the most promising factor for angiogenic gene therapy because it can stimulate angiogenesis without induction of inflammation and vascular permeability [23,24]. Recently two double blind placebo-controlled phase II clinical trials demonstrated that HGF plasmid-based gene therapy significantly improved primary end-points and tissue oxygenation in critical limb ischemia compared to placebo [25,26].

Basing on these findings we hypothesized that plasmid-based gene therapy by HGF may be a promising approach to treat traumatic PNI due to neuroprotective, angiogenic, anti-inflammatory and antifibrotic activity of HGF. In present study we report efficacy of gene therapy by non-viral gene delivery of human HGF to alleviate consequences of traumatic PNI in a mouse model of this lesion.

Section snippets

Animal strain and ethical approval

We used 9–10 week-old C57/Bl6 male mice (purchased from “Andreevka” animal husbandry facility, Russia) for nerve traumatic injury model. After acclimation animals received standard food and water ratios according to in-house rules of husbandry. All animals were narcotized by intraperitoneal injection of avertin (300 μl of 2.5% solution) before surgery. Euthanasia was conducted under isoflurane narcosis by secondary cervical dislocation. Surgical manipulations and euthanasia procedures were

Human HGF is expressedin vivo after plasmid-based gene transfer

Prior to assessment of hHGF influence on nerve repair we evaluated expression of hHGF in explant culture of m. tibialis anterior as described in our previous work [[36], [37], [38]]. After gene delivery of human HGF we detected its production in explant culture of skeletal muscles injected with 100 and 200 μg of pC4W-hHGF on day 3 after plasmid administration. Maximal level of hHGF (1.1 ± 0.163 ng/ml) was detected in conditioned medium from explants of muscles injected with 200 μg of pC4W-hHGF.

Discussion

Traumatic nerve injury model used in present study closely resembles axonotomy when nerve fibers are damaged while connective tissue sheath of the nerve remains intact. This type of damage destroys nerve fibers distal to the injury site and nerve conduction is blocked but subsequent axonal re-growth over the site of injury provides gradual restoration of nerve conductivity.

Peripheral nerve injury (PNI) leads to rapid and robust expression and secretion of neurotrophic and growth factors to

Acknowledgement

This study was supported by Russian Science Foundation (RSF Grant no. 16-45-03007).

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