Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer

Atypical changes in DRG neuron excitability and complex pain phenotype associated with a Nav1.7 mutation that massively hyperpolarizes activation

Abstract Sodium channel Nav1.7 plays a central role in pain-signaling: gain-of-function Nav1.7 mutations usually cause severe pain and loss-of-function mutations produce insensitivity to pain. The Nav1.7 I234T gain-of-function mutation, however, is linked to a dual clinical presentation of episodic pain, together with absence of pain following fractures, and corneal anesthesia. How a Nav1.7 mutation that produces gain-of-function at the channel level causes clinical loss-of-function has remained enigmatic. We show by current-clamp that expression of I234T in dorsal root ganglion (DRG) neurons produces a range of membrane depolarizations including a massive shift to >−40 mV that reduces excitability in a small number of neurons. Dynamic-clamp permitted us to mimic the heterozygous condition via replacement of 50% endogenous wild-type Nav1.7 channels by I234T, and confirmed that the I234T conductance could drastically depolarize DRG neurons, resulting in loss of excitability. We conclude that attenuation of pain sensation by I234T is caused by massively depolarized membrane potential of some DRG neurons which is partly due to enhanced overlap between activation and fast-inactivation, impairing their ability to fire. Our results demonstrate how a Nav1.7 mutation that produces channel gain-of-function can contribute to a dual clinical presentation that includes loss of pain sensation at the clinical level

Subtype-selective small molecule inhibitors reveal a fundamental role for Nav1.7 in nociceptor electrogenesis, axonal conduction and presynaptic release

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release

<div><p>Human genetic studies show that the voltage gated sodium channel 1.7 (Na_v1.7) is a key molecular determinant of pain sensation. However, defining the Na_v1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Na_v1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Na_v1.7’s role in nociceptor physiology. We report that Na_v1.7 is the predominant functional TTX-sensitive Na_v in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Na_v1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Na_v1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.</p></div

Potency of PF-05089771 across hNa_v1.7 splice variants.

<p>Potency of PF-05089771 across hNa_v1.7 splice variants.</p

PF-05198007 acts peripherally and centrally to influence neurotransmitter release.

<p>A. Upper: representative evoked EPSCs during control (blue) and after 30 mins PF-05198007 application (red). Lower: representative synaptically evoked action potential trace (blue) recorded in SG neurons of the dorsal horn following dorsal root stimulation. PF-05198007 (20 mins) abolished synaptically evoked action potentials (red). B. Example time course of EPSC block following PF-05198007 application to the whole preparation. C. Action potentials induced <i>via</i> current injection steps in SG neurons were not abolished by PF-05198007 (30 nM). Representative voltage traces are shown following current injection steps of -20, 0 and 50 pA before (blue traces) and after (red traces) PF-05198007 application. Line chart shows change in firing frequency (Hz) during control and after application of PF-05198007 for all neurons tested (n = 5, p > 0.05, paired t-test). D. Example time course of EPSC block following PF-05198007 application to the dorsal root alone. E. Representative EPSC traces and summary bar graph showing that the application of PF-05198007 (30 nM) to the dorsal root alone inhibited C-fibre mediated EPSCs and resulted in a significant conduction delay (n = 7, * p < 0.05; ANOVA on Ranks). F. PF-05198007 (30 nM; n = 15: 100 nM; n = 19) reduced veratridine evoked CGRP release in spinal cord synaptosomes. Reduction was compared with mexilitine (100 μM; n = 19), Ca²⁺ free conditions (n = 8) and TTX (500 nM; n = 6) (Data are shown ±SEM; * p < 0.05; ANOVA on Ranks).</p

Activity of PF-05089771 at human voltage-gated sodium channel isoforms.

<p>Activity of PF-05089771 at human voltage-gated sodium channel isoforms.</p

Evidence for functional Na_v1.7 in human DRG neurons.

<p>A. Representative TTX-S current traces (recorded in the presence of 1 μM A-803467 and following graded voltage steps from -110 mV to 10 mV. B. Voltage dependence of activation (red, n = 4 for each voltage) generated from the protocol described in A and steady state fast inactivation (blue) generated by conditioning 500 msec prepulses to voltages between -110 mV and +10 mV followed by a test pulse to 0 mV from a holding potential of -110 mV (n = 4 for each voltage). Both datasets are fitted with Boltzmann functions. C. Representative timecourse relationship for peak TTX-S current following the application of 100 nM PF-05089771 and 500 nM TTX. D. Concentration-response relationship for PF-05089771 block of TTX-S current (IC₅₀, slope: 8.4 nM, 1.1; n = 3–6 per concentration) E. Example voltage traces from a current clamp recording. Single action potentials were evoked by a 20 ms suprathreshold current step at 0.1 Hz. The scale bar refers to the voltage traces whereas the start-to-start interval is 10 s. F. Summary pie charts showing that the application of 30 and 100 nM PF-05089771 resulted in action potential block in 3/7 and 5/8 DRG neurons respectively.</p

PF-05198007 reduces the capsaicin flare response in WT, but not Na_v1.7^Nav1.8Cre mice.

<p>A, B.Time-course plots showing the effects of PF-05198007 on skin blood flow measured before and after topical capsaicin application for WT (A) and Nav1.7^Nav1.8Cre (B) mice (for each genotype, n = 8 per group). C, D. Corresponding summary bar graphs showing flare response measured as area under the curve for WT (C) and Nav1.7^Nav1.8Cre (D) mice before and after PF-05198007 treatment. 1 mg/kg and 10 mg/kg PF-05198007 significantly reduced capsaicin-induced flare in WT mice (C, both 1 mg/kg and 10 mg/kg, p < 0.01, ANOVA) but had no effect in Na_v1.7^Nav1.8Cre mice (D, both 1 mg/kg and 10 mg/kg, p > 0.05, ANOVA).</p

PF-05198007 increases action potential rheobase in small diameter mDRG neurons.

<p>A. Overlayed representative voltage traces in response to graded current step injections before (blue) and after PF-05198007 application (red). Current step stimulations are shown below. B. Example timecourse of change in rheobase following PF-05198007 application and washout. C. Summary bar graph, n = 8 neurons, ** p < 0.01, ANOVA. Data are shown ±SEM.</p