key: cord-1004222-0l9w49r1
authors: Moutal, Aubin; Martin, Laurent F.; Boinon, Lisa; Gomez, Kimberly; Ran, Dongzhi; Zhou, Yuan; Stratton, Harrison J.; Cai, Song; Luo, Shizhen; Gonzalez, Kerry Beth; Perez-Miller, Samantha; Patwardhan, Amol; Ibrahim, Mohab M.; Khanna, Rajesh
title: SARS-CoV-2 spike protein co-opts VEGF-A/neuropilin-1 receptor signaling to induce analgesia
date: 2020-10-01
journal: Pain
DOI: 10.1097/j.pain.0000000000002097
sha: f8a0096374ab509f0ca9ca326c0a1ee19d65d49f
doc_id: 1004222
cord_uid: 0l9w49r1

Global spread of severe acute respiratory syndrome coronavirus 2 continues unabated. Binding of severe acute respiratory syndrome coronavirus 2's spike protein to host angiotensin-converting enzyme 2 triggers viral entry, but other proteins may participate, including the neuropilin-1 receptor (NRP-1). Because both spike protein and vascular endothelial growth factor-A (VEGF-A)—a pronociceptive and angiogenic factor, bind NRP-1, we tested whether spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuron firing was blocked by spike protein and NRP-1 inhibitor EG00229. Pronociceptive behaviors of VEGF-A were similarly blocked through suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A “silencing” of pain through subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a coronavirus disease that, as of August 24, has infected more than 23.5 million people and caused nearly 810,000 deaths worldwide. 15 Most patients infected with SARS-CoV-2 report mild to severe respiratory illness with symptoms such as fever, cough, and shortness of breath. 30 However, a subset of patients diagnosed by a positive nucleic acid test are either asymptomatic or minimally symptomatic. 30 Increasing evidence shows that asymptomatic individuals can spread the virus efficiently, and the emergence of these silent spreaders of SARS-CoV-2 has limited control of the pandemic. 14, 41 Pain is a rising concern in symptomatic patients, likely emanating from a direct attack of SARS-CoV-2 on cells and the "cytokine storm" unleashed by affected cells. 51, 67 Whether asymptomatic or minimally symptomatic individuals have reduced pain thresholds, or whether their pain is silenced is unknown, but either could contribute to increased disease transmission dynamics.

The surface expressed angiotensin-converting enzyme 2 (ACE2) has been lionized as the main receptor for uptake of SARS-CoV-2. 22, 60, 64 Emerging evidence points to a subset of ACE2-expressing sensory neurons 48 that synapse with spinal and brainstem CNS neurons to produce neurological effects, including headache and nerve pain. 32, 34 Curiously, ACE2 is not present in most neurons, 48 despite increasing reports of neurological symptoms being common in COVID-19 patients. 32 Paradoxically, although the levels of ACE2 expression decline with age, 49 increased COVID-19 severity was noted in older patient populations, such as that of Italy's, 2 supporting the contention that ACE2 is not the sole gateway for entry of SARS-CoV-2. 1 Two recent reports demonstrated that the SARS-CoV-2 spike protein can bind to the b1b2 domain of the neuropilin-1 receptor (NRP-1). This interaction occurs through a polybasic amino acid sequence ( 682 RRAR 685 ), not conserved in SARS and Middle East Respiratory Syndrome (MERS), termed the "C-end rule" (CendR) motif, which significantly potentiates its entry into cells. 6, 11 Importantly, "omic" analyses revealed a significant upregulation of NRP-1 in biological samples from COVID-19 patients compared to healthy controls. 6 Using vascular endothelial growth factor-A (VEGF-A), a physiological ligand for the b1b2 pocket in NRP-1, we interrogated whether the spike protein, the major surface antigen of SARS-CoV-2, could block VEGF-A/ NRP-1 signaling to affect pain behaviors. Given parallels between the pronociceptive effects of VEGF-A in rodents 4, 58 and humans 23, 58 and clinical findings demonstrating increased VEGF-A levels in bronchial alveolar lavage fluid from COVID-19

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. patients 47 coupled with substantially lower levels in the sera of asymptomatic individuals compared to symptomatic patients, 30 a secondary question was to test whether spike protein could confer analgesia. We found that VEGF-A sensitizes nociceptor activity-a hallmark of neuropathic pain, 59 which was blocked by the spike protein and NRP-1 inhibitor EG00229. 26 Furthermore, we identify a novel analgesic role for spike protein, which is mirrored by NRP-1 inhibition.

An expanded version of all Methods is available in the Supplementary Information section (available online at http:// links.lww.com/PAIN/B190).

Pathogen-free adult male and female Sprague-Dawley rats (225-250 g; Envigo, Indianapolis, IN) were housed in temperature-controlled (23 6 3˚C) and light-controlled (12hour light/12-hour dark cycle; lights on 07:00-19:00) rooms with standard rodent chow and water available ad libitum. The Institutional Animal Care and Use Committee of the College of Medicine at the University of Arizona approved all experiments. All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health. Animals were randomly assigned to treatment or control groups for the behavioral experiments. Animals were initially housed 3 per cage but individually housed after the intrathecal cannulation. All behavioral experiments were performed by experimenters who were blinded to the experimental groups and treatments.

Plates (96-well, Nunc Maxisorp; Thermo Fisher Scientific, Waltham, MA) were coated with human Neuropilin-1-Fc (10 ng per well, Cat# 50-101-8343, Fisher, Hampton, NH) and incubated at room temperature overnight. The following day, the plates were washed and blocked with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) to minimize nonspecific adsorptive binding to the plates. SARS-CoV2 spike protein (S1 domain aa16-685, Cat# Z03485; Genscript, Piscataway, NJ) was added at concentrations ranging from 500 to 0.07 nM. As a negative control, some wells received PBS containing 3% BSA. The plates were incubated at room temperature with shaking for 3 hours. Next, the plates were washed with PBS to eliminate unbound protein. Bound SARS-CoV-2 Spike was detected by anti-His probe HRP (Cat#15165; Thermo Fisher Scientific). Tetramethylbenzidine (Cat#DY999; R&D Systems, St. Louis, MO) was used as the colorimetric substrate. The optical density of each well was determined immediately, using a microplate reader (Multiskan Ascent; Thermo Fisher Scientific) set to 450 nm with a correction wavelength of 570 nm. Data were analysed by nonlinear regression analysis using GraphPad Prism 8 (GraphPad, San Diego, CA).

Dorsal root ganglia (DRG) from all levels were acutely dissociated using methods as described previously. 18 Rat DRG neurons were isolated from 100 g female Sprague-Dawley rats using previously developed procedures. 36 2.4. Culturing primary dorsal root ganglia neurons and microelectrode array analysis Dissociated DRG neurons were maintained in media containing Neurobasal (Cat# 21103049; Thermo Fisher), 2% B-27 (Cat# 17504044; Thermo Fisher), 1% penicillin/streptomycin sulfate from 10,000 mg/mL stock, 30 ng/mL nerve growth factor, and 10% fetal bovine serum (Hyclone R&D Systems, Minneapolis, MN). Collected cells were resuspended in DRG media and seeded as a 10-mL drop on the poly-D-lysine coated electrodes of the microelectrode array (MEA) (24-well plate, Cat# MED-Q2430L, Alpha Med Scientific, Osaka, Japan). The cells were allowed to adhere for 30 minutes and then flooded with DRG media. The next day, cells were analyzed on a MED64 presto where 24 wells, with each containing 16 electrodes, could be recorded simultaneously. Cells were treated with the indicated neuropilin 1 ligands Semaphorin 3A (3 nM; Cat#5926-S3-025; R&D Systems), VEGF-A 165 (1 nM, Cat#P4853; Abnova), or VEGF-B (1 nM, Cat#RPU44324; Biomatik) for 30 minutes before recording. Treatments with either spike (100 nM, Cat#Z03485; Genscript) or the NRP-1 inhibitor EG-00229 (30 mM, Cat#6986; Tocris Bioscience [Bristol, United Kingdom]) were conducted 30 minutes before adding VEGF-A. The data were analyzed on MEA symphony and Mobius offline toolkit to extract the firing rate of the active electrodes before and after treatment. Firing rate is shown as Hz (event per second) for the electrodes that showed spontaneous activity. Recordings were obtained from acutely dissociated DRG neurons as described previously. 25, 37 The solutions and protocols for measuring sodium currents were exactly as described. 35 The protocol for isolating N-type calcium currents was previously described by Khanna et al. 10, 28 To isolate the N-type channel in DRG, contributions of the high-and low-voltage-activated calcium channel subtypes were blocked with the following subunit-selective blockers (all purchased from Alomone Labs, Jerusalem): Nifedipine (10 mM, Ltype); v-agatoxin GIVA (200 nM, P/Q-type) 33 ; SNX-482 (200 nM, Rtype) 39 ; and TTA-P2 (1 mM, T-type). 10 Dorsal root ganglia neurons were interrogated with current-voltage (I-V) and activation/ inactivation voltage protocols as previously described. 16, 18, 35 To determine the effect of VEGF-A application on voltagegated sodium and calcium currents, we incubated recombinant rat VEGF-A (1 nM in PBS) with DRG neurons for 30 minutes before whole-cell recordings. In addition, recombinant spike protein (100 nM in PBS) and the neuropilin 1 (NRP-1) blocker EG00229 (30 mM in DMSO, Cat. No. 6986; Tocris Bioscience) were also applied to the culture medium for 30 minutes before recording. For experiments where the proteins were tested in combination, the spike protein was added first for 30 minutes, followed by VEGF-A and EG00229 before recording commenced. The control conditions used either PBS, dimethyl sulfoxide (DMSO), or both to match the solutions used in the experimental conditions. The proteins and blocker were included at the same concentrations in the extracellular recording solution during all data acquisition.

As described before, 65 transverse 380-mm thick transverse slices were prepared from young rats (postnatal 10-14 days) for electrophysiological recordings at room temperature. VEGFA (1 nM), NRP-1 inhibitor (EG00229, 30 mM), and spike protein were added directly to the recording solution as indicated. Slices were treated for 30 minutes before the recordings.

Substantia gelatinosa neurons were recorded using previously described methods. 65 

PBS vehicle (NaCl 137 mM, KCl 2.5 mM, Na 2 HPO 4 10 mM, and KH 2 PO 4 1.8 mM), VEGF-A 165 (10 nM), spike (100 nM), and EG00229 (30 mM) were injected subcutaneously, alone or in combination, in the dorsum of the left hind paw. Rats were gently restrained under a fabric cloth, and 50 mL was injected using 0.5-mL syringes (27-G needles).

For intrathecal (i.t.) drug administration, rats were chronically implanted with catheters as described by Yaksh and Rudy. 63 

The assessment of tactile allodynia (ie, a decreased threshold to paw withdrawal after probing with normally innocuous mechanical stimuli) using a series of calibrated fine (von Frey) filaments was performed as described in Chaplan et al. 8 

Nerve injury was inflicted as described previously. 12 Rats were tested on day 10 after the surgery, at which time they had developed stable and maximal mechanical allodynia.

Adult rats were anesthetized using isoflurane and decapitated. Spinal cords were removed, and the dorsal horn of the spinal cord was dissected because this structure contains the synapses arising from the DRG. Synaptosome isolation was performed as described previously. 42 Integrity of nonpostsynaptic density (non-PSD) and postsynaptic density fractions was verified by immunoblotting. PSD95 was enriched in the postsynaptic density fractions, whereas synaptophysin was enriched in non-PSD fractions (not shown). BCA protein assay was used to determine protein concentrations.

Protein concentrations were determined using the BCA protein assay (Cat# PI23225; Thermo Fisher Scientific). Indicated samples were loaded on 4% to 20% Novex gels (Cat# EC60285BOX; Thermo Fisher Scientific). Proteins were transferred for 1 hour at 120 V using TGS (25 mM Tris pH 5 8.5, 192 mM glycine, 0.1% (mass/vol) SDS), 20% (vol/vol) methanol as transfer buffer to polyvinylidene difluoride membranes 0.45 mm (Cat# IPVH00010; Millipore, Billerica, MA), preactivated in pure methanol. After transfer, the membranes were blocked at room temperature for 1 hour with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), 5% (mass/vol) nonfat dry milk, then incubated separately in the primary antibodies VEGFR2 (Cat#PA5-16487; Thermo Fisher), pY1175 VEGFR2 (Cat#PA5-105167; Thermo Fisher), Flotillin (Cat#F1180; Sigma-Aldrich, St Louis, MO), or neuropilin-1 (Cat#sc-5307; Santa Cruz Biotechnology, Dallas, TX) in TBST, 5% (mass/vol) BSA, overnight at 4˚C. After incubation in horseradish peroxidase-conjugated secondary antibodies from Jackson ImmunoResearch, blots were revealed by enhanced luminescence (WBKLS0500; Millipore) before exposure to photographic film. Films were scanned, digitized, and quantified using Un-Scan-It gel version 7.1 scanning software by Silk Scientific Inc.

2.14. Docking of vascular endothelial growth factor-A spike protein and EG00229 to NRP-1

Peptide from C-terminus of furin cleaved SARS-CoV-2 spike protein 681-PRRAR-685 (blue sticks) was docked to NRP-1-b1 domain (white surface with binding site in red; PDB 6fmc 46 ) using Glide (Schrödinger 19 ). Molecular figures generated with PyMol 1.8 (Schrödinger, LLC.).

All data were first tested for a Gaussian distribution using a D'Agostino-Pearson test (Prism 8 Software, Graphpad, San Diego, CA). The statistical significance of differences between mean values was determined by a parametric analysis of variance followed by Tukey post hoc or a nonparametric Kruskal-Wallis test followed by Dunn post hoc test depending on whether data sets achieved normality. Behavioral data with a time course were analyzed by two-way analysis of variance with Sidak post hoc test. Differences were considered significant if P # 0.05. Error bars in the graphs represent mean 6 SEM. All data were plotted in Prism 8.

Initially, we assessed the involvement of spike and NRP-1 in the VEGF-A/NRP-1 pathway. An interaction between spike (S1 domain aa 16-685, containing the CendR motif 682 RRAR 685 ) and the extracellular portion of NRP-1 was confirmed by enzymelinked immunosorbent assay (Fig. 1A) . We calculated an equilibrium constant of dissociation (Kd) for this interaction to be ;166.2 nM (Fig. 1A) . Next, we plated sensory neurons on multiwell MEAs, an approach enabling multiplexed measurement of spontaneous, as well as stimulus-evoked extracellular action potentials from large populations of cells. 9 Vascular endothelial growth factor-A increased spontaneous firing of DRG neurons, which was blocked by the S1 domain of the spike protein and by the NRP-1 inhibitor EG00229 (Fig. 1A) . In contrast, ligands VEGF-B (ligand for VEGFR1-a co-receptor for NRP-1 31 ) and semaphorin 3A (Sema3A, ligand for plexin receptor-also a coreceptor for NRP-1 13,53 ) did not affect the spontaneous firing of nociceptors (Figs. 1B and C). The lack of effect of VEGF-B and Sema3A rules out a role for VEGF-R1 and plexin, respectively, thus implicating a novel ligand-, VEGF-A, and receptor-, NRP-1, specific pathway driving nociceptor firing (Fig. 1D) .

Because both VEGF-A and spike protein share a common binding pocket on NRP-1 (Fig. 1C) -H and Table S1 , http://links.lww.com/PAIN/ B190). Preventing VEGF-A from binding to NRP-1 with the NRP-1 inhibitor EG00229 or spike from activating VEGF-A/NRP-1 signaling blunted the mechanical allodynia and thermal hyperalgesia induced by VEGF-A alone ( Fig. 2 and Table S1 , http://links.lww.com/PAIN/B190). Neither Spike nor EG00229 alone had any effect on these behaviors ( Fig. 2 and Table S1 , http://links.lww.com/PAIN/B190) in either sex. Together, these data provide functional evidence that VEGF-A/NRP-1 signaling promotes a pain-like phenotype by sensitizing nociceptor activity (Fig. 1D) .

To gain insight into the mechanism by which VEGF-A contributed to increased nociceptor activity, we postulated that ion channels in DRG may be affected because these contribute to nociceptive plasticity. 61 Typical families of Na 1 currents from small-diameter DRG neurons are shown in Figure 3A . Vascular endothelial growth factor-A facilitated a 1.9-fold increase in total Na 1 currents compared to vehicle (PBS)-treated DRG, which was completely blocked by spike protein (Figs. 3B and C) . Spike protein alone did not affect Na 1 currents (Figs. 3B and C and Table S1 , http://links.lww.com/PAIN/B190). Because this decreased current could arise from changes in channel gating, we determined whether activation and inactivation kinetics of DRG Na 1 currents were affected. Half-maximal activation and inactivation (V 1/2 ), as well as slope values (k) for activation and inactivation, were not different between the conditions tested (Figs. 3D and E and Tables S1, S2, http://links.lww.com/PAIN/ B190), except for an ;8-mV hyperpolarizing shift in sodium channel inactivation induced by cotreatment of VEGF-A and EG00229 (Table S2 , http://links.lww.com/PAIN/B190). Similar results were obtained for the NRP-1 inhibitor EG00229, which also inhibited the VEGF-A-mediated increase in total Na 1 currents (Figs. 3F-H and Table S1 , http://links.lww.com/PAIN/ B190) but had no effect on the biophysical properties (Figs. 3I and J and Tables S1, S2, http://links.lww.com/PAIN/B190).

Because calcium channels play multiple critical roles in the transmission and processing of pain-related information within the primary afferent pain pathway, 61 we evaluated whether they were affected. We focused on N-type (Ca v 2.2) channels because these mediate neurotransmitter release at afferent fiber synapses in the dorsal horn and are critical in the pain matrix. 50 Vascular endothelial growth factor-A facilitated a 1.8-fold increase in total Ca 21 currents compared to vehicle (PBS)treated DRG, which was completely blocked by spike protein (Figs. 4A-C and Table S1, http://links.lww.com/PAIN/B190). Spike protein alone did not affect Ca 21 currents (Figs. 4A-C). In addition, we did not observe any changes in activation and inactivation kinetics between the conditions tested (Figs. 4D and E and Tables S1 and S2, http://links.lww.com/PAIN/ B190). Similar results were obtained for the NRP-1 inhibitor EG00229, which inhibited the VEGF-A-mediated increase in N-type Ca 21 currents (Figs. 4F-H and Table S1, http://links. lww.com/PAIN/B190) but had no effect on the biophysical properties (Figs. 4I and J and Tables S1 and S2, http://links. lww.com/PAIN/B190). These data implicate spike protein and NRP-1 in Na 1 and Ca 21 (Ca v 2.2) channels in VEGF-A/NRP-1 signaling.

The spinal cord is an integrator of sensory transmission where incoming nociceptive signals undergo convergence and modulation. 57 Spinal presynaptic neurotransmission relies on DRG neuron action potential firing and neurotransmitter release. From these fundamental physiological principles, as well as the results described above, we were prompted to evaluate whether synaptic activity was affected in the lumbar dorsal horn. The amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) of neurons in the substantia gelatinosa region of the lumbar dorsal horn were not affected by VEGF-A (Figs. 5A and B and Table S1, http://links.lww.com/PAIN/ B190). In contrast, VEGF-A application increased sEPSC frequency by ;3.6-fold, which was reduced by ;57% by inhibition of NRP-1 with EG00229 and ;50% by spike protein (Figs. 5A and C and Table  S1 , http://links.lww.com/PAIN/B190). Amplitude and interevent interval cumulative distribution curves for sEPSCs are shown in Figures  5D and E . When compared to vehicle controls, VEGF-A, with or without NRP-1 inhibitor or spike protein, had no effect on the cumulative amplitude distribution of the spontaneous EPSCs ( Fig. 5D and Table S1 , http://links.lww.com/PAIN/B190) but changed the cumulative frequency distribution of spontaneous EPSCs with significantly longer interevent intervals (Fig. 5E and Table S1 , http:// links.lww.com/PAIN/B190). Together, these data suggest a presynaptic mechanism of action for spike protein and NRP-1.

We used the spared nerve injury (SNI) model of neuropathic pain, chosen because it produces a reliable and consistent increase in pain sensitivity, 12 to evaluate potential disruption of the VEGF-A/NRP-1 pathway to reverse nociception. Vascular endothelial growth factor-A triggers autophosphorylation of VEGFR2 at Y1175, 54 thereby serving as a proxy for activation of VEGF-A signaling. In rats with SNI, intrathecal application of spike decreased the phosphorylation of VEGFR2 (Y1175) on both the contralateral (noninjured) and ipsilateral (injured) sides (Figs. 6A  and B) . This shows that spike can inhibit VEGF-A signaling in a rat model of chronic neuropathic pain. Spared nerve injury Table S1 , http://links.lww.com/PAIN/B190. Panels B, F, D, and H are the area under the curve for 0 to 24 hours. Data are shown as mean 6 SEM and were analyzed by nonparametric two-way analysis of variance where time was the within-subject factor and treatment was the between-subject factor (post hoc: Sidak), *P , 0.05. Areas under the curve were compared by a one-way analysis of variance with Kruskal-Wallis post hoc test. The experiments were analyzed by an investigator blinded to the treatment. For full statistical analyses, see Table S1 , http://links.lww.com/PAIN/B190. January 2021 · Volume 162 · Number 1 www.painjournalonline.com efficiently reduced PWTs (mechanical allodynia, Fig. 6C and Table S1 , http://links.lww.com/PAIN/B190) 10 days after injury. Spinal administration of spike protein significantly increased PWTs (Fig. 6C and Table S1 , http://links.lww.com/ PAIN/B190), in a dose-dependent manner, for 5 hours. Analysis of the area under the curve confirmed the dosedependent reversal of mechanical allodynia (Fig. 6D and Table  S1 , http://links.lww.com/PAIN/B190) compared to vehicletreated injured animals. Similar results were seen with female rats injected with spike ( Figs. 6E and F) . Finally, inhibition of NRP-1 signaling with EG00229 also reversed paw withdrawal thresholds (Figs. 6G and H and Table S1 , http://links.lww. com/PAIN/B190).

Our data show that SARS-CoV-2 spike protein subverts VEGF-A/NRP-1 pronociceptive signaling. Relevant to chronic neuropathic pain, spike negated VEGF-A-mediated increases in: (1) voltage-gated sodium and N-type calcium current densities in DRG neurons; (2) spontaneous firing in DRG neurons; (3) spinal neurotransmission; and (4) mechanical allodynia and thermal hyperalgesia. Consequently, spike protein was analgesic in a nerve injury rat model. Based on the reported increase in VEGF-A levels in COVID-19 patients, 30 one would expect to observe increased pain-related symptoms. However, our data suggest that the SARS-CoV-2 Table S2 , http://links.lww.com/PAIN/B190. P values of comparisons between treatments are as indicated; for full statistical analyses, see Table S1 , http://links.lww.com/PAIN/B190. Table S2 , http://links.lww.com/PAIN/B190. P values of comparisons between treatments are as indicated; for full statistical analyses, see Table S1 , http://links.lww.com/PAIN/B190. Clinical findings that VEGF-A contributes to pain are supported by observations that in osteoarthritis, increased VEGF expression in synovial fluids has been associated with higher pain scores. 55 Vascular endothelial growth factor-A has been reported to enhance pain behaviors in normal, nerve-injured, and diabetic animals. 23, 58 Blocking VEGF-A has been shown to reduce nociception in rodents and to exert a neuroprotective effect by improving neuronal restoration and conduction, decreasing proapoptotic Caspase-3 levels in sensory neurons, preventing neural perfusion and epidermal sensory fiber loss. 24, 52, 66 Alternative splicing of the VEGF-A gene produces several isoforms of the mature protein containing between 121 and 206 amino acid residues, with VEGF-A165a being pronociceptive 23 through sensitization of transient receptor potential Table S1 , http://links.lww.com/PAIN/ B190. sEPSC, spontaneous excitatory postsynaptic current. January 2021 · Volume 162 · Number 1 www.painjournalonline.com 249 channels 24 and ATP-gated purinergic P2X2/3 receptors 27 in DRG neurons. VEGF-A165b is a VEGFR2 partial agonist that competes with VEGF-A165a for binding to VEGFR2. The VEGF-A165a isoform also binds to the NRP-1 coreceptor, whereas VEGFA165b does not. 44 Thus, it is a balance of the splice isoforms (VEGF-Aa/b) that likely determines the nociceptive impact on sensory neurons. Our data show that VEGF-A elicits long-lasting (up to 24 hours) mechanical allodynia and thermal hyperalgesia in male and female naive rats, thus supporting the premise that VEGF-A is pronociceptive. Vascular endothelial growth factor is augmented in serum of rheumatoid arthritis patients. 4, 40 The levels of VEGF-A were substantially lower in the sera of asymptomatic individuals compared to symptomatic individuals and matched those found in healthy controls. 30 Conversely, transcript levels of the VEGF-A coreceptor NRP-1 were increased in COVID-19 patients compared to healthy controls. 6 NRP-1 is a dimeric transmembrane receptor that regulates pleiotropic biological processes, including axon guidance, angiogenesis, and vascular permeability. 21, 45, 56 In chronic neuropathic pain, a concomitant increase of NRP-1 and VEGF-A has been reported in DRG neurons. 29 We observed that pharmacological antagonism of the VEGF-A binding b1b2 domain of NRP-1 using EG00229 resulted in decreased mechanical allodynia in SNI. A corollary to this, VEGF-Ainduced DRG sensitization and the related allodynia/ hyperalgesia in naive rats was annulled by NRP-1 antagonism. This work identifies a heretofore unknown role of VEGF-A/NRP-1 signaling in pain. Our working model shows that VEGF-A engagement of NRP-1 is blocked by spike protein, consequently decreasing activities of two key nociceptive voltage-gated sodium, likely Na v 1.7, and calcium (Ca v 2.2) channels (Fig. 1D) . The resulting decrease in spontaneous DRG neuronal firing by spike protein translates into a reduction in pain (Fig. 1D) . Characterization of the molecular cascade downstream of VEGF-A/NRP-1 signaling awaits further work.

Altogether, our data suggest that interfering with VEGF-A/ NRP-1 using SARS-CoV-2 spike or the NRP-1 inhibitor EG00229 is analgesic. For cancer, this pathway has been extensively targeted for antiangiogenesis. A monoclonal antibody targeting VEGF-A (bevacizumab, Avastin) has been used as a cancer treatment. 20 A phase 1a clinical trial of NRP-1 antibody MNRP1685A (Vesencumab), targeting the VEGF-binding site of NRP-1, was well tolerated in cancer patients, 62 but neuropathy was noted. Possible reasons for this include the scenario that anti-VEGF-A therapies may target the alternatively spliced VEGF-A165b isoform, which has a neuroprotective role. It is important to note that the VEGF-A165b isoform binds to the VEGFR2 binding site with equal affinity to VEGF165a, but does not bind to the coreceptor neuropilin 1 (NRP-1), 7 thus acting as an inhibitor of the VEGF-A-NRP-1 pathway. 3 Anti-VEGF-A therapies may simultaneously inhibit both proalgesic and antialgesic pathways involving the multiple VEGF-A isoforms, thus an ensuing loss of balance may favor nociceptor sensitization. Here, we are specifically studying the downstream actions after the binding of VEGF-A165a to the b1 domain of NRP-1. Targeting this interaction may be more specific compared to broad VEGF-A inhibitors that have been developed for cancer treatment. Thus, our preclinical work provides a rationale for targeting the VEGF-A/NRP-1 pronociceptive signaling axis in future clinical trials.

R. Khanna is the cofounder of Regulonix LLC, a company developing nonopioid drugs for chronic pain. In addition, Figure 6 . Severe acute respiratory syndrome coronavirus 2's spike protein and neuropilin-1 receptor (NRP-1) antagonism reverses SNI-induced mechanical allodynia. Spared nerve injury (SNI) elicited mechanical allodynia 10 days after surgery. (A) Representative immunoblots of NRP-1, total and pY1175 VEGF-R2 levels at presynaptic sites in rat spinal dorsal horn after SNI. Tissues were collected 3 hours (ie, at peak antiallodynia) after intrathecal injection of the receptor binding domain of the spike protein (2.14 mg/5 mL). (B) Bar graph with scatter plots showing the quantification of n 5 4 samples as in (A) (*P , 0.05, Kruskal-Wallis test). Paw withdrawal thresholds for SNI rats (male) administered saline (vehicle) or 4 doses of the receptor binding domain of the spike protein (0.00214-2.14 mg/5 mL) intrathecally (i.t.); n 5 6 to 12) (C) 2.14 mg/5 mL dose of spike in female rats, or the NRP-1 inhibitor EG00229 in male rats (2 mg/ 5 mL; n 5 6) (E). (D, F, H) Summary of data shown in panels C, E, and G plotted as area under the curve (AUC) for 0 to 5 hours. P values, vs control, are indicated. Data are shown as mean 6 SEM and were analyzed by nonparametric two-way analysis of variance where time was the withinsubject factor and treatment was the between-subject factor (post hoc: Sidak). AUCs were compared by Mann-Whitney test. The experiments were analyzed by an investigator blinded to the treatment. P values of comparisons between treatments are as indicated; for full statistical analyses, see Table S1 , http:// links.lww.com/PAIN/B190. 

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Neurological manifestations of COVID-19 and other coronavirus infections: a systematic review

Studies on CRMP2 SUMOylation-deficient transgenic mice identify sex-specific NaV1.7 regulation in the pathogenesis of chronic neuropathic pain

)-lacosamide inhibition of CRMP2 phosphorylation reduces postoperative and neuropathic pain behaviors through distinct classes of sensory neurons identified by constellation pharmacology

Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides

Dissecting the role of the CRMP2-neurofibromin complex on pain behaviors

Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas

Expression of pro-and antiangiogenic isoforms of VEGF is differentially regulated by splicing and growth factors

Prevalence of asymptomatic SARS-CoV-2 infection: a narrative review

Neuronal pentraxins modulate cocaine-induced neuroadaptations

Structural basis for selective vascular endothelial growth factor-A (VEGF-A) binding to neuropilin-1

Molecular pharmacology of VEGF-A isoforms: binding and signalling at VEGFR2

Neuropilin regulation of angiogenesis, arteriogenesis, and vascular permeability

Small molecule neuropilin-1 antagonists combine antiangiogenic and antitumor activity with immune modulation through reduction of transforming growth factor beta (TGFbeta) production in regulatory T-cells

A pharmacological interactome between COVID-19 patient samples and human sensory neurons reveals potential drivers of neurogenic pulmonary dysfunction

ACE2 expression in human dorsal root ganglion sensory neurons: implications for SARS-CoV-2 virus-induced neurological effects

Decoding SARS-CoV-2 hijacking of host mitochondria in pathogenesis of COVID-19

Targeting chronic and neuropathic pain: the N-type calcium channel comes of age

Pain: a potential new label of COVID-19

Neutralization of schwann cell-secreted VEGF is protective to in vitro and in vivo experimental diabetic neuropathy

Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors

A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-Adependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells

Vascular endothelial growth factor expression and their action in the synovial membranes of patients with painful knee osteoarthritis

C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration

Neuronal circuitry for pain processing in the dorsal horn

Systemic anti-vascular endothelial growth factor therapies induce a painful sensory neuropathy

Adaptive mechanisms driving maladaptive pain: how chronic ongoing activity in primary nociceptors can enhance evolutionary fitness after severe injury

Structural and functional basis of SARS-CoV-2 entry by using human ACE2

Regulating excitability of peripheral afferents: emerging ion channel targets

A phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors

Chronic catheterization of the spinal subarachnoid space

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Phosphorylated CRMP2 regulates spinal nociceptive neurotransmission

Transplantation of recombinant vascular endothelial growth factor (VEGF)189-Neural stem cells downregulates transient receptor potential vanilloid 1 (TRPV1) and improves motor outcome in spinal cord injury

Viral and host factors related to the clinical outcome of COVID-19