The nociceptin/orphanin FQ peptide receptor (NOP) (also called opioid receptor-like 1, ORL1 receptor) is a G-protein-coupled receptor sharing high sequence identity with the 3 classically recognised opioid receptor types, μ, δ, and κ, also termed MOP, DOP, and KOP, respectively.26,39 The endogenous ligand for the NOP receptor, known as nociceptin37 or orphanin FQ,46 is a 17-amino acid peptide whose N-terminal tetrapeptide sequence is related to that of the opioid peptides (FGGF or YGGF), and whose basic core is similar to dynorphin, the endogenous peptide for the κ receptor. In spite of these similarities, the NOP receptor does not recognize the majority of opioid ligands, and nociceptin/orphanin FQ (N/OFQ) itself has low affinity for the classic opioid receptors.46 NOP receptor agonists are highly effective analgesics in a range of preclinical pain models and provide the prospect of new treatments for chronic pain without abuse liability in humans.33
The NOP receptor couples to the same Gi/Go protein-mediated second-messenger systems as the opioid receptors and produces inhibition of adenylate cyclase, activation of an inwardly rectifying K+ conductance, and inhibition of voltage-sensitive Ca2+ channels.36 These intracellular effectors generally act to inhibit cellular excitability, and suggest a role for the N/OFQ/NOP system in the modulation of neuronal activity and transmitter release.22,41
Activation of the NOP receptor is effective in several animal models of neuropathic pain and visceral hypersensitivity disorders. To illustrate, both peripheral42 and spinal15,42,59 NOP activation reduced mechanical allodynia in the rat chronic constriction injury model, and peripheral NOP receptor activation reduced capsaicin-induced thermal nociception in nonhuman primates.25 In rats receiving TNBS instillation, N/OFQ inhibited colonic hyperalgesia after intraperitoneal application.1 Clinically, therapeutic effectiveness of N/OFQ instillation has been reported in patients with detrusor overactivity (DO),28 as well as in patients with painful bladder syndrome.30
Most of the data on NOP receptor expression are derived from rodents, and using mRNA expression analysis.38 NOP is expressed both in the central nervous system and in peripheral tissues. However, little is known about the localization of the NOP receptor in human tissues, and information about any changes in expression levels in human disease is virtually absent. Therefore, we investigated NOP receptor levels in tissues from humans with pain and hypersensitivity disorders; furthermore, we investigated NOP receptor signalling in cultured rat and human dorsal root ganglion (DRG) neurons using calcium imaging.
The aim of our studies was to validate the NOP receptor as a target in tissues from clinical pain disorders, and identify patient groups with chronic pain and hypersensitivity in which activation of NOP may yield a therapeutic benefit.
2. Materials and methods
2.1. Clinical tissues
A range of clinical tissues including sensory ganglia (DRG), peripheral nerve, and urinary bladder were studied. This study was done with approval from the Local Research Ethics Committees, and full informed consent was obtained from all tissue donors.
2.2. Urinary bladder
Bladder tissue specimens were obtained from control subjects (n = 20), patients with overactive bladder (DO, n = 20) and painful bladder syndrome (PBS) (n = 8). All participants underwent clinical examination and their medical history was reviewed. The control subjects were under investigation for asymptomatic microscopic haematuria.60 Subjects with overactive bladder demonstrated symptomatic urinary urgency and urge incontinence assessed using the ICIQ-LUTS QOL questionnaire, >10 voids per day on a minimum 3-day bladder diary, and systolic DO with no cough stress leakage during cystometry. Urodynamic stress incontinence controls demonstrated symptomatic stress incontinence assessed using the ICIQ-LUTS QOL questionnaire, <6 voids per day on a minimum 3-day bladder diary, absence of systolic or provoked DO during cystometry, cystometric capacity >450 mL, and positive cough stress leakage at video cystourethrography. Patients with painful bladder syndrome met the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) research criteria for interstitial cystitis, as described by us previously.60 Flexible or rigid cystoscopic bladder biopsies were obtained from a consistent site just above and lateral to the ureteric orifices. A urine specimen was sent for culture before each cystoscopy to confirm that all patients had sterile urine cultures at the time of cystoscopy. A full-thickness PBS bladder specimen was also obtained during surgery.60
2.3. Dorsal root ganglia
Control DRG (n = 10, 5 lumbar and 5 sacral) of human cadavers were obtained from the Netherlands Brain Bank with consent and described methodology.45 Cervical avulsed DRG were available from 2 young male patients who had surgical repair of traumatic brachial plexus injuries, as described.50
2.4. Peripheral nerves
Specimens of injured limb nerves (n = 10) and painful neuromas (n = 7) were collected from patients during surgery for painful neuroma excision/relocation or peripheral nerve repair. Uninjured nerve trimmings (n = 12), from nerves used for grafting in nerve repairs during surgery, served as controls.
An antibody to the NOP receptor (sc-15309) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and used initially at dilutions from 1:50 to 1:5000 (final dilution 1:100). The NOP receptor antibody (sc-15309) is a rabbit polyclonal antibody with an epitope corresponding to amino acids 161 to 245 mapping to an internal region of the NOP receptor (KOR-3) of human origin. In our studies, this antibody reacted with neuronal fibres and DRG neurons in human tissues (Figs. 1–3). Specificity controls for immunostaining were satisfactory: nerve structures were immunostained in a titre-specific manner (Fig. 2A), and no immunostaining was observed when the primary antibody was omitted. Additionally, no staining was observed in the human cerebellum (Fig. 2B), a tissue in which absence or only very limited presence of both NOP receptor transcripts and N/OFQ binding has been demonstrated,11,44,57 whereas neurofilament cocktail antibodies showed positive neuronal cell bodies and nerve fibres in serial sections. Similar immunoblotting results were reported with this antibody compared with other antibodies directed against the C or N terminus of the receptor.23 This antibody has been previously shown to immunostain rat myenteric neurons,61 and both immunostain and Western blot-cultured rat astrocytes21 and spinal cord20 with the expected band of ∼40 kDa, at dilutions similar to those of our studies. Western blotting studies with this antibody has also revealed that NOP receptor expression of an expected 40-kDa band in distal colonic samples of TNBS-treated rats.1 An affinity-purified rabbit antibody to TRPV1 (C22, unrestricted gift from GlaxoSmithKline, Stevenage, United Kingdom, final dilution 1:10,000) against a synthetic peptide sequence of human TRPV1 sequence was used in this study as previously described.50 We have previously demonstrated the specificity of this TRPV1 antibody in the bowel13 and bladder.10 A cocktail of monoclonal antibodies to the phosphorylated and nonphosphorylated neurofilaments of size 200 kDa (Clone N52; Sigma-Aldrich, Dorset, United Kingdom) and the 57-kDa type III filament, peripherin (Novocastra Laboratories, Newcastle, United Kingdom) were used at final titres of 1:20,000 and 1:500, respectively, as structural neuronal markers.
Tissues specimens were snap-frozen in liquid nitrogen and stored at −70°C until use. Tissues were supported in optimum cutting tissue medium (RA Lamb Ltd, Eastbourne, United Kingdom). Tissue sections (15 μm thick) were collected onto poly-l-lysine (Sigma, Poole, United Kingdom)-coated glass slides, and postfixed in 4% wt/vol paraformaldehyde in 0.15 M phosphate-buffered saline for 30 minutes. Endogenous peroxidase was blocked by incubation in industrial methylated spirits containing 0.3% wt/vol hydrogen peroxide for 30 minutes. After rehydration with phosphate-buffered saline buffer, sections were incubated overnight with primary antibody at dilutions listed above. Sites of primary antibody attachment were revealed using nickel-enhanced, avidin–biotin peroxidase (ABC–Vector Laboratories, Peterborough, United Kingdom) as described. Sections were counterstained for nuclei in 0.1% wt/vol aqueous neutral red, dehydrated, and mounted in xylene-based mountant (DPX; BDH/Merck, Poole, United Kingdom), before photomicrography.
2.6. Image analysis
For urinary bladder studies, images were captured (×40 magnification) in the suburothelium of each tissue section. Five fields per tissue section were scanned, and the mean value was used in subsequent statistical analysis. For DRG neurons, NOP receptor and TRPV1-immunoreactive nucleated neurons were counted visually, their diameter was measured using a calibrated microscope eyepiece graticule and results were expressed as % of total number of neurons. The peripheral nerve analyses were based on the density of nerve fibres, as described in our previous published studies.3,13 Immunoreactive fibres were quantified using computerised software, whereas analogue images were captured through video link to an Olympus BX50 microscope and converted into digital monochrome images, and analysed using analySIS (version 5.0) software. The gray-shade detection threshold was set at a constant level to allow detection of positive immunostaining and the area of highlighted immunoreactivity obtained as a percentage (% area) of the field scanned. The analyses were performed in a blinded manner. The Mann–Whitney test was used for statistical analysis (P values < 0.05 were considered statistically significant).
2.7. Rat dorsal root ganglion neuron cultures
Bilateral DRG from all levels were harvested from 5 adult female Wistar rats (weight: 250 g; Charles River, Margate, Kent), enzyme-digested in 0.5% dispase/0.2% collagenase for 3 hours, followed by 30 minutes in papain (0.1%, 12 U/mL; Sigma), and mechanically dissociated in BSF2 medium containing soybean trypsin inhibitor/DNAse to obtain a single-cell suspension, as previously described.7,8 DRG neurons at a density of 5000 neurons/mL were incubated in BSF2 medium (containing 2% HIFCS, 0.1 mg/mL transferrin, 60 ng/mL progesterone, 0.16 μg/mL sodium selenite, 3 mg/mL bovine serum albumin [BSA], penicillin/streptomycin at 100 μg/mL each, 16 μg/mL putrescine, 10 μg/mL insulin), and the neurotrophic factors nerve growth factor (NGF) (100 ng/mL), glial cell line–derived neurotrophic factor (GDNF) (50 ng/mL), and neurotrophin 3 (NT3) (50 ng/mL), for 48 hours before being studied.
2.8. Human dorsal root ganglion neuron cultures
Avulsed cervical DRG were obtained from 4 patients with brachial plexus avulsion injury undergoing reconstruction surgery at the RNOH Stanmore, United Kingdom, with patient consent and approval of the local ethics committee. Avulsed DRG were collected in Ham F12 containing penicillin and streptomycin (100 μg/mL each), minced, enzyme-digested in Ham F12 containing 0.2% collagenase/0.5% dispase for 3 hours, followed by 30 minutes in papain (0.1%, 12 U/mL; Sigma, United Kingdom), and mechanically dissociated to obtain a single-cell suspension.7 Neurons were plated on collagen/laminin-coated MatTek dishes (MatTek Corp, Ashland, MA), in Ham F12 nutrient medium containing 10% heat-inactivated fetal calf serum, and antibiotics, and incubated at 37°C in a humid environment.
2.9. Calcium imaging
Functional effects of acute N/OFQ (orphanin FQ; Merck Millipore, San Diego, CA) and DAMGO ([D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin, Tocris Bioscience, United Kingdom) treatment to capsaicin responses were determined in Fura2 AM (Molecular Probes)-loaded neurons as previously described.6,7,9 Responses to paired capsaicin stimuli, with and without N/OFQ or DAMGO, were measured as a change from the baseline 340/380-nm excitation ratio. Experiments were conducted at 37°C in a humidified environment on an inverted Nikon microscope (Diaphot 300), and alternately excited at 340 and 380 nm wavelengths. Images were captured every 2 seconds in each of the 3 channels (phase, 340 and 380 nm), and recordings of intracellular changes in bound and unbound Ca2+ ratio were obtained before, during, and after the addition of test compounds. This provided baseline recordings and intracellular changes in Ca2+ levels in response to added compounds. Cells were uniformly loaded with the dye, and no intracellular compartmentalisation of the loaded dye was observed. Images were acquired with a Hamamatsu Orca CCD Camera and analysed with AQM Advance Kinetic imaging software. Individual cells under study were highlighted as regions of interest for calculating the mean ratios of bound to unbound calcium. In each experiment, neurons were exposed to capsaicin for a maximum of only 2 applications, first to identify capsaicin sensitivity and second to test the effect of the added drugs after the washout period. Because capsaicin stimulation is known to cause desensitisation, we used a published protocol in which a minimum concentration (200 nM) and a brief period of application (15 seconds) were used to identify capsaicin-sensitive neurons (demonstrating a rapid increase in 340/380 ratio and sustained response), which was followed by washout of the medium and a rest period of 30 minutes before the second challenge.9 Responses were measured as the difference between baseline and peak ratio change, and the second response was normalised to the first response; subtracting the percent response from 100 gave the value for percent inhibition. Average % inhibition was compared between groups. Calculation of percent inhibition: for each experiment, response to 200 nM capsaicin = R1, response to 1 μM capsaicin (with or without added compounds) = R2, % response = R2/R1 × 100, % inhibition = 100 − % response.
3.1 Urinary bladder
Many intensely stained nerve fibres were seen in the urothelium and suburothelium in bladder tissue from patients with PBS and DO, fewer in controls (Figs. 1A–C). Image analysis of NOP-positive nerve fibre staining within the suburothelium showed a significant increase in tissues both from patients with DO (P < 0.0001) and PBS (P = 0.0014), compared with controls (Fig. 1D).
3.2 Dorsal root ganglia
The NOP receptor antibody immunostained subpopulations of small-diameter (≤50 μm) and large-diameter (>50 μm) neuronal cells in avulsed hDRG, increasing dilutions of antibody diminished immunostaining (Fig. 2A). Similar immunostaining was observed in control postmortem sacral and lumbar DRG (Fig. 2C).
Serial section colocalisation studies at optimal antibody dilutions showed that the NOP receptor was expressed in most of the TRPV1-positive small DRG cells (arrowed, Fig. 3A, NOP left panel, TRPV1 right panel). NOP and TRPV1-positive cell counts in DRG are shown in Figures 3B and C. The total number of nucleated neurons counted positive for NOP ≤50 μm and >50 μm, respectively, were 636 and 45 for lumbar, 542 and 54 for sacral, 402 and 55 for avulsed cervical human DRG.
3.3. Peripheral nerves
NOP receptor nerve fibre staining was seen in all control (Fig. 4A) and injured limb nerves and painful neuroma specimens (Figs. 4C, E); there were fewer nerve fibres immunostained than with neurofilament cocktail antibodies in the same specimens (Figs. 4B, D, and F). Image analysis of NOP receptor-positive nerve fibres showed a significant decrease in the injured nerve group compared with control nerves (Fig. 5 top, P = 0.0004), and also vs painful neuroma specimens (P = 0.025). Neurofilament cocktail staining was similar in these groups (Fig. 5 middle). The ratio of the % area NOP receptor to neurofilament cocktail antibody immunostaining was significantly decreased in the injured group (Fig. 5 bottom, P = 0.0004), and in the painful neuroma group (P = 0.011), compared with controls.
3.4. Effect of N/OFQ on capsaicin responses in cultured rat dorsal root ganglion neurons
Individual neurons were tested for capsaicin sensitivity with a 200-nM test dose of capsaicin. This was followed by washout and change of medium and a rest period of 30 minutes, after which the same neurons were stimulated with 1 μM capsaicin. Responses were measured as the difference in amplitude from baseline to peak response. In the absence of added drugs (control), the average value of the second response was reduced because of tachyphylaxis to 83 ± 5% of the first response, as expected in rat DRG neurons and published previously (Fig. 6).5–7,9 After the rest period, when cells were incubated with N/OFQ for 10 minutes, a concentration-dependent inhibition of the second capsaicin challenge responses was observed (Fig. 6). A robust calcium signal after ionomycin addition at the end of the experiment confirmed that the neurons were viable.
Preincubation with 0.01 nM N/OFQ (10 minutes incubation) significantly reduced the response to 1 μM capsaicin, whereas a subsequent response to 2 μM ionomycin was still present indicating cell viability. Preincubation with 0.1 nM N/OFQ led to an almost complete abolition of capsaicin-induced calcium flux. In rDRG neurons, capsaicin responses were concentration-dependently inhibited in the presence of N/OFQ, with an IC50 of 8.6 pM (95% CI 4.4-17 pM) (Fig. 6). Dose-dependent inhibition of capsaicin responses in rDRG neurons were also observed in the presence of DAMGO, with maximum inhibition at 0.1 nM DAMGO and an IC50 of 1.2 pM (95% CI 0.26-5.2 pM) (Fig. 6).
Although the maximum inhibition of capsaicin responses was observed with 0.1 nM of either N/OFQ or DAMGO, the inhibition due to N/OFQ was greater (100%; 95% CI 90%-111%) than that due to DAMGO (55%; 95% CI 50%-60%), suggesting a much higher efficacy for the NOP receptor system in inhibiting TRPV1 receptor signalling. The maximum inhibition by morphine, a prototypical nonpeptide MOP agonist, was previously shown to be similar (67.22 ± 4.3% at a saturating concentration of 10 μM5) to that of DAMGO, which suggests that the effect in this system may be independent of the agonist and a property of the receptor, or downstream receptor coupling.
3.5. N/OFQ inhibition of capsaicin-mediated calcium influx in human dorsal root ganglion neurons
In human dorsal root ganglion (hDRG) neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (P < 0.001, n = 3 neurons), which is in the same order of magnitude as the inhibition observed with rat DRG. In the absence of N/OFQ, inhibition observed by the second stimulus due to tachyphylaxis was 8.7% (n = 5 neurons).
The NOP receptor is involved in a range of physiological systems, including pain pathways.26 As previous studies of receptor distribution and function were mainly preclinical,38 and NOP mRNA splice variants were described in human and rat DRG,58 we set out to determine the presence of the NOP receptor protein in tissues from patients with chronic pain and hypersensitivity disorders. We focused on peripheral nerve and DRG tissue, and innervation of visceral organs. Importantly, we also examined the functional effects of the endogenous NOP receptor ligand N/OFQ in cultured rat and human DRG neurons.
The NOP receptors were present in the majority of both small- and large-diameter neurons in DRG, at the cervical, lumbar, and sacral levels. This finding is supported by knock-in mice studies with fluorescent-tagged NOP receptors in brain, spinal cord, and DRG neurons.43 Although NOP colocalised with TRPV1 in small-diameter neurons, its presence (unlike TRPV1) in large-diameter neurons suggests a potential role in addition to nociception. Further detailed quantitative studies of NOP receptor expression in somatic and visceral DRG neurons are required, including colocalisation of NOP receptors with TRPV1 and other pain targets. Overall, NOP receptor expression was clearly reduced in injured limb nerves and painful neuromas, which suggests that retrogradely transported neurotrophic factors may be involved in regulating its expression, as for a number of nociceptor-related neuropeptides, ion channels, and receptors. However, in a rodent study of partial sciatic nerve transection, and an inflammatory pain model of complete Freund adjuvant injection into the hind paw, a modest increase of both N/OFQ and its receptor immunoreactivity was observed in neurons after 7 days; the time course of changes and species differences thus deserve further study.14 Residual or spouting nerve fibres in injured nerves may mediate hypersensitivity and which may be ameliorated by NOP receptors. In accord, peripheral42 and spinal15,42,59 NOP activation reduced mechanical allodynia in the rat chronic constriction injury model, and activation of peripheral NOP and MOP receptors exerted antihypersensitivity effects in a rodent pain model of diabetic polyneuropathy.48 The mechanism of action of N/OFQ is likely to include inhibition of neurotransmitter release from peripheral nerve terminals.22,41 The downregulation of NOP should be interpreted with caution as it does not necessarily support a role for this receptor in the pathogenesis of neuropathic pain, nor does it suggest a potential therapeutic target for clinical treatment.
Unlike injured limb nerves, a marked and significant increase in NOP receptor immunoreactive nerve fibres was observed in bladder specimens from patients with overactive bladder and with bladder pain syndrome. In rats, NOP receptors are present at several sites for the integration of the micturition reflex, and their activation has both excitatory or inhibitory effects, depending on the route of administration and the experimental conditions.32 In humans, it has been shown that bladder instillation of N/OFQ in patients with neurogenic/overactive bladder disorder alleviates the symptoms, increases bladder capacity, and elicits a robust acute inhibitory effect on the micturition reflex27–29; effectiveness in bladder pain syndrome has been reported as well,30 supporting the use of NOP agonists as a novel drug for the treatment of painful bladder disorders. Preclinical models have provided further evidence that NOP receptor activation may be antinociceptive in visceral pain.31,32 Small-molecule NOP receptor agonists are effective in a mouse model of TNBS-induced colitis,51 of mustard oil–induced IBS with diarrhea,19 and rat colonic hyperalgesia.1 BU08070, a mixed NOP/MOP receptor agonist, significantly reduced the severity of colitis in TNBS-treated mice.63 Thus the N/OFQ system is an attractive target for novel drugs, which may be effective in the treatment of inflammatory or functional visceral disorders.2,52,63
Previous studies have shown the sensitizing effects of neurotrophic factors on neuronal sensitivity in rodents24 and humans,5–8 including TRPV1. As the tissue studies above showed colocalization of NOP and TRPV1 in DRG neurons, we investigated the effects of NOP receptor ligand activation on TRPV1 receptor-mediated Ca2+ signaling in primary human and rat DRG neurons cultured in the presence of neurotrophic factors, an in vitro model of neuronal sensitization.7 The endogenous ligand N/OFQ was used to activate NOP receptors, which in both rat and human DRG neurons potently inhibited capsaicin-induced Ca2+ signaling with an IC50 of around 0.01 nM, which is in the order of magnitude observed for N/OFQ binding to rat brain membranes, Kd ∼0.02 nM.4 The MOP receptor peptide agonist DAMGO inhibited TRPV1 receptor-mediated Ca2+ signaling with a similar potency; however, N/OFQ proved to be much more efficacious than DAMGO or morphine5 for maximum inhibition. As the NOP receptor couples to the same Gi/Go protein-mediated second-messenger systems as the opioid receptors to inhibit adenylate cyclase, this mechanism is likely to affect TRPV1 signalling by dephosphorylation, leading to its desensitization.12,18 Similarly, the MOP agonist morphine has been demonstrated to act through inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses.55 Our study used a previously described model of neurotrophic factor–mediated neuronal hypersensitivity to capsaicin for analysing the effects of N/OFQ. Our results indicate that N/OFQ inhibits TRPV1 signaling with a high potency, compared with the nanomolar values generally obtained in biochemical studies performed in cells, and may be due to the sensitizing effects of the neurotrophic factors. However, this could be the result of efficient signal amplification in native cells used by us compared with recombinant systems. Future studies with specific NOP antagonists will be useful to confirm specificity of the action.
The diverse potential of NOP receptor agonists for acute and chronic pain treatment in rodent and nonhuman primates has been recently reviewed.16 AT-200, a high-affinity N/OFQ receptor agonist, with low efficacy at MOP, ameliorated chronic hypoxia-induced mechanical, thermal, and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice.54 Although previous preclinical models have shown opposing effects of NOP activation in the CNS,40,62 reviewed by Schroder et al.,49 recent studies using spinally administered bifunctional NOP/MOP ligands have shown attenuation of neuropathic and inflammatory pain, suggesting a promising profile as spinal analgesics.53,56 Spinally administered PWT2-N/OFQ, a tetrabranched derivative of N/OFQ, inhibited nociceptive and neuropathic pain in mice and nonhuman primate models; the PWT derivative was more potent than the natural peptide, and elicited long-lasting effects in nonhuman primates.47 Intracisternal N/OFQ and morphine also produced antinociceptive effects.17 Peripherally mediated effects show promise, including a study describing efficacy of NOP receptor agonist SCH 486757 in suppressing capsaicin-induced cough in a guinea pig model,34 currently being evaluated in a clinical trial.35
In summary, we have demonstrated the expression of the NOP receptor in human peripheral nerve and visceral tissues, with a marked increase of NOP-positive nerve fibres in urinary bladder syndromes. N/OFQ and DAMGO produced dose-dependent inhibition of capsaicin responses in human and rat DRG neurons, in an in vitro model of neuronal sensitization. NOP receptor activation is thus a promising strategy for therapeutic intervention in clinical pain conditions, especially visceral hypersensitivity and overactivity disorders.
Conflict of interest statement
P. Anand has been a consultant to Grünenthal GmbH, Germany. P. Hein is an employee of Grünenthal GmbH, Germany. The other authors have no conflicts of interest to declare.
The authors are grateful to Grünenthal GmbH, Germany, for financial support.
Authors' contributions: P. Anand and P. Hein conceived the original study, its design and coordination, and helped write the manuscript. Y. Yiangou participated in immunohistology studies and helped draft the manuscript. U. Anand performed the in vitro experiments, to which Y. E. Korchev contributed, and helped draft the manuscript. G. Mukerji, M. Sinisi, M. Fox, A. McQuillan, and T. Quick collected the clinical tissues, helped with the human tissue studies, and edited the manuscript. All authors read and approved the final manuscript.
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