- Email: [email protected]
3 receptor of primary sensory neurons
Brain Research Bulletin 83 (2010) 284–291
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
VEGF and its receptor-2 involved in neuropathic pain transmission mediated by P2X2/3 receptor of primary sensory neurons Jiari Lin a,1 , Guilin Li a,1 , Xiaorong Den b,1 , Changshui Xu a,1 , Shuangmei Liu a , Yun Gao a , Han Liu a , Jun Zhang a , Xin Li a , Shangdong Liang a,∗ a b
Department of Physiology, Medical College of Nanchang University, Bayi Road 4613, Nanchang, Jiangxi 330006, PR China First Affiliated Hospital, Medical College of Nanchang University, Nanchang, Jiangxi 330006, PR China
a r t i c l e
i n f o
Article history: Received 6 June 2010 Received in revised form 24 July 2010 Accepted 3 August 2010 Available online 10 August 2010 Keywords: VEGF VEGF receptor-2 P2X2/3 receptor Neuropathic pain Primary sensory neuron
a b s t r a c t The pathogenesis of neuropathic pain is complex. P2X2/3 receptor plays a crucial role in nociception transduction of chronic pain. VEGF inhibitors are effective for pain relief. The present study investigated the effects of VEGF and VEGF receptor-2 (VEGFR2) on the pain transmission in neuropathic pain states that mediated by P2X2/3 receptor in primary sensory neurons. Chronic constriction injury (CCI) model was used as neuropathic pain model. Sprague–Dawley rats had been randomly divided into sham group, CCI group and CCI rats treated with anti-rVEGF antibody group. Mechanical withdrawal threshold and thermal withdrawal latency were measured. Expressions of VEGF, VEGFR2 and P2X2/3 in L4–6 dorsal root ganglia (DRG) were detected by immunohistochemistry, RT-PCR and western blot analysis. The mechanical withdrawal threshold and thermal withdrawal latency in CCI group were lower than those in sham group and CCI rats treated with anti-rVEGF antibody group (p < 0.05), while VEGF, VEGFR2 and P2X2/3 receptors’ expressions of L4–6 DRG in CCI group were higher than those in the other two groups (p < 0.05). The expressions of VEGF, VEGFR2 and P2X2/3 in L4–6 DRG of CCI rats treated with anti-rVEGF antibody group were decreased compared with those in CCI group (p < 0.05). The results show that VEGF and VEGFR2 are involved in the pathogenesis of neuropathic pain and VEGF primarily potentiates pain responses mediated by P2X2/3 receptor on DRG neurons. Anti-rVEGF treatment in CCI rats may alleviate chronic neuropathic pain by decreasing the expressions of VEGFR2 and P2X2/3 receptors on DRG neurons to inhibit the transmission of neuropathic pain signaling. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Vascular endothelial growth factor (VEGF) is a selective endothelial cell mitogen that promotes angiogenesis and also increases blood vessel permeability [14,15,38]. VEGF exerts action via high-affinity binding to three types of phosphotyrosine kinase receptors: VEGFR-1 (also known as fms-like tyrosine kinase 1 or flt1), VEGFR-2 (termed kinase insert-domain containing receptor, KDR in humans or fetal liver kinase 1, flk1 in mice), and VEGFR-3 (flt4) [35]. In the nervous system, VEGF mRNA has been found in neurons of brain’s capillary-rich areas [35]. VEGF plays a crucial role in the nervous system. In the peripheral nervous system, some vessels and nerves migrate along the same path alongside each other. VEGF may thus have therapeutic potential in Alzheimer disease, Parkinson’s disease and brain ischemia [1,23,44].
∗ Corresponding author. Tel.: +86 0791 6360552; fax: +86 0791 6360552. E-mail address: [email protected] (S. Liang). 1 Joint first authors. 0361-9230/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2010.08.002
The prokineticin receptors, prokineticin receptor 1 (PKR1) and PKR2 [25], and their activation activated by peptides belonging to Bv8/EG-VEGF (endocrine gland-derived vascular endothelial growth factor) – PK (prokineticin) family suggests an additional novel mechanism of peripheral nociceptor activation and sensitization [33]. PKR activation potentiated mustard oil induced nociceptive responses to heat. PKR1-null mice exhibited impaired development of hyperalgesia after inflammatory injury. The prokineticins released within inflamed tissues augment nociceptor responsiveness by acting on PKR1. Moreover, PKR2, which is expressed by DRG neurons different from those expressing PKR1 and is expressed normally by PKR1-null mice, could participate in specific pain conditions. A possible role of PKR2 receptor in nociceptor sensitization is to punctate stimuli. VEGF inhibitors improved hypertrophic pulmonary osteoarthropathy (HPOA)-related symptoms. VEGF mRNA in calf muscle is increased after exercise-induced pain in patients with intermittent claudication [33]. VEGF inhibitors might be effective for pain relief. Neuropathic pain is defined as “pain caused by primary dysfunction of the nervous system” [22,28]. The pathogenesis of
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
neuropathic pain is complex. The dorsal root ganglion (DRG) contains primary somatosensory neurons receiving nociceptive, mechanical and proprioceptive inputs. ATP has been shown to elicit peripheral nociceptor sensitization through the activation of receptors at nociceptor terminals [6,7,9,10,13]. ATP is implicated in peripheral pain signaling transmission by acting on P2X receptors [5,9,39]. Sensory afferent neurons contain mRNA and protein for multiple P2X receptors (P2X1–6 ). P2X and P2Y receptor subtypes essentially mediate all ATP-mediated responses in DRG neurons [6,8,10,13,20,24]. P2X1–6 as neuronal receptors includes P2X4 , which is non-neuronal (like P2X7 ). P2X3 receptors have received particular attention because of their high level expression in, and selective localization on sensory afferents [6,9,10,20,30]. P2X2 and P2X3 subunits can form homomultimeric P2X2 , homomultimeric P2X3 , or heteromultimeric P2X2/3 receptors. P2X2/3 heteromer receptors also express in sensory neurons [6,8,10]. P2X3 and P2X2/3 receptors in DRG neurons contribute to neuropathic pain [6,9,10,16,26,45–47]. The purpose of this study is to investigate the effects of VEGF and VEGFR2 on neuropathic pain and the relationship between VEGFR2 and P2X2/3 heteromer receptors on DRG sensory neurons in neuropathic pain state. 2. Materials and methods 2.1. Experimental animals and materials Male Sprague–Dawley rats (180–250 g) were provided by the Center of Laboratory Animal Science of Nanchang University. The animals were housed in plastic boxes in a group of three at 21–25 ◦ C. Use of the animals was reviewed and approved by the Animal Care and Use Committee of Medical College of Nanchang University. The IASP’s ethical guidelines for pain research in animals were followed. All animals were treated in accordance with ARVO Statement for the use of Animals in Ophthalmic and Vision Research in China. The chronic constriction injury (CCI) rats were made as the neuropathic pain model. Rats were randomly divided into three groups: sham group, CCI group and CCI rats treated with anti-rVEGF antibody group (anti-rVEGF group) (n = 6 for each group). Anti-rVEGF antibody (1 g/kg) in anti-rVEGF group and phosphate-buffer saline (PBS) in sham group and CCI group were intrathecally injected to rats by puncture every two days intervals (total 6 times), respectively. All drugs were diluted in PBS and 15 l of drugs was injected every time. The injection was initiated 2 h after operation to ensure all rats were awake completely. Then about 30 min later, behavior of all rats was observed. Anti-rat VEGF antibody was the product of R&D Systems, Inc (AF564). Rabbit anti-P2X3 and rabbit anti-P2X2 polyclonal antibody were bought from Chemicon International Company of America. VEGFR2 antibody was bought from Thermo Fisher Scientific (Fremont, CA, USA). Other antibodies and reagents were ascribed as following. 2.2. Chronic constriction injury (CCI) model CCI rat model was prepared [3,46,47]. Each rat was anesthetized with Nembutal [35 mg/kg intraperitoneally (i.p.)] during surgical procedures. The sciatic nerve was exposed at the middle level of rat thigh. Proximal to the sciatic trifurcation, four ligatures (4–0 chromic gut) were performed loosely with microsurgical techniques. Intervals between every two ligatures were about 1 mm. The same investigator created CCI animals to avoid variation. In the sham-operated group, the sciatic nerve was exposed using the same procedure but not ligatured by chromic gut. We assessed nociception on day 0, 1, 4, 7, 10, 13 after CCI by the observation of spontaneous pain behavior, by measurement of changes of latency in paw withdrawal on thermal stimulation, and of paw withdrawal threshold using von Frey filaments to assess mechanical hyperalgesia. 2.3. Measurement of mechanical withdrawal threshold (MWT) Noxious-pressure stimulation was used to evaluate mechanical hyperalgesia. Unrestrained rats were placed inside a clear plastic chamber (22 cm × 12 cm × 22 cm) on a stainless steel mesh floor and allowed to acclimate. Withdrawal responses to mechanical stimulation were determined using calibrated von Frey filaments (BME-403, Tianjin) applied through an opening in the stainless steel mesh floor of the cage (grid 1 cm × 1 cm) to an area adjacent to the paw. Each von Frey filament was applied once starting with 0.13 g and continuing until a withdrawal response occurred or the force reached 20.1 g (the cut-off value). The right hind paws were tested alternately at 2 min intervals. Measurements of three times were taken by using the method up and down on each side and the mean of the three determinations was taken as the threshold values. The filaments were applied in the order of increasing bending force (0.13, 0.20, 0.33, 0.60, 1.30,
285
3.60, 5.00, 7.30, 9.90, 20.1 g), with each applied 10 times at intervals of 15 s to different parts of the midplantar glabrous skin. The strength of the filaments in the series that evoked at least five positive responses among the 10 trials was designated the pain threshold.
2.4. Measurement of thermal withdrawal latency (TWL) Noxious heat stimulation for assessment of thermal hyperalgesia was applied by the Thermal Paw Stimulation System (BME-410C, Tianjin). Rats were placed in a transparent, square, bottomless acrylic box (22 cm × 12 cm × 22 cm), on a glass plate under which a light was located. Radiant heat stimuli were applied by directing a beam of light at the foot pad of each hind paw through the glass plate. The light beam was turned off automatically when the rat lifted the paw, allowing the measurement of time between the beginning of the light beam and the elevation of the foot. This time was designated as the paw withdrawal latency. The hind paws were tested alternately at 5 min intervals. The cut-off time for the heat stimulation was 30 s.
2.5. Immunohistochemistry Immunohistochemical staining was performed using SP-9001 kit (Beijing Zhongshan Biotech Co.) according to the manufacturer’s instruction. Two weeks later, Sprague–Dawley rats in three groups were anesthetized with Nembutal (35 mg/kg i.p.). The lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The L4–6 (lumbar 4–6) DRGs together with dorsal and ventral roots and attached spinal nerves were taken out from the inner side of each half of the dissected vertebrae with fine dissecting forceps. DRG isolated from rats were washed by PBS. After fixed with 4% paraformaldehyde (PFA) for 24 h, the ganglia were dehydrated by 20% sucrose for overnight at 4 ◦ C, and then ganglia were cut into 15 m thick on a cryostat. After washed by PBS for three times, the preparations were incubated in 3% H2 O2 for 10 min to block the endogenous peroxidase activity, then with 10% goat serum for 30 min at room temperature to block non-specific antigen. After rinsed and washed in PBS, the block sections were incubated with primary antibody (P2X2 and P2X3 1:2500 diluted in PBS; others 1:100 diluted in PBS) for overnight at 4 ◦ C. After three rinses in PBS, the sections were incubated with biotinylated goat anti-rabbit secondary antibody (Beijing Zhongshan Biotech Co.) for 1 h at room temperature. The preparations were washed in PBS and then added streptavidin–horseradish peroxidase (Beijing Zhongshan Biotech Co.) for 30 min. After development of the diaminobenzidine chromogen for 2 min, the slides were washed with distilled water and cover slipped. After immunohistochemistry, the Image-Pro Plus 6.0 was used to analyze the changes in stain values (average optical density) of P2X2 , P2X3 , VEGF and VEGFR2 in ganglia. Background was determined by averaging the optical density of 10 random areas.
2.6. RNA preparation and reverse transcriptase (RT)-PCR The rats in three groups were anesthetized by Nembutal (35 mg/kg i.p.). The L4–6 DRG were isolated immediately and flushed with icecold PBS. Total RNA samples were prepared from L4–6 DRG in each group by using of TRNzol Total RNA Reagent (Beijing Tiangen Biotech Co.). Isolated RNA was resuspended in DEPC water and reverse transcribed to cDNA using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions. PCR was performed on cDNA using primers listed below and the following: 4 l of cDNA, 6.5 l DEPC water, 2 l 10 mol primers (equal sense and antisense), and 12.5 l 2× Easy Taq PCR SuperMix (Beijing TransGen Biotech Co.) in 25 l. PCR conditions included hot start at 94 ◦ C for 3 min, 30 s denaturation (94 ◦ C), 30 s annealing (annealing temperatures (AT) listed below), and 30 s extension (72 ◦ C) for 30 amplification rounds and 5 min final extension at 72 ◦ C. PCR products (8 l) were run on 1% agarose gels with EB using standard protocols. Gels were scanned with gel imaging system (Beijing Junyi Co., Ltd) and the intensity of PCR product bands was analyzed using Gel-Pro Analyzer 32. The ratio of VEGF or these receptors band intensity/-actin band intensity was used as the results of half-quantification analysis. Primer P2X2 [6] AT: 57 ◦ C; PS: 273 bp Sense: 5 -CTGCCTCCTCAGGCTACAACTTCA-3 Antisense: 5 -GAGTACGCACCTTGTCGAACTTCT-3 Primer P2X3 [31] AT: 57 ◦ C; PS: 519 bp Sense: 5 -CAACTTCAGGTTTGCCAAA-3 Antisense: 5 -TGAACAGTGAGGGCCTAGAT-3 Primer VEGF [34] AT: 65 ◦ C; PS: 145 bp Sense: 5 -GCTCTCTTGGGTGCACTGGAC-3 Antisense: 5 -ACGGCAATAGCTGCGCTGGTA-3 Primer VEGFR2 [34] AT: 60 ◦ C; PS: 537 bp Sense: 5 -GCCAATGAAGGGGAACTGAAGA-3 Antisense: 5 -CTCTGACTGCTGGTGATGCTGTC-3 Primer -actin [18] AT: 60 ◦ C; PS: 240 bp Sense: 5 -TAAAGACCTCTATGCCAACACAGT-3 Antisense: 5 -CACGATGGAGGGGCCGGACTCATC-3
286
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
Fig. 1. Effects of anti-rVEGF antibody on MWT (mechanical withdrawal threshold) of CCI rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as “**” when p < 0.01 compared with sham group; the significant difference was denoted as “#” when p < 0.05 and “##” when p < 0.01 compared with CCI group.
2.7. Western blot analysis The rats in three groups were anesthetized by Nembutal (35 mg/kg i.p.). The L4–6 DRG were isolated immediately and flushed with icecold PBS. Ganglia were homogenized by mechanical disruption in lysis buffer (50 mmol/L TrisCl, pH 8.0, 150 mmol/L NaCl, 0.1% dodecyl sodium sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 g/mL phenylmethylsulfonyl fluoride, 1 g/mL Aprotinin) and incubated on ice for 40 min. Homogenate was then pelleted at 12,000 rpm for 10 min and supernatant was collected. Using Lowry method, the quantity of total protein was determined in the supernatant. After diluted with sample buffer (100 mmol/L TrisCl, 200 mmol/L dithiothreitol, 4% sodium dodecylsulfate (SDS), 0.2% bromophenol blue, 20% glycerol) and heated to 95 ◦ C for 5 min, samples containing equal amounts of protein (20 l) were separated by SDS-polyacrylamide gel electrophoresis by using Bio-Rad system and 10% gel. In the wake of electrophoretic transfer onto nitrocellulose (NC) membrane using the same system, the membrane was blocked with 5% non-fat dry milk in 25 mmol/L Tris buffered saline, pH 7.2, plus 0.1% Tween 20 (TBST) for 1–3 h at room temperature, and incubated with primary antibodies in blocking buffer for 2 h at room temperature or overnight at 4 ◦ C. The membranes were washed three times with TBST and incubated (1 h, room temperature) with horseradish peroxidase-conjugated secondary antibody [goat anti-rabbit IgG (1:4000), Beijing Zhongshan Biotech Co.] in blocking buffer. After another wash cycle, labeled proteins were visualized by using the enhanced chemiluminescence (ECL) kit (Fremont, CA, USA). Chemiluminescent signals were collected on autoradiography film, and the quantity of band intensity was carried out using Gel-Pro Analyzer 32. The primary antibodies and dilutions used were the following: rabbit polyclonal anti-P2X3 and anti-P2X2 (1:1000), rabbit polyclonal anti-VEGFR2 (1:100), rabbit polyclonal antiVEGF (1:100; Wuhan Boster Co., China) and monoclonal -actin (1:1000; Beijing Zhongshan Biotech Co.). Band densities were normalized to each -actin internal control.
2.8. Statistical analysis Statistical analysis of the data was performed on computer (SPSS 11.5). All results were expressed as mean ± SE. Statistical significance was determined by one factor analysis of variance (ANOVA) followed by the Fisher post hoc test for multiple comparisons. p-Value <0.05 was considered significant.
3. Results 3.1. Effects of anti-rVEGF antibody on mechanical hyperalgesia of CCI rats At day 7 after operation, the mechanical withdrawal threshold (MWT) in CCI group was lower than that in sham group (p < 0.01), and constantly exists from day 7 to day 14. MWT in anti-rVEGF group was lower than that in sham group at days 1–4, but there is no statistical difference (p > 0.05). MWT in anti-rVEGF group from day 7 to day 14 was no statistical difference compared with that in sham group from day 7 to day 14 (p > 0.05) (Fig. 1).
Fig. 2. Effects of anti-rVEGF antibody on (TWL) thermal withdrawal latency of CCI rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as “*” when p < 0.05 and “**” when p < 0.01 compared with sham group; the significant difference was denoted as “##” when p < 0.01 compared with CCI group.
3.2. Effects of anti-rVEGF antibody on thermal hyperalgesia of CCI rats At day 1 after operation, the thermal withdrawal latency (TWL) in CCI group was lower than that in sham group (p < 0.05), and there is extremely significant difference from day 7 to day 14 (p < 0.01). TWL in anti-rVEGF group was lower than that in sham group from day 1 to day 4 (p < 0.05), but there is no statistical difference compared with that in sham group from day 7 to day 14 (p > 0.05). TWL in anti-rVEGF group has statistical significance compared with that in CCI group from day 7 to day 14 (Fig. 2). 3.3. Effects of anti-rVEGF antibody on the expression of P2X2 , P2X3 , VEGF and VEGFR2 immunoreactivities in L4–6 DRG of CCI rats P2X2 , P2X3 , VEGF and VEGFR2 immunoreactivities in L4–6 DRG were detected by immunohistochemistry. The expressions of P2X2 , P2X3 , VEGF and VEGFR2 in the ganglia of each group were examined. The staining of P2X2 , P2X3 , VEGF and VEGFR2 in L4–6 DRG of CCI group was all higher than that in sham group and anti-rVEGF group, respectively (p < 0.05), and there are no significant difference in the staining values of P2X2 , P2X3 , VEGF and VEGFR2 mRNA between sham group and anti-rVEGF group (p > 0.05) (Fig. 3A–E). 3.4. Effects of anti-rVEGF antibody on the expression of P2X2 , P2X3 , VEGF and VEGFR2 mRNA in L4–6 DRG of CCI rats by RT-PCR RT-PCR studies showed that the expressions of P2X2 , P2X3 , VEGF and VEGFR2 mRNA in DRG were abundantly expressed in CCI group in comparison with those in sham group and anti-rVEGF group, respectively (Fig. 4A–D). The relative levels (normalized to each -actin internal control) of P2X2 , P2X3 , VEGF and VEGFR2 mRNA of CCI group were significantly increased compared with those in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), respectively (Fig. 4E). 3.5. Effects of anti-rVEGF antibody on the expression of P2X2 , P2X3 , VEGF and VEGFR2 protein in L4–6 DRG of CCI rats by western blotting The expressions of P2X2 , P2X3 , VEGF and VEGFR2 in protein level were analyzed by western blotting (Fig. 5A–D). By image analysis, the stain values (average optical density) of P2X2 , P2X3 , VEGF and
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
287
Fig. 3. Expression of P2X2 , P2X3 , VEGF and VEGFR2 immunoreactivities in L4–L6 DRG neurons in each group. Photos of protein expression in L4–L6 DRG neurons measured by immunohistochemistry (n = 6 each group). (A) The expression of P2X2 protein in L4–L6 DRG neurons. (B) The expression of P2X3 protein in L4–L6 DRG neurons. (C) The expression of VEGF in L4–L6 DRG neurons. (D) The expression of VEGFR2 in L4–L6 DRG neurons. (E) The bar histograms showed the optical density (mean) of each group on the expression of P2X2 , P2X3 , VEGF and VEGFR2. (Note: Arrows indicate the immunostaining neurons. Data are shown as the means ± S.E.; *p < 0.05, as compared with sham group; # p < 0.05, as compared with CCI group.)
VEGFR2 protein expressions (normalized to each -actin internal control) in DRG of CCI group were significantly enhanced compared with those in sham group (p < 0.05), but the relative levels of P2X2 , P2X3 , VEGF and VEGFR2 protein expressions in anti-rVEGF group were lower than those in CCI group (p < 0.05) (Fig. 5E). 4. Discussion VEGF plays a crucial role in the different functions of nervous system and therapeutic potential of nervous system diseases [35]. Our data showed that the MWT and TWL were increased after CCI rats treated with anti-VEGF antibody. Anti-VEGF antibody treatment might induce analgesia. The prokineticin receptors, prokineticin receptor 1 (PKR1) and PKR2 [25,35,36], and their activation which exerting by peptides belonging to the Bv8/EG-VEGF (endocrine gland-derived vascular endothelial growth factor) – PK (prokineticin) family suggests an additional novel mechanism of peripheral nociceptor activation and sensitization [25,28]. The lit-
erature indicates that cyclooxygenase-2 (COX-2) plays a key role in the production of VEGF [29]. Co-expression of COX-2 and VEGF in all consecutive sections of inflamed pulps indicates a release of VEGF via a COX-2-dependent pathway [29]. Hypertrophic pulmonary osteoarthropathy (HPOA) is a syndrome defined by digital clubbing and distal periostitis in tubular bones [17]. Symptoms may include bone pain, occasionally severe. VEGF inhibition improved HPOA-related symptoms [17]. Systemic administration of an antiVEGF antibody effectively inhibited pathological angiogenesis [36]. The treatment with anti-VEGF antibody markedly reduced characteristic psoriasis-like symptoms observed in the skin lesions of the mice, such as epidermal hyperplasia with altered differentiation, inflammatory cell infiltration, vascular abnormalities, and elevated levels of IL-23/Th17-related cytokines. It is possible that VEGF is involved in nociception or pain. VEGF receptors have been identified as VEGFR-1 (Flt1), VEGFR2 (KDR, Flk1), and VEGFR-3 (Flt4). VEGF binds to VEGFR-1 and VEGFR-2 [14,35]. The expression levels of VEGF and VEGFR2
288
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
Fig. 4. The changes of P2X2 , P2X3 , VEGF and VEGFR2 mRNA level in L4–L6 DRG in each group. (A) The expression of P2X2 mRNA in DRG of CCI group was increased compared with those in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (B) The expression of P2X3 mRNA in DRG of CCI group was increased in comparison with that in sham group (p < 0.05) and that in anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in the anti-rVEGF group was just slightly higher than that in sham group. (C) The expression of VEGF mRNA in DRG of CCI group was increased compared with that in sham group (p < 0.05), anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (D) The expression of VEGFR2 mRNA in DRG of CCI group was significantly higher than that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in sham group just detect a little. (E) Means ± S.E. of RT-PCR product levels quantified by phosphoimager analysis are shown. Data are expressed as the band intensity for each growth factor or receptor listed relative to the -actin level determined in the same PCR reaction. *p < 0.05, as compared with sham group; # p < 0.05, as compared with CCI group.
immunoreactivities, mRNA and protein in DRG of CCI rats were higher than those in sham group and anti-rVEGF group. The expression of VEGF by immunohistochemistry is strongly positive in cells constituting the inflammatory infiltrate of teeth with irreversible pulpitis [2]. Up-regulation of VEGF expression in the dental pulp may result in increased intra-pulpal pressure, and contribute to pain and irreversible tissue damage [27]. The anti-VEGF antibody significantly attenuated the clinical and morphologic features of iodoacetamide-induced colitis [41]. HPOA-related symptoms can be blunted by different VEGF inhibitors [17]. The subcutaneous administration of carrageenan induces a hyperalgesic response that is reduced by nonsteroidal anti-inflammatory drugs. VEGF may be involved in thermal and mechanical hyperalgesia. Up-regulation of VEGF and VEGFR2 expression in DRG of CCI rats reveals that VEGF
and VEGFR-2 participate in the pathogenesis of neuropathic pain. The expressions of VEGF and VEGFR2 in DRG of anti-rVEGF group were reduced compared with those in DRG of CCI rats. VEGF may bind to VEGFR-2 in DRG of CCI rats. The neutralization of endogenous VEGF decreased the MWT and TWL in CCI rats. The results may reveal certain mechanisms responsible for the analgesia induced by anti-VEGF antibody. Reduction of VEGF and VEGFR2 expression in DRG of anti-rVEGF rats may be a novel strategy that can diminish the activation and sensitization of primary afferent nociceptors to produce therapeutic benefit in acute, inflammatory and neuropathic pain conditions. Activation of P2X3,2/3 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia [6,9,10,16,26,45–47]. The essential role of P2X3,2/3 receptors in
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
289
Fig. 5. The changes of P2X2 , P2X3 , VEGF and VEGFR2 protein level in L4–L6 DRG in each group. (A) The expression of P2X2 protein in DRG of CCI group was increased compared with that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and there is no significant difference between sham group and anti-rVEGF group (p < 0.05). (B) The expression of P2X2 protein in DRG of CCI group was increased compared with that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (C) The expression of VEGF protein in DRG of CCI group was significantly higher than that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and there is no significant difference between sham group and anti-rVEGF group (p > 0.05). (D) The expression of VEGFR2 protein in DRG of CCI group was increased compared with that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (E) The bar histograms showed the ratio of P2X2 , P2X3 , VEGF and VEGFR2 protein level to -actin with each group. (Note: Data are shown as the mean ± S.E. *p < 0.05, as compared with sham group; # p < 0.05, as compared with CCI group.)
the development of carrageenan-induced mechanical hyperalgesia is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of TNF-␣ and by a direct sensitization of the primary afferent nociceptors [32,42,43]. Prokineticin receptors (prokineticin receptor 1 (PKR1) and PKR2) and their activation by peptides belonging to the Bv8/EG-VEGF PK (prokineticin) family suggest a novel mechanism of peripheral nociceptor activation and sensitization [28]. PKR1 expression is important in determining sensory thresholds to nociceptive and to acute and chronic inflammatory stimuli. PKR1 is a Gprotein-coupled metabotropic receptor [25] sensitizes the vanilloid receptor TRPV1 to noxious stimuli. The activation of peripheral P2X2/3 receptors may be crucial to VEGF-mediated sensitization of the primary afferent nociceptor. It is broadly accepted that TRPV1positive neurons are nociceptive neurons. An important feature of TRPV1 regulation also concerns the cellular signaling pathways through which G-protein-coupled receptors sensitize TRPV1 to its chemical and physical stimuli. Bv8 and prokineticins may sensitize TRPV1 to vanilloid agonists, heat, and protons. The co-expression of TRPV1 and high levels of P2X3 (and P2X2 ) receptors in sensory neurons supports the involvement of the P2X3 and P2X2/3 receptors in pain [8,9,24]. PKRs transduce the sensitizing signal through kinase dependent signaling pathways or through the release of nocicep-
tive mediators, which in turn activate kinase signaling throughout their receptors. VEGF may sensitize P2X2/3 receptor to P2X receptor agonists in the primary afferent nociceptor. The role of P2X2/3 receptors in the development of thermal and mechanical hyperalgesia may be mediated, at least in part, by VEGFR2 activation in the primary afferent nociceptor. The modulatory role of anti-VEGF antibody treatment in VEGFR2 and P2X2/3 activation is also suggested by thermal and mechanical hypoalgesia. All of these findings agree on a positive interaction between VEGFR2 and P2X2/3 throughout the peripheral nervous system. Therefore, blockade of VEGFR2 and P2X2/3 receptors prevented the development of thermal and mechanical hyperalgesia. Nociceptive neurons can be divided into two major populations, non-myelinated C-fiber and myelinated A-fiber neurons [37]. The P2X3 receptor subunit is expressed on primary afferent neurons and preferentially on nociceptive C-fibers. The expression levels of P2X2 and P2X3 were reduced after CCI rats treated with anti-VEGF antibody. VEGF primarily potentiates responses mediated by the pain receptor P2X2/3 on dorsal root ganglion neurons, an effect blocked by anti-VEGF treatment. Anti-VEGF antibody treatment might neutralizate VEGF to decrease P2X2 and P2X3 receptors’ activities on nociceptive DRG neurons. Extracellular ATP was reported to produce pain sensation in humans
290
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
and to participate in pain pathways in primary sensory neurons [7,9,10,24]. There are native P2X receptors in sensory neuron of DRG. P2X3 and P2X2/3 receptors are expressed on isolectin B4 (IB4) binding subpopulations of small nociceptive neurons [7,40]. Peripheral nerve injury has been shown to regulate the expression of peripheral P2X3 receptors. An increase of the number of P2X3 receptor-immunoreactive on DRG neurons was demonstrated after a chronic constriction injury of the sciatic nerve [3,9,16,26,45–47]. P2X receptors contribute to neuropathic pain. P2X3 and P2X2/3 receptors play a crucial role in nociception transduction of acute and chronic pain [4,11]. P2X3 double-stranded short interfering RNA (siRNA) relieves chronic neuropathic pain [12,19]. A-317491, a potent and selective antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat [21]. P2X2/3 receptors may be targets for anti-rVEGF treatment to relieve the hyperalgesia of neuropathic pain rats. The finding suggests that selective antagonists for the VEGFR2 may decrease peripheral nociceptor activation and sensitization. 5. Conclusions In summary, VEGF and VEGFR2 are involved in the pathogenesis of neuropathic pain. VEGF primarily potentiates pain responses mediated by P2X2/3 receptors on dorsal root ganglion neurons. CCI rats treated with anti-rVEGF antibody can inhibit the activation of P2X2/3 receptor during chronic pain and block primary afferent transmission mediated by VEGFR2 and P2X2/3 receptors to reduce hyperalgesia in chronic pain states. Anti-rVEGF treatment in CCI rats may alleviate chronic neuropathic pain by decreasing the expressions of VEGFR2 and P2X2/3 receptors in DRG neurons. Conflict of interest None. Acknowledgments This work was supported by the grant (Nos. 30860086, 30860333 and 30660048) from National Natural Science Foundation of China, the grant (No. 20070403007) from Doctoral Fund of Ministry of Education of China, the grant (Nos. 0640042, 2007GZY1002 and 2008GZY0029) from Natural Science Foundation of Jiangxi Province and the grant (No. GJJ08049) from the Educational Department of Jiangxi Province. References [1] L. Anderson, M.A. Caldwell, Human neural progenitor cell transplants into the subthalamic nucleus lead to functional recovery in a rat model of Parkinson’ disease, Neurobiol. Dis. 27 (2007) 133–140. [2] L. Artese, C. Rubini, G. Ferrero, M. Fiorini, A. Santinelli, A. Piattelli, Vascular endothelial growth factor (VEGF) expression in healthy and inflamed human dental pulps, J. Endod. 28 (2002) 20–23. [3] G.J. Bennett, Y.K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 (1988) 87–107. [4] E.J. Bradbury, G. Burnstock, S.B. McMahon, The expression of P2X3 purinoceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor, J. Mol. Cell. Neurosci. 12 (1998) 256–268. [5] G. Burnstock, P2X receptors in sensory neurons, Br. J. Anaesth. 84 (2000) 476–488. [6] G. Burnstock, Purinergic P2 receptors as targets for novel analgesics, Pharmacol. Ther. 110 (2006) 433–454. [7] G. Burnstock, Physiology and pathophysiology of purinergic neurotransmission, Physiol. Rev. 87 (2007) 659–797. [8] C.C. Chen, A.N. Akopian, L. Sivilotti, D. Colquhoun, G. Burnstock, J.N. Wood, A P2X purinoceptor expressed by a subset of sensory neurons, Nature 377 (1995) 428–431. [9] B.A. Chizh, P. Illes, P2X R receptors and nociception, Pharmacol. Rev. 53 (2000) 553–568. [10] M.S. Chong, B. Brandner, Neuropathic agents and pain. New strategies, Biomed. Pharmacother. 60 (2006) 318–322.
[11] D.A. Cockayne, P.M. Dunn, Y. Zhong, S.G. Hamilton, G.R. Cain, G.E. Knight, H.Z. Ruan, Y. Ping, P. Nunn, M. Bei, S.B. McMahon, G. Burnstock, A.P.D.W. Ford, P2X2 knockout mice and P2X2 /P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP, J. Physiol. 567 (2005) 621–639. [12] G. Dorn, S. Patel, G. Wotherspoon, M. Hemmings-Mieszczak, J. Barclay, F.J. Natt, P. Martin, S. Bevan, A. Fox, P. Ganju, W. Wishart, J. Hall, siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32 (2004) e49. [13] G. Dussora, H.R. Koerbere, A.L. Oaklanderb, F.L. Ricec, D.C. Molliverd, Nucleotide signaling and cutaneous mechanisms of pain transduction, Brain Res. Rev. 60 (2009) 24–35. [14] N. Ferrara, Vascular endothelial growth factor: basic science and clinical progress, Endocr. Rev. 25 (2004) 581–611. [15] N. Ferrara, T. Davis-Smyth, The biology of vascular endothelial growth factor, Endocr. Rev. 18 (1997) 4–25. [16] Y. Gao, C.S. Xu, S.D. Liang, S.N. Mu, A.X. Zhang, Y.X. Wang, F. Wan, Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X3 receptor in neuropathic pain states, Brain Res. Bull. 77 (2008) 27–32. [17] G. Güven, C. Altun, O. Günhan, T. Gurbuz, F. Basak, E. Akbulut, Z.C. Cehreli, Co-expression of cyclooxygenase-2 and vascular endothelial growth factor in inflamed human pulp: an immunohistochemical study, J. Endod. 33 (2007) 18–20. [18] S.C. Heffelfinger, M. Yan, R.B. Gear, J. Schneider, K. LaDow, D. Warshawsky, Inhibition of VEGFR2 prevents DMBA-induced mammary tumor formation, Lab. Invest. 84 (2004) 989–998. [19] M.F. Jarvis, Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain, Expert Opin. Ther. Targets 7 (2003) 513–522. [20] M.F. Jarvis, C.T. Wismer, E. Schweitzer, H. Yu, T. van Biesen, K.J. Lynch, E.C. Burgard, E.A. Kowaluk, Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X3 allosteric modulator, cibacron blue, Br. J. Pharmacol. 132 (2001) 259–269. [21] M.F. Jarvis, E.C. Burgard, S. McGaraughty, P. Honore, K. Lynch, T.J. Brennan, A317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 17179–17184. [22] T. Kanno, T. Yaguchi, T. Nishizaki, Noradrenaline stimulates ATP release from DRG neurons by targeting beta(3) adrenoceptors as a factor of neuropathic pain, J. Cell. Physiol. 224 (2) (2010) 345–351. [23] R. Kimura, H. Nakase, R. Tamaki, T. Sakaki, Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction, Stroke 36 (2005) 1259–1263. [24] C. Lewis, S. Neidhart, C. Holy, R.A. North, G. Buell, A. Surprenant, Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons, Nature 377 (1995) 432–435. [25] D.C.H. Lin, C.M. Bullock, F.J. Ehlert, J.L. Chen, H. Thian, Q.Y. Zhou, Identification and molecular characterization of two closely related G-protein coupled receptors activated by prokineticins/EG-VEGF, J. Biol. Chem. 277 (2002) 19276–19280. [26] I.J. Llewellyn-Smith, G. Burnstock, Ultrastructural localization of P2X3 receptors in rat sensory neurons, NeuroReport 9 (1998) 2245–2250. [27] A.A.M. Maroto, E.M. Matínez-Quintana, L. Suárez-Castellano, J.L. PérezArellano, Painful hypertrophic osteoarthropathy successfully treated with octreotide. The pathogenetic role of vascular endothelial growth factor (VEGF), Rheumatology 44 (2005) 1326–1327. [28] L. Negri, R. Lattanzi, E. Giannini, A. Metere, M. Colucci, D. Barra, G. Kreil, P. Melchiorri, Nociceptive sensitization by the secretory protein Bv8, Br. J. Pharmacol. 137 (2002) 1147–1154. [29] L. Negri, R. Lattanzi, E. Giannini, M. Colucci, G. Mignogna, D. Barra, F. Grohovaz, F. Codazzi, A. Kaiser, G. Kreil, P. Melchiorri, Biological activities of Bv8 analogues, Br. J. Pharmacol. 146 (2005) 625–632. [30] R.A. North, P2X3 receptors and peripheral pain mechanisms, J. Physiol. 554 (2004) 301–308. [31] S.D. Novakovic, L.C. Kassotakis, I.B. Oglesby, J.A. Smith, R.M. Eglen, A.P. Ford, J.C. Hunter, Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury, Pain 80 (1999) 273–282. [32] M.C.G. Oliveira, A. Pelegrini-da-Silva, C.H. Tambeli, C.A. Parada, Peripheral mechanisms underlying the essential role of P2X3,2/3 receptors in the development of inflammatory hyperalgesia, Pain 141 (2009) 127–134. [33] U. Palmer-Kazen, P. Religa, E. Wahlberg, Exercise in intermittent claudication elicits signs of inflammation and angiogenesis, Eur. J. Vasc. Endovasc. Surg. 38 (2009) 689–696. [34] J.K. Phillips, A.J. McLean, C.E. Hill, Receptors involved in nerve-mediated vasoconstriction in small arteries of the rat hepatic mesentery, Br. J. Pharmacol. 24 (1998) 1403–1412. [35] C. Ruize de Almodovar, D. Lambrechts, M. Mazzone, P. Carmeliet, Role and therapeutic potential of VEGF in the nervous system, Physiol. Rev. 89 (2009) 607–648. [36] H.B. Schonthalera, R. Huggenbergerc, S.K. Wculeka, M. Detmarc, E.F. Wagnera, Systemic anti-VEGF treatment strongly reduces skin inflammation in a mouse model of psoriasis, Proc. Natl. Acad. Sci. U.S.A. 106 (50) (2009) 21264–21269. [37] W.D. Snider, S.B. McMahon, Tackling pain at the source: new ideas about nociceptors, Neuron 20 (1998) 629–632. [38] M. Sondell, G. Lundborg, M. Kanje, Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and schwann cell proliferation in the peripheral nervous system, J. Neurosci. 19 (1999) 5731–5740.
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291 [39] A. Surprenant, R.A. North, Signaling at purinergic P2X receptors, J. Annu. Rev. Physiol. 71 (2009) 333–359. [40] P.D.S. Telles, C.T. Hanks, M.A.A.M. Machado, J.E. Nör, Lipoteichoic acid upregulates VEGF expression in macrophages and pulp cells, J. Dent. Res. 82 (2003) 466–470. [41] G. Tolstanova, T. Khomenko, X.M. Deng, L.C. Chen, A. Tarnawski, A. Ahluwalia, S. Szabo, Z. Sandor, Neutralizing anti-vascular endothelial growth factor (VEGF) antibody reduces severity of experimental ulcerative colitis in rats: direct evidence for the pathogenic role of VEGF, J. Pharmacol. Exp. Ther. 328 (2009) 749–757. [42] H. Uchida, M. Matsumoto, H. Ueda, Profiling of BoNT/C3-reversible gene expression induced by lysophosphatidic acid: ephrinB1 gene up-regulation underlying neuropathic hyperalgesia and allodynia, Neurochem. Int. 54 (3–4) (2009) 215–221. [43] S. Yamaguchi, K. Tanabe, S. Takai, R. Matsushima-Nishiwaki, S. Adachi, H. Iida, O. Kozawa, S. Dohi, Involvement of Rho-kinase in tumor necrosis factor-alpha-
[44]
[45]
[46]
[47]
291
induced interleukin-6 release from C6 glioma cells, Neurochem. Int. 55 (6) (2009) 438–445. S.P. Yang, B.O. Kwon, Y.S. Gho, C.B. Chae, Specific interaction of VEGF165 with beta-amyloid, its protective effect on beta-amyloidinduced neurotoxicity, J. Neurochem. 93 (2005) 118–127. C.P. Zhang, G.L. Li, S.D. Liang, Myocardial ischemic nociceptive signaling mediated by P2X3 receptor in rat stellate ganglion neurons, Brain Res. Bull. 75 (2008) 77–82. A.X. Zhang, C.S. Xu, S.D. Liang, Y. Gao, G.L. Li, F. Wan, S.M. Liu, J.R. Lin, Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor, Neurochem. Int. 53 (2008) 278– 282. A.X. Zhang, Y. Gao, X.Q. Zhong, C.S. Xu, G.L. Li, S.M. Liu, J.R. Lin, X. Li, J. Zhang, H. Liu, S.D. Linag, Effect of sodium ferulate on the hyperalgesia mediated by P2X3 receptor in the neuropathic pain rats, Brain Res. 1313 (2010) 215– 221.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
VEGF and its receptor-2 involved in neuropathic pain transmission mediated by P2X2/3 receptor of primary sensory neurons Jiari Lin a,1 , Guilin Li a,1 , Xiaorong Den b,1 , Changshui Xu a,1 , Shuangmei Liu a , Yun Gao a , Han Liu a , Jun Zhang a , Xin Li a , Shangdong Liang a,∗ a b
Department of Physiology, Medical College of Nanchang University, Bayi Road 4613, Nanchang, Jiangxi 330006, PR China First Affiliated Hospital, Medical College of Nanchang University, Nanchang, Jiangxi 330006, PR China
a r t i c l e
i n f o
Article history: Received 6 June 2010 Received in revised form 24 July 2010 Accepted 3 August 2010 Available online 10 August 2010 Keywords: VEGF VEGF receptor-2 P2X2/3 receptor Neuropathic pain Primary sensory neuron
a b s t r a c t The pathogenesis of neuropathic pain is complex. P2X2/3 receptor plays a crucial role in nociception transduction of chronic pain. VEGF inhibitors are effective for pain relief. The present study investigated the effects of VEGF and VEGF receptor-2 (VEGFR2) on the pain transmission in neuropathic pain states that mediated by P2X2/3 receptor in primary sensory neurons. Chronic constriction injury (CCI) model was used as neuropathic pain model. Sprague–Dawley rats had been randomly divided into sham group, CCI group and CCI rats treated with anti-rVEGF antibody group. Mechanical withdrawal threshold and thermal withdrawal latency were measured. Expressions of VEGF, VEGFR2 and P2X2/3 in L4–6 dorsal root ganglia (DRG) were detected by immunohistochemistry, RT-PCR and western blot analysis. The mechanical withdrawal threshold and thermal withdrawal latency in CCI group were lower than those in sham group and CCI rats treated with anti-rVEGF antibody group (p < 0.05), while VEGF, VEGFR2 and P2X2/3 receptors’ expressions of L4–6 DRG in CCI group were higher than those in the other two groups (p < 0.05). The expressions of VEGF, VEGFR2 and P2X2/3 in L4–6 DRG of CCI rats treated with anti-rVEGF antibody group were decreased compared with those in CCI group (p < 0.05). The results show that VEGF and VEGFR2 are involved in the pathogenesis of neuropathic pain and VEGF primarily potentiates pain responses mediated by P2X2/3 receptor on DRG neurons. Anti-rVEGF treatment in CCI rats may alleviate chronic neuropathic pain by decreasing the expressions of VEGFR2 and P2X2/3 receptors on DRG neurons to inhibit the transmission of neuropathic pain signaling. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Vascular endothelial growth factor (VEGF) is a selective endothelial cell mitogen that promotes angiogenesis and also increases blood vessel permeability [14,15,38]. VEGF exerts action via high-affinity binding to three types of phosphotyrosine kinase receptors: VEGFR-1 (also known as fms-like tyrosine kinase 1 or flt1), VEGFR-2 (termed kinase insert-domain containing receptor, KDR in humans or fetal liver kinase 1, flk1 in mice), and VEGFR-3 (flt4) [35]. In the nervous system, VEGF mRNA has been found in neurons of brain’s capillary-rich areas [35]. VEGF plays a crucial role in the nervous system. In the peripheral nervous system, some vessels and nerves migrate along the same path alongside each other. VEGF may thus have therapeutic potential in Alzheimer disease, Parkinson’s disease and brain ischemia [1,23,44].
∗ Corresponding author. Tel.: +86 0791 6360552; fax: +86 0791 6360552. E-mail address: [email protected] (S. Liang). 1 Joint first authors. 0361-9230/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2010.08.002
The prokineticin receptors, prokineticin receptor 1 (PKR1) and PKR2 [25], and their activation activated by peptides belonging to Bv8/EG-VEGF (endocrine gland-derived vascular endothelial growth factor) – PK (prokineticin) family suggests an additional novel mechanism of peripheral nociceptor activation and sensitization [33]. PKR activation potentiated mustard oil induced nociceptive responses to heat. PKR1-null mice exhibited impaired development of hyperalgesia after inflammatory injury. The prokineticins released within inflamed tissues augment nociceptor responsiveness by acting on PKR1. Moreover, PKR2, which is expressed by DRG neurons different from those expressing PKR1 and is expressed normally by PKR1-null mice, could participate in specific pain conditions. A possible role of PKR2 receptor in nociceptor sensitization is to punctate stimuli. VEGF inhibitors improved hypertrophic pulmonary osteoarthropathy (HPOA)-related symptoms. VEGF mRNA in calf muscle is increased after exercise-induced pain in patients with intermittent claudication [33]. VEGF inhibitors might be effective for pain relief. Neuropathic pain is defined as “pain caused by primary dysfunction of the nervous system” [22,28]. The pathogenesis of
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
neuropathic pain is complex. The dorsal root ganglion (DRG) contains primary somatosensory neurons receiving nociceptive, mechanical and proprioceptive inputs. ATP has been shown to elicit peripheral nociceptor sensitization through the activation of receptors at nociceptor terminals [6,7,9,10,13]. ATP is implicated in peripheral pain signaling transmission by acting on P2X receptors [5,9,39]. Sensory afferent neurons contain mRNA and protein for multiple P2X receptors (P2X1–6 ). P2X and P2Y receptor subtypes essentially mediate all ATP-mediated responses in DRG neurons [6,8,10,13,20,24]. P2X1–6 as neuronal receptors includes P2X4 , which is non-neuronal (like P2X7 ). P2X3 receptors have received particular attention because of their high level expression in, and selective localization on sensory afferents [6,9,10,20,30]. P2X2 and P2X3 subunits can form homomultimeric P2X2 , homomultimeric P2X3 , or heteromultimeric P2X2/3 receptors. P2X2/3 heteromer receptors also express in sensory neurons [6,8,10]. P2X3 and P2X2/3 receptors in DRG neurons contribute to neuropathic pain [6,9,10,16,26,45–47]. The purpose of this study is to investigate the effects of VEGF and VEGFR2 on neuropathic pain and the relationship between VEGFR2 and P2X2/3 heteromer receptors on DRG sensory neurons in neuropathic pain state. 2. Materials and methods 2.1. Experimental animals and materials Male Sprague–Dawley rats (180–250 g) were provided by the Center of Laboratory Animal Science of Nanchang University. The animals were housed in plastic boxes in a group of three at 21–25 ◦ C. Use of the animals was reviewed and approved by the Animal Care and Use Committee of Medical College of Nanchang University. The IASP’s ethical guidelines for pain research in animals were followed. All animals were treated in accordance with ARVO Statement for the use of Animals in Ophthalmic and Vision Research in China. The chronic constriction injury (CCI) rats were made as the neuropathic pain model. Rats were randomly divided into three groups: sham group, CCI group and CCI rats treated with anti-rVEGF antibody group (anti-rVEGF group) (n = 6 for each group). Anti-rVEGF antibody (1 g/kg) in anti-rVEGF group and phosphate-buffer saline (PBS) in sham group and CCI group were intrathecally injected to rats by puncture every two days intervals (total 6 times), respectively. All drugs were diluted in PBS and 15 l of drugs was injected every time. The injection was initiated 2 h after operation to ensure all rats were awake completely. Then about 30 min later, behavior of all rats was observed. Anti-rat VEGF antibody was the product of R&D Systems, Inc (AF564). Rabbit anti-P2X3 and rabbit anti-P2X2 polyclonal antibody were bought from Chemicon International Company of America. VEGFR2 antibody was bought from Thermo Fisher Scientific (Fremont, CA, USA). Other antibodies and reagents were ascribed as following. 2.2. Chronic constriction injury (CCI) model CCI rat model was prepared [3,46,47]. Each rat was anesthetized with Nembutal [35 mg/kg intraperitoneally (i.p.)] during surgical procedures. The sciatic nerve was exposed at the middle level of rat thigh. Proximal to the sciatic trifurcation, four ligatures (4–0 chromic gut) were performed loosely with microsurgical techniques. Intervals between every two ligatures were about 1 mm. The same investigator created CCI animals to avoid variation. In the sham-operated group, the sciatic nerve was exposed using the same procedure but not ligatured by chromic gut. We assessed nociception on day 0, 1, 4, 7, 10, 13 after CCI by the observation of spontaneous pain behavior, by measurement of changes of latency in paw withdrawal on thermal stimulation, and of paw withdrawal threshold using von Frey filaments to assess mechanical hyperalgesia. 2.3. Measurement of mechanical withdrawal threshold (MWT) Noxious-pressure stimulation was used to evaluate mechanical hyperalgesia. Unrestrained rats were placed inside a clear plastic chamber (22 cm × 12 cm × 22 cm) on a stainless steel mesh floor and allowed to acclimate. Withdrawal responses to mechanical stimulation were determined using calibrated von Frey filaments (BME-403, Tianjin) applied through an opening in the stainless steel mesh floor of the cage (grid 1 cm × 1 cm) to an area adjacent to the paw. Each von Frey filament was applied once starting with 0.13 g and continuing until a withdrawal response occurred or the force reached 20.1 g (the cut-off value). The right hind paws were tested alternately at 2 min intervals. Measurements of three times were taken by using the method up and down on each side and the mean of the three determinations was taken as the threshold values. The filaments were applied in the order of increasing bending force (0.13, 0.20, 0.33, 0.60, 1.30,
285
3.60, 5.00, 7.30, 9.90, 20.1 g), with each applied 10 times at intervals of 15 s to different parts of the midplantar glabrous skin. The strength of the filaments in the series that evoked at least five positive responses among the 10 trials was designated the pain threshold.
2.4. Measurement of thermal withdrawal latency (TWL) Noxious heat stimulation for assessment of thermal hyperalgesia was applied by the Thermal Paw Stimulation System (BME-410C, Tianjin). Rats were placed in a transparent, square, bottomless acrylic box (22 cm × 12 cm × 22 cm), on a glass plate under which a light was located. Radiant heat stimuli were applied by directing a beam of light at the foot pad of each hind paw through the glass plate. The light beam was turned off automatically when the rat lifted the paw, allowing the measurement of time between the beginning of the light beam and the elevation of the foot. This time was designated as the paw withdrawal latency. The hind paws were tested alternately at 5 min intervals. The cut-off time for the heat stimulation was 30 s.
2.5. Immunohistochemistry Immunohistochemical staining was performed using SP-9001 kit (Beijing Zhongshan Biotech Co.) according to the manufacturer’s instruction. Two weeks later, Sprague–Dawley rats in three groups were anesthetized with Nembutal (35 mg/kg i.p.). The lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The L4–6 (lumbar 4–6) DRGs together with dorsal and ventral roots and attached spinal nerves were taken out from the inner side of each half of the dissected vertebrae with fine dissecting forceps. DRG isolated from rats were washed by PBS. After fixed with 4% paraformaldehyde (PFA) for 24 h, the ganglia were dehydrated by 20% sucrose for overnight at 4 ◦ C, and then ganglia were cut into 15 m thick on a cryostat. After washed by PBS for three times, the preparations were incubated in 3% H2 O2 for 10 min to block the endogenous peroxidase activity, then with 10% goat serum for 30 min at room temperature to block non-specific antigen. After rinsed and washed in PBS, the block sections were incubated with primary antibody (P2X2 and P2X3 1:2500 diluted in PBS; others 1:100 diluted in PBS) for overnight at 4 ◦ C. After three rinses in PBS, the sections were incubated with biotinylated goat anti-rabbit secondary antibody (Beijing Zhongshan Biotech Co.) for 1 h at room temperature. The preparations were washed in PBS and then added streptavidin–horseradish peroxidase (Beijing Zhongshan Biotech Co.) for 30 min. After development of the diaminobenzidine chromogen for 2 min, the slides were washed with distilled water and cover slipped. After immunohistochemistry, the Image-Pro Plus 6.0 was used to analyze the changes in stain values (average optical density) of P2X2 , P2X3 , VEGF and VEGFR2 in ganglia. Background was determined by averaging the optical density of 10 random areas.
2.6. RNA preparation and reverse transcriptase (RT)-PCR The rats in three groups were anesthetized by Nembutal (35 mg/kg i.p.). The L4–6 DRG were isolated immediately and flushed with icecold PBS. Total RNA samples were prepared from L4–6 DRG in each group by using of TRNzol Total RNA Reagent (Beijing Tiangen Biotech Co.). Isolated RNA was resuspended in DEPC water and reverse transcribed to cDNA using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions. PCR was performed on cDNA using primers listed below and the following: 4 l of cDNA, 6.5 l DEPC water, 2 l 10 mol primers (equal sense and antisense), and 12.5 l 2× Easy Taq PCR SuperMix (Beijing TransGen Biotech Co.) in 25 l. PCR conditions included hot start at 94 ◦ C for 3 min, 30 s denaturation (94 ◦ C), 30 s annealing (annealing temperatures (AT) listed below), and 30 s extension (72 ◦ C) for 30 amplification rounds and 5 min final extension at 72 ◦ C. PCR products (8 l) were run on 1% agarose gels with EB using standard protocols. Gels were scanned with gel imaging system (Beijing Junyi Co., Ltd) and the intensity of PCR product bands was analyzed using Gel-Pro Analyzer 32. The ratio of VEGF or these receptors band intensity/-actin band intensity was used as the results of half-quantification analysis. Primer P2X2 [6] AT: 57 ◦ C; PS: 273 bp Sense: 5 -CTGCCTCCTCAGGCTACAACTTCA-3 Antisense: 5 -GAGTACGCACCTTGTCGAACTTCT-3 Primer P2X3 [31] AT: 57 ◦ C; PS: 519 bp Sense: 5 -CAACTTCAGGTTTGCCAAA-3 Antisense: 5 -TGAACAGTGAGGGCCTAGAT-3 Primer VEGF [34] AT: 65 ◦ C; PS: 145 bp Sense: 5 -GCTCTCTTGGGTGCACTGGAC-3 Antisense: 5 -ACGGCAATAGCTGCGCTGGTA-3 Primer VEGFR2 [34] AT: 60 ◦ C; PS: 537 bp Sense: 5 -GCCAATGAAGGGGAACTGAAGA-3 Antisense: 5 -CTCTGACTGCTGGTGATGCTGTC-3 Primer -actin [18] AT: 60 ◦ C; PS: 240 bp Sense: 5 -TAAAGACCTCTATGCCAACACAGT-3 Antisense: 5 -CACGATGGAGGGGCCGGACTCATC-3
286
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
Fig. 1. Effects of anti-rVEGF antibody on MWT (mechanical withdrawal threshold) of CCI rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as “**” when p < 0.01 compared with sham group; the significant difference was denoted as “#” when p < 0.05 and “##” when p < 0.01 compared with CCI group.
2.7. Western blot analysis The rats in three groups were anesthetized by Nembutal (35 mg/kg i.p.). The L4–6 DRG were isolated immediately and flushed with icecold PBS. Ganglia were homogenized by mechanical disruption in lysis buffer (50 mmol/L TrisCl, pH 8.0, 150 mmol/L NaCl, 0.1% dodecyl sodium sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 g/mL phenylmethylsulfonyl fluoride, 1 g/mL Aprotinin) and incubated on ice for 40 min. Homogenate was then pelleted at 12,000 rpm for 10 min and supernatant was collected. Using Lowry method, the quantity of total protein was determined in the supernatant. After diluted with sample buffer (100 mmol/L TrisCl, 200 mmol/L dithiothreitol, 4% sodium dodecylsulfate (SDS), 0.2% bromophenol blue, 20% glycerol) and heated to 95 ◦ C for 5 min, samples containing equal amounts of protein (20 l) were separated by SDS-polyacrylamide gel electrophoresis by using Bio-Rad system and 10% gel. In the wake of electrophoretic transfer onto nitrocellulose (NC) membrane using the same system, the membrane was blocked with 5% non-fat dry milk in 25 mmol/L Tris buffered saline, pH 7.2, plus 0.1% Tween 20 (TBST) for 1–3 h at room temperature, and incubated with primary antibodies in blocking buffer for 2 h at room temperature or overnight at 4 ◦ C. The membranes were washed three times with TBST and incubated (1 h, room temperature) with horseradish peroxidase-conjugated secondary antibody [goat anti-rabbit IgG (1:4000), Beijing Zhongshan Biotech Co.] in blocking buffer. After another wash cycle, labeled proteins were visualized by using the enhanced chemiluminescence (ECL) kit (Fremont, CA, USA). Chemiluminescent signals were collected on autoradiography film, and the quantity of band intensity was carried out using Gel-Pro Analyzer 32. The primary antibodies and dilutions used were the following: rabbit polyclonal anti-P2X3 and anti-P2X2 (1:1000), rabbit polyclonal anti-VEGFR2 (1:100), rabbit polyclonal antiVEGF (1:100; Wuhan Boster Co., China) and monoclonal -actin (1:1000; Beijing Zhongshan Biotech Co.). Band densities were normalized to each -actin internal control.
2.8. Statistical analysis Statistical analysis of the data was performed on computer (SPSS 11.5). All results were expressed as mean ± SE. Statistical significance was determined by one factor analysis of variance (ANOVA) followed by the Fisher post hoc test for multiple comparisons. p-Value <0.05 was considered significant.
3. Results 3.1. Effects of anti-rVEGF antibody on mechanical hyperalgesia of CCI rats At day 7 after operation, the mechanical withdrawal threshold (MWT) in CCI group was lower than that in sham group (p < 0.01), and constantly exists from day 7 to day 14. MWT in anti-rVEGF group was lower than that in sham group at days 1–4, but there is no statistical difference (p > 0.05). MWT in anti-rVEGF group from day 7 to day 14 was no statistical difference compared with that in sham group from day 7 to day 14 (p > 0.05) (Fig. 1).
Fig. 2. Effects of anti-rVEGF antibody on (TWL) thermal withdrawal latency of CCI rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as “*” when p < 0.05 and “**” when p < 0.01 compared with sham group; the significant difference was denoted as “##” when p < 0.01 compared with CCI group.
3.2. Effects of anti-rVEGF antibody on thermal hyperalgesia of CCI rats At day 1 after operation, the thermal withdrawal latency (TWL) in CCI group was lower than that in sham group (p < 0.05), and there is extremely significant difference from day 7 to day 14 (p < 0.01). TWL in anti-rVEGF group was lower than that in sham group from day 1 to day 4 (p < 0.05), but there is no statistical difference compared with that in sham group from day 7 to day 14 (p > 0.05). TWL in anti-rVEGF group has statistical significance compared with that in CCI group from day 7 to day 14 (Fig. 2). 3.3. Effects of anti-rVEGF antibody on the expression of P2X2 , P2X3 , VEGF and VEGFR2 immunoreactivities in L4–6 DRG of CCI rats P2X2 , P2X3 , VEGF and VEGFR2 immunoreactivities in L4–6 DRG were detected by immunohistochemistry. The expressions of P2X2 , P2X3 , VEGF and VEGFR2 in the ganglia of each group were examined. The staining of P2X2 , P2X3 , VEGF and VEGFR2 in L4–6 DRG of CCI group was all higher than that in sham group and anti-rVEGF group, respectively (p < 0.05), and there are no significant difference in the staining values of P2X2 , P2X3 , VEGF and VEGFR2 mRNA between sham group and anti-rVEGF group (p > 0.05) (Fig. 3A–E). 3.4. Effects of anti-rVEGF antibody on the expression of P2X2 , P2X3 , VEGF and VEGFR2 mRNA in L4–6 DRG of CCI rats by RT-PCR RT-PCR studies showed that the expressions of P2X2 , P2X3 , VEGF and VEGFR2 mRNA in DRG were abundantly expressed in CCI group in comparison with those in sham group and anti-rVEGF group, respectively (Fig. 4A–D). The relative levels (normalized to each -actin internal control) of P2X2 , P2X3 , VEGF and VEGFR2 mRNA of CCI group were significantly increased compared with those in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), respectively (Fig. 4E). 3.5. Effects of anti-rVEGF antibody on the expression of P2X2 , P2X3 , VEGF and VEGFR2 protein in L4–6 DRG of CCI rats by western blotting The expressions of P2X2 , P2X3 , VEGF and VEGFR2 in protein level were analyzed by western blotting (Fig. 5A–D). By image analysis, the stain values (average optical density) of P2X2 , P2X3 , VEGF and
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
287
Fig. 3. Expression of P2X2 , P2X3 , VEGF and VEGFR2 immunoreactivities in L4–L6 DRG neurons in each group. Photos of protein expression in L4–L6 DRG neurons measured by immunohistochemistry (n = 6 each group). (A) The expression of P2X2 protein in L4–L6 DRG neurons. (B) The expression of P2X3 protein in L4–L6 DRG neurons. (C) The expression of VEGF in L4–L6 DRG neurons. (D) The expression of VEGFR2 in L4–L6 DRG neurons. (E) The bar histograms showed the optical density (mean) of each group on the expression of P2X2 , P2X3 , VEGF and VEGFR2. (Note: Arrows indicate the immunostaining neurons. Data are shown as the means ± S.E.; *p < 0.05, as compared with sham group; # p < 0.05, as compared with CCI group.)
VEGFR2 protein expressions (normalized to each -actin internal control) in DRG of CCI group were significantly enhanced compared with those in sham group (p < 0.05), but the relative levels of P2X2 , P2X3 , VEGF and VEGFR2 protein expressions in anti-rVEGF group were lower than those in CCI group (p < 0.05) (Fig. 5E). 4. Discussion VEGF plays a crucial role in the different functions of nervous system and therapeutic potential of nervous system diseases [35]. Our data showed that the MWT and TWL were increased after CCI rats treated with anti-VEGF antibody. Anti-VEGF antibody treatment might induce analgesia. The prokineticin receptors, prokineticin receptor 1 (PKR1) and PKR2 [25,35,36], and their activation which exerting by peptides belonging to the Bv8/EG-VEGF (endocrine gland-derived vascular endothelial growth factor) – PK (prokineticin) family suggests an additional novel mechanism of peripheral nociceptor activation and sensitization [25,28]. The lit-
erature indicates that cyclooxygenase-2 (COX-2) plays a key role in the production of VEGF [29]. Co-expression of COX-2 and VEGF in all consecutive sections of inflamed pulps indicates a release of VEGF via a COX-2-dependent pathway [29]. Hypertrophic pulmonary osteoarthropathy (HPOA) is a syndrome defined by digital clubbing and distal periostitis in tubular bones [17]. Symptoms may include bone pain, occasionally severe. VEGF inhibition improved HPOA-related symptoms [17]. Systemic administration of an antiVEGF antibody effectively inhibited pathological angiogenesis [36]. The treatment with anti-VEGF antibody markedly reduced characteristic psoriasis-like symptoms observed in the skin lesions of the mice, such as epidermal hyperplasia with altered differentiation, inflammatory cell infiltration, vascular abnormalities, and elevated levels of IL-23/Th17-related cytokines. It is possible that VEGF is involved in nociception or pain. VEGF receptors have been identified as VEGFR-1 (Flt1), VEGFR2 (KDR, Flk1), and VEGFR-3 (Flt4). VEGF binds to VEGFR-1 and VEGFR-2 [14,35]. The expression levels of VEGF and VEGFR2
288
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
Fig. 4. The changes of P2X2 , P2X3 , VEGF and VEGFR2 mRNA level in L4–L6 DRG in each group. (A) The expression of P2X2 mRNA in DRG of CCI group was increased compared with those in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (B) The expression of P2X3 mRNA in DRG of CCI group was increased in comparison with that in sham group (p < 0.05) and that in anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in the anti-rVEGF group was just slightly higher than that in sham group. (C) The expression of VEGF mRNA in DRG of CCI group was increased compared with that in sham group (p < 0.05), anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (D) The expression of VEGFR2 mRNA in DRG of CCI group was significantly higher than that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in sham group just detect a little. (E) Means ± S.E. of RT-PCR product levels quantified by phosphoimager analysis are shown. Data are expressed as the band intensity for each growth factor or receptor listed relative to the -actin level determined in the same PCR reaction. *p < 0.05, as compared with sham group; # p < 0.05, as compared with CCI group.
immunoreactivities, mRNA and protein in DRG of CCI rats were higher than those in sham group and anti-rVEGF group. The expression of VEGF by immunohistochemistry is strongly positive in cells constituting the inflammatory infiltrate of teeth with irreversible pulpitis [2]. Up-regulation of VEGF expression in the dental pulp may result in increased intra-pulpal pressure, and contribute to pain and irreversible tissue damage [27]. The anti-VEGF antibody significantly attenuated the clinical and morphologic features of iodoacetamide-induced colitis [41]. HPOA-related symptoms can be blunted by different VEGF inhibitors [17]. The subcutaneous administration of carrageenan induces a hyperalgesic response that is reduced by nonsteroidal anti-inflammatory drugs. VEGF may be involved in thermal and mechanical hyperalgesia. Up-regulation of VEGF and VEGFR2 expression in DRG of CCI rats reveals that VEGF
and VEGFR-2 participate in the pathogenesis of neuropathic pain. The expressions of VEGF and VEGFR2 in DRG of anti-rVEGF group were reduced compared with those in DRG of CCI rats. VEGF may bind to VEGFR-2 in DRG of CCI rats. The neutralization of endogenous VEGF decreased the MWT and TWL in CCI rats. The results may reveal certain mechanisms responsible for the analgesia induced by anti-VEGF antibody. Reduction of VEGF and VEGFR2 expression in DRG of anti-rVEGF rats may be a novel strategy that can diminish the activation and sensitization of primary afferent nociceptors to produce therapeutic benefit in acute, inflammatory and neuropathic pain conditions. Activation of P2X3,2/3 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia [6,9,10,16,26,45–47]. The essential role of P2X3,2/3 receptors in
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
289
Fig. 5. The changes of P2X2 , P2X3 , VEGF and VEGFR2 protein level in L4–L6 DRG in each group. (A) The expression of P2X2 protein in DRG of CCI group was increased compared with that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and there is no significant difference between sham group and anti-rVEGF group (p < 0.05). (B) The expression of P2X2 protein in DRG of CCI group was increased compared with that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (C) The expression of VEGF protein in DRG of CCI group was significantly higher than that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and there is no significant difference between sham group and anti-rVEGF group (p > 0.05). (D) The expression of VEGFR2 protein in DRG of CCI group was increased compared with that in sham group (p < 0.05) and anti-rVEGF group (p < 0.05), and the expression of VEGF mRNA in anti-rVEGF group was just slightly higher than that in sham group. (E) The bar histograms showed the ratio of P2X2 , P2X3 , VEGF and VEGFR2 protein level to -actin with each group. (Note: Data are shown as the mean ± S.E. *p < 0.05, as compared with sham group; # p < 0.05, as compared with CCI group.)
the development of carrageenan-induced mechanical hyperalgesia is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of TNF-␣ and by a direct sensitization of the primary afferent nociceptors [32,42,43]. Prokineticin receptors (prokineticin receptor 1 (PKR1) and PKR2) and their activation by peptides belonging to the Bv8/EG-VEGF PK (prokineticin) family suggest a novel mechanism of peripheral nociceptor activation and sensitization [28]. PKR1 expression is important in determining sensory thresholds to nociceptive and to acute and chronic inflammatory stimuli. PKR1 is a Gprotein-coupled metabotropic receptor [25] sensitizes the vanilloid receptor TRPV1 to noxious stimuli. The activation of peripheral P2X2/3 receptors may be crucial to VEGF-mediated sensitization of the primary afferent nociceptor. It is broadly accepted that TRPV1positive neurons are nociceptive neurons. An important feature of TRPV1 regulation also concerns the cellular signaling pathways through which G-protein-coupled receptors sensitize TRPV1 to its chemical and physical stimuli. Bv8 and prokineticins may sensitize TRPV1 to vanilloid agonists, heat, and protons. The co-expression of TRPV1 and high levels of P2X3 (and P2X2 ) receptors in sensory neurons supports the involvement of the P2X3 and P2X2/3 receptors in pain [8,9,24]. PKRs transduce the sensitizing signal through kinase dependent signaling pathways or through the release of nocicep-
tive mediators, which in turn activate kinase signaling throughout their receptors. VEGF may sensitize P2X2/3 receptor to P2X receptor agonists in the primary afferent nociceptor. The role of P2X2/3 receptors in the development of thermal and mechanical hyperalgesia may be mediated, at least in part, by VEGFR2 activation in the primary afferent nociceptor. The modulatory role of anti-VEGF antibody treatment in VEGFR2 and P2X2/3 activation is also suggested by thermal and mechanical hypoalgesia. All of these findings agree on a positive interaction between VEGFR2 and P2X2/3 throughout the peripheral nervous system. Therefore, blockade of VEGFR2 and P2X2/3 receptors prevented the development of thermal and mechanical hyperalgesia. Nociceptive neurons can be divided into two major populations, non-myelinated C-fiber and myelinated A-fiber neurons [37]. The P2X3 receptor subunit is expressed on primary afferent neurons and preferentially on nociceptive C-fibers. The expression levels of P2X2 and P2X3 were reduced after CCI rats treated with anti-VEGF antibody. VEGF primarily potentiates responses mediated by the pain receptor P2X2/3 on dorsal root ganglion neurons, an effect blocked by anti-VEGF treatment. Anti-VEGF antibody treatment might neutralizate VEGF to decrease P2X2 and P2X3 receptors’ activities on nociceptive DRG neurons. Extracellular ATP was reported to produce pain sensation in humans
290
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291
and to participate in pain pathways in primary sensory neurons [7,9,10,24]. There are native P2X receptors in sensory neuron of DRG. P2X3 and P2X2/3 receptors are expressed on isolectin B4 (IB4) binding subpopulations of small nociceptive neurons [7,40]. Peripheral nerve injury has been shown to regulate the expression of peripheral P2X3 receptors. An increase of the number of P2X3 receptor-immunoreactive on DRG neurons was demonstrated after a chronic constriction injury of the sciatic nerve [3,9,16,26,45–47]. P2X receptors contribute to neuropathic pain. P2X3 and P2X2/3 receptors play a crucial role in nociception transduction of acute and chronic pain [4,11]. P2X3 double-stranded short interfering RNA (siRNA) relieves chronic neuropathic pain [12,19]. A-317491, a potent and selective antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat [21]. P2X2/3 receptors may be targets for anti-rVEGF treatment to relieve the hyperalgesia of neuropathic pain rats. The finding suggests that selective antagonists for the VEGFR2 may decrease peripheral nociceptor activation and sensitization. 5. Conclusions In summary, VEGF and VEGFR2 are involved in the pathogenesis of neuropathic pain. VEGF primarily potentiates pain responses mediated by P2X2/3 receptors on dorsal root ganglion neurons. CCI rats treated with anti-rVEGF antibody can inhibit the activation of P2X2/3 receptor during chronic pain and block primary afferent transmission mediated by VEGFR2 and P2X2/3 receptors to reduce hyperalgesia in chronic pain states. Anti-rVEGF treatment in CCI rats may alleviate chronic neuropathic pain by decreasing the expressions of VEGFR2 and P2X2/3 receptors in DRG neurons. Conflict of interest None. Acknowledgments This work was supported by the grant (Nos. 30860086, 30860333 and 30660048) from National Natural Science Foundation of China, the grant (No. 20070403007) from Doctoral Fund of Ministry of Education of China, the grant (Nos. 0640042, 2007GZY1002 and 2008GZY0029) from Natural Science Foundation of Jiangxi Province and the grant (No. GJJ08049) from the Educational Department of Jiangxi Province. References [1] L. Anderson, M.A. Caldwell, Human neural progenitor cell transplants into the subthalamic nucleus lead to functional recovery in a rat model of Parkinson’ disease, Neurobiol. Dis. 27 (2007) 133–140. [2] L. Artese, C. Rubini, G. Ferrero, M. Fiorini, A. Santinelli, A. Piattelli, Vascular endothelial growth factor (VEGF) expression in healthy and inflamed human dental pulps, J. Endod. 28 (2002) 20–23. [3] G.J. Bennett, Y.K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 (1988) 87–107. [4] E.J. Bradbury, G. Burnstock, S.B. McMahon, The expression of P2X3 purinoceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor, J. Mol. Cell. Neurosci. 12 (1998) 256–268. [5] G. Burnstock, P2X receptors in sensory neurons, Br. J. Anaesth. 84 (2000) 476–488. [6] G. Burnstock, Purinergic P2 receptors as targets for novel analgesics, Pharmacol. Ther. 110 (2006) 433–454. [7] G. Burnstock, Physiology and pathophysiology of purinergic neurotransmission, Physiol. Rev. 87 (2007) 659–797. [8] C.C. Chen, A.N. Akopian, L. Sivilotti, D. Colquhoun, G. Burnstock, J.N. Wood, A P2X purinoceptor expressed by a subset of sensory neurons, Nature 377 (1995) 428–431. [9] B.A. Chizh, P. Illes, P2X R receptors and nociception, Pharmacol. Rev. 53 (2000) 553–568. [10] M.S. Chong, B. Brandner, Neuropathic agents and pain. New strategies, Biomed. Pharmacother. 60 (2006) 318–322.
[11] D.A. Cockayne, P.M. Dunn, Y. Zhong, S.G. Hamilton, G.R. Cain, G.E. Knight, H.Z. Ruan, Y. Ping, P. Nunn, M. Bei, S.B. McMahon, G. Burnstock, A.P.D.W. Ford, P2X2 knockout mice and P2X2 /P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP, J. Physiol. 567 (2005) 621–639. [12] G. Dorn, S. Patel, G. Wotherspoon, M. Hemmings-Mieszczak, J. Barclay, F.J. Natt, P. Martin, S. Bevan, A. Fox, P. Ganju, W. Wishart, J. Hall, siRNA relieves chronic neuropathic pain, Nucleic Acids Res. 32 (2004) e49. [13] G. Dussora, H.R. Koerbere, A.L. Oaklanderb, F.L. Ricec, D.C. Molliverd, Nucleotide signaling and cutaneous mechanisms of pain transduction, Brain Res. Rev. 60 (2009) 24–35. [14] N. Ferrara, Vascular endothelial growth factor: basic science and clinical progress, Endocr. Rev. 25 (2004) 581–611. [15] N. Ferrara, T. Davis-Smyth, The biology of vascular endothelial growth factor, Endocr. Rev. 18 (1997) 4–25. [16] Y. Gao, C.S. Xu, S.D. Liang, S.N. Mu, A.X. Zhang, Y.X. Wang, F. Wan, Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X3 receptor in neuropathic pain states, Brain Res. Bull. 77 (2008) 27–32. [17] G. Güven, C. Altun, O. Günhan, T. Gurbuz, F. Basak, E. Akbulut, Z.C. Cehreli, Co-expression of cyclooxygenase-2 and vascular endothelial growth factor in inflamed human pulp: an immunohistochemical study, J. Endod. 33 (2007) 18–20. [18] S.C. Heffelfinger, M. Yan, R.B. Gear, J. Schneider, K. LaDow, D. Warshawsky, Inhibition of VEGFR2 prevents DMBA-induced mammary tumor formation, Lab. Invest. 84 (2004) 989–998. [19] M.F. Jarvis, Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain, Expert Opin. Ther. Targets 7 (2003) 513–522. [20] M.F. Jarvis, C.T. Wismer, E. Schweitzer, H. Yu, T. van Biesen, K.J. Lynch, E.C. Burgard, E.A. Kowaluk, Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X3 allosteric modulator, cibacron blue, Br. J. Pharmacol. 132 (2001) 259–269. [21] M.F. Jarvis, E.C. Burgard, S. McGaraughty, P. Honore, K. Lynch, T.J. Brennan, A317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 17179–17184. [22] T. Kanno, T. Yaguchi, T. Nishizaki, Noradrenaline stimulates ATP release from DRG neurons by targeting beta(3) adrenoceptors as a factor of neuropathic pain, J. Cell. Physiol. 224 (2) (2010) 345–351. [23] R. Kimura, H. Nakase, R. Tamaki, T. Sakaki, Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction, Stroke 36 (2005) 1259–1263. [24] C. Lewis, S. Neidhart, C. Holy, R.A. North, G. Buell, A. Surprenant, Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons, Nature 377 (1995) 432–435. [25] D.C.H. Lin, C.M. Bullock, F.J. Ehlert, J.L. Chen, H. Thian, Q.Y. Zhou, Identification and molecular characterization of two closely related G-protein coupled receptors activated by prokineticins/EG-VEGF, J. Biol. Chem. 277 (2002) 19276–19280. [26] I.J. Llewellyn-Smith, G. Burnstock, Ultrastructural localization of P2X3 receptors in rat sensory neurons, NeuroReport 9 (1998) 2245–2250. [27] A.A.M. Maroto, E.M. Matínez-Quintana, L. Suárez-Castellano, J.L. PérezArellano, Painful hypertrophic osteoarthropathy successfully treated with octreotide. The pathogenetic role of vascular endothelial growth factor (VEGF), Rheumatology 44 (2005) 1326–1327. [28] L. Negri, R. Lattanzi, E. Giannini, A. Metere, M. Colucci, D. Barra, G. Kreil, P. Melchiorri, Nociceptive sensitization by the secretory protein Bv8, Br. J. Pharmacol. 137 (2002) 1147–1154. [29] L. Negri, R. Lattanzi, E. Giannini, M. Colucci, G. Mignogna, D. Barra, F. Grohovaz, F. Codazzi, A. Kaiser, G. Kreil, P. Melchiorri, Biological activities of Bv8 analogues, Br. J. Pharmacol. 146 (2005) 625–632. [30] R.A. North, P2X3 receptors and peripheral pain mechanisms, J. Physiol. 554 (2004) 301–308. [31] S.D. Novakovic, L.C. Kassotakis, I.B. Oglesby, J.A. Smith, R.M. Eglen, A.P. Ford, J.C. Hunter, Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury, Pain 80 (1999) 273–282. [32] M.C.G. Oliveira, A. Pelegrini-da-Silva, C.H. Tambeli, C.A. Parada, Peripheral mechanisms underlying the essential role of P2X3,2/3 receptors in the development of inflammatory hyperalgesia, Pain 141 (2009) 127–134. [33] U. Palmer-Kazen, P. Religa, E. Wahlberg, Exercise in intermittent claudication elicits signs of inflammation and angiogenesis, Eur. J. Vasc. Endovasc. Surg. 38 (2009) 689–696. [34] J.K. Phillips, A.J. McLean, C.E. Hill, Receptors involved in nerve-mediated vasoconstriction in small arteries of the rat hepatic mesentery, Br. J. Pharmacol. 24 (1998) 1403–1412. [35] C. Ruize de Almodovar, D. Lambrechts, M. Mazzone, P. Carmeliet, Role and therapeutic potential of VEGF in the nervous system, Physiol. Rev. 89 (2009) 607–648. [36] H.B. Schonthalera, R. Huggenbergerc, S.K. Wculeka, M. Detmarc, E.F. Wagnera, Systemic anti-VEGF treatment strongly reduces skin inflammation in a mouse model of psoriasis, Proc. Natl. Acad. Sci. U.S.A. 106 (50) (2009) 21264–21269. [37] W.D. Snider, S.B. McMahon, Tackling pain at the source: new ideas about nociceptors, Neuron 20 (1998) 629–632. [38] M. Sondell, G. Lundborg, M. Kanje, Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and schwann cell proliferation in the peripheral nervous system, J. Neurosci. 19 (1999) 5731–5740.
J. Lin et al. / Brain Research Bulletin 83 (2010) 284–291 [39] A. Surprenant, R.A. North, Signaling at purinergic P2X receptors, J. Annu. Rev. Physiol. 71 (2009) 333–359. [40] P.D.S. Telles, C.T. Hanks, M.A.A.M. Machado, J.E. Nör, Lipoteichoic acid upregulates VEGF expression in macrophages and pulp cells, J. Dent. Res. 82 (2003) 466–470. [41] G. Tolstanova, T. Khomenko, X.M. Deng, L.C. Chen, A. Tarnawski, A. Ahluwalia, S. Szabo, Z. Sandor, Neutralizing anti-vascular endothelial growth factor (VEGF) antibody reduces severity of experimental ulcerative colitis in rats: direct evidence for the pathogenic role of VEGF, J. Pharmacol. Exp. Ther. 328 (2009) 749–757. [42] H. Uchida, M. Matsumoto, H. Ueda, Profiling of BoNT/C3-reversible gene expression induced by lysophosphatidic acid: ephrinB1 gene up-regulation underlying neuropathic hyperalgesia and allodynia, Neurochem. Int. 54 (3–4) (2009) 215–221. [43] S. Yamaguchi, K. Tanabe, S. Takai, R. Matsushima-Nishiwaki, S. Adachi, H. Iida, O. Kozawa, S. Dohi, Involvement of Rho-kinase in tumor necrosis factor-alpha-
[44]
[45]
[46]
[47]
291
induced interleukin-6 release from C6 glioma cells, Neurochem. Int. 55 (6) (2009) 438–445. S.P. Yang, B.O. Kwon, Y.S. Gho, C.B. Chae, Specific interaction of VEGF165 with beta-amyloid, its protective effect on beta-amyloidinduced neurotoxicity, J. Neurochem. 93 (2005) 118–127. C.P. Zhang, G.L. Li, S.D. Liang, Myocardial ischemic nociceptive signaling mediated by P2X3 receptor in rat stellate ganglion neurons, Brain Res. Bull. 75 (2008) 77–82. A.X. Zhang, C.S. Xu, S.D. Liang, Y. Gao, G.L. Li, F. Wan, S.M. Liu, J.R. Lin, Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor, Neurochem. Int. 53 (2008) 278– 282. A.X. Zhang, Y. Gao, X.Q. Zhong, C.S. Xu, G.L. Li, S.M. Liu, J.R. Lin, X. Li, J. Zhang, H. Liu, S.D. Linag, Effect of sodium ferulate on the hyperalgesia mediated by P2X3 receptor in the neuropathic pain rats, Brain Res. 1313 (2010) 215– 221.