Substance P (SP) is a neuropeptide, predominantly released from sensory nerve fibers, with a potentially protective role in diabetic corneal epithelial wound healing. However, the molecular mechanism remains unclear. We investigated the protective mechanism of SP against hyperglycemia-induced corneal epithelial wound healing defects, using type 1 diabetic mice and high glucose–treated corneal epithelial cells. Hyperglycemia induced delayed corneal epithelial wound healing, accompanied by attenuated corneal sensation, mitochondrial dysfunction, and impairments of Akt, epidermal growth factor receptor (EGFR), and Sirt1 activation, as well as decreased reactive oxygen species (ROS) scavenging capacity. However, SP application promoted epithelial wound healing, recovery of corneal sensation, improvement of mitochondrial function, and reactivation of Akt, EGFR, and Sirt1, as well as increased ROS scavenging capacity, in both diabetic mouse corneal epithelium and high glucose–treated corneal epithelial cells. The promotion of SP on diabetic corneal epithelial healing was completely abolished by a neurokinin-1 (NK-1) receptor antagonist. Moreover, the subconjunctival injection of NK-1 receptor antagonist also caused diabetic corneal pathological changes in normal mice. In conclusion, the results suggest that SP-NK-1 receptor signaling plays a critical role in the maintenance of corneal epithelium homeostasis, and that SP signaling through the NK-1 receptor contributes to the promotion of diabetic corneal epithelial wound healing by rescued activation of Akt, EGFR, and Sirt1, improvement of mitochondrial function, and increased ROS scavenging capacity.

Among various pathological conditions by diabetes, ocular complications have been a leading cause of blindness in the world, including diabetic retinopathy, cataract, and various ocular surface disorders (1). The most recognized diabetic changes in the cornea, i.e., clinical diabetic keratopathy, include impaired corneal sensation, superficial punctate keratitis, and persistent corneal epithelium defects (2). The uncontrolled impairment of corneal wound healing increases the susceptibility of corneal ulcer, microbial keratitis, and even perforation. Diabetic corneal pathology always exhibits epithelial basement membrane abnormalities, reduced hemidesmosome density, and delayed wound healing (3,4). However, hyperglycemia also directly impairs the cellular metabolism and causes abnormal changes of corneal epithelium, such as Akt-, epidermal growth factor receptor (EGFR)–, and Sirt1-mediated cell responses to environmental challenges (57). Moreover, excess oxidative stress, resulting from enhanced accumulation of reactive oxygen species (ROS) and impaired antioxidant capabilities in response to hyperglycemia, has been postulated as an important pathological mechanism, whereas the reduction of ROS attenuated the progression of various diabetes complications (813), including in the cornea (5,14).

Substance P (SP), released predominantly by peripheral terminal, is an 11–amino acid neuropeptide that acts as a neurotransmitter mediating nociceptive transmission. It mainly functions through the interaction with neurokinin receptors, members of the tachykinin subfamily of G-protein–coupled receptors, among which the neurokinin-1 (NK-1) receptor shows a preferential affinity for SP. SP is currently known as a neuroimmunomodulatory regulator of the immune system (15). In the cornea, SP has been detected in the nerve fibers of naïve cornea (16,17) and in antigen-presenting cells in herpetic stromal keratitis (18). Moreover, SP causes the mobilization of bone marrow–derived stem cells to participate in corneal wound healing (19,20). Interestingly, recent evidences have shown that SP activates the EGFR, mitogen-activated protein kinases (MAPKs), extracellular signal–regulated kinases (ERKs), and phosphoinositide 3-kinase–Akt (PI3K-Akt) signaling pathways, as well as promotes the healing of inflamed colonic epithelium (21) and possesses antiapoptotic effects in colonocytes (22), dendritic cells (23), tenocytes (24), neutrophils (25), and bone marrow–derived stem cells (26).

The cornea is one of the most densely innervated tissues in the body, containing nerve fibers derived from the trigeminal ganglion. Corneal nerve fibers exert important trophic influences and contribute to the maintenance of corneal epithelium homeostasis, whereas the dysfunction of corneal innervation produces an impairment of corneal epithelial wound healing, known as neurotrophic keratitis, as caused by for instance herpetic viral infections, trigeminal nerve damage, or diabetes (2729). Although tear fluid impairment is also involved (30), the neurotrophic deficits may play a major role in the pathogenesis of diabetic keratopathy, as the density of corneal nerve fibers and corneal sensation decreased in diabetic patients (3133). However, although being the major sensory neurotransmitter and neuropeptide released from corneal nerve fibers, SP was shown not to be significantly decreased in diabetic cornea (34). Nevertheless, paradoxically, topical SP application promoted corneal epithelial wound healing in diabetic animals and humans when combined with insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) per group (29). The promoting mechanisms of SP on diabetic corneal epithelial wound healing remain elusive. In this study, we sought to demonstrate the protective mechanism of SP against diabetic corneal epithelial wound healing using streptozotocin-induced type 1 diabetic mice and high glucose–treated corneal epithelial cells.

Animals

Adult male C57BL/6 mice were purchased from the Beijing HFK Bioscience Co. Ltd. (Beijing, China). All animal experiments were carried out in accordance with The Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The mice underwent induction of type 1 diabetes with an intraperitoneal injection of 50 mg/kg streptozotocin (Sigma-Aldrich, St. Louis, MO) in ice-cold citrate–citric acid buffer (pH 4.5) for 5 days, and control mice received equal amount of buffer. Blood glucose levels were monitored with a OneTouch Basic glucometer (LifeScan; Johnson & Johnson, Milpitas, CA). In the current study, diabetic mice were used after 12 weeks of final streptozotocin injection, at which point the HbA1c values were 10.83 ± 0.74% (94.75 ± 7.93 mmol/mol), whereas normal mice had HbA1c values of 4.25 ± 0.1% (22.5 ± 1 mmol/mol). For topical SP application, 5 μL SP (1 mmol/L; Calbiochem, San Diego, CA) in distilled water was dropped on the corneal surface by a 10 μL tip, six times daily per eye for 4 days (for the measurement of corneal sensitivity) or 4 days prescrape and 3 days postscrape (for the measurement of corneal epithelial wound healing). For NK-1 receptor inhibition, NK-1 receptor antagonist L-733,060 (6.6 μg in 5 μL distilled water; Sigma-Aldrich) was injected subconjunctivally 3 days before corneal sensitivity measurement and corneal epithelium scrape in normal mice, or 24 h before corneal epithelium scrape in diabetic mice according to our preliminary experiments. Control mice were treated with distilled water vehicle.

Corneal Sensitivity

Corneal sensation was measured bilaterally by using a Cochet-Bonnet esthesiometer (Luneau Ophtalmologie, Chartres Cedex, France) in unanesthetized control, diabetic, SP-treated diabetic mice, and the NK-1 receptor antagonist–injected mice before the scrape of corneal epithelium. The testing began with the maximal length (6 cm) of nylon filament and shortened by 0.5 cm each time until the corneal touch threshold was found. The longest filament length resulting in a positive response was considered as the corneal sensitivity threshold, which was verified four times.

Corneal Epithelial Wound Healing

Normal, diabetic, and SP-treated diabetic mice were anesthetized by an intraperitoneal injection of xylazine and ketamine followed by topical application of 2% xylocaine. The entire corneal epithelium including limbal region (marked with 3 mm trephine) was scraped with Alger Brush II corneal rust ring remover (Alger Co., Lago Vista, TX) and subsequently applied with ofoxacin eye drops to avoid infection. Usually, one eye was wounded at a time in each animal. The defects of corneal epithelium were visualized at 24, 48, and 72 h by instilling 0.25% fluorescein sodium and photographed under slit lamp (BQ 900; Haag-Streit, Bern, Switzerland). The staining area was analyzed by using ImageJ software and calculated as the percentage of residual epithelial defect.

Corneal Epithelial Cell Culture and Treatment

Mouse corneal epithelial cell line (TKE2) was provided by Dr. Tetsuya Kawakita of Keio University (Tokyo, Japan) (35). Human corneal tissues were handled according to the tenets of the Declaration of Helsinki. Primary human corneal epithelial cells (HCECs) were established from limbal explants of donor corneas according to a previous report (36). For the analysis of Akt, EGFR, and Sirt1 signaling activation, and the staining of ROS, glutathione (GSH), and mitochondria, both cells were starved overnight in bovine pituitary extract–free keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) and subsequently incubated in 30 mmol/L glucose or mannose (osmotic control) for 3 days with or without 1 μmol/L SP.

Cell Proliferation and Migration Analysis

For the proliferation analysis, the TKE2 cells were starved overnight in bovine pituitary extract–free keratinocyte serum-free medium, treated with high glucose for 3 days in the absence or presence of 1 μmol/L SP, and measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. For the migration analysis, the cells were cultured in high glucose until confluence, subsequently wounded with a micropipette tip, and incubated with or without SP for 24 h. Digital images of wound closure were used for quantitative assessment of migration by using ImageJ software. Each assay was conducted in at least triplicate.

Immunofluorescence Staining

Eyeballs were snap frozen in Tissue-Tek optimum cutting temperature compound (Sakura Finetek, Tokyo, Japan). Frozen corneal sections were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with normal serum for 1 h at room temperature. The samples were stained with primary antibodies overnight at 4°C, washed, and incubated with fluorescein-conjugated secondary antibodies at 37°C for 1 h (antibody information as listed in Supplementary Table 1). All staining was observed under a confocal microscope or an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) after counterstaining with DAPI.

Reverse Transcription Quantitative PCR

Total RNA was extracted from mouse corneal epithelium using NucleoSpin RNA kits (BD Biosciences, Palo Alto, CA). cDNAs were synthesized using the PrimeScript First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Real-time PCR was carried out using SYBR Green reagents and the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The specific primers used are listed in Supplementary Table 2. The cycling conditions were 10 s at 95°C followed by 45 two-step cycles (15 s at 95°C and 1 min at 60°C). The quantification data were analyzed with the Sequence Detection System software (Applied Biosystems) using GAPDH as an internal control.

Western Blot Analysis

Total protein was extracted from the lysed samples of mouse corneal epithelium, cultured TKE2 cells, and HCECs in RIPA buffer. Samples (total protein concentration: 40 μg for mouse corneal epithelium and 45 μg for cultured cells) were run on 12% SDS-PAGE gels and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The blots were blocked by nonfat dry milk for at least 1 h and incubated with primary antibodies (Supplementary Table 1) in Tris-buffered saline with Tween for 1 h at room temperature. The blots were washed three times and incubated with a horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ). Finally, the blots were visualized via enzyme-linked chemiluminescence using the ECL kit (Chemicon, Temecula, CA).

Mitochondria Superoxide and Membrane Potential Staining

For the observation of mitochondrial structure, superoxide generation, and membrane potential, the cells were preloaded with 100 nmol/L MitoTracker Green (Beyotime, Haimen, China) for 30 min, 5 μmol/L MitoSOX Red reagent (Beyotime), and 5 µg/mL 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazole-carbocyanide iodine (JC-1; Beyotime), respectively, for 15 min at 37°C. The fluorescence was observed and captured using a Nikon confocal microscope.

Measurement of Intracellular ROS Generation and GSH Content

For the observation of intracellular ROS and GSH staining, fresh corneal cryostat sections and cultured cells were loaded with 10 μmol/L fluorescence probe 2,7-dichlorodihydrofluorescein diacetate, acetyl ester (DCHF-DA; Molecular Probes, Eugene, OR) and 50 μmol/L monochlorobimane (Sigma-Aldrich), respectively, for 30 min at 37°C. The staining was observed and captured using a Nikon confocal microscope. For the measurement of ROS generation, 50,000 cells were harvested and incubated with 5 μmol/L DCHF-DA for 20 min at 37°C. For the measurement of GSH content, 50,000 cells were harvested and freeze-thawed with liquid nitrogen and a 37°C water bath three times. The supernatant was collected and mixed with the provided working buffer and NADPH. The total GSH content was quantified by comparison with known GSH standards according to the manufacturer’s instructions (Beyotime). The ROS and GSH fluorescence intensity was measured using a Multi-Mode Microplate Reader (SpectraMax M2; Molecular Devices, Menlo Park, CA).

Statistical Analysis

Data in this study were representative of at least three different experiments and presented as the means ± SD. Statistical analysis was performed using SPSS 17.0 software (SPSS, Chicago, IL) and one-way ANOVA. Differences were considered statistically significant at P < 0.05.

SP Promotes Corneal Epithelial Wound Healing and Sensitivity Recovery in Diabetic Mice

To assess the effects of SP on diabetic corneal epithelial wound healing, entire corneal epithelium was scraped in age-matched normal mice and diabetic mice with or without topical SP application for 7 days. Punctate fluorescence staining showed that SP was detected in corneal epithelium after topical application, which suggests the applied SP penetrated the apical tight junction barrier in diabetic mice (Supplementary Fig. 1). The corneal epithelial healing rate exhibited a significant difference from 48 h after corneal epithelium scrape (Fig. 1A). The defect size of corneal epithelium in SP-treated diabetic mice (48 h: 28.78 ± 11.78%; 72 h: 4.44 ± 2.29%, n = 6) was significantly improved from that of diabetic mice (48 h: 63.59 ± 7.85%; 72 h: 22.73 ± 9.85%, n = 6) and reached the equal level of normal mice (48 h: 19.59 ± 5.67%; 72 h: 3.08 ± 2.17%, n = 6) (Fig. 1B). Moreover, the attenuated corneal sensitivity in diabetic mice was also restored by topical SP application, although still being lower than that of normal mice (n = 8 per group) (Fig. 1C). In addition, compared with diabetic mice, more inflammatory cell infiltration was found underneath the corneal epithelium margin of SP-treated diabetic mice at 48 h, whereas it was reduced at 72 h after corneal epithelium scrape (Supplementary Fig. 2). The results suggest that topical-applied SP penetrates into corneal epithelium and promotes corneal epithelial wound healing in diabetic mice, accompanied by recovery of corneal sensitivity, and an early inflammatory and resolution response.

Figure 1

SP promotes corneal epithelial wound healing and restores corneal sensitivity in diabetic mice. Topical SP application for 7 days was used to examine the wound healing rate in diabetic corneal epithelium. The corneal epithelial wound was inflicted after 4 days of SP application and then stained with fluorescein sodium at 24, 48, and 72 h after the corneal epithelium scrape, with continuous daily SP administration in the Diabetic+SP group (A). Histogram of residual epithelial defect is presented as the percentage of the original wound (n = 6 per group) (B). Corneal sensitivity was measured in unanesthetized control, diabetic, and SP-treated diabetic mice after 4 days of topical application (n = 8 per group) (C). *P < 0.05.

Figure 1

SP promotes corneal epithelial wound healing and restores corneal sensitivity in diabetic mice. Topical SP application for 7 days was used to examine the wound healing rate in diabetic corneal epithelium. The corneal epithelial wound was inflicted after 4 days of SP application and then stained with fluorescein sodium at 24, 48, and 72 h after the corneal epithelium scrape, with continuous daily SP administration in the Diabetic+SP group (A). Histogram of residual epithelial defect is presented as the percentage of the original wound (n = 6 per group) (B). Corneal sensitivity was measured in unanesthetized control, diabetic, and SP-treated diabetic mice after 4 days of topical application (n = 8 per group) (C). *P < 0.05.

SP Promotes Corneal Epithelial Cell Migration and Proliferation In Vitro

To assess the effect of SP on corneal epithelial wound healing in vitro, mouse corneal epithelial cells were treated with high glucose with equal concentration of mannose as osmotic control. Subsequently, the confluent cells were wounded and treated with or without SP for another 24 h to analyze the migration rate. The results showed that high glucose treatment caused significant delay of corneal epithelial cell migration, whereas SP addition improved the migration capacity of high glucose–treated cells to the same level of normal cells (n = 3 per group) (Fig. 2A and B). In addition, SP promoted the proliferation rate of corneal epithelial cells that was impaired by high glucose treatment for 3 days (n = 3 per group) (Fig. 2C). The experiments were performed three times with similar results.

Figure 2

SP promotes the migration and proliferation of corneal epithelial cells impaired by high glucose. Confluent mouse corneal epithelial cells were wounded after treatment with 30 mmol/L glucose or mannose for 3 days. Cell migration was observed with or without SP treatment for another 24 h (A), and the migration area was analyzed by ImageJ software (n = 3 per group) (B). Cell proliferation was measured using MTT assay after treatment for 3 days with glucose or mannose in the absence or presence of SP (n = 3 per group) (C). *P < 0.05.

Figure 2

SP promotes the migration and proliferation of corneal epithelial cells impaired by high glucose. Confluent mouse corneal epithelial cells were wounded after treatment with 30 mmol/L glucose or mannose for 3 days. Cell migration was observed with or without SP treatment for another 24 h (A), and the migration area was analyzed by ImageJ software (n = 3 per group) (B). Cell proliferation was measured using MTT assay after treatment for 3 days with glucose or mannose in the absence or presence of SP (n = 3 per group) (C). *P < 0.05.

SP Reactivates Akt, EGFR, and Sirt1 Altered by Hyperglycemia

To elucidate the mechanism underlying the promotion of SP on corneal epithelial wound healing, we investigated the effects of SP on the activation of Akt, EGFR, and Sirt1 of diabetic corneal epithelium. Representative phosphorylated Akt (p-Akt), p-EGFR, and Sirt1 staining is shown in Fig. 3A. The expression levels of p-Akt, p-EGFR, and Sirt1 were significantly upregulated in diabetic corneal epithelium after topical SP application for 4 days (n = 3 per group) (Fig. 3B). Furthermore, SP also upregulated the expression levels of p-Akt, p-EGFR, and Sirt1 in both mouse TKE2 cells and primary HCECs, which were impaired by high glucose treatment (Supplementary Fig. 3). The results suggest that SP application in both diabetic mice and cultured corneal epithelial cells reactivates Akt, EGFR, and Sirt1 altered by hyperglycemia, which may in part explain the promoting mechanisms of SP in diabetic corneal epithelial wound healing.

Figure 3

SP reactivates Akt, EGFR, and Sirt1 in diabetic corneal epithelium. Topical SP application for 4 days was used to examine the reactivation of Akt, EGFR, and Sirt1 in diabetic corneal epithelium. SP recovered the positive staining (A) and upregulated the protein levels of phosphorylated Akt (p-Akt), p-EGFR, and Sirt1 in diabetic corneal epithelium, as compared with that of untreated diabetic mice (n = 3 per group) (B). *P < 0.05.

Figure 3

SP reactivates Akt, EGFR, and Sirt1 in diabetic corneal epithelium. Topical SP application for 4 days was used to examine the reactivation of Akt, EGFR, and Sirt1 in diabetic corneal epithelium. SP recovered the positive staining (A) and upregulated the protein levels of phosphorylated Akt (p-Akt), p-EGFR, and Sirt1 in diabetic corneal epithelium, as compared with that of untreated diabetic mice (n = 3 per group) (B). *P < 0.05.

SP Attenuates Oxidative Stress of Corneal Epithelium by Hyperglycemia

To evaluate the effect of SP on the regulation of hyperglycemia-induced oxidative stress in corneal epithelium, corneal sections were loaded with fluorescence probe DCHF-DA and monochlorobimane for the detection of intracellular ROS and GSH. Representative results showed that a significant increased ROS and reduced GSH staining were detected in diabetic mouse corneal epithelium than in that of normal mice. However, a weak ROS and strong GSH staining of corneal epithelium was found after topical SP application in diabetic mice (Fig. 4A). Moreover, the expression of major intracellular free radical scavengers in corneal epithelium, including manganese superoxide dismutase (MnSOD), catalase, NAD(P)H: quinone oxidoreductase 1 (NQO1), thioredoxin (TXN), and heme oxygenase 1 (Hmox1) at the mRNA transcription level, were recovered from the diabetic group after topical SP application (n = 4 per group) (Fig. 4B). In addition, immunofluorescence staining and Western blot revealed that the protein levels of NQO1, catalase, and MnSOD in diabetic corneal epithelium partially recovered after topical SP application (n = 4 per group) (Fig. 4C and D). The results suggest that topical SP application attenuates hyperglycemia-induced oxidative stress in diabetic corneal epithelium, at least via the mechanism of reducing ROS accumulation and increasing intracellular GSH content and antioxidant gene expression.

Figure 4

SP attenuates oxidative stress of diabetic corneal epithelium. Topical SP application for 4 days was used to examine the attenuation of oxidative stress in diabetic corneal epithelium. SP restored the ROS and GSH staining in diabetic corneal epithelium to similar levels as those in normal corneal epithelium (A). SP elevated the mRNA transcript levels of antioxidant genes MnSOD, catalase, NQO1, GCLC, TXN, and Hmox1 (n = 4 per group) (B), as well as the staining density and protein levels of NQO1, catalase, and MnSOD in diabetic corneal epithelium, as compared with that in untreated diabetic corneal epithelium (n = 4 per group) (C and D). *P < 0.05.

Figure 4

SP attenuates oxidative stress of diabetic corneal epithelium. Topical SP application for 4 days was used to examine the attenuation of oxidative stress in diabetic corneal epithelium. SP restored the ROS and GSH staining in diabetic corneal epithelium to similar levels as those in normal corneal epithelium (A). SP elevated the mRNA transcript levels of antioxidant genes MnSOD, catalase, NQO1, GCLC, TXN, and Hmox1 (n = 4 per group) (B), as well as the staining density and protein levels of NQO1, catalase, and MnSOD in diabetic corneal epithelium, as compared with that in untreated diabetic corneal epithelium (n = 4 per group) (C and D). *P < 0.05.

SP Attenuates Mitochondrial Dysfunction Induced by High Glucose

Mitochondrial dysfunction plays an important role in the progress of various diabetes complications. To evaluate the effects of SP on the mitochondrial dysfunction in corneal epithelium induced by hyperglycemia, cultured corneal epithelial cells were treated with high glucose in the presence or absence of SP for 3 days. Exposure to elevated glucose caused increased ROS accumulation and reduced GSH content of corneal epithelial cells in vitro (Fig. 5A and B), similar to the diabetic corneal epithelium in vivo. However, SP treatment significantly reduced the oxidative stress caused by high glucose in corneal epithelial cells, as showed by decreased ROS accumulation and increased GSH content (n = 3 per group) (Fig. 5A and B). The results were also repeated by using the primary HCECs (Supplementary Fig. 4). Furthermore, compared with normal or mannose-treated cells, high glucose–treated cells assumed apparent mitochondrial superoxide staining (MitoSOX staining in Fig. 5C), accompanied by a significant change of mitochondrial structure (MitoTracker staining in Fig. 5C) and loss of mitochondrial membrane potential (red to green fluorescence of JC-1 staining in Fig. 5C). However, the addition of SP attenuated the generation of mitochondrial superoxide and promoted the recovery of mitochondrial structure and membrane potential that was impaired by high glucose treatment in corneal epithelial cells (Fig. 5C).

Figure 5

SP improves mitochondrial functions of corneal epithelial cells triggered by high glucose. Mouse corneal epithelial cells were treated with 30 mmol/L glucose or mannose for 3 days in the presence or absence of SP. SP recovered the staining density and levels of intracellular ROS and GSH (n = 3 per group) (A and B). SP improved the impaired mitochondrial functions by high glucose (C), including the mitochondrial superoxide (MitoSOX staining), mitochondrial structure (MitoTracker staining), and mitochondrial membrane potential (JC-1 staining). *P < 0.05.

Figure 5

SP improves mitochondrial functions of corneal epithelial cells triggered by high glucose. Mouse corneal epithelial cells were treated with 30 mmol/L glucose or mannose for 3 days in the presence or absence of SP. SP recovered the staining density and levels of intracellular ROS and GSH (n = 3 per group) (A and B). SP improved the impaired mitochondrial functions by high glucose (C), including the mitochondrial superoxide (MitoSOX staining), mitochondrial structure (MitoTracker staining), and mitochondrial membrane potential (JC-1 staining). *P < 0.05.

NK-1 Receptor Mediates the Promotion of SP on Diabetic Corneal Epithelial Wound Healing

To assess if the NK-1 receptor mediates the improvements of SP on diabetic corneal epithelial wound healing, the NK-1 receptor–specific antagonist L-733,060 was injected before topical SP application in diabetic mice. In unwounded mouse corneal epithelium, the staining density of p-Akt, p-EGFR, and Sirt1 was attenuated in antagonist-injected SP-treated diabetic mice, as compared with the diabetic mice treated with SP alone (Fig. 6A). In corneal epithelium–scraped mice, the antagonist injection before SP application fully reversed the promotion of SP on diabetic corneal epithelial wound healing, with 28.95 ± 3.89% epithelial defect in antagonist-injected SP-treated mice as compared with only 4.44 ± 2.29% defect in mice treated with SP alone and with 22.73 ± 9.85% defect in untreated diabetic mice at 72 h (n = 5 per group) (Fig. 6B). Moreover, at 72 h postscrape, a stronger staining intensity of p-Akt and proliferation marker Ki-67 was found in the migrating area of SP-treated diabetic corneal epithelium than in that of either untreated or antagonist-injected diabetic corneal epithelium (Fig. 6C). The results suggest that the NK-1 receptor mediates the reactivation of Akt, EGFR, and Sirt1 by SP, and that the promotion of SP on diabetic corneal epithelial wound healing is also NK-1 receptor mediated.

Figure 6

NK-1 receptor antagonist blocks the promotion of SP on diabetic corneal epithelial wound healing. NK-1 receptor antagonist L-733,060 was injected subconjunctivally at 24 h before topical SP application in diabetic mice. In the unwounded corneal epithelium, the elevation of p-Akt, p-EGFR, and Sirt1 staining density by SP application was attenuated in antagonist-injected SP-treated diabetic mice (A). In the corneal epithelium 72 h after scrape, the antagonist injection reversed the promotion of SP on diabetic corneal epithelial wound healing (n = 5 per group) (B) and the staining intensity of p-Akt and the proliferation marker Ki-67 in the regenerated corneal epithelium (C). *P < 0.05.

Figure 6

NK-1 receptor antagonist blocks the promotion of SP on diabetic corneal epithelial wound healing. NK-1 receptor antagonist L-733,060 was injected subconjunctivally at 24 h before topical SP application in diabetic mice. In the unwounded corneal epithelium, the elevation of p-Akt, p-EGFR, and Sirt1 staining density by SP application was attenuated in antagonist-injected SP-treated diabetic mice (A). In the corneal epithelium 72 h after scrape, the antagonist injection reversed the promotion of SP on diabetic corneal epithelial wound healing (n = 5 per group) (B) and the staining intensity of p-Akt and the proliferation marker Ki-67 in the regenerated corneal epithelium (C). *P < 0.05.

Local Injection of NK-1 Receptor Antagonist Causes Diabetic Pathological Changes in Normal Mice

To assess the effects of SP-NK-1 receptor signaling inhibition on corneal epithelium, L-733,060 was injected subconjunctivally in normal mice. After 3 days of antagonist injection, the unwounded corneal epithelium assumed a similar attenuation of p-Akt, p-EGFR, and Sirt1 staining as that in diabetic mice (Fig. 7A), accompanied by decreased corneal sensitivity (n = 5 per group) (Fig. 7B). After 72 h of corneal epithelium scrape, the antagonist-injected mice showed a significant delay of corneal epithelial wound healing, with 31.77 ± 5.07% epithelial defect in antagonist-injected mice as compared with only 3.08 ± 2.17% defect in control mice (n = 5 per group) (Fig. 7C), and attenuated p-Akt and Ki-67 staining in the migrating area of corneal epithelium as compared with that in normal mice (Fig. 7D), which was similar to the pathological changes in diabetic corneal epithelium (Fig. 7C and D). Similar results were also obtained with the injection of another NK-1 receptor antagonist spantide I (data not shown). The results show that local injection of NK-1 receptor antagonist in normal mice causes similar pathological changes in corneal epithelial wound healing and corneal sensitivity as that seen in diabetic mice, suggesting that normal activation of SP-NK-1 receptor signaling plays a critical role in the homeostasis of corneal epithelium and corneal sensation.

Figure 7

Local inhibition of SP-NK-1 receptor signaling causes diabetic-like pathological changes in the cornea of normal mice. NK-1 receptor antagonist L-733,060 was injected subconjunctivally in normal mice. In the unwounded corneal epithelium 3 days after antagonist injection, the staining density of p-Akt, p-EGFR, and Sirt1 (A) and corneal sensitivity (B) (n = 5 per group) were attenuated similarly to that in diabetic mice. In the corneal epithelium 72 h after scrape, the local antagonist injection caused a significant delay of corneal epithelial wound healing (n = 5 per group) (C) as well as attenuated p-Akt and Ki-67 staining density in the regenerated corneal epithelium (D). *P < 0.05.

Figure 7

Local inhibition of SP-NK-1 receptor signaling causes diabetic-like pathological changes in the cornea of normal mice. NK-1 receptor antagonist L-733,060 was injected subconjunctivally in normal mice. In the unwounded corneal epithelium 3 days after antagonist injection, the staining density of p-Akt, p-EGFR, and Sirt1 (A) and corneal sensitivity (B) (n = 5 per group) were attenuated similarly to that in diabetic mice. In the corneal epithelium 72 h after scrape, the local antagonist injection caused a significant delay of corneal epithelial wound healing (n = 5 per group) (C) as well as attenuated p-Akt and Ki-67 staining density in the regenerated corneal epithelium (D). *P < 0.05.

The cornea is one of the most densely innervated parts of the human body, and as such, its sensory nerves play not only a prominent role in nociception but also in providing trophism to the corneal tissue. In diabetes, corneal sensitivity, nerve fiber density, and epithelial wound healing are reduced significantly (27,31,32). However, the mechanisms are not completely understood. In the current study, we found that SP promoted epithelial wound healing, stimulated the reactivation of Akt, EGFR, and Sirt1, as well as attenuated oxidative stress in diabetic corneal epithelium. Furthermore, the study shows that the impairment of SP-NK-1 receptor signaling causes changes of normal corneal epithelium similar to those of diabetic mice. The results suggest that SP-NK-1 receptor signaling regulates the activation of multiple signaling pathways that are needed for corneal epithelial wound healing, whereas this regulation is impaired in diabetic corneal epithelium and rescued by SP via an autoregulatory mechanism (37,38). Moreover, SP restored the corneal sensitivity of diabetic mice close to the same level of normal mice, whereas local inhibition of SP-NK-1 receptor signaling caused decreased corneal sensitivity in normal mice similar to that in diabetic mice. The results suggest that the impairment of SP-NK-1 receptor signaling may also be involved in the attenuation of corneal sensitivity in diabetes, which is supported by the fact that the NK-1 receptor exists in peripheral nerve (39). Taken together, SP, secreted by corneal sensory nerve fibers, may be the key neurotransmitter and neuropeptide that mediates corneal nociception transmission and provides trophism to the corneal epithelium. Furthermore, as for the treatment of diabetic keratopathy, many growth factors, cytokines, and various agents have been evaluated for their capacity to accelerate corneal wound healing (40). However, SP and its functional derivative (FGLM-amide), with the advantages of smaller molecules and higher efficiency, have been shown to be effective for the treatment of persistent corneal epithelial defects in clinical studies (4042). In addition, we found that SP promoted the regeneration of nerve fibers in diabetic corneal epithelium and accelerated trigeminal neuronal growth in vitro that was impaired by high glucose (Supplementary Fig. 5).

The NK-1 receptor, the preferred receptor of SP, mediates a variety of physiological and pathophysiological responses (22,23,43). Although previous studies have confirmed that SP enhances corneal epithelial migration when combined with IGF-1 or EGF (41,44,45), the exact mechanism remains unclear. Here we show that SP reactivates Akt, EGFR, and Sirt1 and promotes ROS scavenging capacity that are impaired by hyperglycemia. The results suggest a molecular basis for the synergistic effects of SP and IGF-1 or EGF on the enhancement of diabetic corneal epithelial wound healing (46,47). Even more interestingly, we found that the local inhibition of the SP-NK-1 receptor signaling in normal mice causes pathological changes of the corneal epithelium similar to those of diabetic corneal epithelium. These results suggest that SP-NK-1 receptor signaling may be involved in the maintenance of corneal epithelium homeostasis and also in the protection from hyperglycemia stress, whereas the impairment of SP-NK-1 receptor signaling may explain the fragility of diabetic corneal epithelium in vivo.

Prolonged hyperglycemia always causes the perturbation of catabolic pathways and the overproduction of ROS in the mitochondria, which in turn plays a critical role in the development of diabetes complications (48), including diabetic keratopathy (5). In the current study, we confirmed that the mitochondrial superoxide level was upregulated in high glucose–treated corneal epithelial cells, accompanied by changes of mitochondria structure and loss of mitochondrial membrane potential. Interestingly, we found that SP attenuates the dysfunction of the mitochondria by high glucose. Moreover, SP also elevates the intracellular GSH level, the main antioxidant in the cells. In addition, although increased Nrf2 expression was detected in diabetic corneal epithelium (data not shown), the expressions of Nrf2 downstream antioxidant genes, including MnSOD, catalase, NQO1, TXN, and Hmox1, were downregulated in diabetic corneal epithelium, whereas they were upregulated after SP application, which suggests that additional regulatory mechanisms may exist between Nrf2 and its downstream antioxidant gene expressions in diabetes (49,50). All things considered, the improvement of oxidative stress by SP via the improved mitochondrial function, elevated GSH level, and upregulated expression of antioxidant genes may also play an important role in the protection of corneal epithelium in diabetes.

In conclusion, our study demonstrates, for the first time, that SP promotes diabetic corneal epithelial wound healing while simultaneously triggering the reactivation of the Akt, EGFR, and Sirt1 signaling of importance for that healing, as well as rescuing corneal sensation, improving mitochondrial function, and decreasing ROS accumulation caused by hyperglycemia. Furthermore, we show that local inhibition of SP-NK-1 receptor signaling abolishes the promotion of SP on corneal epithelial healing in diabetic mice and causes diabetic corneal pathological changes in normal mice. Thus, the SP-NK-1 receptor signaling may play a critical role in the maintenance of corneal epithelium homeostasis, and SP signaling may, through the NK-1 receptor, contribute to the promotion of diabetic corneal epithelial wound healing by the rescued activation of Akt, EGFR, and Sirt1, the improvement of mitochondrial function, and the increased ROS scavenging capacity of corneal epithelium.

Acknowledgments. The authors thank Yangyang Zhang, Wenjie Sui, Qian Wang, Hua Gao, Suxia Li, and Zhaoli Chen (Shandong Eye Institute) for their help with the animal experiments, statistical analysis, and human tissue collection.

Funding. This work was partially supported by the National Basic Research Program of China (2012CB722409) and the National Natural Science Foundation of China (81170816 and 81200665). Q.Z. is partially supported by the Shandong Provincial Excellent Innovation Team Program and Taishan Scholar Program (20081148). P.D. is partially supported by the J.C. Kempe and Seth M. Kempe Memorial Foundations, the Swedish Society of Medicine, the Cronqvist and KMA Foundations, and the National Swedish Research Council (521-2013-2612, Q.Z. coapplicant).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. L.Y. and G.D. contributed to sample testing, data analysis, and study design. X.Q., M.Q., Y.W., and H.D. contributed to sample testing and data analysis. P.D., L.X., and Q.Z. contributed to study design, data analysis, and manuscript preparation. L.X. and Q.Z. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Clark
CM
 Jr
,
Lee
DA
.
Prevention and treatment of the complications of diabetes mellitus
.
N Engl J Med
1995
;
332
:
1210
1217
[PubMed]
2.
Schultz
RO
,
Van Horn
DL
,
Peters
MA
,
Klewin
KM
,
Schutten
WH
.
Diabetic keratopathy
.
Trans Am Ophthalmol Soc
1981
;
79
:
180
199
[PubMed]
3.
Ljubimov
AV
,
Huang
ZS
,
Huang
GH
, et al
.
Human corneal epithelial basement membrane and integrin alterations in diabetes and diabetic retinopathy
.
J Histochem Cytochem
1998
;
46
:
1033
1041
[PubMed]
4.
Rehany
U
,
Ishii
Y
,
Lahav
M
,
Rumelt
S
.
Ultrastructural changes in corneas of diabetic patients: an electron-microscopy study
.
Cornea
2000
;
19
:
534
538
[PubMed]
5.
Xu
KP
,
Li
Y
,
Ljubimov
AV
,
Yu
FS
.
High glucose suppresses epidermal growth factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and attenuates corneal epithelial wound healing
.
Diabetes
2009
;
58
:
1077
1085
[PubMed]
6.
Xu
K
,
Yu
FS
.
Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats
.
Invest Ophthalmol Vis Sci
2011
;
52
:
3301
3308
[PubMed]
7.
Wang
Y
,
Zhao
X
,
Shi
D
, et al
.
Overexpression of SIRT1 promotes high glucose-attenuated corneal epithelial wound healing via p53 regulation of the IGFBP3/IGF-1R/AKT pathway
.
Invest Ophthalmol Vis Sci
2013
;
54
:
3806
3814
[PubMed]
8.
Forbes
JM
,
Cooper
ME
.
Mechanisms of diabetic complications
.
Physiol Rev
2013
;
93
:
137
188
[PubMed]
9.
Gorin
Y
,
Block
K
.
Nox as a target for diabetic complications
.
Clin Sci (Lond)
2013
;
125
:
361
382
[PubMed]
10.
Anderson
EJ
,
Kypson
AP
,
Rodriguez
E
,
Anderson
CA
,
Lehr
EJ
,
Neufer
PD
.
Substrate-specific derangements in mitochondrial metabolism and redox balance in the atrium of the type 2 diabetic human heart
.
J Am Coll Cardiol
2009
;
54
:
1891
1898
[PubMed]
11.
Song
B
,
Scheuner
D
,
Ron
D
,
Pennathur
S
,
Kaufman
RJ
.
Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes
.
J Clin Invest
2008
;
118
:
3378
3389
[PubMed]
12.
Bhatt
MP
,
Lim
YC
,
Hwang
J
,
Na
S
,
Kim
YM
,
Ha
KS
.
C-peptide prevents hyperglycemia-induced endothelial apoptosis through inhibition of reactive oxygen species-mediated transglutaminase 2 activation
.
Diabetes
2013
;
62
:
243
253
[PubMed]
13.
Uruno
A
,
Furusawa
Y
,
Yagishita
Y
, et al
.
The Keap1-Nrf2 system prevents onset of diabetes mellitus
.
Mol Cell Biol
2013
;
33
:
2996
3010
[PubMed]
14.
Kim
J
,
Kim
CS
,
Kim
H
,
Jeong
IH
,
Sohn
E
,
Kim
JS
.
Protection against advanced glycation end products and oxidative stress during the development of diabetic keratopathy by KIOM-79
.
J Pharm Pharmacol
2011
;
63
:
524
530
[PubMed]
15.
Mantelli
F
,
Micera
A
,
Sacchetti
M
,
Bonini
S
.
Neurogenic inflammation of the ocular surface
.
Curr Opin Allergy Clin Immunol
2010
;
10
:
498
504
[PubMed]
16.
Tervo
K
,
Tervo
T
,
Eränkö
L
,
Eränkö
O
.
Substance P immunoreactive nerves in the rodent cornea
.
Neurosci Lett
1981
;
25
:
95
97
[PubMed]
17.
Miller
A
,
Costa
M
,
Furness
JB
,
Chubb
IW
.
Substance P immunoreactive sensory nerves supply the rat iris and cornea
.
Neurosci Lett
1981
;
23
:
243
249
[PubMed]
18.
Twardy
BS
,
Channappanavar
R
,
Suvas
S
.
Substance P in the corneal stroma regulates the severity of herpetic stromal keratitis lesions
.
Invest Ophthalmol Vis Sci
2011
;
52
:
8604
8613
[PubMed]
19.
Hong
HS
,
Lee
J
,
Lee
E
, et al
.
A new role of substance P as an injury-inducible messenger for mobilization of CD29(+) stromal-like cells
.
Nat Med
2009
;
15
:
425
435
[PubMed]
20.
Lan
Y
,
Kodati
S
,
Lee
HS
,
Omoto
M
,
Jin
Y
,
Chauhan
SK
.
Kinetics and function of mesenchymal stem cells in corneal injury
.
Invest Ophthalmol Vis Sci
2012
;
53
:
3638
3644
[PubMed]
21.
Castagliuolo
I
,
Morteau
O
,
Keates
AC
, et al
.
Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor
.
Br J Pharmacol
2002
;
136
:
271
279
[PubMed]
22.
Koon
HW
,
Zhao
D
,
Zhan
Y
,
Moyer
MP
,
Pothoulakis
C
.
Substance P mediates antiapoptotic responses in human colonocytes by Akt activation
.
Proc Natl Acad Sci U S A
2007
;
104
:
2013
2018
[PubMed]
23.
Janelsins
BM
,
Mathers
AR
,
Tkacheva
OA
, et al
.
Proinflammatory tachykinins that signal through the neurokinin 1 receptor promote survival of dendritic cells and potent cellular immunity
.
Blood
2009
;
113
:
3017
3026
[PubMed]
24.
Backman
LJ
,
Danielson
P
.
Akt-mediated anti-apoptotic effects of substance P in Anti-Fas-induced apoptosis of human tenocytes
.
J Cell Mol Med
2013
;
17
:
723
733
[PubMed]
25.
Zhou
Z
,
Barrett
RP
,
McClellan
SA
, et al
.
Substance P delays apoptosis, enhancing keratitis after Pseudomonas aeruginosa infection
.
Invest Ophthalmol Vis Sci
2008
;
49
:
4458
4467
[PubMed]
26.
An
YS
,
Lee
E
,
Kang
MH
, et al
.
Substance P stimulates the recovery of bone marrow after the irradiation
.
J Cell Physiol
2011
;
226
:
1204
1213
[PubMed]
27.
Müller
LJ
,
Marfurt
CF
,
Kruse
F
,
Tervo
TM
.
Corneal nerves: structure, contents and function
.
Exp Eye Res
2003
;
76
:
521
542
[PubMed]
28.
Wang
F
,
Gao
N
,
Yin
J
,
Yu
FS
.
Reduced innervation and delayed re-innervation after epithelial wounding in type 2 diabetic Goto-Kakizaki rats
.
Am J Pathol
2012
;
181
:
2058
2066
[PubMed]
29.
Okada
Y
,
Reinach
PS
,
Kitano
A
,
Shirai
K
,
Kao
WW
,
Saika
S
.
Neurotrophic keratopathy; its pathophysiology and treatment
.
Histol Histopathol
2010
;
25
:
771
780
[PubMed]
30.
Dogru
M
,
Katakami
C
,
Inoue
M
.
Tear function and ocular surface changes in noninsulin-dependent diabetes mellitus
.
Ophthalmology
2001
;
108
:
586
592
[PubMed]
31.
He
J
,
Bazan
HE
.
Mapping the nerve architecture of diabetic human corneas
.
Ophthalmology
2012
;
119
:
956
964
[PubMed]
32.
Tavakoli
M
,
Kallinikos
PA
,
Efron
N
,
Boulton
AJ
,
Malik
RA
.
Corneal sensitivity is reduced and relates to the severity of neuropathy in patients with diabetes
.
Diabetes Care
2007
;
30
:
1895
1897
[PubMed]
33.
Hossain
P
,
Sachdev
A
,
Malik
RA
.
Early detection of diabetic peripheral neuropathy with corneal confocal microscopy
.
Lancet
2005
;
366
:
1340
1343
[PubMed]
34.
Marfurt
CF
,
Echtenkamp
SF
.
The effect of diabetes on neuropeptide content in the rat cornea and iris
.
Invest Ophthalmol Vis Sci
1995
;
36
:
1100
1106
[PubMed]
35.
Kawakita
T
,
Shimmura
S
,
Hornia
A
,
Higa
K
,
Tseng
SC
.
Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell
.
J Cell Mol Med
2008
;
12
:
1303
1316
[PubMed]
36.
Kim
HS
,
Jun Song
X
,
de Paiva
CS
,
Chen
Z
,
Pflugfelder
SC
,
Li
DQ
.
Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures
.
Exp Eye Res
2004
;
79
:
41
49
[PubMed]
37.
Koh
YH
,
Moochhala
S
,
Bhatia
M
.
Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells
.
J Cell Mol Med
2012
;
16
:
1582
1592
[PubMed]
38.
Goode
T
,
O’Connor
T
,
Hopkins
A
, et al
.
Neurokinin-1 receptor (NK-1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P
.
J Cell Physiol
2003
;
197
:
30
41
[PubMed]
39.
Steinhoff
MS
,
von Mentzer
B
,
Geppetti
P
,
Pothoulakis
C
,
Bunnett
NW
.
Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease
.
Physiol Rev
2014
;
94
:
265
301
[PubMed]
40.
Abdelkader
H
,
Patel
DV
,
McGhee
CNj
,
Alany
RG
.
New therapeutic approaches in the treatment of diabetic keratopathy: a review
.
Clin Experiment Ophthalmol
2011
;
39
:
259
270
[PubMed]
41.
Yamada
N
,
Matsuda
R
,
Morishige
N
, et al
.
Open clinical study of eye-drops containing tetrapeptides derived from substance P and insulin-like growth factor-1 for treatment of persistent corneal epithelial defects associated with neurotrophic keratopathy
.
Br J Ophthalmol
2008
;
92
:
896
900
[PubMed]
42.
Chikama
T
,
Fukuda
K
,
Morishige
N
,
Nishida
T
.
Treatment of neurotrophic keratopathy with substance-P-derived peptide (FGLM) and insulin-like growth factor I
.
Lancet
1998
;
351
:
1783
1784
[PubMed]
43.
Quartara
L
,
Maggi
CA
.
The tachykinin NK1 receptor. Part II: Distribution and pathophysiological roles
.
Neuropeptides
1998
;
32
:
1
49
[PubMed]
44.
Nakamura
M
,
Chikama
T
,
Nishida
T
.
Synergistic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea
.
Br J Pharmacol
1999
;
127
:
489
497
[PubMed]
45.
Nakamura
M
,
Nishida
T
,
Ofuji
K
,
Reid
TW
,
Mannis
MJ
,
Murphy
CJ
.
Synergistic effect of substance P with epidermal growth factor on epithelial migration in rabbit cornea
.
Exp Eye Res
1997
;
65
:
321
329
[PubMed]
46.
Yamada
N
,
Yanai
R
,
Inui
M
,
Nishida
T
.
Sensitizing effect of substance P on corneal epithelial migration induced by IGF-1, fibronectin, or interleukin-6
.
Invest Ophthalmol Vis Sci
2005
;
46
:
833
839
[PubMed]
47.
Nakamura
M
,
Ofuji
K
,
Chikama
T
,
Nishida
T
.
The NK1 receptor and its participation in the synergistic enhancement of corneal epithelial migration by substance P and insulin-like growth factor-1
.
Br J Pharmacol
1997
;
120
:
547
552
[PubMed]
48.
Blake
R
,
Trounce
IA
.
Mitochondrial dysfunction and complications associated with diabetes
.
Biochim Biophys Acta
2014;1840:1404–1412
[PubMed]
49.
Cheng
X
,
Chapple
SJ
,
Patel
B
, et al
.
Gestational diabetes mellitus impairs Nrf2-mediated adaptive antioxidant defenses and redox signaling in fetal endothelial cells in utero
.
Diabetes
2013
;
62
:
4088
4097
[PubMed]
50.
He
X
,
Ma
Q
.
Redox regulation by nuclear factor erythroid 2-related factor 2: gatekeeping for the basal and diabetes-induced expression of thioredoxin-interacting protein
.
Mol Pharmacol
2012
;
82
:
887
897
[PubMed]

Supplementary data