Diabetic keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by resiniferatoxin severely impaired corneal wound healing and markedly upregulated proinflammatory gene expression. Exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) partially reversed resiniferatoxin’s effects, with VIP specifically inducing interleukin-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIP type 1 receptor (VIPR1) expression in normal (NL) corneas, but not corneas from mice with diabetes mellitus (DM). Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses, while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (Shh) in a VIP-dependent manner. Downregulating Shh expression in NL corneas decreased while exogenous Shh in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of Shh signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in an Shh-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy.

Diabetic peripheral neuropathy is a common and debilitating complication, with a majority of the patients with diabetes developing altered sensation because of damage to the peripheral sensory nerves (SNs) (1). The cornea is the most densely innervated mammalian tissue by small-diameter C-fiber sensory neurons (1,2). As such, a decrease in corneal sensitivity is observed shortly after the onset of diabetes in some patients (3), and decreases in nerve fiber bundle counts precede the impairment of corneal sensitivity (4,5). This nerve damage, termed diabetic neurotrophic keratopathy (DNK), is known to be directly related to the severity of somatic neuropathy in patients with diabetes (6,7). Corneal nerve fiber loss contributes to the development of corneal epitheliopathy/keratopathy, which entails epithelial edema, fragility, superficial punctate keratitis, and delayed and incomplete wound repair (4,8,9). To date, how SN influences the pathogenesis of DNK remains elusive.

Using an epithelium-debridement wound model, studies have shown that diabetes causes a significant decrease in epithelium healing rate (2,4,10). Diabetes also causes defects in SN, including a decrease in the density of nerve endings and in the numbers of branches that innervate the epithelium (7,11,12). In normal (NL) corneas, SN regeneration is robust, starting near the limbus with newly formed SN fibers/endings arranged in parallel and extending radially toward the center of the cornea (2,7,13). In corneas from mice with diabetes mellitus (DM), SN regeneration is severely delayed, with both fewer nerve insertion sites and tortuous, fragmented regenerating SNs fibers (7), similar to that observed in corneas from humans with diabetes (14). The observed decrease in the density of SN fibers/endings suggests a decrease in the content of neuropeptides, substance P (SP), and calcitonin gene-related peptide (CGRP), as well as vasoactive intestinal peptide (VIP) in DM corneas.

SNs release neuropeptides to support corneal epithelial cells (CECs) and dendritic cells (DCs) in the corneas, thus maintaining corneal homeostasis (15). Wounding is known to increase the expression and release of SP, CGRP, and VIP secreted from SN in different tissues, including the cornea (1619). These factors in turn mediate the epithelium response to wounding. In the cornea, SP was reported to promote epithelial wound healing in NL and DM corneas in the presence of IGF (20). CGRP, in contrast, was shown to have anti-inflammatory effects by regulating DC activation (21), including the inhibition of CD86 and tumor necrosis factor-α expression (22). In the cornea, CGRP increases the rate of corneal re-epithelialization in a whole-mount preparation (23). Unlike the ubiquitously expressed SP and CGRP, VIP expression in dorsal root ganglions is induced by axotomy and further enhanced by nerve growth factor (NGF) (24). VIP promotes the survival of peripheral neurons in vitro and inhibits neuronal cell death after injury (25). In the cornea, VIP exhibits protective effects on wound healing of alkali-burned corneas, microbial keratitis, and corneal allograft survival (2628). As for diabetes, VIP receptor subtype–specific agonists (VIP1Ra) have been suggested as a potential treatment for type 2 diabetes (29). To date, the involvement of VIP in mediating corneal wound healing, including epithelial wound closure and SN regeneration, and the underlying mechanisms therein remain to be determined.

In this study, we first assessed the effects of three neuropeptides in restoring corneal epithelial wound closure in locally denervated corneas and then focused on defining the role of VIP in promoting hyperglycemia-attenuated wound healing in C57BL/6 (B6) mouse corneas. Our results showed that VIP induced the expression of neurotrophic factors NGF and ciliary neurotrophic factor (CNTF) and anti-inflammatory cytokines and suppressed the expression of proinflammatory cytokines in the cornea. Moreover, we demonstrated that Sonic Hedgehog (Shh) is an important downstream signaling molecule of VIP/VIP type 1 receptor (VIPR1) pathway that is required for proper wound healing in the corneas.

Materials

Primary antibodies were Shh (ab19897; Abcam), phosphorylated extracellular signal–regulated kinase (p-ERK) (4370; Cell Signaling Technology), ERK2 (sc65981; Santa Cruz Biotechnology), nonmuscle β-actin and Ly6G-FITC (A1978 and SAB4700622; Sigma-Aldrich), VIP (63269; Cell Signaling Technology), and β-tubulin III (NL557; R&D Systems). All conjugated secondary antibodies were from Jackson ImmunoResearch. VIP (6-28) (catalog number 1905/1), selective VIPR1 antagonist (catalog number 3054), CGRP (catalog number 1161), and SP (catalog number 1156) were purchased from Tocris Bioscience.

Animals and Induction of Diabetes

All animal studies were approved by the Wayne State University Institutional Animal Care and Use Committee. All investigations conformed to the regulations of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the National Institutes of Health (NIH). Eight-week-old female B6 mice purchased from The Jackson Laboratory were induced to develop diabetes following Diabetic Complications Consortium’s Low-Dose Streptozotocin Induction Protocol (mouse, 50 mg streptozotocin [STZ]/kg, daily for 5 days) without fasting prior to STZ injections. Animals with blood glucose levels >350 mg/dL were considered diabetic. If at day 3 post–STZ injection, blood glucose levels were <250 mg/dL, an additional two injections would be performed. Animals with blood glucose levels >350 mg/dL at week 3 after initial STZ injection were considered diabetic. Mice, with age-matched animals as the controls, 10 weeks or thereafter of initial STZ injections will be used for the wound healing study.

Induction of Corneal Denervation and Creation of Epithelial Wound

C57BL/6 mice were treated with one drop of the solution containing 0.1% Tween-80 and 0.05% ascorbic acid with or without resiniferatoxin (RTX; 200 ng/μL) after being anesthetized by an intraperitoneal injection of xylazine (7 mg/kg) and ketamine (70 mg/kg) plus topical proparacaine. Eyes were washed with PBS 10 min after the treatment, and corneal sensitivity was measured using an aesthesiometer (Luneau Ophtalmologie) 1 day after the treatment and processed for whole-mount confocal microscopy (WMCM) of SN density. To make an epithelial wound, 2 mm trephine was used, followed by CEC removal within the circle. The collected cells were marked as unwounded. The progress of wound healing was monitored by fluorescence staining for epithelial defects and photographed. At the end of healing, the corneas were processed for immunohistochemistry, WMCM, quantitative PCR (qPCR), and/or Western blotting.

RNA Extraction and PCR Analysis

RNA was extracted from collected corneas or CECs using an RNeasy Mini Kit (Qiagen). cDNA was generated with an oligo(dT) primer, followed by analysis using real-time quantitative PCR (qPCR) with SYBR Green (StepOnePlus; Applied Biosystems). Primer sequences are listed in Supplementary Table 1.

Western Blotting

Twenty-microgram CEC lysates were separated with 5–15% gradient SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibody, followed by incubation with horseradish peroxidase–conjugated secondary antibody. The bands were visualized with enhanced SuperSignal chemiluminescence and the images acquired using a Kodak Image Station 4000R Pro.

Immunohistochemistry, Whole-Mount Immunostaining, and Quantitation of Innervation and Neutrophil Infiltration

OCT compound–embedded mouse corneas were cut 7-μm thick, mounted to poly-L-lysine–coated glass slides, and fixed in 4% paraformaldehyde. Whole-mount immunostaining and quantitation of innervation and numbers of neutrophils of B6 mouse corneas were performed as described (7). Briefly, corneas were cut radially into six standardized sections and incubated at 37°C in 20 mmol/L EDTA for 30 min, followed by 2-day incubation in 0.025% hyaluronidase and 0.1% EDTA in PBS. The tissues or cryostat sections were blocked at room temperature for 2 h in PBS–Triton X-100 containing 2% BSA, followed by incubation overnight at 4°C with primary antibodies. The following morning, the corneas or sections were rinsed with PBS, followed by an overnight incubation at 4°C with a secondary antibody. The rinsed corneas were mounted and examined under a confocal microscope (TCS SP2; Leica Biosystems, Heidelberg, Germany).

Statistical Analysis

The statistical analyses were performed with GraphPad Prism 6 software. Data were presented as means ± SD. Experiments with more than two conditions were analyzed using one- or two-way ANOVA. A Bonferroni post-test was performed to determine statistically significant differences. Significance was accepted at P < 0.05. Experiments were repeated at least twice to ensure reproducibility.

Data and Resource Availability

Data and critical resources supporting the results reported in the article will be availabile upon request after its official publication. A standard material transfer agreement may be required for related materials to be transferred.

Neuropeptides Augment Epithelial Wound Closure in RTX-Denervated Mouse Corneas

To assess SN contribution to corneal wound healing, B6 mouse eyes were treated with eye drops containing RTX, a potent TRPV1 agonist (∼10 µL, 200 ng/μL). WMCM revealed a reduced density of SN endings (Fig. 1A) in RTX-treated corneas (13.49 ± 2.13% pixel density at the central cornea in RTX-treated eyes vs. 38.05 ± 1.75% pixel density in the controls) (Fig. 1B). Manual counting and measuring revealed 210 nerve endings with average 940 μm in length in controls versus 143 endings with 687 μm in RTX-treated corneas. RTX appeared to have no effects on the subbasal nerve fibers. Consistent with the loss of SN density, corneal sensitivity was undetectable in RTX-treated eyes (Fig. 1C). Corneal denervation also resulted in a decreased epithelial healing rate (47.93 ± 6.90% in RTX-treated vs. 84.11 ± 2.70% coverage of the original wound area in the control corneas). Treatments with CGRP, SP, or VIP resulted in significantly higher healing rates (68.39 ± 4.3%, 69.12 ± 10.94%, and 81.66 ± 3.28% vs. 47.93 ± 6.90%, respectively; P ≤ 0.001) in RTX-treated corneas (Fig. 1D and E). The rate for VIP-treated corneas was significantly higher than in CGRP- and SP-treated corneas (P ≤ 0.05).

Figure 1

Neuropeptides enhance severely impaired epithelial wound healing in the denervated B6 mouse corneas. A: Anesthetized mice were treated with one drop of the vehicle or 200 ng/μL RTX for 10 min. Control (CT) and RTX-treated mouse corneas were collected and subjected to WMCM with β-tubulin III staining. A (top): Whole corneal images of SN fibers/endings. A (bottom): High-magnification images showing the central, marked areas of the corneas. Scale bars are as indicated. B: Nerve densities at the central areas (bottom panel in A) were calculated from the areas covered with β-tubulin III staining with ImageJ and presented as percent areas (mean ± SD; n = 4). ***P < 0.001 (Student t test). Two independent experiments were performed. C: The sensitivity of the CT and RTX-treated corneas was measured using an aesthesiometer and presented as the length of the threads (maximum 6 cm). D: Control or RTX-treated corneas pretreated with recombinant CGRP (100 ng/eye), SP (250 ng/eye), and VIP (250 ng/eye) or BSA as the control 4 h prior to epithelium debridement. At 0 h (0H), the corneas were wounded by epithelium debridement (2-mm diameter). At 22 hpw (22H), the remaining wounds were stained with fluorescein and photographed. E: The wound sizes were calculated using ImageJ and presented as percentage of healed area over the size of the original wound (mean ± SD; n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). RTXW, RTX wounded.

Figure 1

Neuropeptides enhance severely impaired epithelial wound healing in the denervated B6 mouse corneas. A: Anesthetized mice were treated with one drop of the vehicle or 200 ng/μL RTX for 10 min. Control (CT) and RTX-treated mouse corneas were collected and subjected to WMCM with β-tubulin III staining. A (top): Whole corneal images of SN fibers/endings. A (bottom): High-magnification images showing the central, marked areas of the corneas. Scale bars are as indicated. B: Nerve densities at the central areas (bottom panel in A) were calculated from the areas covered with β-tubulin III staining with ImageJ and presented as percent areas (mean ± SD; n = 4). ***P < 0.001 (Student t test). Two independent experiments were performed. C: The sensitivity of the CT and RTX-treated corneas was measured using an aesthesiometer and presented as the length of the threads (maximum 6 cm). D: Control or RTX-treated corneas pretreated with recombinant CGRP (100 ng/eye), SP (250 ng/eye), and VIP (250 ng/eye) or BSA as the control 4 h prior to epithelium debridement. At 0 h (0H), the corneas were wounded by epithelium debridement (2-mm diameter). At 22 hpw (22H), the remaining wounds were stained with fluorescein and photographed. E: The wound sizes were calculated using ImageJ and presented as percentage of healed area over the size of the original wound (mean ± SD; n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). RTXW, RTX wounded.

Close modal

VIP Inhibits Inflammatory Reaction in the Denervated B6 Mouse Corneas

Figure 2 shows the anti-inflammatory effects of neuropeptides in the RTX-treated corneas. While wounding induced the expression of interleukin (Il)-1β, Cxcl2, Nos2, and Il-10, their upregulations were greatly augmented by corneal denervation. The addition of three neuropeptides (CGRP, SP, and VIP) each individually dampened the elevated expressions to different extents. VIP was unique, as it was most effective in downregulating Il1β and upregulating Il-10 expression. Hence, we chose to focus on VIP for further study.

Figure 2

VIP inhibits overexpressed proinflammatory factors in the denervated B6 mouse corneas. B6 mouse corneas were treated with RTX as described in Fig. 1, followed by pretreatment with recombinant CGRP (100 ng/eye), SP (250 ng/eye), and VIP (250 ng/eye) or BSA as the control 4 h prior to epithelium debridement. At 22 hpw, corneas were collected and subjected to qPCR to assess the expression of IL-1β, CXCL2, NOS2, and IL-10. qPCR results were first normalized with the levels of β-actin and then compared with the levels of NL/naive corneas (value = 1), presented as fold changes. The results were presented as fold change (mean ± SD) over the naive corneas (control [CT], unwounded [UW]) set as a value of 1 (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Figure 2

VIP inhibits overexpressed proinflammatory factors in the denervated B6 mouse corneas. B6 mouse corneas were treated with RTX as described in Fig. 1, followed by pretreatment with recombinant CGRP (100 ng/eye), SP (250 ng/eye), and VIP (250 ng/eye) or BSA as the control 4 h prior to epithelium debridement. At 22 hpw, corneas were collected and subjected to qPCR to assess the expression of IL-1β, CXCL2, NOS2, and IL-10. qPCR results were first normalized with the levels of β-actin and then compared with the levels of NL/naive corneas (value = 1), presented as fold changes. The results were presented as fold change (mean ± SD) over the naive corneas (control [CT], unwounded [UW]) set as a value of 1 (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Close modal

Hyperglycemia Suppresses Wound-Induced Upregulation of VIP and VIPR1 in B6 Mouse Corneas

VIP is secreted from neurons and can be detected in corneal nerves and the aqueous humor in both mice and humans (30). Because of the strong background staining in the whole cornea, we removed the epithelium and observed colocalization of VIP with a select group of smaller branching nerve fibers in the stroma of NL corneas (Fig. 3A).

Figure 3

Wound-induced VIP and VIPR1 upregulation was suppressed in diabetic corneas. A: Costaining of VIP and β-tubulin III in NL corneas was performed, and images of the limbal region of corneas were captured (top panels), with high-magnification images in bottom panels. B: STZ-induced diabetic and age-matched NL mice were wounded by epithelium debridement (2-mm diameter), and the epithelial cells (CECs) were collected as the NW. At 22 hpw, CECs that had migrated into the original wounds were scraped off from the corneas and collected as wounded (W) CECs. CECs collected were subjected to RNA isolation for qPCR to assess Vip and Vipr1 expression. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA). C: Samples collected as described in B were also subjected to Western blotting analysis (two samples per lane represent each condition) for VIPR1 expression, with actin as the internal loading control. D: At 22 hpw, wounded corneas along with unwounded as the control were excised, snap-frozen in OCT, and cryostat sectioned. Cornea sections were stained with VIPR1 antibody (green) and counterstained with DAPI showing nuclei (blue). For each set of experiments, optimization of the exposure time and other settings was determined using NL healing corneas, and the same settings were kept during image acquisition. Images shown were representatives of three corneas for each condition. L, limbus; NLW, NL wounded.

Figure 3

Wound-induced VIP and VIPR1 upregulation was suppressed in diabetic corneas. A: Costaining of VIP and β-tubulin III in NL corneas was performed, and images of the limbal region of corneas were captured (top panels), with high-magnification images in bottom panels. B: STZ-induced diabetic and age-matched NL mice were wounded by epithelium debridement (2-mm diameter), and the epithelial cells (CECs) were collected as the NW. At 22 hpw, CECs that had migrated into the original wounds were scraped off from the corneas and collected as wounded (W) CECs. CECs collected were subjected to RNA isolation for qPCR to assess Vip and Vipr1 expression. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA). C: Samples collected as described in B were also subjected to Western blotting analysis (two samples per lane represent each condition) for VIPR1 expression, with actin as the internal loading control. D: At 22 hpw, wounded corneas along with unwounded as the control were excised, snap-frozen in OCT, and cryostat sectioned. Cornea sections were stained with VIPR1 antibody (green) and counterstained with DAPI showing nuclei (blue). For each set of experiments, optimization of the exposure time and other settings was determined using NL healing corneas, and the same settings were kept during image acquisition. Images shown were representatives of three corneas for each condition. L, limbus; NLW, NL wounded.

Close modal

Using STZ-induced diabetic mice (8 weeks post–STZ treatment with average glycosylated HbA1c 10.56 ± 0.56% vs. 5.33 ± 0.11% of the control mice) (Supplementary Fig. 1), we investigated VIP and VIPR1 expression in CECs of NL and DM mice. In unwounded corneas, the levels of Vip and Vipr1 transcripts were lower in DM than in NL corneas, and wounding significantly increased their expression in healing epithelia; this wound-induced upregulation of Vip and Vipr1 was suppressed in DM healing corneas (Fig. 3B).

At the protein levels assessed by Western blotting, an increase in VIPR1 levels was observed in wounded NL CECs, which were suppressed in DM wounded corneas (Fig. 3C). Elevated VIP expression was also observed in healing epithelia of organ-cultured human NL but not DM corneas (Supplementary Fig. 2A) as well as in the infiltrated immune cells, including polymorphonuclear leukocytes (PMNs) in mouse healing corneas (Supplementary Fig. 2B). Moreover, immunohistochemistry revealed that VIPR1 in unwounded corneas is primarily expressed in the corneal limbus and peripheral epithelium. However, in wounded corneas, VIPR1 was expressed in healing epithelia, with weaker staining in DM compared with NL corneas (Fig. 3D).

VIP Promotes Corneal Wound Healing In Vivo

Given the upregulation of VIP and VIPR1 in healing epithelia, we next investigated the role of VIP-VIPR1 in mediating corneal wound healing, which includes epithelial wound closure and SN regeneration. VIP at 250 ng/cornea was effective in promoting diabetic epithelial wound closure (Supplementary Fig. 3). In the NL corneas, the blockade of VIPR1 resulted in a decrease in the healing rate compared with untreated eyes. VIP administration accelerated healing rate compared with the untreated DM corneas (Fig. 4A and C). WMCM showed that the blockade of VIPR1 resulted in a decrease in NL, while exogenous VIP showed an increase in SN regeneration in DM corneas (Fig. 4B). The percentage of area covered by nerve staining in the central cornea (Fig. 4B, bottom panels) was 13.09 ± 0.81% in untreated and 8.25 ± 1.79% in VIP1Ra-treated NL corneas, whereas in diabetic corneas, recombinant VIP treatment increased the covered area from 7.55 ± 1.63% to 11.86 ± 0.64% (Fig. 4D).

Figure 4

VIP accelerates diabetic wound healing and nerve regeneration in healing corneas through VIPR1. NL corneas were pretreated with VIPR1 antagonist or PBS and diabetic corneas with recombinant VIP or BSA as the control 4 h prior to epithelium debridement. At 0 h, the corneas were wounded by epithelium debridement (2-mm diameter). A: At 22 hpw, the remaining wounds were stained with fluorescein and photographed. B: Another set of mice was allowed to heal for 3 days, and the corneas were processed for WMCM with β-tubulin III staining for nerve fibers and endings. The images of whole corneas (top panels) and high-magnification images of central area (bottom panels) were shown. C: The wound sizes from A were calculated and presented as percentage of healed area over the size of original wounds (mean ± SD; n = 6). D: Nerve densities at the central areas were calculated from the areas covered with β-tubulin III staining with ImageJ and presented as percent areas (mean ± SD; n = 3). Two independent experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA).

Figure 4

VIP accelerates diabetic wound healing and nerve regeneration in healing corneas through VIPR1. NL corneas were pretreated with VIPR1 antagonist or PBS and diabetic corneas with recombinant VIP or BSA as the control 4 h prior to epithelium debridement. At 0 h, the corneas were wounded by epithelium debridement (2-mm diameter). A: At 22 hpw, the remaining wounds were stained with fluorescein and photographed. B: Another set of mice was allowed to heal for 3 days, and the corneas were processed for WMCM with β-tubulin III staining for nerve fibers and endings. The images of whole corneas (top panels) and high-magnification images of central area (bottom panels) were shown. C: The wound sizes from A were calculated and presented as percentage of healed area over the size of original wounds (mean ± SD; n = 6). D: Nerve densities at the central areas were calculated from the areas covered with β-tubulin III staining with ImageJ and presented as percent areas (mean ± SD; n = 3). Two independent experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA).

Close modal

VIP-VIPR1 Signal Pathway Mediates Corneal Wound Response by Regulating Neurotrophic Factor and Cytokine Expression

We next investigated the underlying mechanism for the observed effects of VIP by assessing the expression of neurotrophic factors and cytokines (Fig. 5). Presence of VIP1Ra downregulated the expression of the neurotrophic factors NGF and CNTF in NL healing corneas, whereas exogenous VIP partially reversed the suppressing effects of DM in B6 mice (Fig. 5A). As for cytokines (Fig. 5B), wounding induced expression of all six cytokines. The upregulation of the proinflammatory cytokines IL-1β, CXCL2, as well as NOS2, a marker for the M1 macrophage, was augmented by VIP1Ra in NL corneas, but significantly attenuated by exogenous VIP in DM corneas. Anti-inflammatory cytokines IL-1Ra, IL-10, and CXCL5, in contrast, exhibited an opposing pattern of expression of proinflammatory cytokines: VIP1Ra suppressed these in NL corneas, while VIP enhanced their expressions in DM corneas. qPCR revealed that SP and CGRP mRNAs were detected in the trigeminal nerve but not NL and DM corneas, while VIP was found in both trigeminus and corneas with or without wounding (Supplementary Fig. 4).

Figure 5

VIP upregulates neurotrophic factors and anti-inflammatory cytokines and downregulates proinflammatory cytokines in diabetic healing corneas. NL, pretreated with VIP1Ra, and diabetic, pretreated with recombinant VIP, mouse corneas were wounded by epithelium debridement (2-mm diameter). At 22 hpw, healing corneas and corneas from unwounded NL and diabetic mice as the control were subjected to qPCR analysis of the expression of neurotrophic factors (NGF and CNTF) and proinflammatory (IL-1β, CXCL2, and CXCL5) and anti-inflammatory cytokines (IL-1Ra and IL-10). The results are presented as fold change (mean ± SD) over the nonwounded control corneas set as a value of 1 (n = 3) (A and B). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). ●, Unwounded; ▪, wounded corneas pretreated with BSA; ▲, wounded plus treatment with VIP1Ra (NL) or VIP (diabetic).

Figure 5

VIP upregulates neurotrophic factors and anti-inflammatory cytokines and downregulates proinflammatory cytokines in diabetic healing corneas. NL, pretreated with VIP1Ra, and diabetic, pretreated with recombinant VIP, mouse corneas were wounded by epithelium debridement (2-mm diameter). At 22 hpw, healing corneas and corneas from unwounded NL and diabetic mice as the control were subjected to qPCR analysis of the expression of neurotrophic factors (NGF and CNTF) and proinflammatory (IL-1β, CXCL2, and CXCL5) and anti-inflammatory cytokines (IL-1Ra and IL-10). The results are presented as fold change (mean ± SD) over the nonwounded control corneas set as a value of 1 (n = 3) (A and B). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). ●, Unwounded; ▪, wounded corneas pretreated with BSA; ▲, wounded plus treatment with VIP1Ra (NL) or VIP (diabetic).

Close modal

VIP-VIPR1 Signal Pathway Affects Neutrophil Infiltration in Healing Corneas

Neutrophils have been shown to play an important role in corneal wound healing (31,32). We next investigated whether neutrophil infiltration was affected by hyperglycemia and the effects of VIP. WMCM revealed that the infiltration of neutrophils in diabetic corneas was significantly increased compared with NL healing corneas (Fig. 6), with a high density of infiltrated cells migrating out of the epithelium, covering the wound bed in DM corneas (DM wounded [DMW]) (Fig. 6C). VIP1Ra greatly increased in NL corneas (nonwounded control [NW]+VIP1Ra) (Fig. 6C), whereas VIP significantly decreased the numbers of infiltrated PMNs, with few cells found in the remaining wound bed in DM healing corneas (DM+VIP) (Fig. 6C and D).

Figure 6

VIP dampens neutrophil infiltration in NL and diabetic healing corneas. NL (NW), pretreated with VIP1Ra (NW+VIP1Ra), and diabetic (DMW), pretreated with recombinant VIP (DMW+VIP), mouse corneas were wounded by epithelium debridement (2-mm diameter). Healing corneas collected at 22 hpw from NL and diabetic mice were subjected to WMCM using Ly6G-FITC antibody for mouse neutrophil staining. The images of the whole cornea were captured (A), the marked areas of whole corneas were amplified (B), and central areas were shown (C). D: Cell numbers in the whole corneas (A) were calculated with ImageJ and presented (mean ± SD; n = 3). Two independent experiments were performed. *P < 0.05 (one-way ANOVA).

Figure 6

VIP dampens neutrophil infiltration in NL and diabetic healing corneas. NL (NW), pretreated with VIP1Ra (NW+VIP1Ra), and diabetic (DMW), pretreated with recombinant VIP (DMW+VIP), mouse corneas were wounded by epithelium debridement (2-mm diameter). Healing corneas collected at 22 hpw from NL and diabetic mice were subjected to WMCM using Ly6G-FITC antibody for mouse neutrophil staining. The images of the whole cornea were captured (A), the marked areas of whole corneas were amplified (B), and central areas were shown (C). D: Cell numbers in the whole corneas (A) were calculated with ImageJ and presented (mean ± SD; n = 3). Two independent experiments were performed. *P < 0.05 (one-way ANOVA).

Close modal

VIP-VIPR1 Signal Pathway Induces ERK Activation and Shh Expression in Response to Wounding in the Corneas

To elucidate the mechanisms underlying VIP’s effects on corneal wound healing, we screened for signaling pathways affected by VIP using qPCR and by detecting phosphorylated signaling molecules. We found that the levels of Shh and p-ERK are most strongly related to VIP activity. Western blot showed that in unwounded corneas, the band intensities for p-ERK and Shh were weaker in DM compared with NL corneas. Epithelium wounding elevated their levels in NL but not DM corneas (Fig. 7A). VIP1Ra decreased while exogenous VIP increased the expression of p-ERK and Shh in NL and DM corneas, respectively. Similar results of Shh staining were also observed by immunohistochemistry of corneal cryostat sections from NL and DM corneas, with or without wounding (Fig. 7C). Time course in wounded NL corneas (Supplementary Fig. 5) and dose in cultured mouse CECs (Supplementary Fig. 6), dependent ERK activation, and Shh upregulation were also observed. Interestingly, VIP was able to stimulate ERK activation and Shh upregulation in NL and unwounded corneas (Supplementary Fig. 7), consistent with the observation that VIP at 250 ng/cornea promotes corneal epithelial wound closure and SN regeneration (Supplementary Fig. 8).

Figure 7

Exogenous VIP strictly through VIPR1 accelerates epithelial wound healing by regulated ERK activity and Shh expression in diabetic corneas. NL (NW), pretreated with VIP1Ra, and diabetic (DMW), pretreated with recombinant VIP, mouse corneas were wounded by epithelium debridement (2-mm diameter). A: CECs collected during wounding (12 hpw) and from the original wound beds (0 hpw) were subjected to Western blot analysis and p-ERK protein level expression. Two samples for each condition were used, and 20 μg total protein from collected CECs was analyzed for each sample. B: The pixels of bands were analyzed with ImageJ, and the results were presented as fold change (mean ± SD) over the nonwounded control (CT) corneas set as a value of 1 (B). *P < 0.05, **P < 0.001, ***P < 0.001 (two-way ANOVA). C: At 20 hpw, wounded corneas, three per group, along with nonwounded NL and diabetic corneas were excised, snap-frozen in OCT, and cryostat sectioned. Three sections from each cornea were stained with their respective primary antibodies, followed by FITC-conjugated secondary antibodies. The cryostat sections were also counterstained with DAPI showing nuclei (blue). For each set of experiments, optimization of the exposure time and other settings was determined using NL healing corneas, and the exact same settings were kept during the analysis of all samples. Immunofluorescence showing expression (green) of Shh and p-ERK. Arrows mark leading edge of healing epithelium. Two independent experiments were performed. T-ERK, total ERK; UW, unwounded.

Figure 7

Exogenous VIP strictly through VIPR1 accelerates epithelial wound healing by regulated ERK activity and Shh expression in diabetic corneas. NL (NW), pretreated with VIP1Ra, and diabetic (DMW), pretreated with recombinant VIP, mouse corneas were wounded by epithelium debridement (2-mm diameter). A: CECs collected during wounding (12 hpw) and from the original wound beds (0 hpw) were subjected to Western blot analysis and p-ERK protein level expression. Two samples for each condition were used, and 20 μg total protein from collected CECs was analyzed for each sample. B: The pixels of bands were analyzed with ImageJ, and the results were presented as fold change (mean ± SD) over the nonwounded control (CT) corneas set as a value of 1 (B). *P < 0.05, **P < 0.001, ***P < 0.001 (two-way ANOVA). C: At 20 hpw, wounded corneas, three per group, along with nonwounded NL and diabetic corneas were excised, snap-frozen in OCT, and cryostat sectioned. Three sections from each cornea were stained with their respective primary antibodies, followed by FITC-conjugated secondary antibodies. The cryostat sections were also counterstained with DAPI showing nuclei (blue). For each set of experiments, optimization of the exposure time and other settings was determined using NL healing corneas, and the exact same settings were kept during the analysis of all samples. Immunofluorescence showing expression (green) of Shh and p-ERK. Arrows mark leading edge of healing epithelium. Two independent experiments were performed. T-ERK, total ERK; UW, unwounded.

Close modal

Shh Promotes Corneal Wound Healing Downstream of VIP-VIPR1

Shh was shown to promote CEC proliferation in corneal organ culture (33). We next assessed whether Shh plays a role in corneal wound healing by using Sant-1, a potent Shh antagonist, in wounded NL and recombinant mouse Shh in DM corneas. The blockade of Shh signaling resulted in a lower healing rate in NL, whereas administration of Shh resulted in a higher healing rate in DM corneas (Fig. 8A, C, and E). WMCM showed that Shh signaling blockade resulted in a significant decrease (13.42 ± 1.52% in control corneas vs. 9.54 ± 1.33% nerve-staining areas in Sant-1–treated NL corneas), while exogenous Shh increased SN regeneration in diabetic corneas (6.21 ± 1.25% in control corneas vs. 11.03 ± 0.45% in Sant-1–treated DM corneas) (Fig. 8B, D, and F). To determine whether Shh is downstream of the VIP-VIPR1 signal pathway, we treated DM wounded corneas with exogenous VIP or VIP+Sant-1. At 22 h postwounding (hpw), Shh signaling blockade abolished VIP-accelerated wound healing (4.03 ± 0.31% in VIP+Sant-1 DM corneas vs. 12.65.42 ± 0.39% nerve-staining areas in VIP-treated DM corneas) (Fig. 8B, C, E, and F). Manipulation of Shh signaling was also shown to alter epithelial wound healing in NL (5 mmol/L) and high glucose–cultured (30 mmol/L) pig corneas. Sant-1 attenuated corneal epithelial wound closure in 5 mmol/L glucose–cultured corneas, while recombinant murine Shh accelerated it in 30 mmol/L glucose–cultured pig corneas (Supplementary Fig. 8).

Figure 8

Shh accelerates diabetic wound healing and nerve regeneration as a downstream signal pathway of VIP in B6 mouse corneas. NL (NW), pretreated with Sant-1 (250 nmol/L, 5 μL/eye) and diabetic (DMW) corneas, pretreated with recombinant mouse Shh (rmShh) (50 ng/μL, 5 μL/eye), VIP (50 ng/μL, 5 μL/eye), and VIP+Sant-1 (250 nmol/L, 5 μL/eye) 4 h prior to epithelium debridement. At 0 h, the corneas were wounded by epithelium debridement (2-mm diameter). A: At 22 hpw, healing corneas were stained with fluorescein and photographed, and the wound healing was calculated using ImageJ and presented as a percentage of the original wound size (C and E; mean ± SD, n = 5). At 3 days postwounding, epithelium-healed corneas were subjected to WMCM for nerve staining of the whole cornea (B, top panels) and high-magnification images of central areas (B, bottom panels) using β-tubulin III antibody. Each image is a representative of three corneas in a group. Nerve densities at the central areas (bottom panels) were calculated from the areas covered with β-tubulin III staining with ImageJ and presented as percent areas (D and F). Two independent experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).

Figure 8

Shh accelerates diabetic wound healing and nerve regeneration as a downstream signal pathway of VIP in B6 mouse corneas. NL (NW), pretreated with Sant-1 (250 nmol/L, 5 μL/eye) and diabetic (DMW) corneas, pretreated with recombinant mouse Shh (rmShh) (50 ng/μL, 5 μL/eye), VIP (50 ng/μL, 5 μL/eye), and VIP+Sant-1 (250 nmol/L, 5 μL/eye) 4 h prior to epithelium debridement. At 0 h, the corneas were wounded by epithelium debridement (2-mm diameter). A: At 22 hpw, healing corneas were stained with fluorescein and photographed, and the wound healing was calculated using ImageJ and presented as a percentage of the original wound size (C and E; mean ± SD, n = 5). At 3 days postwounding, epithelium-healed corneas were subjected to WMCM for nerve staining of the whole cornea (B, top panels) and high-magnification images of central areas (B, bottom panels) using β-tubulin III antibody. Each image is a representative of three corneas in a group. Nerve densities at the central areas (bottom panels) were calculated from the areas covered with β-tubulin III staining with ImageJ and presented as percent areas (D and F). Two independent experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t test).

Close modal

In this study, we investigated the role of VIP in promoting hyperglycemia-attenuated wound healing in diabetic corneas. We identified that CGRP, SP, and VIP partially restored the impaired epithelial wound closure and inhibited the overexpression of proinflammatory factors in locally denervated B6 mouse corneas. We focused on VIP because, in addition to suppressing proinflammatory cytokine expression, it also augmented IL-10 expression. The expression of both VIP and VIPR1 was upregulated in CECs in response to wounding in NL but not DM corneas. Blockade of VIP-VIPR1 signaling in NL corneas resulted in delayed healing, whereas exogenous VIP ameliorated DM-impaired corneal wound healing in B6 mouse corneas. Moreover, VIP-VIPR signaling suppressed DM-induced proinflammatory cytokine expression, while it also enhanced the expression of anti-inflammatory cytokines and neurotrophic factors. Our study also, for the first time, linked the VIP-VIPR1 and Shh signaling pathways. Importantly, Shh signaling is also downregulated in healing corneas and acts as a downstream signaling molecule of VIP-VIPR1. Taken together, our results revealed the molecular mechanisms of the VIP-VIPR signaling pathway in mediating corneal wound healing and suggested that VIP and/or Shh may be used as therapeutic reagents for treating DNK.

Using RTX to treat mouse corneas, our results revealed that while the density of SN endings was greatly reduced, presence of RTX had minimal effect on the stromal nerve fibers, indicating the temporary nature of the treatment. In diabetic corneas, both SN fibers and endings are reduced (Fig. 1A vs. Fig. 4A). Loss of stroma C-fibers was observed in patients with diabetes at early stages of diabetes and has been used to diagnose manifestation of peripheral neuropathy (34). Using local denervation, our study revealed a critical role of SN in mediating corneal wound healing in vivo. Our previous studies indicated that another residential cell type, DCs, also participates in the regulation of epithelium wound healing through, in part, the release of CNTF (7,35). It should be mentioned that epithelial wound closure occurs in organ culture that lacks neuronal and immune components as effectively as in vivo in mouse and human corneas (3638). Potentially, while DC proliferation and infiltration may represent the inflammatory response, SNs may function as an anti-inflammatory element in vivo. Indeed, RTX-treated corneas showed that the lack of SN in the cornea results in an exacerbated expression of cytokines, which were partially restored by the addition of neuropeptides SP, CGRP, and/or VIP, with the latter markedly upregulating the expression of IL-10 in wounded and denervated corneas.

We previously reported a drastic decrease in density of SN fibers/endings in unwounded and impaired reinnervation in postwounding DM corneas (7). VIP is known as an immunoinhibitory neuropeptide released by SN fibers in the hair follicle (35) and as a key guardian of ocular immune privilege (39,40). In the latter case, the source of VIP remains undetermined. Using WMCM, we detected VIP immunoreactivity in a set of SN fibers in the mouse corneal stroma. In healing corneas, infiltrated neutrophils in the anterior stroma were also VIP positive (Supplementary Fig. 1B). Furthermore, we observed the upregulation of VIP at mRNA levels and VIPR1 at mRNA, protein, and tissue levels, suggesting an amplifying signaling in NL corneas in response to wounding. An increase in VIP immune staining was also observed in healing epithelia of organ-cultured human corneas as well as in immune cells such as neutrophils of B6 mice, suggesting that the source for wound-induced VIP is not limited to the SN. Importantly, diabetes suppresses VIP and VIPR1 expression in NL and healing corneas, lowering VIP/VIPR1 signaling.

Our study revealed that VIP/VIPR1 signaling promotes wound healing and that impaired VIP-VIPR1 signaling contributes to the defects in corneal epithelial wound closure and delayed SN regeneration in diabetic corneas. Other factors, including opioid growth factor, insulin, IGF-1, hepatocyte growth factor, epidermal growth factor, thymosin β4, SP, NGF, and CNTF, have also been shown to have altered expression in DM corneas and promote epithelial wound closure in DM corneas (for excellent reviews, see Ljubimov [4] and Ljubimov and Saghizadeh [41]). Our present study revealed that the levels of NGF and CNTF at mRNA levels were closely associated with the activity of VIP signaling. Hence, we propose a positive-feedback loop for the expression and function of neuropeptides and neurotrophic factors in response to tissue injury: healing CECs secrete NGF and infiltrating DCs produce CNTF to induce and stabilize regenerating SN (7), which in turn releases more neuropeptides to nourish and sustain proliferation and/or migration of CECs and infiltrated DCs. Diabetes disturbs this loop of neuroimmune–epithelium interactions, resulting in DNK. Recently, the U.S. Food and Drug Administration approved the use of a topical recombinant human nerve growth factor (OXERVATE) for the treatment of neurotrophic keratopathy (42). Because of this positive-feedback loop, VIP may be used in combination with NGF to synergistically promote wound healing and treat ulcers of diabetic corneas.

Research in humans and in animal models has identified that chronic inflammation underlies, in large part, the failure of diabetic wounds to heal (43,44). Our study revealed that VIP suppresses proinflammatory IL-1β and CXCL2 and augmented expression of anti-inflammatory cytokine IL-1Ra and IL-10 as well as CXCL5 in healing corneas. The lack of VIP signaling in DM corneas exacerbated proinflammatory and diminished anti-inflammatory results in low-grade inflammation in diabetic corneas.

In the current study, we observed that the blockade of VIP/VIPR1 signaling in NL corneas resulted in a great increase in the numbers of infiltrated PMNs in whole-mount corneas. Remarkably, most cells were accumulated in the nude wound bed, whereas in untreated corneas, most infiltrated cells are migrating behind the leading edge of the healing epithelia (12,45). Interestingly, a high density of infiltrated PMNs was also found outside of the healing epithelial sheet and around the corneal wound margin in the diabetic corneas; treatment with VIP greatly reduced the number of infiltrated neutrophils and limited these cells within the boundary of epithelium-covered area. IL-1β, CXCL2, and CXCL5 are known to stimulate neutrophil infiltration and accumulation in response to wounding (12,46). Interestingly, our study showed that CXCL5 had the reverse pattern of CXCL2, a mouse homolog of human IL-8, the dominant chemotactic factor. CXCL5 was reported to desensitize CXCR2, the receptor for human IL-8 and mouse CXCL1/2, in the lung (46). Thus, VIP-promoted CXCL5 expression may be an additional underlying mechanism for VIP to suppress PMN infiltration. We conclude that elevated PMN infiltration, particularly in the remaining wound region, is a contributing factor for increased inflammation and delayed wound healing and that VIP reverses these pathological processes in the diabetic corneas. Hence, targeting these wound bed–accumulated PMNs, such as through the use of VIP as an adjunctive therapy, might be an effective means to reduce inflammatory response to injury in patients with diabetes.

In the current study, we showed that VIP induces Shh expression in healing corneas and that this expression is impaired by hyperglycemia and suppressed by VIP1Ra in NL corneas. Shh is a morphogen and an axonal guidance cue (47), and its expression is known to be induced in healing corneal epithelia (33,48). Shh expression was also upregulated by alkali burn injury and corneal neovascularization and involved in advanced aniridia-related keratitis (48,49). In this study, we found that Shh upregulation was impaired in diabetic healing corneas and that exogenous Shh accelerated delayed wound healing in diabetic corneas, while blockade of Shh signaling resulted in wound healing delay in NL corneas. Moreover, VIP-accelerated corneal wound healing was mitigated when Shh signaling was blocked. Hence, Shh signaling plays a role in modulating corneal wound healing and is a downstream effector of the VIP-VIPR pathway, particularly as an axon guidance molecule, in a manner similar to Sema3C for SN regeneration (50).

Taken together, our study reveals that VIP-VIPR1 signaling plays a positive role in corneal epithelial wound healing and nerve regeneration in diabetic corneas by upregulating neurotrophic factors, inhibiting excessive inflammatory reaction, and inducing Shh expression. Moreover, we suggest that exogenous supplementation of Shh, as it is downstream of VIP-VIPR signaling, may serve as a new strategy for restoring the impaired epithelial wound closure and SN regeneration in patients with diabetes.

This article contains supplementary material online at https://doi.org/10.2337/figshare.12168084.

Funding. This work was supported by NIH/National Eye Institute grants R01-EY-10869, EY-17960 (to F.-s.X.Y.), EY-025923 (to P.S.Y.L.), and EY-004068 (National Eye Institute Core to Wayne State University) and by Research to Prevent Blindness (to Kresge Eye Institute).

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

Author Contributions. Y.Z. performed laboratory testing and sample collection/analysis and edited and checked accuracy of the manuscript. N.G., L.W., P.S.Y.L., R.M., and C.D. performed laboratory testing and data analysis. L.X. and F.-s.X.Y. were responsible for study design and recruitment, contributed to sample collection and data analysis, and reviewed and edited the manuscript. F.-s.X.Y. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. This study was presented at the 91st Annual Meeting of the Association for Research in Vision and Ophthalmology, Vancouver, British Columbia, Canada, 28 Apr–2 May 2019.

1.
Iqbal
Z
,
Azmi
S
,
Yadav
R
, et al
.
Diabetic peripheral neuropathy: epidemiology, diagnosis, and pharmacotherapy
.
Clin Ther
2018
;
40
:
828
849
2.
Rózsa
AJ
,
Beuerman
RW
.
Density and organization of free nerve endings in the corneal epithelium of the rabbit
.
Pain
1982
;
14
:
105
120
3.
Schwartz
DE
.
Corneal sensitivity in diabetics
.
Arch Ophthalmol
1974
;
91
:
174
178
4.
Ljubimov
AV
.
Diabetic complications in the cornea
.
Vision Res
2017
;
139
:
138
152
5.
Davidson
EP
,
Coppey
LJ
,
Holmes
A
,
Yorek
MA
.
Changes in corneal innervation and sensitivity and acetylcholine-mediated vascular relaxation of the posterior ciliary artery in a type 2 diabetic rat
.
Invest Ophthalmol Vis Sci
2012
;
53
:
1182
1187
6.
Tavakoli
M
,
Mitu-Pretorian
M
,
Petropoulos
IN
, et al
.
Corneal confocal microscopy detects early nerve regeneration in diabetic neuropathy after simultaneous pancreas and kidney transplantation
.
Diabetes
2013
;
62
:
254
260
7.
Gao
N
,
Yan
C
,
Lee
P
,
Sun
H
,
Yu
FS
.
Dendritic cell dysfunction and diabetic sensory neuropathy in the cornea
.
J Clin Invest
2016
;
126
:
1998
2011
8.
Lockwood
A
,
Hope-Ross
M
,
Chell
P
.
Neurotrophic keratopathy and diabetes mellitus
.
Eye (Lond)
2006
;
20
:
837
839
9.
Bikbova
G
,
Oshitari
T
,
Baba
T
,
Yamamoto
S
.
Neuronal changes in the diabetic cornea: perspectives for neuroprotection
.
BioMed Res Int
2016
;
2016
:
5140823
10.
Yin
J
,
Huang
J
,
Chen
C
,
Gao
N
,
Wang
F
,
Yu
FS
.
Corneal complications in streptozocin-induced type I diabetic rats
.
Invest Ophthalmol Vis Sci
2011
;
52
:
6589
6596
11.
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
12.
Yan
C
,
Gao
N
,
Sun
H
, et al
.
Targeting imbalance between IL-1β and IL-1 receptor antagonist ameliorates delayed epithelium wound healing in diabetic mouse corneas
.
Am J Pathol
2016
;
186
:
1466
1480
13.
Zhang
M
,
Zhou
Q
,
Luo
Y
,
Nguyen
T
,
Rosenblatt
MI
,
Guaiquil
VH
.
Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development
.
PLoS One
2018
;
13
:
e0191962
14.
Lagali
N
,
Poletti
E
,
Patel
DV
, et al
.
Focused tortuosity definitions based on expert clinical assessment of corneal subbasal nerves
.
Invest Ophthalmol Vis Sci
2015
;
56
:
5102
5109
15.
Brain
SD
.
Sensory neuropeptides: their role in inflammation and wound healing
.
Immunopharmacology
1997
;
37
:
133
152
16.
Suvas
S
.
Role of substance P neuropeptide in inflammation, wound healing, and tissue homeostasis
.
J Immunol
2017
;
199
:
1543
1552
17.
Yang
L
,
Di
G
,
Qi
X
, et al
.
Substance P promotes diabetic corneal epithelial wound healing through molecular mechanisms mediated via the neurokinin-1 receptor
.
Diabetes
2014
;
63
:
4262
4274
18.
Zhang
Y
,
Xu
J
,
Ruan
YC
, et al
.
Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats
.
Nat Med
2016
;
22
:
1160
1169
19.
Ma
W
,
Bisby
MA
.
Partial and complete sciatic nerve injuries induce similar increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in primary sensory neurons and their central projections
.
Neuroscience
1998
;
86
:
1217
1234
20.
Nakamura
M
,
Kawahara
M
,
Morishige
N
,
Chikama
T
,
Nakata
K
,
Nishida
T
.
Promotion of corneal epithelial wound healing in diabetic rats by the combination of a substance P-derived peptide (FGLM-NH2) and insulin-like growth factor-1
.
Diabetologia
2003
;
46
:
839
842
21.
Mikami
N
,
Sueda
K
,
Ogitani
Y
, et al
.
Calcitonin gene-related peptide regulates type IV hypersensitivity through dendritic cell functions
.
PLoS One
2014
;
9
:
e86367
22.
Harzenetter
MD
,
Novotny
AR
,
Gais
P
,
Molina
CA
,
Altmayr
F
,
Holzmann
B
.
Negative regulation of TLR responses by the neuropeptide CGRP is mediated by the transcriptional repressor ICER
.
J Immunol
2007
;
179
:
607
615
23.
Mikulec
AA
,
Tanelian
DL
.
CGRP increases the rate of corneal re-epithelialization in an in vitro whole mount preparation
.
J Ocul Pharmacol Ther
1996
;
12
:
417
423
24.
Thippeswamy
T
,
Howard
MR
,
Cosgrave
AS
,
Arora
DK
,
McKay
JS
,
Quinn
JP
.
Nitric oxide-NGF mediated PPTA/SP, ADNP, and VIP expression in the peripheral nervous system
.
J Mol Neurosci
2007
;
33
:
268
277
25.
Klimaschewski
L
.
VIP -- a ‘very important peptide’ in the sympathetic nervous system
?
Anat Embryol (Berl)
1997
;
196
:
269
277
26.
Jiang
X
,
McClellan
SA
,
Barrett
RP
,
Zhang
Y
,
Hazlett
LD
.
Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea
.
J Immunol
2012
;
189
:
269
278
27.
Tuncel
N
,
Yildirim
N
,
Gurer
F
, et al
.
Effect of vasoactive intestinal peptide on the wound healing of alkali-burned corneas
.
Int J Ophthalmol
2016
;
9
:
204
210
28.
Satitpitakul
V
,
Sun
Z
,
Suri
K
, et al
.
Vasoactive intestinal peptide promotes corneal allograft survival
.
Am J Pathol
2018
;
188
:
2016
2024
29.
Sanlioglu
AD
,
Karacay
B
,
Balci
MK
,
Griffith
TS
,
Sanlioglu
S
.
Therapeutic potential of VIP vs PACAP in diabetes
.
J Mol Endocrinol
2012
;
49
:
R157
R167
30.
Delgado
M
,
Pozo
D
,
Ganea
D
.
The significance of vasoactive intestinal peptide in immunomodulation
.
Pharmacol Rev
2004
;
56
:
249
290
31.
Gagen
D
,
Laubinger
S
,
Li
Z
, et al
.
ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse
.
Exp Eye Res
2010
;
91
:
676
684
32.
Liu
J
,
Xue
Y
,
Dong
D
, et al
.
CCR2- and CCR2+ corneal macrophages exhibit distinct characteristics and balance inflammatory responses after epithelial abrasion
.
Mucosal Immunol
2017
;
10
:
1145
1159
33.
Saika
S
,
Muragaki
Y
,
Okada
Y
, et al
.
Sonic hedgehog expression and role in healing corneal epithelium
.
Invest Ophthalmol Vis Sci
2004
;
45
:
2577
2585
34.
Shtein
RM
,
Callaghan
BC
.
Corneal confocal microscopy as a measure of diabetic neuropathy
.
Diabetes
2013
;
62
:
25
26
35.
Bertolini
M
,
Pretzlaff
M
,
Sulk
M
, et al
.
Vasoactive intestinal peptide, whose receptor-mediated signalling may be defective in alopecia areata, provides protection from hair follicle immune privilege collapse
.
Br J Dermatol
2016
;
175
:
531
541
36.
Hall
ET
,
Cleverdon
ER
,
Ogden
SK
.
Dispatching sonic hedgehog: molecular mechanisms controlling deployment
.
Trends Cell Biol
2019
;
29
:
385
395
37.
Nacu
E
,
Gromberg
E
,
Oliveira
CR
,
Drechsel
D
,
Tanaka
EM
.
FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration
.
Nature
2016
;
533
:
407
410
38.
Wang
J
,
Cao
J
,
Dickson
AL
,
Poss
KD
.
Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling
.
Nature
2015
;
522
:
226
230
39.
Streilein
JW
,
Okamoto
S
,
Sano
Y
,
Taylor
AW
.
Neural control of ocular immune privilege
.
Ann N Y Acad Sci
2000
;
917
:
297
306
40.
Taylor
AW
,
Streilein
JW
,
Cousins
SW
.
Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor
.
J Immunol
1994
;
153
:
1080
1086
41.
Ljubimov
AV
,
Saghizadeh
M
.
Progress in corneal wound healing
.
Prog Retin Eye Res
2015
;
49
:
17
45
42.
Sheha
H
,
Tighe
S
,
Hashem
O
,
Hayashida
Y
.
Update on cenegermin eye drops in the treatment of neurotrophic keratitis
.
Clin Ophthalmol
2019
;
13
:
1973
1980
43.
Blakytny
R
,
Jude
E
.
The molecular biology of chronic wounds and delayed healing in diabetes
.
Diabet Med
2006
;
23
:
594
608
44.
Baltzis
D
,
Eleftheriadou
I
,
Veves
A
.
Pathogenesis and treatment of impaired wound healing in diabetes mellitus: new insights
.
Adv Ther
2014
;
31
:
817
836
45.
Gao
N
,
Yin
J
,
Yoon
GS
,
Mi
QS
,
Yu
FS
.
Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair
.
Am J Pathol
2011
;
179
:
2243
2253
46.
Mei
J
,
Liu
Y
,
Dai
N
, et al
.
CXCL5 regulates chemokine scavenging and pulmonary host defense to bacterial infection
.
Immunity
2010
;
33
:
106
117
47.
Peng
J
,
Fabre
PJ
,
Dolique
T
, et al
.
Sonic hedgehog is a remotely produced cue that controls axon guidance trans-axonally at a midline choice point
.
Neuron
2018
;
97
:
326
340.e4
48.
Fujita
K
,
Miyamoto
T
,
Saika
S
.
Sonic hedgehog: its expression in a healing cornea and its role in neovascularization
.
Mol Vis
2009
;
15
:
1036
1044
49.
Vicente
A
,
Byström
B
,
Pedrosa Domellöf
F
.
Altered signaling pathways in aniridia-related keratopathy
.
Invest Ophthalmol Vis Sci
2018
;
59
:
5531
5541
50.
Lee
PS
,
Gao
N
,
Dike
M
, et al
.
Opposing effects of neuropilin-1 and -2 on sensory nerve regeneration in wounded corneas: role of Sema3C in ameliorating diabetic neurotrophic keratopathy
.
Diabetes
2019
;
68
:
807
818
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