CCL28-induced CCR10/eNOS Interaction in Angiogenesis and Skin Wound Healing

Abstract

Chemokines and their receptors play important roles in vascular homeostasis, development, and angiogenesis. Little is known regarding the molecular signaling mechanisms activated by CCL28 chemokine via its primary receptor CCR10 in endothelial cells (EC). Here we tested the hypothesis that CCL28/CCR10 signaling plays an important role in regulating skin wound angiogenesis through eNOS/NO dependent Src, PI3K and MAPK signaling. We observed nitric oxide (NO) production in human primary ECs stimulated with exogenous CCL28, which also induced direct binding of CCR10 and eNOS resulting in inhibition of eNOS activity. Knockdown of CCR10 with siRNA lead to reduced eNOS expression and tube formation suggesting the involvement of CCR10 in EC angiogenesis. Based on this interaction, we engineered a myristoylated 7 amino acid CCR10 binding domain (Myr-CBD7) peptide and showed that this can block eNOS interaction with CCR10, but not with calmodulin (CaM), resulting in upregulation of eNOS activity. Importantly, topical administration of Myr-CBD7 peptide on mouse dermal wounds not only blocked CCR10-eNOS interaction, but also enhanced expression of eNOS, CD31, and IL-4 with reduction of CCL28 and IL-6 levels associated with improved healing in mice. These results point to a potential therapeutic strategy to upregulate NO bioavailability, enhance angiogenesis, and improve wound healing by disrupting CCL28-activated CCR10-eNOS interaction.

INTRODUCTION

The endothelium maintains vascular homeostasis by balancing the production of nitric oxide (NO), reactive oxygen species, prostaglandins, endothelium-derived hyperpolarizing factor, and endothelin-1 among others which directly and indirectly regulate vascular tone, adhesivity, and permeability (1). NO is a free radical gas (2) with a biological half-life of several seconds (3). Three NO synthase (NOS) isoforms, neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) catalyze the reaction of molecular oxygen with the amino acid substrate L-arginine to produce L-citrulline and NO (4–6). NOS1 produces NO both in the central and peripheral nervous systems where it functions as a neurotransmitter (7) and also in macrophages where NO plays important regulatory functions on inflammatory gene expression (8). Induction of iNOS occurs mainly during infection, chronic inflammation and in tumors (9). Endothelial nitric oxide synthase (eNOS) is a central regulator of vascular homeostasis (10) via NO dependent modulation of hematopoietic, endothelial, and smooth muscle cell functions (11, 12). Adequate NO production is necessary for effective wound healing, and topical application of an NO donor has been shown to accelerate closure of excisional wounds in diabetic rats (13). Furthermore, eNOS^−/− mice exhibit impaired angiogenesis and wound healing (14).

Chemokines are chemotactic cytokines that control the migratory patterns and positioning of all immune cells (15) can be divided into four subclasses (C, CC, CXC, and CX₃C) on the basis of the location of key cysteine residues. The CC class of chemokines consists of at least 28 members (CCL1–28) that signal through 10 known CC receptors (CCR1–10). The expression and function of chemokines and their receptors during the different stages of wound healing is thought to control the trafficking of specialized cell types to local sites of injury in a time- and context-dependent manner post wounding (16, 17).

Chemokine CCL28 was discovered as a ligand for CCR10, known previously as GPR2 (18, 19). CCL28 is typically secreted from epithelial cells, for example in the gut, lung, breast and salivary gland (18, 20), and CCR10 is expressed in primary human dermal fibroblasts and dermal microvascular endothelial cells (21). In injured BALB/c mice, wound-infiltrating endothelial cells express a broad spectrum of chemokine receptors including CCR10 suggesting the involvement of CCR10 in cutaneous wound healing (21). An elegant study in ovarian cancer cells showed that CCL28 modulated by hypoxia facilitates homing of regulatory T cells that foster ovarian tumor angiogenesis by accentuating VEGF production (22). Recently, we reported the overexpression of chemokine CCL28 and its receptor CCR10 in synovial tissues from patients with rheumatoid arthritis (RA) and discovered that CCL28/CCR10 signaling promotes endothelial cell (EC) migration into RA joints (23). Of note, neutralization of CCL28 in RA synovial fluid (SF) or blockade of CCR10 on human ECs significantly reduced SF-induced EC migration and capillary tube formation, demonstrating that ligation of CCR10 by CCL28 in ECs participates in pathological angiogenesis in RA (23). However, the relationship between CCR10 and eNOS in dermal microvascular ECs and the role of CCL28/CCR10 signaling in wound healing are currently unknown.

In this study, we observed that CCL28-dependent CCR10 activation downregulates and inhibits eNOS expression and function in human ECs. Topical application of a novel myristoylated peptide to block CCR10-eNOS interaction upregulated eNOS-dependent NO production and improved angiogenesis and wound healing in mice.

MATERIALS AND METHODS

Reagents

PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine), the calcium ionophore A23187, N^G-nitro-l-arginine methyl ester (L-NAME), wortmannin, STO-609, U0126, and F-127 pluronic gel were from Sigma (St. Louis, MO). n-Octyl-β-d-glucopyranoside (ODG) was from RPI Corp (Mt Prospect, IL). siRNAs for eNOS, CCR10 and transfection reagent DharmaFect 1 were from Dharmacon (Lafayette, CO). TransIT transfection reagent was from Mirus Bio (Madison, WI). Control siRNA were purchased from Santa Cruz Biotechnologies (Dallas, TX). 4’,6-diamidino-2-phenylindole (DAPI), fluorescently labeled secondary antibodies were purchased from Invitrogen (Carlsbad, CA). Mouse anti-eNOS, mouse anti-β-catenin, rabbit and mouse and mouse anti-actin antibodies, human recombinant CCL28 and Matrigel matrix were from BD Biosciences (San Diego, CA). Mouse anti-Bcl-2, rabbit anti-phospho-eNOS (pSer1177), rabbit anti-phospho-p85 (pTyr458), rabbit anti-phospho-Src (pTyr416), rabbit anti-phospho-ERK (pT202/Y204) and the corresponding total antibodies, and Griess Reagent kit were from Cell Signaling Technology (Danvers, MA). Skin punch biopsy tool was from Acuderm Inc. (Fort Lauderdale, FL). Hematoxylin, eosin, High-Def and Bluing were from StatLab (McKinney, TX). DAF-FM Diacetate, Trizol, and SYBR Green PCR mix were from ThermoFisher Scientific (Waltham, MA). Mouse ELISA kits were purchased from R &D Systems (Minneapolis, MN).

Cell Culture and Transfection

Human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 (Lonza, Walkersville, MD) supplemented with 10% (v/v) FBS. cDNA and siRNA transfection in HUVECs were performed as described previously (23–25).

NO Measurement

NO measurement with DAF-FM Diacetate: Confluent HUVEC monolayers on 96-well plates were loaded with 2.5 μM DAF-FM and incubated for 45 min at 37°C. NO concentration was measured using a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA) with 495 nm excitation and 538 nm emission.

Nitrite measurement by Griess Reagent: Supernatants from confluent ECs on 10-cm dishes with and without stimulation at 37°C were analyzed for nitrite level using a Griess Reagent kit according to the manufacturer’s instructions. Nitrite level was normalized to eNOS expression level in the cells.

Immunoblot and Co-Immunoprecipitation (Co-IP)

After serum starvation, inhibitors were added 45 min prior to stimulation for indicated times at 37°C, and cells were then collected and lysed for Western blotting. After probing for p-eNOS (Ser1177), p-p85 (Tyr458), p-Src (Tyr 416), and p-ERK (T202/Y204), the same blots were stripped and re-probed for total proteins mentioned above. For detection of CCL28-induced protein levels of β-catenin and Bcl-2, near-confluent ECs were incubated with 500 ng/ml CCL28 for 24 hr in EGM-2 medium containing 1% FBS. Co-IP experiments were as described previously (24, 25).

EC Tube Formation

HUVECs were transfected with eNOS (50 nM), CCR10 (100 nM), or scrambled control (100 nM) siRNA and after 48 h, the cells were transferred to 96-well plates preloaded with Matrigel matrix. To examine the CCL28 signaling pathways, HUVECs were pre-incubated with vehicle or specific inhibitors and then stimulated with or without CCL28 as described previously (23).

Confocal Microscopy

For cellular immunostaining, confluent monolayers of cells grown on coverslips were prepared as described previously (24). Fluorescent images were obtained using a Zeiss LSM 880 confocal microscope. The co-localization coefficient in specified regions of interest (ROI) were determined using Zeiss Zen software. Briefly, using the Overlay tool, the desired ROI was drawn along the cell edge and the values for the ROIs tabulated. The scatterplot and table were automatically adjusted to the pixel distribution for the selected ROI. The data from different treatment groups were collected and analyzed for statistical significance.

Synthesis of Myristoylated Peptides

Peptide was synthesized using a stepwise solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on a Wang resin (AnaSpec, Fremont, CA) with a 12 channel multiplex peptide synthesizer (Protein Technologies, Tucson, AZ) as described previously (26). Briefly, peptide synthesis started from the C-terminus. The Fmoc group of the resin was removed with 20% piperidine in N, N-Dimethylformamide (DMF) (5 min x2) followed by washing the resin with DMF (6.5 min). After completion, the N-terminal Fmoc was removed with 20% piperidine in DMF (5 min x 2) followed by washing the resin with DMF (6.5 min). Resins were mixed with 5 mL 50% chloroform in DMF containing 5 g myristic anhydride and kept at 60°C for 1 hr. The crude peptide was then purified on a preparative Kinetex reversed-phase C18 column (Phenomenex, Torrance, CA) using a BioCad Sprint analyzer (Applied Biosystems, Foster City, CA). Elution was performed with a linear gradient from 5% solvent B to 100% solvent B in 30 min. The absorbance of the column effluent was monitored at 230 nm and peak fractions were pooled and lyophilized. The pure peptide fraction was identified by electrospray ionization mass spectrometry (ESI-MS) and lyophilized. Finally, peptides were dissolved in DMSO and 10 mM aliquots stored at −80°C.

Mouse Skin Wound Healing Model

Wild-type (WT) C57BL/6 male mice 8–10 weeks old were purchased from Jackson Laboratory (Bar Harbor, ME). Four 5 mm full thickness excisional wounds were made on the dorsal skin using a standard skin biopsy punch under ketamine (100 mg/kg) and xylazine (5 mg/kg) anesthesia. The wounds were photographed at time zero and daily post wounding. Myristoylated peptides were applied topically to wounds in F-127 pluronic gel (30 μl of 25% gel in saline) after wounding (27). Wound size was determined as previously described (28) and collected at indicated times for ELISA, real-time RT-PCR, and Western blotting analysis.

Hematoxylin and Eosin (H&E) staining

H&E staining was performed as described previously (29). Slides with 5 μm tissue sections were baked at 60°C for 30 min and stained with an Autostainer XL (Leica Microsystems, Wetzlar, Germany) using preset protocol. Briefly, slides were deparaffinized in 3 changes of xylene, rehydrated in 100% and 95% ethanol, incubated for 1.5 min followed by a brief immersion in High-Def and Bluing to obtain crisp nuclear details. Slides were then incubated in eosin for 2 min, dehydrated and mounted with Micromount media (Leica Biosystems). Whole slide images were obtained using Aperio AT2 brightfield scanner (Leica Microsystems, Wetzlar, Germany).

Real-time RT-PCR

Total cellular RNA from mouse skin wounds was extracted using TRIzol. mRNA expression of different genes was examined by real-time RT-PCR that employed a SYBR Green PCR mix. Relative gene expression was determined by the ΔΔC_t method based on GAPDH levels as described previously (30).

ELISA Measurement

Mouse skin wounds were collected, lysed, and prepared for ELISA measurement of mouse CCL28 and IL-6 according to manufacturer’s instructions.

Statistical Analysis

Data are expressed as mean ± SEM from at least 3 independent experiments. Statistical analysis was performed by Student’s t-test or one-way ANOVA using GraphPad InStat software (San Diego, CA). Statistical significance was defined as P < 0.05.

RESULTS

CCL28 activated eNOS-dependent Src, PI3K and MAPK signaling pathways in human endothelial cells

In different human cell types, CCR10 level was found highly expressed in human umbilical vein endothelial cells (HUVECs) and endothelial progenitor cells (EPCs) (Supplemental Fig. 1). We also observed NO production in HUVECs following treatment with 500 ng/ml human recombinant CCL28, the ligand for CCR10 (Fig. 1A). The signaling pathways activated by CCL28 binding to CCR10 in ECs were further investigated. Phosphorylation of eNOS (Ser1177), p85 (Tyr458; PI3K), Src kinase (Tyr418), and ERK (T202/Y204; MAPK) were all significantly increased following CCL28 treatment for up to 60 min (Fig. 1B, C). However, when eNOS was depleted with siRNA, phosphorylation of p85, Src, and MAPK were abolished (Fig. 1D, E) suggesting CCL28 promotes eNOS-dependent Src, PI3K and MAPK signaling pathways in ECs.

Figure 1.

eNOS/NO-dependent signaling pathways activated in HUVECs following stimulation with recombinant human CCL28. A) NO measurement with DAF-FM in HUVECs. Confluent EC monolayers in 96-well plates were loaded with 2.5 μM DAF-FM and incubated for 45 min at 37°C. Cells were then treated with 5 μM A23187 (positive control), 500 ng/ml CCL28, or HBSS alone (NT). The fluorescence intensity difference between 30 min and 0 min in the NT group was set as 1. ** P < 0.01, *** P < 0.001 v NT (n=5). B, C) Following 2 h serum deprivation, confluent ECs were stimulated with 500 ng/ml human recombinant CCL28 for 0 to 60 min and lysates were collected and probed for p-eNOS (Ser1177), p-p85 (Tyr458), p-Src (Tyr 416), and p-ERK (T202/Y204), or loading controls. Normalized values of p-eNOS (n=9), p-p85 (n=5), p-Src (n=5) and p-ERK (n=5) relative to their total protein, respectively. * P < 0.05 v 0’ treatment. D, E) After depletion of eNOS with siRNA for 72 h, ECs were stimulated with 500 ng/ml CCL28, lysed, and blotted for p-eNOS, p-P85, p-Src and p-ERK. E) Normalized values of p-eNOS, p-p85, p-Src and p-ERK relative to their total protein from (D). p-, phosphorylated. t-, total. * P < 0.05, *** P < 0.001 v 0’ treatment (n=4).

We further investigated the role of CCL28/CCR10 signaling pathways in EC angiogenesis. Pretreatment of cells with 10 μM wortmannin (PI3K inhibitor), 5 μM U0126 (MAPK/ERK inhibitor), 1 mM L-NAME (eNOS inhibitor), 10 μM PP2 (Src kinase inhibitor), and 10 μM STO-609 (CaMKII inhibitor) reduced EC tube formation induced by 500 ng/ml CCL28 (Fig. 2A, B). These results suggest eNOS/NO, Src, PI3K and MAPK mediate, at least in part, CCL28-dependent angiogenesis. In addition, CCL28 stimulation of ECs for 24 hr enhanced expression levels of cell adhesion protein β-catenin and anti-apoptotic protein B-cell leukemia/lymphoma-2 (Bcl-2) (Fig. 2C), supporting the proliferation potential of ECs by CCL28.

Figure 2.

eNOS, CaMKII, Src kinase, PI3K and MAPK/ERK inhibition reduced CCL28 activated EC tube formation. A) Photomicrographs of EC tubes from representative wells with no treatment (NT; negative control), VEGF (10 ng/ml, positive control), CCL28 alone (500 ng/ml) or following pretreatment with 10 μM wortmannin (PI3K inhibitor), 5 μM U0126 (MAPK/ERK inhibitor), 1 mM L-NAME (eNOS inhibitor), 10 μM PP2 (Src kinase inhibitor), and 10 μM STO-609 (CaMKII inhibitor) in DMSO. B) Normalized EC tube number from photomicrographs. Note that inhibitors of eNOS, CaMKII, Src kinase, PI3K and MAPK/ERK reduced, at least in part, CCL28-driven EC tube formation. * P < 0.05, ** P < 0.01, *** P < 0.001v CCL28 treatment (n=6). ). C) Enhanced expression levels of β-catenin and Bcl-2 in HUVECs after treatment with 500 ng/ml CCL28 for 24 hr. ** P < 0.01 v NT (n=5).

CCR10 regulation of eNOS-dependent angiogenesis in HUVECs

To further investigate the role of CCR10 and eNOS in EC tube formation, siRNA targeting CCR10 (100 nM) or eNOS (50 nM) were used and tube formation measured in HUVEC. As shown in Fig. 3A, knockdown either of CCR10 or eNOS abolished EC tube formation, indicating both CCR10 and eNOS proteins play a significant role in angiogenesis. We showed by Western blot that eNOS knockdown did not affect CCR10 expression, however, CCR10 knockdown reduced eNOS expression level by 60% (Fig 3B). The regulation of eNOS by CCR10 in ECs was further demonstrated by confocal microscopy (Fig. 3C) in that eNOS (green) co-localized with CCR10 (red) in control siRNA treated cells (upper panel in Fig. 3C). Consistent with Western blotting results (Fig. 3B), CCR10 fluorescence intensity was not affected in eNOS depleted cells (middle panel in Fig. 3C), however, the fluorescence intensity of eNOS dramatically decreased in CCR10 depleted cells (bottom panel in Fig. 3C). These data may indicate that eNOS depletion in ECs treated with CCR10 siRNA reflects the stabilization of the pool of eNOS that co-localizes with CCR10. Taken together, these results suggest CCR10, via regulation of eNOS expression and activation, plays an important role in angiogenesis.

Figure 3.

eNOS-dependent angiogenesis is regulated by CCR10 in HUVECs. A) Representative photomicrographs of EC tubes following treatment of cells treated with EBM-2 medium alone, control siRNA (100 nM), CCR10 siRNA (100 nM), or eNOS siRNA (50 nM); Results are representative of 4 experiments. B) After treatment for 72 h with control, CCR10 or eNOS siRNA, cells were collected and prepared for Western blotting. *** P < 0.001 v ctl-siRNA (n=5). C) Confocal microscopy of ECs after treatment with CCR10 or eNOS siRNA. Note that knockdown of CCR10 (red) resulted in decreased eNOS (green) intensity (bottom panel), compared with ctl-siRNA treatment (upper panel). Images are representative of 4 independent experiments.

Regulation of eNOS expression and function by CCR10

Since eNOS expression can be regulated by CCR10 in primary culture human ECs (Fig. 3B, C), we further investigated the effect of CCR10 expression on eNOS activity in human ECs. After treatment with 100 nM CCR10 siRNA for 72 h, ECs were stimulated with 5 μM calcium ionophore A23187 and cell lysates were collected and prepared for Western blotting (Fig. 4A). Both CCR10 and eNOS expression levels were reduced (Fig. 4B). However, phosphorylation of eNOS was significantly increased compared to control siRNA, even at 0 min (Fig. 4C), suggesting CCR10 binding basally suppresses eNOS activity. We also measured nitrite concentration in cell supernatants and calculated eNOS activity relative to total eNOS expression. Consistent with increased eNOS phosphorylation, NO production was significantly higher per molecule of eNOS in ECs after CCR10 knockdown (Fig. 4D) indicating that CCR10 negatively regulates eNOS expression and activity in ECs.

Figure 4.

eNOS expression and NO production are regulated and inhibited by CCR10 in human ECs. A) A23187-induced increase in p-eNOS in HUVECs was enhanced in ECs transfected with control or CCR10 siRNA (100 nM). B) Normalized expression level of CCR10 and eNOS in (A). ** P < 0.01, *** P < 0.001 (n=4). C) Normalized p-eNOS shown in (A). * P < 0.05, *** P < 0.001 (n=6). D) NO release from ECs transfected with control or CCR10 siRNA. eNOS activity was calculated as nitrite accumulation per min normalized to eNOS expression level. * P < 0.05, ** P < 0.01, *** P < 0.001 (n=6). Note that knockdown of CCR10 in ECs reduced eNOS expression and inhibited its activation.

Ligand-induced interaction between CCR10 and eNOS

To investigate whether CCR10 interacts directly with eNOS, primary culture HUVEC were collected and prepared for co-immunoprecipitation (Co-IP) to assess CCR10-eNOS interaction following stimulation with 5 μM A23187. Enhanced binding of CCR10 to eNOS was observed in ECs stimulated with A23187 based on Co-IP with anti-CCR10 Ab (Fig. 5A) or anti-eNOS Ab (Fig. 5B), where maximum binding was observed 5 min after addition of A23187. Similar results were obtained in ECs following stimulation with 500 ng/ml human recombinant CCL28 (Fig. 5C). CCR10-eNOS interaction was further supported by confocal microscopy and high resolution imaging (Fig. 5D). Upon stimulation with A23187 or CCL28 for 5 min, CCR10 (red) co-localized to a greater extent with eNOS (green) on the plasma membrane (yellow indicated by white arrows in Fig. 5D). Co-localization coefficient in the regions of interest (ROI) in the plasma membrane (right panel in Fig. 5D) were quantified from Zeiss LSM 880 confocal microscope images using Zeiss Zen software. Co-localization coefficient of CCR10 and eNOS was significantly greater following A23187 (81%) and CCL28 (78%) stimulation as compared to untreated control cells (43%; Fig. 5E). These results indicate increased interaction between CCR10 and eNOS at or near the plasma membrane of activated ECs.

Figure 5.

Increased interaction between CCR10 and eNOS in activated HUVECs. A, B) Co-IP experiments revealed direct interaction between CCR10 and eNOS in HUVEC after stimulation with 5 μM A23187. After treatment, the cells were collected and immunoprecipitated (IP) with anti-CCR10 Ab (A, n=5) or anti-eNOS Ab (B, n=4). * P < 0.05, ** P < 0.01, *** P < 0.001 v 0’ treatment. C) IP experiment indicated enhanced binding between CCR10 and eNOS in cells stimulated with 500 ng/ml CCL28. * P < 0.05, *** P < 0.001 v 0’ treatment (n=5). D) Confocal microscopy reveals co-localization (white arrows) between CCR10 (red) and eNOS (green) in ECs stimulated with 5 μM A2318 (middle panel) or 500 ng/ml CCL28 (bottom panel) for 5 min. Scale bar, 10 μm. E) Normalized co-localization coefficient of CCR10 (red) and eNOS (green) in the regions of interest (ROI) analyzed using Zeiss Zen software, as detailed in Materials and Methods. *** P < 0.001 v NT (n=40).

Blockade of eNOS-CCR10 interaction with myristoylated CCR10 binding domain (Myr-CBD) peptides enhances eNOS activity

Results thus far indicate CCR10 may directly interact with eNOS in ECs (Fig. 5), that CCR10 regulates eNOS expression (Fig. 3B, C) and angiogenesis (Fig. 3A) and inhibits eNOS activity (Fig. 4C, D), therefore we investigated the effect of inhibition of CCR10-eNOS interaction using a panel of cell-permeable peptides based on the sequence of the predicted interaction domain on eNOS. First, myristoylated 20 amino acid (aa) peptide (491-TRKKTFKEVANAVKISASLM-510; P1) was synthesized based on the sequence of human eNOS (which is conserved in mouse eNOS). Co-IP experiments in HUVECs indicated that pretreatment with 50 μM cell permeable 20 amino acid myristoylated CCR10 binding domain peptide (Myr-CBD20) significantly reduced CCR10-eNOS interaction in ECs stimulated with 5 μM A23187 for 5 min compared with control peptide (Supplemental Fig. 2). We also evaluated truncated versions of the CBD-derived peptide by synthesizing Myr-CBD −15 (FKEVANAVKISASLM; P2), −12 (VANAVKISASLM; P3) and −7 (KISASLM; P4) (Fig. 6A). Pretreatment with 50 μM P4, or Myr-CBD7, completely blocked CCR10-eNOS interaction in ECs following stimulation with 5 μM A23187 for 5 min (Fig. 6B). We next assessed the effect of Myr-CBD7 peptide on eNOS activity. Co-IP experiments showed significant reduction of CCR10-eNOS interaction in cells treated with Myr-CBD7 compared with scrambled control peptide (Myr-MSIALKS; Fig. 6C). Consistent with reduction in negative regulation by CCR10-dependent binding, eNOS phosphorylation (Ser1177) in presence of Myr-CBD7 was significantly enhanced (Fig. 6D).

Figure 6.

Blockade of eNOS-CCR10 interaction with myristoylated (Myr) CBD7 peptide enhanced eNOS activity in HUVEC. A) Four myristoylated peptides, P1–P4, were synthesized based on human eNOS sequence which is also conserved in mouse. Amino acid, aa. B) HUVEC were pretreated with each of 4 myristoylated peptides (50 μM) for 1 h at 37°C followed by stimulation with 5 μM A23187 for 5 min and the co-IP. The ratio of eNOS/CCR10 with no treatment was set as 1. * P < 0.05, ** P < 0.01 (n=4). C) Co-IP experiments revealed interaction between CCR10 and eNOS in HUVECs after pretreatment with Myr-control peptide (Ctl-P, Myr-MSIALKS) was reduced in cells treated with CBD7 peptide (50 μM) for 1 h prior to addition of 5 μM A23187. * P < 0.05 (n=4). D) Increased phosphorylation of eNOS (Ser1177) in HUVEC pretreated with 50 μM Myr-CBD7 peptide; * P < 0.05, ** P < 0.01 (n=4). E) Myr-CBD7 (50 μM) pretreatment blocked eNOS interaction with CCR10, but not CaM, in HUVEC treated with 500 ng/ml CCL28. * P < 0.05, *** P < 0.001 (n=5).

It was reported previously that calmodulin (CaM) binding to bovine eNOS occurred at residues 493-TRKKTFKEVANAVKISASLM-512 (31, 32). We thus assessed whether Myr-CBD7 affects eNOS-CaM binding by Co-IP. As shown in Fig. 6E, pretreatment of ECs with Myr-CBD7 peptide had no effect on CaM-eNOS binding induced by 5 min treatment with 500 ng/ml CCL28.

Topical administration of Myr-CBD7 peptide improved dermal wound healing in mice

CCL28 (also called mucosa-associated epithelial chemokine) is a recently described CC chemokine that signals via CCR10 as well as CCR3 (20). CCR10 has been reported to have two functional ligands, CCL27 and CCL28, that are involved in the epithelial immunity (33). We observed that CCL28 and CCR10 levels were highly expressed compared to CCL27 and CCR3 in skin of WT C57BL/6 mice (Supplemental Fig. 3). Similar to IL-6, the protein level of CCL28 determined by ELISA was elevated significantly at day 3 and day 7 in mouse wounds (Supplemental Fig. 4). Therefore, we next investigated whether blockade of CCR10-eNOS interaction with Myr-CBD7 peptide has an effect on wound healing in WT C57BL/6 mice. Four 5 mm diameter full thickness excisional wounds were made on the mouse dorsal skin and 30 μl Myr-CBD7 peptide (50 μM) was topically administrated onto the wounds immediately after creating the punch wound. Compared to control peptide treated wounds, Myr-CBD7 peptide significantly reduced wound size by 20% on day 5 and by 35% on day 7 (Fig. 7A). Wound size reduction with Myr-CBD7 treatment was associated with re-epithelization of the skin wound observed in H & E stained wound sections (Fig. 7B).

Figure 7.

Topical administration of Myr-CBD7 peptide improved wound healing in WT C57BL/6 mice. A) Representative photomicrographs of wounds after treatment with 30 μl 50 μM Myr- control peptide (Ctl-P, Myr-MSIALKS) or CBD7 peptide per wound. Four 5 mm full thickness excisional wounds were made on the mouse dorsal skin. Note that CBD7 peptide treatment reduced wound size significantly on day 5 and 7. * P < 0.05 (n=4). B) H&E sections showing re-epithelialization of skin wounds in WT mice treated with Myr-CBD7 or control peptide on day 7. Broken lines denote wound margins. Scale bar corresponds to 1 mm. Wound closure was measured and quantified on day 7 in each condition. * P < 0.05 (n=5). C) Enhanced eNOS expression by CBD7 peptide compared with control peptide in mouse wound at day 7. * P < 0.05, ** P < 0.01 (n=3–4). D) Co-IP experiments revealed reduced CCR10-eNOS interaction in mouse wounds after treatment for 7 days with 50 μM CBD7 peptide. *** P < 0.001 (n=5). Elevated day 7 mRNA level was observed for eNOS (E) and CD31 (F), but not CCR10 (G), in mouse wounds treated with CBD7 peptide by real-time RT-PCR. * P < 0.05, *** P < 0.001 (n≥5). Reduced CCL28 (H) and IL-6 (I) and elevated IL-4 (J) mRNA level by real-time RT-PCR in day 7 mouse wounds treated with Myr-CBD7 peptide compared with control peptide. * P < 0.05 (n=4).

Myr-CBD7 peptide treatment also enhanced eNOS protein level in wounds on day 7 (Fig. 7C) compared to control peptide, and Co-IP showed that Myr-CBD7 peptide treatment reduced CCR10-eNOS interaction in the wounds on day 7 (Fig. 7D). Besides eNOS (Fig. 7E), mRNA level of another EC marker, CD31, was also elevated (Fig. 7G); CCR10 mRNA level, however, did not change (Fig. 7F). In addition, Myr-CBD7 peptide treatment resulted in reduction of mRNA levels of CCL28 (Fig. 7H) and proinflammatory cytokine IL-6 (Fig. 7I) while enhancing anti-inflammatory cytokine IL-4 mRNA (Fig. 7J) in the mouse dermal wounds on day 7. The mRNA level of hepatocyte growth factor (HGF) increased, albeit non-significantly, by 35% in Myr-CBD7 treated wounds (Supplemental Fig. 5). Taken together, these results suggest topical treatment of mouse skin wounds with Myr-CBD7 peptide enhances eNOS/NO levels by blocking CCR10-eNOS interaction thereby facilitating an anti-inflammatory environment that promotes wound angiogenesis and wound healing.

DISCUSSION

Signaling mechanisms downstream of chemokine CCL28 activation of CCR10 receptor in endothelial cells (ECs) are not well established. Here we show CCL28 promotes CCR10 and eNOS/NO-dependent Src/PI3K/MAPK phosphorylation/activation and tube formation in primary human ECs. Further, CCR10 was observed to directly bind to eNOS and negatively regulate eNOS expression and calcium-dependent activation. Importantly, topical application of a novel cell permeable 7-mer CCR10/eNOS binding domain peptide (Fig. 6A; Myr-CBD7) blocked CCR10-eNOS interaction and upregulated eNOS expression and angiogenesis which were associated with improved dermal wound healing in mice. These findings suggest for first time that CCL28/CCR10 signaling regulates angiogenesis of dermal microvessels and may represent a novel therapeutic target for improving skin wound healing.

Angiogenesis is the formation of new blood vessels from pre-existing vasculature (34) and is controlled by endogenous pro- and anti-angiogenic factors (35, 36) that regulate endothelial cell (EC) proliferation, differentiation, migration, and organization into branched tubular networks. Under physiological postnatal conditions, angiogenesis is important for tissue neovascularization in response to ischemia and in repair during wound healing (37).

Chemokines and their receptors were originally characterized as playing a role in cellular trafficking of leukocytes during inflammation and immune surveillance (38). Increasing evidence indicates they are also involved in the promotion and inhibition of angiogenesis (37) in part by promoting recruitment of inflammatory cells which release growth factors and cytokines that facilitate the wound healing process (39). It was reported that CX3CL1 (fractalkine) via CX3CR1 receptor stimulated angiogenesis by activating the Raf-1/MEK/ERK and PI3K/Akt/eNOS-dependent signal pathways (40). Furthermore, CCL2 has been shown to promote the expression of signaling proteins phosphatidylinositol 3-kinase (PI3K), Akt, MAPK and ERK1/2 that increase NO production, promote EC proliferation and migration, and ultimately promote inflammation-driven angiogenesis (41, 42). In addition, CXCL12 has been shown to stimulate the CXCR4/PI3K/Rac1 signaling pathway and enhance endothelial barrier function necessary for vessel maturation (43). CXCL10 binding to its receptor CXCR3-A also induced activation of ERK, Akt, and Src in human vascular pericytes as well as activate p38 MAPK signaling in HEK-293 cells overexpressing CXCR3-B (44). Numerous other studies have shown an important role for PI3K in coordinating chemokine responses via coupling to downstream effectors, including ERK and Akt (45).

Previously, we reported the overexpression of chemokine CCL28 and its receptor CCR10 in synovial tissues from RA patients and discovered that CCL28/CCR10 signaling promotes EC migration into RA joints (23). Here we found that CCR10 was highly expressed in human primary ECs and EPCs (Supplemental Fig. 1), consistent with abundant CCR10 expression noted in dermal microvascular ECs (21). It was also reported previously that human CCL28 was able to stimulate calcium signaling in BaF/3 cells transfected with either human and mouse CCR10 cDNA (18). Since eNOS activity is dependent on intracellular calcium (25) and CCL28 induces Ca²⁺ mobilization in CCR10 transfected human cells (18), it is not surprising that CCL28 induced NO production in human ECs where its receptor CCR10 is highly expressed. eNOS activity is known to be acutely regulated by an increase in the concentration of intracellular calcium upon specific receptor activation by agonists such as bradykinin (46) and vascular endothelial growth factor (VEGF) (47). Consistent with BK, VEGF, and several chemokine signaling pathways, we showed that CCL28 binding to CCR10-activated eNOS/NO dependent Src/PI3K/MAPK signaling in human primary ECs. We also observed that CCL28 stimulation increased the expression levels of cell adhesion protein β-catenin (48) as well as anti-apoptotic protein Bcl-2 (49, 50) in ECs, supporting the proliferation potential of ECs induced by CCL28. Furthermore, pretreatment with inhibitors of these kinases lead to reduced tube formation in ECs stimulated with CCL28, suggesting an important role of CCL28/CCR10 in angiogenesis.

eNOS is expressed in vascular ECs and plays an important role in the regulation of vascular tone, platelet aggregation and angiogenesis (51). eNOS has been shown to interact with a variety of regulatory and structural proteins such as calmodulin (CaM) (52), heat shock protein 90 (HSP90) (53), dynamin-2 (54), caveolin-1 (Cav-1) (24) which regulate eNOS activity and trafficking in a number of physiological and pathophysiological contexts. In this study, we identified CCR10 as a novel eNOS-interacting protein in primary culture human ECs. CCR10 not only inhibited eNOS activity, its expression correlated with eNOS expression in cell culture expression model systems and during angiogenesis and wound healing. We showed that CCR10 binds to eNOS amino acid residues 504-KISASLM-510 which is in the CaM binding region of bovine eNOS (residues 493–512) (31, 32). We further observed that a peptide mimetic of these 7 residues, which we termed Myr-CBD7, specifically blocked interaction of eNOS and CCR10, but not eNOS-CaM, in ECs activated by CCL28, indicating CaM-dependent regulation of eNOS remains intact in presence of the antagonist peptide. However, the identity of the eNOS binding site on CCR10, a GPCR, is not yet known and needs further investigation.

Clinical studies have shown that oral supplementation of L-arginine (the substrate for NO) had a beneficial effect on immune function and wound healing in patients (55, 56). Addition of NO donors such as NO-gel and S-nitroso-N-acetyl-penicillamine (SNAP) to wounds enhanced angiogenesis and accelerated healing (57). Genetic disruption of eNOS or pharmacological inhibition by S-methyl isothiouronium (MITU, a competitive inhibitor of NO synthase) limited angiogenesis during tissue repair resulting in delayed wound closure (58, 59). Further, eNOS knockout mice recover poorly from hind-limb ischemia as a consequence of decreased angiogenesis (60, 61). Transfection of eNOS cDNA in a rat hindlimb ischemia model resulted in a significant increase in peripheral blood flow, capillary number and VEGF level (62). It was reported that eNOS at the mRNA and protein level was induced as early as day 1 post-wounding in a skin repair model in C57BLKS mice (63). Recently, it was reported that over-expression of a transcription factor X-box binding protein 1 splicing (XBP1s) increased eNOS protein and NO production, leading to EC migration and improved wound healing and angiogenesis (64). Thus, elevation of eNOS-dependent NO production may be a clinical strategy for enhancing angiogenesis to improve wound healing. In the current study, we present data showing that CCR10 negatively regulates angiogenesis by repressing the expression and activity of eNOS. In vitro, Myr-CBD7 peptide blocked CCR10-eNOS interaction and elevated eNOS activity in cultured ECs. In vivo, topical application of Myr-CBD7 peptide improved skin wound healing in association with elevated expression of eNOS as well as CD31 (EC marker) suggestive of increase in EC number and angiogenesis.

Previously, we found that proinflammatory factors such as LPS and IL-6 increased the protein level of both CCL28 and CCR10 in endothelial cells (23). Here we found that Myr-CBD7 treatment reduced both CCL28 and proinflammatory cytokine IL-6 in the dermal wounds of mice compared with control peptide. In addition, mRNA level of anti-inflammatory cytokine IL-4 was elevated. Pro-inflammatory classically-activated (M1) macrophages produce cytokines such as IL-6, IL-1, and TNF-α, while alternatively-activated (M2) macrophages produce IL-10, TGF-β (65), and IL-4 (66) which are thought to be associated with tissue repair. In addition, it was reported that IL-4 transgenic mice had delayed wound closure and re-epithelialization accompanied by drastically elevated levels of inflammation (29). Interestingly, Matrigel supplemented with macrophage subsets induced by IL-4 that were injected subcutaneously in C57BL/6J mice resulted in increased numbers of ECs and tubular structures in M2-enriched plugs compared to controls (67). Thus, it may be that Myr-CBD7 peptide treatment also suppressed proinflammatory M1 (IL-6) and facilitated M2 macrophage functions (IL-4) resulting in angiogenesis and improved wound healing.

Wound healing is a complex and dynamic process consisting of four overlapping phases consisting of hemostasis, inflammation, proliferation, and maturation (68, 69). Normal repair in healthy animals was characterized by a moderate induction of eNOS at the mRNA and protein level, whereas impaired healing as in subjects with type II diabetes is clearly associated with reduced eNOS protein expression (63). In patients with diabetic foot ulcers (DFUs), persistent hyperglycemia leads to EC dysfunction (70) which results in decreased pro-angiogenic signaling including NO production (71). Consistent with these findings, we previously reported that eNOS expression level (25), as well as average plasma NO concentration (72), was significantly reduced in human muscle biopsies from patients with type 2 diabetes mellitus compared to lean healthy controls. Therefore, CCL28/CCR10 levels and their roles in regulation of eNOS should be further investigated in chronic wound healing diseases such as DFUs.

In summary, we identified and characterized novel functions of CCL28 and CCR10 in angiogenesis and skin wound healing. Our findings suggest blockade of CCR10/eNOS interaction using novel binding domain inhibitory peptide Myr-CBD7 may represent a potential therapeutic strategy for improving chronic skin wound healing.

ABBRIVATIONS:

CCL28

chemokine ligand 28

CCR10

chemokine receptor 10

NO

nitric oxide

eNOS

endothelial nitric oxide synthase

EC

endothelial cell

A23187

calcium ionophore

Bcl-2

B-cell leukemia/lymphoma-2

Myr

myristoylated

CBD7

CCR10 binding domain 7

GFP

green fluorescent protein

HUVEC

human umbilical vein endothelial cell

HEK

human embryonic kidney

DAF-FM

4-Amino-5-methylamino-2’,7’-difluorofluorescein

PI3K

phosphoinositide 3-kinase

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinase

co-IP

co-immunoprecipitation

CaM

calmodulin

H&E

hematoxylin and eosin

IL-4

interleukin 4

IL-6

interleukin 6

HGF

hepatocyte growth factor

T2DM

type 2 diabetes mellitus

DFU

diabetic foot ulcer