Nitric Oxide Produced by the Antioxidant Activity of Verapamil Improves the Acute Wound Healing Process

Abstract

Background:

Verapamil is used in the treatment of hypertension, angina pectoris, cardiac arrhythmia, hypertrophic scars, and keloids to block transmembrane calcium ion flux. Verapamil has antioxidant activity, which enhances the production of nitric oxide (NO). NO promotes the proliferation of fibroblasts, keratinocytes, endothelial cells, and epithelial cells during wound healing. In this study, we investigated the effect of verapamil and its antioxidant properties on the enhancement of acute wound healing via NO.

Methods:

A full-thickness wound healing model was created on the rat dorsal with a silicone ring. The wound closure rate was estimated every 2 days for 14 days. A histological study was performed to evaluate wound healing. Immunofluorescence staining was analyzed for angiogenesis. The expressions of collagen type I (COL I), collagen type III (COL III), and vascular endothelial growth factor (VEGF) were assessed by Western blot. Real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of endothelial NO synthase and inducible NO synthase, which are related to antioxidant activity in the process of wound healing.

Results:

The wound closure rate was faster in the verapamil group compared to the control and silicone groups. Histologic analysis revealed capillaries and stratum basale in the verapamil group. Immunofluorescence staining was shown vessel formation in the verapamil group. Western blot and qRT-PCR analysis revealed high expression levels of COL I, VEGF, eNOS, and FGF in the verapamil.

Conclusion:

Verapamil’s antioxidant activity enhances NO production in acute wound healing. We suggest that verapamil can be used to promote acute wound healing.

Introduction

Verapamil, a nondihydropyridine calcium channel blocker (CCB), is used in the treatment of hypertension, angina pectoris, and cardiac arrhythmias [1–3]. It is also used to improve hypertrophic scars and keloids via inhibition of proline synthesis and induction of procollagenase synthesis [4–9]. On the other hand, the antioxidant activity of verapamil promote acute wound healing by increases the production of nitric oxide (NO), which regulates collagen synthesis, angiogenesis, and extracellular matrix (ECM) formation [10–14].

The wound healing process consists of hemostasis, inflammation, proliferation, and remodeling [15]. Wound repair and regeneration processes are regulated by the cytokines and growth factors released at the wound site and involve inflammatory cells, fibroblasts, keratinocytes, and epithelial cells [16]. All of these cells are capable of producing NO [17]. NO regulates the proliferation of epithelial cells, endothelial cells, fibroblasts, and keratinocytes by releasing fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and transforming growth factor-beta (TGF-β) [18, 19]. It also regulates collagen synthesis, collagen accumulation, and tissue contraction [20]. An in vivo study demonstrated that eNOS deficiency delayed wound healing [21].

Previous study, there was not demonstrated the effect of verapamil’s antioxidant activity on NO production and wound healing. In this study, we investigated the effects of verapamil on antioxidant activity using a rat acute wound model. We hypothesized that the antioxidant activity of verapamil increases NO, which promotes the proliferation of fibroblasts, keratinocytes, epithelial cells, endothelial cells, and angiogenesis in the process of wound healing.

Materials and methods

Animal experiment

The animal experiment was approved by the Institutional Review Board of the Catholic University of Korea (CUMC-2018–0067-02). Sprague–Dawley rats (n = 27) were randomly divided into control, silicone gel, and 1 mg/g verapamil gel (Genewel, Seongnam-si, Korea) groups. After anesthetization with isoflurane (2% isoflurane, 2 L/min oxygen) and removal of the dorsal hair, a 2.5 cm-diameter full-thickness wound was created on the center of the dorsal. Skin wound contraction can interrupt the evaluation of wound healing potential [16, 22]. A 3 cm inner-diameter silicone ring was attached around the wound. A silicone ring fixed with Vetbond Tissue Adhesive (3 M Animal Care Products, St. Paul, MN, USA) can be used to prevent wound contraction and sutured using 4–0 black silk. The wounds were cleansed with saline and treated with gel daily for 14 days.

Measurement of wound closure

The wound closure rate was evaluated by measuring the wound area every 2 days for 14 days using the ImageJ software (NIH, Bethesda, MD, USA) (Fig. 1). The wound closure rate (%) was calculated as [(initial defect − remaining defect)/(initial defect)] × 100% (Fig. 1) [11].

Fig. 1.

Full-thickness wound on the dorsal side of the rat. The wound closure rate was calculated as [(initial defect − remaining defect)/(initial defect)] × 100%. The black and red lines delineate the initial defect area at the time of surgery and the remaining defect, respectively

Histological analysis

Rats were anesthetized, and the wound tissue was excised and fixed in 10% formalin for at least 24 h. The tissue sample was embedded in paraffin, sectioned at a thickness of 4 μm, and stained with hematoxylin and eosin (H&E). The sample was observed using a slide scanner (Panoramic MIDI; 3DHISTECH, Budapest, Hungary). Fibroblasts and capillaries were counted using a slide image analysis software (QuPath, University of Edinburgh, Edinburgh, UK).

Immunofluorescence staining

Paraffin sections were heated at 60 °C for 1 h then deparaffinization and rehydration. The sections were blocked with goat serum at room temperature for 1 h. After blocking, CD31(Abcam, Cambridge, UK), and VEGF-R(Abcam) were used for primary antibodies to incubate at 4 °C for overnight. The secondary antibody (Alexa486, Abcam) was incubated at room temperature for 2 h. A fluorescence microscope (Zeiss, Oberkochen, Germany) was used for immunofluorescence staining analysis.

Western blot analysis

A 2.5 cm-diameter tissue section of the rat dorsal was homogenized, and the protein was extracted using T-PER reagent (Thermo Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA). The total protein concentration was measured with a Quick Start Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and the SoftMax Pro software (MDS Analytical Technologies, Sunnyvale, USA). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose blotting membrane (Bio-Rad). The membrane was blocked with 5% skim milk (BD Biosciences, Mansfield, MA, USA) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) at room temperature for 1 h. The membrane was incubated with the following primary antibodies at room temperature for 2 h: collagen 1 alpha 1 antibody (Novus Biologicals, Littleton, CO, USA), collagen 3 alpha 1/col3a1 antibody (Novus Biologicals), anti-VEGF antibody (Abcam), or β-actin monoclonal antibody (Thermo Scientific). After three washes with TBS-T, the membranes were incubated with the secondary antibodies (mouse anti-rabbit IgG-HRP antibody and goat anti-mouse IgG-HRP antibody; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 2 h. The proteins were detected with the HRP-conjugate detection reagent Clarity Western ECL Substrate (Bio-Rad) and the LAS 4000 (Fujifilm, Tokyo, Japan). Protein was quantified using the Multi Gauge software (Fujifilm).

Real-time polymerase chain reaction (qRT-PCR)

RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA concentration was measured using a NanoDrop instrument (NanoDrop Technologies, Wilmington, DE, USA). PCR was performed using 1 μg cDNA, 10 pmol/L of each primer and the SYBR Green I master reagent (Roche Diagnostics, Mannheim, Germany). The sequences of primers were as follows: eNOS forward 5′-CACACTGCTAGAGGTGCTG GAA-3′, eNOS reverse 5′-TGCTGAGCTGACAGAGTAGTAC-3′, iNOS forward 5′-GCAG GTTGAGGATTACTTCTTCCA-3′, iNOS reverse 5′-GCCCTTTTTTGCTCCATAGGAAA-3′, VEGF forward 5′-GAGTATATCTTCAAGCCGTCCTGT-3′, VEGF reverse 5′-ATCTG CATAGTGACGTTGCTCTC-3′, FGF forward 5′-GCAGTATAAACTCGGATCCAAAAC-3′, FGF reverse 5′-GCCTGAGAGTGACAGTGTCTAAAG-3′, TGF-β forward 5′-CGAGG. TGACCTGGGCACCATCCATGAC-3′, TGF-β reverse 5′-CTGCTCCACCTTGGGCTTGC.GACCCAC-3′ GAPDH forward 5′-CAAGTTCAACGGCACAGTCAAGG-3′, and GAPDH reverse 5′-ACATACTCAGCACCAGCATCACC-3′. The Light Cylcer 480 software (Roche, Basel, Switzerland) was used to perform quantitative analysis.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) with the GraphPad Prism 5.0 software (GraphPad, La Jolla, CA, USA) (*p < 0.05, **p < 0.01, ***p < 0.001).

Results

Wound healing

The wound closure rates were similar in the control, silicone, and verapamil groups until day 6. At day 14, the wound closure rate in the verapamil group was faster than the control and silicone groups (Fig. 2). But, there was no significant difference.

Fig. 2.

Wound closure rate (%). A Images of healing wounds in the control, silicone, and verapamil groups at post-operative day (POD) 2, 4, 6, 8, 10, 12, and 14. B At POD 14, the wound closure rate was higher in the verapamil (84.7 ± 3.45%) group compared to the control (77.9 ± 2.65%) and silicone (78 ± 2.65%) groups. There was no significant difference between the groups. The wound healing area was quantified using ImageJ

Histological and immunofluorescence staining evaluation

Histological findings with H&E staining revealed that granulation tissues formed in the control, silicone, and verapamil groups (Fig. 3A–C). The stratum basale was observed in the center of the wound bed in the verapamil group (Fig. 3C). The number of fibroblasts in the verapamil group was higher than the other groups. But, there was no significant difference (Fig. 3D). The capillary in the verapamil group and silicone group increased threefold and 2.2-fold compared to the control group. There was significant difference in capillary count results (**p < 0.01, ***p < 0.001, Fig. 3E).

Fig. 3.

A–C Histological analysis of the healing tissue by H&E staining. Granulation tissue formed by collagen synthesis in the control, silicone, verapamil groups. The stratum basale layer was observed only in the verapamil group (yellow arrow). D The number of fibroblasts is higher in the verapamil (260 ± 24) group compare to the control (233 ± 24) and silicone (288 ± 12) group (red arrows). E The capillary in the verapamil (30 ± 1) group was higher than the control (10 ± 2) group and silicone (22 ± 1) group (black arrows). A Control group, B silicone group, C verapamil group. (**p < 0.01, ***p < 0.001)

CD31 and VEGF-R were used to detect endothelial cells by immunofluorescence analysis (Fig. 4A, B). The CD31 was shown to the vascular lining only in the verapamil group (Fig. 4A). VEGF-R was detected in the silicone and verapamil group. But, the more prominent vascular line was observed in the verapamil group (Fig. 4B).

Fig. 4.

Endothelial cells labeled CD31 and VEGF-R antibodies express green fluorescent. CD31 and VEGF-R fluorescent express vascular lines and most prominent in the verapamil group

Expression of COL I, COL III, and VEGF

The protein expressions of COL I, COL III, and VEGF were analyzed by Western blotting (Fig. 5A). COL I expression was higher in the verapamil group compared to the control and silicone groups, but the difference was not significant (Fig. 5B). The expression of COL III was similar in all groups (Fig. 5C). VEGF expression was significantly higher in the verapamil group compared to the control and silicone groups (*p < 0.05, ***p < 0.001, Fig. 5D).

Fig. 5.

A Western blot analysis of COL I, COL III, and VEGF protein expression levels. B Expression of COL I was higher in the verapamil (0.99 ± 0.06) group compared to the control (0.75 ± 0.06) and silicone (0.85 ± 0.18) group. C COL III expression was similar in all groups. D VEGF expression was significantly higher in the verapamil (0.76 ± 0.34) compared to the control (0.29 ± 0.06) and silicone (0.18 ± 0.01) groups. (*p < 0.05, ***p < 0.001)

Expression of eNOS, iNOS, VEGF, TGF-β, and FGF.

The mRNA expression levels of eNOS, iNOS, VEGF, FGF, and TGF-β were analyzed by qRT-PCR (Fig. 6). eNOS, VEGF, and TGF-β expression levels were higher in the verapamil group compared to the control and silicone groups (*p < 0.05, **p < 0.01, Fig. 6A, C, E). The expression level of iNOS was lower in the verapamil group than the other groups (**p < 0.01, Fig. 6B). FGF expression was higher in the verapamil group, but there was no significant difference (Fig. 6D).

Fig. 6.

A–E qRT-PCR analysis of eNOS, iNOS, VEGF, FGF, and TGF-β. mRNA expression levels. Expressions of eNOS, VEGF, and TGF-β were higher in the verapamil (7.17 ± 2.76 fold, 2.62 ± 0.85 fold, and 6.86 ± 2.68 fold) group compared to the control group (A, C, E). iNOS expression level was low in the verapamil group (B). The verapamil group showed a higher level of FGF expression. But, there was no significant difference (D). (*p < 0.05, **p < 0.01)

Discussion

CCBs such as verapamil are used for the treatment of hypertension, angina pectoris, cardiac arrhythmias, hypertrophic scarring, and keloids [1, 2, 23]. Verapamil also has antioxidant activity that increases the production of NO [12, 14, 24]. NO promotes the proliferation of epithelial cells, endothelial cells, fibroblasts, and keratinocytes in the wound healing process [17–20].

Several studies have demonstrated that verapamil increases the activity of collagenases, which reduce the ECM of hypertrophic scars and keloids [23]. On the other hand, the antioxidant activity of verapamil increases NO production, which regulates collagen synthesis, angiogenesis, and ECM formation in acute wound healing.

We hypothesized that the antioxidant activity of verapamil promotes acute wound healing via NO synthesis. In this study, we showed that the antioxidant activity of verapamil enhanced wound healing in a rat acute wound model. To demonstrate our hypothesis, we have analyzed the expression levels of collagen type I and collagen type III which are the major components of ECM by western blot. Angiogenesis in regenerated skin tissue was analyzed through histological analysis. We also performed qRT-PCR analysis of the expression of eNOS, iNOS, and growth factors to demonstrate the relation between NO production by verapamil’s antioxidant activity and wound healing.

NO stimulates the wound healing process [20, 25]. The various cells involved in the wound healing process produce NO via nitric oxide synthases (NOSs) [17]. eNOS accelerates wound healing by promoting cell migration and the proliferation of fibroblasts, epithelial cells, endothelial cells, and keratinocytes [26]. The expression of eNOS was elevated in the verapamil group, thus accelerating wound healing via cell migration and proliferation. iNOS is induced by macrophages, cytokines, and bacteria [17]. iNOS was expressed by the immune response in the early phase of wound healing. The level of iNOS expression was low in the verapamil group. The verapamil group, which is in the remodeling phase, showed a low expression of iNOS, whereas the expression of iNOS was high in the other groups. Because they were not remodeling phase yet [17].

Silicone gel is effective in the treatment of hypertrophic scars and keloids [23]. However, silicone gel does not affect the early wound healing process [27]. Silicone gel was used as a vehicle for verapamil delivery in this study. A previous study showed that a 1 mg/g concentration of verapamil had the most stable release time and was more effective than other concentrations (not shown).

The results of this study demonstrated that verapamil improved the wound healing process. Our results showed differences in wound closure rate, and histological findings between the verapamil group and the other groups. We also demonstrated that verapamil regulated growth factors and NO synthesis during the wound healing process based on expression levels of COL I, COL III, VEGF, eNOS, iNOS, FGF, and TGF-β. The most abundant component in ECM is COL I, and the second most abundant is COL III [28]. In the inflammation and proliferation phase, COL III increases due to the proliferation of fibroblasts and collagen synthesis [29]. Our results showed no significant difference between the groups. The expression of COL III was low in the control group, indicating that collagen synthesis and cell proliferation were delayed compared to the other groups. In the remodeling phase, COL III is replaced by COL I via collagen cross-linking [30]. In the verapamil group, the expression of COL I was higher than in the other groups, suggesting that verapamil promoted wound healing. The expression levels of COL I and COL III showed that the wound healing process was faster in the verapamil group compared to the control and silicone groups. VEGF promotes angiogenesis, epithelialization, and vessel formation [31]. In our study, capillaries and vessel formation were increased in the verapamil group compared to the control and silicone groups. TGF-β and FGF are produced by macrophages, keratinocytes, and fibroblasts and mediate angiogenesis, granulation tissue formation, epithelialization, and matrix formation in wound healing [31]. The expression levels of TGF-β and FGF showed higher in the verapamil group compare to the other groups.

Our results showed that verapamil has antioxidant activity, which enhances the production of NO. NO regulates the expression of growth factors, collagen synthesis, angiogenesis, and re-epithelialization in the wound healing process. In conclusion, verapamil can be used to improve acute wound healing.

Compliance with ethical standards

Conflict of interest

The authors have no financial conflicts of interest.

Ethical statement

The animal studies were performed after receiving approval of the Institutional Animal Care and Use Committee (IACUC) in Catholic University (IACUC approval No. CUMC-2018–0067-02).