Early intervention by Captopril does not improve wound healing of partial thickness burn wounds in a rat model.

The Renin Angiotensin System is involved in fibrotic pathologies in various organs such as heart, kidney and liver. Inhibition of this system by angiotensin converting enzyme antagonists, such as Captopril, has been shown beneficial effects on these pathologies. Captopril reduced the inflammatory reaction but also directly influenced the fibrotic process. Prolonged and excessive inflammatory response is a major cause of hypertrophic scar formation in burns. We therefore evaluated the effect of Captopril on the healing of partial thickness burn wounds in a rat model. Partial thickness contact burns were inflicted on the dorsum of the rats. The rats received either systemic or local treatment with Captopril. The inflammatory reaction and wound healing (scar) parameters were investigated and compared to control animals. In this study we could not detect positive effects of either administration route with Captopril on the inflammatory reaction, nor on wound healing parameters. The local treatment showed reduced wound closure in comparison to the systemic treatment and the control group. Early Captopril treatment of burn wounds did not show the beneficial effects that were reported for fibrotic disorders in other tissues. To influence the fibrotic response Captopril treatment at a later time point, e.g. during the remodeling phase, might still have beneficial effects.

Introduction

Excessive scar formation after partial and full-thickness burn wounds is still an unresolved problem and prevention remains a major challenge. Tightly regulated processes which are necessary for successful regeneration of the skin have become uncontrolled in burn wounds, resulting in fibrosis. The onset of fibrosis in various tissue types is diverse, although common features are also present. One of the common factors, and the main effector of fibrosis, is transforming growth factor beta one (TGF-ß1) [1]. This growth factor is involved in the transition of fibroblasts into myofibroblasts. These myofibroblasts are major contributors to the production of excessive extracellular matrix proteins such as collagens, as well as to the process of wound contraction. Ultimately this process results in red, stiff, contracted and disfiguring scars. To date, the underlying mechanisms leading to scar formation are still a puzzle that needs to be solved and an effective therapy to prevent excessive scarring has not been discovered yet.

A possible other player in the fibrotic process is the Renin Angiotensin System (RAS). The main function of this system is the regulation of blood pressure and electrolyte balance. Several studies have shown the activation of a local RAS (local or tissue RAS: tRAS) upon tissue damage in various tissues [2]. It was shown that tRAS is involved in fibrotic processes of many organs including heart, kidney and liver [2], [3]. In addition tRAS has been linked to scar formation in the skin [4], [5], [6]. The main bioactive component of RAS is angiotensin II (AngII) which is derived from cleavage of angiotensin I by angiotensin converting enzyme (ACE). Subcutaneous administration of AngII in C57BL/6 mice induced skin fibrosis characterised by an increased deposition of collagen, α smooth muscle actin (αSMA) expression and number of inflammatory cells [7]. In addition increased ACE activity was shown in human scars compared to control and wounded skin [4].

AngII exerts its effects by binding to one of the two main RAS receptors; angiotensin receptor I (AT1) and angiotensin receptor II (AT2). AT1 activation is linked to fibrosis through the induction of the production of various cytokines (e.g. TGF-ß1) and extracellular matrix proteins, and by inducing cell proliferation and inflammation [2], [3]. In mice, scars showed an increased expression of AT1 compared to unwounded skin. More contraction of the granulation tissue and enhanced fibroblast migration was observed through AT1 activation [5]. In humans, two days after wounding, both AT1 and AT2 expression are upregulated, while at later time points AT2 expression in skin biopsies was more pronounced compared to AT1 [8]. In contrast to the AT1, the exact biological functions of AT2 are not completely clear yet. It has been suggested that AT2 can counteract the effects of AT1 and thereby has an anti-fibrotic function. However, literature is equivocal about the exact role of AT2 in fibrosis.

Studies on fibrotic processes in other tissues than skin have demonstrated beneficial effects of RAS inhibition. These inhibitors were shown to elicit an anti-fibrotic as well as an anti-inflammatory response to ameliorate fibrosis. One of the methods to inhibit the RAS is inhibiting the formation of AngII by ACE-inhibitors such as Captopril or Enalapril. Systemic treatment with Enalapril was shown to reduce pro-fibrotic collagen III expression and reduce hypertrophic scar formation in the rabbit ear wound model [6]. The effect of ACE-inhibition on burn wound healing and scar formation is largely unknown. One case-study describes the topical treatment of a patient with a 5-month old hypertrophic scald burn scar. Captopril improved the scar by reducing hypertrophy and redness. However, an appropriate placebo-treated scar was missing in this study [9]. Inhibition of AngII production using ACE-inhibitors early during wound healing might be effective to reduce scar formation in burn wounds. In this paper we describe the effects of systemic and topical Captopril administration early during the wound healing process on partial thickness contact burns in a rat model.

Materials and methods

Animals

The experimental protocol of this study was approved by the institutional Animal Experiments Committee of the VU University Medical Center Amsterdam, The Netherlands in accordance with the governmental and international guidelines on animal experimentation. Animals had access to water and food ad libitum. Before the start of the experiment, seventy-eight male Wistar rats of 6–7 weeks old, weighing 280 g ± 11 g, received an acclimatization period of 12 days of which the last three days included handling of the animals. During the acclimatization period animals were housed in groups of 4–5.

After the infliction of the burn wound the animals were individually housed in order to prevent detachment of the bandage by other animals and disturbance of the wound healing process. Two animals died several hours after the bandage procedure at PBD 2 (topical Captopril treatment group) and PBD 8 (systemic Captopril treatment group) due to unknown reasons.

Application of the burn wound

Approximately 30 min before the infliction of the burn wound, animals received the analgesic Temgesic 0.05 mg/kg via subcutaneous injection. Anesthesia was induced by inhalation of 4% isoflurane combined with O2 with a flow rate of 0.3 L/min, the maintenance dose was 2.5% isoflurane. During surgery the animals were placed on a warming pad to prevent hypothermia. The dorsum and abdomen of the animals were shaved from neck till the hind legs with electric clippers. Additionally the dorsum (the area of the burn wound) was shaved wet with a razor blade to remove possible remaining stubbles.

A partial thickness contact burn wound of 2 × 2 cm was inflicted on the dorsum, just behind the forelegs of the animal, with a brass stamp (100 g) of 100 °C for 10 s without applying pressure.

To prevent contamination of the wound and further damage from scratching, the burn wounds were covered with sterile gauze and fixed with adhesive Curafix® (Lohmann & Rauscher GmbH & Co., Neuwied, Germany). Subsequently the Curafix® bandage was covered with an elastic self-adhesive PetFlex® (Andover Healthcare, Inc.; Almelo, The Netherlands) bandage forming a jacket which was fixed with silk tape.

Bandage changes were performed at PBD 1, 4, 7, 11 and 15 under inhalational anesthesia which was induced with 4% isoflurane and maintained with 2.5% isoflurane. On these days the burn wound was covered with Multisorb® (3 × 3 cm) (BSN medical Limited, Willerby, United Kingdom) which was subsequently covered and fixed with adhesive Curafix®. The rest of the bandage procedure was similar to the procedure after burn wound infliction (PBD 0).

Treatments

At PBD 1 the animals were randomly divided in three groups of 26 animals: control, topical and systemic treatment. Since burn wound patients are often not immediately hospitalized after the incident, treatment with Captopril (Sigma–Aldrich, Steinheim, Germany) was started one day post burn in order to mimic the clinical setting. Half of the animals of each group were sacrificed at PBD 7, the other animals were sacrificed at PBD 42.

The Captopril solution was either applied systemically via the drinking water or locally (topically) by covering the wound area with a dressing containing 5% Captopril. Water intake was monitored daily by weighing the water bottle. The 5% Captopril solution for systemic and topical administration were both prepared in Dulbecco’s phosphate buffered saline (PBS) (Gibco®; Thermo Fisher Scientific, Etten-Leur, The Netherlands). Captopril solutions were freshly prepared on the day of bandage change or on the day when the drinking water was refreshed. Captopril was added to the drinking water (final concentration: 0.1 mg/mL) for the treatment of the systemic group; the water containing Captopril was refreshed every four days. For the topical Captopril treatment, 1 ml of 5% Captopril/PBS was applied on the Multisorb® bandage which was subsequently placed on the burn wound. Multisorb® bandages of the control and systemic group were treated with 1 mL PBS in order to create the same moist conditions. This procedure was repeated every dressing change.

The partial thickness burn wounds were treated with Captopril for a period of 6 days for those animals that were sacrificed at PBD 7 or 14 days for the animals that were sacrificed at PBD 42. At the end of the Captopril treatment the wounds were covered with dry Multisorb® bandage (PBD 15). Bandages were completely removed after wound closure (PBD 21).

For macroscopic evaluation of the wounds, pictures were made during bandage change. At the end of the experiment animals were euthanized by CO2 inhalation. The burn wounds were excised and fixed in Kryofix (48% ethanol, 7% polyethylene glycol (PEG)-300) and stored at 4 °C.

Microscopic wound evaluation

Kryofix fixed tissue biopsies were dehydrated, embedded in paraffin and cut in five μm sections. For (immuno)histochemical stainings, sections were deparaffinized and rehydrated.

Tissue sections were stained with hematoxylin (Mayers; DAKO, Glostrup, Denmark) and eosin (Sigma–Aldrich) to evaluate wound closure.

Activated neutrophils were assessed by immunohistochemical staining using antibodies against Myeloperoxidase (MPO) (Abcam, ab9535, dilution 1:50; Cambridge, United Kingdom). Macrophage subtypes M1 and M2 were determined using ED1 and ED2 antibodies (Serotec, dilution 1:200; Oxford, UK). The presence of myofibroblasts and in the wounds was assessed by immunohistochemistry using an antibody against αSMA (DAKO, clone 1A4, dilution 1:500; Glostrup, Denmark). Subsequently the slides were incubated for 30 min at room temperature with Brightvision Poly-HRP anti-Mouse (Immunologic, Duiven, The Netherlands) for αSMA, ED1 and ED2 or BrightVision Poly-HRP anti-rabbit (Immunologic) for MPO. αSMA, ED1, ED2 and MPO staining was visualized by the substrate 3,3′-Diaminobenzidine (DAB) (Immunologic) and were counterstained with hematoxylin. The αSMA, ED1, ED2, and MPO expression was analyzed by selection of the DAB signal in the tissue using the threshold function in NIS Elements (version 4.2; Nikon, Badhoevedorp, The Netherlands). The MPO, ED1 and ED2 staining was analyzed by setting a threshold for the positive DAB signal and expressed as an area fraction. The αSMA expression by myofibroblasts was analyzed by two thresholds: 1. total DAB signal and 2. DAB signal of blood vessels. The threshold for the blood vessels was based on high intensity and area size of the DAB signal, as blood vessels that express αSMA have a dense and intense DAB signal and have a larger area size than myofibroblasts. Expression of αSMA by myofibroblasts was calculated by extraction of the measured αSMA positive blood vessel area (threshold 2) from the total DAB positive area (threshold 1) and expressed as an area fraction.

A toluidine blue staining was performed to determine the presence of mast cells in the tissues at PBD 42. Tissue sections were stained with a 0.1% toluidine blue/1% sodium chloride solution pH 2.3 for 2–3 min at room temperature. Mast cells are indicated by a purple color while the surrounding tissue is stained light-blue. The number of mast cells was determined using NIS elements and expressed as number of cells per μm2.

Statistics

The software program SPSS (version 21.0 MS Windows, SPSS Inc., Chicago, IL, USA) was used to perform all statistical analyses. Statistically significant differences between the three treatment groups were determined by using the nonparametric Kruskal Wallis test. A two-tailed Mann–Whitney U test was performed as post-hoc analysis and statistical significance was considered at a p-value of <0.05. The results are presented in dot plots. Each dot represents one animal and the horizontal line represents the median.

Results

Partial thickness contact burns were inflicted on the dorsum of male Wistar rats in order to study possible effects of Captopril on wound healing and scar formation. Burn wounds that were treated with Captopril systemically or topically for 6 days did not show differences in wound healing at a macroscopic level compared to the control group at PBD 7 (Fig. 1A).

Fig. 1

Partial thickness burn wounds at PBD 7 and 42.

Macroscopic overview of partial thickness contact burn wounds on the dorsum of male Wistar rats. The animals were divided into 3 groups: control (n = 13), systemic Captopril (10 mg/kg/day, n = 13) and topical 5% Captopril (n = 13), Randomly chosen pictures of the burn wounds from 3 animals at PBD 7. All treatment groups showed similar healing of the burn wounds. Within the different treatment groups there is a variety in appearance of the burn wounds, from brown to white-yellow. B) Randomly chosen pictures of the scars from 3 animals at PBD 42. No differences were observed between the scars of the control group and group systemically treated with Captopril for 14 days. However the topical administration seemed to have negative effects on wound healing.

At PBD 42, macroscopic evaluation of scars between control and the systemic group (14 days treatment) revealed no differences (Fig. 1B). However, more animals receiving the topical Captopril treatment still had large scabs, indicating open wounds (6/13 animals, 46%) compared to the systemic group (2/12, 17%) and control (0/13, 0%). HE-stained sections showed a lack of epidermis under the scabs in all animals with open wounds.

We determined whether the inflammatory response was affected by the Captopril treatment by evaluating the infiltrate of neutrophils and macrophages at PBD7. No differences were detected in the amount of neutrophils and in macrophage subtypes (Fig. 2).

Fig. 2

Neutrophils and macrophages at PBD 7. The acute inflammatory response was assessed by staining for MPO (neutrophils), ED1 (M1 macrophages) and ED2 (M2 macrophages). No differences between the different groups were detected.

To investigate whether the reduced healing capacity of the topical treatment group could be related to an (allergic) inflammatory reaction evoked by Captopril we assessed the presence of mast cells at PBD 42 (Fig. 3). Scar tissue of partial thickness burn wounds of the systemic group contained similar numbers of mast cells compared to that of the control group. The group of animals receiving the topical Captopril administration was split in a group with open wounds at PBD 42 and a group of closed wounds. The open wounds showed a statistically significant higher number of mast cells in their scar tissue compared to closed wounds in this group at PBD 42 (p 0.045). However, the mean number of mast cells in the entire group was not statistically significant higher in comparison to that of the control and systemic treatment groups.

Fig. 3

Mast cells in scar tissue of partial thickness burn wounds at PBD 42.

A toluidine blue staining was performed to indicate the presence of mast cells in scar tissue of partial thickness burn wounds in rats at PBD 42. A) Images of mast cells in scar tissue of the different treatment groups: control, systemic and topical. Tissue sections of the group animals receiving the topical Captopril treatment were divided into two groups: open wounds (down left) and closed wounds (down right). Mast cells are indicated by the purple color and the background is stained light blue. B) Statistical significant higher numbers of mast cells were observed in scar tissue of open wounds which received the topical Captopril treatment compared to closed wounds (p = 0.045; Mann–Whitney U). No statistical significant differences were found between all the different treatments.

Beside the effect on the inflammatory reaction, RAS is thought to play a role in fibrotic processes such as scar formation. Key players in scar formation are myofibroblasts; these cells are characterized by αSMA containing stress fibers. The presence of myofibroblasts in the scar was assessed by αSMA staining in scar tissue sections of PBD 42 (Fig. 4A). Systemic and topical treatment of the partial thickness burn wounds with Captopril did not affect the bloodvessel or myofibroblast associated αSMA expression in the burn scar tissue compared to that of the control treatment (Fig. 4B). Also no differences between the open and closed wounds of the topical treatment group at PBD 42 were detected.

Fig. 4

αSMA expression in scars of partial thickness burn wounds at PBD 42.

Presence of myofibroblasts in the scars of the burn wounds was assessed by staining αSMA in tissue sections of PBD 42. A) Images of αSMA expression (brown) in scars of the control, systemic and topical Captopril-treated animals at PBD 42. B) The αSMA-positive signal was analyzed as percentage of the area fraction in the scars of the different treatment groups. Similar αSMA-positive area fractions were measured for all treatments at PBD 42.

Discussion

Manipulation of the Renin Angiotensin System with registered agents such as the ACE-inhibitor Captopril has previously been shown to reduce inflammation and attenuate fibrosis in various organs, e.g. heart, lung, and kidney. In an effort to elucidate the effects of Captopril on wound healing of burn wounds, we performed a study with partial thickness burn wounds in a rat model. Rats were treated either systemically or topically with Captopril. The original human application for the ACE-inhibitor Captopril is to lower the blood pressure. Patients with a large total burned body surface area often suffer from severe body fluid loss by leakage, which directly affects their blood pressure [10], [11], [12]. Systemic administration of Captopril might further lower the blood pressure and could endanger the lives of these patients. Therefore, we also assessed topical treatment of this drug because this might be a safer administration route for patients with severe burn wounds.

We first assessed the effect of Captopril on the acute inflammatory response by assessing the presence of activated neutrophils and macrophage subtypes M1 and M2. It has been shown that Captopril reduced the inflammatory response in other pathologies by reducing neutrophil and macrophage infiltration [13], [14], [15], [16]. Burn wounds are characterized by an excessive inflammatory response and a reduction of the influx of neutrophils and macrophages could improve the outcome of wound healing. However we could not find a reduction of these inflammatory cells in our model by Captopril administration. Moreover this study indicates that early topical administration of Captopril onto partial thickness burn wounds possibly impairs the wound healing process: more animals of the topical treatment group still had open wounds at PBD 42 compared to those of control and systemic treatment. Open wounds could be caused by scratching by the animals, e.g. as a result of itching from irritation of Captopril or by the delayed reepithelialization. Irritation of the skin is often associated with massive infiltration of mast cells and their release of histamine [17]. Mast cell numbers were slightly increased in scars of the topical treatment at PBD 42, but no signs of massive infiltration were observed in the total topical group. However, the number of mast cells in open wounds was significantly higher compared to healed wounds in the topical application group. Trautmann et al. [18] has shown that mast cell numbers significantly increased in human incisional wounds from day 2 to day 10 after wounding, returning to basal levels at day 21, suggesting that during wound closure mast cell numbers are normalized. Thus whether the increased mast cell numbers in the open wounds of the topical treatment at PBD 42 were caused by the delayed wound closure or by irritation and itch cannot be deduced from these experiments.

Furthermore no differences were detected in the number of α-SMA positive myofibroblasts, which are the most important cells in fibrotic processes, between the different groups. Stawski et al. showed that subcutaneous administration of AngII can induce skin fibrosis in mice [7] and suggests a role of RAS in the healing process. The lack of effect of Captopril observed in our study could be caused by the lack of specificity of RAS inhibition by Captopril. Captopril prevents the formation of AngII, which is the biologically active component of the RAS. AngII exerts its effects by binding to one of the AngII receptors. Currently two main AngII receptors have been recognized: AT1 and AT2. Most known functions of AngII, such as regulation of the blood pressure and its role in fibrosis, have been attributed to the activation of AT1. AT2 is thought to counteract the effects of AT1 activation and reduce fibrosis [19]. Inhibition of the formation of AngII will also affect the downstream signaling by the AT2 receptor. Specific inhibition of the fibrosis-associated AT1 receptor might have given better results. Another explanation of the lack of results might be the timing of the treatment. In this study Captopril treatment was administered only during the first two weeks after the infliction of the burn wound; this potentially will mainly affect the acute inflammatory reaction. However we did not find indications for that. To influence the fibrotic process, Captopril administration can start at later time point, e.g. during the remodeling phase. Therefor further research is needed to unravel the role of RAS on scar formation.

In summary: we could not detect beneficial effects of early Captopril treatment on the inflammatory reaction nor on the healing of partial thickness burn wounds.

Conflicts of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

We further confirm that any aspect of the work covered in this manuscript that has involved experimental animals has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.