E‐cadherin and aquaporin‐3 are downregulated in wound edges of human chronic wounds

Chronic wounds are defined as wounds that fail to proceed through the normal phases of wound healing; a complex process involving different dynamic events including migration of keratinocytes in the epidermis. Chronic wounds are estimated to affect 1–2% of the human population worldwide and are a major socioeconomic burden. The prevalence of chronic wounds is expected to increase with the rising number of elderly and patients with diabetes and obesity, who are at high risk of developing chronic wounds. Since E‐cadherin and the water channel aquaporin‐3 are important for both skin function and cell migration, and aquaporin‐3 is furthermore involved in wound healing of the skin demonstrated by impaired wound healing in aquaporin‐3‐null mice, we hypothesized that E‐cadherin and aquaporin‐3 expression may be dysregulated in chronic wounds. Therefore, we investigated the expression of E‐cadherin and aquaporin‐3 in biopsies from the edges of chronic wounds from human patients. This was accomplished by immunohistochemical stainings of E‐cadherin and aquaporin‐3 on serial sections followed by qualitative evaluation of staining patterns, which revealed low expression of both E‐cadherin and aquaporin‐3 at the wound edge. Future studies are needed to reveal if this downregulation is associated with the pathophysiology of chronic wounds.

The skin forms a protective barrier against the external environment and thus, upon injury, the skin must rapidly undergo repair to restore barrier function (1). In the vast majority of cases, wound healing is uncomplicated with efficient healing within a few weeks. However, some wounds fail to proceed through the normal wound healing phases leading to chronic wounds, which is estimated to affect 1-2% of the human population worldwide (2,3). This is especially prevalent in diabetic patients who are at increased risk of developing foot ulcers, which ultimately can lead to amputation. Chronic wounds are a major socioeconomic burden expected to increase with the rising number of diabetic and older patients, increase in obesity and the development of antibiotic resistance (3,4).
Skin wound healing is a complex process that can be divided into three continuous and overlapping phases: inflammation, proliferation and tissue remodeling (5). An important step during the proliferation phase is re-epithelialization, which is responsible for restoring an intact epidermis during wound healing. Re-epithelialization requires coordinated migration and proliferation of keratinocytes located at the wound edge or dermal appendages, including hair follicles and sweat glands. To successfully cover the wound, keratinocytes at the wound edge migrate collectively and coordinately into the wound followed by proliferation of keratinocytes behind the actively migrating cells (6)(7)(8).
The epidermis is a stratified squamous epithelium with epidermal cells tightly connected by different cellular junctions: tight junctions, gap junctions, desmosomes and adherens junctions (9). These junctions are essential for skin barrier formation. E-cadherin is the main transcellular adhesion protein in adherens junctions, and is expressed from stratum basale to stratum granulosum in human epidermal cells, where it is localized to the plasma membrane (10).
Another important protein in the skin is aquaporin-3 (AQP3), which is a channel protein facilitating transport of both water and glycerol across plasma membranes. AQP3 is expressed in human epidermal cells from stratum basale to stratum granulosum, where it is similarly localized to the plasma membrane (11)(12)(13)(14)(15). AQP3-null mice suffered from dry skin; hence, AQP3 is thought to be important for glycerol uptake into keratinocytes and thus, skin hydration (16).
Besides their canonical functions, both E-cadherin and AQP3 are involved in multiple cellular processes including cell migration and proliferation and for AQP3, also cell-cell adhesion (17). In vitro studies of migrating epithelial cell sheets have shown that adherens junctions are essential for cells to migrate collectively (18), and that E-cadherin is tightly regulated in collective cell migration during embryonal development, tumor metastasis, and wound healing (19,20). In an acute wound model using human xenografts, a downregulation of E-cadherin is observed in the wound front (21). AQPs are pivotal players in collective cell migration and it is thought that the facilitated water permeability aids in cell shape changes required for cell migration in restricted 3D environments (22). Moreover, AQPs at the leading edge of migrating cell sheets facilitate local cell swelling thought to provide space for actin polymerization and thereby increasing cell migration (22). AQP3-deficient human keratinocytes display decreased collective cell migration and cell proliferation in vitro and delayed wound healing is observed in AQP3-null mice (23). Also, in wounds on diabetic rats, both delayed wound healing and impaired AQP3 expression are observed (24).
To our knowledge, no previous studies have investigated the expression of E-cadherin and AQP3 in human chronic wounds. Since both Ecadherin and AQP3 are important for skin function and collective cell migration, we hypothesized that the expression may be dysregulated. Thus, we examined the expression of E-cadherin and AQP3 in the epidermis in biopsies taken at the wound edge of human chronic wounds. The staining was performed on serial sections to qualitatively evaluate staining patterns of E-cadherin and AQP3 in the same regions. Immunohistochemical analysis revealed a decreased expression of both E-cadherin and AQP3 in the edges of human chronic wounds compared to the expression in the epidermis near the wound edge.

Patient tissue and ethics statement
Clinical samples were obtained through a prospective cohort study of patients suffering from chronic wounds planned for surgical debridement followed by splitthickness skin grafting (STSG). The collection of samples from patients with chronic wounds was approved by the Danish Board of National Health ethics, protocol no.: H-20032214.
The samples (n = 9) were obtained as punch biopsies (4-6 mm) positioned at the wound edge so that the biopsy includes wound tissue with a brim of surrounding intact skin. The biopsies were obtained before surgical debridement and STSG. Biopsies were fixed in formalin and embedded in paraffin following standard operating procedures at the Department of Biomedical Sciences, University of Copenhagen, Denmark. All biopsies were cut with the maintained organization from the surrounding brim of skin toward the wound center and used for investigation of E-cadherin and AQP3.
Formalin fixed, paraffin-embedded human control skin (n = 2) was obtained from the Department of Pathology, Aarhus University Hospital, Denmark. The Danish Act on Processing of Personal Data and Health was complied with, and since the material encompassed fully anonymized tissue, informed consent from patients was not required.

Immunohistochemistry
Paraffin-embedded skin sections were deparaffinized overnight in xylene, rehydrated and endogenous peroxidase activity was blocked in 0.3% hydrogen peroxide in methanol. This was followed by heat-mediated antigen retrieval in TEG buffer (10 mM Tris pH 9, 0.5 mM EGTA) and blocking of aldehyde groups in 50 mM NH 4 Cl in PBS for 30 min. The sections were washed in 1% bovine serum albumin (BSA), 0.2% gelatin and 0.05% saponin in PBS and labeled with mouse anti-E-cadherin (1:100, sc-8426, Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-AQP3 (1:400, AQP-003, Alomone Labs, Jerusalem, Israel) antibodies diluted in 0.1% BSA and 0.3% Triton X-100 in PBS in a humidity chamber at 4°C overnight. The following day, the sections were allowed to adjust to room temperature, washed in 0.1% BSA, 0.2% gelatin and 0.05% saponin in PBS and incubated for 60 min in secondary horseradish peroxidase-conjugated goat antimouse (1:500, P0447, Agilent Technologies, Santa Clara, CA, USA) or goat anti-rabbit antibodies (1:500, P0448, Agilent Technologies) and developed with 3,3 0diaminobenzidine (DAB; K3468, Agilent Technologies). Finally, the sections were counterstained in Mayer's hematoxylin before dehydration and mounting. Staining of serial sections was performed to enable qualitative evaluation of staining patterns. No DAB reactivity was observed on negative control sections without incubation with primary antibodies. For histological evaluation, skin sections were stained with Mayer's hematoxylin and eosin.

Microscopy
Control skin sections were imaged on a Leica DMLB bright-field microscope equipped with a 209/0.40 air objective and an Invenio 6EIII camera, which was controlled by Deltapix software. Wound edge skin sections were imaged on a Zeiss LSM 710 microscope equipped with a 109/0.30 air objective and an Axiocam MRc camera, which was controlled by Zen Blue software. Image preparation was performed in ImageJ (25).

Patient data
All samples from chronic wounds were obtained from lower extremity ulcers all located below knee level. Of the six patients included four were male (66.7%) and two were female (33.3%). The mean (SD) age was 75.7 (6.7) years with a range of 68-89 years. Diabetes was present in two (33.3%) of six patients. No patients were active smokers, four were former smokers and two had never smoked. All patients reported alcohol consumption within limits of the national recommendations by the Danish health government (<7 units/week). The mean (SD) wound age was 24.8 (SD 13.8) months. Four wounds were primarily attributed to chronic venous insufficiency (66.7%), one to arterial disease (16.7%), and one wound had a venous and an arterial component (16.7%).

Histological evaluation of human chronic wounds
Sections of skin biopsies from the edge of human chronic wounds (n = 9) were stained with hematoxylin and eosin and histologically evaluated. A clear transition from intact epidermis to the wound edge, defined in this study by loss of epidermal cells, was observed in six out of nine chronic wound samples (Fig. 1A). The remaining three samples showed no loss of epidermis, indicating that the wound edge was not present in these samples, and therefore, they were excluded from the study (data not shown). Furthest from the wound edge, in the surrounding skin, the epidermis was intact and well organized with clear basal cell organization, which was in contrast to the decreased organization and thinner epidermis closer toward the wound edge. At the wound edge, the epidermis was disorganized prior to complete loss in the actual wound (Fig. 1A).
In the intact epidermis near the wound edge, different histopathological characteristics were observed (Fig. 1B). The epidermis near the wound edge was intact and well organized, but showed epidermal hyperplasia (5 of 6) characterized by increased thickness of the epidermis due to an increased number of epidermal cells. In most of the samples, elongated, thickened, and/or irregular rete ridges were present (5 of 6). Hyperkeratosis, characterized by increased thickness of the stratum corneum, was observed in all samples with the presence of both non-nucleated (orthokeratosis, 2 of 6) and nucleated (parakeratosis, 5 of 6) keratinocytes, indicating incomplete keratinocyte differentiation. Lastly, we observed different levels of epidermal intercellular edema (spongiosis, 5 of 6), characterized by widened intercellular spaces between epidermal cells with elongation of the intercellular junctions, and dermal inflammation (6 of 6; Fig. 1B).
Our histopathological observations are consistent with previous studies of chronic wounds, reporting loss of the intact epidermis at the wound edge (26) and hyperplasia, hyperkeratosis and spongiosis in the intact epidermis near the wound edge (26)(27)(28).

The expression of E-cadherin and AQP3 is decreased in the epidermis of human chronic wound edges
To investigate the expression of E-cadherin and AQP3 in the wound edge of human chronic wounds, serial sections of control skin biopsies and chronic wound edge biopsies were immunohistochemically labeled with antibodies directed against E-cadherin and AQP3. In control skin, E-cadherin and AQP3 were localized to the plasma membrane of epidermal cells from stratum basale to stratum granulosum, as previously reported (10-12) ( Fig. 2A). Lower expression of E-cadherin and AQP3 was observed in stratum granulosum, whereas no immunolabeling was detected in stratum corneum and in the dermis ( Fig. 2A), similarly to previous reports (10)(11)(12).
In the intact epidermis near the wound edge of all the human chronic wounds, E-cadherin and AQP3 were observed in the plasma membrane of epidermal cells from stratum basale to stratum granulosum (6 of 6), but in some samples, low expression was observed in stratum basale (3 of 6). Closer to the wound edge, the epidermis was thinner and less organized, but plasma membrane localization of E-cadherin and AQP3 could still be observed. At the wound edge, the expression of both E-cadherin and AQP3 was markedly decreased in all samples (6 of 6; Fig. 2B). The wound edges were histologically very different, but common to all of the wounds was the decrease of E-cadherin and AQP3 expression at the wound edge (Fig. 2C). Interestingly, regulation of E-cadherin and AQP3 expression may be interdependent in skin since E-cadherin is decreased in human keratinocytes after siRNAmediated knockdown of AQP3 (29).
Like adherens junctions, tight junctions are also essential in collective cell migration in skin wound healing (30). Loss of tight junction proteins claudin-1 and occludin has been observed in wound edges of human chronic wounds, whereas they were present in human acute wounds (31). We speculate if downregulation of tight junction proteins combined with adherens junctions at the wound edge of human chronic wounds might contribute to the pathophysiology of the chronic wounds by impairing skin repair due to decreased migratory capacity of the epidermal cells as well as reduced barrier function. However, E-cadherin expression has been   found to be almost absent in the wound front in an acute wound model using human skin xenograft (21). These observations are made in the wound front with intact epidermis, without any observations from the specific wound edge (transition from intact epidermis to loss of epidermal cells). In this study, we observe a downregulation of E-cadherin in the wound edge of human chronic wounds. Further studies are needed to confirm if E-cadherin is also downregulated at the wound edge of acute wounds. Since AQP3-deficient human keratinocytes show decreased collective cell migration and cell proliferation in vitro and delayed wound healing is observed in AQP3-null mice, reduced AQP3 expression may decrease migration of cells into the wound thereby contributing to the impaired wound healing in the chronic wounds. Besides defective cell migration, dysregulation of AQP3 expression has been shown to be involved in many different skin conditions. AQP3 expression is increased in the human epidermis of burn wounds and atopic eczema (32,33) and mislocalized in human epidermis of psoriatic lesions (11).

CONCLUSION
In conclusion, we show a downregulation of Ecadherin and AQP3 in the wound edge of human chronic wounds. Since there are no reports about AQP3 in the wound edge of human acute wounds, and E-cadherin is downregulated in the wound front, not wound edge, of human acute wounds, further studies are needed to determine if the downregulation of E-cadherin and AQP3 in chronic wounds is involved in the wounds failure to heal. If so, it should be determined if E-cadherin and AQP3 are potential intervention targets for wound healing in chronic wounds and other skin diseases associated with dysregulation of adherens junctions and AQP3.
The authors would like to express their gratitude to the nursing and medical staff at the Copenhagen Wound Healing Center, Bispebjerg Hospital and the patients who offered their participation and made it possible to collect samples from chronic wounds. We are also grateful to consultant pathologist, associate clinical professor, Henrik Hager, Department of Pathology, Aarhus University Hospital, for patient skin samples, and to Vibeke Secher Dam for technical assistance.

FUNDING
Collection of samples from patients with chronic wounds was performed with financial support from Novo Nordisk Challenge Program (NNF19O C0056411) that also supported MD. A.K.S. Iversen. Novo Nordisk foundation (Tandem Program 2019 #0054390) generously provided financial support for Asst. Prof. L. Bay and Professor T. Bjarnsholt. This project was supported by a grant from the Graduate School of Health (ClinFO), Aarhus University, Denmark to C. Ernstsen.