Influence of Birch Bark Triterpenes on Keratinocytes and Fibroblasts

Article pubs.acs.org/jnp

Influence of Birch Bark Triterpenes on Keratinocytes and Fibroblasts from Diabetic and Nondiabetic Donors Tina Wardecki,†,# Philipp Werner,†,# Maria Thomas,‡ Markus F. Templin,§ Gudula Schmidt,⊥ Johanna M. Brandner,∥ and Irmgard Merfort*,† †

Pharmaceutical Biology and Biotechnology and ⊥Institute for Experimental and Clinical Pharmacology and Toxicology, Albert-Ludwigs-University Freiburg, Freiburg, Germany ‡ Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Tübingen, Germany § Institute of Natural and Medical Sciences at the University of Tübingen, Reutlingen, Germany ∥ Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Hamburg, Germany S Supporting Information *

ABSTRACT: Impaired wound healing is one of the main risk factors associated with diabetes mellitus. Few options are available to treat diabetic wounds, and therefore efficient remedies are urgently needed. An interesting option might be an extract of birch bark (TE) that has been clinically proven to accelerate acute wound healing. We investigated the effects of TE and its main components betulin and lupeol in cultured normal keratinocytes and dermal fibroblasts from diabetic and nondiabetic donors. These in vitro models can provide insights into possible beneficial effects in wound healing. TE and betulin treatment led to increased mRNA levels of chemokines, proinflammatory cytokines, and mediators important in wound healing, e.g., IL-6, TNFα, IL-8, and RANTES. We observed a pronounced upregulation of MIF, IL-8, and RANTES on the protein level. Furthermore, a shape change of the actin cytoskeleton was seen in keratinocytes and fibroblasts, and the Rho-GTPases and p38-MAPK were found to be activated in keratinocytes. On the basis of our results, TE is worthy of further study as a potential option to influence wound-healing processes under diabetic conditions. These first insights need to be confirmed by clinical studies with diabetic patients.

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collagen is actively remodeled to type I collagen, which strengthens the repaired tissue.4,5 In diabetic patients, the complex wound-healing process is often disturbed due to hyperglycemia, chronic inflammation, micro- and macrocirculatory dysfunction, hypoxia, autonomic and sensory neuropathy, and impaired neuropeptide signaling.3 Narrowing or occlusion of blood vessels in the wound area can result in poor blood, nutrient, and oxygen supply and is worsened by the aberrant formation of new blood vessels after injury. Moreover, inflammation and new tissue formation phases are affected. A low-grade inflammatory state and a prolonged inflammatory response are caused by various changes at the cellular and molecular level. Thus, it has been reported that patients with diabetes have an increased number of inflammatory cells in the dermis and around vessels as well as in poorly healing wounds.3 Furthermore, neutrophils and macrophages have defects in chemotactic, phagocytotic, and microbicidal activities, resulting in an elevated risk for wound infection.2,6 Neutrophils and macrophages can continuously release pro-inflammatory cytokines, chemokines, and degrading enzymes such as MMP-9 (matrix metalloproteinase 9),

iabetes mellitus is one of the major health problems worldwide. In 2013, 382 million people suffered from diabetes, and this number is estimated to increase to 592 million by 2035.1 This disease and the associated complications cause a tremendous financial burden to the public health system. About 25% of patients with diabetes suffer from a foot ulcer also called diabetic foot syndrome. Peripheral neuropathy, peripheral arterial disease, and trauma are the main factors responsible for its development. Impaired wound healing is its main complication, which is deemed chronic once delayed beyond 8−12 weeks.2,3 Cutaneous wound healing is a highly complex process that involves a large array of molecular factors and that can be divided into three overlapping phases: inflammation, new tissue formation, and remodeling. The inflammatory phase starts immediately after wounding with hemostasis and initiation of a controlled inflammatory response. Inflammatory cells such as neutrophils and monocytes/macrophages are recruited. They remove dead tissue and bacteria and release growth factors. Approximately 2 days after injury new tissue formation begins with the proliferation and migration of keratinocytes and fibroblasts. New blood vessels are formed, and extracellular matrix is produced. In the final stage of wound healing type III © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 11, 2016

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DOI: 10.1021/acs.jnatprod.6b00027 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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cultivated under hyperglycemic conditions.17,18 Alternatively, fibroblasts were cultivated on 3-deoxyglucosone-collagen, imitating an extracellular matrix altered by advanced glycation end products.19,20 Another approach is the use of keratinocytes and fibroblasts derived from diabetic donors.12,21 Although all conditions mentioned above can only partly reflect the diabetic status in patients, we here used primary human keratinocytes (NHK = normal human keratinocytes) and fibroblasts (HDF = human dermal fibroblasts) from nondiabetic and diabetic donors (NHK-nd, HDF-nd and NHKd, HDF-d, respectively) of different ages (young and adult) cultured under the same conditions. By doing so, a condition was simulated where diabetic patients have normal glucose levels. The aim of this study was to evaluate whether keratinocytes from diabetic donors show the same behavior toward birch bark extract (TE) and its triterpenes as previously reported for nondiabetic donors. Moreover, we extended our studies to fibroblasts. Using all these cells, gene and protein arrays were performed to study the effect of TE and its main constituent, betulin, on the expression of a variety of genes and proteins important in wound healing. Additionally, the effect of TE and its triterpenes on the actin cytoskeleton in these cells was compared, as changes in the shape are a prerequisite for migration. In general, human primary keratinocytes and fibroblasts from nondiabetic and diabetic donors were similarly influenced by TE and its triterpenes, indicating that TE may also have beneficial effects in diabetic wounds. This has to be proven in vivo.

establishing an exacerbated inflammatory and proteolytic environment, preventing the healing process.2,7 Interestingly, macrophages in diabetes show a decrease in the release of cytokines.8 Additionally, keratinocytes failed to upregulate the expression of growth factors, including those that promote angiogenesis.6 New tissue formation, such as reepithelialization and the formation of granulation tissue, is perturbed.6,9 Fibroblasts from diabetic wounds exhibited a phenotypic change and decreased migration and proliferation.10 Keratinocytes showed an absence of migration, hyperproliferation, and incomplete differentiation at the edge of chronic wounds from diabetic patients,10,11 whereas keratinocytes derived from diabetic donors demonstrated a reduced proliferation and migration in vitro.12 The guideline-compliant treatment for DFU (diabetic foot ulcers) includes off-loading of pressure, debridement, administration of moist wound dressings, and, if needed, infection control. Additional treatment options such as hyaluronic acid or protease-modulating products are mentioned, but not further evaluated.13 Hence, up to now, only a few treatment possibilities are available, and new, easily accessible therapeutic options are urgently needed. Besides the conventional remedies, phytomedicines may be an effective alternative or addendum to beneficially influence wound healing including diabetic wounds. Interestingly, an open, blind evaluated, controlled, prospective, randomized phase II clinical trial including 24 healthy patients demonstrated that a birch bark (Betula pendula, B. pubescens, and hybrids of both species, Betulaceae family) preparation significantly accelerated reepithelialization of surgical skin lesions.14 Moreover, birch extract also showed beneficial effects in the treatment of two healthy patients suffering from second degree burns.15 In 2015, an oleogel-containing birch bark extract received approval for the treatment of mid-dermal wounds by the European Medicines Agency (EMA). However, studies with wounds from diabetic patients have not yet been performed. The reported studies on nondiabetic patients were conducted with a preparation that contained a lipophilic extract of the outer bark of birch (TE: triterpene extract).16 This extract consists of 97% pentacyclic triterpenes with betulin as the main compound (87%) and minor concentrations of the triterpenes lupeol, betulinic acid, oleanolic acid, and erythrodiol. Previously, we have published work designed to increase our understanding of the molecular mechanism of the clinically proven wound-healing effect of birch bark in euglycemic conditions.16 We demonstrated with primary human keratinocytes that the extract (TE) influences the inflammatory and the new tissue formation phase. Concerning the pro-inflammatory effects, only betulin was responsible for the transient upregulation of some pro-inflammatory cytokines, chemokines, cyclooxygenase-2 (Cox-2), and growth factors. Notably, the most pronounced effects were observed with the chemokines. Furthermore, a migration-promoting effect was detected for TE and its triterpenes. Part of the underlying process is the activation of Rho-GTPases and an alteration of the shape of the actin cytoskeleton, resulting in the formation of filopodia, lamellipodia, and stress fibers.16 The question arises whether TE and its triterpenes could also be advantageous in a diabetic context. Here, the use of in vitro models is an interesting option and can provide novel insight in advance of clinical studies with diabetic patients. Different in vitro models are used to simulate diabetic conditions in the skin. Keratinocytes or fibroblasts have been

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RESULTS In Cells Derived from Nondiabetic and Diabetic Patients, TE and Betulin Influence the Expression of Genes Involved in the Inflammatory Phase. Recently, we demonstrated that the expression of a variety of genes is markedly enhanced in primary human keratinocytes treated with TE and betulin.16 We now extended our gene panel to 48 genes encoding for proteins that cover different aspects in the complex wound-healing process: genes for pro-inflammatory proteins, chemokines, and growth factors regulating migration, infiltration, proliferation, and differentiation of primary human keratinocytes and primary human dermal fibroblasts;22 antiinflammatory factors essential for the shift from the inflammatory phase to the new tissue formation phase and preventing chronic inflammation;23,24 MMPs that degrade the matrix and are known to be increased in DFU, whereas TIMPs (tissue inhibitor of metalloproteinases) are known to be downregulated;9,25 antioxidants regulating the redox environment in healing skin and associated with impaired healing when their levels are reduced;26 CAMs (cell adhesion molecules) such as VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) involved in the infiltration of leukocytes in inflammatory tissues27,28 and important in wound healing, as ICAM-1-deficient mice showed delayed wound healing;29 cellular connections, such as gap junctions crucial for tightening the epithelial barrier and for communication via intercellular channels.30,31 Distribution patterns of gap junction proteins Cx43 and Cx26 (connexins 43 and 26) are altered during wound healing and are upregulated in chronic wounds.31,32 Treatment of NHK cells with two different concentrations of either TE or betulin for 6 h either up- or downregulated the expression of several genes. Betulin was studied in the concentration in which it occurs in 1 and 5 μg/mL TE. B



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Figure 1. Birch bark extract (TE) and betulin influence mRNA expression of various genes in primary human keratinocytes (NHK-nd and NHK-d). The mRNA expression in primary human keratinocytes derived from nondiabetic young and adult (NHK-nd) and diabetic adult (NHK-d) donors was measured after treatment with TE (1 and 5 μg/mL) or betulin (0.87 μg/mL = 1.96 μM and 4.35 μg/mL = 9.81 μM) for 6 h with the Fluidigm’s Biomark high-throughput qPCR chip platform. The results are expressed as fold change (= fold) in relation to the solvent control (DMSO 0.1%). Results from independent experiments (2−4) of two different donors per group are given, and altogether 5−7 independent experiments were performed. SD was calculated from the average. The figure lists the genes where at least one value was influenced significantly. a: p < 0.05; b: p < 0.01; c: p < 0.001 compared to control group by one-way ANOVA followed by Bonferroni’s post-test. The colors indicate the extent of change in mRNA expression in relation to the control. Blue: fold change 4.

Figure 2. Birch bark extract (TE) and betulin influence mRNA expression of different genes in fibroblasts (HDF-nd and HDF-d). mRNA expression in primary human fibroblasts derived from a nondiabetic young and an adult (HDF-nd) and a diabetic adult (HDF-d) donor was measured after treatment with TE (1 and 5 μg/mL) or betulin (0.87 μg/mL = 1.96 μM and 4.35 μg/mL = 9.81 μM) for 6 h with the Fluidigm’s Biomark highthroughput qPCR chip platform. The results are expressed as fold change (= fold) in relation to the solvent control (DMSO 0.1%). Independent experiments were performed four times. mRNA of the adhesion molecules ICAM-1 and VCAM-1 was measured by standard qRT-PCR (n = 3). SD was calculated from the average, respectively. The figure lists the genes where at least one value was statistically significantly influenced. a: p < 0.05; b: p < 0.01; c: p < 0.001 compared to control group by one-way ANOVA followed by Bonferroni’s post-test. The colors indicate the extent of change in mRNA expression in relation to the control. Blue: fold change 4. NT: not tested.

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Figure 3. Betulin affects the production of different mediators important in wound healing in human keratinocytes. Keratinocytes (NHK-nd and NHK-d) were treated with betulin (0.87 μg/mL = 1.96 μM) or DMSO (0.1%) for 24 h. Protein levels of the mediators were determined in the supernatant using a bead-based suspension array technology (xMAP, Luminex Corp., Austin, TX, USA). Values represent means of three independent experiments from one diabetic and one nondiabetic donor + SD. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control group by two-way ANOVA followed by Bonferroni’s post-test. #One of the three values was below the detection limit and was substituted by the value for the detection limit. ##Two of the three values were below the detection limit and were substituted by the value for the detection limit. ###All values were below the detection limit and were substituted by the value for the detection limit.

expression levels of IL-1β and its receptor IL1R1 were slightly decreased. Expression of the chemokines IP-10 (interferon γ induced protein 10), IL-8, and RANTES (regulated upon activation, normal T-cell expressed and secreted) was strongly upregulated, but MIP-2 (macrophage inflammatory protein 2) mRNA expression level was not affected. mRNA expression of the gap junction protein Cx26 and aquaporin 3 was slightly

NHK-nd from young and adult donors and NHK-d from adult donors exhibited similar responses to the treatment. Figure 1 lists the genes where at least one value was significantly influenced. Figure S1 (Supporting Information) lists genes where the change was not statistically significant, but often showed a trend. mRNA expression of the pro-inflammatory factors IL-6 and TNFα were upregulated, whereas the D



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Figure 4. Betulin affects the production of different mediators important in wound healing in human fibroblasts. Fibroblasts (HDF-nd and HDF-d) were treated with betulin (0.87 μg/mL = 1.96 μM) or DMSO (0.1%) for 24 h. Protein levels of the mediators were determined in the supernatant using the Luminex assay. Values represent means of three independent experiments from one diabetic and one nondiabetic donor + SD. *p < 0.05; **p < 0.01; ***p < 0.001 compared to control group by two-way ANOVA followed by Bonferroni’s post-test. ###All values were below the detection limit and were substituted by the value for the detection limit.

whereas downregulation occurred rarely. By comparing the mRNA of HDF cells from different donors the most pronounced effects were visible in HDF-nd from the adult donor. Notably, HDF cells were more susceptible to TE or betulin treatment than NHK cells. Specifically, mRNA of the pro-inflammatory mediators Cox-2, IL-1β, IL1R1, and IL-6 were strongly upregulated in HDF cells, whereas in NHK cells, mRNA levels of IL-1β and its receptor IL1R1 were moderately downregulated by either TE or betulin. The expression of the mRNA of chemokines MCP-1 (synonymous with CCL2, chemokine (C-C motif) ligand 2) and IL-8 increased in HDF cells. mRNA of MIP-2 was only slightly influenced in HDF cells, whereas no effect could be observed in NHK cells. Additionally, an effect on the mRNA levels of the growth factors FGF2, GDF-15, and VEGF-A were most pronounced with the 5 μg/mL TE treatment. In accordance with the findings in NHK cells, TGF-β1 mRNA expression was not affected. Furthermore, mRNA of the anti-inflammatory factors IκBα and SOCS3 (suppressor of cytokine signaling 3) and antioxidants such as Nrf2 (nuclear factor-like 2) and SOD2 were upregulated. mRNA expression of MMPs, TIMPs, and proteins involved in the formation of ECM was not affected. The treatment of HDF-nd with betulin resulted in a strong elevation in VCAM-1 and ICAM-1 mRNA expression levels. However, only a tendency of increased mRNA expression could be observed in HDF-d cells. Some genes such as IL-1β and MCP-1 were upregulated only in HDF cells, but not in NHK cells.

downregulated by TE and betulin but not dose-dependently. Studies on growth factors revealed a slight increase in GDF-15 (growth differentiation factor 15) and VEGF-A (vascular endothelial growth factor A) mRNA expression but only at higher concentrations. mRNA of other growth factors such as FGF2 (basic fibroblast growth factor), TGF-β1 (transforming growth factor β1), and PDGF-2 (platelet derived growth factor 2) were not affected under the tested conditions. Only treatment with 5 μg/mL TE or 4.35 μg/mL (= 9.81 μM) betulin resulted in moderate elevation of mRNA levels of the anti-inflammatory factor IκBα (inhibitor of nuclear factor of kappa-light-chain-enhancer in B-cells, alpha), the inhibitory subunit of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), the antioxidative enzyme SOD2 (superoxide dismutase 2), and the transcription factor c-Jun. No effect was observed at the lower concentrations. Genes involved in the formation or restructuring of ECM (extracellular matrix) such as FN1 (fibronectin 1) or MMP-2 were not affected. mRNA expression of ICAM-1 and VCAM-1 was not significantly elevated in NHK-nd cells. Slightly enhanced mRNA levels of ICAM-1 could be detected only in NHK-d cells, but were not statistically significant. HDF-nd cells from a young and an adult donor and HDF-d cells from an adult donor were treated identically to the NHK cells with TE (1 and 5 μg/mL) or betulin (0.87 μg/mL = 1.96 μM and 4.35 μg/mL = 9.81 μM) for 6 h. The data presented in Figure 2 are partly statistically significant, and those presented in Figure S2 (Supporting Information) often show a tendency, but are not statistically significant. Gene expression trended up, E



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Figure 5. Influence of TE, betulin, and lupeol on the actin cytoskeleton and Rho-GTPases in keratinocytes. (A) Shape change of the actin cytoskeleton of keratinocytes from a diabetic donor (NHK-d) after treatment with the positive control CNF1 (1 nM), TE (5.1 ng/mL), or the triterpenes betulin (10 nM) and lupeol (10 nM) for 2 h. For inhibition of p38-MAPK, inhibitor LN 950 (100 nM) was used 30 min before adding the test compounds. Representative photomicrographs are shown. (B) Influence of TE and the triterpenes betulin and lupeol on activated GTPbound Rho-GTPases and the total amount of Rho-GTPases of keratinocytes from a diabetic donor (NHK-d) measured by pulldown experiments followed by immunoblotting. For RhoA, incubation time was 3 h; for Cdc42 and Rac1, incubation time was 20 min. As positive control the strong RhoA activator CNFY (3 nM) or the RhoA/Rac/Cdc42 activator CNF1 (2 nM), respectively, was used. Representative immunoblots are shown, n = 3.

Considering the effects of TE and betulin on NHK and HDF cells of nondiabetic and diabetic donors the question arises how these results are influenced by differences in baseline mRNA expression. Therefore, we studied the differences in baseline mRNA expression with the same panel of genes as we used for the cells treated with TE or betulin. The results indicated that only a few significant differences can be observed between

NHK cells from young and adult and nondiabetic and diabetic origin (Figure S3, Supporting Information). Interestingly, SOD2, responsible for the removal of ROS (reactive oxygen species), was slightly upregulated in NHK-nd cells from adult donors. The gene for the antibacterial active defensin HBD3 (human beta defensin 3) was upregulated in NHK-d cells, although a higher risk for bacterial infections is known for F



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Figure 6. Influence of TE (5.1 ng/mL), betulin (10 nM), or lupeol (10 nM) on the actin cytoskeleton in fibroblasts. (A) Fibroblasts from a nondiabetic donor (HDF-nd) and (B) a diabetic donor (HDF-d) after treatment with the positive control PDGF or the test compounds for 2 h. Representative photomicrographs are shown.

diabetes patients.7 In contrast, the comparative studies with HDF cells show that age and diabetes significantly influence gene expression of several different genes involved in the wound-healing process (Figure S3, Supporting Information). Thus, HDF-nd cells from the young donor showed a much higher baseline mRNA expression compared to HDF-nd cells from the adult donor. Here, the pro-inflammatory IL-1β and the chemokine IP-10 exhibited the most pronounced effects. Baseline mRNA expression was higher in HDF-d cells compared to HDF-nd cells. Again, the chemokine IP-10 showed the highest fold increase. Betulin Influences Protein Levels of Various Mediators Important in the Wound-Healing Processes. To validate altered mRNA levels in cells exposed to betulin or vehicle, protein levels were evaluated using multiplex Luminex assays (Table D in S1 Protocol, Supporting Information). Proteins whose mRNAs had not been evaluated above included the soluble forms of the adhesion molecule sE-selectin, receptors IL-6sR, sTNF-R1, sTNF-R2, sgp130 (soluable glycoprotein 130), and sFas. Treatment of NHK cells led to significantly altered protein expression levels of various mediators playing a role in the wound-healing process (Figure 3). The chemokines RANTES, IL-8, and MIP-1α, the proinflammatory cytokines TNFα as well as MIF, and the adhesion

molecule sE-selectin were found to be upregulated in NHK-nd and NHK-d cells. The anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) was also elevated in both NHK cell types. Levels of the chemokine MCP-1 were increased in NHK-nd, but decreased in NHK-d cells, whereas the growth factor GMCSF (granulocyte-macrophage colony-stimulating factor) and the soluble adhesion molecule sICAM-1 were increased in NHK-nd, but remained unaltered in NHK-d cells after betulin stimulation. The amount of soluble receptors IL-6 sR and sTNF-R1 decreased after treatment with betulin, as was also the case for sFas and the adhesion molecule sVCAM-1 in NHK-nd and NHK-d cells. The protein sgp130, part of the IL-6 receptor, was only slightly increased in NHK-nd cells. Expression of some proteins, such as IL-1β, IL-2, IL-4, IL-10, IL-12p40, IL17A, IFNγ (interferon γ), sTNF-R2, sRAGE (soluble receptor of advanced glycation end products), PLA2G7 (phospholipase A2G7), MIP-1β, sCD40L (soluble cluster of differentiation 40 ligand), FGF23, insulin, leptin, osteocalcin, and osteopontin was below the detection limit. Results for GROα were not significant (Figure S4A). In HDF cells, fewer proteins were significantly influenced by the treatment with betulin (Figure 4). Elevated protein levels were observed for the chemokine RANTES, the proinflammatory cytokine MIF, the soluble receptor sTNF-R1, G



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GTPase RhoA after treatment with TE, betulin, and lupeol, a slight activation of the Rho-GTPase Cdc42, but no activation of Rac1 after treatment with TE and betulin in young NHK-nd.16 In keratinocytes of diabetic donors, all three Rho-GTPases were activated after treatment with TE, betulin, or lupeol in nanomolar concentrations (Figure 5B). For the measurement of RhoA, the cells were incubated for 3 h with TE, betulin, or lupeol. For the measurement of Cdc42 and Rac1, a shorter time period of 20 min was chosen, as these Rho GTPases are known to be activated more rapidly. To further study whether p38-MAP-kinase is involved in the observed shape change necessary for migratory processes, the effect of TE on p38-MAPK in NHK-d cells was examined. TE treatment caused a slight activation of p38-MAPK (Figure 7), as also observed by Ebeling et al. in NHK-nd.16

and the adhesion molecules sICAM-1 and sVCAM-1 in HDFnd and HDF-d. This was the same for the chemokines MCP-1 and IL-8, but these results were statistically not significant (Figure S4B, Supporting Information). The secreted amount of the soluble receptor IL-6sR and sFas was decreased in both HDF cell types. The expression of the following proteins was below the detection limit: sE-selectin, PLA2G7, sRAGE, GMCSF, IFNγ, IL-1β, IL-2, IL-4, IL-10, IL-12p40, IL-17A, MIP-1α, MIP-1β, sCD40L, TNFα, FGF23, insulin, leptin, osteocalcin, and osteopontin. Altogether, we could confirm that proteins that showed an upregulation of mRNA expression associated with betulin treatment, such as RANTES, IL-8, MCP-1, and TNFα, also showed increased protein levels. Comparing the protein expression changes in NHK and HDF, it is obvious that mostly the same proteins are affected in a similar manner by betulin. Exceptions are the soluble forms of TNF-R1 and sVCAM-1, where the concentrations were upregulated by betulin in HDF but downregulated in NHK cells. TE and Its Triterpenes Induce Change of the Actin Cytoskeleton in Cells from Nondiabetic and Diabetic Donors. Important steps in the new tissue formation phase are migration and proliferation of keratinocytes and fibroblasts.4 A shape change of the actin cytoskeleton is a prerequisite for cell migration.33 The migratory phenotype in keratinocytes is characterized by a polarized cell shape with protrusions called lamellipodia and filopodia and with the formation of stress fibers.34 The moving fibroblast also shows stress fibers and lamellipodia and changes from an outspread cell body toward a fan-shaped or elongated one.35 Recently we could show a migration-promoting effect of TE on primary human keratinocytes in scratch assay experiments and an effect of TE and its triterpenes betulin and lupeol on the actin cytoskeleton.16 To study if NHK from diabetic donors behave similarly, subconfluent cells were cultivated on glass coverslips, treated with TE or the single triterpenes for 2 h, and finally stained with rhodamine-phalloidin. Cells treated with the solvent control DMSO exhibited the sessile cell shape. Keratinocytes of diabetic donors treated with CNF1 (cytotoxic necrotizing factor 1) as positive control, TE, betulin, and lupeol showed remarkable cellular protrusions (Figure 5A). Fibroblasts from diabetic and nondiabetic donors exhibited a change toward a thin elongated shape when treated with PDGF as positive control, TE, betulin, or lupeol (Figure 6). Furthermore, betulinic acid, but not oleanolic acid, induced a slight shape change of the actin cytoskeleton of HDF-nd and HDF-d cells (Figure S5, Supporting Information). In our previous paper, we discussed a possible role of p38 MAPK in the shape change of the actin cytoskeleton for nondiabetic keratinocytes.16 Here, we confirmed this assumption for NHK-d cells from adult (Figure 5A) and NHK-nd cells from young and adult donors (Figure S6, Supporting Information). In these cells, pretreatment with the p38MAPK inhibitor LN 950 led to the absence of marked protrusions comparable to cells treated with the solvent control. In fibroblasts treated with LN 950 the shape change was not reversed. TE, Betulin, and Lupeol Activate p38-MAPK and RhoGTPases in NHK-d. Rho-GTPases are involved in the reorganization of the actin cytoskeleton. More precisely, RhoA participates in the formation of stress fibers, Rac1 in the formation of lamellipodia and Cdc42 in the formation of filopodia.36 Ebeling et al. showed an activation of the Rho-

Figure 7. Influence of TE (5.1 ng/mL) on p38-MAPK in keratinocytes. The influence of the TE on phosphorylated and unphosphorylated p38-MAPK in keratinocytes from a diabetic donor (NHK-d) measured by immunoblotting. IL-1ß was used as positive control. DMSO: 0.1%. A representative immunoblot is shown, n = 3.

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DISCUSSION The objective of this study was to determine if TE, which has been clinically proven to accelerate wound healing in euglycaemic wounds, could also be an option to benefit wound healing in a diabetic context. We used primary human keratinocytes and fibroblasts isolated from nondiabetic and diabetic donors because they are the main cell populations present in the epidermis and dermis and thus play a key role in wound healing. Our data show that a variety of factors important in the wound-healing process are affected by the treatment with TE and betulin in both diabetic and nondiabetic cells. The strongest effects could be observed on mRNA and protein expression levels of pro-inflammatory factors and chemokines. TE and betulin led to a strong increase in IL-6 and TNFα mRNA expression in NHK cells, whereas the Cox-2 expression was only slightly increased. HDF cells similarly responded to the treatment and enhanced mRNA levels of IL-6 and Cox-2, but also induced mRNA levels of IL-1β and IL-1R1, albeit with a high standard deviation. The importance of IL-6 in the wound-healing process was proven in IL-6 knockout mice, which showed a delayed reduction of wound area with attenuated leukocyte infiltration, reepithelialization, angiogenesis, and collagen accumulation.37 In the wound fluid of streptozytocin-induced diabetic mice, IL6 levels were found to be reduced, and it was suggested that the delayed healing is associated with these alterations.38 Studies with diabetic rabbits showed an increased baseline gene expression of IL-6, but postinjury the increase over baseline gene expression was significantly less in diabetic wounds compared with nondiabetic wounds.39 H



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keratinocytes from diabetic donors concentrate on alterations in the MMP-1, -2, -9, TIMP-1, -2, Cx26, Cx43, IL-1β, and TNFα mRNA expression levels.12,21 Our results indicate that the baseline expression of only a few genes is significantly different in NHK-d compared to NHK-nd cells. Furthermore, our data also indicate that the age of the donors influences baseline mRNA expression. mRNA expression of MMP-1 and MMP-2 and of the anti-inflammatory factor SOCS3 was slightly increased in NHK-nd cells, and the expression levels of HBD3 and aquaporin 3 were elevated in NHK-d cells. However, the differences we observed between the mRNA baseline expressions are significant, but small. Brandner et al. did not find significant differences for MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, TNFα, Cx26, and Cx43 mRNA expression in cultured keratinocytes from nondiabetic and diabetic origin,21 even though there were significant differences for MMP-2, IL-1β, and TNFα in skin biopsies. Of note, when NHK cells were cultured under hyperglycemic conditions, MMP-2 and MMP-9 mRNA levels were diminished compared to NHK cells cultured under euglycemic conditions.17 Interestingly, MMP-2 protein levels were increased in diabetic wound tissues.53 SOCS3 mRNA was found to be elevated in wounds of diabetic mice, whereas aquaporin 3 mRNA expression was decreased in wounds of diabetic rats.54,55 We observed more pronounced baseline differences in HDF cells derived from nondiabetic and diabetic patients. The expression of 11 different genes was significantly increased in HDF-d compared to HDF-nd cells. The genes with altered expression levels belong to groups such as the ECM, cell junctions, pro- and anti-inflammatory mediators, chemokines, antioxidants, and TIMPs. The largest differences in mRNA baseline expression was evident for the chemokine IP-10. This result is in line with reports that serum IP-10 levels are significantly elevated in type 1 diabetes and that transgenic mice constitutively expressing IP-10 show impaired formation of granulation tissue and delayed wound healing.56,57 Furthermore, baseline levels of the chemokines MCP-1 and IL-8 mRNA were elevated in HDF-d compared to HDF-nd cells. Higher MCP-1 levels correlate with results from Wetzler et al., who published that MCP-1 mRNA and protein was maintained longer in genetically diabetic mice during healing.44 Heterogenous results have been reported regarding IL-8 mRNA levels in diabetes. While IL-8 is downregulated in chronic diabetic foot ulcers,9 the baseline expression of IL-8 in diabetic rabbits was significantly increased.39 We also found elevated IL-6 and SOCS3 baseline mRNA expression in cultured HDF-d cells but not in NHK-d cells. Notably, Rieusset et al. reported that elevated IL-6 correlates with induced SOCS3 expression in skeletal muscle of type 2 diabetic patients and that high glucose concentrations enhanced IL-6-induced SOCS3 mRNA expression in cultured human muscle cells.58 It may be that in HDF-d cells a similar correlation exists. Furthermore, we observed elevated Cx43 mRNA levels in HDF-d compared to HDF-nd cells. In line with these results, Abdullah et al. measured a higher rate of gap junctional intercellular communication in cultured HDF-d in comparison to HDF-nd cells.59 However, Pollok et al. could not observe differences in Cx43 protein levels in cultured HDF-d compared to HDF-nd cells, but they observed impaired susceptibility to a Cx43 mimetic peptide in HDF-d cells.12 These differences may be explained by variability between diabetic donors. Cell migration is connected to changes in the actin cytoskeleton.33 Recently, we have reported that TE and its

The proinflammatory enzyme Cox-2 is rapidly upregulated in response to injury, and its inhibition delayed reepithelialization and angiogenesis in the early phase of wound healing.40 Furthermore, inhibition with celecoxib delayed wound closure and reepithelialization in mice with uniform full-thickness wounds.41 These findings underline the importance of Cox-2 in wound healing. Few studies exist that deal with the expression of Cox-2 in a diabetic context. Kämpfer et al. found elevated levels of Cox-2 in chronic wounds of diabetic mice, although the diabetes-impaired healing could not be associated with the increased levels.42 Our results show that after treatment with TE or betulin the mRNA and protein levels of the pro-inflammatory cytokine TNFα were significantly upregulated only in NHK-nd cells, partly so in NHK-d cells, but not in HDF cells. Interestingly, we could not detect IL-1β protein in any of the cells studied. mRNA of IL-1β showed either a high variability in HDF cells or no regulation in NHK cells, suggesting that IL-1β may not play as prominent a role as TNFα, which is a key player in the initiation of inflammatory responses in wound healing.22,43 However, both pro-inflammatory mediators are also reported to remain longer in wound tissue from genetically diabetic mice.44 The important role of chemokines in wound healing has often been addressed.39,45,46 On one hand, they play a pivotal role in the attraction of immune cells to the wound area; on the other hand, prolonged recruitment and local persistence of immune cells are believed to promote impaired wound healing. Treatment of HDF and NHK cells with either TE or betulin differentially upregulated chemokines IL-8, RANTES, MCP-1, IP-10, and MIP-2. The mRNA and protein levels of IL-8 were upregulated in HDF and NHK cells independent of the donor characteristics. This chemokine is known to enhance wound healing as the major bioactive chemoattractant for granulocytes and as a potent promoter of angiogenesis.47 Furthermore, keratinocytes and endothelial cells in chronic diabetic foot ulcers lack IL-8 upregulation, but not in the wound margin.46 The chemokines MCP-1 and RANTES, which are more upregulated in cells from diabetic donors in our studies, play a critical role in macrophage recruitment.48 Although it has been reported that neutrophils and macrophages persist locally in poorly healing wounds of diabetic patients,3 an adequate macrophage response is necessary in the early stage of wound repair. Other studies revealed that macrophages isolated from the wounds of diabetic mice have an impaired function, which is associated with a prolonged inflammatory response.2,49 Interestingly, a one-time treatment with MCP-1 significantly stimulated healing in diabetic wounds by restoring the macrophage response.50 The impact of TE and betulin on the mRNA of growth factors was only marginal in NHK and HDF cells. In contrast, mRNA expression of the cell adhesion molecules VCAM-1 and ICAM-1 was highly upregulated in HDF-nd cells. In HDF-d, NHK-d, and NHK-nd cells, we could observe a tendency for only a slight upregulation on mRNA level. The importance of ICAM-1 in wound healing was demonstrated in ICAM-1deficient mice, which showed delayed wound healing.29 In some instances, the baseline expression of mRNA differs in skin cells from diabetic and nondiabetic donors. Thus far, few in vitro studies exist comparing cells derived from diabetic and nondiabetic donors. The existing studies that used fibroblasts focused on variations in cell response to growth factors such as EGF (epidermal growth factor), IGF-1 (insulinlike growth factor 1), FGF2, and PDGF.51,52 Studies with I



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(Glaswarenfabrik Karl Hecht, Sondheim, Germany); LN950 (Prof. S. Laufer, University of Tübingen, Germany); GST-Rhotekin and GST-PAK (immobilized to glutathione-sepharose beads) (Pharmacia Biotech, Cambridge, MA, USA), CNF1, CNFY (Prof. G. Schmidt, Albert-Ludwigs-University Freiburg, Germany); Bradford Quick Start dye (Bio-Rad Laboratories, Munich, Germany); penicillin−streptomycin, 0.45 μm PVDF membrane (Roche, Mannheim, Germany); 0.45 μm PVDF membrane, Cdc42 antibody (17-299) (Millipore, Bedford, MA, USA); Tropix i-Block: TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA, USA); HRP juice (pjk, Kleinblittersdorf, Germany); Amersham ECL Western blotting detection reagents (GE Healthcare, Little Chalfont, UK); p38-(9212) and pp38-MAPK (9211) antibody (Cell Signaling, Danvers, MA, USA); RhoA (sc418) and Rac1 (05-389) antibody (Santa Cruz, CA, USA); anti-rabbit (111-035-144) and anti-mouse (115-035-146) antibody (Jackson Immuno Research, Newmarket, UK); β-actin (69100) (MP Biomedicals, Santa Ana, CA, USA); human interleukin-1β (R&D Systems, Minneapolis, MN, USA); RNeasy Plus kit, QuantiTect reverse transcription kit (Qiagen, Venlo, The Netherlands). Cells. Primary human keratinocytes and fibroblasts from nondiabetic and diabetic patients were obtained from different sources. The information on the used keratinocytes and fibroblasts concerning age, gender, diabetic status, biopsy site, and classification is summarized in Table A of the S1 Protocol (Supporting Information). The use was approved by the ethics committees of the Aerztekammer Hamburg, Germany (06.09.2000: 060900, 17.09.2013: PV4198) and Freiburg (21.02.2010: 45/03). A written informed consent from the donor or the next of kin was obtained for the use of the samples in research. In the case of the keratinocytes derived from the foreskin, the ethics committee waived the need for consent. Isolation and cultivation of primary human keratinocytes (NHK-nd and NHK-d) and primary human dermal fibroblasts (HDF-nd and HDF-d) are described in the S1 Protocol (Supporting Information). RNA Isolation, cDNA Synthesis, and Quantitative RT-PCR Analysis. Total RNA was isolated and purified from fibroblasts and keratinocytes using the RNeasy Plus kit according to the manufacturer’s instructions. A total of 400 000 keratinocytes or 320 000 fibroblasts were used for each sample. The concentration of RNA was determined by measuring the UV absorption with the NanoDrop 2000 (Thermo Fischer Scientific, Waltham, MA, USA) at 260 nm. The ratio of absorbance at 260 and 280 nm was used to assess the purity of RNA. The QuantiTect reverse transcription kit was used to synthesize first-strand cDNA from 1000 ng of total RNA according to the manufacturer’s protocol. qRT-PCR was performed with a LightCycler 480 (Roche, Basel, Switzerland). The primer and probes for the TaqMan probe-based assays were designed with the online tool OligoArchitect and purchased from Sigma-Aldrich. The sequences of the used primers and probes are listed in Table B in the S1 Protocol (Supporting Information). The analysis of mRNA expression profiles is described in detail in the S1 Protocol. Fluidigm’s Biomark 48.48 high-throughput qRT-PCR chip platform (Fluidigm Corporation, San Francisco, CA, USA) with predesigned gene expression assays from Applied Biosystems was used according to the manufacturer’s instructions.63 The system was used to analyze 48 different genes (Table C in S1 Protocol, Supporting Information). For the high-throughput qRT-PCR 100 μL of RNA was reverse transcribed into cDNA with the TaqMan reverse transcription kit according to the manufacturer’s protocol. The gene expression data were normalized to the expression of GAPDH, and the relative expression levels were determined by the ΔΔCt method. For comparison of the baseline gene expression, the normalized 2−ΔCt values of the untreated controls were averaged and put in relation to each other. The genes that were below the detection limit and the control genes were excluded in the figures. Soluble Inflammation-Related Proteins. Cell culture supernatant was obtained from 500 000 fibroblasts or 650 000 keratinocytes that were cultured for 30 h in 8 mL of starvation medium (see the S1 Protocol, Supporting Information). The concentration of 32 different inflammation-related proteins (Table D in the S1 Protocol) was

triterpenes betulin and lupeol alter the actin cytoskeleton in NHK-nd cells from young donors.16 In the current study, we observed the same effect in NHK-d cells, whereas HDF-nd and HDF-d cells adopted a thin elongated shape. Ebeling et al. have shown that treatment of NHK-nd cells from young donors with TE, betulin, or lupeol causes activation of the Rho-GTPases RhoA and treatment with TE and betulin causes activation of Cdc42.16 In the present study, we found that treatment of NHK-d cells with TE, betulin, or lupeol activates all three RhoGTPases (RhoA, Cdc42, Rac1). These small differences between the reaction of NHK-nd and NHK-d cells might be explained by differences in incubation times. Whereas Ebeling et al. used an incubation for 3 h prior to examining the activity of Cdc42 and Rac1, we used a shorter time period of 20 min, as these two Rho-GTPases were shown to be rapidly and transiently activated.16,60,61 However, it cannot be excluded that these differences are due to diabetic donors, the age of the donors, or variability of the NHK cells. Moreover, Ebeling et al. demonstrated that treatment of NHK-nd cells with TE or betulin caused activation of p38-MAPK. TE-activated p38MAPK has been shown to enhance the mRNA stability of mediators important in the inflammatory phase of wound healing, and involvement in cell migration has been discussed.16 In the current study, TE moderately activated p38-MAPK in NHK-d cells. Beyond that, our results support the hypothesis that p38-MAPK plays a role in keratinocyte migration in response to treatment with TE. The ability of the p38-MAPK inhibitor LN 950 to block the change in cell shape induced by TE and the triterpenes in NHK-nd and NHK-d cells suggests the involvement of p38-MAPK in this process. However, the p38-MAPK inhibitor could not influence the shape change induced by TE and the triterpenes in HDF-nd or HDF-d cells. Therefore, p38-MAPK does not seem to be involved in the actin polymerization processes in these cells. This might be due to cell-type-specific signaling pathways62 Considering the results obtained with the in vitro model of keratinocytes and fibroblasts derived from diabetic donors, which are similar to those received with the respective cells from nondiabetic donors, TE can be regarded as an option to influence the wound-healing processes under diabetic conditions. Regarding the increased levels of pro-inflammatory mediators and chemokines that are upregulated by TE and betulin, use may be appropriate only for wounds in which inflammation has already ceased. While our model provides insight into how skin cells such as NHKs and HDFs may be affected, the efficacy of TE will have to be proven in clinical studies with diabetic patients.

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EXPERIMENTAL SECTION

Test Compounds. The birch bark extract (TE) with its main components betulin (86.9%) and lupeol (3.9%) as well as the single triterpenes betulin, lupeol, oleanolic acid, and betulinic acid were received from Birken AG, Niefern-Ö schelbronn, Germany. These materials were dissolved in DMSO (dimethyl sulfoxide) for the preparation of the stock solutions and diluted with the medium to obtain the respective dilution for the different assays. Antibodies, Reagents, and Cell Culture Consumables. Antibiotic-Antimycotic 100×, phalloidin−rhodamine, Prolong Gold antifade reagent, Keratinocyte SFM and supplements (human recombinant EGF, bovine pituitary extract), DMEM + GlutaMAX, FCS, trypsin/EDTA 0.05%/0.02%, human PDGF, Antibiotic-Antimycotic (Thermo Fischer Scientific, Waltham, MA, USA); Ciprobay 400 mg (Bayer, Leverkusen, Germany); collagen type 1 solution from rat tail (Sigma, Steinheim, Germany); 10 mm glass coverslips J



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analyzed using the Luminex assay platform as has been described in detail.64 Immune mediators were detected using the commercially available Milliplex human cytokine assay system (Millipore) to detect 16 proteins (GM-CSF, GRO-α, INF-γ, IL-10, IL-12(p40), IL-17A, IL1β, IL-1RA, IL-2, IL-4, IL-8, MCP-1, MIP-1α, MIP-1β, sCD40L, TNFα); FGF23 and leptin were measured using the Milliplex human bone panel. A multiplexed Luminex-based immunoassay for soluble receptor proteins (sRAGE, sgp130, sTNF-R1, sTNF-R2, IL-6sR), soluble adhesion molecules (sICAM, sVCAM, sE-Selectin), sFas, RANTES, MIF, and PLA2G7 was developed and validated in-house as described in detail by Hsu et al.65 Staining of the Actin Cytoskeleton. Cells were plated on glass coverslips. For fibroblasts, the coverslips were coated with collagen type I solution for 2 h before plating. Keratinocytes were starved (for conditions see S1 Protocol, Supporting Information) for 2 days prior to stimulation. After administration of test substances, cells were stained with rhodamine phalloidin (dilution 1:25 with PBS) and examined by fluorescence microscopy (Zeiss) as described in the S1 Protocol. P38-MAPK inhibitor LN 950 was added 30 min prior to administration of test substances. Rho-GTPase Pulldown Experiments. Rho-GTPase pulldown experiments were conducted as described previously.16 Briefly, protein extracts were obtained by scraping in lysis buffer (10% glycerol, 50 mM Tris pH 7.4, 100 mM NaCl, 1% NP-40, 2 mM MgCl2, and 1 mM PMSF) and subsequent centrifugation at 4 °C. The supernatant was utilized to determine the total amount of Rho-GTPase as well as to perform the pulldown experiment. In the latter case, the supernatant was incubated at 4 °C for 1 h under agitation with glutathionesepharose beads bound to GST-Rhotekin for binding RhoA or GSTPAK for binding Cdc42 or Rac1. Afterward, the beads bound to the active form of the Rho-GTPase were washed and separated by centrifugation. Finally, Rho-GTPases were analyzed by immunoblotting. Immunoblot Analysis. The protein amount was determined by using Bradford Quick Start dye. A 40 μg amount of protein was separated by SDS-PAGE. For p38-MAPK a 12% gel and for RhoGTPases a 13% gel was used. Subsequently, the proteins were transferred to a 0.45 μm PVDF membrane. The membrane was blocked with 5% milk powder (for p38-MAPK) or 0.2% Tropix i-Block (for Rho-GTPases) in TBST and subsequently incubated overnight with the primary antibody at 4 °C with agitation. After washing and incubation with horseradish peroxidase labeled secondary antibody, the proteins were detected with an enhanced chemiluminescence detection reagent. The antibodies were used in the following concentrations: Phosphop38- and p38-MAPK 1:1.000 in 5% BSA in TBST; RhoA 1:500 in 0.2% Tropix i-Block in TBST; Rac1 1:2.000 in 0.2% Tropix i-Block in TBST; Cdc42 1:500 in 0.2% Tropix i-Block in TBST; β-actin 1:10.000 in 1% BSA in TBST; anti-rabbit antibody 1:5.000 in 5% BSA in TBST; anti-mouse antibody 1:3.750 in 0.2% Tropix i-Block in TBST. Statistical Analysis. Values are expressed as mean + or ± standard deviation (SD). Statistical analyses were performed with GraphPad Prism 5 (GraphPad Software Inc.) and Excel 2013 (Microsoft). Statistical analysis was determined using Student’s t-test or ANOVA with Bonferroni’s post hoc test as indicated in the figure legend. pValues were calculated, and p < 0.05 was considered statistically significant. Wherever significance has been proven, it is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0049-761-203-8373. Fax: 0049-761-203-8383. E-mail: [email protected]. Author Contributions #

T. Wardecki and P. Werner contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors are grateful to Prof. Dr. S. Laufer, University of Tübingen, Germany, for the p3-MAPK inhibitor LN 950, to Birken AG, Niefern-Ö schelbronn for TE and the triterpenes, to Prof. Dr. L. Bruckner-Tuderman for the primary keratinocytes and fibroblasts, to G. Ertinger, Institute of Pharmaceutical Sciences, Albert-Ludwigs University Freiburg, for her assistance regarding the staining of the actin cytoskeleton of fibroblasts, and to A. Döttinger, Institute of Natural and Medical Sciences at the University of Tübingen, Reutlingen, Germany, for technical assistance in performing the Luminex analysis. The study was financially supported by the Federal Ministry for Economic Affairs and Energy on the basis of a decision by the German Bundestag. M.T. gratefully acknowledges financial support by the Robert Bosch Foundation, Stuttgart, Germany.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00027. Additional figures and experimental details (PDF) K



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Influence of Birch Bark Triterpenes on Keratinocytes and Fibroblasts

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