Current Approaches to Wound Repair in Burns: How far Have we Come From Cover to Close? A Narrative Review.

Burn injuries are a significant global health concern, with more than 11 million people requiring medical intervention each year and approximately 180,000 deaths annually. Despite progress in health and social care, burn injuries continue to result in socioeconomic burdens for victims and their families. The management of severe burn injuries involves preventing and treating burn shock and promoting skin repair through a two-step procedure of covering and closing the wound. Currently, split-thickness/full-thickness skin autografts are the gold standard for permanent skin substitution. However, deep burns treated with split-thickness skin autografts may contract, leading to functional and appearance issues. Conversely, defects treated with full-thickness skin autografts often result in more satisfactory function and appearance. The development of tissue-engineered dermal templates has further expanded the scope of wound repair, providing scar reductive and regenerative properties that have extended their use to reconstructive surgical interventions. Although their interactions with the wound microenvironment are not fully understood, these templates have shown potential in local infection control. This narrative review discusses the current state of wound repair in burn injuries, focusing on the progress made from wound cover to wound closure and local infection control. Advancements in technology and therapies hold promise for improving the outcomes for burn injury patients. Understanding the underlying mechanisms of wound repair and tissue regeneration may provide new insights for developing more effective treatments in the future.

Unravelling heterogeneous effects of cancer­associated fibroblasts on poor prognosis markers in breast cancer EM­G3 cell line: In vitro­targeted treatment (anti­IL-6, anti­VEGF-A, anti­MFGE8) based on transcriptomic profiling.

Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer­associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co­expression of keratins­8/­14 in the EM­G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin­8, ­14, ­18, ­19) and epithelial­mesenchymal transition­associated markers (SLUG, SNAIL, ZEB1, E­/N­cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGF­A and MFGE8 attenuated the modulatory effect of CAFs on EM­G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM­G3 cells in vitro. CAFs of different origins support the pro­inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.

Heterogeneous response to TGF-ß1/3 isoforms in fibroblasts of different origins: implications for wound healing and tumorigenesis.

Identification of therapeutic targets for treating fibrotic diseases and cancer remains challenging. Our study aimed to investigate the effects of TGF-ß1 and TGF-ß3 on myofibroblast differentiation and extracellular matrix deposition in different types of fibroblasts, including normal/dermal, cancer-associated, and scar-derived fibroblasts. When comparing the phenotype and signaling pathways activation we observed extreme heterogeneity of studied markers across different fibroblast populations, even within those isolated from the same tissue. Specifically, the presence of myofibroblast and deposition of extracellular matrix were dependent on the origin of the fibroblasts and the type of treatment they received (TGF-ß1 vs. TGF-ß3). In parallel, we detected activation of canonical signaling (pSMAD2/3) across all studied fibroblasts, albeit to various extents. Treatment with TGF-ß1 and TGF-ß3 resulted in the activation of canonical and several non-canonical pathways, including AKT, ERK, and ROCK. Among studied cells, cancer-associated fibroblasts displayed the most heterogenic response to TGF-ß1/3 treatments. In general, TGF-ß1 demonstrated a more potent activation of signaling pathways compared to TGF-ß3, whereas TGF-ß3 exhibited rather an inhibitory effect in keloid- and hypertrophic scar-derived fibroblasts suggesting its clinical potential for scar treatment. In summary, our study has implications for comprehending the role of TGF-ß signaling in fibroblast biology, fibrotic diseases, and cancer. Future research should focus on unraveling the mechanisms beyond differential fibroblast responses to TGF-ß isomers considering inherent fibroblast heterogeneity.

The sweet side of wound healing: galectins as promising therapeutic targets in hemostasis, inflammation, proliferation, and maturation/remodeling.

INTRODUCTION: Understanding the molecular and cellular processes involved in skin wound healing may pave the way for the development of innovative approaches to transforming the identified natural effectors into therapeutic tools. Based on the extensive involvement of the ga(lactoside-binding)lectin family in (patho)physiological processes, it has been well established that galectins are involved in a wide range of cell-cell and cell-matrix interactions. AREAS COVERED: In the present paper, we provide an overview of the biological role of galectins in repair and regeneration, focusing on four main phases (hemostasis, inflammation, proliferation, and maturation/remodeling) of skin repair using basic wound models (open excision vs. sutured incision). EXPERT OPINION: The reported data make a strong case for directing further efforts to treat excisional and incisional wounds differently. Functions of galectins essentially result from their modular presentation. In fact, Gal-1 seems to play a role in the early phases of healing (anti-inflammatory) and wound contraction, Gal-3 accelerates re-epithelization and increases tensile strength (scar inductor). Galectins have also become subject of redesigning by engineering to optimize the activity. Clinically relevant, these new tools derived from the carbohydrate recognition domain platform may also prove helpful for other purposes, such as potent antibacterial agglutinins and opsonins.

Agrimonia eupatoria L. Aqueous Extract Improves Skin Wound Healing: An In Vitro Study in Fibroblasts and Keratinocytes and In Vivo Study in Rats.

BACKGROUND/AIM: We have previously shown that the water extract of Agrimonia eupatoria L. (AE) is a valuable source of polyphenols with excellent antioxidant properties and has clinical potential for the prevention and/or adjuvant therapy of cardiovascular complications associated with diabetes. Inspired by our previously published data, in the present study we examined whether AE improves skin wound healing in a series of in vitro and in vivo experiments. MATERIALS AND METHODS: In detail, we investigated the ability of the AE extract to induce fibroblast to myofibroblast conversion, extracellular matrix (ECM) deposition, and keratinocyte proliferation/differentiation, in vitro. In parallel, in an animal model, we measured wound tensile strength (TS) and assessed the progression of open wounds using basic histology and immunofluorescence. RESULTS: The AE extract induced the myofibroblast-like phenotype and enhanced ECM deposition, both in vitro and in vivo. Furthermore, the wound TS of skin incisions and the contraction rates of open excisions were significantly increased in the AE-treated group. CONCLUSION: The present data show that AE water extract significantly improves the healing of open and sutured skin wounds. Therefore, our data warrant further testing in animal models that are physiologically and evolutionarily closer to humans.

Aesculus hippocastanum L. extract differently modulates normal human dermal fibroblasts and cancer-associated fibroblasts from basal/squamous cell carcinoma.

Fibroblasts are actively involved in the formation of granulation tissue and/or tumor stroma. These cells possess the potential to differentiate into myofibroblasts acquiring a highly contractile phenotype characterized by the expression of α-smooth muscle actin (SMA). Considering TGF-ß1 as the main inducer of myofibroblast differentiation and horse chestnut extract (HCE) as an effective modulator of the wound healing, we have new evidence to demonstrate canonical TGF-ß1/SMAD and non-canonical/non-SMAD signaling in normal fibroblasts, isolated from healthy human skin (human dermal fibroblasts - HDFs), and their malignant counterparts (CAFs) isolated from basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) using western blot and immunofluorescence. Our study revealed that HCE stimulated the deposition of fibronectin by BCC fibroblasts (BCCFs), an effect not seen in other studied fibroblasts. Moreover, HCE in combination with TGF-ß1 showed a synergic effect on the presence of polymerized SMA-stress fibers, particularly visible in CAFs. Interestingly, the TGF-ß1 exposure led to activation of the canonical SMAD signaling in HDFs and BCCFs, whereas treatment of SCC fibroblasts (SCCFs) resulted in activation of the non-canonical AKT and/or ERK1/2 signaling. In conclusion, we observed specific differences in signaling between HDFs and CAFs that should be considered when developing new therapeutic approaches targeting wound/tumor microenvironments.

Pro-Apoptotic Potential of Pseudevernia furfuracea (L.) Zopf Extract and Isolated Physodic Acid in Acute Lymphoblastic Leukemia Model In Vitro.

Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed type of leukemia among children. Although chemotherapy is a common treatment for cancer, it has a wide range of serious side effects, including myelo- and immunosuppression, hepatotoxicity and neurotoxicity. Combination therapies using natural substances are widely recommended to attenuate the adverse effects of chemotherapy. The aim of the present study was to investigate the anti-leukemic potential of extract from the lichen Pseudevernia furfuracea (L.) Zopf (PSE) and isolated physodic acid (Phy) in an in vitro ALL model. A screening assay, flow cytometry and Western blotting were used to analyze apoptosis occurrence, oxidative stress, DNA damage and stress/survival/apoptotic pathway modulation induced by the tested substances in Jurkat cells. We demonstrate for the first time that PSE and Phy treatment-induced intrinsic caspase-dependent cell death was associated with increased oxidative stress, DNA damage and cell cycle arrest with the activation of cell cycle checkpoint proteins p53, p21 and p27 and stress/survival kinases p38 MAPK, JNK and PI3K/Akt. Moreover, using peripheral T lymphocytes, we confirmed that PSE and Phy treatment caused minimal cytotoxicity in normal cells, and therefore, these naturally occurring lichen secondary metabolites could be promising substances for ALL therapy.

Molecular Changes Underlying Genistein Treatment of Wound Healing: A Review.

Estrogen deprivation is one of the major factors responsible for many age-related processes including poor wound healing in postmenopausal women. However, the reported side-effects of estrogen replacement therapy (ERT) have precluded broad clinical administration. Therefore, selective estrogen receptor modulators (SERMs) have been developed to overcome the detrimental side effects of ERT on breast and/or uterine tissues. The use of natural products isolated from plants (e.g., soy) may represent a promising source of biologically active compounds (e.g., genistein) as efficient alternatives to conventional treatment. Genistein as natural SERM has the unique ability to selectively act as agonist or antagonist in a tissue-specific manner, i.e., it improves skin repair and simultaneously exerts anti-cancer and chemopreventive properties. Hence, we present here a wound healing phases-based review of the most studied naturally occurring SERM.

Desmoplastic Crosstalk in Pancreatic Ductal Adenocarcinoma Is Reflected by Different Responses of Panc-1, MIAPaCa-2, PaTu-8902, and CAPAN-2 Cell Lines to Cancer-associated/Normal Fibroblasts.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) still represents one of the most aggressive cancers. Understanding of the epithelial-mesenchymal crosstalk as a crucial part of the tumor microenvironment should pave the way for therapies to improve patient survival rates. Well-established cell lines present a useful and reproducible model to study PDAC biology. However, the tumor-stromal interactions between cancer cells and cancer-associated fibroblasts (CAFs) are still poorly understood. MATERIALS AND METHODS: We studied interactions between four PDAC cell lines (Panc-1, CAPAN-2, MIAPaCa-2, and PaTu-8902) and conditioned media derived from primary cultures of normal fibroblasts/PDAC-derived CAFs (PANFs). RESULTS: When the tested PDAC cell lines were stimulated by PANF-derived conditioned media, the most aggressive behavior was acquired by the Panc-1 cell line (increased number and size of colonies, remaining expression of vimentin and keratin 8 as well as increase of epithelial-to-mesenchymal polarization markers), whereas PaTu-8902 cells were rather inhibited. Of note, administration of the conditioned media to MIAPaCa-2 cells resulted in an inverse effect on the size and number of colonies, whereas CAPAN-2 cells were rather stimulated. To explain the heterogeneous pattern of the observed PDAC crosstalk at the in vitro level, we further compared the phenotype of primary cultures of cells derived from ascitic fluid with that of the tested PDAC cell lines, analyzed tumor samples of PDAC patients, and performed gene expression profiling of PANFs. Immuno-cyto/histo-chemical analysis found specific phenotype differences within the group of examined patients and tested PDAC cell lines, whereas the genomic approach in PANFs found the key molecules (IL6, IL8, MFGE8 and periostin) that may contribute to the cancer aggressive behavior. CONCLUSION: The desmoplastic patient-specific regulation of cancer cells by CAFs (also demonstrated by the heterogeneous response of PDAC cell lines to fibroblasts) precludes simple targeting and development of an effective treatment strategy and rather requires establishment of an individualized tumor-specific treatment protocol.

Molecular Changes Underlying Hypertrophic Scarring Following Burns Involve Specific Deregulations at All Wound Healing Stages (Inflammation, Proliferation and Maturation).

Excessive connective tissue accumulation, a hallmark of hypertrophic scaring, results in progressive deterioration of the structure and function of organs. It can also be seen during tumor growth and other fibroproliferative disorders. These processes result from a wide spectrum of cross-talks between mesenchymal, epithelial and inflammatory/immune cells that have not yet been fully understood. In the present review, we aimed to describe the molecular features of fibroblasts and their interactions with immune and epithelial cells and extracellular matrix. We also compared different types of fibroblasts and their roles in skin repair and regeneration following burn injury. In summary, here we briefly review molecular changes underlying hypertrophic scarring following burns throughout all basic wound healing stages, i.e. during inflammation, proliferation and maturation.

Human galectin­3: Molecular switch of gene expression in dermal fibroblasts in vitro and of skin collagen organization in open wounds and tensile strength in incisions in vivo.

Understanding the molecular and cellular processes in skin wound healing can pave the way for devising innovative concepts by turning the identified natural effectors into therapeutic tools. Based on the concept of broad­scale engagement of members of the family of galactoside­binding lectins (galectins) in pathophysiological processes, such as cancer or tissue repair/regeneration, the present study investigated the potential of galectins­1 (Gal­1) and ­3 (Gal­3) in wound healing. Human dermal fibroblasts, which are key cells involved in skin wound healing, responded to galectin exposure (Gal­1 at 300 or Gal­3 at 600 ng/ml) with selective changes in gene expression among a panel of 84 wound­healing­related genes, as well as remodeling of the extracellular matrix. In the case of Gal­3, positive expression of Ki67 and cell number increased when using a decellularized matrix produced by Gal­3­treated fibroblasts as substrate for culture of interfollicular keratinocytes. In vivo wounds were topically treated with 20 ng/ml Gal­1 or ­3, and collagen score was found to be elevated in excisional wound repair in rats treated with Gal­3. The tensile strength measured in incisions was significantly increased from 79.5±17.5 g/mm2 in controls to 103.1±21.4 g/mm2 after 21 days of healing. These data warrant further testing mixtures of galectins and other types of compounds, for example a combination of galectins and TGF­ß1.

Aesculus hippocastanum L. Extract Does Not Induce Fibroblast to Myofibroblast Conversion but Increases Extracellular Matrix Production In Vitro Leading to Increased Wound Tensile Strength in Rats.

The ability of horse chestnut extract (HCE) to induce contraction force in fibroblasts, a process with remarkable significance in skin repair, motivated us to evaluate its wound healing potential in a series of experiments. In the in vitro study of the ability of human dermal fibroblasts to form myofibroblast-like cells was evaluated at the protein level (Western blot and immunofluorescence). The in vivo study was conducted on male Sprague-Dawley rats with inflicted wounds (one open circular and one sutured incision) on their backs. Rats were topically treated with two tested HCE concentrations (0.1% and 1%) or sterile water. The control group remained untreated. The incisions were processed for wound tensile strength (TS) measurement whereas the open wounds were subjected to histological examination. On the in vitro level the HCE extract induced fibronectin-rich extracellular matrix formation, but did not induced α-smooth muscle actin (SMA) expression in dermal fibroblasts. The animal study revealed that HCE increased wound TS and improved collagen organization. In conclusion, the direct comparison of both basic wound models demonstrated that the healing was significantly increased following HCE, thus this extract may be found useful to improve healing of acute wounds. Nevertheless, the use of an experimental rat model warrants a direct extrapolation to the human clinical situation.

Overexpression of Galectin-3 in Chronic Lymphocytic Leukemia Is Associated With 17p Deletion: A Short Report.

BACKGROUND/AIM: Galectins belong to the family of galactose-binding proteins known to play an important role in the processes of cell proliferation, differentiation, migration and neoplastic progression. Herein, we studied the expression of galectin-3 (Gal-3) in chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: The expression of Gal-3 was analyzed by means of multiparametric flow cytometry in normal and pathological B-cells from peripheral blood and bone marrow samples of 67 patients with CLL. RESULTS: Pathological B-cells expressed significantly higher levels of cytoplasmic Gal-3 than normal B-cells. Moreover, overexpression of cytoplasmic Gal-3 was observed in the prognostically poorest subgroup of CLL patients, namely those with 17p deletion. CONCLUSION: Our results indicate a possible role of galectin-3 in CLL pathophysiology and its potential value as a prognostic marker and therapeutic target.

Derivatization of Rosmarinic Acid Enhances its in vitro Antitumor, Antimicrobial and Antiprotozoal Properties.

On its own, rosmarinic acid possesses multiple biological activities such as anti-inflammatory, antimicrobial, cardioprotective and antitumor properties, and these are the consequence of its ROS scavenging and inhibitory effect on inflammation. In this study, two quaternary phosphonium salts of rosmarinic acid were prepared for the purpose of increasing its penetration into biological systems with the aim of improving its antimicrobial, antifungal, antiprotozoal and antitumor activity. The synthetized molecules, the triphenylphosphonium and tricyclohexylphosphonium salts of rosmarinic acid, exhibited significantly stronger inhibitory effects on the growth of HCT116 cells with IC50 values of 7.28 or 8.13 µM in comparison to the initial substance, rosmarinic acid (>300 µM). For the synthesized derivatives, we detected a greater than three-fold increase of activity against Acanthamoeba quina, and a greater than eight-fold increase of activity against A. lugdunensis in comparison to rosmarinic acid. Furthermore, we recorded significantly higher antimicrobial activity of the synthetized derivatives when compared to rosmarinic acid itself. Both synthetized quaternary phosphonium salts of rosmarinic acid appear to be promising antitumor and antimicrobial agents, as well as impressive molecules for further research.

Evolution of Cancer Progression in the Context of Darwinism.

Our review compares evolution of cancer in the human body to the origin of new species from a common ancestor organism with respect to the theory of Charles Darwin. Moreover, the functional role of the tumor microenvironment as a selective pressure actively participating in cancer progression is also demonstrated. Evolutionary aspects of tumor growth and invasion from the point of view of modern therapeutic challenges and opportunities in precision personalized medicine are also discussed.

Genistein Improves Skin Flap Viability in Rats: A Preliminary In Vivo and In Vitro Investigation.

Selective estrogen receptor modulators (SERMs) have been developed to achieve beneficial effects of estrogens while minimizing their side effects. In this context, we decided to evaluate the protective effect of genistein, a natural SERM, on skin flap viability in rats and in a series of in vitro experiments on endothelial cells (migration, proliferation, antioxidant properties, and gene expression profiling following genistein treatment). Our results showed that administration of genistein increased skin flap viability, but importantly, the difference is only significant when treatment is started 3 days prior the flap surgery. Based on our in vitro experiments, it may be hypothesized that the underlying mechanism may rather by mediated by increasing SOD activity and Bcl-2 expression. The gene expression profiling further revealed 9 up-regulated genes (angiogenesis/inflammation promoting: CTGF, CXCL5, IL-6, ITGB3, MMP-14, and VEGF-A; angiogenesis inhibiting: COL18A1, TIMP-2, and TIMP-3). In conclusion, we observed a protective effect of genistein on skin flap viability which could be potentially applied in plastic surgery to women undergoing a reconstructive and/or plastic intervention. Nevertheless, further research is needed to explain the exact underlying mechanism and to find the optimal treatment protocol.

ß-Escin Effectively Modulates HUVECS Proliferation and Tube Formation.

In the present study we evaluated the anti-angiogenic activities of ß-escin (the major active compound of Aesculus hippocastanum L. seeds). Human umbilical-vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying the anti-angiogenic effect of ß-escin. We investigated the in vitro effects on proliferation, migration, and tube formation of HUVECs and in vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) angiogenesis assay. Moreover, the effect on gene expressions was determined by the RT2 ProfilerTM human angiogenesis PCR Array. It was found that ß-escin exerts inhibitory effect on the basic fibroblast growth factor (bFGF)-induced proliferation, migration and tube formation, as well as CAM angiogenesis in vivo. The inhibition of critical steps of angiogenic process observed with ß-escin could be partially explained by suppression of Akt activation in response to bFGF. Moreover, the anti-angiogenic effects of ß-escin could also be mediated via inhibition of EFNB2 and FGF-1 gene expressions in endothelial cells. In conclusion, ß-escin affects endothelial cells as a negative mediator of angiogenesis in vitro and in vivo and may therefore be considered as a promising candidate for further research elucidating its underlying mechanism of action.