Treatment of murine partial thickness scald injuries with multipotent adult progenitor cells decreases inflammation and promotes angiogenesis leading to improved burn injury repair.

Stem cells have been shown to have potential as a new therapy for burns and promote wound healing through decreasing inflammation and increasing angiogenesis. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of cytokines which can affect cellular activities. This article aimed to examine the effects of MAPC cells treatment on burn injury repair using a mouse model of partial thickness burn injury. The immunomodulatory effect of MAPC cells was investigated in vitro using a simultaneous T-cell proliferation assay. Partial thickness burns were created on the dorsal surface of mice and MAPC cells were administered via intradermal injection to the wound margins 24 h post-burn injury. The burn tissues were analysed macroscopically to determine wound area and histologically assessed to determine wound width and rate of re-epithelialisation. Immunohistochemistry and ELISA were employed to assess cell proliferation, inflammation and angiogenesis and collagen deposition in the burn area. MAPC cells inhibit the proliferation of stimulated T cells in culture. Burns intradermally injected with MAPC cells showed a significant reduction in the macroscopic wound area, histologic wound width and had an increased rate of re-epithelialisation. Immunohistochemistry and ELISA analysis of burn tissues showed dampened inflammation evidenced by a reduction in neutrophilic infiltration and modulation of inflammatory cytokines. Angiogenesis within the burn area was also improved in MAPC cell treated mice. However, no significant effect of MAPC cell treatment was observed on extracellular matrix production. Treatment of burns with MAPC cells improved burn injury repair with reduced time to healing, decreased inflammation and increased angiogenesis. These findings demonstrate the promising effects of MAPC cells on burn injury repair and suggest MAPC cells as a candidate source for clinical cell therapies.

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/ß-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/-, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/- embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and ß-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.

Mesenchymal Stem Cell Secretome as an Emerging Cell-Free Alternative for Improving Wound Repair.

The use of mesenchymal stem cells (MSC) for the treatment of cutaneous wounds is currently of enormous interest. However, the broad translation of cell therapies into clinical use is hampered by their efficacy, safety, manufacturing and cost. MSCs release a broad repertoire of trophic factors and immunomodulatory cytokines, referred to as the MSC secretome, that has considerable potential for the treatment of cutaneous wounds as a cell-free therapy. In this review, we outline the current status of MSCs as a treatment for cutaneous wounds and introduce the potential of the MSC secretome as a cell-free alternative for wound repair. We discuss the challenges and provide insights and perspectives for the future development of the MSC secretome as well as identify its potential clinical translation into a therapeutic treatment.

Attenuation of Flightless I Increases Human Pericyte Proliferation, Migration and Angiogenic Functions and Improves Healing in Murine Diabetic Wounds.

Pericytes are peri-vascular mural cells which have an important role in the homeostatic regulation of inflammatory and angiogenic processes. Flightless I (Flii) is a cytoskeletal protein involved in regulating cellular functions, but its involvement in pericyte activities during wound healing is unknown. Exacerbated inflammation and reduced angiogenesis are hallmarks of impaired diabetic healing responses, and strategies aimed at regulating these processes are vital for improving healing outcomes. To determine the effect of altering Flii expression on pericyte function, in vitro and in vivo studies were performed to assess the effect on healing, inflammation and angiogenesis in diabetic wounds. Here, we demonstrated that human diabetic wounds display upregulated expression of the Flii protein in conjunction with a depletion in the number of platelet derived growth factor receptor ß (PDGFRß) +/ neural glial antigen 2 (NG2) + pericytes present in the dermis. Human pericytes were found to be positive for Flii and attenuating its expression in vitro through siRNA knockdown led to enhanced proliferation, migration and angiogenic functions. Genetic knockdown of Flii in a streptozotocin-induced murine model of diabetes led to increased numbers of pericytes within the wound. This was associated with dampened inflammation, an increased rate of angiogenic repair and improved wound healing. Our findings show that Flii expression directly impacts pericyte functions, including proliferation, motility and angiogenic responses. This suggests that Flii regulation of pericyte function may be in part responsible for the changes in pericyte-related processes observed in diabetic wounds.

Reforming the Barrier: The Role of Formins in Wound Repair.

The restoration of an intact epidermal barrier after wound injury is the culmination of a highly complex and exquisitely regulated physiological process involving multiple cells and tissues, overlapping dynamic events and protein synthesis and regulation. Central to this process is the cytoskeleton, a system of intracellular proteins that are instrumental in regulating important processes involved in wound repair including chemotaxis, cytokinesis, proliferation, migration, and phagocytosis. One highly conserved family of cytoskeletal proteins that are emerging as major regulators of actin and microtubule nucleation, polymerization, and stabilization are the formins. The formin family includes 15 different proteins categorized into seven subfamilies based on three formin homology domains (FH1, FH2, and FH3). The formins themselves are regulated in different ways including autoinhibition, activation, and localization by a range of proteins, including Rho GTPases. Herein, we describe the roles and effects of the formin family of cytoskeletal proteins on the fundamental process of wound healing and highlight recent advances relating to their important functions, mechanisms, and regulation at the molecular and cellular levels.

Wound Healing from an Actin Cytoskeletal Perspective.

Wound healing requires a complex cascade of highly controlled and conserved cellular and molecular processes. These involve numerous cell types and extracellular matrix molecules regulated by the actin cytoskeleton. This microscopic network of filaments is present within the cytoplasm of all cells and provides the shape and mechanical support required for cell movement and proliferation. Here, an overview of the processes of wound healing are described from the perspective of the cell in relation to the actin cytoskeleton. Key points of discussion include the role of actin, its binding proteins, signaling pathways, and events that play significant roles in the phases of wound healing. The identification of cytoskeletal targets that can be used to manipulate and improve wound healing is included as an emerging area of focus that may inform future therapeutic approaches to improve healing of complex wounds.

Flightless I Negatively Regulates Macrophage Surface TLR4, Delays Early Inflammation, and Impedes Wound Healing.

TLR4 plays a pivotal role in orchestrating inflammation and tissue repair. Its expression has finally been balanced to initiate the early, robust immune response necessary for efficient repair without excessively amplifying and prolonging inflammation, which impairs healing. Studies show Flightless I (Flii) is an immunomodulator that negatively regulates macrophage TLR4 signalling. Using macrophages from Flii+/-, WT, and FliiTg/Tg mice, we have shown that elevated Flii reduces early TLR4 surface expression, delaying and reducing subsequent TNF secretions. In contrast, reduced Flii increases surface TLR4, leading to an earlier robust TNF peak. In Flii+/- mice, TLR4 levels peak earlier during wound repair, and overall healing is accelerated. Fewer neutrophils, monocytes and macrophages are recruited to Flii+/- wounds, leading to fewer TNF-positive macrophages, alongside an early peak and a robust shift to M2 anti-inflammatory, reparative Ym1+ and IL-10+ macrophages. Importantly, in diabetic mice, high Flii levels are found in plasma and unwounded skin, with further increases observed in their wounds, which have impaired healing. Lowering Flii in diabetic mice results in an earlier shift to M2 macrophages and improved healing. Overall, this suggests Flii regulation of TLR4 reduces early inflammation and decreases the M2 macrophage phenotype, leading to impaired healing.

Plasma-polymerized pericyte patches improve healing of murine wounds through increased angiogenesis and reduced inflammation.

Pericytes have the potential to be developed as a cell therapy for the treatment of wounds; however, the efficacy of any cell therapy relies on the successful delivery of intact and functioning cells. Here, the effect of delivering pericytes on wound repair was assessed alongside the development of a surface-functionalized pericyte patch. Plasma polymerization (PP) was used to functionalize the surface of silicone patches with heptylamine (HA) or acrylic acid (AA) monomers. Human pericytes were subsequently delivered to murine excisional wounds by intradermal injection or using the pericyte-laden patches and the comparative effects on wound healing, inflammation and revascularization determined. The AA surface provided the superior transfer of the cells to de-epidermized dermis. Excisional murine wounds treated either with pericytes injected directly into the wound or with the pericyte-laden AA patches showed improved healing with decreased neutrophil infiltration and reduced numbers of macrophages in the wounds. Pericyte delivery also enhanced angiogenesis through a mechanism independent of VEGF signalling. Pericytes, when delivered to wounds, improved healing responses by dampening inflammation and promoting angiogenesis. Delivery of pericytes using PP-AA-functionalized patches was equally as effective as direct injection of pericytes into wounds. Pericyte-functionalized dressings may therefore be a clinically relevant approach for the treatment of wounds.

Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms.

Healing of the skin and oral mucosa utilises similar mechanisms of tissue repair, however, scarring and the rate of wound closure is vastly superior in the oral cavity suggesting differences between these two environments. One key difference is the phenotype of dermal fibroblasts compared to fibroblasts of gingival tissues. Human gingival fibroblasts (hGFs) are undifferentiated cells with multi-differentiation and self-renewal capacities. This study aimed to examine if delivering hGFs or their secretome, contained in hGF-conditioned media (hGF-CM), would improve healing of the skin and recapitulate features of oral healing. Human fibroblasts, keratinocytes and endothelial cells were first treated with hGF-CM and showed improved migration, proliferation and angiogenic functions. A significant reduction in macroscopic wound area and histologic dermal wound width, as well as an increased rate of re-epithelialisation, were observed in both hGFs and hGF-CM treated murine excisional wounds. This improvement was associated with reduced inflammation, increased angiogenesis and elevated collagen deposition. These findings demonstrate that treatment of dermal wounds with either hGFs or hGF-CM may provide beneficial gingival-like properties to dermal wounds and may be a potential opportunity for improving healing of the skin.

Human multipotent adult progenitor cell-conditioned medium improves wound healing through modulating inflammation and angiogenesis in mice.

BACKGROUND: Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. METHODS: The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice (n = 8/group) and 100 µL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. RESULTS: Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. CONCLUSION: These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.

Crab Shell Extract Improves Sperm Parameters and Antioxidant Status in Testes of Diabetic Rats.

Crab shell (CS), which contains antioxidant compounds, has been used as a promising nutritional and medical compound in traditional medicine. Oxidative stress plays a critical role in the development of diabetes. This study aims to investigate the effects of CS extract (CSE) on sperm parameters, antioxidant status, and histopathology changes of testes in diabetic rats. In this experimental study, 40 Wistar male rats were investigated in five groups (n = 8/group): diabetic rats treated with different CS concentrations (i.e., 100, 200, and 400 mg/kg), diabetic group, and nondiabetic control. To induce diabetes, a single dose (60 mg/kg) of streptozotocin (STZ) was injected intraperitoneally; three days later, treatment with CSE was begun and conducted for 14 days. The fasting blood glucose, testes weight, and viability, number, and motility of sperm were assessed. In addition, the levels of ferric reducing antioxidant power (FRAP) and nitric oxide (NO) were estimated in the testes. Testes were examined using histological analysis. The results of this study revealed that sperm number, motility, and serum testosterone levels of CSE-treated diabetic rats increased significantly (p = .000) compared with the untreated diabetic group in a dose-depended manner while the number of immotile sperm decreased significantly (p = .017). CS also reduced the testicular level of nitric oxide and fasting blood glucose; however, it led to significant growth in the FRAP levels of testes (p = .002). Our results suggest that CSE improves sperm parameters and protects the testicular tissue against the oxidative stress damage induced by diabetes.

Treatment of undifferentiated colorectal cancer cells with fish-oil derived docosahexaenoic acid triggers caspase-3 activation and apoptosis.

BACKGROUND: The effectiveness of chemotherapy is often limited by the side effects on normal tissues. Consequently, the search for new therapeutic agents with minimal toxicity is of particular interest in cancer management. Many studies have shown that docosahexaenoic acid (DHA) have cytotoxic effects against different kinds of cancer cells. However, little attention has been paid to explore the effect of DHA on undifferentiated colorectal cancer cells. In this study, the effects of DHA on LS174T cells as an early stage of tumor initiation were investigated. MATERIALS AND METHODS: Tumor cells were treated to various concentrations of DHA and proliferation, survivin expression, caspase-3 activation, and apoptosis were evaluated by different cellular and molecular techniques. RESULTS: Following 48 h treatment, proliferation was measured to be 73 ± 4.5% (P = 0.000), 53 ± 5.7% (P = 0.000) and 26.3 ± 3.5% (P = 0.000) for 50, 100, and 150 µM DHA, respectively compared to untreated cells. This molecule induced 63% (P = 0.001) and 46% (P = 0.000) decrease in survivin messenger ribonucleic acid (mRNA) level as well as 1.8 (P = 0.001) and three-fold (P = 0.000) increase in caspase-3 activation for 50 and 100 µM DHA, respectively compared to untreated cells. Our evidence showed that survivin mRNA is expressed at the early stage of colorectal cancer cells and DHA-treated cells expressed markedly a lower survivin mRNA compared to untreated cells. CONCLUSIONS: DHA is an attractive repressor of survivin expression, increases caspase-3 and apoptosis in colorectal cancer cells and may provide a novel approach to the treatment of colorectal cancer at the early stage of tumor initiation.

Treatment of LS174T colorectal cancer stem-like cells with n-3 PUFAs induces growth suppression through inhibition of survivin expression and induction of caspase-3 activation.

PURPOSE: Colorectal cancer stem cells (CCSCs) are thought to contribute to tumor initiation, progression, metastasis, chemo-resistance and therapy failure. Therefore, assessment of the effectiveness of agents with anti-proliferative activities against CCSCs is warranted. Several studies have shown that different tumorigenic steps, ranging from initiation to metastasis, can be affected by n-3 polyunsaturated fatty acids (PUFAs). Here, we evaluated the effects of the PUFA components docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), alone or in combination, on LS174T cells that serve as a model for colorectal cancer initiating cells with stem cell-like properties. METHODS: LS174T cells were treated with 50, 100 and 150 µM DHA and EPA, or equal mixtures of DHA/EPA (i.e., 25/25, 50/50 and 75/75 µM), after which cell number, viability, growth inhibition, survivin expression, caspase-3 activation and apoptotic rate were evaluated. RESULTS: We found that treatment of LS174T cells with increasing PUFA concentrations significantly increased growth inhibition in a dose- and time-dependent manner. After a 72 h treatment with 150 µM DHA and EPA, or their combination (75/75 µM), growth rates were inhibited by 80.3 ± 5.5%, 79.3 ± 5% and 71.1 ± 1%, respectively, compared to untreated cells. We also found that treatment for 48 h with 100 µM DHA and EPA, or their combination (50/50 µM), resulted in 2.9-, 3- and 2.6-fold increases in caspase-3 activation, as well as 54, 62.4 and 100% decreases in survivin mRNA expression levels, respectively, compared to untreated cells. Low survivin mRNA levels combined with high caspase-3 activity levels were found to correlate with a higher growth inhibition in PUFA-treated cells. DHA appears to be a more potent growth inhibitor than EPA and the DHA/EPA combination. An increase in the number of apoptotic cells (early + late), ranging from 12.9 to 44.7%, was observed with increasing DHA doses. CONCLUSION: From our data we conclude that PUFAs induce growth inhibition via targeting survivin expression in LS174T cells, which serve as a model for CCSCs.