ADSC-Exos containing MALAT1 promotes wound healing by targeting miR-124 through activating Wnt/ß-catenin pathway.

Cutaneous wound is a soft tissue injury that is difficult to heal during aging. It has been demonstrated that adipose-derived stem cells (ADSCs) and its secreted exosomes exert crucial functions in cutaneous wound healing. The present study aimed to elucidate the mechanism of exosomes derived from ADSCs (ADSC-Exos) containing MALAT1 in wound healing. ADSCs were isolated from human normal subcutaneous adipose tissues and identified by flow cytometry analysis. Exosomes were extracted from ADSC supernatants and MALAT1 expression was determined using qRT-PCR analysis. HaCaT and HDF cells were exposed to hydrogen peroxide (H2O2) for simulating the skin lesion model. Subsequently, CCK-8, flow cytometry, wound healing and transwell assays were employed to validate the role of ADSC-Exos containing MALAT1 in the skin lesion model. Besides, cells were transfected with sh-MALAT1 to verify the protective role of MALAT1 in wound healing. The binding relationship between MALAT1 and miR-124 were measured by dual-luciferase reporter assay. ADSC-Exos promoted cell proliferation, migration, and inhibited cell apoptosis of HaCaT and HDF cells impaired by H2O2. However, the depletion of MALAT1 in ADSC-Exos lose these protective effects on HaCaT and HDF cells. Moreover, miR-124 was identified to be a target of MALAT1. Furthermore, ADSC-Exos containing MALAT1 could mediate H2O2-induced wound healing by targeting miR-124 and activating Wnt/ß-catenin pathway. ADSC-Exos containing MALAT1 play a positive role in cutaneous wound healing possibly via targeting miR-124 through activating the Wnt/ß-catenin pathway, which may provide novel insights into the therapeutic target for cutaneous wound healing.

Silibinin Induced Human Glioblastoma Cell Apoptosis Concomitant with Autophagy through Simultaneous Inhibition of mTOR and YAP.

Silibinin, also known as silybin, is the major flavonolignan isolated from Silybum marianum. Although previous reports demonstrated that silibinin exhibits significant tumor suppressor activities in various cancers by promoting cell apoptosis, it was also shown to trigger autophagy to counteract apoptosis induced by exogenous stresses in several types of cells. However, there is no report to address the role of silibinin induced autophagy in human A172 and SR glioblastoma cells. Our study showed that silibinin treatment not only inhibited the metabolic activities of glioblastoma cells but also promoted their apoptosis through the regulation of caspase 3 and PARP-1 in concentration- and time-dependent manners. Meanwhile, silibinin induced autophagy through upregulation of microtubule-associated protein a light chain 3- (LC3-) II. And autophagy inhibition with chloroquine, a lysosomotropic agent, significantly enhanced silibinin induced glioblastoma cell apoptosis. Moreover, silibinin dose-dependently downregulated the phosphorylation levels of mTOR at Ser-2448, p70S6K at Thr-389, and 4E-BP1 at Thr-37/46. Furthermore, the expression of YAP, the downstream effector of Hippo signal pathway, was also suppressed by silibinin. These results suggested that silibinin induced glioblastoma cell apoptosis concomitant with autophagy which might be due to simultaneous inhibition of mTOR and YAP and silibinin induced autophagy exerted a protective role against cell apoptosis in both A172 and SR cells.

Growth Factor-Reinforced ECM Fabricated from Chemically Hypoxic MSC Sheet with Improved In Vivo Wound Repair Activity.

MSC treatment can promote cutaneous wound repair through multiple mechanisms, and paracrine mediators secreted by MSC are responsible for most of its therapeutic benefits. Recently, MSC sheet composed of live MSCs and their secreted ECMs was reported to promote wound healing; however, whether its ECM alone could accelerate wound closure remained unknown. In this study, Nc-ECM and Cc-ECM were prepared from nonconditioned and CoCl2-conditioned MSC sheets, respectively, and their wound healing properties were evaluated in a mouse model of full-thickness skin defect. Our results showed that Nc-ECM can significantly promote wound repair through early adipocyte recruitment, rapid reepithelialization, enhanced granulation tissue growth, and augmented angiogenesis. Moreover, conditioning of MSC sheet with CoCl2 dramatically enriched its ECM with collagen I, collagen III, TGF-ß1, VEGF, and bFGF via activation of HIF-1α and hence remarkably improved its ECM's in vivo wound healing potency. All the Cc-ECM-treated wounds completely healed on day 7, while Nc-ECM-treated wounds healed about 85.0% ± 8.6%, and no-treatment wounds only healed 69.8% ± 9.6% (p < 0.05). Therefore, we believe that such growth factor-reinforced ECM fabricated from chemically hypoxic MSC sheet has the potential for clinical translation and will lead to a MSC-derived, cost-effective, bankable biomaterial for wound management.

Evaluation of xenogeneic extracellular matrix fabricated from CuCl2-conditioned mesenchymal stem cell sheets as a bioactive wound dressing material.

The extracellular matrix has drawn considerable interest in tissue engineering not only acts as a bioactive three-dimensional scaffold but also regulates cell behaviors through providing biochemical signals. Extracellular matrix-based biomaterials, mainly derived from xenogeneic tissues, have shown positive outcomes in promoting cutaneous wound healing. However, such extracellular matrices only contain low doses of growth factors, which limit their therapeutic efficiency. Recent reports demonstrated that cell sheets made from mesenchymal stem cell can accelerate wound repair through enhanced re-epithelialization and angiogenesis, but its clinical translation is hindered by several limitations, such as the risk of aberrant immune responses and cost implications. In this study, acellular extracellular matrices were prepared from CuCl2-conditioned mesenchymal stem cell sheets and their in vivo wound healing properties were evaluated in a mouse model of full-thickness skin defect. We found that extracellular matrices derived from CuCl2-conditioned mesenchymal stem cell sheets have a compact surface with thick solid-like cross-sectional structure. Moreover, CuCl2 dramatically enriched the extracellular matrices with collagen I, collagen III, transforming growth factor-ß1, vascular endothelial growth factor, and basic fibroblast growth factor via hypoxia-inducible factor-1α activation. And as a consequence, the resulting extracellular matrices showed markedly improved in vivo wound healing potency through early adipocyte mobilization, enhanced granulation tissues formation, rapid re-epithelialization, and augmented angiogenesis. Therefore, we consider that the extracellular matrix derived from CuCl2-conditioned mesenchymal stem cell sheets has the potential for clinical translation and may lead to a novel strategy for wound management.

Histone deacetylase inhibitor reduces hypertrophic scarring in a rabbit ear model.

BACKGROUND: Hypertrophic scars result from excessive collagen deposition at sites of healing dermal wounds and could be functionally and cosmetically problematic. The authors tested the ability of the histone deacetylase inhibitor trichostatin A to reduce hypertrophic scar formation in a rabbit ear model. METHODS: The authors have developed a reliable rabbit model that results in hypertrophic scarring. Four 1-cm, full-thickness, circular wounds were made on each ear. After the wounds reepithelialized, 0.02% trichostatin A was injected intradermally into the wounds in the treatment group. Expression of collagen I and fibronectin was detected by reverse transcription polymerase chain reaction and Western blot analysis at postoperative day 23. Scar hypertrophy was quantified by measurement of the scar elevation index at postoperative day 45. RESULTS: Compared with the control group, injection of trichostatin A led to much more normal-appearing scars in the rabbit ear. The scar elevation index at postoperative day 45 was significantly decreased after injection of trichostatin A compared with untreated scars. Furthermore, the authors confirmed the decreased expression of collagen I and fibronectin at postoperative day 23 (after the rabbits had been treated with trichostatin A for 1 week) in the treated scars compared with the control scars according to reverse transcription polymerase chain reaction and Western blot analysis. CONCLUSIONS: The introduction of trichostatin A can result in the decreased formation of hypertrophic scars in a rabbit ear model, which is corroborated by evidence of decreased collagen I and fibronectin synthesis.

Rolling autogenetic dermis up to form a tube may be used as scaffold in tissue-engineered blood vessels.

Coronary and peripheral artery bypass grafting are widely being used to deal with vascular deficiencies currently, and a man-made synthetic tube or autogenous arteries or veins are needed a lot. But one's autogenous arteries or veins are limited, and artificial graft substitute isn't yet available in clinical applications because of many disadvantages. Various polymeric materials have been used as scaffolds, but without satisfying results due to intimal hyperplasia and the rate of degradation. Autogenetic dermis, which has the advantages of resistance to immunogenicity, good biocompatibility, and appropriate mechanical and physiological properties, has gained our attention to use it as a scaffold for tissue-engineered blood vessels. What is more, autogenetic dermis can be harvested easily. So we postulate that autogenetic dermis rolled up to form a tube may be an ideal scaffold for tissue-engineered blood vessels.

Wedge-shaped excision and modified vertical mattress suture fully buried in a multilayered and tensioned wound closure.

BACKGROUND: A successful deep multilayered wound suture should provide a firm tension-relieving closure, good wound-edge eversion, hemostasis, and minimal intradermal extraneous materials. However, this is not always achieved with a single standard technique. The authors describe their modification of a wound closure method that can rapidly and reliably achieve these results. METHODS: A wedge-shaped excision was adopted to obtain a trapezoid pattern transect, after which a modified fully buried vertical mattress suture technique was used to close the wound. These techniques were compared with the standard excision and suture techniques used for the same patient at different times after surgery. RESULTS: The wedge-shaped excision can facilitate good wound-edge eversion, and the modified fully buried vertical mattress suture can provide firm tension relief and optimal apposition. Compared with conventional excision and suture techniques, the described techniques brought about a better outcome in terms of hypertrophic scar prevention. CONCLUSION: The described modified technique seems to be more efficient than conventional procedures used to prevent hypertrophic scar formation.

Inhibition of tumor angiogenesis by HMGB1 A box peptide.

High mobility group box 1 protein (HMGB1) is a highly conserved, ubiquitous non-histone nuclear protein, which participates in maintaining nucleosome structure, regulation of gene transcription, and modulating the activity of steroid hormone receptors. Substantial evidence demonstrated that HMGB1 could be secreted into the extracellular milieu, acts as a proinflammatory cytokine and mediates the downstream inflammatory responses in endotoxemia, arthritis and sepsis. Recently, several reports suggested that HMGB1 plays a key role in tumor angiogenesis through multiple mechanisms, including up-regulation of proangiogenic factors, promoting endothelial progenitor cells homing to ischemic tumor tissues and induction of endothelial cell migration and sprouting. And blockade of HMGB1 binding to the receptor for advanced glycation end products (RAGE) with anti-HMGB1 antibody, soluble RAGE or anti-RAGE neutralizing antibody has been proved to inhibit angiogenesis efficiently. Since HMGB1 A box peptide could antagonize the HMGB1 whole length protein by competitively binding to RAGE and has been considered as a HMGB1 specific antagonist, we postulate that the HMGB1 A box peptide could function as an anti-angiogenic agent to inhibit tumor angiogenesis. In our opinion, if the hypothesis proved to be practical, HMGB1 A box peptide could be widely used in clinical settings to treat malignant tumors.

Enhancing skin flap survival by a cell-permeable wild-type survivin.

Skin grafts, including skin flaps, are widely used in plastic and reconstructive surgery to cover wounds and tissue defects resulting from mechanic or burn injury. Ischemic necrosis is the main complication in skin graft surgery due to inefficient revascularization. Though the surgical delay procedure has been proved to be the only effective technique to prevent skin flap ischemic necrosis by mechanism of inducing adaption to hypoxia, but it is time consuming, costly, and having high risk of infection due to repeated surgery. Recent research demonstrated that, in addition to protecting cells against apoptosis, the expression of survivin correlates with intratumoral microvessel density in several different types of tumors and survivin could upregulate several proangiogenic factors, including VEGF, Egr-1 and Siah-1. Moreover, Survivin DeltaEx3, one of the survivin alternative splice variants, is necessary for activating the small GTPase Rac1 during endothelial tube formation and required for in vivo endothelial cell invasion. Therefore, we postulate that intracellular delivery of survivin or Survivin DeltaEx3 by fusion with protein transduction domain would enhance skin flap survival through accelerating revascularization by both inhibiting the apoptosis of microvascular endothelial cells and promoting skin flap angiogenesis. If the hypothesis was proved to be practical, the fusion proteins would be widely used in plastic and reconstructive surgery to prevent skin flap from ischemic necrosis in the future.

Cell-permeable hypoxia-inducible factor-1 (HIF-1) antagonists function as tumor radiosensitizers.

Hypoxia is a common phenomenon in human solid tumors and has been considered as an important, independent negative prognostic factor for response to treatment and survival of tumor patients. Hypoxia-inducible factor-1 (HIF-1) is the central transcription factor which is activated by hypoxia and modulates the expression of many genes involved in cell metabolism, proliferation, apoptosis, angiogenesis. Recently, it has been reported that HIF-1 contributes to tumor radioresistance by upregulating survivin expression under hypoxic conditions. Moreover, in hypoxic tumor cells, HIF-1 dependent signal transduction pathway is activated and could be further enhanced by radiation, thereby providing survival signals to adjacent vascular endothelial cells by upregulation of VEGF and bFGF and resulting in tumor radioresistance through vascular radioprotection. Recent research revealed that the stability of HIF-1alpha, one of the two subunits of HIF-1, determines the whole HIF-1 activity and the C-terminal transactivation domain of HIF-1alpha could reduce HIF-1 activity when overexpressed in tumor cells by disruption of the assembly of HIF-1 transcription complex. Therefore, we postulate that fusion with protein transduction domains would overcome the inability of C-terminal transactivation domain of HIF-1alpha to cross cellular membrane. Thus the recombinant fusion proteins could serve as cell-permeable HIF-1 antagonists, function as both inhibitors of tumor angiogenesis and tumor radiosensitizers, and would be widely used in clinical settings to improve tumor response to radiotherapy.

Ex vivo expanding hematopoietic stem cells by intracellular delivery of Cdx4 fusion proteins.

Hematopoietic stem cells transplantation has been wildly used in clinical settings and has shown exciting results in treating a wide variety of diseases. However, the relative small number of hematopoietic stem cells in bone marrow, even lower in peripheral or umbilical cord blood limits its clinical utility. There are several protocols, which have been developed to expand hematopoietic stem cells to reach clinical goal. With the recent insights into the mechanisms of hematopoietic stem cells self-renewal, we postulate that Cdx4, which is coded by one of the caudal related homeobox genes and regulates the expression of hox genes, could fuse with protein transduction domains, thereby get the ability to cross cellular membrane. And the recombinant fusion proteins could be used in expanding hematopoietic stem cells. Meanwhile, Cdx4 fusion proteins would be more efficient than other methods that had been developed for it can up-regulate a cocktail of hox genes, which has been proved to be capable of amplifying hematopoietic stem cells. It would provide us an alternative protocol to amplify hematopoietic stem cells if the hypothesis proved to be practical.

Enhancing tumor radiosensitivity by intracellular delivery of survivin antagonists.

Radiotherapy is frequently applied to control local tumors by mechanisms of direct killing tumor cells and inducing tumor vascular endothelial cells apoptosis. Recently, it has been demonstrated that survivin, an intracellular molecule with anti-apoptotic function, is widely expressed in human malignancies and its expression correlates with radioresistance in several tumors. Moreover, VEGF, which is highly expressed in solid tumors and further up-regulated by irradiation, has been shown to induce survivin expression in both tumor cells and vascular endothelial cells. Thus provide a survival signal to these cells and induce radioresistance to the subsequent irradiation exposure. Knocking down the expression of survivin by RNA interference or transfecting with a gene coding for a dominant negative survivin has been proved to be efficient in enhancing tumor cell radiosensitivity and improving tumor response to radiotherapy. The development of protein transduction technology made it possible to deliver large molecules into mammalian cells. We postulate that dominant negative mutants of survivin could fuse with protein transduction domain and the fusion proteins could cross cellular membranes and generate their biological activity to serve as tumor radiosensilizers. If the hypothesis proved to be practical, it would provide us an alternate method to enhance tumor radiosensitivity and the fusion proteins would be widely applicated in clinical settings because they were safer than gene therapy.

[Experimental study of transplanted endothelial progenitor cells transfected with VEGF165 gene augment the survival volume of transplanted fat tissue].

OBJECTIVE: To investigate the feasibility of transplanting endothelial progenitor cells (EPCs) transfected with VEGF165 gene to free transplanted fat tissue for increasing neovascularization and the survival. METHODS: EPCs isolated from human cord blood were cultured in vitro and identified by immunocytochemistry. After transfection by VEGF165 gene, the expression of VEGF was assessed using ELISA. Then EPCs with (VEGF gene transfection group) and without VEGF165 gene transfection (EPCs group) were transplanted to free transplanted fat tissue at 18 nude mice's back, and nine nude mice transplanted with free fat tissue were injected with M199 (control group). CM-DiI was used to trace the transplanted cells. The capillary density of transplanted fat tissue was detected by CD34 immunohistochemistry. RESULTS: EPCs expressed cell markers CD34, KDR and CD133. After transfection, the expression of VEGF was positive. Transplanted EPCs survived and proliferated, and transplanted EPCs were incorporated into the capillary networks in the transplanted fat tissue. The percent of survival volume of transplanted fat tissue of VEGF gene transfection group was (96.2 +/- 8.6)%, significantly higher than that of the EPCs group [(75.3 +/- 6.8)%, P < 0.05) and M199 group [(40.2 +/- 2.5)%, P < 0.05). The capillary density of transplanted fat tissue of VEGF gene transfection group was significantly higher than those of the EPCs group and M199 group (P < 0.05). CONCLUSIONS: EPCs from human cord blood can increase free transplanted fat tissue neovascularization and the survival volume, and the ability of promoting neovascularization of EPCs transfected with VEGF165 gene is more potent than EPCs alone.

[Promotion of the survival of ischemic skin flap by transplanted endothelial progenitor cells transfected with VEGF165 gene: an experimental study with mice].

OBJECTIVE: To investigate the feasibility of transplanted endothelial progenitor cells transfected with VEGF165 gene to ischemic flap with increased neovascularization and augmented the survival areas. METHODS: EPCs were isolated from human cord blood and cultured in vitro. Plasmid PcDNA3.1(-)/VEGF165 containing VEGF gene was transfected into the EPCs. EPCs transfected with blank plasmid, and EPCs without transfection were used as controls. ELISA was used to detect the expression of VEGF protein in the culture fluids. The EPCs were dyed with CM-DiI 7 days later. Ischemic skin flaps were made on the backs of 27 nude mice. The mice were randomly divided into 3 equal groups with their skin flaps being transplanted with EPCs transfected with 3.1(-)/VEGF165 plasmid, EPCs not transfected with 3.1(-)/VEGF165 plasmid, and injected with M199 medium at the basal part. Four days after the peduncles of the skin flaps were cut. Seven days after the cutting-off of the peduncles the survival rate of skin flap was observed and the blood perfusion was observed with laser Doppler flowmetry, 10 days after the density of capillary arteries were observed with microcirculation microscope. Three specimens of skin flap were taken 7 and 11 days after the skin flaps were made to undergo histological examination to detect the density of capillary arteries by CD34 immunohistochemistry and to observe the proliferation of EPCs with fluorescence microscopy. Peripheral blood samples were collected 1, 4, 7, 14, and 28 days after the skin flaps were made to undergo ELISA to detect the levels of VEGF protein RESULTS: The VEGF levels in the culture supernatants of the groups A, B, and C were 352 ng/L +/- 35 ng/L, 45 ng/L +/- 5 ng/L, and 0 ng/L respectively with significant difference between any 2 groups (all P < 0.05). The skin flap survival rates of the three groups were 97.2%, 60.3%, and 34.2% respectively with significant difference between any 2 groups (all P < 0.05) and the survival quality of the group A was the best. The capillary density of the group A was greater than those of the groups B and C. The VEGF levels at any time point of the group A were all significantly higher than those of the group B and C (all P < 0.05) and there was not a significant difference between the groups B and C. The capillary density levels at different time points decreased progressively in the order of groups A, B, and C with significant difference between any 2 groups (all P < 0.05). No EPC was shown by fluorescence microscopy in the skin flaps of the group C. The EPC density in skin flap 7 and 11 days after the flaps were made were 136 +/- 10 and 75 +/- 6/mm(2) and 305 +/- 26 and 199 +/- 18/mm(2) respectively with significant differences between the groups A and B. (both P < 0.05). CONCLUSION: The EPCs from human cord blood, especially those transfected with VEGF165 gene increases the neovascularization in ischemic skin flaps and augments their survival rate.