Plant-derived human collagen scaffolds for skin tissue engineering.

Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission.

Cutaneous wound healing after treatment with plant-derived human recombinant collagen flowable gel.

Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds.

MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue.

AIMS: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Encoded in the genome of most eukaryotes, miRNAs have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. For the first time, we sought to test how myocardial ischaemia-reperfusion (IR) changes miR expression. METHODS AND RESULTS: Following 2 and 7 h of IR or sham operation, myocardial tissue was collected and subjected to miRNA expression profiling and quantification using a Bioarray system that screens for human-, mice-, rat-, and Ambi-miR. Data mining and differential analyses resulted in 13 miRs that were up-regulated on day 2, 9 miRs that were up-regulated on day 7, and 6 miRs that were down-regulated on day 7 post-IR. Results randomly selected from expression profiling were validated using real-time PCR. Tissue elements laser-captured from the infarct site showed marked induction of miR-21. In situ hybridization studies using locked nucleic acid miR-21-specific probe identified that IR-inducible miR-21 was specifically localized in the infarct region of the IR heart. Immunohistochemistry data show that cardiac fibroblasts (CFs) are the major cell type in the infarct zone. Studies with isolated CFs demonstrated that phosphatase and tensin homologue (PTEN) is a direct target of miR-21. Modulation of miR-21 regulated expression of matrix metalloprotease-2 (MMP-2) via a PTEN pathway. Finally, we noted a marked decrease in PTEN expression in the infarct zone. This decrease was associated with increased MMP-2 expression in the infarct area. CONCLUSION: This work constitutes the first report describing changes in miR expression in response to IR in the mouse heart, showing that miR-21 regulates MMP-2 expression in CFs of the infarct zone via a PTEN pathway.

Evidence for the involvement of miRNA in redox regulated angiogenic response of human microvascular endothelial cells.

OBJECTIVE: A Dicer knockdown approach was used to test the significance of miRNA in regulating the redox state and angiogenic response of human microvascular endothelial cells (HMECs). METHODS AND RESULTS: Lowering of miRNA content by Dicer knockdown induced vascular endothelial growth factor expression but diminished the angiogenic response of HMECs as determined by cell migration and Matrigel tube formation. Such impairment of angiogenic response in the Matrigel was rescued by exogenous low micromolar H2O2. Dicer knockdown HMECs demonstrated lower inducible production of reactive oxygen species (ROS) when activated with either phorbol ester, tumor necrosis factor-alpha, or vascular endothelial growth factor. Limiting the production of ROS by antioxidant treatment or NADPH oxidase knockdown approaches impaired angiogenic responses. Experiments to identify how ROS production is limited by Dicer knockdown identified lower expression of p47phox protein in these cells. This lowering of cellular miRNA content induced expression of the transcription factor HBP1, a suppressor transcription factor that negatively regulates p47phox expression. Knockdown of HBP1 restored the angiogenic response of miRNA-deficient HMECs. CONCLUSIONS: This study provides the first evidence that redox signaling in cells is subject to regulation by miRNA. Specifically, p47phox of the NADPH oxidase complex has been identified as one target that regulates the angiogenic properties of endothelial cells.

Selenium supplementation increases liver MnSOD expression: molecular mechanism for hepato-protection.

Selenium is recognized as essential in animal and human nutrition. Several hypotheses have been advanced for its biological activity. The aim of this study was to investigate the in vivo effect of selenium on rat liver manganese superoxide dismutase (MnSOD), a key antioxidant enzyme, under naïve and inflammatory conditions. Rats received sodium selenite supplementation and LPS injection. Whole-liver samples, isolated hepatocytes, Kupffer cells and blood samples were subjected to protein, RNA and biochemical analysis. Liver enrichment with selenium increased whole-liver MnSOD levels due to an increase in MnSOD transcription in hepatocytes. This was due to an increase in the ratio of specificity protein 1 to activating enhancer binding protein 2 DNA-binding activity. The inflammatory stimulus further elevated MnSOD levels in the whole-liver that was abrogated in sodium selenite supplementation due to reduced transcription of MnSOD in Kupffer cells. Moreover, selenium enrichment decreased Kupffer cells IL-6 transcription in LPS-injected animals. Anti-inflammatory activity of selenium was demonstrated by normalized blood levels of ALT and IL-6 in LPS-injected animals. In conclusion, selenium up-regulates hepatocytes MnSOD expression, probably improving their anti-oxidant defense, while decreasing MnSOD and IL-6 transcription in Kupffer cells in the presence of inflammatory stimuli, attenuating their inflammatory response. This selective mechanism may explain the anti-inflammatory and hepato-protective effect of selenium.

MicroRNA in cutaneous wound healing: a new paradigm.

Repair of a defect in the human skin is a highly orchestrated physiological process involving numerous factors that act in a temporally resolved synergistic manner to re-establish barrier function by regenerating new skin. The inducible expression and repression of genes represents a key component of this regenerative process. MicroRNAs (miRNAs) are approximately 22-nucleotide-long endogenously expressed non-coding RNAs that regulate the expression of gene products by inhibition of translation and/or transcription in animals. miRNAs play a key role in skin morphogenesis and in regulating angiogenesis. The vascular endothelial growth factor signaling path seems to be under repressor control by miRNAs. Mature miRNA-dependent mechanisms impair angiogenesis in vivo. It is critically important to recognize that the understanding of cutaneous wound healing is incomplete without appreciating the functional significance of wound-induced miRNA. Ongoing work in our laboratory has led to the observation that the cutaneous wound healing process involves changes in the expression of specific miRNA at specific phases of wound healing. We hypothesize that dysregulation of specific miRNA is critical in derailing the healing sequence in chronic problem wounds. If tested positive, this hypothesis is likely to lead to completely novel diagnostic and therapeutic strategies for the treatment of problem wounds.

Selenium attenuates expression of MnSOD and uncoupling protein 2 in J774.2 macrophages: molecular mechanism for its cell-death and antiinflammatory activity.

Selenium can activate cell death. However, the mechanism of action is not yet fully defined. We hypothesized that selenium may impede mitochondrial superoxide dismutation to H2O2 and O2, leading to cell death in macrophages and that this effect may be relevant to antiinflammatory treatment by selenium. In this study, the mechanism of action of selenium was investigated in nonactivated and activated (immune-stimulated) J774.2 macrophages. Sodium selenite treatment decreased dichlorodihydrofluorescein-reacting intracellular reactive oxygen species (ROS) (mainly peroxides and hydroxyl radicals), with no correlation to glutathione peroxidase activity. However, selenite decreased the transcription and expression of manganese superoxide dismutase (MnSOD) and uncoupling protein 2 (UCP2). This cellular effect was due to inhibition of specificity protein-1 (Sp1) binding to its DNA binding site. Following immune stimulation of macrophages using lipopolysaccharides plus interferon-gamma, MnSOD was up-regulated. Activated macrophages showed higher mitochondrial membrane potential, intracellular ROS levels, and cellular resistance to cell death. Selenite treatment attenuated all of these parameters. Selenite prevented nuclear factor-kappaB (NF-kappaB) activation as a mechanism of its inhibitory activity on MnSOD expression in the immune-stimulated cells. In addition, overexpression of human MnSOD protected against death induced by selenite treatment. It is therefore concluded that selenium at high nanomolar to low micromolar concentrations shifts the balance between inflammatory response and cell death toward the latter, through a direct effect on the transcription factors Sp1 and NF-kappaB, and down-regulation of MnSOD and UCP2.

Selenite activates caspase-independent necrotic cell death in Jurkat T cells and J774.2 macrophages by affecting mitochondrial oxidant generation.

Sodium selenite, a common dietary form of selenium, is recognized as essential in animal and human nutrition. Mechanisms regulating the inflammatory response of the immune system involve regulation of apoptosis and control of reactive oxygen species (ROS) production. In this study, the effect of sodium selenite on ROS production and cell-death rates in macrophages and T cells was investigated. Exposing Jurkat T cells or J774.2 macrophages to >5 micro M sodium selenite induced cell death. In both Jurkat T cells and J774.2 macrophages, rapid loss of the cell's capacity to generate dichlorofluorescein-sensitive ROS preceded cell death. The main cellular source of ROS was found to be the mitochondria electron-transfer chain. DEVDase activity in the cells remained unchanged and even decreased with time, as well as DNA fragmentation level, which was almost unaffected, indicating cell death with necrotic characteristics. tert-Butyl hydroperoxide at a concentration of 5 micro M was beneficial in attenuating the rate of cell death. The superoxide scavenger Tiron was tested for its ability to protect the cells against selenium. Tiron completely protected the J774.2 macrophage cell line against selenium and attenuated the cell death effect in Jurkat T cells. In the presence of the superoxide dismutase-mimicking compound tempol, selenium's macrophage-killing effect was inhibited. Therefore, our results show that, at least in vitro, selenite induces changes in the balance between mitochondrial superoxide and hydrogen peroxide production, which can facilitate cell death in immune system cells. This may be one mechanism by which selenium down-regulates the immune system's inflammatory response and protects against overproduction of peroxides.

Selenite sensitizes mitochondrial permeability transition pore opening in vitro and in vivo: a possible mechanism for chemo-protection.

There is a known connection between selenium supplementation and chemo-protective anti-cancer activity. This biological phenomenon may be due to the ability of selenium to instigate cellular apoptosis. However, the mechanism by which selenium promotes cellular apoptosis is still obscure. The present study shows that sodium selenite, a common dietary form of selenium, promotes the mitochondrial permeability transition (MPT) in isolated rat liver mitochondria both in vitro and following in vivo supplementation. A low selenium concentration (0.1-10 microM) strongly induced cyclosporin A-sensitive mitochondrial swelling. Selenium also promoted both calcium release from the matrix of isolated mitochondria and uncoupled respiration. The MPT-inducing effect of selenium provoked the release of cytochrome c, a pro-apoptotic factor, into the incubation medium. Selenium did not increase intra-mitochondrial peroxide production, but did consume endogenous mitochondrial glutathione. Moreover, the effect of MPT induction was greatly potentiated in the presence of thiol-bearing antioxidants, e.g. N -acetylcysteine and lipoamide. During MPT progression, selenium induced NADH oxidation via electron acceptance from complex I. Supplementation for 20 days with 16 p.p.m. selenium in the drinking water of rats increased the propensity of mitochondria to undergo the MPT. More marked mitochondrial swelling in response to calcium and lower calcium-uptake capacity were observed, in the absence of liver damage or the intensive oxidation of reduced glutathione. Therefore selenite facilitates MPT pore opening via its thiol- and NADH/complex I-dependent reduction, and thereby may provide chemo-protection by potentiation of the capacity of the mitochondria to regulate programmed cell death. Data from the present study suggest that selenium can regulate important mitochondrial functions both in vivo and in vitro.

Membrane depolarization of isolated rat liver mitochondria attenuates permeability transition pore opening and oxidant production.

It has been suggested that one key feature of mitochondrial permeability transition (PT) regulation is its control by the proton electrochemical gradient and that depolarization favors pore opening, swelling, and reactive oxygen species (ROS) production. Moreover, ROS have been suggested to facilitate the process of mitochondrial PT pore opening. The aim of this study was to show that collapsing the mitochondrial membrane potential with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), at concentrations of up to 10 microM, does not induce mitochondrial swelling and, in fact, stabilizes mitochondria exposed to oxidant, protecting them from tert-butyl hydroperoxide (TBH)-induced high-amplitude swelling. FCCP decreased polyethylene glycol-induced mitochondrial contraction following exposure to TBH, indicating closing of the PT mega-channel. In the presence of the calcium uniporter inhibitor ruthenium red, FCCP induced PT due to suppression of calcium efflux. Under PT-favorable conditions, ROS production was evaluated in mitochondria following treatments with TBH, inorganic phosphate, or FCCP (with or without ruthenium red). FCCP alone and in combination with ruthenium red attenuated mitochondria-derived ROS production. FCCP also decreased the augmented ROS production induced by inorganic phosphate. It is concluded that mitochondrial depolarization protects and prevents high-amplitude swelling and PT-derived ROS production.