Jiongyu Hu

CXCL12/CXCR4 axis promotes mesenchymal stem cell mobilization to burn wounds and contributes to wound repair

BACKGROUND Bone marrow-derived mesenchymal stem cells (BM-MSCs) play a crucial role in tissue repair. Their role in thermal burn wound regeneration and the relevant mechanism, however, is rarely studied. METHODS BM-MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice. Twenty-one days later, the female mice were inflicted with burn wounds. The size of the burned area was measured by an in vivo fluorescence imaging system, and BM-MSC chemotaxis and epithelialization were estimated by fluorescence in situ hybridization and immunofluorescence technology. The expression of CXCL12 and CXCR4 in the wound margin was detected by enzyme-linked immunosorbent assay and immunohistochemistry. The importance of CXCL12/CXCR4 signaling in BM-MSC chemotaxis was further estimated by blocking CXCR4 in vivo and in vitro. RESULTS In vivo imaging results showed that BM-MSCs migrated to the injured margins. Fluorescence in situ hybridization and immunofluorescence technology revealed that Y chromosome-positive cells derived from green fluorescent protein transgenic mice were detected to be colocalized with keratin protein. Enzyme-linked immunosorbent assay revealed increased levels of CXCL12 and CXCR4 protein in the wound sites of BM-MSC-treated chimeric mice after burn. Immunohistochemistry also disclosed that CXCL12 levels were elevated at postburn day 7 compared with day 0. Furthermore, pretreatment of the BM-MSCs with the CXCR4 antagonist AMD3100 significantly inhibited the mobilization of BM-MSCs in vitro and in vivo, which attenuated wound closure. CONCLUSION BM-MSC migration to the burned margins promotes the epithelialization of the wound, and mobilization of BM-MSCs is mediated by CXCL12/CXCR4 signaling.

ASTRAGALOSIDE IV ATTENUATES HYPOXIA-INDUCED CARDIOMYOCYTE DAMAGE IN RATS BY UPREGULATING SUPEROXIDE DISMUTASE-1 LEVELS

1 Astragaloside IV (AST‐IV) is purified from a natural plant product. Previous studies have shown that AST‐IV has anti‐oxidant activity. In the present study, we investigated the effect and mechanism of action AST‐IV on rat cardiomyocytes subjected to hypoxic conditions (up to 12 h). 2 Cardiomyocytes were prepared from neonatal rats and cultured under normoxic or hypoxic conditions in the absence or presence of AST‐IV (12.5, 25 or 50 µg/mL). Cell viability, malondialdehyde (MDA) levels, activity and expression of superoxide dismutase (SOD)‐1 (mRNA and protein levels determined by reverse transcription–polymerase chain reaction and western blotting, respectively) and reactive oxygen species (ROS; determined by 2′,7′‐dichlorodihydrofluorescein diacetate) were investigated under these culture conditions. Intracellular localization of AST‐IV was tested using fluorescein isothiocyanate‐labelled AST‐IV. 3 Hypoxic culture reduced the viability of cardiomyocytes, which was improved following treatment with 25 or 50 µg/mL AST‐IV. Under hypoxic conditions, MDA levels were double those under control conditions. Astragaloside IV (25 and 50 µg/mL) dose‐dependently reduced the increase in MDA seen in hypoxic cardiomyocytes. 4 Fluorescein isothiocyanate‐labelled AST‐IV entered cardiomyocytes and was localized mainly within the cytoplasm. 5 Under hypoxic conditions, SOD‐1 activity was decreased, but mRNA and protein expression increased, compared with normoxia. Following treatment with 25 µg/mL AST‐IV, SOD‐1 activity and expression were increased under both normoxic and hypoxic conditions. The ROS scavenging effect of AST‐IV was abolished in the presence of the SOD inhibitor sodium diethyl dithiocarbamate (25 µmol/L). 6 These in vitro results show that AST‐IV protects cardiomyocytes from oxidative stress‐mediated injury under hypoxic conditions. A major part of this action is achieved by upregulation of SOD‐1 content and activity within the cell cytoplasm.

The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells

In both cardiomyocytes and HeLa cells, hypoxia (1% O2) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.

miR-27b Represses Migration of Mouse MSCs to Burned Margins and Prolongs Wound Repair through Silencing SDF-1a

Background Interactions between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXCR4 are crucial for the recruitment of mesenchymal stem cells (MSCs) from bone marrow (BM) reservoirs to damaged tissues for repair during alarm situations. MicroRNAs are differentially expressed in stem cell niches, suggesting a specialized role in stem cell regulation. Here, we gain insight into the molecular mechanisms involved in regulating SDF-1α. Methods MSCs from green fluorescent protein transgenic male mice were transfused to irradiated recipient female C57BL/6 mice, and skin burn model of bone marrow-chimeric mice were constructed. Six miRNAs with differential expression in burned murine skin tissue compared to normal skin tissue were identified using microarrays and bioinformatics. The expression of miR-27b and SDF-1α was examined in burned murine skin tissue using quantitative real-time PCR (qPCR) and immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA). The Correlation of miR-27b and SDF-1α expression was analyzed by Pearson analysis Correlation. miRNAs suppressed SDF-1α protein expression by binding directly to its 3′UTR using western blot and luciferase reporter assay. The importance of miRNAs in MSCs chemotaxis was further estimated by decreasing SDF-1α in vivo and in vitro. Results miR-23a, miR-27a and miR-27b expression was significantly lower in the burned skin than in the normal skin (p<0.05). We also found that several miRNAs suppressed SDF-1α protein expression, while just miR-27a and miR-27b directly bound to the SDF-1α 3′UTR. Moreover, the forced over-expression of miR-27a and miR-27b significantly reduced the directional migration of mMSCs in vitro. However, only miR-27b in burn wound margins significantly inhibited the mobilization of MSCs to the epidermis. Conclusion miR-27b may be a unique signature of the stem cell niche in burned mouse skin and can suppress the directional migration of mMSCs by targeting SDF-1α by binding directly to its 3′UTR.

P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury

Excessive activation of inflammation and the accompanying lung vascular endothelial barrier disruption are primary pathogenic features of acute lung injury (ALI). Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures. However, both the involvement and exact mechanism of MAP4 in the development of endothelial barrier disruption in ALI remains unknown. In this study, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) were applied to human pulmonary microvascular endothelial cells (HPMECs) to mimic the endothelial damage during inflammation in vitro. We demonstrated that the MAP4 (Ser696 and Ser787) phosphorylation increased concomitantly with the p38/MAPK pathway activation by the LPS and TNF-α stimulation of HPMECs, which induced MT disassembly followed by hyperpermeability. Moreover, the application of taxol, the overexpression of a MAP4 (Ala) mutant, or the application of the p38/MAPK inhibitor SB203580 inhibited the MT disruption and the intracellular junction dysfunction. In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability. Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.

p38 MAP kinase mediates burn serum-induced endothelial barrier dysfunction: involvement of F-actin rearrangement and L-caldesmon phosphorylation.

The aim of this study was to test the hypothesis that circulating factors released after a severe burn cause endothelial barrier dysfunction by triggering endothelial cell (EC) contraction through a p38 mitogen-activated protein (MAP) kinase-dependent mechanism. Human umbilical vein ECs (ECV304 cell line) were cultured to create a monolayer of cells that were then cultured with 20% human normal or burn serum. Monolayer permeability was measured by the influx of labeled albumin across the cells. Endothelial cells contraction was determined by alterations of cell surface area and formation of intracellular gaps. P38 MAP kinase activation, F-actin arrangement, and L-caldesmon phosphorylation were assessed by Western blots or immunofluorescence staining. These studies showed that exposure to burn serum resulted in a significant increase in endothelial permeability in a time-dependent manner, which was paralleled by a rapid and persistent activation of p38 MAP kinases. Morphologically, increased intercellular gaps, reduced cell surface area, and a unique rearrangement of F-actin cytoskeleton were observed in burn serum-treated ECs. Inhibition of p38 MAP kinase suppressed the rearrangement of F-actin cytoskeleton, reduced the occurrence of burn serum-induced formation of intercellular gaps, and ameliorated endothelial hyperpermeability. Further study showed that phosphorylation of L-caldesmon was enhanced in burn serum-treated cells via p38 MAP kinase; overexpression of L-caldesmon by adenovirus transfection, however, attenuated the increase in endothelial permeability by burn serum challenge. Collectively, these results have demonstrated for the first time that p38 MAP kinase is an important participant in mediating burn serum-induced endothelial barrier dysfunction through rearrangement of the F-actin cytoskeleton and phosphorylation of L-caldesmon. Inhibition of p38 MAP kinase in vivo, thus, would be a promising therapeutic strategy in ameliorating burn shock development.

MAP4 Mechanism that Stabilizes Mitochondrial Permeability Transition in Hypoxia: Microtubule Enhancement and DYNLT1 Interaction with VDAC1

Mitochondrial membrane permeability has received considerable attention recently because of its key role in apoptosis and necrosis induced by physiological events such as hypoxia. The manner in which mitochondria interact with other molecules to regulate mitochondrial permeability and cell destiny remains elusive. Previously we verified that hypoxia-induced phosphorylation of microtubule-associated protein 4 (MAP4) could lead to microtubules (MTs) disruption. In this study, we established the hypoxic (1% O2) cell models of rat cardiomyocytes, H9c2 and HeLa cells to further test MAP4 function. We demonstrated that increase in the pool of MAP4 could promote the stabilization of MT networks by increasing the synthesis and polymerization of tubulin in hypoxia. Results showed MAP4 overexpression could enhance cell viability and ATP content under hypoxic conditions. Subsequently we employed a yeast two-hybrid system to tag a protein interacting with mitochondria, dynein light chain Tctex-type 1 (DYNLT1), by hVDAC1 bait. We confirmed that DYNLT1 had protein-protein interactions with voltage-dependent anion channel 1 (VDAC1) using co-immunoprecipitation; and immunofluorescence technique showed that DYNLT1 was closely associated with MTs and VDAC1. Furthermore, DYNLT1 interactions with MAP4 were explored using a knockdown technique. We thus propose two possible mechanisms triggered by MAP4: (1) stabilization of MT networks, (2) DYNLT1 modulation, which is connected with VDAC1, and inhibition of hypoxia-induced mitochondrial permeabilization.

Pigment epithelium-derived factor regulates microvascular permeability through adipose triglyceride lipase in sepsis.

The integrity of the vascular barrier, which is essential to blood vessel homoeostasis, can be disrupted by a variety of soluble permeability factors during sepsis. Pigment epithelium-derived factor (PEDF), a potent endogenous anti-angiogenic molecule, is significantly increased in sepsis, but its role in endothelial dysfunction has not been defined. To assess the role of PEDF in the vasculature, we evaluated the effects of exogenous PEDF in vivo using a mouse model of cecal ligation and puncture (CLP)-induced sepsis and in vitro using human dermal microvascular endothelial cells (HDMECs). In addition, PEDF was inhibited using a PEDF-monoclonal antibody (PEDF-mAb) or recombinant lentivirus vectors targeting PEDF receptors, including adipose triglyceride lipase (ATGL) and laminin receptor (LR). Our results showed that exogenous PEDF induced vascular hyperpermeability, as measured by extravasation of Evans Blue (EB), dextran and microspheres in the skin, blood, trachea and cremaster muscle, both in a normal state and under conditions of sepsis. In control and LR-shRNA-treated HDMECs, PEDF alone or in combination with inflammatory mediators resulted in activation of RhoA, which was accompanied by actin rearrangement and disassembly of intercellular junctions, impairing endothelial barrier function. But in ATGL-shRNA-treated HDMECs, PEDF failed to induce the aforementioned alterations, suggesting that PEDF-induced hyperpermeability was mediated through the ATGL receptor. These results reveal a novel role for PEDF as a potential vasoactive substance in septic vascular hyperpermeability. Furthermore, our results suggest that PEDF and ATGL may serve as therapeutic targets for managing vascular hyperpermeability in sepsis.

Identification of mitochondria translation elongation factor Tu as a contributor to oxidative damage of postburn myocardium

Mitochondrial damage plays an important role in mediating postburn cardiac injury. To elucidate the pivotal mitochondrial proteins and pathways underlying postburn cardiac injury, mitochondria were purified from control and postburn rat hearts. 2-dimensional gel electrophoresis (2-DE) and HPLC-chip-MS/MS analyses revealed 9 differentially expressed proteins, 3 of which were further validated by Western blotting. The differential expression of these mitochondrial proteins was accompanied by increased levels of oxidative cardiac damage and decreased levels of cardiac output. One of the differentially expressed proteins, mitochondria translation elongation factor Tu (EF-Tumt), was hypothesized to contribute crucially to postburn oxidative cardiac damage. The small interfering RNA (siRNA)-mediated downregulation of EF-Tumt in cultured rat cardiomyocytes increased reactive oxygen species (ROS) generation and protein carbonyl levels, and led to cell damage. The potential pathway of this process was associated with respiratory chain complex I deficiency. Together, these results demonstrate the mitochondrial responses to severe burn, and indicate a pathway by which decreased EF-Tumt expression mediates oxidative damage in postburn myocardium.

Phosphorylation of DYNLT1 at serine 82 regulates microtubule stability and mitochondrial permeabilization in hypoxia

Hypoxia-induced microtubule disruption and mitochondrial permeability transition (mPT) are crucial events leading to fatal cell damage and recent studies showed that microtubules (MTs) are involved in the modulation of mitochondrial function. Dynein light chain Tctex-type 1 (DYNLT1) is thought to be associated with MTs and mitochondria. Previously we demonstrated that DYNLT1 knockdown aggravates hypoxia-induced mitochondrial permeabilization, which indicates a role of DYNLT1 in hypoxic cytoprotection. But the underlying regulatory mechanism of DYNLT1 remains illusive. Here we aimed to investigate the phosphorylation alteration of DYNLT1 at serine 82 (S82) in hypoxia (1% O2). We therefore constructed recombinant adenoviruses to generate S82E and S82A mutants, used to transfect H9c2 and HeLa cell lines. Development of hypoxia-induced mPT (MMP examining, Cyt c release and mPT pore opening assay), hypoxic energy metabolism (cellular viability and ATP quantification), and stability of MTs were examined. Our results showed that phosph-S82 (S82-P) expression was increased in early hypoxia; S82E mutation (phosphomimic) aggravated mitochondrial damage, elevated the free tubulin in cytoplasm and decreased the cellular viability; S82A mutation (dephosphomimic) seemed to diminish the hypoxia-induced injury. These data suggest that DYNLT1 phosphorylation at S82 is involved in MTs and mitochondria regulation, and their interaction and cooperation contribute to the cellular hypoxic tolerance. Thus, we provide new insights into a DYNLT1 mechanism in stabilizing MTs and mitochondria, and propose a potential therapeutic target for hypoxia cytoprotective studies.