Xiaohua Shi

Mitochondrial chaperone tumour necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxic injury by regulating mitochondrial permeability transition pore opening

Tumour necrosis factor receptor‐associated protein 1 (TRAP1) is a mitochondrial chaperone that plays a role in maintaining mitochondrial function and regulating cell apoptosis. The opening of the mitochondrial permeability transition pore (MPTP) is a key step in cell death after hypoxia. However, it is still unclear whether TRAP1 protects cardiomyocytes from hypoxic damage by regulating the opening of the pore. In the present study, primary cultured cardiomyocytes from neonatal rats were used to investigate changes in TRAP1 expression after hypoxia treatment as well as the mechanism and effect of TRAP1 on hypoxic damage. The results obtained showed that TRAP1 expression increased after 1 h of hypoxia and continued to increase for up to 12 h of treatment. Hypoxia caused an increase in cell death and decreased cell viability and mitochondrial membrane potential; overexpressing TRAP1 prevented hypoxia‐induced damage to cardiomyocytes. The silencing of TRAP1 induced an increase in cell death and decreased both cell viability and mitochondrial membrane potential in cardiomyocytes under normoxic and hypoxic conditions. Furthermore, cell damage induced by the silencing of TRAP1 was prevented by the mitochondrial permeability transition pore inhibitor, cyclosporin A. These data demonstrate that hypoxia induces an increase in TRAP1 expression in cardiomyocytes, and that TRAP1 plays a protective role by regulating the opening of the mitochondrial permeability transition pore.

Requirement of Gαi1/3-Gab1 signaling complex for keratinocyte growth factor-induced PI3K-AKT-mTORC1 activation

Keratinocyte growth factor (KGF), also termed as fibroblast growth factor-7, promotes proliferation, migration, and adhesion of skin keratinocytes via binding to keratinocyte growth factor receptor (KGFR) and subsequent activation of downstream signaling including the PI3K-AKT-mTORC1 pathway. Here, we found that the α-subunits of the G proteins (Gαi1/3) and growth factor receptor binding 2-associated binding protein 1 (Gab1) are required for this activation process. With KGF stimulation, Gαi1/3 formed a complex with KGFR and was required for subsequent Gab1 recruitment, phosphorylation, and following PI3K-p85 activation. In addition, Gαi1/3 short hairpin RNA knockdown largely inhibited KGF-induced cell proliferation, migration, and the accumulation of cyclin D1/fibronectin in cultured skin keratinocytes. Furthermore, we observed increased expression of Gαi1/3 in wounded human skin and keloid skin tissues, suggesting the possible involvement of Gαi1/3 in wound healing and keloid formation. Overall, we suggest that Gαi1/3 proteins lie downstream of KGFR, but upstream of Gab1-mediated activation of PI3K-AKT-mTORC1 signaling, thus revealing a role for Gαi proteins in mediating KGFR signaling, cell migration, and possible wound healing.

Myocardial Autophagy after Severe Burn in Rats

Background Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn. Methods Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function. Results Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function. Conclusion Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction.

Activation of the prolyl‐hydroxylase oxygen‐sensing signal cascade leads to AMPK activation in cardiomyocytes

The proline hydroxylase domain‐containing enzymes (PHD) act as cellular oxygen sensors and initiate a hypoxic signal cascade to induce a range of cellular responses to hypoxia especially in the aspect of energy and metabolic homeostasis regulation. AMP‐activated protein kinase (AMPK) is recognized as a major energetic sensor and regulator of cardiac metabolism. However, the effect of PHD signal on AMPK has never been studied before. A PHD inhibitor (PHI), dimethyloxalylglycine and PHD2‐specific RNA interference (RNAi) have been used to activate PHD signalling in neonatal rat cardiomyocytes. Both PHI and PHD2‐RNAi activated AMPK pathway in cardiomyocytes effectively. In addition, the increased glucose uptake during normoxia and enhanced myocyte viability during hypoxia induced by PHI pretreatment were abrogated substantially upon AMPK inhibition with an adenoviral vector expressing a dominant negative mutant of AMPK‐α1. Furthermore, chelation of intracellular Ca2+ by BAPTA, inhibition of calmodulin‐dependent kinase kinase (CaMKK) with STO‐609, or RNAi‐mediated down‐regulation of CaMKK α inhibited PHI‐induced AMPK activation significantly. In contrast, down‐regulation of LKB1 with adenoviruses expressing the dominant negative form did not affect PHI‐induced AMPK activation. We establish for the first time that activation of PHD signal cascade can activate AMPK pathway mainly through a Ca2+/CaMKK‐dependent mechanism in cardiomyocytes. Furthermore, activation of AMPK plays an essential role in hypoxic protective responses induced by PHI.

Osteopontin (OPN) Is an Important Protein to Mediate Improvements in the Biocompatibility of C Ion-Implanted Silicone Rubber

Medical device implants are drawing increasing amounts of interest from modern medical practitioners. However, this attention is not evenly spread across all such devices; most of these implantable devices can cause adverse reactions such as inflammation, fibrosis, thrombosis, and infection. In this work, the biocompatibility of silicone rubber (SR) was improved through carbon (C) ion implantation. Scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) results confirmed that these newly generated carbon-implanted silicone rubbers (C-SRs) had large, irregular peaks and deep valleys on their surfaces. The water contact angle of the SR surface decreased significantly after C ion implantation. C ion implantation also changed the surface charge distribution, silicone oxygen rate, and chemical-element distribution of SR to favor cell attachment. The dermal fibroblasts cultured on the surface C-SR grew faster and showed more typical fibroblastic shapes. The expression levels of major adhesion proteins, including talin-1, zyxin, and vinculin, were significantly higher in dermal fibroblasts cultured on C-SR coated plates than in dermal fibroblasts cultured on SR. Those same dermal fibroblasts on C-SRs showed more pronounced adhesion and migration abilities. Osteopontin (OPN), a critical extracellular matrix (ECM) protein, was up-regulated and secreted from dermal fibroblasts cultured on C-SR. Matrix metalloproteinase-9 (MMP-9) activity was also increased. These cells were highly mobile and were able to adhere to surfaces, but these abilities were inhibited by the monoclonal antibody against OPN, or by shRNA-mediated MMP-9 knockdown. Together, these results suggest that C ion implantation significantly improves SR biocompatibility, and that OPN is important to promote cell adhesion to the C-SR surface.

Biofunctionalization of silicone rubber with microgroove-patterned surface and carbon-ion implantation to enhance biocompatibility and reduce capsule formation

Purpose Silicone rubber implants have been widely used to repair soft tissue defects and deformities. However, poor biocompatibility can elicit capsule formation, usually resulting in prosthesis contracture and displacement in long-term usage. To overcome this problem, this study investigated the properties of silicone rubber materials with or without a microgroove-patterned surface and with or without carbon (C)-ion implantation. Materials and methods Atomic force microscopy, X-ray photoelectron spectroscopy, and a water contact angle test were used to characterize surface morphology and physicochemical properties. Cytocompatibility was investigated by a cell adhesion experiment, immunofluorescence staining, a Cell Counting Kit-8 assay, and scanning electron microscopy in vitro. Histocompatibility was evaluated by studying the inflammatory response and fiber capsule formation that developed after subcutaneous implantation in rats for 7 days, 15 days, and 30 days in vivo. Results Parallel microgrooves were found on the surfaces of patterned silicone rubber (P-SR) and patterned C-ion-implanted silicone rubber (PC-SR). Irregular larger peaks and deeper valleys were present on the surface of silicone rubber implanted with C ions (C-SR). The silicone rubber surfaces with microgroove patterns had stable physical and chemical properties and exhibited moderate hydrophobicity. PC-SR exhibited moderately increased dermal fibroblast cell adhesion and growth, and its surface microstructure promoted orderly cell growth. Histocompatibility experiments on animals showed that both the anti-inflammatory and antifibrosis properties of PC-SR were slightly better than those of the other materials, and there was also a lower capsular contracture rate and less collagen deposition around implants made from PC-SR. Conclusion Although the surface chemical properties, dermal fibroblast cell growth, and cell adhesion were not changed by microgroove pattern modification, a more orderly cell arrangement was obtained, leading to enhanced biocompatibility and reduced capsule formation. Thus, this approach to the modification of silicone rubber, in combination with C-ion implantation, should be considered for further investigation and application.

Effect of microtubule-associated protein-4 on epidermal cell migration under different oxygen concentrations

After skin trauma, regional epidermal cell migration mediates the re‐epithelialization of the wound surface, which is an important step for wound healing, yet the underlying molecular regulatory mechanism is unclear. In the current study, HaCaT cells were maintained under different oxygen concentrations (1%, 21%, 40% and 65%). Technologies including immunofluorescence staining, wound scratch, transwell invasion, western blot and low‐expression lentiviral vector were utilized to observe the changes in microtubule dynamics and the microtubule‐associated protein (MAP)4 expression. MAP4s effect on cell migration under different oxygen concentrations was also studied. The results showed that under hyperoxic (40% and 65%) and hypoxic (1%) conditions, HaCaT cells were able to regulate cell microtubule dynamics by MAP4, thus promoting cell migration. On the other hand, MAP4 silencing through targeted shRNA attenuated HaCaT cell migration under the above oxygen concentrations. These results imply that MAP4 plays an important role in epidermal cell migration under different oxygen concentrations.

Carbon Ion Implantation: A Good Method to Enhance the Biocompatibility of Silicone Rubber.

Background: Silicone rubber and silicone rubber–based materials have been used as medical tissue implants in the field of plastic surgery for many years, but there are still some reports of adverse reactions to long-term implants. Earlier studies have shown that ion implantation could enhance the biocompatibility of biomaterials. However, whether ion implantation has a good effect on silicone rubber is unknown. Methods: Three types of carbon ion silicone rubber were obtained by implanting three doses of carbon ions. Then, the antibacterial adhesion properties and the in vivo host responses were evaluated. The antibacterial adhesion properties were examined by plate colony counting, fluorescence staining, and scanning electron microscopic observation. The host responses were evaluated by surveying inflammation and fiber capsule formation that developed after subcutaneous implantation in Sprague-Dawley rats for 7, 30, 90, and 180 days. In addition, the possible mechanism by which ion implantation enhanced the biocompatibility of the biomaterial was investigated and discussed. Results: Carbon ion silicone rubber exhibits less bacterial adhesion, less collagen deposition, and thinner and weaker tissue capsules. Immunohistochemical staining results for CD4, tumor necrosis factor-&agr;, &agr;-smooth muscle actin, and elastin showed the possible mechanism enhancing the biocompatibility of silicone rubber. These data indicate that carbon ion silicone rubber exhibits good antibacterial adhesion properties and triggers thinner and weaker tissue capsules. In addition, high surface roughness and high zeta potential may be the main factors that induce the unique biocompatibility of carbon ion silicone rubber. Conclusion: Ion implantation should be considered for further investigation and application, and carbon ion silicone rubber could be a better biomaterial to decrease silicone rubber–initiated complications.

Hydroxyapatite-Coated Sillicone Rubber Enhanced Cell Adhesion and It May Be through the Interaction of EF1β and γ-Actin

Silicone rubber (SR) is a common soft tissue filler material used in plastic surgery. However, it presents a poor surface for cellular adhesion and suffers from poor biocompatibility. In contrast, hydroxyapatite (HA), a prominent component of animal bone and teeth, can promote improved cell compatibility, but HA is an unsuitable filler material because of the brittleness in mechanism. In this study, using a simple and economical method, two sizes of HA was applied to coat on SR to counteract the poor biocompatibility of SR. Surface and mechanical properties of SR and HA/SRs confirmed that coating with HA changes the surface topology and material properties. Analysis of cell proliferation and adhesion as well as measurement of the expression levels of adhesion related molecules indicated that HA-coated SR significantly increased cell compatibility. Furthermore, mass spectrometry proved that the biocompatibility improvement may be related to elongation factor 1-beta (EF1β)/γ-actin adjusted cytoskeletal rearrangement.

MAP4 regulates Tctex-1 and promotes the migration of epidermal cells in hypoxia

After acute wound formation, the oxygen supply is reduced, which results in the formation of an acute hypoxic microenvironment; whether this hypoxic microenvironment enhances epidermal cell migration and the underlying regulatory molecular mechanism of this effect are unclear. In this study, HaCaT cells were maintained under hypoxic (1% oxygen) or normoxic conditions. Methods including immunofluorescence staining, wound scratch assays, transwell assays, Western blotting and high‐ and low‐expression lentiviral vector transfection were utilized to observe the changes in cell migration, microtubule dynamics and the expression levels of microtubule‐associated protein (MAP) 4 and the light chain protein DYNLT1 (Tctex‐1). The possible mechanisms were studied and discussed. The results showed that epidermal cell migration was enhanced during early hypoxia. Further experiments revealed that MAP4 regulates microtubule dynamics and promotes epidermal cell migration through Tctex‐1. MAP4 and Tctex‐1 play important roles in regulating the migration of epidermal cells under hypoxia. This evidence will provide a basis for further revealing the cellular and molecular mechanisms of local wound hypoxia and for promoting wound healing.