Yinghua Chen

Mesenchymal stem cell-laden anti-inflammatory hydrogel enhances diabetic wound healing

The purpose of this study was to permit bone marrow mesenchymal stem cells (BMSCs) to reach their full potential in the treatment of chronic wounds. A biocompatible multifunctional crosslinker based temperature sensitive hydrogel was developed to deliver BMSCs, which improve the chronic inflammation microenvironments of wounds. A detailed in vitro investigation found that the hydrogel is suitable for BMSC encapsulation and can promote BMSC secretion of TGF-β1 and bFGF. In vivo, full-thickness skin defects were made on the backs of db/db mice to mimic diabetic ulcers. It was revealed that the hydrogel can inhibit pro-inflammatory M1 macrophage expression. After hydrogel association with BMSCs treated the wound, significantly greater wound contraction was observed in the hydrogel + BMSCs group. Histology and immunohistochemistry results confirmed that this treatment contributed to the rapid healing of diabetic skin wounds by promoting granulation tissue formation, angiogenesis, extracellular matrix secretion, wound contraction, and re-epithelialization. These results show that a hydrogel laden with BMSCs may be a promising therapeutic strategy for the management of diabetic ulcers.

Activin B Promotes Epithelial Wound Healing In Vivo through RhoA-JNK Signaling Pathway

Background Activin B has been reported to promote the proliferation and migration of keratinocytes in vitro via the RhoA-JNK signaling pathway, whereas its in vivo role and mechanism in wound healing process has not yet been elucidated. Principal Findings In this study, we explored the potential mechanism by which activin B induces epithelial wound healing in mice. Recombinant lentiviral plasmids, with RhoA (N19) and RhoA (L63) were used to infect wounded KM mice. The wound healing process was monitored after different treatments. Activin B-induced cell proliferation on the wounded skin was visualized by electron microscopy and analyzed by 5′-bromodeoxyuridine (BrdU) incorporation assay. Protein expression of p-JNK or p-cJun was determined by immunohistochemical staining and immunoblotting analysis. Activin B efficiently stimulated the proliferation of keratinocytes and hair follicle cells at the wound area and promoted wound closure. RhoA positively regulated activin B-induced wound healing by up-regulating the expression of p-JNK and p-cJun. Moreover, suppression of RhoA activation delayed activin B-induced wound healing, while JNK inhibition recapitulated phenotypes of RhoA inhibition on wound healing. Conclusion These results demonstrate that activin B promotes epithelial wound closure in vivo through the RhoA-Rock-JNK-cJun signaling pathway, providing novel insight into the essential role of activin B in the therapy of wound repair.

Activin B regulates adipose-derived mesenchymal stem cells to promote skin wound healing via activation of the MAPK signaling pathway

Adipose-derived stem cells (ADSCs) are multipotent stromal cells that can differentiate into a variety of cell types, including skin cells, and they can provide an abundant source of cells for skin tissue engineering and skin wound healing. The purpose of this study is to explore the therapeutic effects of activin B in combination with ADSCs and the possible signaling mechanism. In this study, we found that activin B was able to promote ADSC migration by inducing actin stress fiber formation in vitro. In vivo, activin B in combination with ADSCs was capable of enhancing α-SMA expression and wound closure. This combined treatment also promoted fibroblast and keratinocyte proliferation and accelerated re-epithelialization and collagen deposition. Moreover, activin B in combination with ADSCs boosted angiogenesis in the wound area. Further study of the mechanism revealed that activation of JNK and ERK signaling, but not p38 signaling, were required for activin B-induced ADSC actin stress fiber formation and cell migration. These results showed that activin B was able to activate JNK and ERK signaling pathways to induce actin stress fiber formation and ADSC migration to promote wound healing. These results suggest that combined treatment with activin B and ADSCs is a promising therapeutic strategy for the management of serious skin wounds.

Peptide-modified Chitosan Hydrogel accelerates skin wound healing by promoting fibroblast proliferation, migration and secretion.

Skin wound healing is a complicated process that involves a variety of cells and cytokines. Fibroblasts play an important role in this process and participate in transformation into myofibroblasts, the synthesis of extracellular matrix (ECM) and fibers, and the secretion of a variety of growth factors. This study assessed the effects of peptide Ser-Ile-Lys-Val-Ala-Val (SIKVAV)--modified chitosan hydrogels on skin wound healing. We investigated the capability of peptide SIKVAV to promote cell proliferation and migration, the synthesis of collagen, and the secretion of a variety of growth factors using fibroblasts in vitro. We also treated skin wounds established in mice using peptide SIKVAV-modified chitosan hydrogels. Hematoxylin and eosin staining showed that peptide-modified chitosan hydrogels enhanced the reepithelialization of wounds compared with negative and positive controls. Masson’s trichrome staining demonstrated that more collagen fibers were deposited in the wounds treated with peptide-modified chitosan hydrogels compared with the negative and positive controls. Immunohistochemistry revealed that the peptide-modified chitosan hydrogels promoted angiogenesis in the skin wound. Taken together, these results suggest that peptide SIKVAV-modified chitosan hydrogels may be useful in wound dressing and the treatment of skin wounds.

mDia1 and Cdc42 regulate Activin B‐induced migration of bone marrow‐derived mesenchymal stromal cells

In a previous study, we have shown that Activin B is a potent chemoattractant for bone marrow‐derived mesenchymal stromal cells (BMSCs). As such, the combination of Activin B and BMSCs significantly accelerated rat skin wound healing. In another study, we showed that RhoA activation plays a key role in Activin B‐induced BMSC migration. However, the role of the immediate downstream effectors of RhoA in this process is unclear. Here, we demonstrated that mammalian homolog of Drosophila diaphanous‐1 (mDia1), a downstream effector of RhoA, exerts a crucial function in Activin B‐induced BMSC migration by promoting membrane ruffling, microtubule morphology, and adhesion signaling dynamics. Furthermore, we showed that Activin B does not change Rac1 activity but increases Cdc42 activity in BMSCs. Inactivation of Cdc42 inhibited Activin B‐stimulated Golgi reorientation and the cell migration of BMSCs. Furthermore, knockdown of mDia1 affected Activin B‐induced BMSC‐mediated wound healing in vivo. In conclusion, this study demonstrated that the RhoA‐mDia1 and Cdc42 pathways regulate Activin B‐induced BMSC migration. This study may help to optimize clinical MSC‐based transplantation strategies to promote skin wound healing. Stem Cells 2019;37:150–161

Endothelial Cdc42 deficiency impairs endothelial regeneration and vascular repair after inflammatory vascular injury

BackgroundEndothelial cell (EC) regeneration is essential for inflammation resolution and vascular integrity recovery after inflammatory vascular injury. Cdc42 is a central regulator of cell survival and vessel formation in EC development. However, it is unknown that whether Cdc42 could be a regulating role of EC repair following the inflammatory injury in the lung. The study sought to test the hypothesis that Cdc42 is required for endothelial regeneration and vascular integrity recovery after LPS-induced inflammatory injury.Methods and resultsThe role of Cdc42 for the regulation of pulmonary vascular endothelial repair was tested in vitro and in vivo. In LPS-induced acute lung injury (ALI) mouse models, knockout of the Cdc42 gene in ECs increased inflammatory cell infiltration and pulmonary vascular leakage and inhibited vascular EC proliferation, which eventually resulted in more severe inflammatory lung injury. In addition, siRNA-mediated knockdown of Cdc42 protein on ECs disrupted cell proliferation and migration and tube formation, which are necessary processes for recovery after inflammatory vascular injury, resulting in inflammatory vascular injury recovery defects.ConclusionWe found that Cdc42 deficiency impairs EC function and regeneration, which are crucial in the post-inflammatory vascular injury repair process. These findings indicate that Cdc42 is a potential target for novel treatments designed to facilitate endothelial regeneration and vascular repair in inflammatory pulmonary vascular diseases, such as ALI/ARDS.

Antioxidative Effect of Quetiapine on Acute Ultraviolet-B-Induced Skin and HaCaT Cell Damage

Quetiapine is a new type of antipsychotic drug, with effective protection of pheochromocytoma PC12 cells from oxidative stress-induced apoptosis. Ultraviolet-B radiation can increase reactive oxygen species (ROS) production, resulting in significant inflammatory responses in damaged skin. Thus, the purpose of this study is to explore whether quetiapine protects the skin from intermediate-wave ultraviolet (UVB)-induced damage through antioxidant stress. In vivo, we found quetiapine treatment was able to significantly decrease skin thickness, erythema, and edema, as well as inflammation compared to control group. Moreover, quetiapine treatment increased the activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). In addition, it reduced the production of malondialdehyde (MDA), a kind of oxidized lipid. In vitro, we found that quetiapine blocked UVB-induced intracellular ROS generation and maintained the cell activity at a normal level. Furthermore, we tested the phosphorylation of p38 both in vivo and in vitro, and we found that quetiapine could inhibit phosphorylation of p38, which is caused by UVB irradiation. We concluded that quetiapine was able to relieve UVB-induced skin damage through its antioxidative properties. These effects might be associated with p38 MAPK signaling pathway.

Deletion of epithelial cell-specific Cdc42 leads to enamel hypermaturation in a conditional knockout mouse model

Recent evidence suggests that GTPases Rho family plays an important role in tooth development; however, the role of Cdc42 in tooth development remains unclear. We aimed to investigate the function of Cdc42 in tooth development and amelogenesis. We generated an epithelial cell-specific K5-Cdc42 knockout (KO) mouse to evaluate post-eruption dental phenotypes using a K5-Cre driver line. This model overcomes the previously reported perinatal lethality. Tooth phenotypes were analyzed by micro X-ray, micro-computed tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wear rate, shear strength, and a microhardness test. Enamel matrix protein expression was determined by immunohistochemistry. KO mice displayed a hypomaturation phenotype, including incisors that lacked yellow pigmentation and were abnormally white, rapid attrition of molars following eruption, and decreased micro-hardness and shearing strength. Micro-CT data revealed that of incisor and molar enamel volumes were smaller in the KO than in wild-type (WT) mice. SEM analysis showed that the enamel prism structure was disordered. In addition, HE staining indicated a remarkable difference in the ameloblast morphology and function between KO and WT mice, and immunohistochemistry showed increased expression of amelogenin, ameloblastin, matrix metallopeptidase 20, kallikrein-related peptidase 4 and amelotin in the KO mice teeth. Our results suggest epithelium cell-specific Cdc42 deletion leads to tooth hypomaturation and transformation of the enamel prism structure that is likely due to altered ameloblast morphology and the secretion of enamel matrix proteins and proteases. This is the first in vivo evidence suggesting that Cdc42 is essential for proper tooth development and amelogenesis.

Bioinformatics analysis of gene expression alterations conferring drug resistance in tumor samples from melanoma patients with EGFR-activating BRAF mutations

Melanoma is a highly malignant tumor of the skin melanocytes. Patients with this cancer have a high frequency (~50%) of oncogenic BRAF mutations, particularly BRAF V600E. Treatments for melanoma often target BRAF mutations or involve mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitors. A major challenge in melanoma treatment is resistance to BRAF inhibitor treatment, which may be enhanced by the BRAF mutation itself and/or epidermal growth factor receptor (EGFR) activation, leading to poor prognosis. However, no effective clinical treatment exists for patients with EGFR-activating feedback. The aim of the present study was to analyze gene expression changes in tumors from patients with EGFR-activating BRAF mutations during development of drug resistance. RNA-seq data was downloaded from the Gene Expression Omnibus (GEO) database for pre- and post-treatment tumor samples from three melanoma patients with EGFR-activating BRAF V600E mutations, and from The Cancer Genome Atlas (TCGA) melanoma database for tumor and non-tumor samples from patients with the BRAF V600E mutation and unknown EGFR activation status. Using functional enrichment and KEGG pathway analyses, the present study analyzed differentially expressed genes (DEGs) between pre- vs. post-treatment data from the GEO database and tumor or non-tumor sample data from the TCGA database. The results of the present study indicated that functional and structural changes to the plasma membrane may be associated with drug resistance. The present study identified 9 DEGs that were significantly different between tumor and non-tumor samples and also between prior to and following treatment. Thus, it was confirmed that patients with EGFR-activating BRAF V600E mutations undergo gene expression changes during disease development, and during therapy. These findings may provide potential directions for melanoma-specific therapy.

RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling

BACKGROUND In our previous study, Activin B induced actin stress fiber formation and cell migration in Bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. However, the underlying molecular mechanisms are not well studied. RhoA is recognized to play a critical role in the regulation of actomyosin cytoskeletal organization and cell migration. METHODS Pull-down assay was performed to investigate the activity of RhoA. The dominant-negative mutants of RhoA (RhoA(N19)) was used to determine whether RhoA has a role in Activin B-induced cytoskeleton organization and cell migration in BMSCs. Cytoskeleton organization was examined by fluorescence Rhodamine-phalloidin staining, and cell migration by transwell and cell scratching assay. Western blot was carried out to investigate downstream signaling cascade of RhoA. Inhibitor and siRNAs were used to detect the role of downstream signaling in stress fiber formation and/or cell migration. RESULTS RhoA was activated by Activin B in BMSCs. RhoA(N19) blocked Activin B-induced stress fiber formation and cell migration. ROCK inhibitor blocked Activin B-induced stress fiber formation but enhanced BMSCs migration. Activin B induced phosphorylation of LIMK2 and Cofilin, which was abolished by ROCK inhibition. Both of siRNA LIMK2 and siRNA Cofilin inhibited Activin B-induced stress fiber formation. CONCLUSIONS RhoA regulates Activin B-induced stress fiber formation and migration of BMSCs. A RhoA-ROCK-LIMK2-Cofilin signaling node exists and regulates actin stress fiber formation. RhoA regulates Activin B-induced cell migration independent of ROCK. GENERAL SIGNIFICANCE Better understanding of the molecular mechanisms of BMSCs migration will help optimize therapeutic strategy to target BMSCs at injured tissues.