Retracted: Paeoniflorin promotes angiogenesis and tissue regeneration in a full-thickness cutaneous wound model through the PI3K/AKT pathway

The treatment of wounds remains a clinical challenge because of poor angiogenesis under the wound bed, and increasingly, the patients' need for functional and aesthetically pleasing scars. For the wound healing process, new blood vessels which can deliver nutrients and oxygen to the wound area are necessary. In this study, we investigated the pro-angiogenesis ability and mechanism in wound healing of paeoniflorin (PF), which is a traditional Chinese medicine. In our in vitro results, the ability for proliferation, migration and in vitro angiogenesis in human umbilical vein endothelial cells was promoted by coculturing with PF (1.25–5 μM). Meanwhile, molecular docking studies revealed that PF has excellent binding abilities to phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT), and consistent with our western blot results, that PF suppressed PI3K and AKT phosphorylation. Furthermore, to investigate the healing effect of PF in vivo, we constructed a full-thickness cutaneous wound model in rats. PF stimulated the cellular proliferation status, collagen matrix deposition and remodeling processes in vitro and new blood vessel formation at the wound bed resulting in efficient wound healing after intragastric administration of 10 mg·kg⁻¹·day⁻¹ in vivo. Overall, PF performed the pro-angiogenetic effect in vitro and accelerating wound healing in vivo. In summary, the capacity for angiogenesis in endothelial cells could be enhanced by PF treatment via the PI3K/AKT pathway in vitro and could accelerate the wound healing process in vivo through collagen deposition and angiogenesis in regenerated tissue. This study provides evidence that application of PF represents a novel therapeutic approach for the treatment of cutaneous wounds.     

Primordial follicle activation is affected by the absence of Sohlh1 in mice

Previous studies have reported that only primordial follicles and empty follicles can be found in 7.5 days postparturition (dpp) Sohlh1^?/? mouse ovaries and females are infertility. There appears to be a defect in follicle development during the primordial‐to‐primary follicle transition in Sohlh1^?/? mouse ovaries. However, detailed analyses of these phenomena have not been performed. In this study, we used Sohlh1^?/? transgenic mice to explore the role of Sohlh1 in folliculogenesis. The results showed that only primordial follicles and empty follicles can be observed in Sohlh1^?/? ovaries from 0.5 to 23.5 dpp. The expression of Foxo3 and FOXO3 was downregulated; nucleocytoplasmic shuttling of FOXO3 was normal in 7.5‐dpp Sohlh1^+/+ but not Sohlh1^?/? ovaries; and primordial follicle activation (PFA) was not observed in 7.5‐dpp Sohlh1^?/? mice. The expression levels of KIT, AKT, and P308‐AKT were downregulated (p < 0.05), whereas that of P473‐AKT was not significantly changed (p > 0.05). The KIT/PI3K/AKT pathway was inhibited. Furthermore, we conducted a dual luciferase assay and chromatin immunoprecipitation. The results showed that SOHLH1 can upregulate the Kit gene by binding to the ?3698 bp E‐box motif. The absence of Sohlh1 may affect PFA in mouse ovaries via downregulation of Kit and inhibition of the KIT/PI3K/AKT pathway.     

Divergent cellular phenotypes of human and mouse cells lacking the Werner syndrome RecQ helicase

Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn^?/? mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn^?/? mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts. These analyses confirmed previously reported cellular phenotypes of WRN-mutant and WRN-deficient human fibroblasts, and demonstrated that the human WRN-deficient cellular phenotype can be detected in cells grown in 5% or in 20% oxygen. In contrast, we did not identify prominent cellular phenotypes present in WRN-deficient human cells in Wrn^?/? mouse fibroblasts. Our results indicate that human and mouse fibroblasts have different functional requirements for WRN protein, and that the absence of a strong cellular phenotype may in part explain the failure of Wrn^?/? mice to develop an organismal phenotype resembling Werner syndrome.     

Deletion of the FHL2 gene attenuates intima‐media thickening in a partially ligated carotid artery ligated mouse model

The four and a half LIM domain protein 2 (FHL2) is a member of the four and a half LIM domain (FHL) gene family, and it is associated with cholesterol‐enriched diet‐promoted atherosclerosis. However, the effect of FHL2 protein on vascular remodelling in response to hemodynamic alterations remains unclear. Here, we investigated the role of FHL2 in a model of restricted blood flow‐induced atherosclerosis. To promote neointimal hyperplasia in vivo, we subjected FHL2^+/+ and FHL2^?/? mice to partial ligation of the left carotid artery (LCA). The expression of p‐ERK and p‐AKT was decreased in FHL2^?/? mice. FHL2 bound to AKT regulated AKT phosphorylation and led to Rac1‐GTP inactivation. FHL2 silencing in human aortic smooth muscle cells down‐regulated the PDGF‐induced phosphorylation of ERK and AKT. Furthermore, FHL2 silencing reduced cytoskeleton conformational changes and caused cell cycle arrest. We concluded that FHL2 is essential for the regulation of arterial smooth muscle cell function. FHL2 modulates proliferation and migration via mitogen‐activated protein kinase (MAPK) and PI3K‐AKT signalling, leading to arterial wall thickening and thus neointimal hyperplasia.     

Hepatocyte growth factor plays a dual role in tendon-derived stem cell proliferation,migration, and differentiation

Heterotopic ossification is common in tendon healing after trauma, but the detailed mechanisms remain unknown. Tendon-derived stem cells (TDSCs) are a type of progenitor cell found in the tendon niche, and their incorrect differentiation after trauma may lead to tendon calcification. The expression of hepatocyte growth factor (HGF) presents drastic fluctuations in serum/tissue after trauma and was found to activate quiescent stellate cells and contribute to wound healing; however, its potential role in TDSCs remains elusive. In this study, TDSCs isolated from rats were cultured in media containing HGF with or without a signaling inhibitor, and the proliferation, migration, and differentiation ability of TDSCs were measured to determine the role and mechanism of HGF in TDSCs. We showed that HGF promotes TDSC proliferation and migration but inhibits TDSC osteogenic differentiation ability. HGF activated-HGF/c-Met, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, which was positively correlated with TDSCs proliferation and migration but negatively related to TDSC osteogenic differentiation ability. The phosphorylation of Smad1/5/8 was also negatively related to HGF/c-Met, MAPK/ERK1/2, and PI3K/AKT signaling, which demonstrated that the inhibition of osteogenic differentiation was dependent on BMP/Smad1/5/8 signaling. Overall, we showed that HGF could promote TDSCs proliferation and migration and inhibit osteogenic differentiation in vitro, suggesting a potential role for HGF as a cytokine treatment of tendon trauma.     

Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN‐deficient human fibroblasts

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double‐strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short‐term rapamycin treatment. Long‐term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near‐normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.     

Platelet derived growth factor B and epithelial mesenchymal transition of peritoneal mesothelial cells

Platelet derived growth factor (PDGF) is involved in wound healing in various organ systems. Its potential role in the context of peritoneal injury following long-term peritoneal dialysis is unclear. We used an adenovirus expressing the B chain of PDGF (AdPDGF-B) to assess its effect on pro-fibrotic pathways in the peritoneal membrane. To assess the transforming growth factor (TGF) β independent effects of PDGF, we over-expressed PDGF-B in the peritoneum of either wild-type mice (Smad3^+/+) or those with a deletion of the TGFβ signaling protein Smad3 (Smad3^?/?). PDGF-B induced sustained angiogenesis in both Smad3^+/+ and Smad3^?/? mice. Despite increased collagen gene expression, collagen accumulation was transient and fibrogenesis was associated with induction of collagenase activity. We observed epithelial to mesenchymal transition (EMT) involving the peritoneal mesothelial cells, as shown by increased SNAIL and decreased E-Cadherin expression with evidence of mesothelial cells expressing both epithelial and mesenchymal markers. Unlike TGFβ-induced EMT, PDGF-B exposure did not lead to mobilization of the mesothelial cells; they remained as a single monolayer throughout the observation period. This “non-invasive” EMT phenomenon is a novel finding and may have implications concerning the role of EMT in peritoneal fibrosis and injury to other organ systems. The observed effects were similar in Smad3^?/? and Smad3^+/+ animals, suggesting that the PDGF-B effects were independent of TGFβ or Smad signaling.     

Deferoxamine promotes mesenchymal stem cell homing in noise‐induced injured cochlea through PI3K/AKT pathway

Objective

Over 5% of the world's population suffers from disabling hearing loss. Stem cell homing in target tissue is an important aspect of cell‐based therapy, which its augmentation increases cell therapy efficiency. Deferoxamine (DFO) can induce the Akt activation, and phosphorylation status of AKT (p‐AKT) upregulates CXC chemokine receptor‐4 (CXCR4) expression. We examined whether DFO can enhance mesenchymal stem cells (MSCs) homing in noise‐induced damaged cochlea by PI3K/AKT dependent mechanism.

Materials and Methods

Mesenchymal stem cells were treated with DFO. AKT, p‐AKT protein and hypoxia inducible factor 1‐ α (HIF‐1α) and CXCR4 gene and protein expression was evaluated by RT‐ PCR and Western blot analysis. For in vivo assay, rats were assigned to control, sham, noise exposure groups without any treatment or receiving normal, DFO‐treated and DFO +LY294002 (The PI3K inhibitor)‐treated MSCs. Following chronic exposure to 115 dB white noise, MSCs were injected into the rat cochlea through the round window. Number of Hoechst‐ labelled cells was determined in the endolymph after 24 hours.

Results

Deferoxamine increased P‐AKT, HIF‐1α and CXCR4 expression in MSCs compared to non‐treated cells. DFO pre‐conditioning significantly increased the homing ability of MSCs into injured ear compared to normal MSCs. These effects of DFO were blocked by LY294002.

Conclusions

Pre‐conditioning of MSCs by DFO before transplantation can improve stem cell homing in the damaged cochlea through PI3K/AKT pathway activation.

     

The clinical characteristics of Werner syndrome: molecular and biochemical diagnosis

Werner syndrome (WS) is an adult onset segmental progeroid syndrome caused by mutations in the WRN gene. The WRN gene encodes a 180 kDa nuclear protein that possesses helicase and exonuclease activities. The absence of WRN protein leads to abnormalities in various DNA metabolic pathways such as DNA repair, replication and telomere maintenance. Individuals with WS generally develop normally until the third decade of life, when premature aging phenotypes and a series of age-related disorders begin to manifest. In Japan, where a founder effect has been described, the frequency of Werner heterozygotes appears to be as high as 1/180 in the general population. Due to the relatively non-specific nature of the symptoms and the lack of awareness of the condition, this disease may be under-diagnosed in other parts of the world. Genetic counseling of WS patients follows the path of other autosomal recessive disorders, with special attention needed for cancer surveillance in relatives. Molecular diagnosis of WS is made by nucleotide sequencing and, in some cases, protein analysis. It is also of potential interest to measure WRN activities in WS patients. More than 50 different disease-causing mutations in the WRN gene have been identified in WS patients from all over the world. All but one of these cases has mutations that result in the premature termination of the protein. Here we describe the clinical, molecular and biochemical characteristics of WS for use by medical professionals in a health care setting. Additional information is available through the International Registry of WS ().     

A role for LFA‐1 in delaying T‐lymphocyte egress from lymph nodes

Lymphocytes use the integrin leukocyte function‐associated antigen‐1 (LFA‐1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA‐1 dependent. We show that LFA‐1 mediates prolonged LN residence as LFA‐1^?/? CD4 T cells have significantly decreased dwell times compared with LFA‐1^+/+ T cells, a distinction lost in hosts lacking the major LFA‐1 ligand ICAM‐1. Intra‐vital two‐photon microscopy revealed that LFA‐1^+/+ and LFA‐1^?/? T cells reacted differently when probing the ICAM‐1‐expressing lymphatic network. While LFA‐1^+/+ T cells returned to the LN parenchyma with greater frequency, LFA‐1^?/? T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA‐1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T‐cell response to antigen. Thus, we identify a novel function for LFA‐1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.     

Up‐regulated basigin‐2 in microglia induced by hypoxia promotes retinal angiogenesis

Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well‐characterized mouse model of oxygen‐induced retinopathy, which recapitulated hypoxia‐induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up‐regulation of PI3K‐AKT and IGF‐1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro‐angiogenic role, providing new insights into microglia‐promoting retinal angiogenesis.     

Histamine deficiency delays ischaemic skeletal muscle regeneration via inducing aberrant inflammatory responses and repressing myoblast proliferation

Histidine decarboxylase (HDC) catalyses the formation of histamine from L‐histidine. Histamine is a biogenic amine involved in many physiological and pathological processes, but its role in the regeneration of skeletal muscles has not been thoroughly clarified. Here, using a murine model of hindlimb ischaemia, we show that histamine deficiency in Hdc knockout (Hdc^?/?) mice significantly reduces blood perfusion and impairs muscle regeneration. Using Hdc‐EGFP transgenic mice, we demonstrate that HDC is expressed predominately in CD11b⁺Gr‐1⁺ myeloid cells but not in skeletal muscles and endothelial cells. Large amounts of HDC‐expressing CD11b⁺ myeloid cells are rapidly recruited to injured and inflamed muscles. Hdc^?/? enhances inflammatory responses and inhibits macrophage differentiation. Mechanically, we demonstrate that histamine deficiency decreases IGF‐1 (insulin‐like growth factor 1) levels and diminishes myoblast proliferation via H3R/PI3K/AKT‐dependent signalling. These results indicate a novel role for HDC‐expressing CD11b⁺ myeloid cells and histamine in myoblast proliferation and skeletal muscle regeneration.     

Comparison of proliferation and genomic instability responses to WRN silencing in hematopoietic HL60 and TK6 cells

Background

Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis.

Methodology/Principal Findings

We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges.

Conclusions/Significance

Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency.     

Mesenchymal stromal cells enhance wound healing by ameliorating impaired metabolism in diabetic mice

Background aimsMesenchymal stromal cells (MSCs) have been documented to improve delayed wound healing in diabetes, but the underlying mechanism remains obscure. We aimed to investigate whether the therapeutic effects on wounds was associated with metabolic alterations by paracrine action of MSCs.MethodsMSCs from mice with high-fat diet/streptozotocin–induced diabetes or wild-type C57BL/6 mice were evaluated for their paracrine potential in vitro using enzyme-linked immunosorbent assay and immunohistochemical staining assay. MSCs were then evaluated for their therapeutic potential in vivo using an excisional cutaneous wound model in mice with diabetes. Metabolic alterations and glucose transporter four (GLUT4) as well as PI3K/Akt signaling pathway expression after wounding were also examined.ResultsMSCs from normal mice expressed even more insulin-like growth factor-1 (IGF-1) than mice with diabetes, suggesting putative paracrine action. Furthermore, compared with IGF-1 knockdown MSCs, normal MSCs markedly accelerated wound healing, as revealed by higher wound closure rate and better healing quality at 21 days post-wound. By contrast, MSCs administration increased the level of insulin as well as GLUT4 and PI3K/Akt signaling pathway expression but repressed the biochemical indexes of glucose and lipid, resulting in obvious metabolic improvement.ConclusionsThese findings suggest that IGF-1 is an important paracrine factor that mediates the therapeutic effects of MSCs on wound healing in diabetes, and the benefits of MSCs may be associated with metabolism improvements, which would provide a new target for treatment.     

Sodium 5,6‐benzylidene‐L‐ascorbate induces oxidative stress,autophagy, and growth arrest in human colon cancer HT‐29 cells

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT‐29 cells with SAA and SBA, and found a significant exposure time‐dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC₅₀ = 5 mM) than SBA (IC₅₀ = 10 mM). A short‐term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase‐dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21^+/+ and p21^?/?), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co‐incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N‐acetyl‐L‐cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3‐II were found in SBA‐treated HT‐29 cells; implicating that SBA induced AKT phosphorylation‐autophagy and p21‐growth arrest in colon cancer HT‐29 cells through an NAC‐inhibitable oxidative stress pathway. J. Cell. Biochem. 111: 412–424, 2010. © 2010 Wiley‐Liss, Inc.