Targeting the Glycolytic Enzyme PGK1 to Inhibit the Warburg Effect: A New Strategy for Keloid Therapy.

BACKGROUND: Aerobic glycolysis (the Warburg effect) may play an important role in keloid pathogenesis, which may be aggravated by the hypoxic microenvironment in keloids. Phosphoglycerate kinase 1 (PGK1), a key glycolytic enzyme, is essential for cellular aerobic glycolysis, but its role in keloid formation remains unknown. This study aimed to detect PGK1 expression in keloid tissue and investigate the effects of inhibiting PGK1 expression on keloid fibroblasts (KFbs) under hypoxia and normoxia. METHODS: Normal skin and keloid samples were separated into two parts, one was used for immunohistochemistry, and one for primary cell culture. PGK1 tissue expression was detected by immunohistochemistry. Reverse-transcriptase polymerase chain reaction and Western blotting were used to detect PGK1, GLUT1, LDHA, and COL1 expression, and glucose uptake and lactate production were detected with a microplate reader. Cell proliferation and apoptosis were investigated with IncuCyte and flow cytometry. Cell migration and invasion were detected with Transwell assays. Glycolytic function was explored with the Seahorse XF96 system. RESULTS: Immunohistochemistry showed PGK1 overexpression in keloid tissue compared with normal skin tissue ( P < 0.05). Consistently, PGK1 expression was significantly higher in KFbs than in normal skin fibroblasts (NFbs), and hypoxia stimulated PGK1 expression in KFbs and NFbs ( P < 0.05). PGK1 knockdown significantly inhibited KFb glycolysis, proliferation, migration, invasion, glucose consumption, and lactate production ( P < 0.05). Furthermore, GLUT1, LDHA, and COL1 expression was decreased in KFbs compared with NFbs ( P < 0.05). In addition, suppressing PGK1 may mediate the PI3K/AKT pathway to down-regulate GLUT1, LDHA, and COL1 expression ( P < 0.05). CONCLUSIONS: These findings provide new evidence that suppressing PGK1, inhibiting glycolysis, reduces KFb proliferation, migration, invasion, and type I collagen expression. Targeting PGK1 to inhibit the Warburg effect may be a new therapeutic strategy for keloids. CLINICAL RELEVANCE STATEMENT: This article may provide new suggestions into the pathogenesis and treatment of keloids. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

PI3K/AKT pathway promotes keloid fibroblasts proliferation by enhancing glycolysis under hypoxia.

Our previous study demonstrated altered glucose metabolism and enhanced phosphorylation of the PI3K/AKT pathway in keloid fibroblasts (KFb) under hypoxic conditions. However, whether the PI3K/AKT pathway influences KFb cell function by regulating glucose metabolism under hypoxic conditions remains unclear. Here, we show that when PI3K/AKT pathway was inactivated with LY294002, the protein expression of glycolytic enzymes decreased, while the amount of mitochondria and mitochondrial membrane potential increased. The key parameters of extracellular acidification rate markedly diminished, and those of oxygen consumption rate significantly increased after inhibition of the PI3K/AKT pathway. When the PI3K/AKT pathway was suppressed, the levels of reactive oxygen species (ROS) and mitochondrial ROS (mitoROS) were significantly increased. Meanwhile, cell proliferation, migration and invasion were inhibited, and apoptosis was increased when the PI3K/AKT pathway was blocked. Additionally, cell proliferation was compromised when KFb were treated with both SC79 (an activator of the PI3K/AKT pathway) and 2-deoxy-d-glucose (an inhibitor of glycolysis), compared with the SC79 group. Moreover, a positive feedback mechanism was demonstrated between the PI3K/AKT pathway and hypoxia-inducible factor-1α (HIF-1α). Our data collectively demonstrated that the PI3K/AKT pathway promotes proliferation and inhibits apoptosis in KFb under hypoxia by regulating glycolysis, indicating that the PI3K/AKT signalling pathway could be a therapeutic target for keloids.

Comparing the Efficacy and Safety of Intralesional Verapamil With Intralesional Triamcinolone Acetonide in Treatment of Hypertrophic Scars and Keloids: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Clinical treatment of hypertrophic scars (HSs) and keloids is often unsatisfactory. Intralesional injections of triamcinolone acetonide (TAC) and verapamil are widely used to treat HSs and keloids, but their efficacy and safety are controversial. OBJECTIVES: The aim of this study was to conduct a meta-analysis of the effectiveness and safety of verapamil and TAC in the treatment of HSs and keloids. METHODS: Embase, Google Scholar, and PubMed were searched for randomized controlled trials (RCTs) from inception to February 2020. RCTs that evaluated treatment effects with the Vancouver Scar Scale or reported adverse effects were included. The continuous data and the dichotomous variables were analyzed as mean difference (MD) and relative risk (RR), respectively. RESULTS: Seven RCTs (461 patients) were included. Compared with verapamil, TAC rapidly changed the ∆height (MD = 0.07; P < 0.05) and ∆pliability (MD = 0.23; P < 0.05) after the first session, but subsequent treatments resulted in no significant differences in the ∆height, ∆pigmentation, ∆vascularity, and ∆pliability. Although total adverse effects (RR = 0.42; P = 0.1) were not significantly different, in the subgroup analysis the incidence of telangiectasia (RR = 0.04; P < 0.05) and skin atrophy (RR = 0.10; P < 0.05), but not pain (RR = 1.27; P = 0.77), was significantly lower with verapamil than with TAC. CONCLUSIONS: Verapamil may be an effective substitute for TAC. Although total adverse effects did not change, the incidence of telangiectasia and skin atrophy was lower with verapamil than with TAC.

Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia.

Keloids exhibit metabolic reprogramming including enhanced glycolysis and attenuated oxidative phosphorylation. Hypoxia induces a series of protective responses in mammalian cells. However, the metabolic phenotype of keloid fibroblasts under hypoxic conditions remains to be elucidated. The present study aimed to investigate glycolytic activity, mitochondrial function and morphology, and the HIF1α and PI3K/AKT signaling pathways in keloid fibroblasts (KFB) under hypoxic conditions. Our results showed that hypoxia promoted proliferation, migration invasion and collagen synthesis and inhibited apoptosis in KFB. The mRNA levels, protein expressions and enzyme activities of glycolytic enzymes in KFB were higher than those in normal skin fibroblasts (NFB) under normoxia. Moreover, hypoxia remarkedly upregulated glycolysis in KFB. Decreased activities of mitochondrial complexes and abnormal mitochondria were detected in KFB under normoxic conditions and the damage was aggravated by hypoxia. An intracellular metabolic profile assay suggested hypoxia increased glycolytic parameters except glycolytic reserve but inhibited the key parameters of mitochondrial function apart from H+ leak. Protein levels of HIF1α and phosphorylation levels of the PI3K/AKT signaling pathway were upregulated in the context of 3% oxygen. Enhanced total reactive oxygen species (ROS), mitochondrial ROS (mitoROS) and antioxidant activities of KFB were observed in response to hypoxia. Additionally, autophagy was induced by hypoxia. Our data collectively demonstrated potentiated glycolysis and attenuated mitochondrial function under hypoxia, indicating that altered glucose metabolism regulated by hypoxia could be a therapeutic target for keloids.

Associations of LRP5 Gene With Bone Mineral Density, Bone Turnover Markers, and Fractures in the Elderly With Osteoporosis.

Objective: The study aimed to explore the associations of rs4988300 and rs634008 in the low-density lipoprotein receptor-related protein 5 (LRP5) gene with bone mineral density (BMD), bone turnover markers (BTM), and fractures in elderly patients with osteoporosis (OP). Methods: Our study included 328 unrelated OP patients with or without fractures. Genomic DNA was extracted for genotyping. BTM levels were assessed by electrochemiluminescence (ECL). Dual-energy X-ray absorptiometry (DXA) was employed to measure BMD in the lumbar spine (LS) and proximal femur. Basic features between the OP and fracture groups were analyzed using the t-test. The Chi-square test was performed to analyze the differences in allele and genotype frequencies. The associations of single-nucleotide polymorphisms (SNPs) with BMD and BTM in the subgroups were investigated by the analysis of covariance (ANCOVA) adjusted for confounding factors. Results: In both females and males, individuals with fractures exhibited higher BTM levels and lower BMD values than those with OP (P < 0.05). The allele and genotype frequencies of rs4988300 in the subgroups were significantly different (P < 0.05). In both females and males suffering from OP, participants with rs4988300 GG or rs634008 TT presented lower procollagen I N-terminal propeptide (PINP) levels (P < 0.05). Women with OP carrying rs4988300 GG exhibited lower BMD values at FN and TH (P < 0.05). In both females and males with fractures, individuals carrying rs4988300 GG genotype or rs634008 TT genotype exhibited lower PINP levels and BMD values at FN and TH than those with other genotypes (P < 0.05). Conclusions: Rs4988300 and rs634008 polymorphisms in the LRP5 gene are associated with bone phenotypes in the elderly with OP or fractures.

Lower Metal Element Levels in Hypertrophic Scars: A Potential Mechanism of Aberrant Cicatrix Hyperplasia.

BACKGROUND We investigated levels of the metal elements Ca, Mg, Zn, Fe, and Cu in blood, normal skin (NS), and different types of scar tissue and aimed to elucidate the pathogenesis of hypertrophic scars (HS). MATERIAL AND METHODS Tissue specimens were excised from 3 groups of research participants: scar-free, flat scar (FS), and HS groups. Levels of the study elements were measured in blood, NS, and scar tissues with a spectrophotometer. The levels in plasma or in different types of specimens were compared among subgroups. In the FS and HS groups, levels were compared between the scar tissue and NS of each individual. In addition, element differences in exposed and unexposed areas of NS were investigated in the scar-free group. HS fibroblasts (HFB) were cultured in medium with various reduced levels of metal elements to determine the influence of metal elements on fibroblast growth. RESULTS Levels of trace elements, including Zn, Fe, and Cu, were significantly lower in HS than in FS. The levels of Ca, Zn, Fe, and Cu were markedly lower in HS than in the patients' own NS, while the Cu/Zn ratio was higher. However, no such difference was observed in the FS group. No significant difference in element levels was found in either plasma or NS among the 3 groups. Reduced levels of the elements promoted HFB proliferation within 24 h while an inhibition effect was observed at 72 h. CONCLUSIONS Our findings indicate reduced levels of metal elements in part of the healing microenvironment, suggesting that decreased metal levels may be involved in the pathogenesis of HS.

Efficacy and safety of topical nitroglycerin in the prevention of mastectomy flap necrosis: a systematic review and meta-analysis.

Flap necrosis is a common complication after mastectomy, and nitroglycerin (NTG) ointment has been used successfully to treat it. However, it is not clear whether topical NTG can completely prevent the occurrence of flap necrosis after breast cancer surgery, and it is also unclear whether this treatment may cause side effects. Three randomized controlled trials (RCTs) and two retrospective cohort studies (RCSs) were included in our investigation. This meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We found that NTG significantly reduced the rates of mastectomy flap necrosis, full-thickness flap necrosis, and debridement as well as the rate of early complications other than flap necrosis. However, there was no significant difference in drug-related adverse reactions, explantation, superficial flap necrosis, infection, hematoma or seroma between the NTG and placebo groups.

Shared genetic susceptibilities for irritable bowel syndrome and depressive disorder in Chinese patients uncovered by pooled whole-exome sequencing.

Irritable bowel syndrome (IBS) is the most prevalent functional gastrointestinal disorder presenting a high comorbidity with depressive disorder (DD). Many studies have confirmed that these two disease share the similar pathophysiological process, but evidence of the genetic risks is limited. This study aimed to analyze the genetic susceptibilities for IBS and DD in Chinese patients. Pooled whole-exome sequencing (pooled-WES) was performed to identify the candidate variants in the group of diarrhea predominant IBS (IBS-D) patients, DD patients, and healthy controls (HC). Then, targeted sequencing was used to validate the candidate variants in three additional cohorts of IBS-D, DD, and HC. Four variants associated with both IBS-D and DD were identified through pooled-WES, and three of them were validated in targeted sequencing. SYT8 rs3741231 G allele and SSPO rs12536873 TT genotype were associated with both IBS-D and DD. The genes of these variants are important in neurogenesis and neurotransmission. In addition, we found COL6A1 rs13051496, a unique risk variation for IBS-D. It increased the IBS-D risk and had a positive correlation with the scores of abdominal bloating and dissatisfaction of bowel habits. Through the results of this study, it provides a genetic basis for the high comorbidity of IBS-D and DD.

Exosomes from Adipose-Derived Stem Cells (ADSCs) Overexpressing miR-21 Promote Vascularization of Endothelial Cells.

In the past few years, exosomes released from adipose-derived stem cells (abbreviated as ADSCs) have shown promises to provide therapeutic benefits in the fields of regenerative medicine. miRNAs, existing in exosomes, are endogenous, small noncoding RNAs that play important roles in a variety of cellular functions and tumor development. Emerging evidences have indicated that miR-21 is one of the important miRNAs associated with tumor angiogenesis. In this study, we identified the role of exosomes from ADSCs overexpressing miR-21 in regulating/promoting vascularization of endothelial cells. Experimental data indicated an elevated miR-21 level in exosomes released by ADSCs overexpressing miR-21. In vitro matrigel angiogenesis assay showed that exosomes secreted by ADSCs overexpressing miR-21 significantly promoted the vascularization of HUVEC cells (an endothelial cell line). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) revealed an upregulation of HIF-1α, VEGF, SDF-1, p-Akt, p-ERK1/2 and downregulation of PTEN in response to miR-21 overexpression, indicating that miR-21 enriched exosomes induced angiogenesis through Akt and ERK activation and also HIF-1α and SDF-1 expression. Our work suggests that exosomes from ADSCs that overexpressing miR-21 can potentially promote vascularization and therefore the transplantation of exosomes from their culture may be suitable for clinical effort in regenerative medicine.

Circulating microRNAs as potential cancer biomarkers: the advantage and disadvantage.

MicroRNAs are endogenous single-stranded non-coding small RNA molecules that can be secreted into the circulation and exist stably. They usually exhibit aberrant expression under different physiological and pathological conditions. Recently, differentially expressed circulating microRNAs were focused on as potential biomarkers for cancer screening. We herein review the role of circulating microRNAs for cancer diagnosis, tumor subtype classification, chemo- or radio-resistance monitoring, and outcome prognosis. Moreover, circulating microRNAs still have several issues hindering their reliability for the practical clinical application. Future studies need to elucidate further potential application of circulating microRNAs as specific and sensitive markers for clinical diagnosis or prognosis in cancers.

In vitro Flow Perfusion Maintaining Long-term Viability of the Rat Groin Fat Flap: A Novel Model for Research on Large-scale Engineered Tissues.

BACKGROUND: Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. METHODS: A fat flap based on the superficial inferior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was performed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. RESULTS: Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. CONCLUSIONS: Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.

Metabolic reprogramming in keloid fibroblasts: Aerobic glycolysis and a novel therapeutic strategy.

Keloids, tumor-like fibroproliferative cutaneous lesions, were reported in metabolic disturbance. However, the metabolic character remains unclear. The purpose of this study is to determine if glycolytic reprogramming is important for the pathogenesis of keloids and to assess the inhibition potential of glycolysis in keloid treatment. An intracellular metabolic profile assay was used to compare metabolic phenotypes between normal skin fibroblasts and keloid fibroblasts (NFs and KFs). Our data indicated that KFs underwent reprogramming of their metabolic phonotype from oxidative phosphorylation to aerobic glycolysis (Warburg effect) with augmented glycolysis and glycolytic capacity. Both gene and protein assays showed that the expression of glycolytic enzymes was upregulated in KFs compared to NFs. Our data showed higher glucose influx and lactate production in KFs compared to NFs. Furthermore, the proliferation of KFs was suppressed in a dose-dependent and time-dependent manner after inhibition of glycolysis with 2-deoxy-glucose (2-DG). Taken together, these findings suggested that keloids underwent a reprogrammed metabolic phenotype of aerobic glycolysis. This was essential for keloid hyperplasia, and glycolytic inhibitors might provide a potential treatment for keloids.

[Different effects of simvastatin on keloid fibroblasts under hypoxia and TGF-ß1 treatment].

Objective: To explore the effect of simvastatin on the proliferation, apoptosis and protein expressions of keloid fibroblasts under normoxia,hypoxia or TGF-ß1 treatment. Methods: Keloid fibroblasts (KFs) were isolated by explants culture method. KFs were treated with different concentrations of simvastatin under normoxia or hypoxia (2％ O2) for 24 h and 48 h. The effects of simvastatin on cell proliferation were detected by CCK-8.Flow cytometer was used to detect the apoptosis of KFs treated with 10 µ mol/L simvastatin for 24 h or 48 h under normoxia, hypoxia or 10 ng/ml TGF-ß1 treatment. Then the expressions of keloid-related proteins were analyzed by Western Blot. Results: It showed that simvastatin could inhibit the proliferation of KFs in a concentration-and time-dependent manner with the concentration range of 10-500 µ mol/L for 24 h and 0.1-500 µ mol/L for 48 h. This inhibitory effect could be significantly enhanced when cells were incubated under hypoxia for 48h with 10-500 µ mol/L simvastatin.10 µ mol/L simvastatin could not influence the apoptosis of KFs under normoxia or TGF-ß1 treatment, neither incubated for 24 h nor 48 h.When incubated under hypoxia,10 µ mol/L simvastatin could significantly induce the apoptosis of KFs, with the rate of 155.6％ for 24 h and 478.8％ for 48 h, compared with no-drug control. There are no significant influences on the expression of type Ⅰ collagen, CTGF or TIMP-1 when KFs were treated with 10 µ mol/L simvastatin under normoxia for 48 h. When incubated with 10 ng/ml TGF-ß1 together with 10 µmol/L simvastatin for 48 h, the expression of CTGF was significantly inhibited. KFs treated with 10 µ mol/L simvastatin under hypoxia for 48 h showed a significant decrease of type Ⅰ collagen and CTGF, and a significant increase of TIMP-1. Conclusions: Simvastatin has different effects on the proliferation, apoptosis and protein expressions of KFs in a dosedependent manner under different conditions. The effects are enhanced under hypoxia.

[Mitochondrial dysfunctions of keloid fibroblasts and it' s effects on cell metabolic functions].

Objective: To compare the differences of mitochondrial functions between keloid fibroblasts and normal skin fibroblasts and explore its relationship with cell proliferation. Methods: Keloid fibroblasts (KFb) and normal skin fibroblasts (NFb) were isolated by explants culture method. KFb and NFb were cultured under normoxia or hypoxia (2％ O2).Differences of cell proliferation were detected by CCK-8.Flow cytometer was used to detect the content of mitochondria and reactive oxygen species (ROS) in KFb and NFb. Ultra-structures of mitochondria in KFb and NFb were observed by transmission electron microscope (TEM).Mitochondria fusion/fission related genes MFN1,MFN2 and FIS1 were detected by RT-PCR. Oxygen consumption rate, lactate production and ATP contents were determined by spectrophotometry. Results: KFb showed a higher proliferation rate compared with NFb, especially under hypoxia. The oxygen consumption rate, ATP content, lactate production and ROS of KFb were lower than NFb under normoxia. After incubated under hypoxia, there was a significant increase in oxygen consumption, ATP content, lactate production and ROS in KFb, while NFb showed less increase compared with KFb. KFb had 15.33％ more mitochondrion than NFb, and expressions of MFN1, MFN2, FIS1 in KFb were 33.27％,113.39％ and 20.34％ higher compared with NFb. Under TEM, KFb showed an increase of enlarged mitochondrion, with disrupted inner membrane and loss of cristae. Conclusions: KFb may have dysfunctions of mitochondrion which lead to changes of cell metabolism and continuous proliferation of KFb.

Upregulated periostin promotes angiogenesis in keloids through activation of the ERK 1/2 and focal adhesion kinase pathways, as well as the upregulated expression of VEGF and angiopoietin­1.

Periostin, a secreted extracellular matrix protein, is highly expressed in wound healing and in various types of human cancer and is involved in angiogenesis. Keloids, considered dermal benign tumors, are granulomatous lesions characterized by capillary proliferation. However, the underlying regulatory mechanism of angiogenesis in keloids remains to be elucidated. The present study aimed to examine the effect of periostin on angiogenesis in keloids. The expression of periostin was upregulated and the vessel density was higher in human keloids compared with normal tissue, observed following staining with CD31 and CD105. Periostin demonstrated a markedly positive correlation with blood vessel density, which was assessed using CD31 staining (r=0.711; P<0.01) and a weak correlation was observed using CD105 staining (r=0.251; P<0.01). Conditioned medium from keloid fibroblasts (KFs) promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs) compared with normal fibroblasts and this effect may have been abrogated by the short hairpin RNA knockdown of periostin. Treatment with recombinant human periostin promoted the migration and tube formation of HUVECs by activating the extracellular signal­regulated kinase 1/2 and focal adhesion kinase signaling pathway. In addition, periostin increased the secretion of vascular endothelial growth factor and angiopoietin­1 in the KFs. In conclusion, these data suggested that upregulation in the level of periostin may promote angiogenesis directly and indirectly in keloids and may be a key factor in keloid development. Periostin may, therefore, be a promising therapeutic target in the treatment of keloids and other angioproliferative diseases.