Traumatic acid inhibits ACSL4 associated lipid accumulation in adipocytes to attenuate high-fat diet-induced obesity.

Obesity is a major health concern that lacks effective intervention strategies. Traumatic acid (TA) is a potent wound-healing agent in plants, considered an antioxidant food ingredient. This study demonstrated that TA treatment significantly reduced lipid accumulation in human adipocytes and prevented high-fat diet induced obesity in zebrafish. Transcriptome sequencing revealed TA-activated fatty acid (FA) degradation and FA metabolism signaling pathways. Moreover, western blotting and quantitative polymerase chain reaction showed that TA inhibited the expression of long-chain acyl-CoA synthetase-4 (ACSL4). Overexpression of ACSL4 resulted in the reversal of TA beneficiary effects, indicating that the attenuated lipid accumulation of TA was regulated by ACSL4 expression. Limited proteolysis-mass spectrometry and microscale thermophoresis were then used to confirm hexokinase 2 (HK2) as a direct molecular target of TA. Thus, we demonstrated the molecular basis of TA in regulating lipid accumulation and gave the first evidence that TA may function through the HK2-ACSL4 axis.

Differences in the metabolic profiles of brown and white adipocytes based on secretomics.

Brown adipocytes and white adipocytes play important roles in systemic metabolism and energy homeostasis. Recent studies have demonstrated that white adipocytes and brown adipocytes secrete numerous adipokines and thus act as endocrine cells. However, differences in the metabolites secreted from white adipocytes and brown adipocytes have never been reported. In this study, we assessed the metabolites secreted from white adipocytes and brown adipocytes. In total, the levels of 47 metabolites in brown adipocytes were significantly different from those in white adipocytes, with 31 high and 16 low in brown adipocytes as compared with those in white adipocytes. We classified these secreted metabolites as amino acids and peptides, fatty acids, and conjugates, glycerophosphocholines, furanones, and trichloroacetic acids. In addition, we identified the glycerophospholipid metabolism activated in white adipocytes, and these differentially expressed metabolites were associated with the mitogen-activated protein kinase pathway and Janus kinase-signal transducer and activator of transcription signaling pathway according to the Ingenuity Pathway Analysis (IPA) software analysis. This study revealed novel metabolites secreted from brown adipocytes and white adipocytes, and these metabolites from adipocytes may perform specific biological functions based on the type of adipocyte that secretes them, and this forms the material basis of the interaction between adipocytes and other cells.

Brown adipocyte activation mediates lipid metabolism through exosomal tRNA-derived fragments.

Human obesity is related with intrinsic impairments of adipocyte lipolysis and ectopic lipid accumulation. Small regulatory RNAs, such as tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs), are enriched in exosomes and play a crucial role in lipid metabolism. To determine certain tRFs for lipolysis, brown adipocytes were treated with forskolin. Using tRFs sequencing, 207 different expressed exosomal tRFs were determined. In forskolin samples, 145 downregulated and 62 upregulated tRFs were identified. Further, qRT-PCR validated that three notably upregulated tRFs (tRF-Gly-GCC-007, tRF-Gly-GCC-008, and tRF-Gly-GCC-009) were in accordance with the sequencing result. Target genes of tRFs were involved in positive regulation of protein phosphorylation and cell adhesion process by significantly downregulating UCHL1 expression, which might participate in lipolysis. This study might provide therapeutic targets and potential diagnostic biomarkers for obesity treatment.

Transcriptomics profiling reveal the heterogeneity of white and brown adipocyte.

The marker genes associated with white adipocytes and brown adipocytes have been previously identified; however, these markers have not been updated in several years, and the differentiation process of preadipocytes remains relatively fixed. Consequently, there has been a lack of exploration into alternative differentiation schemes. In this particular study, we present a transcriptional signature specific to brown adipocytes and white adipocytes. Notably, our findings reveal that ZNF497, ZIC1, ZFY, UTY, USP9Y, TXLNGY, TTTY14, TNNT3, TNNT2, TNNT1, TNNI1, TNNC1, TDRD15, SOX11, SLN, SFRP2, PRKY, PAX3KLHL40, PAX3, INKA2-AS1, SOX11, and TDRD15 exhibit high expression levels in brown adipocytes. XIST, HOXA10, PCAT19, HOXA7, PLSCR3, and AVPR1A exhibited high expression levels in white adipocytes, suggesting their potential as novel marker genes for the transition from white to brown adipocytes. Furthermore, our analysis revealed the coordinated activation of several pathways, including the PPAR signaling pathway, focal adhesion, retrograde endocannabinoid signaling, oxidative phosphorylation, PI3K-Akt signaling pathway, and thermogenesis pathways, in brown adipocytes. Moreover, in contrast to prevailing culture techniques, we conducted a comparative analysis of the differentiation protocols for white preadipocytes and brown preadipocytes, revealing that the differentiation outcome remained unaffected by the diverse culture schemes employed. However, the expression levels of certain marker genes in both adipocyte types were found to be altered. This investigation not only identified potential novel marker genes for adipocytes but also examined the impact of different differentiation methods on preadipocyte maturation. Consequently, these findings offer significant insights for further research on the differentiation processes of diverse adipocyte subtypes.

Methionine sulfoxide suppresses adipogenic differentiation by regulating the mitogen-activated protein kinase signaling pathway.

In this study, methionine sulfoxide (MetO) was identified as an active metabolite that suppresses adipogenesis after screening obese individuals versus the normal population. MetO suppressed the gene and protein expression of CCAAT/enhancer binding protein (C/EBP) α, adipocyte fatty acid binding protein 4 (FABP4), and the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) during human preadipocyte (HPA) differentiation. Adipogenesis decreased following MetO treatment; however, the preadipocyte number, proliferation, and apoptosis were unaffected. The activity of phosphorylated extracellular signal-related kinase (P-ERK) of the mitogen-activated protein kinase (MAPK) pathway was significantly inhibited in HPA after MetO treatment. Furthermore, treatment of preadipocytes with the selective P-ERK1/2 agonist Ro 67-7476 abolished the effect of MetO against adipogenesis suggesting that MetO function is dependent on the MAPK pathway. The mechanistic insights of adipogenesis suppression by MetO presented in this study shows its potential as an antiobesity drug.

Metabonomics profile analysis in inflammation-induced preterm birth and the potential role of metabolites in regulating premature cervical ripening.

BACKGROUND: Preterm birth (PTB) is the primary cause of infant morbidity and mortality. Moreover, previous studies have established that PTB is related to premature cervical ripening. However, the underlying mechanism remains to be elucidated. This study sought to identify differentially expressed metabolites and investigate their potential biological functions in PTB. METHODS: Pregnant C57BL/6 J mice were treated with either LPS or normal saline and cervical alterations before labor were detected by staining. Metabolic profiles in the plasma of PTB and control mice were examined through non-targeted metabonomics analyses, quantitative polymerase chain reaction and immunofluorescence staining were performed on human cervical smooth cells. RESULTS: The study demonstrated that the mRNA and protein levels of α-SMA, SM-22, and calponin in cervical smooth muscle cells of PTB mice were lower while OR was higher at both mRNA and protein levels compared to the CTL group. A total of 181 differentially expressed metabolites were analyzed, among them, 96 were upregulated, while 85 were downregulated in the PTB group. Differentially expressed metabolites may play a role in STAT3, RhoA, mTOR, TGF-ß, and NK-κB signaling pathways. Furthermore, when treated with taurine, the levels of α-SMA and SM-22 in human cervical smooth muscle cells were elevated, whereas that of connexin-43 was decreased. CONCLUSION: Our study highlighted the changes of metabolites in the peripheral blood changed prior to PTB and revealed that these differentially expressed metabolites might participate in the development of premature cervical ripening. Taurine was identified as an important metabolite may modulate human cervical smooth muscle cells. Our study provided new insights into the mechanism underlying premature cervical ripening in PTB.

Liposome-encapsulated peptide PDBSN ameliorates high-fat-diet-induced obesity and improves metabolism homeostasis.

In recent years, the obese and overweight population has increased rapidly, which has become a worldwide public health problem. However, effective medication is lacking. Our previous study identified a novel peptide, PDBSN (GLSVADLAESIMKNL), that could significantly restrict adipocyte differentiation in vitro, but its in vivo function has not been determined. Thus, in this study, we encapsulated the peptide into liposomes attached with two ligands (visceral-adipose-tissue-targeting peptide and cell-penetrating peptide) to improve stability and specificity. We then tested the peptide's function in HFD (high-fat diet)-induced obese mice and found that PDBSN could reduce weight gain and improve insulin resistance as well as lipid homeostasis. These results suggest that PDBSN may be a potential candidate for anti-obesity drug discovery.

Effect of helmet therapy in the treatment of positional head deformity.

AIM: Most positional head deformities can be treated conservatively with postural correction training or a head orthosis ('helmet'). We aimed to investigate whether infants with helmet therapy have cosmetic improvement in head deformity. METHODS: A total of 376 infants at age 2-40 months who were diagnosed with mild-moderate-severe positional head deformity were enrolled. Among these infants, 101 infants were treated with helmet therapy or postural correction training. After matching by infant's age and time of therapy, three retrospective cohort studies of 56 infants were conducted for infants with plagiocephaly, brachycephaly and asymmetrical brachycephaly, respectively. The cephalic ratio (CR), radial symmetry index (RSI), cranial vault asymmetry (CVA) and cranial vault asymmetry index (CVAI) were compared between two groups before and after treatment. RESULTS: Before treatment, no significant differences in CR, RSI, CVA and CVAI between groups were found. After treatment, compared with the postural correction training group, the helmet therapy group had significant improvements in CR, RSI, CVA or CVAI (Plagiocephaly: PCVA = 0.017, PCVAI = 0.028; Brachycephaly: PCR = 0.002; Asymmetrical brachycephaly: PRSI = 0.002, PCVA < 0.001, PCVAI < 0.001). Moreover, there was no significant difference in head circumference growth between the groups. CONCLUSIONS: Helmet therapy may be more effective in the treatment of mild-moderate-severe positional head deformity than postural correction training in infants. And helmet therapy may not hinder head circumference growth.

The long non-coding RNA Gm10768 activates hepatic gluconeogenesis by sequestering microRNA-214 in mice.

Overactivated hepatic gluconeogenesis contributes to the pathogenesis of metabolic disorders, including type 2 diabetes. Precise control of hepatic gluconeogenesis is thus critical for maintaining whole-body metabolic homeostasis. Long non-coding RNAs (lncRNAs) have been shown to play key roles in diseases by regulating diverse biological processes, but the function of lncRNAs in maintaining normal physiology, particularly glucose homeostasis in the liver, remains largely unexplored. We identified a novel liver-enriched long non-coding RNA, Gm10768, and examined its expression patterns under pathophysiological conditions. We further adopted gain- and loss-of-function strategies to explore the effect of Gm10768 on hepatic glucose metabolism and the possible molecular mechanism involved. Our results showed that the expression of Gm10768 was significantly increased in the liver of fasted mice and was induced by gluconeogenic hormonal stimuli. Functionally, overexpression of Gm10768 activated hepatic gluconeogenesis in a cell-autonomous manner. In contrast, depletion of Gm10768 suppressed hepatic glucose production both in vitro and in vivo Adenovirus-mediated hepatic knockdown of Gm10768 improved glucose tolerance and hyperglycemia of diabetic db/db mice. Mechanistically, Gm10768 sequestrated microRNA-214 (miR-214) to relieve its suppression on activating transcription factor 4 (ATF4), a positive regulator of hepatic gluconeogenesis. Taken together, we identified Gm10768 as a new lncRNA activating hepatic gluconeogenesis through antagonizing miR-214 in mice.

Identification of intracellular peptides associated with thermogenesis in human brown adipocytes.

OBJECTIVES: Currently, brown adipose tissue (BAT) is a therapeutic target in obesity and diabetes, but the mechanism of BAT activation remains unclear. Because increasing emphasis has been placed on the role of intracellular peptides in biological processes, we conducted a study to gain insight into the mechanism of BAT activation by using a peptidomic approach and then attempted to identify peptides that are capable of activating BAT. METHODS: In the present study, we generated the peptidomic profile of the intracellular peptides in brown adipocytes treated with forskolin (FSK) using a peptidomic approach. Then, the differentially expressed peptides were evaluated via Gene Ontology (GO) enrichment, KEGG pathway, and protein-protein interaction (PPI) network analysis. Finally, we selected candidate peptides for further validation via assessing the expression levels of UCP-1 and PGC-1α in brown adipocytes exposed to the peptides. RESULTS: A total of 4,370 peptides were identified, of which 951 were upregulated and 379 were downregulated after FSK treatment. Bioinformatic analysis demonstrated that the ECM-receptor interaction GO term was the most enriched and that collagen alpha-related proteins exhibited the highest degree of PPI. Four peptides separately derived from TSC22 domain family protein 1 (T22D1), bromodomain and WD repeat-containing protein 1 (BRWD1), protein piccolo (PCLO), and collagen alpha-1 (III) chain (CO3A1) increased the expression levels of UCP-1 and PGC-1α. CONCLUSIONS: ECM-receptor interaction may play an important role in the process of FSK-stimulated BAT activation, and the pT22D1tide, pBRWD1tide, pPCLOtide, and pCO3A1tide peptides potentially promote BAT thermogenesis.

A novel peptide suppresses adipogenic differentiation through activation of the AMPK pathway.

Obesity rates have risen rapidly over the past several decades and obesity is now a global public health challenge. The reduction of excessive adipogenesis is thought to be an effective intervention for obesity and obesity-related metabolic diseases such as type 2 diabetes. In this study, a novel peptide PDBSN was identified that functions to suppress adipogenesis. In both human preadipocytes and mouse adipose-derived stem cells (ADSCs), PDBSN exhibited a suppressive effect on the accumulation of lipids and the expression of genes as well as their corresponding proteins (CCAAT/enhancer binding protein (C/EBP)ß, C/EBPα and nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ)) relevant to adipogenic cell differentiation. Although adipogenesis decreased, the preadipocyte number and proliferation were not influenced by the PDBSN treatment. Apoptosis and the cell cycle were also determined to not have a role in the action of PDBSN. Mechanistically, the activity of the AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway was markedly increased upon PDBSN treatment. Moreover, treatment of preadipocytes with compound C, a selective AMPK inhibitor, abolished the effect of PDBSN in anti-adipogenesis, suggesting that the function of PDBSN relied on the AMPK pathway. These results suggest an effective role for PDBSN in suppressing adipogenesis and show potential for anti-obesity drug discovery.

Peptidomic analysis of zebrafish embryos exposed to polychlorinated biphenyls and their impact on eye development.

Polychlorinated biphenyls (PCBs), a class of persistent organic pollutant, are closely related to abnormal eye development in children. However, little is known regarding the role of peptides in the development of PCB-induced ocular dysplasia. To characterize the nature of PCB exposure on peptides involved in the development of the ocular system, we used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to detect differential expression of peptides between normal and PCB-exposed zebrafish embryos. A total of 7900 peptides were analyzed, 90 of which were differentially expressed, with 29 being up-regulated and 61 down-regulated. These peptides were investigated using ingenuity pathway analysis (IPA) and gene ontology (GO) analysis to explore their role in eye development. This study identified 18 peptides associated with the development of the optic nerve and ocular system in the PCB-exposure group, as well as 10 peptides that are located in the functional domain of their precursor proteins. These peptides provide potential biomarkers for the treatment of ocular dysplasia caused by PCBs and may help us understand the mechanism of abnormal eye development caused by organic pollutants.

The sonic hedgehog signaling pathway is suppressed following PCB1254 exposure during retinal development.

Polychlorinated biphenyl (PCB) has been reported to have detrimental effects on retinal development. In order to explore the role of Shh signaling in retinal development after PCB1254 exposure in vivo and in vitro, zebrafish and RGC-5 retinal cell line were used. Compared with the controls, PCB exposure inhibited proliferation and increased the apoptosis levels. The expression of Shh mRNA decreased in the PCB1254 -treated groups both in vivo and in vitro compared with that of the controls. The ptch2 mRNA expression increased in the experimental groups. The expression of gli2 mRNA decreased in the PCB1254 -treated groups. Immunofluorescence and western blotting assays confirmed that the expression of Shh proteins decreased in PCB1254 -treated groups compared with control groups. Moreover, ptch2 protein levels increased in the PCB1254 -treated groups as well as the decreased protein expressions of gli1 and gli2. These results demonstrated that Shh signaling pathway may participate in the damage of retinal development caused by PCB1254 exposure, providing evidence that eye diseases could be caused by environmental pollutants.

Fluorometric determination of the CCAAT/enhancer binding protein alpha by using gold nanoparticles and a labeled protein-binding DNA.

A method is described for the determination of the CCAAT/enhancer binding protein alpha (C/EBPα) which is a regulator in adipocyte differentiation. The method is based on quenching of the red fluorescence (with excitation/emission maxima at 548/562 nm) of Cy3-labeled DNA if it becomes adsorbed on positively charged gold nanoparticles (AuNPs). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored. The fluorescent signal increases linearly in the 0.05 to 600 ng·mL-1 µM C/EBPα concentration range, and the detection limit is 29 pg·mL-1. The method is specific and was applied to analyze cell lysates and in-situ. Graphical abstractSchematic representation of a fluorometric method for determination of the CCAAT/enhancer binding protein alpha (C/EBPα). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored.

GM13133 is a negative regulator in mouse white adipocytes differentiation and drives the characteristics of brown adipocytes.

Obesity is tightly associated with the disturbance of white adipose tissue storing excess energy. Thermogenic adipocytes (brown and beige) exert a critical role of oxidizing nutrients at the high rates through non-shivering thermogenesis. The recruitment of brown characteristics in white adipocytes, termed browning, has been considered as a promising strategy for treating obesity and associated metabolic complications. Recently, long noncoding RNAs play a crucial role in regulating tissue development and participating in disease pathogenesis, yet their effects on the conversion of white into brown-like adipocytes and thermogenic function were not totally understood. Here, we identified a mouse brown adipose specific expressed lncRNA, termed GM13133. Moreover, a considerable amount of GM13133 is expressed in adipocytes and actively modulated by cold, ß3 -adrenergic agonist and cAMP stimuli, implying a potential role in the conversion from white to brown adipocytes. Overexpression of GM13133 did not affect the proliferation of mouse white pre-adipocytes, but inhibited white adipocyte differentiation by decreasing lipid accumulation. The forced expression of GM13133 also significantly drove the conversion of white into brown-like adipocytes with the enhanced mitochondrial biogenesis and the induced expression of brown adipocytes specific markers. A global mRNA analysis further indicated the possible regulatory role of cAMP signaling pathway in GM13133 mediated white-to-brown adipocytes conversion. Our results identified a lncRNA-mediated modulation in primary mouse white adipocyte differentiation and indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.

High folate intake contributes to the risk of large for gestational age birth and obesity in male offspring.

Folate supplementation is recommended before and during early pregnancy to prevent neural tube defects, but the effect of red blood cell (RBC) folate on large for gestational age (LGA) is still unknown. We performed a nested case-control study including 542 LGA cases and 1,084 appropriate for gestational age (AGA) controls to examine the association of RBC folate concentrations with risk of LGA. Then, male offspring of dams fed basic folic acid (2 mg/kg, control) or 10-fold folic acid (20 mg/kg, HFol) diet before and during pregnancy were used to explore the effect of high folate intake on birth weight and long-term effects. We observed higher RBC folate concentrations in the cases compared to controls (p = 0.039). After adjustment for maternal age, BMI at enrollment, gestational weeks at enrollment, gestational weeks at delivery and infant gender, higher RBC folate levels were significantly associated with increased risk of LGA (Ptrend = 0.003). Interestingly, male offspring of HFol dams showed the higher birth weight, elevated levels of post loading blood glucose at 9 and 13 weeks post-weaning and increased triglyceride (TG) and total cholesterol (TC) levels at 17 weeks post-weaning. Furthermore, we observed that high folate intake increased the proliferation and differentiation of adipose cells. Our results suggest that maternal high folate intake confers the risk of LGA birth and accelerates the development of obesity in male offspring.

Identification and characterization of metformin on peptidomic profiling in human visceral adipocytes.

To gain insight into the effect of metformin on losing weight from peptidomic perspective and to screen potential active peptides for reducing fat lipid deposition. After determining the proper concentration of metformin on human primary visceral adipocytes, we constructed a comparative peptidomic profiling between control and metformin treatment group (n = 3) using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by liquid chromatography tandem mass spectrometry. We identified and quantified 3065 non-redundant peptides, 304 of which were differentially expressed after metformin treatment, 206 peptides were up regulated and 98 peptides were down regulated significantly. Gene ontology (GO) enrichment and pathway analysis were performed to study differentially peptides though their precursor proteins. We concluded three peptides located within the functional domains of their precursor proteins could be candidate bioactive peptides for obesity. On one hand, these results confirmed the versatile effects of metformin on adipocyte and advance our current understanding of metformin, on the other hand, these identified peptides might play putative roles in treatment of obesity.

Genome-wide analysis reveals that altered methylation in specific CpG loci is associated with childhood obesity.

Over the past decades, the epidemic of childhood obesity has greatly increased, and it has recently become a global public health concern. Methylation, serving as a crucial regulator of the gene-environment interaction, has exhibited a strong association with obesity. In this study, we aimed to evaluate the relationship between DNA methylation and childhood obesity, and further uncover the potential association of aberrantly methylated genes with obesity. DNA samples of peripheral blood leukocytes from three obese subjects (mean BMI: 21.67) and 4 age/sex matched controls (mean BMI: 14.92) were subjected to Infinium Human Methylation 450 Bead Array analysis. A total of more than 4 85 000 methylation sites were identified across the genome, and 226 methylated CpGs (DMCpGs) were differentially methylated between these two groups. Subsequent Gene Ontology (GO) and KEGG Pathway analyses showed that these DMCpGs were mainly engaged in immunity and lipoprotein metabolism, indicating their physiological significance. Further verification of the candidate CpG sites within the HDAC4, RAX2, APOA5, CES1, and SLC25A20 gene loci, were performed using bisulfite sequencing PCR (BSP) in a cohort of 42 controls and 39 obese cases. The results revealed that methylation levels within HDAC4 and RAX2 loci were positively associated with obesity, while the methylation levels of loci within APOA5 and CES1 loci were negatively correlated with obesity. Thus, alterations in methylation of CpG sites of specific genes may contribute to childhood obesity, which provide novel insights into the aetiology of obesity.

Maternal Vitamin D Status and Risk of Gestational Diabetes: a Meta-Analysis.

BACKGROUND/AIMS: Whether maternal vitamin D deficiency is associated with gestational diabetes remains controversial. This meta-analysis aimed to systematically evaluate published evidence on the association between maternal vitamin D status and the risk of gestational diabetes. METHODS: We retrieved relevant articles from the PubMed, Medline and Embase databases up to May 2017 for observational studies investigating the association between vitamin D status and the risk of gestational diabetes. Odds ratios (OR) or risk ratios (RR) from individual studies were pooled using the fixed and random effect models. RESULTS: The meta-analysis of 29 observational studies included 28,982 participants, of which 4,634 were diagnosed with gestational diabetes, and showed that maternal vitamin D insufficiency was associated with a significantly increased risk of gestational diabetes by 39% (pooled OR = 1.39, 95%CI = 1.20-1.60) with moderate heterogeneity (I2 = 50.2%; P = 0.001). Moreover, the 25(OH)D level was significantly lower in gestational diabetes cases than in controls with a pooled effect of -4.79 nmol/L (95% CI = -6.43, -3.15). Significant heterogeneity was also detected (I2 = 65.0%, P < 0.001). Further subgroup analysis indicated that this association was also evident in most subpopulations. CONCLUSION: This meta-analysis indicated a significant association between vitamin D insufficiency and increased risk of gestational diabetes. Further well-designed large-scale clinical trials are essential to verify this association.

Autocrine VEGF and IL-8 Promote Migration via Src/Vav2/Rac1/PAK1 Signaling in Human Umbilical Vein Endothelial Cells.

BACKGROUND/AIMS: Pro-angiogenic factors VEGF and IL-8 play a major role in modulating the migratory potential of endothelial cells. The goal of this study was to investigate the effect of autocrine VEGF and IL-8 in the form of self-conditioned medium (CM) on human umbilical vein endothelial cells (HUVECs). METHODS: Enzyme-linked immunosorbent assay (ELISA) examined the automatic secretion of VEGF and IL-8 protein by HUVECs. Western blot, small interfering RNA (siRNA), pulldown and Transwell assays were used to explore the role and the mechanism of autocrine VEGF and IL-8 in migration of HUVECs. RESULTS: Neutralizing VEGF and IL-8 in CM significantly abrogated CM-induced migration of HUVECs. Autocrine VEGF and IL-8 increased Src phosphorylation, Rac1 activity and PAK1 phosphorylation in a time dependent manner. Additionally, blocking Rac1 activity with Rac1 siRNA largely abolished autocrine VEGF and IL-8-induced cell migration. Vav2 siRNA suppressed autocrine VEGF and IL-8-induced Rac1 activation and cell migration. Furthermore, blocking Src signaling with PP2, a specific inhibitor for Src, markedly prevented autocrine VEGF and IL-8-induced Vav2 and Rac1 activation as well as consequently cell migration. PAK1 siRNA also significantly abolished autocrine VEGF and IL-8-induced cell migration. CONCLUSIONS: We demonstrated for the first time that autocrine VEGF and IL-8 promoted endothelial cell migration via the Src/Vav2/Rac1/PAK1 signaling pathway. This finding reveals the molecular mechanism in the increase of endothelial cell migration induced by autocrine growth factors and cytokines, which is expected to provide a novel therapeutic target in vascular diseases.