The role of altered fatty acid in pathological scars and their dermal fibroblasts.

PURPOSE: The proposed pathological mechanism for scar formation is controversial, and increased attention has been paid to the fatty acids (FAs) in the formation of pathological scars. Notably, FAs are known to be important in inflammation and mechanotransduction, which is closely related to scar formation. Therefore, it is necessary to clarify the roles of FA in scar formation. METHODS: Hypertrophic scar and keloid formed for more than a year and without other treatment, as well as normal skin samples were obtained from patients who underwent plastic surgery. Finally, keloids (n = 10), hypertrophic scars (n = 10), and normal skin samples (n = 10) were collected under informed consent. Primary dermal fibroblasts were isolated and cultured. The amount and variety of FAs were detected by lipid chromatography-mass spectrometry. Immunohistochemistry, real-time PCR, and western blotting were used to verify the expression of sterol regulatory element-binding protein-1 (SREBP1) and fatty acid synthase (FASN) in the samples and their fibroblasts. Student's t-test, ANOVA, and orthogonal partial least square discriminant analysis were performed for statistical analysis (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). RESULTS: Compared with full-thickness normal skin, there were 27 differential FAs in keloids and 15 differential FAs in hypertrophic scars (∗p < 0.05 and variable influence on projection >1.0). The expression of SREBP1 and FASN was lower in pathological scars both at mRNA and protein levels (all ∗p < 0.05). However, the mRNA levels of SREBP1 (∗∗∗p = 0.0002) and FASN (∗∗∗p = 0.0021) in keloid-derived fibroblasts were higher than that in normal skin fibroblasts (NFBs), while the expression in hypertrophic scar-derived fibroblasts was lower than that in NFBs (both ∗p < 0.05). Whereas there was no significant difference in FASN protein expression between keloid-derived fibroblasts and NFBs (p > 0.05). CONCLUSION: FAs involved in pathological scars are abnormally changed in scar formation. Thus, fatty acid-derived inflammation and de novo synthesis pathway of FA may play a key role in the formation of pathological scars.

Yin Yang-1 suppresses CD40 ligand-CD40 signaling-mediated anti-inflammatory cytokine interleukin-10 expression in pulmonary adventitial fibroblasts by promoting histone H3 tri-methylation at lysine 27 modification on interleukin-10 promoter.

During the pathogenesis of early pulmonary arterial hypertension (PAH), pulmonary arterial adventitial fibroblast act as an initiator and mediator of inflammatory processes that predispose vessel walls to excessive vasoconstriction and pathogenic vascular remodeling. Emerging studies report that Yin Yang-1 (YY-1) plays important roles in inflammatory response and vascular injury. Our recent study finds that activation of CD40 ligand (CD40L)-CD40 signaling promotes pro-inflammatory phenotype of pulmonary adventitial fibroblasts. However, whether YY-1 is involved in CD40L-CD40 signaling-triggered inflammatory response in pulmonary adventitial fibroblasts and its underlying mechanism is still unclear. Here, we show that soluble CD40L (sCD40L) stimulation promotes YY-1 protein expression and suppresses anti-inflammatory cytokine, interleukin 10 (IL-10) expression in pulmonary adventitial fibroblasts, while YY-1 knockdown prevents sCD40L-mediated reduction of IL-10 expression via enhancing IL-10 gene transactivation. Further, we find that sCD40L stimulation significantly increases histone H3 tri-methylation at lysine 27 (H3K27me3) modification on IL-10 promoter in pulmonary adventitial fibroblasts, and YY-1 knockdown prevents the effect of sCD40L on IL-10 promoter by reducing the interaction with enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, binding to IL-10 promoter. Moreover, we find that sCD40L stimulation promotes YY-1 protein, but not messenger RNA (mRNA) expression, via decreasing N6-methyladenosine methylation on YY-1 mRNA to suppress YTHDF2-medicated mRNA decay. Overall, this in-depth study shows that the activation of CD40L-CD40 signaling upregulates YY-1 protein expression in pulmonary adventitial fibroblasts, which results in increasing YY-1 and EZH2 binding to the IL-10 promoter region to enhance H3K27me3 modification, eventually leading to suppression of IL-10 transactivation. This study first uncovers the roles of YY-1 on CD40L-CD40 signaling-triggered inflammatory response in pulmonary adventitial fibroblasts.

RNA-binding protein SFPQ cooperates with HDAC1 to suppress CD40 transcription in pulmonary adventitial fibroblasts.

Pulmonary artery adventitial fibroblasts, the most abundant cellular constituent of adventitia, are often the first to be activated and reprogrammed to then influence the tone and structure of the vessel wall in pulmonary arterial hypertension (PAH). Our previous study found that interruption of CD40 ligand (CD40L)-CD40 signaling improves the efficacy of transplanted endothelial progenitor cells in monocrotaline induced-PAH. However, whether CD40L-CD40 signaling is involved in the activation of adventitial fibroblasts in PAH and whether Drosophila behavior human splicing (DBHS) protein family members have any roles during adventitial fibroblasts activation are completely unclear. Here, we show that soluble CD40L (sCD40L) stimulation progressively increases pro-inflammatory activity, proliferation, and migration of pulmonary adventitial fibroblasts. Besides, sCD40L stimulation decreases splicing factor proline- and glutamine-rich protein (SFPQ) protein (one member of DBHS protein family) expression, while SFPQ overexpression suppresses sCD40L stimulation-induced proliferation and migration of pulmonary adventitial fibroblasts by repressing CD40 transcription. Moreover, ChIP assays found that sCD40L stimulation promotes histone H3 tri-methylation at lysine 4 (H3K4me3), H3K36me3, and H3K27 acetylation (H3K27ac) modifications on CD40 promoter region in pulmonary adventitial fibroblasts, while SFPQ overexpression decreases H3K36me3 modification and increases H3K36ac on CD40 promoter region by interacting with histone deacetylase-1 (HDAC1) to inhibit CD40 transcription. This in-depth study shows that CD40L-CD40 signaling promotes activation of pulmonary adventitial fibroblasts by increasing proliferation, migration, and pro-inflammatory activity of adventitial fibroblasts, and SFPQ could inhibit CD40 transcription though switching H3K36me3 to H3K36ac modifications on its promoter by interacting with HDAC1. This study, first, uncovers the roles of SFPQ on CD40L-CD40 signaling-mediated activation of pulmonary adventitial fibroblasts.

The Role of RhoA in Neovascular-Related Functions of Endothelial Progenitor Cells Induced by Angiotensinâ ¡.

BACKGROUND/AIMS: Interference with endothelial progenitor cell (EPC) neovascularization is a novel therapeutic target for neovascular-related diseases. Angiotensin Ⅱ (Ang Ⅱ) was found to enhance new vessel formation and aggravated neovascular-related diseases. In this study, we investigated the effects of Ang Ⅱ on EPC neovascular-related functions and explored the underlying mechanisms. METHODS: EPCs were cultured from bone marrow derived mononuclear cells. The effects of Ang Ⅱ on EPC proliferation, adhesion, migration, and in vitro tube formation were investigated using the MTT assay, adhesion assay, transwell chamber assay, and in vitro tube formation assay respectively. The underlying mechanisms were explored using Western blotting assay. RESULTS: EPC adhesion, migration and in vitro tube formation were promoted by Ang Ⅱ, and the effects were reversed by RhoA/Rho-associated kinases (ROCK) signaling pathway inhibitors including C3 exoenzyme, GGTI-286 and Y-27632. The active form of RhoA was up-regulated by Ang Ⅱ and this effect was abolished by C3 exoenzyme. Moreover, RhoA silencing resulted in a notable inhibition of EPC adhesion, migration and in vitro tube formation, suggesting that RhoA activation played a pivotal role in Ang Ⅱ angiogenic effect. The results also demonstrated that phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun-NH2 kinase was elevated by Ang Ⅱ and attenuated by C3 exoenzyme, GGTI-286 and Y-27632. The enhancing effects of Ang Ⅱ on EPC adhesion, migration and in vitro vasculogenesis were reversed by p38 inhibitor SB202190 and JNK inhibitor SP600125. CONCLUSION: Ang Ⅱ may enhance EPC neovascular-related functions through activating RhoA/ ROCK and MAPK signaling pathway.

Tanshinone II A Attenuates TNF-α-Induced Expression of VCAM-1 and ICAM-1 in Endothelial Progenitor Cells by Blocking Activation of NF-κB.

BACKGROUND/AIMS: Tanshinone IIA (Tan IIA) is effective in the treatment of inflammation and atherosclerosis. The adhesion of inflammatory cells to vascular endothelium plays important role in atherogenic processes. This study examined the effects of Tan IIA on expression of adhesion molecules in tumor necrosis factor-α (TNF-α)-induced endothelial progenitor cells (EPCs). METHODS: EPCs were pretreated with Tan IIA and stimulated with TNF-α. Mononuclear cell (MNC) adhesion assay was performed to assess the effects of Tan IIA on TNF-α-induced MNC adhesion. Expression of vascular cell adhesion molecule-1 (VCAM-1)/intracellular adhesion molecule-1 (ICAM-1) and activation of Nuclear factor κB (NF-κB) signaling pathway were measured. RESULTS: The results showed that the adhesion of MNCs to TNF-α-induced EPCs and expression of VCAM-1/ICAM-1 in EPCs were promoted by TNF-α, which were reduced by Tan IIA. TNF-α increased the amount of phosphorylation of NF-κB, IκB-α and IKKα/ß in cytosolic fractions and NF-κB p65 in nucleus, while Tan IIA reduced its amount. CONCLUSION: This study demonstrated a novel mechanism for the anti-inflammatory/anti-atherosclerotic activity of Tan IIA, which may involve down-regulation of VCAM-1 and ICAM-1 through partial blockage of TNF-α-induced NF-κB activation and IκB-α phosphorylation by the inhibition of IKKα/ß pathway in EPCs.

Alteration of mevalonate pathway related enzyme expressions in pressure overload-induced cardiac hypertrophy and associated heart failure with preserved ejection fraction.

BACKGROUND: Abnormalities of the mevalonate pathway, an important cellular metabolic pathway, are common in many diseases including cardiovascular disease. The mevalonate pathway related enzyme expressions in pressure overload-induced cardiac hypertrophy and associated diastolic dysfunction remains largely unknown. This study aims to investigate whether the expression of mevalonate pathway related enzyme is altered during the progression of cardiac hypertrophy and associated diastolic dysfunction induced by pressure overload. METHODS: Male Sprague-Dawley (SD) rats were randomly divided into two groups: the suprarenal abdominal aortic coarctation (AAC) group and the sham group. RESULTS: Histological and echocardiographic assessments showed that there was a significant cardiovascular remodeling in the AAC group compared with the sham group after 3 weeks post-operatively, and the left ventricular (LV) diastolic function was reduced at 8 and 14 weeks post-operatively in the AAC group, without any change in systolic function during the study. The tissue of the heart and the abdominal aorta proximal to the coarctation showed over-expression of several enzymes, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FDPS), farnesyltransferase-α (FNTA), farnesyltransferase-ß (FNTB), geranylgeranyltransferase type I (GGTase-I) and the activation of their downstream proteins was enhanced. CONCLUSIONS: AAC induced compensatory LV hypertrophy to decompensatory diastolic dysfunction, accompanied by altered expression of several key enzymes in the mevalonate pathway.

Superparamagnetic iron oxide nanoparticles may affect endothelial progenitor cell migration ability and adhesion capacity.

BACKGROUND AIMS: Cell labeling with superparamagnetic iron oxide (SPIO) nanoparticles enables non-invasive tracking of transplanted cells. The aim of this study was to investigate whether SPIO nanoparticles have an effect on endothelial progenitor cell (EPC) functional activity and the feasibility of a protocol for labeling swine- and rat-origin EPC using SPIO nanoparticles at an optimized low dosage. METHODS: EPC were isolated from the peripheral blood of swine and bone marrow of rat and characterized. After ex vivo cultivation, EPC were labeled with SPIO nanoparticles (to make a series of final concentrations, 50, 100, 200 and 400 microg/mL) or vehicle control. We also investigated the long-term effects of 200 microg/mL SPIO nanoparticles on EPC (4, 8, 12 and 16 days after labeling). The labeling efficiency was tested through Prussian blue (PB) staining and the intracellular iron uptake was also measured quantitatively and confirmed. EPC proliferation and migration were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes and then counting the adherent cells. EPC apoptosis was evaluated using an Annexin V-FITC apoptosis kit. RESULTS: SPIO nanoparticles impaired EPC migration and promoted EPC adhesion. EPC proliferation and apoptosis were not affected. SPIO nanoparticles could label EPC efficiently at 200 microg/mL overnight without significantly affecting EPC functional activity. CONCLUSIONS: SPIO nanoparticles impaired the EPC migration ability and promoted the EPC adhesion capacity. EPC could be labeled efficiently at an appropriate concentration (200 microg/mL) without significantly affecting their functional activity.

Endothelial progenitor cells may inhibit apoptosis of pulmonary microvascular endothelial cells: new insights into cell therapy for pulmonary arterial hypertension.

BACKGROUND AIMS: Endothelial apoptosis underlies the pathophysiology of pulmonary arterial hypertension (PAH). Some factors/cytokines released by endothelial progenitor cells (EPC) have been revealed as potent inhibitors of apoptosis. The aim of this study was to investigate the effects of EPC on pulmonary microvascular endothelial cell (PMVEC) survival with the PAH condition. METHODS: PMVEC apoptosis was induced by high shear stress (HSS) with serum starvation or pro-inflammatory factors in an artificial capillary system. EPC were delivered into monocrotaline-induced PAH nude rats. RESULTS: PMVEC apoptosis under HSS and serum starvation conditions was significantly inhibited by EPC conditioned medium (CM). It was attenuated by vascular endothelial growth factor (VEGF)-A or -B blocking. EPC CM promoted PMVEC proliferation, which was weakened by VEGF-A or interleukin (IL)-8 blocking. The EPC CM caused less apoptosis of PMVEC induced by HSS plus pro-inflammatory factors. The anti-apoptotic effect of EPC CM was attenuated by blockade of either vascular endothelial growth factor receptor (VEGFR)-1 or -2. However, the pro-proliferating effect appeared to be weakened only by VEGFR-2 blocking. Both Erk1/2 and Akt phosphorylation were enhanced by EPC CM. VEGFR-2 blockage led to significant inhibition of Erk1/2 and Akt activation; VEGFR-1 blockage only of Erk1/2 activation. Human-origin VEGF co-localized with incorporated EPC in small pulmonary arterioles, and EPC transplantation resulted in down-regulation of caspase-3 expression. CONCLUSIONS: The VEGF-A/B-VEGFR-1/2-Erk1/2 signal pathway took major responsibility for the anti-apoptotic effects of EPC on PMVEC, and VEGF-A-VEGFR-2-Akt for pro-proliferating effects. Growth factors, secreted in a paracrine manner by transplanted EPC, inhibited cell apoptosis in PAH lung.

Perilipin gene 1237 T > C polymorphism is not associated with obesity risk in northern Chinese Han adults.

OBJECTIVE: To identify the association between PLIN 1237 polymorphism and obesity in Chinese Han adults. METHODS: A total of 994 adults (157 obese subjects, 322 overweight subjects, and 515 normal controls) were recruited from two rural communities. PLIN 1237 polymorphism was genotyped by polymerase chain reaction-restriction-fragment-length-polymorphism (PCR-RFLP). Association between PLIN polymorphisms and obesity status was estimated by ordinal logistic regression. RESULTS: The three genotypes of PLIN 1237 were detected with a percentage of 54.3%, 37.1%, and 8.6% in TT, TC, and CC genotypes, respectively. For the PLIN 1237 polymorphism locus, the frequency of alleles T and C was 0.73 and 0.27, respectively. The PLIN 1237 polymorphisms were in Hardy-Weinberg equilibrium. PLIN 1237 polymorphism was not associated with obesity. The odds ratio for overweight or obesity for the CC+TC genotype was 0.8 (0.4, 1.4) in women (P = 0.4) and 0.6 (0.3, 1.3) in men (P = 0.2) after adjustment for age, education, household income and alcohol consumption, smoking, and physical activity. CONCLUSION: Chinese Han adults have a lower frequency of variant-allele C in PLIN 1237. PLIN 1237 T > C polymorphism is not significantly associated with obesity in northern Chinese adults.

Endothelial progenitor cells in age-related vascular remodeling.

Accumulating evidence has demonstrated that endothelial progenitor cells (EPCs) could facilitate the reendothelialization of injured arteries by replacing the dysfunctional endothelial cells, thereby suppressing the formation of neointima. Meanwhile, other findings suggest that EPCs may be involved in the pathogenesis of age-related vascular remodeling. This review is presented to summarize the characteristics of EPCs and age-related vascular remodeling. In addition, the role of EPCs in age-related vascular remodeling and possible solutions for improving the therapeutic effects of EPCs in the treatment of age-related diseases are discussed.

Protective effects of tanshinone â ¡A on endothelial progenitor cells injured by tumor necrosis factor-α.

Tanshinone ⅡA (Tan ⅡA) is a Traditional Chinese Medicine commonly used in Asian and Western countries for the prevention and treatment of cardiovascular disorders, such as atherosclerosis. Endothelial dysfunction and associated inflammatory processes have a critical role in the development of atherosclerosis. Endothelial progenitor cells (EPCs) have been demonstrated to be involved in certain aspects of the endothelial repair process. The present study aimed to investigate the putative protective effects of Tan ⅡA on EPCs injured by tumor necrosis factor­α (TNF­α). The potential effects of Tan ⅡA on TNF-α-stimulated EPC proliferation, migration, adhesion, in vitro tube formation ability and paracrine activity were investigated in the current study. The results indicated that TNF­α impaired EPC proliferation, migration, adhesion capacity and vasculogenesis ability in vitro as well as promoted EPC secretion of inflammatory cytokines, including monocyte chemoattractant protein­1 (MCP­1), interleukin­6 (IL­6) and soluble CD40 ligand (sCD40L). However, Tan ⅡA was able to reverse these effects. In conclusion, these findings demonstrated that Tan ⅡA may have the potential to protect EPCs against damage induced by TNF­α. Therefore, these results may provide evidence for the pharmacological basis of Tan ⅡA and its potential use in the prevention and treatment of early atherosclerosis associated with EPC and endothelial damage.

Endothelial progenitor cell-based therapy for pulmonary arterial hypertension.

A growing body of evidence in animal models and clinical studies supports the concept that endothelial progenitor cell (EPC)-mediated therapy ameliorates pulmonary arterial hypertension (PAH) and thus may represent a novel approach to treat it. Conversely, several experimental findings suggest that EPCs may be involved in PAH pathogenesis and disease progression. These discrepant results confuse the application of EPC transplantation as an effective treatment strategy for PAH. To improve the study of EPC transplantation in PAH therapy, it is high time that we resolve this dilemma. In this review, we examine the pathobiological changes of PAH, the characteristics of EPCs, and the underlying mechanisms of EPC effects on PAH.

Zoledronate attenuates angiogenic effects of angiotensin II-stimulated endothelial progenitor cells via RhoA and MAPK signaling.

BACKGROUND: New vessel formation plays a pivotal role in the pathogenesis of neovascular-related diseases. Endothelial progenitor cells (EPCs) were found to contribute to neovascular-related diseases and interference with EPC neovascularization may be a novel target for these diseases. Zoledronate (Zol) was reported to exhibit anti-angiogenic effect. Basing on these evidences, we proposed that Zol may affect EPC function to exert novel anti-angiogenic effect. In this study, we therefore investigated the effects of Zol on multiple aspects of EPC function and explored the underlying mechanisms involved. METHODOLOGY/PRINCIPAL FINDINGS: EPCs were cultured from bone marrow derived mononuclear cells. The potential effects of Zol on Angiotensin II (Ang II)-stimulated EPC proliferation, migration, adhesion, in vitro tube formation were investigated. The results showed that Ang II (1 µM) enhanced EPC migration, adhesion, in vitro tube formation but had no effect on cell proliferation. Zol (75 and 100 µM) inhibited proliferation of EPCs and 50 µM geranylgeranyol (GGOH) could reverse the decrease of EPC proliferation. We found for the first time that Zol (50-100 µM) dose dependently attenuated migration, adhesion, and in vitro tube formation of EPCs stimulated by Ang II. GGOH could reverse the attenuation of EPC function induced by Zol. However, Zol did not induce EPC apoptosis. In addition, the underlying mechanisms were determined. The results revealed that Zol markedly down-regulated active RhoA stimulated by Ang II and inhibited the phosphorylation of Erk1/2 and JNK. Moreover, RhoA silencing resulted in a notable inhibition of EPC in vitro tube formation, suggesting that RhoA suppression played a pivotal role in Zol antiangiogenic effect. CONCLUSIONS/SIGNIFICANCE: These findings suggested that Zol attenuated the promotion of EPC function stimulated by Ang II and exhibited novel antiangiogenic effect via RhoA and MAPK signaling. Thus, Zol may be served as a novel therapeutic agent for neovascular-related diseases treatment.

Interleukin-1 beta increases activity of human endothelial progenitor cells: involvement of PI3K-Akt signaling pathway.

Interleukin-1ß (IL-1ß) is a multifunctional proinflammatory cytokine upregulated in acute phase of heart ischemic disease. Controversial effects of IL-1ß have been demonstrated on endothelial progenitor cells (EPCs) functional activity. The aim of this study was to investigate the in vitro effect of IL-1ß on activity of human origin EPCs and the possible mechanism involved. EPCs were isolated from peripheral blood of healthy volunteers without cardiovascular risk factors and characterized. After ex vivo cultivation, EPCs were stimulated with a series of final concentrations (0, 0.1, 1, and 10 ng/ml) of IL-1ß for 24 h. In some other experiments, EPCs were pretreated with 10 µM LY294002 (Akt inhibitor) for 30 min and then stimulated with 1 ng/ml IL-1ß for 24 h. Cell proliferation, apoptosis, adhesion, and migration were determined, respectively, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, annexin V/propidium iodide binding assay, adhesion assay, and transwell migration assay. In addition, the vascular endothelial vascular growth factor-A (VEGF-A) production has been examined using quantitative real-time RT-PCR and ELISA assay. Furthermore, the total and phosphorylation level of Akt was determined by Western blot. IL-1ß significantly stimulated EPC proliferation, migration, and adhesion and upregulated the angiogenic growth factor VEGF-A at mRNA and protein level, while exerted no influence on cell apoptosis. However, pretreatment with LY294002 significantly diminished IL-1ß-induced proliferation, migration, adhesion, and VEGF-A production. One nanogram per milliliter IL-1ß for 15 min activated phosphorylation of Akt. These results suggest a potent role for IL-1ß in upregulating EPCs functions. The phosphatidyl-inositol-3-kinase-Akt signaling pathway could be involved in the regulation of EPCs functions induced by IL-1ß.