Roberta Melchionna

MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand ephrin-A3

MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.

NEGATIVE REGULATION OF BETA ENOLASE GENE TRANSCRIPTION IN EMBRYONIC MUSCLE IS DEPENDENT UPON A ZINC FINGER FACTOR THAT BINDS TO THE G-RICH BOX WITHIN THE MUSCLE-SPECIFIC ENHANCER

We have previously identified a muscle-specific enhancer within the first intron of the human β enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed β enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the β enolase gene transcription in transient transfection assays. Using fusion polypeptides of β enolase repressor factor 1 and the yeast GAL4 DNA-binding domain, we have identified an amino-terminal region responsible for the transcriptional repression activity, whereas a carboxyl-terminal region was shown to contain a potential transcriptional activation domain. The expression of this protein decreases in developing skeletal muscles, correlating with lack of binding activity in nuclear extract from adult skeletal tissue, in which novel binding activities have been detected. These results suggest that in addition to the identified factor, which functionally acts as a negative regulator and is enriched in embryonic muscle, the G-rich box binds other factors, presumably exerting a positive control on transcription. The interplay between factors that repress or activate transcription may constitute a developmentally regulated mechanism that modulates β enolase gene expression in skeletal muscle.

Laminar shear stress inhibits CXCR4 expression on endothelial cells: functional consequences for atherogenesis

Laminar shear stress (LSS) represents a major athero‐protective stimulus. However, the mechanisms for this effect are poorly characterized. As chemokine receptors modulate endothelial cell functions, we hypothesized that at least some LSS effects on endothelial cells (ECs) may be due to LSS‐dependent changes in chemokine receptor expression and function. Exposure of Human umbilical vein endothelial cells (HUVECs) to 15 dynes/cm2/sec−1LSS strongly inhibited CXC chemokine receptor 4 (CXCR4) expression at the transcriptional level and impaired stromal‐derived factor (SDF)‐1/CXCL12‐driven chemotaxis. On the contrary, low shear stress (SS; 4 dynes/cm2/sec−1) only marginally affected CXCR4 expression when compared with static control cells. Differently from CXCR4, the expression of SDF‐1 mRNA was not affected by LSS treatment. CXCR4 overexpression induced a dose‐dependent endothelial cell apoptosis that was enhanced by SDF‐1 treatment and was caspase‐dependent. CXCR4 overexpression inhibited the LSS‐mediated antiapoptotic effect on ECs and was associated to impairment of LSS‐induced ERK1/2 phosphorylation. These findings suggest that LSS‐induced CXCR4 down‐regulation may contribute to endothelial cell survival. Interestingly, the expression of the proatherogenic chemokines MCP‐1 and IL‐8 was induced by SDF‐1 treatment and by CXCR4 overexpression in HUVECs. Further, the known LSS‐induced inhibition of MCP‐1 expression was impaired in CXCR4 overexpressing ECs. Finally, CXCR4 was abundantly expressed by human atherosclerotic plaque endothelium that is exposed to low/absent shear stress, while it was poorly expressed by minimally diseased carotid artery endothelium. In conclusion, LSS‐dependent CXCR4 down‐regulation may contribute to atheroprotection by favoring the integrity of the endothelial barrier and by inhibiting MCP‐1 and IL‐8 expression.

Gene expression profiles in peripheral blood mononuclear cells of chronic heart failure patients

The present study was aimed at identifying chronic heart failure (CHF) biomarkers from peripheral blood mononuclear cells (PBMCs) in patients with ischemic (ICM) and nonischemic dilated (NIDCM) cardiomyopathy. PBMC gene expression profiling was performed by Affymetrix in two patient groups, 1) ICM (n = 12) and 2) NIDCM (n = 12) New York Heart Association (NYHA) III/IV CHF patients, vs. 3) age- and sex-matched control subjects (n = 12). Extracted RNAs were then pooled and hybridized to a total of 11 microarrays. Gene ontology (GO) analysis separated gene profiling into functional classes. Prediction analysis of microarrays (PAM) and significance analysis of microarrays (SAM) were utilized in order to identify a molecular signature. Candidate markers were validated by quantitative real-time polymerase chain reaction. We identified a gene expression profiling that distinguished between CHF patients and control subjects. Interestingly, among the set of genes constituting the signature, chemokine receptor (CCR2, CX(3)CR1) and early growth response (EGR1, 2, 3) family members were found to be upregulated in CHF patients vs. control subjects and to be part of a gene network. Such findings were strengthened by the analysis of an additional 26 CHF patients (n = 14 ICM and n = 12 NIDCM), which yielded similar results. The present study represents the first large-scale gene expression analysis of CHF patient PBMCs that identified a molecular signature of CHF and putative biomarkers of CHF, i.e., chemokine receptor and EGR family members. Furthermore, EGR1 expression levels can discriminate between ICM and NIDCM CHF patients.

Induction of myogenic differentiation by SDF-1 via CXCR4 and CXCR7 receptors.

The stromal cell–derived factor (SDF)‐1/CXC receptor 4 (CXCR4) axis has been shown to play a role in skeletal muscle development, but its contribution to postnatal myogenesis and the role of the alternate SDF‐1 receptor, CXC receptor 7 (CXCR7), are poorly characterized. Western blot analysis and real‐time polymerase chain reaction (PCR) were performed to evaluate in vitro the effect of SDF‐1 and CXCR4 and CXCR7 inhibition on myogenic differentiation. Proliferating myoblasts express CXCR4, CXCR7, and SDF‐1; during myogenic differentiation, CXCR4 and CXCR7 levels are downregulated, and SDF‐1 release is decreased. SDF‐1 anticipates myosin heavy chain accumulation and myotube formation in both C2C12 myoblasts and satellite cells. Interestingly, inhibition of CXCR4 and CXCR7 signaling, either by drugs or RNA interfererence, blocks myogenic differentiation. Further, the CXCR4 antagonist, 4F‐benzoyl‐TN14003, inhibits myoblast cell cycle withdrawal and decreases the retinoblastoma gene (pRb) product accumulation in its hypophosphorylated form. Our experiments demonstrate that SDF‐1 regulates myogenic differentiation via both CXCR4 and CXCR7 chemokine receptors. Muscle Nerve 000:000–000, 2010

Increase of plasma IL-9 and decrease of plasma IL-5, IL-7, and IFN-γ in patients with chronic heart failure

BackgroundSeveral cytokines are associated with the development and/or progression of chronic heart failure (CHF). Our aim was to look more closely at the cytokine networks involved in CHF, and to assess whether disease etiology affects cytokine expression. The study population was comprised of a) 69 patients with stable CHF, New York Heart Association (NYHA) II/IV classes, secondary to ischaemic (ICM) and non ischaemic dilated (NIDCM) cardiomyopathy and b) 16 control subjects. We analyzed and compared the plasma levels of 27 pro- and anti-inflammatory mediators, in the study population and assessed for any possible correlation with echocardiographic parameters and disease duration.Methods27 cytokines and growth factors were analyzed in the plasma of ICM- (n = 42) and NIDCM (n = 27) NYHA class II-IV patients vs age- and gender-matched controls (n = 16) by a beadbased multiplex immunoassay. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test for multiple comparison.ResultsMacrophage inflammatory protein (MIP)-1β, Vascular endothelial growth factor (VEGF), interleukin (IL)-9, Monocyte chemotactic protein (MCP)-1, and IL-8 plasma levels were increased in both ICM and NIDCM groups vs controls. In contrast, IL-7, IL-5, and Interferon (IFN)-γ were decreased in both ICM and NIDCM groups as compared to controls. Plasma IL-6 and IL-1 β were increased in ICM and decreased in NIDCM, vs controls, respectively.IL-9 levels inversely correlated, in ICM patients, with left ventricular ejection fraction (LVEF) while IL-5 plasma levels inversely correlated with disease duration, in NYHA III/IV ICM patients.This is the first time that both an increase of plasma IL-9, and a decrease of plasma IL-5, IL-7 and IFN-γ have been reported in ICM as well as in NIDCM groups, vs controls. Interestingly, such cytokines are part of a network of genes whose expression levels change during chronic heart failure. The altered expression levels of MIP-1 β, VEGF, MCP-1, IL-1 β, IL-6, and IL-8, found in this study, are in keeping with previous reports.ConclusionsThe increase of plasma IL-9, and the decrease of plasma IL-5, IL-7 and IFN-γ in ICM as well as in NIDCM groups vs controls may contribute to get further insights into the inflammatory pathways involved in CHF.

C/EBPγ Regulates Wound Repair and EGF Receptor Signaling

We aimed at identifying novel regulators of skin wound healing (WH), in an epidermal scratch WH assay, by a small interfering RNA (siRNA) silencing approach. Several transcription factors have been previously reported to affect wound repair. We here show that gene silencing of the transcription factor CAAT enhancer-binding protein γ (C/EBPγ), STAT3, REL, RELA, RELB, SP1, and NFkB impaired WH in vitro, in keratinocytes, whereas E2F and CREBBP silencing accelerated the WH process. We further characterized C/EBPγ, as its silencing yielded the maximal impairment (52.2 ± 12.5%) of scratch wounding (SW). We found that C/EBPγ silencing inhibited both EGF- and serum-induced keratinocyte migration, whereas C/EBPγ overexpression enhanced cell migration to EGF and to serum via the EGFR. Further, C/EBPγ silencing impaired scratch-induced Y1068 and Y1173 EGFR phosphorylation, as well as Y118 paxillin phosphorylation, key molecules regulating cell migration and epidermal WH. Moreover, C/EBPγ levels were induced in keratinocytes, following both SW and EGF stimulation. C/EBPγ siRNA silencing in vivo impaired WH at 3, 5, 7, and 14 days following excisional wounding in mice inhibited both re-epithelialization and granulation tissue formation, and induced a decrease of arteriole number. In conclusion, we here report that C/EBPγ positively regulates wound repair both in vitro and in vivo, at least in part, by affecting EGFR signaling.

Role of HIF-1α in proton-mediated CXCR4 down-regulation in endothelial cells

AIMS Acidification is associated with a variety of pathological and physiological conditions. In the present study, we aimed at investigating whether acidic pH may regulate endothelial cell (EC) functions via the chemokine receptor CXCR4, a key modulator of EC biological activities. METHODS AND RESULTS Exposure of ECs to acidic pH reversibly inhibited mRNA and protein CXCR4 expression, CXCL12/stromal cell-derived factor (SDF)-1-driven EC chemotaxis in vitro, and CXCR4 expression and activation in vivo in a mouse model. Further, CXCR4 signalling impaired acidosis-induced rescue from apoptosis in ECs. The inhibition of CXCR4 expression occurred transcriptionally and was hypoxia-inducible factor (HIF)-1alpha-dependent as demonstrated by both HIF-1alpha and HIF-1alpha dominant negative overexpression, by HIF-1alpha silencing, and by targeted mutation of the -29 to -25 hypoxia response element (HRE) in the -357/-59 CXCR4 promoter fragment. Moreover, chromatin immunoprecipitation (ChIP) analysis showed endogenous HIF-1alpha binding to the CXCR4 promoter that was enhanced by acidification. CONCLUSION The results of the present study identify CXCR4 as a key player in the EC response to acidic pH and show, for the first time, that HRE may function not only as an effector of hypoxia, but also as an acidosis response element, and raise the possibility that this may constitute a more general mechanism of transcriptional regulation at acidic pH.

Transcriptional control of skin reepithelialization

The wound healing process is characterized by a series of overlapping phases, such as coagulation, inflammation, reepithelialization/granulation tissue generation and remodeling. It is important to obtain a deeper insight into the cutaneous wound repair mechanisms, in order to develop novel pharmacological tools for the treatment of chronic non-healing ulcers which are a frequent and high morbidity complication of diabetes, ischaemia, venous insufficiency, and other local or systemic factors. Several transcription factors, many of which belong to gene families, are known to play a role in cutaneous wound repair through the orchestration of cellular responses which promote the reconstitution of skin integrity. The aim of this review is to provide an updated analysis of the transcription factor role in the reepithelialization process, in the context of skin wound repair.

Ex vivo acidic preconditioning enhances bone marrow ckit+ cell therapeutic potential via increased CXCR4 expression

Aims The chemokine receptor CXCR4 modulates endothelial progenitor cell migration, homing, and differentiation, and plays a key role in cardiovascular regeneration. Here we examined the effect of ex vivo acidic preconditioning (AP) on CXCR4 expression and on the regenerative potential of mouse bone marrow (BM) ckit+ cells. Methods and results Acidic preconditioning was achieved by exposing BM ckit+ cells to hypercarbic acidosis (pH 7.0) for 24 h; control cells were kept at pH 7.4. Acidic preconditioning enhanced CXCR4 and stromal cell-derived factor 1 (SDF-1) mRNA levels, as well as CXCR4 phosphorylation. Acidic preconditioning ability to modulate CXCR4 expression depended on cytosolic calcium [Ca2+]i mobilization and on nitric oxide (NO), as determined by [Ca2+]i buffering with BAPTA, and by treatment with the NO donor (DETA/NO) and the NO synthase inhibitor (L-NAME). Further, AP increased SDF-1-driven chemotaxis, transendothelial migration, and differentiation toward the endothelial lineage in vitro. In a mouse model of hindlimb ischaemia, control and AP ckit+ cells were transplanted into the ischaemic muscle; AP cells accelerated blood flow recovery, increased capillary, and arteriole number as well as the number of regenerating muscle fibres vs. control. These effects were abolished by treating AP cells with L-NAME. Conclusion Acidic preconditioning represents a novel strategy to enhance BM ckit+ cell therapeutic potential via NO-dependent increase in CXCR4 expression.