Elisa Ghelfi

Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells

Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte‐ and macrophage‐specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF‐A and bFGF treatment. The effect of VEGF‐A on FABP4 expression was inhibited by chemical inhibition or short‐hairpin (sh) RNA‐mediated knockdown of VEGFreceptor‐2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs.—Elmasri, H., Karaaslan, C., Teper, Y., Ghelfi, E., Weng, M., Ince, T. A., Kozakewich, H., Bischoff, J., Cataltepe, S. Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells. FASEB J. 23, 3865–3873 (2009). www.fasebj.org

Cardiac Oxidative Stress and Electrophysiological Changes in Rats Exposed to Concentrated Ambient Particles are Mediated by TRP-Dependent Pulmonary Reflexes

Previous studies suggest that, through the stimulation of pulmonary nervous endings, ambient particles modulate the autonomic tone on the heart leading to cardiac oxidant stress and dysfunction. In this paper we investigated the effect of blockade of vanilloid receptor 1 (Transient Receptor Potential Vanilloid Receptor 1 [TRPV1]) on concentrated ambient particles (CAPs)-induced cardiac oxidative stress and dysfunction in a rat model of inhalation exposure. Capsazepine (CPZ), a selective antagonist of TRPV1, was given ip or as an aerosol immediately before exposure to CAPs. Control and CPZ-treated rats were exposed to filtered air or CAPs aerosols for 5 h using the Harvard Ambient Particle Concentrator (mean PM(2.5) mass concentration: 218 +/- 23 mug/m(3)). At the end of the exposure we measured cardiac oxidative stress (in situ chemiluminescence [CL]), lipid peroxidation (thiobarbituric acid reactive substances [TBARS]), and tissue edema. Cardiac function was monitored throughout the exposure. CPZ (ip or aerosol) decreased CAPs-induced CL, lipid TBARS, and edema in the heart, indicating that blocking TRP receptors, systemically or locally, decreases heart CL. CAPs exposure led to significant decreases in heart rate (CAPs 350 +/- 32 bpm, control: 370 +/- 29), and in the length of the QT, RT, Pdur and Tpe intervals. These changes were observable immediately upon exposure and were maintained throughout the 5 h of CAPs inhalation. Changes in cardiac rhythm and electrocardiogram morphology were prevented by CPZ. These data suggest that current abnormalities in CAPs-exposed rats alter the action potentials leading to changes in conduction velocity and ventricular repolarization, and that triggering of TRPV1-mediated autonomic reflexes in the lung is essential for the observed changes in cardiac rhythms.

Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway

Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4−/− mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis.

Fatty acid binding protein 4 is expressed in distinct endothelial and non-endothelial cell populations in glioblastoma

O. Cataltepe, M. C. Arikan, E. Ghelfi, C. Karaaslan, Y. Ozsurekci, K. Dresser, Y. Li, T. W. Smith and S. Cataltepe (2012) Neuropathology and Applied Neurobiology38, 400–410

Cardiac Oxidative Stress and Dysfunction by Fine Concentrated Ambient Particles (CAPs) are Mediated by Angiotensin-II

Inhalation exposure to fine concentrated ambient particles (CAPs) increases cardiac oxidants by mechanisms involving modulation of the sympathovagal tone on the heart. Angiotensin-II is a potent vasoconstrictor and a sympatho-excitatory peptide involved in the regulation of blood pressure. We hypothesized that increases in angiotensin-II after fine particulate matter (PM) exposure could be involved in the development of cardiac oxidative stress. Adult rats were treated with an angiotensin-converting enzyme (ACE) inhibitor (benazepril®), or an angiotensin receptor blocker (ARB; valsartan®) before exposure to fine PM aerosols or filtered air. Exposures were carried out for 5 hours in the chamber of the Harvard fine particle concentrator (fine PM mass concentration: 440 ± 80 μg/m3). At the end of the exposure the animals were tested for in situ chemiluminescence (CL) of the heart, thiobarbituric acid reactive substances (TBARS) and for plasma levels of angiotensin-II. Also, continuous electrocardiogram (ECG) measurements were collected on a subgroup of exposed animals. PM exposure was associated with statistically significant increases in plasma angiotensin concentrations. Pre-treatment with the ACE inhibitor effectively lowered angiotensin concentration, whereas ARB treatment led to increases in angiotensin above the PM-only level. PM exposure also led to significant increases in heart oxidative stress (CL, TBARS), and a shortening of the T-end to T-peak interval on the ECG that were prevented by treatment with both the ACE inhibitor and ARB. These results show that ambient fine particles can increase plasma levels of angiotensin-II and suggest a role of the renin–angiotensin system in the development of particle-related acute cardiac events.

Fatty Acid–Binding Proteins and Peribronchial Angiogenesis in Bronchopulmonary Dysplasia

Inflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia (BPD). Fatty acid-binding proteins (FABPs) 4 and 5 regulate the inflammatory activity of macrophages. Whether FABPs 4 and 5 could play a role in the pathogenesis of BPD via the promotion of macrophage inflammatory activity is unknown. This study sought to examine whether the expression levels of FABP4 and FABP5 were altered in bronchoalveolar lavage fluid and lung tissue in a baboon model of BPD. This study also sought to characterize the cell types that express these proteins. Real-time PCR, immunoblotting, immunohistochemistry, and double immunofluorescence were used to examine the expression of FABPs in samples of BPD. Morphometric analysis was used to quantify FABP4-positive peribronchial blood vessels in lung sections. FABP4 was primarily expressed in macrophages in samples of BPD. In addition, FABP4 was expressed in the endothelial cells of blood vessels in peribronchial areas and the vasa vasorum, but not in the alveolar vasculature in samples of BPD. FABP4 concentrations were significantly increased in lungs and bronchoalveolar lavage fluid samples with BPD. An increased density of FABP4-positive peribronchial blood vessels was evident in both baboon and human BPD sections. FABP5 was expressed in several cell types, including alveolar epithelial cells and macrophages. FABP5 concentrations did not show any significant alterations in BPD. In conclusion, FABP4 but not FABP5 levels are increased in BPD. FABP4 is differentially expressed in endothelial cells of the bronchial microvasculature, which demonstrates a previously unrecognized expansion in BPD.

Pulmonary inflammation by ambient air particles is mediated by superoxide anion.

Lung inflammation is a key response to increased levels of particulate air pollution (PM); however, the cellular mechanisms leading to this response remain poorly understood. We have previously shown that oxidants are critical mediators of the inflammatory response elicited by inhalation of ambient air particles. Here we tested the possible role of a specific oxidant, superoxide anion, by using the membrane-permeable analog of superoxide dismutase, Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP). Adult Sprague-Dawley rats were instilled with either urban air particles (UAP) or saline. MnTBAP-treated rats received 10 mg/kg (ip) MnTBAP 2 h prior to exposure to UAP. Recruitment of inflammatory cells into bronchoalveolar lavage was evaluated 4 h after instillation. Rats exposed to UAP showed significant increases in the total cell number (8.9 ± 0.6× 106; sham: 5.1 ± 0.6 × 106, p < .02), the numbers of polymorphonuclear leukocytes (26 ± 4%; sham: 6 ± 1%, p < .0001), protein levels (1.2 ± 0.5 mg/ml, sham:0.4 ± 0.1 mg/ml, p < .001), and a trend of increase in myeloperoxidase levels (5 ± 1; sham: 2 ± 1 mU/ml) in bronchoalveolar lavage (BAL). Pretreatment with MnTBAP at a dose that prevented UAP-induced increases in oxidants effectively prevented increase in BAL cells (2.7 ± 0.6 × 106, p < .0001 vs. UAP), PMN influx into the lungs (4 ± 3%, p < .0001 vs. UAP), and increase in myeloperoxidase (2 ± 1 mU/ml) and protein levels in BAL (0.1 ± 0.1 mg/ml). These data indicate that superoxide anion is a critical mediator of the inflammatory response elicited by PM deposition in the lung.

Fatty Acid Binding Protein 4 Deficiency Protects against Oxygen-Induced Retinopathy in Mice

Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4−/− mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4−/− OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies.

Genetic Polymorphisms Associated with Hearing Threshold Shift in Subjects during First Encounter with Occupational Impulse Noise

Noise-induced hearing loss (NIHL) is the most significant occupational health issue worldwide. We conducted a genome-wide association study to identify single-nucleotide polymorphisms (SNPs) associated with hearing threshold shift in young males undergoing their first encounter with occupational impulse noise. We report a significant association of SNP rs7598759 (p < 5 x 10-7; p = 0.01 after permutation and correction; Odds Ratio = 12.75) in the gene coding for nucleolin, a multifunctional phosphoprotein involved in the control of senescence and protection against apoptosis. Interestingly, nucleolin has been shown to mediate the anti-apoptotic effect of HSP70, a protein found to prevent ototoxicity and whose polymorphisms have been associated with susceptibility to NIHL. Increase in nucleolin expression has also been associated with the prevention of apoptosis in cells undergoing oxidative stress, a well-known metabolic sequela of noise exposure. To assess the potential role of nucleolin in hearing loss, we tested down-regulation of nucleolin in cochlear sensory cells HEI-OC1 under oxidative stress conditions and report increased sensitivity to cisplatin, a chemotherapeutic drug with ototoxic side effects. Additional SNPs were found with suggestive association (p < 5 x 10-4), of which 7 SNPs were located in genes previously reported to be related to NIHL and 43 of them were observed in 36 other genes previously not reported to be associated with NIHL. Taken together, our GWAS data and in vitro studies reported herein suggest that nucleolin is a potential candidate associated with NIHL in this population.

Comparative global immune-related gene profiling of somatic cells, human pluripotent stem cells and their derivatives: implication for human lymphocyte proliferation

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications.