Juan Miguel Redondo

Activation of PPARβ/δ Induces Endothelial Cell Proliferation and Angiogenesis

Objective—The role of the nuclear receptor peroxisome-proliferator activated receptor (PPAR)-&bgr;/&dgr; in endothelial cells remains unclear. Interestingly, the selective PPAR&bgr;/&dgr; ligand GW501516 is in phase II clinical trials for dyslipidemia. Here, using GW501516, we have assessed the involvement of PPAR&bgr;/&dgr; in endothelial cell proliferation and angiogenesis. Methods and Results—Western blot analysis indicated PPAR&bgr;/&dgr; was expressed in primary human umbilical and aortic endothelial cells, and in the endothelial cell line, EAHy926. Treatment with GW501516 increased human endothelial cell proliferation and morphogenesis in cultures in vitro, endothelial cell outgrowth from murine aortic vessels in vitro, and angiogenesis in a murine matrigel plug assay in vivo. GW501516 induced vascular endothelial cell growth factor mRNA and peptide release, as well as adipose differentiation-related protein (ADRP), a PPAR&bgr;/&dgr; target gene. GW501516-induced proliferation, morphogenesis, vascular endothelial growth factor (VEGF), and ADRP were absent in endothelial cells transfected with dominant-negative PPAR&bgr;/&dgr;. Furthermore, treatment of cells with cyclo-VEGFI, a VEGF receptor1/2 antagonist, abolished GW501516-induced endothelial cell proliferation and tube formation. Conclusions—PPAR&bgr;/&dgr; is a novel regulator of endothelial cell proliferation and angiogenesis through VEGF. The use of GW501516 to treat dyslipidemia may need to be carefully monitored in patients susceptible to angiogenic disorders.

An Essential Role of the Nuclear Factor of Activated T Cells in the Regulation of the Expression of the Cyclooxygenase-2 Gene in Human T Lymphocytes

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions −117 and −58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca2+/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression.

ABSTRACT Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified κB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-κB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-κB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

Cyclooxygenase-2: a therapeutic target in angiogenesis

Angiogenesis has a role in the pathogenesis of several disorders, including cancer, chronic inflammatory diseases and retinopathies. Recent evidence demonstrates that the production of prostanoids by cyclooxygenase-2 (COX-2) promotes the expression of pro-angiogenic factors. Furthermore, inhibition of COX-2 by non-steroidal anti-inflammatory drugs leads to restricted angiogenesis and downregulated production of pro-angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor. These findings suggest that COX enzymes could be important therapeutic targets in the treatment of pathological angiogenesis.

Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.

Dendrite branching and spine formation determines the function of morphologically distinct and specialized neuronal subclasses. However, little is known about the programs instructing specific branching patterns in vertebrate neurons and whether such programs influence dendritic spines and synapses. Using knockout and knockdown studies combined with morphological, molecular, and electrophysiological analysis, we show that the homeobox Cux1 and Cux2 are intrinsic and complementary regulators of dendrite branching, spine development, and synapse formation in layer II-III neurons of the cerebral cortex. Cux genes control the number and maturation of dendritic spines partly through direct regulation of the expression of Xlr3b and Xlr4b, chromatin remodeling genes previously implicated in cognitive defects. Accordingly, abnormal dendrites and synapses in Cux2(-/-) mice correlate with reduced synaptic function and defects in working memory. These demonstrate critical roles of Cux in dendritogenesis and highlight subclass-specific mechanisms of synapse regulation that contribute to the establishment of cognitive circuits.

Inhibitors of the Calcineurin / NFAT Pathway

The well known calcium-sensitive phosphatase calcineurin is implicated in many eukaryotic activation and developmental programmes, including lymphocyte activation, heart-valve morphogenesis, angiogenesis, and neural and muscle development. The importance of this phosphatase is graphically illustrated by the observation that the immunosuppressive actions of the microbial drugs Cyclosporin A (CsA) and FK506 arise from their inhibition of calcineurin. As substrates of calcineurin, transcription factors of the NFAT family play an essential role in lymphocyte activation, and it follows that their function is also inhibited by CsA and FK506. Although the use of these drugs has been crucial for the success of organ transplantation, their therapeutic use is associated with severe side effects. There is, therefore a need to develop better, less toxic immunosuppressive agents. In recent years, a number of endogenous calcineurin inhibitor proteins have been identified that bind calcineurin and block its phosphatase activity. In some cases the calcineurin interaction domains of these proteins, or their corresponding docking sites on calcineurin, have been described. However, their mode of action and regulatory mechanisms are not completely known. In a more recent development, specific amino acidic sequences implicated in the interaction between calcineurin and NFAT have been identified. It is of special interest that specific disruption of this pathway has been obtained through the expression of peptides based on some of these sequences. A more profound analysis of these issues could open up new perspectives in immunosuppressive therapy; promising compounds with features of endogenous calcineurin inhibitors (and thus likely to have fewer toxic effects than CsA and FK506), or selective blockers of calcineurin-NFAT interactions that would not alter the functioning of other calcineurin substrates.

DOXORUBICIN INDUCES APOPTOSIS AND CD95 GENE EXPRESSION IN HUMAN PRIMARY ENDOTHELIAL CELLS THROUGH A p53-DEPENDENT MECHANISM

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox), a drug used in antitumor therapy, triggered a marked accumulation of p53 and induced CD95gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore, the p53 inhibitor pifithrin-α (PFT-α) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However, Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-α and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally, Dox triggered Bcl-2 down-regulation, cytochrome c release from mitochondria, and the activation of caspases 9 and 3, suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox, related to its ability to induce apoptosis in proliferating endothelial cells.

Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin.

Kelvin J. A. Davies,* Gennady Ermak,* Beverley A. Rothermel, Melanie Pritchard, Joseph Heitman, Joohong Ahnn, Flavio Henrique-Silva, Dana Crawford, Silvia Canaider,** Pierluigi Strippoli,** Paolo Carinci,** Kyung-Tai Min, Deborah S. Fox, Kyle W. Cunningham, Rhonda Bassel-Duby, Eric N. Olson, Zhuohua Zhang, R. Sanders Williams, Hans-Peter Gerber,*** Merce Perez-Riba, Hisao Seo, Xia Cao, Claude B. Klee, Juan Miguel Redondo, Lois J. Maltais, Elspeth A. Bruford, Sue Povey, Jeffery D. Molkentin,**** Frank D. McKeon, Elia J. Duh, Gerald R. Crabtree,§§§§ Martha S. Cyert, Susana de la Luna, and Xavier Estivill

Reciprocal Relationship Between Reactive Oxygen Species and Cyclooxygenase-2 and Vascular Dysfunction in Hypertension

AIMS This study evaluates a possible relationship between reactive oxygen species (ROS) and cyclooxygenase (COX)-2-derived products in conductance and resistance arteries from hypertensive animals. Angiotensin II (Ang II)-infused mice or spontaneously hypertensive rats treated with the NAD(P)H Oxidase inhibitor apocynin, the mitochondrion-targeted SOD2 mimetic Mito-TEMPO, the superoxide dismutase analog tempol, or the COX-2 inhibitor Celecoxib were used. RESULTS Apocynin, Mito-TEMPO, and Celecoxib treatments prevented Ang II-induced hypertension, the increased vasoconstrictor responses to phenylephrine, and the reduced acetylcholine relaxation. The NOX-2 inhibitor gp91ds-tat, the NOX-1 inhibitor ML171, catalase, and the COX-2 inhibitor NS398 abolished the ex vivo effect of Ang II-enhancing phenylephrine responses. Antioxidant treatments diminished the increased vascular COX-2 expression, prostanoid production, and/or participation of COX-derived contractile prostanoids and thromboxane A(2) receptor (TP) in phenylephrine responses, observed in arteries from hypertensive models. The treatment with the COX-2 inhibitor normalized the increased ROS production (O(2)·(-) and H(2)O(2)), NAD(P)H Oxidase expression (NOX-1, NOX-4, and p22phox) and activity, MnSOD expression, and the participation of ROS in vascular responses in both hypertensive models. Apocynin and Mito-TEMPO also normalized these parameters of oxidative stress. Apocynin, Mito-TEMPO, and Celecoxib improved the diminished nitric oxide (NO) production and the modulation by NO of phenylephrine responses in the Ang II model. INNOVATION This study provides mechanistic evidence of circuitous relationship between COX-2 products and ROS in hypertension. CONCLUSION The excess of ROS from NAD(P)H Oxidase and/or mitochondria and the increased vascular COX-2/TP receptor axis act in concert to induce vascular dysfunction and hypertension.

Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers.

We have previously shown that the delta E3 site is an essential element for transcriptional activation by the human T-cell receptor (TCR) delta enhancer and identified two factors, NF-delta E3A and NF-delta E3C, that bound to overlapping core (TGTGGTTT) and E-box motifs within delta E3. In this study, we show that protein binding to the core motif is necessary but not sufficient for transcriptional activation by the delta E3 element. In contrast, protein binding to the E-box motif does not contribute significantly to enhancer activity. A similar core motif present within the enhancers of T-cell-tropic murine retroviruses has been shown to contribute to transcriptional activity of the viral long terminal repeat in T lymphocytes and to viral T-cell tropism. We therefore determined the relationship between the nuclear factors that bind to the TCR delta and Moloney murine leukemia virus core motifs. On the basis of electrophoretic mobility shift binding and competition studies, biochemical analysis of affinity-labeled DNA-binding proteins, and the binding of a purified core binding factor, the proteins that bound to the TCR delta core site were indistinguishable from those that bound to the murine leukemia virus core site. These data argue that DNA-binding proteins that interact with the core site of murine leukemia virus long terminal repeats and contribute to viral T-cell tropism also play an essential role in the T-cell-specific expression of cellular genes.