Biomedical subjects

Marco Lopez-Ilasaca

Publications and source records attributed to Marco Lopez-Ilasaca.

8 recordsLinked to original sources

PubMed2006

Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation.

We previously reported that intramyocardial injection of bone marrow-derived mesenchymal stem cells overexpressing Akt (MSC-Akt) efficiently repaired infarcted rat myocardium and improved cardiac function. Controversy still exists over the mechanisms by which MSC contribute to tissue repair. Herein, we tested if cellular fusion of MSC plays a determinant role in cardiac repair. We injected MSC expressing Cre recombinase, with or without Akt, into Cre reporter mice. In these mice, LacZ is expressed only after Cre-mediated excision of a loxP-flanked stop signal and is indicative of fusion. MSC engraftment within infarcted myocardium was transient but significantly enhanced by Akt. MSC fusion with cardiomyocytes was observed as early as 3 days, but was infrequent, and we found a low rate of differentiation of MSC into cardiomyocytes. MSC-Akt decreased infarct size at 3 days and restored early cardiac function. In conclusion, MSC-Akt improved early repair despite transient engraftment, low levels of cellular fusion, and differentiation. These new observations further confirm our recently reported data that early paracrine mechanisms mediated by MSC are responsible for enhancing the survival of existing myocytes and that Akt could alter the secretion of various cytokines and growth factors.

Animals↗

PubMed2005

Identification of calcium-modulating cyclophilin ligand (CAML) as transducer of angiotensin II-mediated nuclear factor of activated T cells (NFAT) activation.

Angiotensin II (Ang II) plays a central role in cardiovascular physiology and disease. Ang II type I receptor (AT1) is thought to mediate most actions of Ang II. A novel AT1 receptor intracellular partner called AT1 receptor-associated protein (ATRAP) was identified, but its exact function has not been elucidated. A yeast two-hybrid screen using ATRAP as bait identified calcium-modulating cyclophilin ligand (CAML) as an ATRAP partner. Yeast two-hybrid and coimmunoprecipitation analysis demonstrated that the N-terminal hydrophilic domain of CAML (amino acids (aa) 1-189) mediates a specific interaction between ATRAP and CAML. Our analysis also showed that aa 40-82 of ATRAP contribute to this interaction. Bioluminescence resonance energy transfer and intracellular colocalization analysis by immunofluorescence in HEK293 cells verified this association within endoplasmic reticulum vesicular structures. Functionally, transcriptional reporter assays and RNA interference ATRAP experiments demonstrated that ATRAP knockdown increased nuclear factor of activated T cells (NFAT) activity. Overexpression of ATRAP decreased Ang II- or CAML-induced NFAT transcriptional activation, whereas an ATRAP-interacting domain of CAML (aa 1-189) sensitized NFAT activation in response to Ang II. These results indicate that CAML is an important signal transducer for the actions of Ang II in regulating the calcineurin-NFAT pathway and suggest that the interaction of CAML with ATRAP may mediate the Ang II actions in vascular physiology.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Liver X receptors alpha and beta regulate renin expression in vivo.

The renin-angiotensin-aldosterone system controls blood pressure and salt-volume homeostasis. Renin, which is the first enzymatic step of the cascade, is critically regulated at the transcriptional level. In the present study, we investigated the role of liver X receptor alpha (LXR(alpha)) and LXR(beta) in the regulation of renin. In vitro, both LXRs could bind to a noncanonical responsive element in the renin promoter and regulated renin transcription. While LXR(alpha) functioned as a cAMP-activated factor, LXR(beta) was inversely affected by cAMP. In vivo, LXRs colocalized in juxtaglomerular cells, in which LXR(alpha) was specifically enriched, and interacted with the renin promoter. In mouse models, renin-angiotensin activation was associated with increased binding of LXR(alpha) to the responsive element. Moreover, acute administration of LXR agonists was followed by upregulation of renin transcription. In LXR(alpha) mice, the elevation of renin triggered by adrenergic stimulation was abolished. Untreated LXR(beta) mice exhibited reduced kidney renin mRNA levels compared with controls. LXR(alpha)LXR(beta) mice showed a combined phenotype of lower basal renin and blunted adrenergic response. In conclusion, we show herein that LXR(alpha) and LXR(beta) regulate renin expression in vivo by directly interacting with the renin promoter and that the cAMP/LXR(alpha) signaling pathway is required for the adrenergic control of the renin-angiotensin system.

Animals↗

PubMed2005

Searching for transcriptional regulators of Ang II-induced vascular pathology.

Ang II plays a key role in cardiovascular regulation and participates in vascular pathobiology, including inflammation and remodeling. Whether these tissue effects are mediated by direct Ang II actions or indirectly as a result of its influence on hemodynamics is being debated. In vitro data have shown that Ang II induces vascular cellular transcriptional activation and gene expression, but the mechanisms explaining its long-term tissue effects in vivo are relatively unknown. Do the multiple in vivo vascular activities elicited by Ang II (such as inflammation, fibrosis, and vascular cell hypertrophy/proliferation) occur via independent pathways, or do common transcription mechanisms mediate these multiple effects? In this issue, Zhan et al. identify Ets-1 as a critical downstream transcriptional mediator of vascular inflammation and remodeling in vivo; their data suggest that Ets-1 may be a common denominator of a complex process that involves multiple pathways previously considered to be mechanistically independent. Characterization of the critical transcription programs activated by Ang II in vivo and determination of the hierarchy of responses are vital to the understanding of the mechanism of vascular disease and to the development of therapies targeted at inhibiting the common transcription effectors of vascular pathology.

Angiotensin II↗

PubMed2004

Discs large (Dlg1) complexes in lymphocyte activation.

T cell antigen recognition involves the formation of a structured interface between antigen-presenting and T cells that facilitates the specific transmission of activating and desensitizing stimuli. The molecular machinery that organizes the signaling molecules and controls their disposition in response to activation remains poorly understood. We show here that in T cells Discs large (Dlg1), a PDZ domain-containing protein, is recruited upon activation to cortical actin and forms complexes with early participants in T cell activation. Transient overexpression of Dlg1 attenuates basal and Vav1-induced NFAT reporter activation. Reduction of Dlg1 expression by RNA interference enhances both CD3- and superantigen-mediated NFAT activation. Attenuation of antigen receptor signaling appears to be a complex, highly orchestrated event that involves the mutual segregation of important elements of the early signaling complex.

Actins↗

PubMed2004

The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death.

The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. Herein we demonstrate a novel aspect of CNK1 function, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) have been reported previously. In this study, we examined the hypothesis that overexpression of vasculoprotective gene endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) in EPCs enhances their ability to inhibit neointimal hyperplasia. METHODS AND RESULTS: EPCs were isolated from rabbit peripheral blood, expanded in culture, and transduced with pseudotyped retroviral vectors expressing human eNOS (eNOS-EPCs), HO-1 (HO-1-EPCs), or green fluorescent protein (GFP-EPCs). Transduction efficiency of EPCs ex vivo was >90%. Four groups of rabbits (n=5 to 6 per group) were subjected to balloon angioplasty of the common carotid artery. Immediately after injury, approximately 5x10(6) autologous eNOS-EPCs or HO-1-EPCs were transplanted into the injured vessel. Control animals received an equivalent number of GFP-EPCs or Ringer's saline. Two weeks after transplantation, eNOS and HO-1 transgene transcripts and proteins were detected in the transduced rabbit vessels. Endothelialization was enhanced in the EPC-transplanted vessels independently of gene transfer. Neointimal thickening was significantly reduced in the GFP-EPC-treated vessels relative to the saline control. Neointima size was further reduced in vessels treated with eNOS-EPCs. Surprisingly, no additional reduction was seen in vessels treated with HO-1-EPCs relative to GFP-EPCs. Thrombosis occurred in approximately 50% of the saline-treated vessels but was virtually absent in all EPC-transplanted vessels. CONCLUSIONS: We conclude that transplantation of autologous EPCs overexpressing eNOS in injured vessels enhances the vasculoprotective properties of the reconstituted endothelium, leading to inhibition of neointimal hyperplasia. This cell-based gene therapy strategy may be useful in treatment of vascular disease.

Angioplasty, Balloon↗

PubMed2003

The angiotensin II type I receptor-associated protein, ATRAP, is a transmembrane protein and a modulator of angiotensin II signaling.

Our group identified angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) in a yeast two-hybrid screen for proteins that bind to the carboxyl-terminal cytoplasmic domain of the AT1. In this work, we characterize ATRAP as a transmembrane protein localized in intracellular trafficking vesicles and plasma membrane that functions as a modulator of angiotensin II-induced signal transduction. ATRAP contains three hydrophobic domains at the amino-terminal end of the protein, encompassing the amino acid residues 14-36, 55-77, and 88-108 and a hydrophilic cytoplasmic carboxyl-terminal tail from residues 109-161. Endogenous and transfected ATRAP cDNA shows a particulate distribution; electron microscopy reveals the presence of ATRAP in prominent perinuclear vesicular membranes; and colocalization analysis by immunofluorescence shows that ATRAP colocalizes in an intracellular vesicular compartment corresponding to endoplasmic reticulum, Golgi, and endocytic vesicles. Real-time tracking of ATRAP vesicles shows constitutive translocation toward the plasma membrane. Using epitope-tagged forms of ATRAP at either the amino or carboxyl end of the molecule, we determined the orientation of the amino end as being outside the cell. Mutant forms of ATRAP lacking the carboxyl end are unable to bind to the AT1 receptor, leading to the formation of prominent perinuclear vesicle clusters. Functional analysis of the effects of ATRAP on angiotensin II-induced AT1 receptor signaling reveals a moderate decrease in the generation of inositol lipids, a marked decrease in the angiotensin II-stimulated transcriptional activity of the c-fos promoter luciferase reporter gene, and a decrease in cell proliferation.

Adaptor Proteins, Signal Transducing↗