Structural and agonist properties of XCL2, the other member of the C-chemokine subfamily.

Known for its unusual metamorphic native state structure, XCL1 has been the focus of most efforts to elucidate the structural, functional, and physiological properties of chemokines in the C subfamily. By comparison, its closely related paralog XCL2 remains virtually uncharacterized. Based on the importance of the chemokine N-terminus in receptor activation, it was hypothesized that two amino acid differences in XCL2 would alter its agonist activity relative to XCL1 for their shared receptor XCR1. This present study reveals several properties of XCL2 that were unexamined until now. Structurally, XCL1 and XCL2 are very similar, exchanging between the monomeric chemokine fold and an unrelated dimeric state under physiological NaCl and temperature conditions. Ca(2+) flux, chemotaxis, and heparin binding assays showed that the monomer form of XCL2 is responsible for G protein-coupled receptor activation while the dimeric form is important for GAG binding. Despite their high structural similarity, XCL2 displays a slightly higher affinity for heparin than XCL1. Because their in vitro functional profiles are virtually identical, distinct physiological roles for XCL1 and XCL2 are probably encoded at the level of expression.

Protein composition of plasminogen activator inhibitor type 1-derived endothelial microparticles.

Endothelial microparticles (EMPs) are small vesicles released from the plasma membrane of endothelial cells in response to cell injury, apoptosis, or activation. Low levels of MPs are shed into the blood from the endothelium, but in some pathologic states, the number of EMPs is elevated. The mechanism of MP formation and the wide-ranging effects of elevated EMPs are poorly understood. Here, we report the protein composition of EMPs derived from human umbilical cord endothelial cells stimulated with plasminogen activator inhibitor type 1 (PAI-1). Two-dimensional gel electrophoresis followed by mass spectrometry identified 58 proteins, of which some were verified by Western blot analysis. Gene Ontology database searches revealed that proteins identified on PAI-1-derived EMPs are highly diverse. Endothelial microparticles are composed of proteins from different cellular components that exhibit multiple molecular functions and are involved in a variety of biological processes. Important insight is provided into the generation and protein composition of PAI-1-derived EMPs.

Structure of the SCAN domain from the tumor suppressor protein MZF1.

The SCAN domain mediates interactions between members of a subfamily of zinc-finger transcription factors and is found in more than 60 C2H2 zinc finger genes in the human genome, including the tumor suppressor gene myeloid zinc finger 1 (MZF1). Glutathione-S-transferase pull-down assays showed that the MZF1 SCAN domain self-associates, and a Kd value of 600 nM was measured by intrinsic tryptophan fluorescence polarization. The MZF1 structure determined by NMR spectroscopy revealed a domain-swapped dimer. Each monomer consists of five alpha helices in two subdomains connected by the alpha2-alpha3 loop. Residues from helix 3 of each monomer compose the core of the dimer interface, while the alpha1-alpha2 loop and helix 2 pack against helices 3 and 5 from the opposing monomer. Comprehensive sequence analysis is coupled with the first high-resolution structure of a SCAN dimer to provide an initial view of the recognition elements that govern dimerization for this large family of transcription factors.

The molecular basis of congenital heart disease.

Clinically relevant congenital heart disease affects 1% of all live births. It is the leading cause of birth defects-related death in the United States, claiming more than 6000 lives per year. Despite the many advances in our understanding of cardiac development, the fundamental etiology for the majority of cases of congenital heart disease (CHD) remains unknown. Although causal links have been established, including maternal diabetes, exposure to drugs, and genetic variants in a few genes, these, at best, explain a small fraction of cases. Elucidating the molecular basis of CHD presents several challenges. While CHD has an increased risk of recurrence within families, suggesting genes are at play, CHD occurs with variable expressivity. Several chromosomal abnormalities clearly associate with CHD; however, many children with these same chromosomal abnormalities have normal hearts. Thus, the etiology cannot be explained by simple Mendelian genetics. Abnormal cardiac development occurs through a process that is complex, possibly involving both genetic and environmental risk factors. Because the majority of cases occur without known cause, the molecular basis of CHD is an active and evolving discussion.

Endothelium-derived microparticles inhibit human cardiac valve endothelial cell function.

Elevated numbers of endothelium-derived microparticles (EMPs) in the circulation are found in a variety of critical illnesses. EMPs have been associated with vascular dysfunction, including thrombotic complications and loss of normal vascular reactivity, common responses associated with cardiac valve injury. However, the exact mechanisms of this dysfunction and the potential impact on cardiac endothelium are unknown. We hypothesize that pathologic levels of circulating EMPs negatively regulate proliferation and migration of valvular endothelial cells (ECs), leading to downstream endothelial dysfunction. EMPs were generated from plasminogen activation inhibitor 1-stimulated human umbilical vein endothelial cells (HUVECs). Human mitral valve endothelial cells (HMVECs) were isolated and characterized by platelet endothelial cell-derived adhesion molecule-1 (PECAM-1, or CD31) and von Willebrand factor immunocytochemistry. HMVECs were treated with increasing EMP doses, and then, the effects of EMPs on growth factor-induced proliferation and migration were tested. Proliferation was assessed by H-thymidine incorporation. EC migration was assayed by photographing microtubules of HMVECs and HUVECs in fibrin gel incubated with EMPs +/- growth factors for 48 h. The EMP effects on non-valve HUVECs were tested in parallel. EMPs inhibited HMVEC proliferation at high doses but stimulated HUVEC proliferation at all doses. In HMVECs, EMPs inhibited basic fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and migration. Taken together, these data suggest EMPs regulate valvular EC proliferation in a dose-dependent manner and, furthermore, modulate growth factor signaling in ECs. These results implicate EMPs as a possible source of downstream EC dysfunction in disease states. EMPs may play a role in valvular leaflet injury in human disease by inhibiting normal growth and repair of endothelium.

Rosiglitazone antagonizes vascular endothelial growth factor signaling and nuclear factor of activated T cells activation in cardiac valve endothelium.

Nuclear factor of activated T cells, Cytoplasmic 1 (NFATc1) is required for heart valve formation. Vascular endothelial growth factor (VEGF) signaling, mediated by NFATc1 activation, positively regulates growth of valvular endothelial cells. However, regulators of VEGF/NFATc1 signaling in valve endothelium are poorly understood. Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits NFATc1 activity in T cells and cardiomyocytes, but it is not known if PPARgamma controls NFATc1 function in endothelial cells. The authors hypothesize PPARgamma antagonizes VEGF signaling in valve endothelium by inhibiting NFATc1. Endothelial cells isolated from human valve leaflet tissue were shown by immunocytochemistry to express the endothelial-specific markers von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule (PECAM)-1. VEGF-induced proliferation and migration of human pulmonary valve endothelial cells (HPVECs) were inhibited by rosiglitazone (ROSI), a specific ligand of PPARgamma activation, suggesting that PPARgamma disrupts VEGF signaling in the valve endothelium. ROSI also antagonized VEGF-mediated NFATc1 nuclear translocation in HPVECs, suggesting that PPARgamma inhibits VEGF signaling of NFATc1 activation in the valve. The effect of ROSI on nonvalve human umbilical vein endothelial cells (HUVECs) was tested in parallel and a similar inhibition of NFATc1 activation was observed. These data provide the first demonstration that ROSI negatively regulates VEGF signaling in the valve endothelium by a mechanism involving NFATc1 activation and nuclear translocation.

Molecular and cellular basis of congenital heart disease.

The cellular and molecular basis of congenital heart disease (CHD) is an evolving area of rapid discovery. This article introduced the basic mechanisms underlying cardiac development and CHD in order to permit a clear understanding of current diagnostics and therapeutics and their future development. It is clear that although significant advances have been made in understanding mechanisms controlling heart formation, the direct causes of CHD remain poorly defined. Future studies tha delineate the complexity of these mechanisms are required to provide a comprehensive understanding of the etiologies of CHD. Such understanding will lead to the development of novel approaches to prevention and therapy.

Characterization of the SCAN box encoding RAZ1 gene: analysis of cDNA transcripts, expression, and cellular localization.

The SCAN box (SRE-ZBP; CT-fin51; AW-1; Number 18) is a highly-conserved 80-amino-acid domain identified in a subset of C(2)H(2) zinc finger proteins. We and others have recently demonstrated that the SCAN box is a protein association domain that mediates hetero- and homo-protein associations with SCAN box containing proteins. RAZ1 (SCAN-related protein associated with MZF1B) is a novel gene identified in a yeast two hybrid genetic screen for binding to the MZF1B SCAN box. RAZ1 maps to chromosome 20q11 at a region frequently disrupted in various leukemias. We characterized the RAZ1 gene by analysing cDNA transcripts, mRNA expression, and cellular localization of the expressed protein. RAZ1 mRNA expression was detected in various human tissues and cell lines by Northern blot analysis and multiple tissue expression arrays. Highest levels of expression are in prostate, testis, thyroid, liver, and kidney. The RAZ1 gene produces two transcripts with variant 5'-untranslated regions containing identical open reading frames that express a 28 kDa protein in vitro. RAZ1 transcription start sites were mapped by primer extension and confirmed by identification of the RAZ1 promoter in the 5' flanking genomic DNA. RAZ1 protein fused to the green fluorescent protein (GFP) localizes to the nucleus in a diffuse pattern and the carboxyl terminus containing the SCAN-related domain is sufficient for nuclear localization. These data suggest that RAZ1 is a widely expressed nuclear protein that may function as a key regulator of zinc finger transcription factor function.

The SCAN domain defines a large family of zinc finger transcription factors.

The SCAN domain is a highly conserved dimerization motif that is vertebrate-specific and found near the N-terminus of C(2)H(2) zinc finger proteins (SCAN-ZFP). Although the function of most SCAN-ZFPs is unknown, some have been implicated in the transcriptional regulation of growth factors, genes involved in lipid metabolism, as well as other genes involved in cell survival and differentiation. Here we utilize a bioinformatics approach to define the structures and gene locations of the 71 members of the human SCAN domain family, as well as to assess the conserved syntenic segments in the mouse genome and identify potential orthologs. The genes encoding SCAN domains are clustered, often in tandem arrays, in both the human and mouse genomes and are capable of generating isoforms that may affect the function of family members. Twenty-three members of the mouse SCAN family appear to be orthologous with human family members, and human-specific cluster expansions were observed. Remarkably, the SCAN domains in lower vertebrates are not associated with C(2)H(2) zinc finger genes, but are contained in large retrovirus-like polyproteins. Collectively, these studies define a large family of vertebrate-specific transcriptional regulators that may have rapidly expanded during recent evolution.

An in vivo murine model of rosiglitazone use in pregnancy.

OBJECTIVE: To identify the effects of rosiglitazone use during murine pregnancy. DESIGN: The effect of rosiglitazone on blastocyst development was determined by culturing two-cell mouse embryos with rosiglitazone for 72 hours. From January to June 2005, five independent groups of ICR/CD1 female mice were treated with rosiglitazone during pregnancy, from the time of identification of seminal plugs until delivery of pups. SETTING: Controlled animal facility. ANIMAL(S): Two-cell mouse embryos and an outbred line of mice, ICR/CD1. INTERVENTION(S): Two-cell embryos were cocultured with rosiglitazone (10 microM) for 72 hours and scored. Ten-week-old female ICR mice were mated. Females with seminal plugs then were randomized to rosiglitazone (10 or 0.1 mg/kg per day) or to carrier alone, by gavage, until delivery. Weekly weights were obtained, and pregnancy outcomes were documented. MAIN OUTCOME MEASURE(S): Blastocyst development, number of pups and pup weights, and morphological changes. RESULT(S): Embryos exposed to rosiglitazone progressed to the blastocyst stage within 72 hours. Pregnant animals demonstrated normal weight gain throughout pregnancy. Postnatal growth and litter size were not statistically different between groups. No changes in normal mouse neonate development were observed. CONCLUSION(S): Rosiglitazone did not impair murine blastocyst development in vitro or cause phenotypic harm to the mouse fetus when administered during pregnancy, suggesting potential safety for rosiglitazone use in pregnancy.

NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells.

Mice deficient for the transcription factor NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent of some types of congenital human heart disease. We examined the mechanisms by which NFATc1 is activated and translocated to the nucleus in human pulmonary valve endothelial cells to gain a better understanding of its potential role(s) in post-natal valvular repair as well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both cyclosporin A and the calcineurin-specific peptide inhibitor reduced VEGF-induced human pulmonary valve endothelial cell proliferation, indicating a functional role for NFATc1 in endothelial growth. In contrast, VEGF-induced proliferation of human dermal microvascular and human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac valve maintenance and function by enhancing endothelial proliferation.