Biomedical subjects

T Quertermous

Publications and source records attributed to T Quertermous.

At least 19 recordsLinked to original sources

PubMed1999

Cloning of a unique lipase from endothelial cells extends the lipase gene family.

A new lipoprotein lipase-like gene has been cloned from endothelial cells through a subtraction methodology aimed at characterizing genes that are expressed with in vitro differentiation of this cell type. The conceptual endothelial cell-derived lipase protein contains 500 amino acids, including an 18-amino acid hydrophobic signal sequence, and is 44% identical to lipoprotein lipase and 41% identical to hepatic lipase. Comparison of primary sequence to that of lipoprotein and hepatic lipase reveals conservation of the serine, aspartic acid, and histidine catalytic residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein endothelial cells, human coronary artery endothelial cells, and murine endothelial-like yolk sac cells by Northern blot. In addition, Northern blot and in situ hybridization analysis revealed expression of the endothelial-derived lipase in placenta, liver, lung, ovary, thyroid gland, and testis. A c-Myc-tagged protein secreted from transfected COS7 cells had phospholipase A1 activity but no triglyceride lipase activity. Its tissue-restricted pattern of expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions in lipoprotein metabolism and in vascular disease.

Amino Acid Sequence↗

PubMed1999

Del1 induces integrin signaling and angiogenesis by ligation of alphaVbeta3.

Del1 is a novel extracellular matrix protein encoding three Notch-like epidermal growth factor repeats, an RGD motif, and two discoidin domains. Del1 is expressed in an endothelial cell-restricted pattern during early development. In studies reported here, recombinant baculovirus Del1 protein was shown to promote alphavbeta3-dependent endothelial cell attachment and migration. Attachment of endothelial cells to Del1 was associated with clustering of alphavbeta3, the formation of focal complexes, and recruitment of talin and vinculin into these complexes. These events were shown to be associated with phosphorylation of proteins in the focal complexes, including the time-dependent phosphorylation of p125(FAK), MAPK, and Shc. When recombinant Del1 was evaluated in an in ovo chick chorioallantoic membrane assay, it was found to have potent angiogenic activity. This angiogenic activity was inhibited by a monoclonal antibody directed against alphavbeta3, and an RAD mutant Del1 protein was inactive. Thus Del1 provides a unique autocrine angiogenic pathway for the embryonic endothelium, and this function is mediated in part by productive ligation of integrin alphavbeta3.

Amino Acid Sequence↗

PubMed1998

Regional variability in preproEndothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension. Participation Of arterial smooth muscle cells.

We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.

Animals↗

PubMed1998

Functional analysis of the endothelial cell-specific Tie2/Tek promoter identifies unique protein-binding elements.

To investigate the molecular basis of endothelial cell-specific gene expression, we have examined the DNA sequences and the cognate DNA-binding proteins that mediate transcription of the murine tie2/tek gene. Reporter transfection experiments conformed with earlier findings in transgenic mice, indicating that the upstream promoter of Tie2/Tek is capable of activating transcription in an endothelial cell-specific fashion. These experiments have also allowed the identification of a single upstream inhibitory region (region I) and two positive regulatory regions (regions U and A) in the proximal promoter. Electrophoretic mobility-shift assays have allowed further characterization of three novel DNA-binding sequences associated with these regions and have provided preliminary characterization of the protein factors binding to these elements. Two of the elements (U and A) confer increased transcription on a heterologous promoter, with element U functioning in an endothelial-cell-selective manner. By employing embryonic endothelial-like yolk sac cells in parallel with adult-derived endothelial cells, we have identified differences in functional activity and protein binding that may reflect mechanisms for specifying developmental regulation of tie2/tek expression. Further study of the DNA and protein elements characterized in these experiments is likely to provide new insight into the molecular basis of developmental- and cell-specific gene expression in the endothelium.

Animals↗

PubMed1998

Cloning and characterization of developmental endothelial locus-1: an embryonic endothelial cell protein that binds the alphavbeta3 integrin receptor.

We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the alphavbeta3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the alphavbeta3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.

Amino Acid Sequence↗

PubMed1998

Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries.

The basic helix-loop-helix (bHLH) class of transcription factors have been linked to a variety of cellular differentiation processes, including myogenesis, neurogenesis and hematopoiesis. Here we report the cloning of a new member of this family of factors, capsulin. Capsulin was shown to be expressed as early as 9.5 days of mouse development, with expression in mesodermal cells that are progenitors of the epicardium and the coronary arteries. At later stages of development, expression is seen in mesenchymal cells that are closely associated with the epithelium of the developing lung, gut and kidney. In the proepicardial organ, and in the organs where it is expressed in later development, capsulin is expressed in cells that give will give rise to smooth muscle. Given the likely expression of capsulin in smooth muscle cell progenitors, and significant sequence similarity through the bHLH domain, capsulin may be a functional ortholog of a Drosophila gene that is expressed in cells that give rise to the longitudinal visceral muscle. Capsulin alone or in combination with other bHLH proteins, was shown to function as a transcription factor by its ability to transactivate both a synthetic and a native promoter, each of which contains multiple E-boxes. These studies extend the growing family of bHLH factors that are expressed in the early mesoderm, and suggest that capsulin may have a functional role in development of the coronary vasculature and organs containing epithelial lined tubular structures.

3T3 Cells↗

PubMed1997

Identification of an endothelial cell-specific regulatory region in the murine endothelin-1 gene.

Endothelin-1 is a 21-amino acid peptide first characterized as a potent vasoactive compound synthesized by endothelial cells. Because of its high level cell-restricted pattern of expression, we have employed this gene as a model for investigating the DNA and protein elements that mediate endothelial cell-specific gene expression. In this study we have identified a complex positive regulatory region located at base pairs -364 to -320 in the murine endothelin-1 gene. This region consists of three functionally dependent elements, ETE-C, ETE-D, and ETE-E, which are all required for full activity. When a 43-base pair fragment containing these three elements was employed in heterologous promoter experiments, this sequence was capable of increasing transcriptional activity in an endothelial cell-specific fashion. None of the elements contains a recognized consensus sequence known to bind transcriptional regulatory proteins in higher eukaryotes; however, each element does appear to mediate protein binding. The combination of all three elements promotes binding of a protein complex that is endothelial cell-specific. This is the first evidence for an endothelial cell-specific DNA regulatory element and cognate binding proteins.

Animals↗

PubMed1997

Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension.

The human endothelin-1 (ET-1) gene under the control of its natural promoter was transferred into the germline of mice. The transgene was expressed predominantly in the brain, lung, and kidney. Transgene expression was associated with a pathological phenotype manifested by signs such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis leading to a progressive decrease in glomerular filtration rate. This pathology developed in spite of only slightly elevated plasma and tissue ET-1 concentrations. Blood pressure was not affected even after the development of an impaired glomerular filtration rate. Therefore, these transgenic lines provide a new blood pressure-independent animal model of ET-1-induced renal pathology leading to renal fibrosis and fatal kidney disease.

Animals↗

PubMed1997

Functional activity of the CFTR Cl- channel in human myocardium.

The cyclic AMP (cAMP)-dependent chloride channel in the heart has been identified in various species as the cystic fibrosis transmembrane conductance regulator (CFTR). Although functional expression of the channel in the human atrium has been reported, we could not induce any cAMP-dependent chloride conductance in the atrial cells even with maximal cAMP stimulation, whereas the conductance could be induced in rabbit ventricular cells. To clarify the discrepancy between the results, we examined the level of CFTR mRNA expression in both conductance-positive (human colonic epithelium and rabbit ventricle) and -negative (human atrium) tissues. Total RNA samples prepared from these tissues were subjected to the reverse transcription-polymerase chain reaction (RT-PCR). While CFTR transcripts were amplified from the conductance-positive samples, no amplified products could be detected from the conductance-negative sample. A nested PCR performed on the RT-PCR products of the conductance-negative sample resulted in successful amplification of the transcripts, indicating that the level of the CFTR mRNA expression in human atrium is extremely low compared with that in colonic epithelium and rabbit ventricle. The same molecular results were observed in human ventricular tissues. A nucleotide sequencing of the amplified transcripts showed that exon 5 of the CFTR gene was not alternatively spliced in human atrium and ventricle, and both the exon 5 spliced and unspliced isoforms were expressed in rabbit ventricle, unlike the findings of previous reports. Our data suggest that the amount of CFTR expressed in human myocardium might be physiologically insufficient to activate detectable cAMP-dependent chloride conductance.

Adolescent↗

PubMed1996

Endogenous endothelin-1 mediates cardiac hypertrophy and switching of myosin heavy chain gene expression in rat ventricular myocardium.

OBJECTIVES: We investigated the role of endogenous endothelin-1 in the development of cardiac hypertrophy in vivo under pressure overload conditions. BACKGROUND: Endothelin-1, a potent vasoconstrictor peptide, has recently been shown to act as a growth factor of myocardial cells in culture. METHODS: We examined the effect of an endothelin-A receptor antagonist (FR139317) on the development of right ventricular hypertrophy in rats with monocrotaline-induced pulmonary hypertension. Three groups of rats were studied: those given monocrotaline alone or monocrotaline plus FR139317 and those given vehicle alone (control group). RESULTS: The ratio of right ventricular systolic pressure to aortic systolic pressure was similarly elevated in rats treated with monocrotaline and monocrotaline plus FR139317. The right ventricular/left ventricular weight ratio was increased in monocrotaline-treated rats but lower in rats treated with monocrotaline plus FR139317 than in those treated with monocrotaline alone (p < 0.01). As a biochemical marker of hypertrophy, the isoform ratio of beta-myosin heavy chain protein was determined for the right ventricular tissue samples. This ratio was increased in all monocrotaline-treated rats but was lower (p < 0.01) in rats given monocrotaline plus FR139317 than in those given monocrotaline alone. The isoform ratio of beta-myosin heavy chain messenger ribonucleic acid quantitated by S1 nuclease mapping also was lower (p < 0.025) in rats receiving monocrotaline plus FR139317 than in those receiving monocrotaline alone. CONCLUSIONS: These data suggest that blocking the action of endothelin-1 with a receptor antagonist ameliorates cardiac hypertrophy in this model system, and that this action is not mediated by ameliorating hemodynamic changes.

Animals↗

PubMed1996

Role of endothelin in a rabbit model of acute myocardial infarction: effects of receptor antagonists.

The role of endothelin (ET) in acute myocardial infarction and proarrhythmic potential was investigated in a rabbit model. One group of rabbits underwent 30 min of circumflex occlusion and 3 h of reperfusion with measurements of myocardial blood flow and myocardial levels of ET-1 messenger RNA (mRNA). In a second group, the systemic and coronary effects of exogenous ET were studied in animals pretreated with either saline, FR139317, an ETA-receptor antagonist, or PD145065, an ETA-and ETB-receptor antagonist. In a third study, animals undergoing 30 min of circumflex occlusion followed by 48 h of reperfusion were treated with exogenous ET-1, FR139317, PD145065, or saline. Arrhythmias were recorded and infarct size measured at 48 h. These studies revealed that ischemia and reperfusion was followed by a progressive microcirculatory failure ("no-reflow phenomenon") in rabbits. This was associated with a 2.6-fold elevation in levels of myocardial ET-1 mRNA in the ischemic zone in comparison to the nonischemic zone (p = 0.04). Exogenous ET-1 caused elevation in coronary and systemic vascular resistance that was significantly blocked by antagonism of the ETA receptor. In rabbits subjected to myocardial ischemia and reperfusion, ET-1 infusion led to a higher incidence of ventricular arrhythmias, whereas ET-receptor antagonism with PD145065 significantly reduced ventricular arrhythmias. Exogenous ET-1 and FR139317 failed to alter infarct size (AN) of the area at risk (AR) compared with control [AN/AR(%) was 46 +/- 8, 55 +/- 9, and 47 +/- 7, respectively]. However, PD145065 significantly decreased AN/AR (22 +/- 7; p < or = 0.02). The increased production of ET-1, resulting from increased levels of mRNA after reperfusion, may contribute to the no-reflow phenomenon. Although the vasoconstrictor effects of ET-1 can be blocked by ETA-receptor antagonism alone, only blockade of both the ETA and ETB receptors significantly reduced infarct size. These data suggest that production of ET increases in the heart during ischemia and is deleterious to the reperfused myocardium.

Animals↗

PubMed1995

Genomic organization and chromosomal localization of the gene TCF15 encoding the early mesodermal basic helix-loop-helix factor bHLH-EC2.

bHLH-EC2 is a recently characterized member of a growing family of basic helix-loop-helix transcription factors. This family includes bHLH factors such as twist, which appear to be primarily involved in early mesodermal differentiation, and bHLH factors such as TAL-1, which have been characterized through their association with chromosomal breakpoints associated with T-cell leukemias. To provide for studies aimed at understanding the genetic regulation of bHLH-EC2, we have characterized the organization of this gene and conducted preliminary studies of the transcriptional activity of the upstream promoter region. The mouse bHLH-EC2 gene was found to consist of two exons separated by a 5-kb intron, an organization pattern similar to the mouse twist gene. The transcription initiation site was identified by RNase protection assay and primer extension analysis. Linked promoter-reporter gene transfection experiments in cultured cells indicated that while the identified upstream sequence can function to promote transcription, it does not function in a cell-specific fashion. To investigate the possible association of bHLH-EC2 with hematological malignancy, the chromosomal location of this gene in the human was mapped by fluorescence in situ hybridization and assigned to chromosome band 20p13.

Animals↗

PubMed1995

Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.

We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.

Alternative Splicing↗

PubMed1995

Directed endothelial differentiation of cultured embryonic yolk sac cells in vivo provides a novel cell-based system for gene therapy.

Cultured murine yolk sac cells transfected with the cytomegalovirus immediate early promoter/human growth hormone (CMVIE-hGH) fusion gene, expressing high levels of hGH in culture, and suspended in Matrigel were subcutaneously (s.c.) injected into experimental mice. The injected cells were shown to form discrete vesicular structures within the Matrigel implant, suggesting directed differentiation of the embryonic yolk sac cells into endothelial tissue. Human growth hormone radioimmune assay of these mice showed sustained physiologically significant levels of hGH in their serum for beyond four months. These results confirmed that long-term cultured murine embryonic yolk sac cells can be induced to differentiate into endothelial cells both in vivo and in vitro and suggested a novel approach to the delivery to the circulation of therapeutic proteins for the treatment of inherited and acquired diseases.

Animals↗

PubMed1995

Cooperative interaction of GATA-2 and AP1 regulates transcription of the endothelin-1 gene.

Endothelin-1 (ET-1) is a 21-amino-acid vasoactive peptide initially characterized as a product of endothelial cells. Reporter gene transfection experiments have indicated that a GATA site and an AP1 site are essential for ET-1 promoter function in endothelial cells, and GATA-2 appears to be the active GATA factor which regulates ET-1 expression. To look for interactions between AP1 and GATA-2, transactivation experiments were performed with expression vectors encoding c-Jun, c-Fos, and GATA-2. Cooperativity between the AP1 complex and GATA-2 was observed as a synergistic increase in transcriptional activity of the ET-1 reporter plasmid. In addition, AP1 was able to potentiate the action of GATA-2 on reporter constructs lacking a functional AP1 site. In a similar fashion, GATA-2 was able to potentiate the action of AP1 despite deletion of the GATA site. Experiments with GATA-1 and GATA-3 expression vectors provided evidence that this capacity to interact with AP1 may be a characteristic of all GATA family members. Biochemical evidence for AP1-GATA interaction was provided by immunoprecipitation experiments. A GATA-2-specific antiserum was shown to immunoprecipitate in vitro-synthesized Jun and Fos protein from reticulocyte lysate. Also, antisera directed against Jun and Fos were able to immunoprecipitate from nuclear extracts a GATA-binding protein, indicating the association of AP1 and GATA proteins in vivo.

Base Sequence↗

PubMed1995

Regional and maturation-associated expression of endothelin 2 in rat gastrointestinal tract.

Endothelin 2 (ET2), also referred to as vasoactive intestinal contractor peptide, is a member of a family of vasoactive peptides. ET2 is a potent constrictor of intestinal smooth muscle, and the mRNA that encodes it has been detected in murine intestinal extracts. To further investigate the potential physiological roles of ET2, we characterized the cellular distribution of ET2 gene expression in adult rat gastrointestinal tract. Using an RNAse protection assay, an overall proximal to distal gradient of increasing ET2 gene expression was observed from stomach to colon. In situ hybridization studies confirmed this finding and demonstrated ET2 mRNA localized in lamina propria stromal cells. Moreover, ET2 gene expression in stromal cells increased from crypt to villous tip. The results demonstrate that ET2 is produced by stromal cells in villi throughout the intestine. Increased ET2 gene expression at the villous tip is associated with more mature overlying epithelial cells, suggesting a possible role for this vasoactive peptide in intestinal epithelial differentiation or secretory activity.

Animals↗

PubMed1994

Cloning and characterization of a basic helix-loop-helix protein expressed in early mesoderm and the developing somites.

Basic helix-loop-helix (bHLH) heterodimer protein complexes regulate transcription of genes during the processes of differentiation and development. To study the molecular basis of early mesodermal differentiation, we sought to identify bHLH proteins from cells of mesodermal origin. By using an interaction cloning strategy with a radiolabled recombinant bHLH protein, E12, a clone encoding a potential heterodimer partner was isolated from an endothelial cell library. This gene (bHLH-EC2) is most homologous to Twist but shares similarity within the bHLH domain with TAL1 and other members of this family. bHLH-EC2 is expressed in cultured endothelial cells and in embryonic stem cell, erythroleukemia, and muscle cell lines in a differentiation-dependent manner. In situ hybridization studies of mouse embryos reveal that bHLH-EC2 is expressed throughout the primitive mesoderm as early as 7.5 days postcoitum. Expression then becomes restricted to the paraxial mesoderm and to the dermamyotome of the developing somite. Expression of bHLH-EC2 in cells destined to become myoblasts thus predates expression of myogenic bHLH factors. bHLH-EC2 is expressed in early endothelial and hematopoietic cells in vivo, as shown by RNA studies of embryonic yolk sac and cultured cells derived from yolk sac explants. These findings suggest that bHLH-EC2 plays a role in the development of multiple cell types derived from the primitive mesoderm.

Amino Acid Sequence↗

PubMed1994

The immunoreactive region in a novel autoantigen contains a nuclear localization sequence.

Antibodies in the serum of patients with autoimmune diseases have been used to identify human autoantigens. Because autoantibodies often recognize active sites within corresponding protein antigens, autoantibodies have facilitated the functional characterization of these polypeptides. In the present study, serum from a patient with Sjögren's syndrome was used to identify a novel autoantigen which was designated Ge-1. Using the patient's serum, a 4.8-kb cDNA encoding Ge-1 was identified. Fragments of the cDNA were ligated into prokaryotic expression vectors, expressed in Escherichia coli, and used to produce recombinant Ge-1 fusion proteins. Fusion proteins containing different portions of Ge-1 were used to identify a 58 amino acid immunoreactive region within the protein. This immunoreactive region contained the protein's putative nuclear localization sequence (NLS). To demonstrate that the immunoreactive region was capable of functioning as a NLS, a eukaryotic expression plasmid was constructed to encode the immunoreactive region fused to the cytoplasmic protein, chicken muscle pyruvate kinase. After transfection of this plasmid into COS-1 cells, the fusion protein was detected in the nucleus. The presence of the NLS motif within the immunoreactive region of Ge-1 and other nuclear autoantigens suggests that the NLS may be a target of human autoantibodies.

Amino Acid Sequence↗