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Philip A Marsden

Publications and source records attributed to Philip A Marsden.

36 records · Page 2Linked to original sources
PubMed2004

The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation.

The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episome-based eNOS promoters, we examined the methylation status of 5'-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.

Animals↗
PubMed2004

The fgl2 prothrombinase/fibroleukin gene is required for lipopolysaccharide-triggered abortions and for normal mouse reproduction.

Increased fgl2 prothrombinase activity in maternal decidua and fetal trophoblasts may trigger abortions by proinflammatory cytokines induced by bacterial lipopolysaccharide (LPS) in mice and is implicated in human recurrent miscarriages and pre-eclampsia. Defining the physiological and pathological role of the fgl2/fibroleukin gene required an fgl2-knockout mouse and data on normal pattern of fgl2 expression during pregnancy. Expression of fgl2 protein was determined by immunostaining with specific antibody. Fgl2 knockout mice were generated and typed by PCR for presence of the altered gene. Immunostaining of timed CBAxDBA/2 mouse matings in a low-abortion-rate colony showed a distinct pattern of development of fgl2 protein expression in maternal decidua, and in embryonic tissues in early pregnancy. Outbred (mixed background) heterozygous fgl2 +/-x+/- matings with a similar low abortion rate showed selective occult loss of both +/- and, to a greater extent, -/- embryos prior to gestation day 11.5, in association with haemorrhage at the anti-mesometrial pole of fgl2-deficient embryo. LPS injected on day 6.5 caused classical abortions at mid-pregnancy in fgl2 +/+x+/+ matings, but not -/-x-/- matings. Physiological expression of fgl2 in fetal trophoblast may prevent occult loss in early pregnancy, along with other coagulation factors, but fgl2 expression is required for LPS to induce abortion pathology.

Abortion, Spontaneous↗
PubMed2003

Role of the 3'-untranslated region of human endothelin-1 in vascular endothelial cells. Contribution to transcript lability and the cellular heat shock response.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978-987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.

3' Untranslated Regions↗
PubMed2003

Endothelial nitric oxide synthase: a new paradigm for gene regulation in the injured blood vessel.

Advances in our understanding of the molecular mechanisms involved in the constitutive and regulated expression of endothelial nitric oxide synthase (eNOS) mRNA expression present a new level of complexity to the study of endothelial gene regulation in health and disease. Recent studies highlight the contribution of both transcription and RNA stability to net steady-state mRNA levels of eNOS in vascular endothelium, introducing a new paradigm to gene regulation in the injured blood vessel. Constitutive eNOS expression is dependent on basal transcription machinery in the core promoter, involving positive and negative protein-protein and protein-DNA interactions. Chromatin-based mechanisms and epigenetic events also regulate expression of eNOS at the transcriptional level in a cell-restricted fashion. Although constitutively active, important physiological and pathophysiologic stimuli alter eNOS gene transcription rates. For instance, eNOS transcription rates increase in response to lysophosphatidylcholine, shear stress, and TGF-beta, among others. Under basal conditions, eNOS mRNA is extremely stable. Surprisingly, posttranscriptional mechanisms have emerged as important regulatory pathways in the observed decreases in eNOS expression in some settings. In models of inflammation, proliferation/injury, oxidized low-density lipoprotein treatment, and hypoxia, eNOS mRNA destabilization plays a significant role in the rapid downregulation of eNOS mRNA levels.

Animals↗
PubMed2003

Abnormalities in villin gene expression and canalicular microvillus structure in progressive cholestatic liver disease of childhood.

BACKGROUND: The molecular basis of clinical cholestasis is a subject of intense investigation. Villin is an actin binding, bundling, and severing protein needed for maintenance of structural integrity of canalicular microvilli, in which membrane transporters required for bile secretion are located. We aimed to investigate the role of canalicular cytoskeletal proteins in three genetically unrelated children with a biliary atresia-like clinical disorder, each of whom developed liver failure requiring liver transplantation. METHODS: Explanted livers from the three patients were examined by standard pathological methods followed by transmission and cryoimmunoelectron microscopy. With archival tissue samples, a panel of cytoskeletal proteins was investigated by immunohistochemistry and western blotting, with purified canalicular membrane preparations. Villin mRNA analyses were undertaken on liver homogenates, with primers from coding regions of the human villin gene. Classic biliary atresia, other types of cholestasis, and normal livers served as controls. FINDINGS: In patients, pronounced ultrastructural deformities of canaliculi and especially of their microvilli were noted, which correlated with absence of villin protein by immunostaining of liver tissue sections and by western blot analysis. Additionally, villin mRNA was strikingly reduced or absent. These results differed greatly from those in controls. INTERPRETATION: These results suggest that the disorder described mimics biliary atresia, but structural and molecular pathological findings differ. We propose that a functional abnormality in villin gene expression is key to the mechanism of cholestasis in patients with progressive cholestasis and hepatic failure.

Biliary Atresia↗
PubMed2003

MAP kinase kinase 6-p38 MAP kinase signaling cascade regulates cyclooxygenase-2 expression in cardiac myocytes in vitro and in vivo.

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in delayed prostaglandin biosynthesis. The purpose of this study was to evaluate the role of the MAP kinase kinase 6 (MKK6)-p38 MAPK signaling cascade in the regulation of myocardial COX-2 gene expression, in vitro and in vivo. RT-PCR analysis identified p38alpha and p38beta2 MAPK mRNA in rat cardiac myocytes. Interleukin-1beta induced the phosphorylation of p38alpha and p38beta2 MAPK in cardiomyocytes and stimulated RNA polymerase II binding to the COX-2 promoter, COX-2 transcription, COX-2 protein synthesis, and prostaglandin E2 (PGE2) release. Infecting cardiomyocytes with adenoviruses that encode mutant, phosphorylation-resistant MKK6 or p38beta2 MAPK inhibited interleukin-1beta-induced p38 MAPK activation, COX-2 gene expression, and PGE2 release. Treatment with the p38alpha and p38beta2 MAPK inhibitor, SB202190, attenuated interleukin-1beta-induced COX-2 transcription and accelerated the degradation of COX-2 mRNA. Cells infected with adenoviruses encoding wild-type or constitutively activated MKK6 or p38beta2 MAPK, in the absence of interleukin-1beta, exhibited increased cellular p38 MAPK activity, COX-2 mRNA expression, and COX-2 protein synthesis, which was blocked by SB202190. In addition, elevated levels of COX-2 protein were identified in the hearts of transgenic mice with cardiac-restricted expression of wild-type or constitutively activated MKK6, in comparison with nontransgenic littermates. These results provide direct evidence that MKK6 mediated p38 MAPK activation is necessary for interleukin-1beta-induced cardiac myocyte COX-2 gene expression and PGE2 biosynthesis in vitro and is sufficient for COX-2 gene expression by cardiac myocytes in vitro and in vivo.

Animals↗
PubMed2003

Perturbations in paracrine control of the circulation: role of the endothelial-derived vasomediators, endothelin-1 and nitric oxide.

Central to the control of vascular resistance in the systemic and pulmonary circulation and at the solid organ level is the function of the endothelial-derived vasomediators, endothelin-1 (ET-1) and nitric oxide. Regulation of steady-state levels of ET-1 and endothelial nitric oxide synthase (eNOS) mRNAs represents an early and influential step in their biosynthesis and is highly responsive to exogenous stimuli. ET-1 is expressed from a rapidly inducible promoter to generate a highly labile mRNA. Conversely, the eNOS promoter generates a constitutive level of a very stable mRNA and utilizes posttranscriptional mechanisms to modulate mRNA expression. The response of these genes in models of cellular activation commonly reflects a reciprocal pattern of regulation, namely, transcriptional induction of ET-1 and destabilization of the eNOS mRNA. Elucidating the mechanisms influencing ET-1 and eNOS mRNA is providing novel insight into endothelial gene regulation and providing opportunities for future therapeutic strategies.

Animals↗
PubMed2003

Gene transcription of fgl2 in endothelial cells is controlled by Ets-1 and Oct-1 and requires the presence of both Sp1 and Sp3.

The immune coagulant fgl2/fibroleukin has been previously shown to play a pivotal role in the pathogenesis of murine and human fulminant hepatitis and fetal loss syndrome. Constitutive expression of fgl2 transcripts at low levels are seen in cytotoxic T cells, endothelial, intestinal and trophoblast cells, while specific factors (such as virus and cytokines) are required to induce high levels of fgl2 expression in other cell types including monocytes/macrophages. To address the transcriptional mechanisms that regulate constitutive expression of fgl2, murine genomic clones were characterized and the transcription start site was defined by 5'-RACE and primer extension. A comprehensive assessment of basal fgl2 promoter activity in murine vascular endothelial cells defined a minimal 119 bp region responsible for constitutive fgl2 transcription. A complex positive regulatory domain (PRD) spanning a 39-bp sequence from -87 to -49 (relative to the transcription start site) was identified. Electrophoretic mobility shift assay studies in vascular endothelial cells revealed that the nucleoprotein complexes that form on this positive regulatory domain (PRD) contain Sp1/Sp3 family members, Oct-1, and Ets-1. Heterologous expression studies in Drosophila Schneider cells confirmed that the constitutive expression of this gene is controlled by Ets-1 and requires the presence both of the Sp1 and Sp3 transcription factors. The presence of this complex multicomponent PRD in the fgl2 proximal promoter is consistent with the observation that, in vivo, fgl2 expression is tightly regulated. Moreover, viral induced fgl2 expression also requires the presence of this PRD. These results clearly demonstrate that multiple cis DNA elements in a clustered region work cooperatively to regulate constitutive fgl2 expression and interact with inducible elements to regulate viral-induced fgl2 expression in endothelial cells.

Animals↗
PubMed2003

The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis.

Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of murine hepatitis virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in murine viral hepatitis we generated a Fgl2/fibroleukin-deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin-/- mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral hepatitis we studied patients with minimal and marked chronic hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for Fgl2/fibroleukin in the pathophysiology of experimental and human viral hepatitis.

Adult↗
PubMed2002

Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

5' Untranslated Regions↗
PubMed2002

Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity.

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent V(max) value of 6 mol/min/mol fgl2 and an apparent K(m) value for prothrombin of 8.3 microM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V(max) value to 3670 mol/min/mol fgl2 and decreasing the apparent K(m) value for prothrombin to 7.2 microM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.

Animals↗
PubMed2002

Regulation of Toll-like receptor 4 expression in the lung following hemorrhagic shock and lipopolysaccharide.

The Toll-like receptor 4 (TLR4) has recently been shown to function as the major upstream sensor for LPS. In this study, a rodent model of lung injury following resuscitated hemorrhagic shock was used to examine the regulation of TLR4 gene and protein expression in vivo and in vitro. Intratracheal LPS alone induced a rapid reduction in whole lung TLR4 mRNA, an effect which is also observed in recovered alveolar macrophages. This effect appeared to be due to a lowering of TLR4 mRNA stability by approximately 69%. By contrast, while shock/resuscitation alone had no effect on TLR4 mRNA levels, it markedly altered the response to LPS. Specifically, antecedent shock prevented the LPS-induced reduction in TLR4 mRNA levels. This reversal was explained by the ability of prior resuscitated shock both to prevent the destabilization of TLR4 mRNA by LPS and also to augment LPS-stimulated TLR4 gene transcription compared with LPS alone. Oxidant stress related to shock/resuscitation appeared to contribute to the regulation of TLR4 mRNA, because supplementation of the resuscitation fluid with the antioxidant N-acetylcysteine reversed the ability of shock/resuscitation to preserve TLR4 mRNA levels following LPS. TLR4 protein levels in whole lung mirrored the changes seen for TLR4 mRNA. Considered in aggregate, these data suggest that levels of tlr4 expression are controlled both transcriptionally as well as posttranscriptionally through altered mRNA stability and that antecedent shock/resuscitation, a form of global ischemia/reperfusion, might influence regulation of this gene.

Animals↗
PubMed2002

An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells.

Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of beta-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. beta-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P(450)scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, beta-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of beta-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, beta-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of the male gonad suggests an important role in the regulation of testosterone release and represents an intriguing model with which to dissect the molecular basis of Leydig cell-specific gene expression.

Animals↗
PubMed2002

Familial risk of preeclampsia in Newfoundland: a population-based study.

This study sought to quantify the familial risk of preeclampsia (proteinuric hypertension) in Newfoundland and to identify characteristics in probands that predict increased familial risk. Reviewed were 5173 obstetric charts from 10 hospitals, representing 99% of deliveries on the island of Newfoundland for a 1-yr period from April 1996 to March 1997; pregnancy-induced hypertension was diagnosed according to strict criteria. Family obstetric histories were obtained from identified probands with preeclampsia, and sisters and mothers of probands were interviewed. In addition, the obstetric charts from sisters and mothers were reviewed to identify preeclampsia. The incidence of preeclampsia in the population was 5.6% (n = 292), and in primiparous women it was 7.9%. Factors independently associated with increased risk of preeclampsia included primiparous delivery, multiple gestation, pregestational and gestational diabetes, maternal age of more than 35 yr, and region of the province. Of 330 sisters identified, 217 had 445 pregnancies, with 331 charts located for review. The incidence of preeclampsia (based on chart review) in 163 primiparous sisters was 20.2%. The relative risk of preeclampsia in primiparous sisters of probands with preeclampsia compared with primiparous women in the population was 2.6 (95% confidence interval, 1.8 to 3.6). Factors in probands independently associated with a higher risk of preeclampsia in sisters included at least 2+ proteinuria and region of the province. This population-based study, which used unbiased ascertainment and strict diagnostic criteria, demonstrated a significantly higher risk of preeclampsia in sisters of probands with preeclampsia, particularly when probands were defined by severity of preeclampsia and by geographic region.

Adult↗