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G J Clesham

Publications and source records attributed to G J Clesham.

15 recordsLinked to original sources
PubMed2000

Identification and characterisation of transforming growth factor beta-regulated vascular smooth muscle cell genes.

Transforming growth factor beta (TGFbeta) is thought to play an important role in the development and/or progression of a number of vascular disorders through its numerous effects on vascular smooth muscle cells (VSMCs). In this study we sought to identify and characterize TGFbeta-regulated VSMC genes using differential mRNA display (DD-RT-PCR) analysis of RNA isolated from TGFbeta-stimulated cultured rat aortic VSMCs. Northern blot analysis was used to demonstrate that five of 19 differentially displayed bands identified represented VSMC transcripts differentially expressed by TGFbeta. DNA sequencing revealed that three of these TGFbeta regulated genes were novel whilst the remaining two were identified through homologies to known genes. One TGFbeta upregulated transcript represented the protease cathepsin B. Since cathepsins may play a role in TGFbeta activation, an enzyme-linked immunosorbent assay (ELISA) for active TGFbeta1 was used to demonstrate an effect of cathepsin B on TGFbeta1 activation in vitro using both recombinant and human serum platelet-derived latent TGFbeta1 as substrate. These results suggest that induction of cathepsin B by TGFbeta, and its ability to activate TGFbeta1, may represent a mechanism whereby the autocrine action of TGFbeta is facilitated through expression of a protein which can process its latent form.

Animals↗
PubMed1998

Expression of endoglin mRNA and protein in human vascular smooth muscle cells.

Endoglin, the gene linked to the autosomal dominant vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1), encodes a 95-kDa membrane-bound proteoglycan which binds TGF beta 1 and regulates signaling via the type I and II TGF beta receptors on the surface of vascular endothelial cells. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis we have shown that endoglin mRNA is expressed in both cultured human VSMCs and VSMCs freshly isolated from human aortas. Northern blot analysis was also used to demonstrate that endoglin expression decreased in serum-stimulated cultured human VSMCs but could be maintained by exogenous TGF beta 1. Endoglin protein expression in human VSMCs was shown by immunocytochemistry. These data, the first describing the existence of endoglin in VSMCs, suggest that through regulating TGF beta 1 signaling endoglin may mediate the effects of TGF beta 1 on VSMC behavior in vitro and in vivo.

Antigens, CD↗
PubMed1998

High adenoviral loads stimulate NF kappaB-dependent gene expression in human vascular smooth muscle cells.

Replication-deficient adenoviral vectors have been widely used for gene transfer with the aim of delivering genes of interest to investigate their function and potentially to treat human disease. The ability to critically evaluate the biological role of a gene of interest, using adenovirus-based vectors, has been hampered by the development of local inflammation at the site of delivery. We have demonstrated that high multiplicity infection of human VSMCs with a replication-deficient adenoviral vector expressing no transgene leads to activation of the transcription factor NF kappa B. Activation of NF kappa B by this mechanism was able to augment gene expression from the human cytomegalo-virus immediate-early promoter (CMV-IEP) and induce expression of the adhesion molecule ICAM-1 in human VSMCs. These effects were inhibited by pretreatment with N alpha-p-tosyl1-L-lysine chloromethyl ketone (TLCK), a serine protease inhibitor known to inhibit the activation of NF kappa B. This important effect of the vector itself may have profound implications when replication-deficient adenoviral vectors are used for experimental gene transfer at a high multiplicity of infection.

Adenoviridae↗
PubMed1997

Echocardiographic assessment of cardiac involvement in systemic AL amyloidosis in relation to whole body amyloid load measured by serum amyloid P component (SAP) clearance.

The severity of cardiac infiltration in AL amyloidosis is unrelated to whole body amyloid load as measured by serum amyloid P (SAP) tracer studies. Radiolabeled SAP and echocardiography permit identification of patients with severe cardiac disease with a low whole body load who may be the best candidates for transplantation.

Adult↗
PubMed1996

Enhancer stimulation unmasks latent gene transfer after adenovirus-mediated gene delivery into human vascular smooth muscle cells.

Recombinant adenoviral vectors are being used increasingly for gene transfer studies in mammalian cells and gene therapy protocols in humans. High adenoviral titers are often required for successful transduction of vascular smooth muscle cells (VSMCs), defined as uptake and detectable expression of the foreign gene, but the relative contributions of efficiency of viral uptake and control of transcription are poorly understood. To explore the extent to which a lack of detectable gene expression may be due to inefficient transcription of a successfully transferred gene, we have used a replication-deficient adenovirus expressing beta-galactosidase (RAd35 beta-Gal), under the control of the human cytomegalovirus major immediate-early promoter (CMV-IEP), which contains cAMP and nuclear factor-kappa B response elements, to investigate constitutive and inducible gene expression after gene transfer into human VSMCs. Histochemical staining with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal), a quantitative spectrophotometric assay, SDS-PAGE, Western blotting, and Northern analysis were used to evaluate beta-galactosidase expression in infected cells. After infection with RAd35 beta-Gal at 30, 100, and 1000 plaque-forming units per cell (pfu/cell), expression of beta-galactosidase was augmented up to 17-, 19-, and 23-fold, respectively, in human VSMCs treated with forskolin and phorbol ester compared with unstimulated cells. After infection, the proportion of detectably transduced cells was increased by enhancer stimulation from 58% to 100% at 100 pfu/cell and from 9% to 62% at 10 pfu/cell, indicating quiescent viral DNA in unstimulated cells. At high adenoviral titers (1000 pfu/cell), the recombinant gene became the most abundant protein in cell extracts. These findings demonstrate that in human VSMCs, limited constitutive expression from the CMV-IEP, rather than failure of translocation of adenoviral DNA, may be responsible for the apparent failure of transduction at a low multiplicity of infection.

Adenoviridae↗
PubMed1993

Sarcoid, amyloid and heart failure.

We report the case of an adult West Indian patient who presented with heart failure 20 years after an initial diagnosis of pulmonary sarcoidosis. Endomyocardial biopsy revealed AL type amyloid which was later found to be secondary to an underlying multiple myeloma.

Amyloid↗