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Biomedical subjects

P D Flynn

Publications and source records attributed to P D Flynn.

11 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

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

Thrombin generation by apoptotic vascular smooth muscle cells.

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 +/- 17 nmol x min/L and a peak thrombin (PT) of 154 +/- 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 +/- 15 nmol x min/L, P = .3; PT of 172 +/- 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 +/- 8 nmol x min/L; PT of 103 +/- 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 +/- 2 nmol x min/L; PT of 128 +/- 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

Adenovirus E1A Proteins↗
PubMed1996

Is platelet phospholipid-dependent thrombin generation altered by acute myocardial infarction or aspirin?

The ability of unactivated and calcium ionophore activated platelets to support thrombin generation in defibrinated plasma was measured by a chromogenic substrate assay in the absence of clot formation. Platelet phospholipid-dependent thrombin generation (Platelet-TG) could be measured using platelets isolated from blood collected into either sodium citrate or EDTA as anticoagulant. Measurements were stable in samples kept at room temperature for 24 hours after venesection. There was no significant difference in either the unactivated or activated platelet-TG with platelets collected into either anticoagulant (mean difference 10.12 nmol/min unactivated and 10.60 nmol/min activated). There was no correlation between unactivated and activated platelet-TG and patient age. Platelet-TG was 179 (153-237) nmol/min (median and inter quartile range) for unactivated and 489 (462-508) nmol/min for activated platelets from healthy volunteer subjects (median age 31, range 20-40 years). Platelet-TG was the same in subjects from a population-based cohort study (median age 58, range 45-70 years [162 (142-193) nmol/min and 527 (490-551) nmol/min, unactivated and activated platelets respectively] as compared to patients admitted with acute myocardial infarction (median age 68, range 36-85 years [179 (146-200) nmol/min and 473 (440-517) nmol/min unactivated and activated platelets respectively] (p = 0.497 for comparison between unactivated platelet-TGs and p = 0.487 for comparison between activated platelet-TGs in the two groups). Aspirin inhibited platelet aggregation but did not affect platelet-TG using either unactivated or activated platelets exposed to aspirin in vitro; or in vivo, using platelets obtained from individuals after ingestion of aspirin. In conclusion these results show that for measurement of platelet-TG, blood samples can be anticoagulated with EDTA as well as sodium citrate for up to 24 hours after venesection and that this measurement is not affected by subject age, aspirin treatment or the acute stage of myocardial infarction.

Adult↗