Joanne S Lymn

Thrombospondin-1 Is a Potent Mitogen and Chemoattractant for Human Vascular Smooth Muscle Cells

Thrombospondin-1 (TSP-1) is a matricellular protein that is present in negligible amounts in normal human vasculature but occurs in significant amounts in diseased vessels. In this study, we examined the effect of TSP-1 on DNA synthesis, proliferation, and migration in human vascular smooth muscle cells grown from saphenous vein. TSP-1 (0.1 to 30 micrograms/mL) elicited a concentration-dependent increase in DNA synthesis under serum-free conditions. In combination with platelet-derived growth factor, TSP-1 induced a synergistic effect on DNA synthesis that was significantly higher than the additive effect of both agents. In proliferation assays, TSP-1 increased cell numbers by 50% relative to the serum-free controls over 14 days. In migration assays, conducted using modified Boyden chambers, TSP-1 (> or = 10 micrograms/mL) elicited marked chemotaxis to a degree equivalent to platelet-derived growth factor. The chemotactic response to TSP-1 (10 micrograms/mL) was abolished by the GRGDSP peptide but unaffected by the control GRGESP peptide, whereas neither peptide inhibited DNA synthesis stimulated by TSP-1. Inhibition of tyrosine kinase activity with genistein or tyrphostin A23 abolished DNA synthesis induced by TSP-1, and a neutralizing antibody to platelet-derived growth factor had no effect on DNA synthesis. Similarly, migration in response to TSP-1 was largely inhibited by these tyrosine kinase inhibitors. TSP-1 is a strong mitogen and chemoattractant for human vascular smooth muscle cells under serum-free conditions. The novel finding that TSP-1 is mitogenic for human cells contrasts with previous studies that have not shown any significant effect of TSP-1 itself on the growth of animal-derived smooth muscle cells. TSP-1 may play an important modulatory role in the local regulation of vascular smooth muscle function in vascular pathologies in humans.

Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60c-src kinase activity

Tyrosine kinases have been proposed as regulators of voltage‐operated calcium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage‐operated calcium channel currents (IBa) and pp60c‐src kinase (c‐src) activity in vitro. IBa was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage‐clamp techniques. The activity of purified human c‐src was measured in vitro using a non‐radioactive assay. Bath application of tyrphostin‐23 and genistein (non‐selective PTK inhibitors), bistyrphostin (a receptor‐PTK‐selective inhibitor) and PP1 (a src family‐selective inhibitor) inhibited IBa in a concentration‐dependent manner over a range of test membrane potentials. Intracellular application of peptide‐A, a peptide inhibitor of c‐src also inhibited currents. Inhibitor potency series against IBa was PP1 > genistein > tyrphostin 23 > bistyrphostin. Tyrphostin‐23, genistein, PP1, and peptide‐A shifted the steady‐state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no significant effects on IBa activation calculated from current‐voltage relationships. The agents inhibited c‐src activity in a concentration‐dependent manner. The order of potency was PP1 > genistein > peptide‐A > tyrphostin‐23 > bistyrphostin. The IC50 for inhibition of c‐src activity was similar to the IC50 for inhibition of IBa in all cases. Western blot analysis with a specific antibody to c‐src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. A range of structurally dissimilar inhibitors of PTKs inhibit IBa and c‐src activity with similar potency. These data provide further evidence implicating endogenous c‐src in the modulation of L‐type calcium channels in vascular smooth muscle cells.

Stakeholders' views of UK nurse and pharmacist supplementary prescribing

Objectives: Supplementary prescribing (SP) by pharmacists and nurses in the UK represents a unique approach to improving patients’ access to medicines and better utilizing health care professionals’ skills. Study aims were to explore the views of stakeholders involved in SP policy, training and practice, focusing upon issues such as SP benefits, facilitators, challenges, safety and costs, thereby informing future practice and policy. Method: Qualitative, semi-structured interviews were conducted with 43 purposively sampled UK stakeholders, including pharmacist and nurse supplementary prescribers, doctors, patient groups representatives, academics and policy developers. Analysis of transcribed interviews was undertaken using a process of constant comparison and framework analysis, with coding of emergent themes. Results: Stakeholders generally viewed SP positively and perceived benefits in terms of improved access to medicines and fewer delays, along with a range of facilitators and barriers to the implementation of this form of non-medical prescribing. Stakeholders’ views on the economic impact of SP varied, but safety concerns were not considered significant. Future challenges and implications for policy included SP being potentially superseded by independent nurse and pharmacist prescribing, and the need to improve awareness of SP. Several potential tensions emerged including nurses’ versus pharmacists’ existing skills and training needs, supplementary versus independent prescribing, SP theory versus practice and prescribers versus non-prescribing peers. Conclusion: SP appeared to be broadly welcomed by stakeholders and was perceived to offer patient benefits. Several years after its introduction in the UK, stakeholders still perceived several implementation barriers and challenges and these, together with various tensions identified, might affect the success of supplementary and other forms of non-medical prescribing.

Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level

Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking αvβ3 antibody, a function-blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking α3β1 antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis.

Effect of inhibition of tyrosine phosphatases on voltage-operated calcium channel currents in rabbit isolated ear artery cells

The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage‐operated calcium channel currents in vascular smooth muscle cells. In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 μM) and peroxyvanadate (100 μM orthovanadate+1 mM H2O2) increased voltage‐operated calcium channel currents by 56% and 83%, respectively. Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 μM) and dephostatin (50 μM) also increased voltage‐operated calcium channel currents by 48% and 52%, respectively. The selective tyrosine kinase inhibitor, tyrphostin‐23 (100 μM) reduced calcium channel currents by 41%. Pre‐incubation with tyrphostin‐23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels. Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate. These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage‐operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.

Platelet-Derived Growth Factor β-Receptors Can Both Promote and Inhibit Chemotaxis in Human Vascular Smooth Muscle Cells

Abstract The effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on migration was investigated in cultured human saphenous vein smooth muscle cells. The modified Boyden chamber technique yielded efficacies BB>>AB, AA=0. However, the BB concentration-response relationship displayed a pronounced peak, occurring between 1 and 10 ng/mL, with no response above this range. Checkerboard analysis showed that the promotion of migration at low concentrations was chemotactic in nature but that the downturn was independent of gradient. Furthermore, at high concentrations BB was able to prevent chemotaxis induced by fetal calf serum and epidermal growth factor (EGF). Experiments using low concentrations of BB in combination with high concentrations of AA to saturate PDGF α-receptors in the presence and absence of a neutralizing antibody to α-receptors revealed that α-receptor activation induced partial inhibition of chemotaxis but this did not account for the inhibition of migration by high concentrations of BB. Despite possessing no significant chemotactic action itself, high concentrations of the AB isoform completely inhibited BB induced chemotaxis. Taken together these results suggest that the chemotactic signal induced by PDGF is dominated by PDGF β-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [ 3 H] thymidine incorporation) yielded saturable responses for the AB and BB isoforms, with similar efficacy and weak or no response for the AA isoform. Concentration-dependent patterns of tyrosine phosphorylation of certain proteins mirrored the form of the chemotactic response and suggest one possible underlying regulatory mechanism to account for the disparity between PDGF-induced chemotaxis and DNA synthesis.

Phosphatidylinositol 3-Kinase and Focal Adhesion Kinase Are Early Signals in the Growth Factor–Like Responses to Thrombospondin-1 Seen in Human Vascular Smooth Muscle

Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1-stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1-induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure.

Supplementary prescribing by community and primary care pharmacists: an analysis of PACT data, 2004–2006

Background and objective:  Pharmacist prescribing is a relatively new intiative in the extension of prescribing responsibilities to non‐medical healthcare professionals. Pharmacist supplementary prescribing was introduced in 2003 and allowed prescribing in accordance with a clinical management plan agreed with a medical practitioner and patient to improve patient access to medicines and better utilize the skills of healthcare professionals. The objective of this research was to examine the volume, cost and trends in pharmacist prescribing in community and primary care using Prescription Analysis and Cost (PACT) data and to suggest possible reasons for the trends.

Effect of serum withdrawal on the contribution of L-type calcium channels (CaV1.2) to intracellular Ca2+ responses and chemotaxis in cultured human vascular smooth muscle cells.

1 Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L‐type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. 2 hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle α‐actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin‐coated plates was also quantified. The number and affinity of dihydropyridine‐binding sites was assessed using [5‐methyl‐3H]PN 200‐110 binding. 3 In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet‐derived growth factor‐BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF‐induced migration. 4 In serum‐deprived cells, smooth muscle α‐actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF‐induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF‐induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum‐deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF‐induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum‐deprived hVSMC. 5 Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine‐binding sites (Bmax) and a decrease in affinity (KD). 6 Serum deprivation of hVSMC results in increased expression of smooth muscle α‐actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.

Regulation of phospholipase C-delta by GTP-binding proteins-rhoA as an inhibitory modulator

The regulation of Phospholipase C (PLC)delta activity remains obscure. These studies show that PLCdelta1 activity is significantly enhanced by both guanosine thiotriphosphate (GTPgammaS) and Clostridium botulinum exoenzyme C3 (C3) but not by aluminium fluoride. C3 ADP ribosylated a 21-kDa protein in the PLCdelta1 preparation and Western blotting identified rhoA in these samples. RhoA acts as an inhibitory modulator of PLCdelta activity.