Dr Laurice Fretwell

Job: Senior Lecturer

Faculty: Health and Life Sciences

School/department: School of Allied Health Sciences

Address: De Montfort, University, The Gateway, Leicester, LE1 9BH.

T: +44 (0)116 250 6297

E: lfretwell@dmu.ac.uk

W: http://www.dmu.ac.uk/hls

 

Personal profile

  • Cardiovascular cell signalling
  • Physiology
  • Pharmacology
  • Potassium channels
  • GPCRs
  • VEGFRs

Research group affiliations

  • Cell Signalling

Publications and outputs 

  • Effects of novel chalcone derivatives on human endothelial cell proliferation and migration
    Effects of novel chalcone derivatives on human endothelial cell proliferation and migration Hussain, A.; Fretwell, L.; Ruparelia, K. C.; Beresford, Kenneth J. M.; Singh, Harprit Introduction: Angiogenesis is the formation of new blood vessels from the pre-existing vasculature and is essential for some physiological processes including wound healing and menstrual cycles. Unregulated angiogenesis can lead to vascular related diseases such as age related macular degeneration, cancer and rheumatoid arthritis. Chalcones (1,3-diphenylpropenones) are naturally occurring phenolic compounds found in a variety of plants and fruits. Their simple molecular structures have demonstrated an array of pharmacological activity including antioxidant and anti-vascular. Due to Chalcones having an easily modifiable scaffold, they are widely used as parent compounds in drug discovery studies. Endothelial cell proliferation and migration are essential for angiogenic growth, so compounds that possess anti-endothelial activity could be developed further as potential inhibitors of angiogenesis. With this in mind, we report the anti-endothelial activity of two novel analogues (AH1 and AH9) derived from the parent compound 2-chloro-2’5’-dihydroxychalcone. Method: Chalcones were synthesised via the Claisen-Schmidt condensation and verified via nuclear magnetic resonance (NMR) and mass spectrometry (MS). Primary Human umbilical vein endothelial cells (HUVECs) were cultured according to manufactures protocol. Cells were seeding into 96-well plates (3500 cells/well) and, 24 hours later, treated either with AH1, AH9 or Sorafenib, a known anti-angiogenic drug for 72hr at a concentration of 10 µM. Effects of the compounds on cell proliferation was assessed by quantifying the colorimetric conversion of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) to purple formazan product (measured at 560 nm). Data are expressed as mean % inhibition of cell proliferation ± SEM (n=4). Anti-migratory activity was assessed via the wound healing assay and the effects of the compounds at 3 µM on the width of the scratch across an 8 hour period were compared to untreated control. Analysis was performed by one-way ANOVA with Tukey-Kramer’s multiple comparisons test. Data are expressed as mean % of maximum migration ± SEM (n=3). Results: AH1 and AH9 displayed significant anti-proliferative activity with inhibitory values of 94.94 ± 1.64% and 79.62 ± 4.45% compared to Sorafenib, 69.95 ± 4.12%, respectively. Effects on HUVEC migration showed that AH9 limited migration to 16.19 ± 14.44% (P<0.05 vs. control). AH1 (47.23 ± 6.8%) showed similar anti-migratory activity to Sorafenib (52.68 ± 3.32%). Conclusion: This preliminary data suggests that AH9 has potential antiangiogenic properties and could be developed further as a potential antiangiogenic or vascular remodelling agent. Further studies will elucidate the molecular mechanism of AH9 action in cells stimulated with known angiogenic agent, VEGF.
  • Promoting internationalisation in human health degrees
    Promoting internationalisation in human health degrees Pena-Fernandez, A.; Evans, M. D.; Fretwell, L.; Pena, M. A.
  • Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats.
    Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats. Carter, Joanne J.; Fretwell, L.; Woolard, J. VEGF inhibitors, including receptor tyrosine kinase inhibitors, are used as adjunct therapies in a number of cancer treatments. An emerging issue with these drugs is that most cause hypertension. To gain insight into the physiological mechanisms involved, we evaluated their regional hemodynamic effects in conscious rats. Male Sprague Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes (renal and mesenteric arteries, and the descending abdominal aorta) and catheters (jugular vein, peritoneal cavity, and distal abdominal aorta). Regional hemodynamics were measured over 4 d, before and after daily administration of cediranib (3 and 6 mg/kg, 3 and 6 mg/kg/h for 1 h, i.v.), sorafenib (10 and 20 mg/kg, 10 and 20 mg kg/h for 1 h, i.v.), pazopanib (30 and100 mg/kg, i.p.), or vandetanib (12.5 and 25 mg/kg, i.p.). All drugs evoked significant increases (P < 0.05; n = 7-8) in mean arterial pressure, which were generally accompanied by significant mesenteric and hindquarters, but not renal, vasoconstrictions. The hypertensive effects of cediranib were unaffected by losartan (10 mg/kg/h), bosentan (20 mg/kg/h), or a combination of phentolamine and propranolol (each 1 mg/kg/h), suggesting a need for new strategies to overcome them. This work was completed at the University of Nottingham. This is an Open Access article
  • Effects of Novel Chalcone Derivatives upon H9c2 and MDCK Cell Viability
    Effects of Novel Chalcone Derivatives upon H9c2 and MDCK Cell Viability Hussain, A.; Wright, Jack; Ruparelia, K. C.; Beresford, Kenneth J. M.; Fretwell, L. Many compounds with potential anti-cancer activity fail to reach the latter stages of clinical trials due to adverse effects, often causing cardiac and renal toxicity. Here, we synthesised a group of novel chalcone compounds, thought to have potential anti-cancer activity4 and assessed their effects upon cardiac and renal cell viability. Data revealed that all compounds produced minimal short term toxicity. Further work will be performed to assess the long term effects of these compounds on cell viability, leading to mechanistic studies and structure activity relationship analyses.
  • Cardiovascular responses to retigabine in conscious rats - under normotensive and hypertensive conditions.
    Cardiovascular responses to retigabine in conscious rats - under normotensive and hypertensive conditions. Fretwell, L.; Woolard, J. BACKGROUND AND PURPOSE: Retigabine is a recently approved anti-epileptic agent which activates Kv7.2-7.5 potassium channels. An important role for these channels in vascular regulation is emerging, but the vascular effects of retigabine in the conscious state are unknown. In the present study the regional haemodynamic responses to retigabine were assessed in conscious rats. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats were chronically instrumented with pulsed Doppler flow probes to measure regional haemodynamic responses to retigabine under control conditions and during acute hypertension induced by infusion of angiotensin II (AII) and arginine vasopressin (AVP). Further experiments were performed to elucidate the roles of β-adrenoceptor activation in the responses to retigabine in vivo and in vitro. KEY RESULTS: Under normotensive conditions, retigabine caused dose-dependent hypotension and hindquarters vasodilatation, with small, transient renal and mesenteric vasodilatations. In the acutely hypertensive state, the renal and mesenteric, but not hindquarters, vasodilatations were enhanced. The hindquarters response to retigabine was mediated, in part, by β2 -adrenoceptors. However, in vitro experiments confirmed that retigabine did not act as a β-adrenoceptor agonist. CONCLUSIONS AND IMPLICATIONS: These results, showing differential vasodilator effects of retigabine, broadly support other studies identifying Kv7 channels as mediators of vascular tone, highlighting the different responses under normotensive and hypertensive conditions, and a role for β-adrenoceptor activation.
  • Role of large-conductance Ca2+-activated K+ channels in adenosine A1 receptor-mediated pharmacological postconditioning in H9c2 cells
    Role of large-conductance Ca2+-activated K+ channels in adenosine A1 receptor-mediated pharmacological postconditioning in H9c2 cells Fretwell, L.; Dickinson, J. Ischaemic postconditioning is a phenomenon whereby short periods of ischaemia applied during the start of reperfusion protect the myocardium from the damaging consequences of reperfusion. As such, pharmacological-induced postconditioning represents an attractive therapeutic strategy for reducing reperfusion injury during cardiac surgery and following myocardial infarction. The primary aim of this study was to determine the role of large-conductance Ca2+-activated potassium channels (BKCa channels) in adenosine A1 receptor-induced pharmacological postconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. H9c2 cells were exposed to 6 h hypoxia (0.5% O2) followed by 18 h reoxygenation (H/R) after which cell viability was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. The adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 100 nmol/L) or the BKCa channel opener NS1619 (10 µmol/L) were added for 30 min at the start of reoxygenation following 6 h hypoxic exposure. Where appropriate, cells were treated (15 min) before pharmacological postconditioning with the BKCa channel blockers paxilline (1 µmol/L) or iberiotoxin (100 nmol/L). Pharmacological postconditioning with CPA or NS1619 significantly reduced H/R-induced LDH release. Treatment with paxilline or iberiotoxin attenuated adenosine A1 receptor and NS1619-induced pharmacological postconditioning. These results have shown for the first time that BKCa channels are involved in adenosine A1 receptor-induced pharmacological postconditioning in a cell model system.
  • Predicting in vivo cardiovascular properties of blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses
    Predicting in vivo cardiovascular properties of blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses Baker, J. G.; Kemp, P.; March, J.; Fretwell, L.; Hill, S. J.; Gardiner, S. M. Beta -Adrenoceptor antagonists differ in their degree of partial agonism. In vitro assays have provided information on ligand affinity, selectivity, and intrinsic efficacy. However, the extent to which these properties are manifest in vivo is less clear. Conscious freely moving rats, instrumented for measurement of heart rate ( 1; HR) and hindquarters vascular conductance ( 2; HVC) were used to measure receptor selectivityand ligand efficacy in vivo. CGP 20712A caused a dose-dependent decrease in basal HR (P<0.05, ANOVA) at 5 doses between 6.7 and 670 g/kg (i.v.) and shifted the dose-response curve for isoprenaline to higher agonist concentrations without altering HVC responses. In contrast, at doses of 67 g/kg (i.v.) and above, ICI 118551 substantially reduced the HVC response to isoprenaline without affecting HR responses. ZD 7114, xamoterol, and bucindolol significantly increased basal HR ( HR: 122 12, 129 11, and 59 11 beats/min, respectively; n 6), whereas other -blockers caused significant reductions (all at 2 mg/kg i.v.). The agonist effects of xamoterol and ZD 7114 were equivalent to that of the highest dose of isoprenaline. Bucindolol, however, significantly antagonized the response to the highest doses isoprenaline. An excellent correlation was obtained between in vivo and in vitro measures of 1-adrenoceptor efficacy(R2 0.93; P<0.0001).
  • Role of large-conductance Ca2+ -activated potassium channels in adenosine A1 receptor-mediated pharmacological preconditioning in H9c2 cells
    Role of large-conductance Ca2+ -activated potassium channels in adenosine A1 receptor-mediated pharmacological preconditioning in H9c2 cells Fretwell, L.; Dickenson, J. Large-conductance Ca2+-activated potassium channels, located on the inner mitochondrial membrane, have recently been implicated in cytoprotection. Therefore, the primary aim of this study was to determine the role of large-conductance Ca2+-activated potassium channels in adenosine A1 receptor-induced pharmacological preconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. For pharmacological preconditioning, H9c2 cells were exposed to the adenosine A1 receptor agonist N6-cyclopentyladenosine (100 nM) or the Ca2+-activated potassium channel opener NS1619 (10 µM) for 30 min prior to 6 h hypoxia (0.5% O2) in glucose-free and serum-free media. Where appropriate cells were treated (15 min) before pharmacological preconditioning with the Ca2+-activated potassium channels blockers paxilline (1 µM) or iberiotoxin (100 nM). Cell viability following 6 h hypoxia was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. Ca2+-activated potassium channel subunit protein expression and cell survival protein kinase (ERK1/2 and PKB/Akt) activation were assessed by Western blotting. The results demonstrate that the adenosine A1 receptor is functionally expressed in H9c2 cells and when activated protects against hypoxia-induced LDH release and caspase-3 activation. Treatment with paxilline or iberiotoxin attenuated adenosine A1 receptor and NS1619-induced pharmacological preconditioning. Large-conductance Ca2+-activated potassium channel α and β4 protein subunits were detected in mitochondrial fractions isolated from H9c2 cells. NS1619 (10 µM) induced no significant changes in ERK1/2 or PKB phosphorylation. These results have shown for the first time that large-conductance Ca2+-activated potassium channels are involved in adenosine A1 receptor-induced pharmacological preconditioning in a cell model system.

Click here for a full listing of Laurice Fretwell's publications and outputs.

Research interests/expertise

  • Cell-based assays
  • Pressure myography
  • Organ bath studies
  • Western blot
  • Molecular biology
  • Regional haemodynamics
  • Statistics and experimental design

Areas of teaching

  • Organ Systems Physiology
  • Pharmacology
  • Statistics

Qualifications

  • BSc (Hons) Physiology and Pharmacology – Nottingham Trent University (2002-2005)
  • PhD in Cardiovascular Cell Signalling – Nottingham Trent University (2005-2009)
  • Career Development Fellow in Cardiovascular Pharmacology and Drug Discovery – The University of Nottingham (2010-2013)

Honours and awards

  • British Pharmacological Society Diploma in Advanced Pharmacology (2012)
  • WinSET Award (2012): Represented the School of Biomedical Sciences at the Women in Science, Engineering and Technology event (University of Nottingham)

Membership of professional associations and societies

  • British Pharmacological Society
  • The Physiological Society

Conference attendance

  • British Pharmacology Winter Meeting, 2011: Cardiovascular effects of the KCNQ channel opener, retigabine, in conscious rats
  • British Pharmacology Winter Meeting, 2011: Cardiovascular responses to tetraethylammonium in conscious rats under normotensive and pharmacologically-induced hypertensive conditionsEPHAR, 2008: Role of Ca2+-activated K+ channels in adenosine A1 receptor-mediated protection against hypoxia-induced cell death in myocardiac H9c2 cells.
  • Life Sciences, 2007: Functional expression of the adenosine A1 and κ-opioid receptor in myocardiac H9c2 cells.
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