Regulation of Vascular Inwardly Rectifying Potassium Channels in Health and Disease

Lead Research Organisation: University College London
Department Name: Medicine

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Publications

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Buckley JF (2006) Role of KATP channels in sepsis. in Cardiovascular research

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Falcetti E (2010) Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension. in American journal of respiratory and critical care medicine

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Falcetti E (2007) IP receptor-dependent activation of PPARgamma by stable prostacyclin analogues. in Biochemical and biophysical research communications

 
Description Glibenclaimde as a treatment for life-threatening hyperkalemia in critically ill patients
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description BHF Funding (Molecular and biophysical properties of inward rectifier potassium)
Amount £134,357 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2003 
End 09/2006
 
Description BHF funding (Does actin cytoskeletal disruption account for KATP channel)
Amount £148,575 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2003 
End 09/2006
 
Description BHF funding (Regulation of vascular KATP channels by nitric oxide and sepsis)
Amount £177,145 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2007 
End 06/2010
 
Description Impact PhD Studentship (Interaction of prostacyclin analogues with PDE inhibitors in advanced models of pulmonary hypertension and fibrosis)
Amount £55,000 (GBP)
Organisation Pfizer Ltd 
Sector Private
Country United Kingdom
Start 09/2010 
End 02/2014
 
Description Impact PhD Studentship (Novel biomarkers of vascular remodelling and inflammation in patients with pulmonary arterial hypertension)
Amount £63,627 (GBP)
Organisation United Therapeutics 
Sector Academic/University
Country United States
Start 01/2012 
End 01/2015
 
Description Industrial Collaborative Research Grant (Role of non IP receptor-dependent mechanisms in prostacyclin analogue responses)
Amount £39,279 (GBP)
Organisation United Therapeutics 
Sector Academic/University
Country United States
Start 05/2010 
End 07/2011
 
Description MRC Research Grant (The UCL sepsis group)
Amount £343,705 (GBP)
Funding ID G0500712 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2005 
End 04/2008
 
Description Mechanism of vasopressin hypersensitivity in septic shock
Amount £171,705 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2004 
End 09/2007
 
Description Unrestricted Educational Grant (Interaction of treprostinil with endothelin and cGMP pathways in pulmonary hypertension)
Amount £30,000 (GBP)
Organisation United Therapeutics 
Sector Academic/University
Country United States
Start 09/2009 
End 06/2012
 
Title Development of a prostacyclin (IP) receptor antibody 
Description We designed a peptide that corresponded to the C terminus of the IP receptor (RRDPRAPSAVGKE). This was synthesized and conjugated to hemocyanin and injected into rabbits by Eurogentec (Seraing, Belgium). The bleeds were assayed for activity using a standard ELISA assay, and those showing reactivity were affinity purified in our laboratory. Specficity of this antibody was tested in stable lines either expressing the IP receptor or empty vector (see PMID:20622039). 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact In our hands, all the commercially available IP receptor antibodies did not specfically stain the target for which they were developed. We showed specificity of our antibody, both immunohostologically and by Western blot using our unique resource (stable cell lines expressing IP receptor). This antibody has been used to assess the impact of pulmonary hypertension and treatment on expression of this receptor in cells and whole blood vessels, both in our laboratory and those of Professor Norel in Paris, France. 
URL http://europepmc.org/abstract/MED/20622039
 
Title Human pulmonary arterial smooth muscle cell lines derived from PAH patients 
Description Cell lines derived from patients with pulmonary arterial hyptension. One cells caused by pulmonary occulsive venous disease is particularly rare form of pulmonary hypertension, and will thus act as a valuable research tool for other groups interested in this disease. 
Type Of Material Cell line 
Year Produced 2011 
Provided To Others? Yes  
Impact Worked on assessing novel combination of drugs that might provided a novel therapy to treat pulmonary hypertension 
 
Description Biochemistry and functional roles of calcineurin isoforms in vascular smooth muscle 
Organisation University of Nevada
Department Department of Physiology and Cell Biology
Country United States 
Sector Academic/University 
PI Contribution Dr Rita Jabr, Dr Nelson Orie and myself brought to the collaboration a wealth of expertise in smooth muscle electrophysiology and the role of potassium channels in regulating smooth muscle tone. Our research team made the initial observation upon which the collaboration was formed. We discovered that calcineurin, a ubiquitously expressed calcium-dependent protein phosphatase, was a key modulator of vascular ATP-sensitive potassium channels at physiological calcium levels (PMID: 11090547). This observation has completely revised our understanding of how this channel is regulated under physiological conditions, providing a mechanism of regulation independent of cell metabolism.
Collaborator Contribution Dr Brian Perrino provided us with calcineurin constructs, including cDNAs of mutant proteins, purified constitutively active proteins and made fusion proteins of calcineurin fragments involved in NFAT (nuclear activator factor of T cells) binding. In addition Dr Perrino assisted us in the setting up of phosphatase assays, Western blotting and RT-PCR studies of calcineurin A isoforms in vascular smooth muscle. This was indeed a muli-disciplinary approach Together we have identified novel splice variants of CnAß, and have shown that different calcineurin A isoforms have distinct cellular functions in smooth muscle.
Impact Together we have identified novel splice variants of CnAß, and have shown that different calcineurin A isoforms have distinct cellular functions in smooth muscle; CnAa being a major regulator of ATP-sensitive potassium channel function (PMID:19422382) while CnAß is crucial for controlling smooth muscle cell proliferation in response to certain growth factors (PMID: 17303652). In other work published with Dr Perrino, we were the first to show that CaN heterodimers composed of either CnAa or CnAß exhibit differences in substrate selectivity and sensitivity to immunophilin/immunosuppressant inhibition (PMID 12135494). This was a multi-disciplinary colloaboration since it brought together a world expert in calcineurin biochemistry (Dr Perrino) with a leading expert in the electrophysiology of vascular smoth muscle potassium channels.
 
Description Prostacyclin receptor dependent activation of PPAR? 
Organisation Pasteur Institute, Lille
Country France 
Sector Charity/Non Profit 
PI Contribution Prostacyclin analogues (but not cicaprost) bind to and activate PPAR a and ß, though before our investigation, it was readily assumed that PPAR? was not a target for these analogues. However, we made stable lines expressing either the IP receptor and showed, using a luciferase reporter assay, that these agents(including cicaprost) could activate a PPAR? in an IP receptor dependent manner.
Collaborator Contribution In order to show that prostacyclin analogues could activate PPAR?, we needed to have a PPAR? specific reporter construct and an appropriate control. Both these constructs were supplied to us from Dr Bart Stales (Lille University, France), who also helped us to modify them because our investigational drugs were able to drive luciferase expression of the control construct. This technical problem was circumvented by reducing the size of the promotor region of the luciferase gene from 700 to 150 base pairs.
Impact Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues were able to activate PPAR? only in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented this activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPAR? antagonist, GW9662. This work was the first demonstration that PPAR? could be activated through the IP receptor via a cyclic AMP-independent mechanism. We also showed that this pathway could contribute to the anti-growth effects of prostacyclin analogues (PMID 17624303). Thus PPAR? could be a clinically relevant target for prostacyclin analogues which are used in the treatment of pulmonary hypertension.
 
Description Reversal of life-threatening, drug-related potassium-channel syndrome by glibenclamide 
Organisation University College London
Department Bloomsbury Institute of Intensive Care Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution In our previous work published in Circulation Resreach we showed that the immunosupressant cyclosporin was a good activator of ATP-sensitive potassium channels in smooth muscle cells and thought that such an effect might be responsible for the side effects of cyclosporinin use, namel global raised potassium levels. Thus we came up with the idea that glibenclamide a known inhibitor of these channels might reverse the effects of cyclosporin and other situations where drug-induced excessive ATP-sensitive potassium channel activation was found to be associated with hyperkalaemia.
Collaborator Contribution My collaborators were all clinicians. They described a syndrome in critically ill patients who received drugs with ATP-sensitive potassium channel opening properties and subsequently developed severe life-threatening complications, including hyperkalaemia (raised potassium in the plasma) and cardiovascular disturbances. Administration of the anti-diabetic agent, glibenclamide, an inhibitor of these potassium channel proteins, promptly reversed these complications. Over three years they saw this syndrome and response in two post-operative cardiothoracic patients taking nicorandil, two immunosuppressed patients taking cyclosporin, and one severely asthmatic patient given prolonged administration of the general anaesthetic, isoflurane, suggesting this disorder arises more frequently than is currently realised.
Impact 1. Our results were published in the Lancet (Singer M et al, 2005 PMID: 15924984) 2. The use of glibenclamide appears to be a safe and effective way to treat certain forms of hyperkalaemia in the critically ill patient. This novel treatment is being used as a vital backup when more convential treatment fails. 3. Our results pose questions about how to treat hyperkalaemia, particularly in relation to immunosuppressant therapy. This phenomena is widely thought to be due to poor excretion of potassium from the kidney, but our results suggest it is coming from muscle, thus challenging prevails views and the what is the appropriate treatment.
 
Description Role of the IP receptor in regulating smooth muscle cell growth in pulmonary arterial hypertension 
Organisation Great Ormond Street Hospital (GOSH)
Department NIHR Great Ormond Street Biomedical Research Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution In previous studies from this laboratory (PMID:11804870), we showed that replication of normal pulmonary artery smooth muscle cells (PASMCs) was reduced by prostacyclin analogs in a largely cyclic AMP-dependent manner. However, we wished to investigate the extent to which the prostacyclin (IP) receptor mediated the effects of prostacyclin analogs in cells derived from patients with pulmonary arterial hypertension, and also to determine the role of teh transcription factor peroxisome proliferator-activated receptor ? (PPAR?). All cell proliferation, cyclic AMP and PPAR? assays in human cell lines were performed in our laboratory. In addition we designed, had custom made and affinity purifed an IP receptor antibody for these studies to assess the impact of disease on the expression of this receptor.
Collaborator Contribution Professor Haworth (GOSH) and Dr Hall (Institute of Child Health) provided under ethical approval histological samples of human lung from normal children and from patients with IPAH who had either not been treated or had undergone transplant after failed treatment. Dr Hall, was responsible for making primary cell lines of human PASMCs from distal blood vessels and for characterising them. She also performed most of the immunohistochemistry and immunofluorescence studies to assess the expression of the IP receptor and PPAR? in lungs or isolated smooth muscle cells. Professor Morrell and Dr Phillips from Cambridge, UK were responsible for deriving cell lines of distal mouse PASMCs from homozygous IP receptor-deficient and wild-type mice and for performing cell proliferation and cyclic AMP assays to assess the role of the IP receptor in mediating the antiproliferative effects of prostacyclin anlogues in smooth muscle.
Impact In this translational study, involving clinicians and basic scientists, we found that down-regulation of the IP receptor occurred in idopathic PAH, an effect which related to chronic treatment with prostacyclin rather than the disease process itself. Despite this finding, the ability of prostacyclin analogues to inhibit pulmonary smooth muscle cell proliferation from these patients was not impaired. However the surprising result was the mechanism differed from normal cells because neither the IP receptor nor cyclic AMP-mediated these effects but was largely mediated by the transcription factor PPAR?. Thus, PPAR? may represent a previously unrecognised therapeutic target in PAH. The results of this study were published in Am J Crit Care Medicine (PMID:11804870).
 
Description Role of the IP receptor in regulating smooth muscle cell growth in pulmonary arterial hypertension 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution In previous studies from this laboratory (PMID:11804870), we showed that replication of normal pulmonary artery smooth muscle cells (PASMCs) was reduced by prostacyclin analogs in a largely cyclic AMP-dependent manner. However, we wished to investigate the extent to which the prostacyclin (IP) receptor mediated the effects of prostacyclin analogs in cells derived from patients with pulmonary arterial hypertension, and also to determine the role of teh transcription factor peroxisome proliferator-activated receptor ? (PPAR?). All cell proliferation, cyclic AMP and PPAR? assays in human cell lines were performed in our laboratory. In addition we designed, had custom made and affinity purifed an IP receptor antibody for these studies to assess the impact of disease on the expression of this receptor.
Collaborator Contribution Professor Haworth (GOSH) and Dr Hall (Institute of Child Health) provided under ethical approval histological samples of human lung from normal children and from patients with IPAH who had either not been treated or had undergone transplant after failed treatment. Dr Hall, was responsible for making primary cell lines of human PASMCs from distal blood vessels and for characterising them. She also performed most of the immunohistochemistry and immunofluorescence studies to assess the expression of the IP receptor and PPAR? in lungs or isolated smooth muscle cells. Professor Morrell and Dr Phillips from Cambridge, UK were responsible for deriving cell lines of distal mouse PASMCs from homozygous IP receptor-deficient and wild-type mice and for performing cell proliferation and cyclic AMP assays to assess the role of the IP receptor in mediating the antiproliferative effects of prostacyclin anlogues in smooth muscle.
Impact In this translational study, involving clinicians and basic scientists, we found that down-regulation of the IP receptor occurred in idopathic PAH, an effect which related to chronic treatment with prostacyclin rather than the disease process itself. Despite this finding, the ability of prostacyclin analogues to inhibit pulmonary smooth muscle cell proliferation from these patients was not impaired. However the surprising result was the mechanism differed from normal cells because neither the IP receptor nor cyclic AMP-mediated these effects but was largely mediated by the transcription factor PPAR?. Thus, PPAR? may represent a previously unrecognised therapeutic target in PAH. The results of this study were published in Am J Crit Care Medicine (PMID:11804870).
 
Description Vasopressin receptors as a potential therapeutic target in sepsis 
Organisation Ferring Pharmaceuticals
Country Switzerland 
Sector Private 
PI Contribution We described hypersensitivity to the contactile effects of vasopressin was associated with potentiation of V1 but not V2 receptor function in a long term model of sepsis. We also found that the mechanism by which calcium is mobilised by vasopressin and norepinephrine differs between the two agonists in small mesenteric vessels, providing a possible explanation why these two agents have differential effects on vascular function is sepsis.
Collaborator Contribution Providing novel (not commerically available) vasopressin agonists and antagonists for use in our sepsis research
Impact Strengthened the rationale for the use of V1 (vasopressin) receptor agonists in the treatment of sepsis. This work was published in Crit Care Med (Barrett et al 2007; PMID: 17944022). Ferring Pharmaceuticals is proeceeding with clinical tials of a vasopressin agonist in spesis.
Start Year 2006
 
Description Bayer Advisory Board Meeting on sGC-stimulation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A working group consisting of 25 scientists/ healthcare professionals from Bayer and experts in the pulmonary hypertension field gathered together in Halle, Germany. The reason was to dicuss the therapeutic potential and advantages of soluble guanylate cyclase stimulation over PDE5-inhibition. I provided a short presentation covereing my experience from own my research/ data, giving my expert opinion on how to proceed to further differentiate the novel approach of sGC stimulation in pulmonary hypertension.


The working group as a whole were able to advise Bayer on how to appropriately test whether their novel compound would have significant advantages over existing PDE5 (e.g. sildenafil) therapies in the treatment of pulmonary hypertension.
Year(s) Of Engagement Activity 2011
 
Description School visit Cranleigh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Gave a talk to Cranleigh scholars (15 of them, aged between 13-14) about how a scientist develops a hypothesis, what and how I carry out research into sepsis and pulmonarty hypertension. Talked about how potassium channels are defective in thse two diseasesand my involvement in drug development to combat these potentially fatal conditions.

Increased awareness of how important science is to advancing human health.
Year(s) Of Engagement Activity 2009,2010,2011,2012
 
Description United Therapeutics Corp 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Health professionals
Results and Impact Had extensive discussions with medical officers and other healthcare professionals about the mechanism of action of their patented drug used for treating pulmonary arterial hypertension

Design of further clinical research
Year(s) Of Engagement Activity 2009,2010,2011,2012