Imperial College London

DrBlerinaAhmetaj-Shala

Faculty of MedicineNational Heart & Lung Institute

Research Fellow
 
 
 
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Contact

 

+44 (0)20 7351 8137b.ahmetaj

 
 
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Location

 

Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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42 results found

Ahmetaj-Shala B, Marei I, Kawai R, Rothery S, Pericleous C, Mohamed N, Gashaw H, Bokea K, Samuel J, Vandenheste A, Shala F, Kirkby N, Mitchell Jet al., 2021, Activation and contraction of human ‘vascular’ smooth muscle cells grown from circulating blood progenitors, Frontiers in Cell and Developmental Biology, Vol: 9, Pages: 1-6, ISSN: 2296-634X

Blood outgrowth smooth muscle cells offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering and personalised medicine. However, little is known about these cells which has meant their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of blood outgrowth smooth muscle cells and vessel derived smooth muscle cells to sense the thromboxane mimetic U46619 bymeasuring intracellular calcium elevation and contraction. U46619 (10 26 -6M) increased cytosolic calcium in blood outgrowth smooth muscle cells fibroblasts. Increased calcium signal peaked between 10-20 seconds after U46619 in both smoothmuscle cell types. Importantly, U46619 (10-9 to 10-6M) induced concentration-dependent contractions of both blood outgrowth smooth muscle cells and vascular smooth muscle cells but not in fibroblasts. In summary, we show that functional responses of blood outgrowth smooth muscle cells are in line with vascular smooth muscle cells providing critical evidence of their application in biomedical research.

Journal article

Mohamed NA, Abou-Saleh H, Kameno Y, Marei I, de Nucci G, Ahmetaj-Shala B, Shala F, Kirkby NS, Jennings L, Al-Ansari DE, Davies RP, Lickiss PD, Mitchell JAet al., 2021, Studies on metal-organic framework (MOF) nanomedicine preparations of sildenafil for the future treatment of pulmonary arterial hypertension, Scientific Reports, Vol: 11, Pages: 1-8, ISSN: 2045-2322

Pulmonary arterial hypertension (PAH) is an incurable disease, although symptoms are treated with a range of dilator drugs. Despite their clinical benefits, these drugs are limited by systemic side-effects. It is, therefore, increasingly recognised that using controlled drug-release nanoformulation, with future modifications for targeted drug delivery, may overcome these limitations. This study presents the first evaluation of a promising nanoformulation (highly porous iron-based metal–organic framework (MOF); nanoMIL-89) as a carrier for the PAH-drug sildenafil, which we have previously shown to be relatively non-toxic in vitro and well-tolerated in vivo. In this study, nanoMIL-89 was prepared and charged with a payload of sildenafil (generating Sil@nanoMIL-89). Sildenafil release was measured by Enzyme-Linked Immunosorbent Assay (ELISA), and its effect on cell viability and dilator function in mouse aorta were assessed. Results showed that Sil@nanoMIL-89 released sildenafil over 6 h, followed by a more sustained release over 72 h. Sil@nanoMIL-89 showed no significant toxicity in human blood outgrowth endothelial cells for concentrations up to100µg/ml; however, it reduced the viability of the human pulmonary artery smooth muscle cells (HPASMCs) at concentrations > 3 µg/ml without inducing cellular cytotoxicity. Finally, Sil@nanoMIL-89 induced vasodilation of mouse aorta after a lag phase of 2–4 h. To our knowledge, this study represents the first demonstration of a novel nanoformulation displaying delayed drug release corresponding to vasodilator activity. Further pharmacological assessment of our nanoformulation, including in PAH models, is required and constitutes the subject of ongoing investigations.

Journal article

Mitchell JA, Kirkby NS, Ahmetaj-Shala B, Armstrong P, Crescente M, Ferreira P, Pires MEL, Vaja RK, Warner TDet al., 2021, Cyclooxygenases and the cardiovascular system, Pharmacology and Therapeutics, Vol: 217, Pages: 1-11, ISSN: 0163-7258

Cyclooxygenase (COX)-1 and COX-2 are centrally important enzymes within the cardiovascular system with a range of diverse, sometimes opposing, functions. Through the production of thromboxane, COX in platelets is a pro-thrombotic enzyme. By contrast, through the production of prostacyclin, COX in endothelial cells is antithrombotic and in the kidney regulates renal function and blood pressure. Drug inhibition of COX within the cardiovascular system is important for both therapeutic intervention with low dose aspirin and for the manifestation of side effects caused by nonsteroidal anti-inflammatory drugs. This review focuses on the role that COX enzymes and drugs that act on COX pathways have within the cardiovascular system and provides an in-depth resource covering COX biology and pharmacology. The review goes on to consider the role of COX in both discrete cardiovascular locations and in associated organs that contribute to cardiovascular health. We discuss the importance of, and strategies to manipulate, the thromboxane: prostacyclin balance. Finally within this review the authors discuss testable COX-2-hypotheses intended to stimulate debate and facilitate future research and therapeutic opportunities within the field.

Journal article

Ahmetaj-Shala B, Peacock TP, Baillon L, Swann OC, Gashaw H, Barclay WS, Mitchell JAet al., 2020, Resistance of endothelial cells to SARS-CoV-2 infection in vitro

<jats:title>Abstract</jats:title><jats:sec><jats:title>Rationale</jats:title><jats:p>The secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium activating thrombotic pathways, disrupting barrier function and allowing access of the virus to other organs of the body. However, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed.</jats:p></jats:sec><jats:sec><jats:title>Objective</jats:title><jats:p>To determine infectibility of primary endothelial cell lines with live SARS-CoV-2 and pseudoviruses expressing SARS-CoV-2 spike protein.</jats:p></jats:sec><jats:sec><jats:title>Methods and Results</jats:title><jats:p>Expression of ACE2 and BSG pathways genes was determined in three types of endothelial cells; blood outgrowth, lung microvascular and aortic endothelial cells. For comparison nasal epithelial cells, Vero E6 cells (primate kidney fibroblast cell line) and HEK 293T cells (human embryonic kidney cells) transfected with either ACE2 or BSG were used as controls. Endothelial and Vero E6 cells were treated with live SARS-CoV-2 virus for 1 hour and imaged at 24 and 72 hours post infection. Pseudoviruses containing SARS-CoV-2, Ebola and Vesicular Stomatis Virus glycoproteins were generated and added to endothelial cells and HEK 239Ts for 2 hours and infection measured using luminescence at 48 hours post infection. Compared to nasal epithelial cells, endothelial cells expressed low or undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG, PPIA and PPIB. Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudovirus (but showed susceptibility to Ebola and Vesicular Stomatitis Virus). Overexpression of ACE2 but not BSG in HEK 239T cells conferred SARS-CoV-2 pseudovirus entry. Endoth

Journal article

Ahmetaj-Shala B, Ricky V, Santosh A, Peter G, Nicholas K, Jane Met al., 2020, Cardiorenal tissues express SARS-CoV-2 entry genes and basigin (BSG/CD147) increases with age in endothelial cells, JACC: Basic to Translational Science, Vol: 5, Pages: 1111-1123, ISSN: 2452-302X

Objectives: To obtain mechanistic insight into COVID-19 within a cardiovascular setting.Background: Thrombosis and vascular dysfunction are part of the complex pathology seen in severe COVID-19 and advancing age is the most significant risk factor. Little is known about age and expression of pathways utilised by the COVID-19 virus, SARS-CoV-2, in cardiovascular tissues.Methods: We used publicly available databases (GTEx, GEO and Array Express) to investigate gene expression levels, in adult tissues, of the two putative SARS-CoV-2 receptors, ACE2 and BSG along with a selected range of genes thought to be involved in virus binding/processing. Our analysis included; vessels (aorta and coronary artery), heart (atrial appendage and left ventricle), kidney (cortex), whole blood, lung, colon and spleen along with endothelial cells, nasal and bronchial epithelium and peripheral blood mononuclear cells. Gene expression levels were then analysed for age associations.Results: We found: (i) cardiovascular tissues/endothelial cells express the required genes for SARS-CoV-2 infection, (ii) SARS-CoV-2 receptor pathways, ACE2/TMPRSS2 and BSG/PPIB(A) polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of host genes are relatively stable with age and (iv) notable exceptions are ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells.Conclusion: Our data identifies a positive correlation of BSG with age in endothelial cells. Since BSG is utilised by other pathogens and is implicated in a range of cardiovascular disease, our observations may have relevance to our understanding of mechanisms associated with other pathogens and in the diseases associated with aging respectively.

Journal article

Kirkby N, Raouf J, Ahmetaj-Shala B, Liu B, Mazi S, Edin M, Geoffrey Chambers M, Korotkova M, Wang X, Wahli W, Zeldin D, Nusing R, Zhou Y, Jakobsson P-J, Mitchell Jet al., 2020, Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis, Cardiovascular Research, Vol: 116, Pages: 1972-1980, ISSN: 0008-6363

Aims:Cardiovascular side effects caused by non-steroidal anti-inflammatory drugs (NSAIDs), which all inhibit cyclooxygenase (COX)-2, have prevented development of new drugs that target prostaglandins to treat inflammation and cancer. Microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors have efficacy in the NSAID arena but their cardiovascular safety is not known. Our previous work identified asymmetric dimethylarginine (ADMA), an inhibitor of eNOS, as a potential biomarker of cardiovascular toxicity associated with blockade of COX-2. Here we have used pharmacological tools and genetically modified mice to delineate mPGES-1 and COX-2 in the regulation of ADMA.Methods and Results:Inhibition of COX-2 but not mPGES-1 deletion resulted in increased plasma ADMA levels. mPGES-1 deletion but not COX-2 inhibition resulted in increased plasma prostacyclin levels. These differences were explained by distinct compartmentalisation of COX-2 and mPGES-1 in the kidney. Data from prostanoid synthase/receptor knockout mice showed that the COX-2/ADMA axis is controlled by prostacyclin receptors (IP and PPARβ/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors.Conclusions:These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.

Journal article

Mohamed N, Abou Saleh H, Kameno Y, Marei I, de Nucci G, Ahmetaj-Shala Bet al., 2020, Novel sildenafil nanoformulation as a potential therapy for pulmonary arterial hypertension, Meeting of the British-Pharmacological-Society, Publisher: WILEY, Pages: 2508-2509, ISSN: 0007-1188

Conference paper

Kirkby N, Gashaw H, Perikleous A, Ferreira P, Ahmetaj-Shala B, Mitchell Jet al., 2020, Novel arginine formulations of celecoxib fully retain COX-2 inhibitory and anti-cancer activity and reverse L-NAME-induced endothelial dysfunction, Meeting of the British-Pharmacological-Society, Publisher: WILEY, Pages: 2491-2492, ISSN: 0007-1188

Conference paper

Latifi-Pupovci H, Selmonaj M, Ahmetaj-Shala B, Dushi M, Grajqevci Vet al., 2020, Incidence of haematological malignancies in Kosovo-A post "uranium war" concern, PLOS ONE, Vol: 15, ISSN: 1932-6203

Journal article

Ahmetaj-Shala B, Kawai R, Marei I, Nikolakopoulou Z, Shih C-C, Konain B, Reed DM, Mongey R, Kirkby NS, Mitchell JAet al., 2020, A bioassay system of autologous human endothelial, smooth muscle cells and leucocytes for use in drug discovery, phenotyping and tissue engineering, The FASEB Journal, Vol: 34, Pages: 1745-1754, ISSN: 0892-6638

Purpose: Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leucocytes to generate a same donor ‘vessel in a dish’ bioassay. Basic procedures: Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs) and leucocytes were obtained from 4 donors. Cells were treated in mono and cumulative co-culture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumour necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines.Main findings: Co-cultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels.Principle conclusions: For the first time, we report a proof of concept study where autologous blood outgrowth ‘vascular’ cells and leucocytes were studied alone and in co-culture. This novel bioassay has utility in vascular biology research, patient phenotyping, drug testing and tissue engineering.

Journal article

Ahmetaj-Shala B, Kawai R, Marei I, Bhatti F, Gashaw H, Kirkby NS, Mitchell JAet al., 2019, A bioassay system of autologous human endothelial and smooth muscle cells for use in cardiovascular drug discovery and patient phenotyping, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3040-3041, ISSN: 0007-1188

Conference paper

Marei I, Al Shammari H, Latif N, Ahmetaj-Shala B, Yacoub MH, Chester AH, Mitchell JAet al., 2019, Effect of toll-like receptor antagonists on side specific aortic valve endothelial cells, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 2999-3000, ISSN: 0007-1188

Conference paper

Mazi SI, Ahmetaj-Shala B, Warner TD, Mitchell JA, Kirkby NSet al., 2019, Omic profiling in healthy volunteers taking celecoxib reveals novel biomarkers regulated by cyclooxygenase-2, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 1628-1628, ISSN: 0306-5251

Conference paper

Kawai R, Ahmetaj-Shala B, Shih CC, Marei I, Bhatti K, Kirkby NS, Mitchell JAet al., 2018, Development of a human autologous 3-cell cytokine release assay that models the vascular wall in vitro, 54th Congress of the European-Societies-of-Toxicology (EUROTOX) - Toxicology Out of the Box, Publisher: ELSEVIER IRELAND LTD, Pages: S114-S114, ISSN: 0378-4274

Conference paper

Ahmetaj-Shala B, Olanipekun M, Tesfai A, MacCallum N, Kirkby N, Qunilan G, Shih C-C, Kawai R, Mumby S, Paul-Clark M, Want E, Mitchell JAet al., 2018, Development of a novel UPLC-MS/MS-based platform to quantify amines, amino acids and methylarginines for applications in human disease phenotyping, Scientific Reports, Vol: 8, ISSN: 2045-2322

Amine quantification is an important strategy in patient stratification and personalised medicine. This is because amines, including amino acids and methylarginines impact on many homeostatic processes. One important pathway regulated by amine levels is nitric oxide synthase (NOS). NOS is regulated by levels of (i) the substrate, arginine, (ii) amino acids which cycle with arginine and (iii) methylarginine inhibitors of NOS. However, biomarker research in this area is hindered by the lack of a unified analytical platform. Thus, the development of a common metabolomics platform, where a wide range of amino acids and methylarginines can be measured constitutes an important unmet need. Here we report a novel high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) platform where ≈40 amine analytes, including arginine and methylarginines can be detected and quantified on a molar basis, in a single sample of human plasma. To validate the platform and to generate biomarkers, human plasma from a well-defined cohort of patients before and after coronary artery bypass surgery, who developed systemic inflammatory response syndrome (SIRS), were analysed. Bypass surgery with SIRS significantly altered 26 amine analytes, including arginine and ADMA. Consequently, pathway analysis revealed significant changes in a range of pathways including those associated with NOS.

Journal article

Yen I-C, Shi L-S, Chung M-C, Ahmetaj-Shala B, Chang T-C, Lee S-Yet al., 2018, Antrolone, a Novel Benzoid Derived from Antrodia cinnamomea, Inhibits the LPS-Induced Inflammatory Response in RAW264.7 Macrophage Cells by Balancing the NF-kappa B and Nrf2 Pathways, AMERICAN JOURNAL OF CHINESE MEDICINE, Vol: 46, Pages: 1297-1313, ISSN: 0192-415X

Journal article

Kirkby NS, Sampaio W, Etelvino G, Alves DT, Anders KL, Temponi R, Shala F, Nair AS, Ahmetaj-Shala B, Jiao J, Herschman HR, Wang X, Wahli W, Santos RA, Mitchell JAet al., 2018, Cyclooxygenase-2 Selectively Controls Renal Blood Flow Through a Novel PPAR beta/delta-Dependent Vasodilator Pathway (vol 71, pg 297, 2018), HYPERTENSION, Vol: 71, Pages: e10-e10, ISSN: 0194-911X

Journal article

Kirkby NS, Sampaio W, Etelvino G, Alves D, Anders KL, Temponi R, Shala F, Nair AS, Ahmetaj-Shala B, Jiao J, Herschman HR, Xiaomeng W, Wahli W, Santos RA, Mitchell JAet al., 2018, Cyclooxygenase-2 selectively controls renal blood flow through a novel PPARβ/δ-dependent renal vasodilator pathway, Hypertension, Vol: 71, Pages: 297-305, ISSN: 0194-911X

Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2–dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPARβ/δ (peroxisome proliferator-activated receptor-β/δ). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPARβ/δ-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPARβ/δ axis as a therapeutic target in renal disease.

Journal article

Mitchell JA, Benson J, Shala F, Ahmetaj-Shala B, Kirkby NSet al., 2017, Vascular Prostanoids Paradoxically Amplify Vasoconstriction During Platelet Activation, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322

Conference paper

Mitchell JA, Shala F, Ahmetaj-Shala B, Jiao J, Armstrong PC, Chan MV, Crescente M, Warner TD, Herschman HR, Kirkby NSet al., 2017, Novel Tissue-specific Cyclooxygenase-1 Knockout Mice Demonstrate a Dominant Role for Endothelial Cyclooxygenase-1 in Prostacyclin Production, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322

Conference paper

Lee S-Y, Tsai W-C, Lin J-C, Ahmetaj-Shala B, Huang S-F, Chang W-L, Chang T-Cet al., 2017, Astragaloside II promotes intestinal epithelial repair by enhancing L-arginine uptake and activating the mTOR pathway, SCIENTIFIC REPORTS, Vol: 7, ISSN: 2045-2322

Astragaloside II (AS II) extracted from Astragalus membranaceus has been reported to promote tissue wound repair. However, the effect of AS II on inflammatory bowel disease is unknown. We investigated the effects and mechanism of AS II on intestinal wound healing in both in vitro and in vivo models. Human intestinal Caco-2 cells were treated with multiple concentrations of AS II to assess cell proliferation, scratch wound closure, L-arginine uptake, cationic amino acid transporter activity, and activation of the mTOR signaling pathway. These effects were also measured in a mouse model of colitis. AS II promoted wound closure and increased cell proliferation, L-arginine uptake, CAT1 and CAT2 protein levels, total protein synthesis, and phosphorylation of mTOR, S6K, and 4E-BP1 in Caco-2 cells. These effects were suppressed by lysine or rapamycin treatment, suggesting that the enhanced arginine uptake mediates AS II-induced wound healing. Similar results were also observed in vivo. Our findings indicate that AS II can contribute to epithelial barrier repair following intestinal injury, and may offer a therapeutic avenue in treating irritable bowel disease.

Journal article

Tesfai A, MacCallum N, Kirkby NS, Gashaw H, Gray N, Quinlan G, Mumby S, Leiper JM, Paul-Clark M, Ahmetaj-Shala B, Mitchell JAet al., 2017, Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways, PLoS ONE, Vol: 12, ISSN: 1932-6203

RationaleNitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known.ObjectiveOur objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity.Methods and measurements34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay.Main resultsOf all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis.ConclusionsIn early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo.

Journal article

Huang L-Y, Yen I-C, Tsai W-C, Ahmetaj-Shala B, Chang T-C, Tsai C-S, Lee S-Yet al., 2017, Rhodiola crenulata Attenuates High Glucose Induced Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells, AMERICAN JOURNAL OF CHINESE MEDICINE, Vol: 45, Pages: 1201-1216, ISSN: 0192-415X

Journal article

Mohamed NA, Davies RP, Lickiss PD, Ahmetaj-Shala B, Reed DM, Gashaw HH, Saleem H, Freeman GR, George PM, Wort SJ, Morales-Cano D, Barreira B, Tetley TD, Chester AH, Yacoub MH, Kirkby NS, Moreno L, Mitchell JAet al., 2017, Chemical and biological assessment of metal organic frameworks (MOFs) in pulmonary cells and in an acute in vivo model: relevance to pulmonary arterial hypertension therapy, Pulmonary Circulation, Vol: 7, Pages: 1-11, ISSN: 2045-8940

Pulmonary arterial hypertension (PAH) is a progressive and debilitating condition. Despite promoting vasodilation, current drugs have a therapeutic window within which they are limited by systemic side effects. Nanomedicine uses nanoparticles to improve drug delivery and/or reduce side effects. We hypothesize that this approach could be used to deliver PAH drugs avoiding the systemic circulation. Here we report the use of iron metal organic framework (MOF) MIL-89 and PEGylated MIL-89 (MIL-89 PEG) as suitable carriers for PAH drugs. We assessed their effects on viability and inflammatory responses in a wide range of lung cells including endothelial cells grown from blood of donors with/without PAH. Both MOFs conformed to the predicted structures with MIL-89 PEG being more stable at room temperature. At concentrations up to 10 or 30 µg/mL, toxicity was only seen in pulmonary artery smooth muscle cells where both MOFs reduced cell viability and CXCL8 release. In endothelial cells from both control donors and PAH patients, both preparations inhibited the release of CXCL8 and endothelin-1 and in macrophages inhibited inducible nitric oxide synthase activity. Finally, MIL-89 was well-tolerated and accumulated in the rat lungs when given in vivo. Thus, the prototypes MIL-89 and MIL-89 PEG with core capacity suitable to accommodate PAH drugs are relatively non-toxic and may have the added advantage of being anti-inflammatory and reducing the release of endothelin-1. These data are consistent with the idea that these materials may not only be useful as drug carriers in PAH but also offer some therapeutic benefit in their own right.

Journal article

Ahmetaj-Shala B, Tesfai A, Constantinou C, Leszczynski R, Chan MV, Gashaw HH, Galaris G, Mazi SI, Warner TD, Kirkby NS, Mitchell JAet al., 2017, Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing, Biochemical and Biophysical Research Communications, Vol: 484, Pages: 762-766, ISSN: 1090-2104

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l-arginine. The ibuprofen salt, ibuprofen arginate (Spididol®) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo. Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety.

Journal article

Duluc L, Ahmetaj-Shala B, Mitchell J, Abdul Salam VB, Mahomed AS, Aldabbous L, Oliver E, Iannone L, Dubois OD, Storck EM, Tate EW, Zhao L, Wilkins MR, Wojciak-Stothard Bet al., 2017, Tipifarnib prevents development of hypoxia-induced pulmonary hypertension, Cardiovascular Research, Vol: 113, Pages: 276-287, ISSN: 1755-3245

Aims.RhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and Results.The analysis of lung tissues from rodent models and pulmonary hypertensive patientsshowed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerisation and increased eNOS expression in vitroand in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB) and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator.Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins.Conclusions.Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodeling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.

Journal article

Kirkby NS, Tesfai A, Ahmetaj-Shala B, Gashaw H, Sampaio W, Etelvino G, Miricéia Leão N, Santos RA, Mitchell JAet al., 2016, Ibuprofen arginate retains eNOS substrate activity and reverses endothelial dysfunction: implications for the COX-2/ADMA axis, The FASEB Journal, Vol: 30, Pages: 4172-4179, ISSN: 0892-6638

: Nonsteroidal antiinflammatory drugs, including ibuprofen, are among the most commonly used medicationsand produce their antiinflammatory effects by blocking cyclooxygenase (COX)-2. Their use is associatedwith increased risk of heart attacks caused by blocking COX-2 in the vasculature and/or kidney, with our recent workimplicating the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), a cardiotoxic hormone whoseeffects can be prevented by L-arginine. The ibuprofen salt ibuprofen arginate (Spididol) was created to increasesolubility but we suggest that it could also augment the NO pathway through codelivery of arginine. Here weinvestigated the idea that ibuprofen arginate can act to simultaneously inhibit COX-2 and preserve the NO pathway.Ibuprofen arginate functioned similarly to ibuprofen sodium for inhibition of mouse/human COX-2, but onlyibuprofen arginate served as a substrate for NOS. Ibuprofen arginate but not ibuprofen sodium also reversed theinhibitory effects of ADMA and NG-nitro-L-arginine methyl ester on inducible NOS (macrophages) and endothelialNOS in vitro (aorta) and in vivo (blood pressure). These observations show that ibuprofen arginate provides, in onepreparation, a COX-2 inhibitor and NOS substrate that could act to negate the harmful cardiovascular consequencesmediated by blocking renal COX-2 and increased ADMA. While remarkably simple, our findings arepotentially game-changing in the nonsteroidal antiinflammatory drug arena.—Kirkby, N. S., Tesfai, A., AhmetajShala,B., Gashaw, H. H., Sampaio, W., Etelvino, G., Leão, N. M., Santos, R. A., Mitchell, J. A. Ibuprofen arginateretains eNOS substrate activity and reverses endothelial dysfunction: implications for the COX-2/ADMA axis.

Journal article

Mohamed NA, Ahmetaj-Shala B, Duluc L, Mackenzie LS, Kirkby NS, Reed DM, Lickiss PD, Davies RP, Freeman GR, Wojciak-Stothard B, Chester AH, El-Sherbiny IM, Mitchell JA, Yacoub MHet al., 2016, A New NO-Releasing Nanoformulation for the Treatment of Pulmonary Arterial Hypertension., Journal of Cardiovascular Translational Research, Vol: 9, Pages: 162-164, ISSN: 1937-5395

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease which continues to carry an unacceptably high mortality and morbidity. The nitric oxide (NO) pathway has been implicated in the pathophysiology and progression of the disease. Its extremely short half-life and systemic effects have hampered the clinical use of NO in PAH. In an attempt to circumvent these major limitations, we have developed a new NO-nanomedicine formulation. The formulation was based on hydrogel-like polymeric composite NO-releasing nanoparticles (NO-RP). The kinetics of NO release from the NO-RP showed a peak at about 120 min followed by a sustained release for over 8 h. The NO-RP did not affect the viability or inflammation responses of endothelial cells. The NO-RP produced concentration-dependent relaxations of pulmonary arteries in mice with PAH induced by hypoxia. In conclusion, NO-RP drugs could considerably enhance the therapeutic potential of NO therapy for PAH.

Journal article

Raouf J, Kirkby NS, Ahmetaj-Shala B, Liu B, Mazi SI, Korotkova M, Zhou Y, Mitchell JA, Jakobsson PJet al., 2016, mPGES-1 DELETION INCREASES PROSTACYCLIN AND EVADES THE ELEVATED SYSTEMIC ADMA ASSOCIATED WITH COX-2 INHIBITORS: RELEVANCE TO CARDIOVASCULAR SAFETY OF mPGES-1 INHIBITORS, 36th European Workshop for Rheumatology Research (EWRR), Publisher: BMJ PUBLISHING GROUP, Pages: A11-+, ISSN: 0003-4967

Conference paper

Kirkby NS, Chan MV, Zaiss AK, Garcia-Vaz E, Jiao J, Berglund LM, Verdu EF, Ahmetaj-Shala B, Wallace JL, Herschman HR, Gomez MF, Mitchell JAet al., 2016, Systematic study of constitutive cyclo-oxygenase-2 expression: role of NFκB and NFAT transcriptional pathways, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 434-439, ISSN: 1091-6490

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.

Journal article

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