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Dr Harprit Singh

Job: Associate Professor/Reader; Faculty Head of Research Students

Faculty: Health and Life Sciences

School/department: School of Allied Health Sciences

Address: ²ÝÁñÊÓƵ, The Gateway, Leicester, LE1 9BH.

T: +44 (0)116 257 7779

E: harprit.singh@dmu.ac.uk

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Personal profile

A senior researcher with a well-established niche in vascular cell signalling. My work focuses on better understanding the protective role that certain growth factors play in maintaining human blood vessel integrity and how dietary components influence their function during adulthood.

I teach in the BSc (Hons) Biomedical Science and BMedSci (Hons) Medical Science programmes. In my role as Faculty Head of Research Students, I manage provisions related to Postgraduate Research Students, including recruitment, timely completions, and research training

Research group affiliations

 

Publications and outputs


  • dc.title: Phenolic Metabolites Protocatechuic Acid and Vanillic Acid Improve Nitric Oxide Bioavailability via the Akt-eNOS Pathway in Response to TNF-α Induced Oxidative Stress and Inflammation in Endothelial Cells dc.contributor.author: Festa, Joseph; Hussain, Aamir; Al-Hareth, Zakia; Bailey, Stephen J.; Singh, Harprit; Da Boit, Mariasole dc.description.abstract: Background/Objectives: Reduced nitric oxide (NO) bioavailability secondary to excess-superoxide-driven oxidative stress is central to endothelial dysfunction. Previous studies suggest that phenolic metabolites may improve NO bioavailability, yet limited research is available in response to an inflammatory mediator. Therefore, we assessed the effects of cyanidin-3-glucoside (C3G) and its phenolic metabolites protocatechuic acid (PCA) and vanillic acid (VA) on NO bioavailability in a TNF-α induced inflammatory environment. Methods: Primary human umbilical vein endothelial cells (HUVECs) were supplemented with either C3G, PCA, or VA at 1 μM for 24 h before being stimulated with TNF-α 20 ng/mL for an additional 24 h. Measurements included cell viability, apoptosis, reactive oxygen species (ROS), nitrite concentrations, and endothelial nitric oxide synthase (eNOS) and Akt at the mRNA and protein level. Results: Phenolic metabolites did not increase the eNOS expression or nitrite levels in the unstimulated environment; rather, the metabolites mediated NO bioavailability in response to TNF-α induced oxidative stress, with increased viability, eNOS mRNA, phosphorylation, and nitrite levels. Conclusions: Phenolic metabolites, in the presence of TNF-α, can improve NO bioavailability at physiologically relevant concentrations via the Akt-eNOS pathway. This demonstrates that the induction of inflammation is a prerequisite for phenolic metabolites to promote protective properties in endothelial cells by activating the Akt-eNOS pathway. dc.description: open access article

  • dc.title: Elderberry extract inhibits tumour necrosis factor induced monocyte adhesion to endothelial cells via modulation of the NF-κB pathway dc.contributor.author: Festa, Joseph; Singh, Harprit; Hussain, Aamir; Da Boit, Mariasole

  • dc.title: The Effects of SARS-CoV-2 on the Angiopoietin/Tie Axis and the Vascular Endothelium dc.contributor.author: Janchivlamdan, Dolgormaa; Shivkumar, Maitreyi; Singh, Harprit dc.description.abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause potentially life-threatening coronavirus disease (COVID-19). COVID-19 is a multisystem disease and is associated with significant respiratory distress, systemic hyperinflammation, vasculitis, and multi-organ failure. SARS-CoV-2 causes the deterioration of numerous systems, with increasing evidence implying that COVID-19 affects the endothelium and vascular function. The endothelium is important for preserving vascular tone and homeostasis. The overactivation and dysfunction of endothelial cells are significant outcomes of severity in patients with COVID-19. The Angiopoietin 1/Tie 2 pathway plays an important role in endothelium quiescence and vessel stability. The disruption of Angiopoietin/Tie balance affects the vessel contact barrier and leads to vessel leakage, and this in turn causes endothelial dysfunction. Although vascular instability through SARS-CoV-2 is associated with endothelial dysfunction, it is still not understood if the virus affects the Angiopoietin/Tie axis directly or via other mechanisms such as cytokine storm and/or immune response associated with the infection. This review provides an overview of the impact SARS-CoV-2 has on endothelial function and more specifically on the Angiopoietin/Tie pathway. dc.description: open access article

  • dc.title: Elderberry extract improves molecular markers of endothelial dysfunction linked to atherosclerosis dc.contributor.author: Festa, Joseph; Hussain, Aamir; Hackney, Amon; Desai, Unmesh; Sahota, T. S.; Singh, Harprit; Da Boit, Mariasole dc.description.abstract: Endothelial dysfunction (ED), secondary to diminished nitric oxide (NO) production and oxidative stress, is an early subclinical marker of atherosclerosis. Reduced NO bioavailability enhances the adhesion of monocytes to endothelial cells and promotes atherosclerosis. Elderberry extract (EB) is known to contain high levels of anthocyanins which could exert vascular protective effects. Specifically, we investigated the functional capacity of EB on various markers of ED. Human umbilical vein endothelial cells (HUVEC) were pretreated with EB 50 μg/mL and stimulated with TNF-α 10 ng/mL. Cell viability, apoptosis, oxidative stress; eNOS, Akt, Nrf2, NOX-4, and NF-κB at the protein level were measured. A co-culture model was used to determine whether EB could prevent the adhesion of monocytes (THP-1) to HUVECs. Moreover, the expression of adhesion molecules and pro-inflammatory cytokines were also measured. It was demonstrated that EB prevented TNF-α induced apoptosis and reactive oxygen species production in HUVECs. Additionally, EB upregulated Akt and eNOS activity, and Nrf2 expression in response to TNF-α, whereas it decreased NOX-4 expression and NF-κB activity. EB prevented the adhesion of monocytes to HUVECs, as well as reduced IL-6 and MCP-1 levels, which was associated with inhibition of VCAM-1 expression. Our results demonstrate that EB upregulates key cellular markers of endothelial function and ameliorates markers of ED. EB could be used as a potential nutritional aid for preventing atherosclerosis progression. dc.description: open access article

  • dc.title: Anthocyanins and Vascular Health: A Matter of Metabolites dc.contributor.author: Festa, Joseph; Hussain, Aamir; Al-Hareth, Zakia; Singh, Harprit; Da Boit, Mariasole dc.description.abstract: Anthocyanins are a subgroup of flavonoid polyphenols previously investigated for improv ing cardiovascular health and preventing the development of endothelial dysfunction. However, their poor bioavailability raises the question of whether the observed biological activity is due to their metabolites. Phenolic metabolites can reach higher plasma concentrations and can persist in the circulation for periods much longer than their original anthocyanin form; therefore, the biological activity and health promoting effects of anthocyanins may differ from their metabolites. To address this, recent studies have facilitated different cell models, in vivo studies and explored physiologically relevant concentrations to better understand their mechanisms of action. The criteria were chosen based on previous reports demonstrating that anthocyanins can improve endothelial function via modulation of the Akt-endothelial nitric oxide synthase pathway and transcription factors Nrf2 and NF-κB, which made it critical to assess the phenolic metabolites’ modes of action via these pathways. This review demonstrates how phenolic metabolites differ in bioactivity from their precursor an thocyanin, demonstrating improved endothelial function in response to inflammatory mediators at concentrations that are tolerated in vivo. The review highlights the crucial need for further studies to focus on improving the bioavailability of metabolites in isolation and explore the effect of metabolites in mixtures. dc.description: open access article

  • dc.title: C2- linked alkynyl poly-ethylene glycol(PEG) adenosine conjugates as water-soluble adenosine receptor agonists dc.contributor.author: Ferguson, Lindsay; Madieh, Nasrin Shokrzadeh; Brucoli, Federico; Vaideanu, Alexandra; Schatzlein, Andreas; Festa, Joseph; Singh, Harprit; Wells, Geoffrey; Bhakta, Sanjib dc.description.abstract: A series of 12 novel polyethylene-glycol(PEG)-alkynyl C2-adenosine(ADN) conjugates were synthesized using a robust Sonogashira coupling protocol and characterized by NMR spectroscopy and mass spectrometry analysis. The ADN-PEG conjugates showed null to moderate toxicity in murine macrophages and 12c was active against Mycobacterium aurum growth (MIC = 62.5 mg/L). The conjugates were not active against Mycobacterium bovis BCG. Conjugates 10b and 11b exhibited high water solubility with solubility values of 1.22 and 1.18 mg/ml, respectively, in phosphate buffer solutions at pH 6.8. Further, 10b and 11b induced a significant increase in cAMP accumulation in RAW264.7 cells comparable with that induced by adenosine. Analogues 10c, 11c and 12c were docked to the A1, A2A, A2B and A3 adenosine receptors (ARs) using crystal-structures and homology models. ADN-PEG-conjugates bearing chains with up to five ethyleneoxy units could be well accommodated within the binding sites of A1, A2A and A3 ARs. Docking studies showed that compound 10b and 11b were the best A2A receptor binders of the series, whereas 12c was the best binder for A1 AR. In summary, introduction of hydrophilic PEG substituents at the C2 of adenine ring significantly improved water solubility and did not affect AR binding properties of the ADN-PEG conjugates. dc.description: open access article

  • dc.title: Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate dc.contributor.author: Sahota, T. S.; Vu, Tam; Taylor, M. Joan; Singh, Harprit; Bottrill, Andrew; Bilmoria, Jay dc.description.abstract: Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7–9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications. dc.description: The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.

  • dc.title: Elderberries as a potential supplement to improve vascular function in a SARS-CoV-2 environment dc.contributor.author: Festa, Joseph; Singh, Harprit; Hussain, Aamir; Da Boit, Mariasole dc.description.abstract: Coronavirus disease 2019 (COVID-19) pandemic has been triggered by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). Although recent studies demonstrate that SARS-CoV-2 possibly does not directly infect endothelial cells (EC), the endothelium may be affected as a secondary response due to the damage of neighboring cells, circulating pro-inflammatory cytokines, and/or other mechanisms. Long-term COVID-19 symptoms specifically nonrespiratory symptoms are due to the persistence of endothelial dysfunction (ED). Based on the literature, anthocyanins a major subgroup of flavonoid polyphenols found in berries, have been well researched for their vascular protective properties as well as the prevention of cardiovascular disease (CVD)-related deaths. Elderberries have been previously used as a natural remedy for treating influenza, cold, and consequently cardiovascular health due to a high content of cyanidin-3-glucoside (C3G) a major anthocyanin found in the human diet. The literature reported many studies demonstrating that EE has both antiviral and vascular protective properties that should be further investigated as a nutritional component used against the (in)direct effect of SARS-CoV-2 in vascular function. dc.description: The Publisher's final version can be found by following the DOI link.. Open access article.

  • dc.title: Potential Benefits of Berry Anthocyanins on Vascular Function dc.contributor.author: Festa, Joseph; Hussain, Aamir; Da Boit, Mariasole; Singh, Harprit dc.description: The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.

  • dc.title: BS20 The opposing effects of chronic interleukin-1β on TIE2:TIE1 ratio and angiopoietin1 induced PI3 Kinase/AKT signaling in endothelial cells dc.contributor.author: Singh, Harprit; Bilimoria, Jay dc.description.abstract: Introduction Angiopoietin-1 (Ang-1) is a growth factor that plays a crucial role in maintaining normal vascular function. The main role of Ang-1 is to maintain endothelial survival. Ang-1 exerts its protective effect by activating Tie-2 receptors and subsequently the Phosphatidylinositol 3-kinase (PI3Kinase)/ AKT pathway. Tie-1 regulates Ang-1 signalling with high levels of the receptor reducing Ang-1-induced Tie-2 activation. Proinflammatory cytokines including Tumor Necrotic factor (TNF-a) Interleukin 1b (IL-1b) have been implicated in a range of vascular pathologies including vascular inflammation and atherosclerosis. While TNF-a has shown to regulate Tie receptors, the chronic impact IL-1b has on Ang-1/Tie receptor signalling pathway and vascular function has not been investigated. Aim To examine the impact IL-1b has on Tie receptor expression and Angiopoietin-1 induced PI3Kinase/AKT activity in endothelial cells. Method Primary Human Umbilical Vein Endothelial Cells (HUVEC) were stimulated with 25ng/ml of IL-1b in the presence or absence of 100ng/ml of human recombinant Ang-1. The treatment times ranged from 0 to 48h. Cell lysates from the treated cells were then subjected to Western blotting to analyze Tie receptor levels and phospho-AKT (pAKT), a signaling molecule associated with Ang-1 cellular transduction. The levels of target proteins were compared between reactions by quantifying mean intensity of bands. In addition, cells were treated with Ang-1 in the absence or presence of IL-1b at various time points. The cell viability assay was performed on the treated cells by following the manufactures protocol. The mean percentage of live to dead cells was calculated from three random fields for each treatment. Data for the Tie receptor and AKT analysis is presented as means and SEM of three independent experiments. Statistical significance represented with p<0.05 using Student’s t-test. Data for the cell viability assay is presented as means and SEM of two independent experiments. Results A significant reduction in the levels of Tie-1 receptor was observed at 3h (58.6±8.6%) in HUVECs treated with IL1b. The cytokine was able to maintain significant low levels of Tie1 up until 48h of treatment, whereas the changes in levels of Tie-2 were insignificant. Interestingly, IL-1b significantly reduced Ang1-induced pAKT activity from 3h onwards with maximum reduction of 73.4±12.8% observed at 48h. The cell viability assays showed reduction in the percentage live to dead cells between Ang-1 and Ang1 + IL-1b for chronic time points tested. Conclusion Long term exposure of Interleukin-1b is capable of altering Tie-1 levels and increasing the Tie-2: Tie-1 ratio in endothelial cells. In contracts, IL-1b reduces Ang-1-PI3Kinase/ AKT signalling and endothelium cell viability. This opposing phenomenon observed where IL-1b impairs Ang-1 protective ability suggests a different mechanism of regulation, independent of the Tie-1 receptor.

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Research interests/expertise

Research expertise include:

  • Cardiovascular Disease
  • Nutrition and cardiovascular function
  • Endothelial biology
  • Cell signalling
  • Growth hormones and their Receptors
  • Molecular markers of vascular disease

Areas of teaching

  • Endocrinology
  • Cardiovascular 
  • Human Anatomy and Physiology
  • Angiogenesis

Qualifications

  • PhD in Biochemistry
  • PGCert in Teaching and Learning in Higher Education
  • BSc (Hons) Medical Biochemistry

²ÝÁñÊÓƵ taught

  • Medical Science (BMed Sci)
  • Biomedical Science (BSc)
  • Adv Biomedical Science (MSc)

Honours and awards

  • Fellow of the Higher Education Academy (FHEA)

Membership of professional associations and societies

  • The British Society for Cardiovascular Research
  • European Society of Cardiology Working Group Atherosclerosis & Vascular Biology

Consultancy work

Scientific consultant for Emerald Nutrition ()

Current research students

  • Dolgormaa Janchivlamdan, PhD. The effect of OC43 virus on TIE2 signalling pathway in endothelial cells
  • Koddus Ali, PhD. The role of a pharmacist in screening, communicating and reporting the risks of cardiovascular disease associated with non-steroidal anti-inflammatory drugs and the perception of the population 

Externally funded research grants information

 

Successfully Supervised PhD Students

Joseph Festa, 2019-2023

Nasrin Madieh, 2018-2023

Tam Ngoc Minh Vu, 2016-2021 

Jay Bilimoria, 2016-2020

Aamir Hussain, 2015-2019

Rasmieh Alziedan, 2012-2016

 

Journal Peer Reviewer

  • Nature communications
  • Frontiers in Physiology: Vascular Physiology
  • BMC Cardiovascular disorders
  • Journal of Biomedical Research
  • Cancer Letters
  • Human Cell

Grant Peer Reviewer

Medical Research Council

Diabetes UK