School of Molecular & Cell Biology (ETDs)
Permanent URI for this communityhttps://hdl.handle.net/10539/38016
Browse
37 results
Search Results
Item Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer(University of the Witwatersrand, Johannesburg, 2019) Saha, Sourav Taru; Kaur, MandeepCancer cells have an increased need for cholesterol, which is required for cell membrane integrity. Cholesterol accumulation has been described in various malignancies including breast cancer. Cholesterol has also been known to be the precursor of estrogen and vitamin D, both of which play a key role in the histology of breast cancer. Elevated cholesterol levels have been linked to breast cancer therefore depleting cholesterol levels in cancer cells can be a viable strategy for treatment. 2-hydroxypropyl-β-cyclodextrin (HPβCD) is a cholesterol depleting compound which is a cyclic amylose oligomer composed of glucose units. It solubilizes cholesterol and is proven to be toxicologically benign in humans. This led us to hypothesise that it might deplete cholesterol from cancer cells and may prove to be a clinically useful compound. Our work provides experimental evidences to support this hypothesis. We identified the potency of HPβCD in vitro against two breast cancer cell lines: MCF7 (Estrogen positive, ER+), MDA-MB-231 [Triple negative breast cancer (TNBC)], and compared the results against two normal cell lines: MRC-5 (Normal Human Lung Fibroblasts) and HEK-293 (Human embryonic kidney) using cytotoxic, apoptosis and cholesterol based assays. HPβCD treatment reduced intracellular cholesterol resulting in significant breast cancer cell growth inhibition through apoptosis. The results hold true for both ER+ and TNBC. We have also tested HPβCD in vivo in MF-1 mice xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late, intermediate and early stage TNBC. These data suggest that HPβCD can prevent cholesterol accumulation in breast cancer cells and is a promising anti- cancer agentItem Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer(University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, MandeepThe cellular epithelial-mesenchymal transition (EMT) process is a complex labyrinth dependent on subversion of critical cellular signalling pathways, which crosstalk extensively to confer cancer cells with characteristics that mediate metastasis. Based on the pleotropic role of cholesterol in the cell, it is not surprising that cancer cells have evolved several mechanisms to facilitate cholesterol dyshomeostasis. In addition to meeting the increased metabolic demands of cancer cells, deregulated cholesterol metabolism also facilitates increased cellular cholesterol availability which is crucial to regulating the activity of protein intermediates in EMT-related signalling pathways. Despite evidence indicating that cholesterol directly regulates signalling pathways related to EMT, no publication to date has attempted to address the effect of EMT induction on cellular cholesterol levels in cancer. To shed light on the dynamics of cholesterol in the relationship between hypoxia and EMT, cholesterol content in MCF-7 cells pre- and post-hypoxia induced EMT was assessed. This dissertation presents findings indicating increased levels of free cholesterol, cholesteryl esters as well as lipid raft cholesterol in MCF-7 cells following hypoxia-induced EMT. Interestingly, MCF-7 cells post- EMT induction displayed increased sensitivity to treatment with cholesterol targeting agents and presented with reversion to an epithelial state as evidenced by the increased expression of epithelial markers, decreased expression of mesenchymal markers and also reduced invasive potential. Importantly, treatment with cholesterol targeting agents is also seen to abrogate the drug resistant potential following hypoxia-induced EMT. Based on these observations, it is proposed that targeting cellular cholesterol could be a promising area to invest in the search for novel therapeutics effective in combatting cancer metastasisItem Insights into silver(I) phosphine complexes in targeting cell death and metastatic mechanisms in malignant cell lines(University of the Witwatersrand, Johannesburg, 2023-09) Roberts, Kim Elli; Engelbrecht, Zelinda; Cronjé, Marianne J.Cancer is the leading cause of death worldwide, with 18.1 million new cases and 9.6 million deaths reported annually. Cisplatin, a popular chemotherapeutic drug, exhibits certain limitations in terms of selectivity and efficacy. This emphasizes the necessity for novel therapeutic approaches in addressing a variety of cancer types. Multiple studies have shown that silver-based compounds suppress cancer cell proliferation and induce apoptosis. Thirteen novel silver(I) mono-dentate phosphine complexes were investigated for their anticancer effects on seven different human malignant cell lines; A375 non-pigmented melanoma, A549 lung adenocarcinoma, HEP-G2 hepatocellular carcinoma, HT-29 colorectal adenocarcinoma, MCF-7 and MDA-MB-231 breast adenocarcinoma, and SNO oesophageal squamous cell carcinoma. Two non-malignant human cell lines, HEK-293 embryonic kidney cells and MRHF foreskin fibroblast cells, were used to assess the selectivity of the complexes. Cisplatin and the efficient silver(I) phosphine complexes were selected for dose-response experiments to determine IC50 concentrations for the respective cell lines. On the basis of these screening results (chapter two), five difficult-to-treat cancer cell lines, and their most efficient complexes were selected for further investigation. Various cellular characteristics were investigated in chapter three (A549, HEP-G2, HT-29); these included morphological changes, ATP levels, GAPDH levels, Ptd-L-Ser externalization, mitochondrial membrane potential, oxidative stress levels, and the activity of a metabolic enzyme, cytochrome P450 isoform CYP1B1. The antimetastatic activity of the selected complexes was assessed by evaluating their ability to impede the migration of A549 cells. The fourth chapter examines the anticancer effect of selected complexes on hormone-dependent (MCF-7) versus triple-negative (MDA-MB-231) breast cells. Changes in morphology, Ptd-L-Ser externalization, alterations in mitochondrial membrane potential, oxidative stress levels, cytochrome c release, and DNA damage were studied. Furthermore, in chapter five, molecular docking simulations were used to determine whether the most potent silver(I) phosphine complex across all cell lines bonds to estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Seven of the thirteen silver(I) phosphine complexes significantly reduced cell viability in malignant cell lines while being less toxic to non-malignant cells. Complex 4 best targeted all cancer types, with IC50 values ranging from 5.75 to 10.80 µM across malignant cell lines. In the malignant treated cells, morphological changes, reactive oxygen species production, mitochondrial membrane depolarization, and Ptd-L-Ser externalization were observed. Complexes 1 and 4 repressed cell migration in the A549 cells. The presence of damaged nuclei, metabolically inactive mitochondria and cytochrome c translocation from the mitochondria’ intermembrane to the cytosol in MCF-7 cells were observed. These findings suggest that complexes 2, 4 and 7 induced apoptotic cell death. Furthermore, in silico computational predictions suggested a promising interaction between complex 4, and ER-α and ER-β. Overall, this study demonstrates the potential of silver(I) phosphine complexes as anticancer agents, with promising effects on various cancer cell lines.Item Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)(University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.Item HIV-1 subtype C protease: enzyme kinetics, thermodynamics, and X-ray crystal structure(University of the Witwatersrand, Johannesburg, 2023-09) Dlamini, Nozinhle Precious; Sayed, YasienHuman immunodeficiency virus (HIV), a precursor for AIDS is still one of the most devastating pandemics in history. In 2021 alone there were 650 000 deaths associated with the virus and the number of people living with the infection was recorded to be 38.4 million globally. Sub-Saharan Africa suffers the most burden of the virus with approximately 8.3 million people living with virus, HIV-1 subtype C is the main driver of the disease in South Africa and accounts for 46% of global infections. Even with these alarming statistics this subtype is not the main focus point for the majority of HIV-1 research which mainly focuses on subtype B though it only accounts for 12% of infections globally. There is no vaccine or cure against HIV; however, great strides have been made in suppressing the virus. Viral suppression drugs have been developed to target different stages of viral replication such as those targeting the three important enzymes (protease, reverse transcriptase and integrase). In this study the focus will be on HIV-1 subtype C protease. This is a homodimeric aspartyl protease with 99 amino acids in each monomer. It plays a crucial role in the replication cycle of HIV-1 by producing mature infectious virions through cleavage of the Gag and Gag-Pol polyproteins. The subtype C protease differs from subtype B protease in that it has eight naturally occurring polymorphisms which are substitution mutations, some occurring in different regions of the protease with some in the fulcrum (T12S, I15V and L19I), others in the hinge region (M36I and R41K), with H69K and L89M found in the loops and I93L in the α helix. In this study, structural and functional characterisation of HIV-1 subtype C protease was carried out. The secondary structure was characterised using far-UV CD, which is a technique that measures the difference in left and right circularly polarised light. The subtype C protease was estimated to be predominantly β-sheeted, with spectra showing a maximum at 195 nm and a minimum between 215-225 nm. Tertiary structure characterisation of protease was performed using fluorescence spectroscopy. The maximum emission at 347 nm close to that of water (350 nm), demonstrated that the tertiary conformation of the HIV-1 protease was conserved, and that the tryptophan residues within the protease are solvent exposed. SE-HPLC was used to characterise the quaternary structure of the protease and the homodimeric size was determined to be approximately 22 kDa. Steady-state enzyme kinetics to assess the catalytic activity of the subtype C protease was performed using a fluorogenic substrate. The activity of the enzyme was confirmed, with the specific activity of 24.22±1.72 µmol. min-1.mg-1 and the binding of the substrate to the HIV-1 protease was demonstrated by the KM value of 79.546±6.491 µM. This correlates to literature indicating that the substrate was weakly bound and that a high substrate concentration will be required to reach the maximum velocity (Vmax), and Vmax was determined to be 0.036±0.003 µmol. min-1. Enzyme kinetics was coupled with displacement isothermal titration calorimetry for determination of thermodynamics parameters using second generation PIs (atazanavir, darunavir and lopinavir). Thermodynamic studies indicated that the HIV-1 protease has a high affinity for LPV (Kd = 1 nM), compared to ATV (Kd = 18.57nM) and DRV (Kd = 42.26 nM) and binding reactions were all spontaneous with ΔG values(ATV = -43.39 kJ/mol, DRV = -41.39 kJ/mol and LPV = -50.51 kJ/mol). The values also indicated that LPV complexed with HIV-1 is more a stable complex. Also, all the binding reactions were exothermic as indicated by the negative ΔH values of ATV = -45.54 kJ/mol, DRV = -55.62 kJ/mol and LPV = -54.71 kJ/mol. The entropy of all the reactions were determined to be unfavourable with the -T∆S of DRV = 14.23 kJ/mol followed by LPV: 4.2 kJ/mol and ATV: 2.15 kJ/mol. Overall this suggested that all the binding reactions were enthalpically driven. Furthermore, the three-dimensional structure of the HIV-1 subtype C protease was elucidated using X-ray crystallography. The three-dimensional structure the HIV-1 CSA (PDB ID: 8CI7) was solved at a 2.4 Å resolution which is better than the 2.7 Å (PDB ID: 3U71) initially solved in our lab. The high-resolution three-dimensional structure of the protease will provide precise information about the arrangement of atoms within the protease molecule, thus enabling the design and development of protease inhibitors that will be specific for the subtype C protease. This study emphasised the significance of investigating subtype C protease in the context of enzyme kinetics, thermodynamics and detailed X-ray crystallography.Item The ligandin activity of Schistosoma 26-kDa and 28-kDa glutathione transferases towards 17β-Hydroxyandrost-4-ene-3-one from a biophysical perspective(University of the Witwatersrand, Johannesburg, 2023) Makumbe, Hattie Hope; Achilonu, Ikechukwu AnthonySchistosomiasis, caused by helminth worms, ranks second amongst parasitic diseases and accounts for over 220 million fatalities globally. Statistics show that in South Africa, schistosomiasis (bilharzia) has infected approximately 4 million individuals. Currently, there are parasite resistance challenges with the sole available remedy. The World Health Organisation (WHO) acknowledges the need for new effective drugs. The 26-kDa Schistosoma bovis/haematobium (Sbh26GST) and 28-kDa Schistosoma haematobium (Sh28GST) are parasite Glutathione S-transferases (GSTs) which consist of two identical subunits that perform a vital role in mitigating the adverse effects of harmful electrophilic substances within the parasite since the parasite is devoid of the neutralizing cytochrome P-450. This automatically renders these parasite GSTs as potential therapeutic targets for schistosomiasis. Testosterone, the major hormone responsible for sexual characteristics and growth in males, can be repurposed as a drug target against schistosomiasis. In this study, we examined the structural, stability and functional interactions between the parasite GSTs and testosterone. After confirmation of inhibition, IC50 experiments were performed. The enzymes were overexpressed in Escherichia coli (E.coli) and then purified through a single-step nickel ion-immobilized metal affinity chromatography (IMAC). Extrinsic fluorescence spectroscopy was also done to provide evidence for the binding of the recombinant GSTs with testosterone. The GST activity was measured by employing 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Additionally, we investigated if the enzyme activity was influenced by the presence of testosterone. To analyse the stability of the enzymes, a SYPRO Orange-based thermal shift assay was used in the presence and absence of testosterone. In addition to empirical investigations, computational modelling, molecular docking, and molecular dynamic simulations were used to provide complementary insights to show binding affinities, prediction of binding modes and stability of the GST-testosterone complex. The secondary structural composition was found to be predominantly alpha-helical. Insights into tertiary structure analysis revealed the presence of buried solvent exposed tryptophan residues. The findings from spectroscopy with 8-anilino-1-naphthalenesulfonate (ANS) indicated that both Human GST-mu and parasite GSTs bound to ANS. Enzyme kinetic studies show that testosterone is a potent inhibitor of the parasite GSTs, with a specific activity that decreases from 16 μmol min-1mg-1 to 0.03 μmol min-1mg-1 and IC50 in the nanomolar range of 20 µM for Sh28GST. Sbh26GST exhibited a specific activity that decreased from 20 μmol min-1mg-1 to 0.14 μmol min-1mg-1, and a testosterone IC50 of 23 µM. The thermal stability assay confirmed Sh28GST to be more stable than Sbh26GST, and this stability of Sh28GST intensified when the enzyme bound to testosterone and GSH. Steady state kinetics towards glutathione (GSH) revealed a Km of 4.2mM and 6.6 mM for Sh28GST and Sbh26GST respectively. The present study has practical implications for novel application of the enzymes to serve as a basis for future studies aimed at development of inhibitors with potential therapeutic benefits through rational drug design.Item Evaluating the in vitro anti-metastatic effects of silver(I) phosphine complexes on malignant breast cancer cell lines(University of the Witwatersrand, Johannesburg, 2023-08) Ferreira, Mizan; Engelbrecht, Zelinda; Cronje, Marianne JacquelineBreast cancer is the most diagnosed cancer type among females worldwide. Metastasis, the spread of cancer cells from the primary tumour and establishment of macroscopic secondary tumours, is regarded as the most dangerous characteristic of cancer cells as it is responsible for over 90% of cancer-related deaths. Globally there is a lack of drugs available to specifically target or prevent either the dissemination of cells from the primary tumour or the establishment of distant metastases. The purpose of this study was to ascertain whether a series of silver(I) phosphine complexes, which have previously been shown to display anti-cancer properties in vitro, are also effective as anti-metastatic compounds. The migration, invasion and adhesive abilities of two malignant breast cancer cell lines, MCF-7 and MDA-MB-231, in response to silver(I) phosphine treatment were evaluated. In addition, the colony-forming abilities of cells under both anchorage-dependent and -independent conditions were investigated. Furthermore, the effects of silver(I) phosphine treatment on the expression and activities of key metastatic proteins, matrix metalloproteinases (MMPs), were studied. Of the nine complexes evaluated, all of them showed the ability to reduce one or more metastatic steps namely cell migration, invasion through collagen towards a chemoattractant or adhesion to collagen. In addition, a selected number of complexes reduced the colony-forming abilities of MCF-7 and/or MDA-MB-231 cells in culture plates as well as in soft agar. Moreover, three of these complexes increased the in vitro invasion and colony formation of breast cancer cells. Further investigation into complexes showing anti-metastatic abilities revealed that, apart from one complex on MDA-MB-231 cells, anti-metastatic effects were not achieved through a reduction in MMP levels or activities. The findings presented here show the potential for silver(I) phosphine complexes to reduce the in vitro metastatic abilities of breast cancer cells, warranting further investigations into these complexes for their use as anti-metastatic drugs.Item Elucidating the Structure-Function Relationships of Enterococcus faecium Nicotinate-Nucleotide Adenylyltransferase through X-Ray Crystallography, Computational Modelling and Binding Studies(University of the Witwatersrand, Johannesburg, 2024) Jeje, Olamide Adetomi; Pandian, Ramesh; Achilonu, Ikechukwu A.Nicotinate nucleotide adenylyltransferase (NNAT) is a vital enzyme at the heart of NAD biosynthesis, catalysing a crucial reaction that leads to the formation of pyridine dinucleotides. NAD+ is an essential coenzyme in numerous metabolic processes, DNA repair, and cellular signalling. Given its pivotal role, NNAT has emerged as a compelling drug target, particularly for its potential to disrupt the survival mechanisms of bacterial pathogens. By inhibiting NNAT, it is possible to undermine the metabolic integrity of these pathogens, making NNAT a promising focal point in the fight against bacterial infections and antibiotic resistance. However, understanding the structure-function relationship of Enterococcus faecium NNAT (EfNNAT) has remained elusive. Hence, this study aimed to address this gap bycharacterising EfNNAT and validating its potential as a druggable target. EfNNAT was overexpressed and purified using the Escherichia coli system and IMAC purification technique. Subsequently, biophysical characterisation was performed, followed by the determination of the three-dimensional structure in both apo and liganded forms using X-ray crystallography. High-throughput virtual screening, along with SP and XP docking, was conducted using a library of synthesizable flavonoids. Molecular dynamic simulation and fluorescence studies were employed to establish and validate the binding of identified inhibitors to EfNNAT. Successful expression and purification of EfNNAT yielded approximately 101 mg per 7.8 g of wet E. coli cells, with a purity exceeding 98%. High-resolution crystal structures of EfNNAT in native, adenine-bound, and NMN-bound forms were determined at 1.90 Å, 1.82 Å, and 1.84 Å, respectively. These structures provided insights into EfNNAT's substrate preference and revealed a potential allosteric site at the dimer interface of the NMN-bound structure. Virtual screening identified quercetin 3-O-beta-D-glucose- 7-O-beta-D-gentiobioside as the only potential inhibitor from the flavonoid library used. A 500 ns atomistic molecular dynamics simulation showed the compound interacted through hydrogen bonding and water bridges, albeit unstable within the receptor. ANS and mant-ATP fluorescence spectroscopy confirmed quercetin binding, while thermal shift assay revealed minimal impact of the inhibitor on the protein stability and structure. This study establishes a pipeline from expression and purification to structure solution and potential inhibitor identification for EfNNAT, validating its druggability. The mechanistic insights offer a foundation for advancing drug discovery efforts targeting EfNNAT and other bacterial NNAT enzymes.Item Comparison of Saccharomyces cerevisiae and the novel wild yeast used in beer fermentation and their future in industrial biotechnology(University of the Witwatersrand, Johannesburg, 2024) Zviuya, Patience; Moodley, Sanchia; Rumbold, KarlThe alcohol industry has grown over centuries due to the increase in alcohol demand by customers. Beer consumers now understand the brewing process and they well understand the role of yeast in fermentation. The alcohol industry now has a lot of customers because of the growth that this sector has made, and this has resulted in an increased demand for new beer styles and high flavor profile beers. Due to this demand, research has been conducted on unconventional wild yeasts that can be employed in making beer, demonstrating the variety of fermentation yeasts that are available and capable of enhancing beer quality and producing a wide range of new beer varieties. The commercial S. cerevisiae yeast and eight wild yeasts (Samson’s Saison, Ragnarok, Dark knight, B. brusc, Neipa, The Proletariat, La Trappist and B. clauss) were used to ferment pale ale in different fermentation vessels. The commercial yeast was the control of this research because this yeast has been used for generations in the brewing industry. The wild yeast strains used were identified using sanger sequencing and seven of these yeasts were S. cerevisiae wild species with similarity index of more than 80% and one was B. bruxellensis strain similarity index more than 95% before and after fermentation. The research outcomes demonstrated that most of the wildyeast performed the same as the commercial yeast in terms of physical and chemical parameters however most of the wild yeast produced more volatiles and esters as compared to the commercial brewing yeast. Commercial S. cerevisiae produced the highest alcohol content 4.5% and the average alcohol content for wild yeast was 3.4% because they are challenging to regulate during fermentation and have low alcohol tolerance unlike the commercial yeast that has been harvested and used for generations. Overall, the utilization of unconventional wild yeast to make beer was identified as a promising alternative to produce beers with exotic flavours and alcohol-free beers. Future work identifying specific yeast that suit different fermentation processes and beer types are recommended.Item Using ChIP-seq and Gene Expression Microarray data to explore transcriptional dysregulation of PXDN and PXDNL in cardiovascular diseases(University of the Witwatersrand, Johannesburg, 2024) Naidoo, Shiven; Gentle, Nikki; Mavri-Damelin, DemetraBackground: Cardiovascular diseases (CVDs) remain one of the leading causes of death globally. The genes PXDN and PXDNL are both expressed in the cardiovascular system, and their dysregulation has been linked to various disorders, including CVDs, but little is known of their transcriptional regulation in the cardiovascular system or their roles in CVD pathogenesis. Methods: This study developed two custom bioinformatics pipelines in R to mine and analyse ChIP-seq data from ChIP-Atlas and gene expression microarray data from the Gene Expression Omnibus (GEO). The first pipeline used ChIPseeker to identify regulatory transcription factors (TFs) of PXDN and PXDNL in cardiovascular cells and tissues. ChIP-seq data from 400 experiments across 63 TFs was filtered to isolate TFs with high confidence binding peaks in the promoter and first intron of PXDN and PXDNL. The second pipeline used R Bioconductor packages to explore the expression profiles of PXDN, PXDNL, and their TFs in seven microarray datasets across three CVD-related contexts: cardiomyopathies, heart failure and TNF-α stimulation. Results and discussion: This study identified 27 TFs binding to PXDN and 18 TFs binding to PXDNL in cardiovascular cells. Sixteen of these TFs were shared by both PXDN and PXDNL, suggesting potential coregulatory mechanisms in cardiovascular cells where they are both expressed. Unique TFs were also identified for PXDN (11) and PXDNL (2). Differential gene expression analysis revealed no significant change in expression (log2FC > 0.5; p.adj < 0.05) for PXDN, PXDNL and many of their identified TFs in the CVD-related conditions investigated, suggesting that changes at the transcript level may not contribute to the progression of these conditions. Conclusions: This study advances our understanding of the transcriptional regulation of PXDN and PXDNL in healthy cardiovascular cells as well as their expression levels in the investigated CVD-related contexts. This study also contributes a bioinformatics pipeline which can be further developed and applied to analysing data from ChIP-Atlas and GEO. Future research can elucidate the roles of each TF in regulating PXDN and PXDNL in healthy and diseased cell lines