Browsing by Author "Thakur, Shori"

Now showing 1 - 4 of 4
  • Thumbnail Image
    ItemOpen Access
    The Impact of Semicarbazide Sensitive Amine Oxidase Activity on Rat Aortic Vascular Smooth Muscle Cells.
    (MDPI, 2023-03-03) Manasieva, Vesna; Thakur, Shori; Lione, Lisa; Baydoun, Anwar R; Skamarauskas, John
    Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while βAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and βAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.
  • Thumbnail Image
    ItemOpen Access
    Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells
    (MDPI, 2022-10-26) Manasieva, Vesna; Thakur, Shori; Lione, Lisa; Patel, Jessal; Baydoun, Anwar; Skamarauskas, John
    Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. βAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). βAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.
  • Thumbnail Image
    ItemOpen Access
    Serum cytokine levels as markers of paralytic ileus following robotic radical prostatectomy at different pneumoperitoneum pressures.
    (Wolters Kluwer, 2021-05-26) Hampson, Alexander; Raj, Nidhin; Lingamanaicker, Vidhya; Thakur, Shori; Shan, Gowrie Mohan; Prasad, Venkat; Baydoun, Anwar; Vasdev, Nikhil
    Background: To evaluate intraoperative and postoperative cytokines in patients who underwent robotic prostatectomy (RP) at a pressure of 12 or 15mmHg, and the risk of postoperative ileus. Materials and methods: We presented the first series evaluating intraoperative and postoperative cytokines in patients undergoing RP at a pressure of 12 or 15mmHg by a single surgeon. Changes in cytokine concentrations were shown to correlate with surgical outcomes and pathological states. The study investigated the changes in cytokine concentrations (interferon-g, tumor necrosis factor-a, interleukin-1b [IL-1b], IL-2, IL-4, IL-6, IL-12, and IL-17) at different pneumoperitoneum pressures and their potential role in the development of postoperative ileus. Results: The data on 10 consecutive patients confirmed that a lower pneumoperitoneum pressure was associated with lower cytokine levels and a lower risk of ileus. There were increased levels of postoperative interferon-g, tumor necrosis factor-a, IL-12p70, IL-1b, IL-2, IL-4, and IL-17a at 15mmHg when compared to 12mmHg. Conclusions: The data indicated that lower pressure RP reduced intra-/postoperative cytokine levels confirming our hypothesis. Larger patient numbers are required to further validate this but the implications of this data will benefit not only urological patients but also other speciality patients undergoing minimally invasive surgery.
  • Thumbnail Image
    ItemOpen Access
    Uremic serum induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2
    (Portland Press, 2019-07-24) Patidar, Ashish; Singh, Dhruv; Thakur, Shori; Farrington, Ken; Baydoun, Anwar
    Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving trans-differentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of RUNX2, SM22a and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater ( p<0.01 ) than that of controls, though not influenced by CKD stage. MDRD-4 eGFR ( p<0.001 ), serum phosphate ( p= 0.042 ), RANKL ( p= 0.001 ), PTH ( p= 0.014 ) and HDL/Cholesterol ratio ( p= 0.026 ) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7mM] and β glycerophosphate [7mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression ( p<0.01 ) in human SMCs and decreased SM22a expression ( p<0.05 ). Co-incubating control but not CKD serum with CB further increased RUNX2 expression ( p<0.01 ). Both SM22a and Klotho expression decreased significantly ( p<0.01 ) in the presence of CKD serum, and were virtually abolished with Stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22a and Klotho.