Numerous natural compounds including Nigella sativa (N. sativa) demonstrate anti-inflammatory and anti-diabetic antiangiogenic properties. Lipopolysaccharide (LPS) mediated inflammation is regarded as an important contributor to the inflammation that is associated with the development of arteriosclerosis. In this study, it was hypothesised that N. sativa Extract (NSE) and its main active component Thymoquinone (TQ) could potentially inhibit LPS mediated inflammatory cytokine secretion and monocyte recruitment factors and monocyte in Human Vascular Endothelial Cell (HECV) lines. In addition the Ten-Eleven Translocation (TET-2) an epigenetic regulator, increasingly regarded has having a major role in both the regulation of cytokine secretion and in the development of atherosclerosis through its ability to inhibit the inflammasome Nod-like Receptor Protein 3 (NLRP3) and Interleukin-1b (IL-1b) secretion was investigated. NSE significantly inhibited the production of both IL-6 and-8 and both NSE and TQ inhibited the gene expression of vascular endothelial growth factor and monocyte chemotactic protein-1 in HECV cells. NSE and TQ inhibited the gene expression of NLRP3 and IL-1b and significantly upregulated the gene expression of TET-2 in the presence of LPS. To conclude, NSE and TQ attenuated inflammatory and monocyte recruitment response and also demonstrate a potentially important role in regulating both NLRP3 and TET-2 expression.
Introduction: Lack of efficient collateralisation during vascular occlusion leads to tissue ischemia in coronary, peripheral and cerebrovascular disease. Patients with peripheral, coronary, and cerebrovascular disease have elevated levels of the anti-angiogenic isoform of VEGF (VEGF-A 165 b), which correlate with poorer outcomes. In experimental animals, VEGF-A 165 b neutralisation improves blood flow and collateralisation after femoral or coronary artery ligation. Hypothesis: We tested the hypothesis that a potent, high affinity, humanised neutralising antibody to VEGF-A 165 b could have therapeutic potential in models of cardiovascular disease. Methods: A mouse monoclonal antibody to VEGF-A 165 b was humanised by cloning and site directed mutagenesis. Multiple clones were screened, and one with high affinity (measured by biolayer interferometry) was amplified. The efficacy in vitro was tested using endothelial cell migration assays and in vivo using the hindlimb ischemia (HLI) model in C57Bl6 mice fed a high fat high sucrose (HFHS) diet, and in Zucker Diabetic Fatty rats. Results: One clone (HC4LC2) had a VEGF-A 165 b affinity of 600pM. 0.3μg/ml of this clone reversed the inhibitory effect of recombinant human VEGF-A 165 b (31±4%, N=6 of VEGF-A 165 a alone) on migration of endothelial cells to 40ng/ml rhVEGF-A 165 a (89±9% N=3). Human monocyte dependent inhibition of VEGF-A 165 a mediated migration was dose dependently reversed by HC4LC2 (EC50, 55ng/ml, N=9 per concentration). HLI in HFHS mice impaired blood flow after 28 days in IgG treated mice (70±7.7% of pre-ischemic blood flow, N=6), which was restored to normal (93.3±5.5, p<0.05, N=8) by intraperitoneal injection of 1mg/kg HC4LC2. Blood flow recovery after HLI was also seen in ZDF rats treated with HC4LC2 (106±16% of pre-ischemic flow, N=5) compared with IgG treated animals (76±.9%, N=5, p<0.05, two-way ANOVA, all values mean ±SEM). Conclusions: These results indicate that HC4LC2 is a humanised, high affinity neutralising antibody to VEGF-A 165 b that could have therapeutic potential for patients with cardiovascular disease, by enhancing collateral formation.
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