Studies on vascular response to full superantigens and superantigen derived peptides:Possible production of novel superantigen variants with less vasodilation effect for tolerable cancer immunotherapy

Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release and causing symptoms similar to sepsis, e.g. hypotension and subsequent hyporeactivity. We investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of Nitric Oxide (NO) and potentially hyperpolarization, which is dependent on the K + channel activation, were also explored. The data show that SEA and SPEA have direct vasodilatory effects that were in part NO-dependent, but completely dependent on activation of K + channels. Our work also identified the functional regions of one of the superantigens, SPEA, that are involved in causing the vasodilation and possible hypotension. A series of 20 overlapping peptides, spanning the entire sequence of SPEA, were designed and synthesized. The vascular response of each peptide was measured, and the active peptides were identified. Our results implicate the regions, (61–100), (101–140) and (181–220) which cause the vasodilation and possible hypotension effects of SPEA. The data also shows that the peptide 181–220 exert the highest vasodilation effect. This work therefore, demonstrates the direct effect of SAgs on vascular tone and identify the active region causing this vasodilation. We propose that these three peptides could be effective novel antihypertensive drugs. We also overexpressed, in E.coli, four superantigens from codon optimized genes

Downregulation of CYP17A1 by 20-hydroxyecdysone: plasma progesterone and its vasodilatory properties

Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC–MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation

Comparative Effect of Type 1 and Type 2 Diabetes Mellitus on Vascular Responses of Rat Thoracic Aorta to Potassium Ion Channel Openers

Background: Diabetes mellitus is associated with many cardiovascular dysfunction and impairment of potassium channel function. Aim: We compared the vascular reactivity in aorta from streptozotocin-induced and Goto-Kakizaki (GK) diabetic rats to potassium channel openers. Methodology: Diabetes mellitus (DM) was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin (STZ) at 65 mg/kg body weight. After four weeks of DM, vascular reactivity of the aortic rings from STZ-induced Sprague Dawley and age-matched GK and control rats to phenylephrine, acetylcholine, levcromakalim and naringenin was studied using standard organ bath procedure. Results: The phenylephrine-induced contraction was significantly (P<0.05) increased in STZ-diabetic aortic rings [2.03 ±0.07 g] when compared with GK rats [1.47±0.14 g] and STZ-control [1.42±0.21 g]. Maximal relaxation and potency to acetylcholine, levcromakalim and (+/-)-naringenin were significantly (P<0.05) decreased in STZdiabetic aorta when compared with GK-diabetic and control groups. Conclusion: The phenylephrine-induced contraction, endothelium-dependent relaxation, KATP - and (+/-)-naringenin-induced vasorelaxation are not altered in the early stages of Type 2 diabetes whereas there is exaggerated contractile response and a relaxant dysfunction involving the endothelium, KATP in Type 1 diabetes mellitus

Depot- and diabetes-specific differences in norepinephrine-mediated adipose tissue angiogenesis, vascular tone, collagen deposition and morphology in obesity

Aims: Norepinephrine (NE) is a known regulator of adipose tissue (AT) metabolism, angiogenesis, vasoconstriction and fibrosis. This may be through autocrine/paracrine effects on local resistance vessel function and morphology. The aims of this study were to investigate, in human subcutaneous and omental adipose tissue (SAT and OAT): NE synthesis, angiogenesis, NE-mediated arteriolar vasoconstriction, the induction of collagen gene expression and its deposition in non-diabetic versus diabetic obese subjects. Materials and methods: SAT and OAT from obese patients were used to investigate tissue NE content, tyrosine hydroxylase (TH) density, angiogenesis including capillary density, angiogenic capacity and angiogenic gene expression, NE-mediated arteriolar vasoconstriction and collagen deposition. Key findings: In the non-diabetic group, NE concentration, TH immunoreactivity, angiogenesis and maximal vasoconstriction were significantly higher in OAT compared to SAT (p < 0.05). However, arterioles from OAT showed lower NE sensitivity compared to SAT (10−8 M to 10–7.5 M, p < 0.05). A depot-specific difference in collagen deposition was also observed, being greater in OAT than SAT. In the diabetic group, no significant depot-specific differences were seen in NE synthesis, angiogenesis, vasoconstriction or collagen deposition. SAT arterioles showed significantly lower sensitivity to NE (10−8 M to 10–7.5 M, p < 0.05) compared to the non-diabetic group. Significance: SAT depot in non-diabetic obese patients exhibited relatively low NE synthesis, angiogenesis, tissue fibrosis and high vasoreactivity, due to preserved NE sensitivity. The local NE synthesis in OAT and diabetes desensitizes NE-induced vasoconstriction, and may also explain the greater tissue angiogenesis and fibrosis in these depots

Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35&ndash;36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p &lt; 0.05 and met &ge; 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT

20-hydroxyecdysone dilates muscle arterioles in a nitric oxide-dependent, estrogen ER-β receptor-independent manner

Background: 20-hydroxyecdysone is an ecdysteroid which is abundant in plants and insects and has anabolic potentials in mammals. It was recently shown to have affinity for estrogen ER-β receptor, which could potentially make it vasodilatory. Yet this possibility has not been previously investigated. Such an activity in muscle arterioles could have huge implications for muscle blood flow and performance. Hypothesis/Purpose: We hypothesized that 20-hydroxyecdysone would dilate muscle arterioles by activating estrogen ER-β receptors. To test this, we investigated its vasodilatory properties in ovine muscle arterioles and further explored the mechanisms in human tissues and cells. Study Design: The study was carried out experimentally, employing functional recording of arteriolar reactivity in intact ovine muscle arterioles and gene and protein expression analysis in human tissues and cells. Methods: Direct effects of the compound on arteriolar tone were assessed by wire myography in abdominal muscle and mesenteric arterioles isolated from samples obtained from male sheep. The roles of endothelial nitric oxide synthase (NOS3), cyclooxygenase (COX) and estrogen ER-β receptor (ER-β), and their effects were determined with specific blockers. The NOS3 mRNA and protein expressions were analyzed in human coronary artery endothelial cells (HCAECs) and humanized liver of uPA+/+-SCID mice, before and after 20-hydroxyecdysone administration. Results: Comparable dose-dependent relaxations were recorded for 20-hydroxyecdysone in both muscle and mesenteric arterioles with maximum relaxations of 46.94 ± 5.84% and 56.88 ± 7.04% respectively, which were not statistically different. Similar relaxation was recorded for β-estradiol in both arterioles. NOS inhibition with 100 µM L-NAME attenuated the relaxation to 20-hydroxyecdysone (p < 0.001) and β-estradiol (p < 0.001) in muscle arterioles. Neither COX inhibition with 10 µM indomethacin nor ER blockade with 1 µM PHTPP or 1 µM ICI182780 had any noticeable effect on 20-hydroxyecdysone relaxation in these arterioles. Transcriptome analysis revealed elevated NOS3 gene in the humanized liver of 20-hydroxyecdysone-treated mice, and, elevation of both NOS3 mRNA and protein in HCAECs treated with 20-hydroxyecdysone. Conclusion: The data suggest that 20-hydroxyecdysone has a nitric oxide-dependent, but ERβ-independent, vasodilatory property in muscle arterioles. The benefits to muscle blood flow would however be dependent on the impact of its effects on other vascular beds

Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35&ndash;36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p &lt; 0.05 and met &ge; 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT