Innovative assessment of lipid-induced oxidative stress and inflammation in harvested human endothelial cells.

Studying acute changes in vascular endothelial cells in humans is challenging. We studied ten African American women and used the J-wire technique to isolate vein endothelial cells before and after a four-hour lipid and heparin infusion. Dynamic changes in lipid-induced oxidative stress and inflammatory markers were measured with fluorescence-activated cell sorting. We used the surface markers CD31 and CD144 to identify human endothelial cells. Peripheral blood mononuclear cells isolated from blood were used as a negative control. The participants received galantamine (16 mg/day) for 3 months. We previously demonstrated that galantamine treatment effectively suppresses lipid-induced oxidative stress and inflammation. In this study, we infused lipids to evaluate its potential to increase the activation of endothelial cells, as assessed by the levels of CD54+ endothelial cells and expression of Growth arrest-specific 6 compared to the baseline sample. Further, we aimed to investigate whether lipid infusion led to increased expression of the oxidative stress markers IsoLGs and nitrotyrosine in endothelial cells. This approach will expedite the in vivo identification of novel pathways linked with endothelial cell dysfunction induced by oxidative stress and inflammatory cytokines. This study describes an innovative method to harvest and study human endothelial cells and demonstrates the dynamic changes in oxidative stress and inflammatory markers release induced by lipid infusion.

Opuntia monacantha: Validation of the anti-inflammatory and anti-arthritic activity of its polyphenolic rich extract in silico and in vivo via assessment of pro- and anti-inflammatory cytokines.

ETHNOPHARMACOLOGICAL RELEVANCE: Opuntia monacantha belongs to the cactus family Cactaceae and is also known by cochineal prickly pear, Barbary fig or drooping prickly pear. It was traditionally used to treat pain and inflammation. O. monacantha cladodes showed pharmacological effects such as antioxidant potential owing to the presence of certain polysaccharides, flavonoids, and phenols. AIM OF THE STUDY: This research aimed to evaluate the anti-inflammatory as well as the anti-arthritic potential of ethanol extract of Opuntia monacantha (E-OM). MATERIALS AND METHODS: In vivo edema in rat paw was triggered by carrageenan and used to evaluate anti-inflammatory activity, while induction of arthritis by Complete Freund's Adjuvant (CFA) rat model was done to measure anti-arthritic potential. In silico studies of the previously High performance liquid chromatography (HPLC) characterized metabolites of ethanol extract was performed by using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) within active pocket of glutaminase 1 (GLS1) (PDB code: 3VP1; 2.30 Å). RESULTS: EOM, particularly at 750 mg/kg, caused a reduction in the paw edema significantly and decreased arthritic score by 80.58% compared to the diseased group. It revealed significant results when histopathology of ankle joint was examined at 28th day as it reduced inflammation by 18.06%, bone erosion by 15.50%, and pannus formation by 24.65% with respect to the diseased group. It restored the altered blood parameters by 7.56%, 18.47%, and 3.37% for hemoglobin (Hb), white blood count (WBC), and platelets, respectively. It also reduced rheumatoid factor RF by 13.70% with concomitant amelioration in catalase (CAT) and superoxide dismutase (SOD) levels by 19%, and 34.16%, respectively, in comparison to the diseased group. It notably decreased mRNA expression levels of COX-2, IL-6, TNF-α, IL-1, NF-κß and augmented the levels of IL-4 and IL-10 in real time PCR with respect to the diseased group and piroxicam. HPLC analysis previously performed showed that phenolic acids and flavonoids are present in E-OM. Molecular docking studies displayed pronounced inhibitory potential of these compounds towards glutaminase 1 (GLS1), approaching and even exceeding piroxicam. CONCLUSIONS: Thus, Opuntia monacantha could be a promising agent to manage inflammation and arthritis and could be incorporated into pharmaceuticals.

Identifying and treating ROBO1-ve /DOCK1+ve prostate cancer: An aggressive cancer subtype prevalent in African American patients.

BACKGROUND: There is a need to develop novel therapies which could be beneficial to patients with prostate cancer (CaP) including those who are predisposed to poor outcome, such as African-Americans. This study investigates the role of ROBO1-pathway in predicting outcome and race-based disparity in patients with CaP. METHODS AND RESULTS: Aided by RNA sequencing-based DECIPHER-testing and immunohistochemical (IHC) analysis of tumors we show that ROBO1 is lost during the progressive stages of CaP, a prevalent feature in African-Americans. We show that the loss of ROBO1 predicts high-risk of recurrence, metastasis and poor outcome of androgen-deprivation therapy in radical prostatectomy-treated patients. These data identified an aggressive ROBO1deficient /DOCK1+ve sub-class of CaP. Combined genetic and IHC data showed that ROBO1 loss is accompanied by DOCK1/Rac1 elevation in grade-III/IV primary-tumors and Mets. We observed that the hypermethylation of ROBO1-promoter contributes to loss of expression that is highly prevalent in African-Americans. Because of limitations in restoring ROBO1 function, we asked if targeting the DOCK1 could be an ideal strategy to inhibit progression or treat ROBO1deficient metastatic-CaP. We tested the pharmacological efficacy of CPYPP, a selective inhibitor of DOCK1 under in vitro and in vivo conditions. Using ROBO1-ve and ROBO1+ve CaP models, we determined the median effective concentration of CPYPP for growth. DOCK1-inhibitor treatment significantly decreased the (a) Rac1-GTP/ß-catenin activity, (b) transmigration of ROBO1deficient cells across endothelial lining, and (c) metastatic spread of ROBO1deficient cells through the vasculature of transgenicfl Zebrafish model. CONCLUSION: We suggest that ROBO1 status forms as predictive biomarker of outcome in high-risk populations such as African-Americans and DOCK1-targeting therapy has a clinical potential for treating metastatic-CaP.