Dose Response of Bumetanide on Aquaporins and Angiogenesis Biomarkers in Human Retinal Endothelial Cells Exposed to Intermittent Hypoxia

Aquaporins (AQPs) are important for regulating cellular water, solute transport, and balance. Recently, AQPs have also been recognized as playing a key role in cell migration and angiogenesis. In the retina, hypoxia induces vascular endothelial growth factor (VEGF), a potent angiogenic and vascular permeability factor, resulting in retinal edema, which is facilitated by AQPs. Bumetanide is a diuretic agent and AQP 1–4 blocker. We tested the hypothesis that bumetanide suppression of AQPs ameliorates intermittent hypoxia (IH)-induced angiogenesis and oxidative stress in human microvascular retinal endothelial cells (HMRECs). HMRECs were treated with a low-dose (0.05 µg/mL) or high-dose (0.2 µg/mL) of bumetanide and were exposed to normoxia (Nx), hyperoxia (50% O2), or IH (50% O2 with brief hypoxia 5% O2) for 24, 48, and 72 h. Angiogenesis and oxidative stress biomarkers were determined in the culture media, and the cells were assessed for tube formation capacity and AQP-1 and -4 expression. Both doses of bumetanide significantly decreased oxidative stress and angiogenesis biomarkers. This response was reflected by reductions in tube formation capacity and AQP expression. These findings confirm the role of AQPs in retinal angiogenesis. Therapeutic targeting of AQPs with bumetanide may be advantageous for IH-induced aberrant retinal development.

Sensitivity and Specificity of Vascular Endothelial Growth Factor A (VEGF-A) against Endometriosis

Background. Endometriosis is a benign disorder defined by the presence of endometrial glands and stroma outside the uterus. Endometriosis occurs in 10-15% of women during their reproductive years. Angiogenesis and the inflammatory response are important factors in the development of endometriosis. The formation of a new blood supply is a crucial step in the formation of endometrial lesions. Angiogenesis is induced by a growth factor peptide, namely vascular endothelial growth factor A (VEGF-A). VEGF-A is known as a vascular permeability factor that plays an important role in the pathological angiogenesis process and is a more specific and prominent angiogenesis factor among the VEGF family. The purpose of this study was to determine the sensitivity and specificity of examination VEGF-A of menstrual blood in diagnosing endometriosis compared to laparoscopy. Methods. This diagnostic test research has been carried out at the gynecology polyclinic, especially the division of Fertility, Endocrinology and Reproduction, Faculty of Medicine, Sriwijaya University- Dr. Central General Hospital. Mohammad Hoesin from August to November 2018, there were 45 patients who met the inclusion criteria. VEGF-A examination in instrumental blood based on ELISA examination. Data analysis to measure sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy using Medcalc statistics. Results. From the results of this study, it is known that the majority of the study samples were aged 31.69 years (24-38 years) 75.6%, body mass index (BMI) 51.1% with normal BMI, experienced infertility by 82.2% of the sample, most of the samples did not smoke (95.6%) and most experienced mild pain 55.6% using the VAS pain scale. From this study, it is known that 84.4% or 38 of the study samples had endometriosis with a cut off point value of VEGF-A > 347 pg/mL. From the results of this study, it is known that the sensitivity of VEGF-A in diagnosing endometriosis is 84.2%, specificity is 85.7%, positive predictive value is 97%, negative predictive value is 50% and accuracy is 84%. Conclusion. VEGF-A menstrual blood can be used as a diagnostic tool for endometriosis.

Reconciling VEGF With VPF: The Importance of Increased Vascular Permeability for Stroma Formation in Tumors, Healing Wounds, and Chronic Inflammation

It is widely believed that vascular endothelial growth factor (VEGF) induces angiogenesis by its direct mitogenic and motogenic actions on vascular endothelial cells. However, these activities are only detected when endothelial cells are cultured at very low (0.1%) serum concentrations and would not be expected to take place at the much higher serum levels found in angiogenic sites in vivo. This conundrum can be resolved by recalling VEGF’s original function, that of an extremely potent vascular permeability factor (VPF). In vivo VPF/VEGF increases microvascular permeability such that whole plasma leaks into the tissues where it undergoes clotting by tissue factor that is expressed on tumor and host connective tissue cells to deposit fibrin and generate serum. By providing tissue support and by reprogramming the gene expression patterns of cells locally, fibrin and serum can together account for the formation of vascular connective tissue stroma. In sum, by increasing vascular permeability, VPF/VEGF triggers the “wound healing response,” setting in motion a fundamental pathophysiological process that induces the mature stroma that is found not only in healing wounds but also in solid tumors and chronic inflammatory diseases. Once initiated by increased vascular permeability, this response may be difficult to impede, perhaps contributing to the limited success of anti-VEGF therapies in treating cancer.

Increased serum peripheral C-reactive protein is associated with reduced small-molecule brain perfusion in healthy volunteers and subjects with major depressive disorder

ABSTRACTThe relationship between peripheral and central immunity and how these ultimately may cause depressed behaviour has been the focus of a number of imaging studies conducted with Positron Emission Tomography (PET). These studies aimed at testing the immune-mediated model of depression that proposes a direct effect of peripheral cytokines and immune cells on the brain to elicit a neuroinflammatory response via a leaky blood-brain barrier and ultimately depressive behaviour. However, studies conducted so far using PET radioligands targeting the neuroinflammatory marker 18 kDa translocator protein (TSPO) in patient cohorts with depression have demonstrated mild inflammatory brain status but no correlation between central and peripheral immunity.To gain a better insight into the relationship between heightened peripheral immunity and neuroinflammation, we estimated blood-to-brain and blood-to-CSF perfusion rates for two TSPO radiotracers collected in two separate studies, one large cross-sectional study of neuroinflammation in normal and depressed cohorts and a second study where peripheral inflammation in healthy controls was induced via subcutaneous injection of interferon (IFN)-α. In both studies we observed a consistent negative association between peripheral inflammation, measured with c-reactive protein P (CRP), and radiotracer perfusion into and from the brain parenchyma and CSF. Importantly, there was no association of this effect with the marker of BBB leakage S100β, that was unchanged, but there was an association between the reduction of tracer perfusion in volunteers injected with interferon (IFN)-α and VEGF, a potent vascular permeability factor.These results support a different model of peripheral-to-central immunity interaction whereas peripheral inflammation causes a “stiffening” of the healthy BBB with consequent reduction of small molecule trafficking to and from the blood into the brain and CSF. This effect, on the long term, is likely to disrupt brain homeostasis and induce depressive behavioural symptoms. Moreover, given the molecular similarity between the TSPO ligands and antidepressant, this phenomenon may underlie treatment resistance in depressive cohorts with heightened peripheral status.

Several reasons for the development of proteinuria in nephrotic syndrome

The review discusses some of the causes of proteinuria in nephrotic syndrome due to extrarenal mechanisms. Autoantibodies identified in recent years are involved in the violation of the selective permeability of the filtration barrier in membranous nephropathy. The direct relationship between the level of hyperglycemia and proteinuria in diabetic nephropathy is analyzed. The role of reactive oxygen species, end products of glycation, angiotensin II, transforming growth factor β-1, epithelial-mesenchymal transformation of podocytes, Rho GTPases, intracellular signaling pathway mTOR, Wnt/β-catenin signaling cascade is emphasized. Particular attention is paid to the problem of searching and identifying circulating permeability factors in the pathogenesis of idiopathic nephrotic syndrome in patients with minimal changes and focal segmental glomerulosclerosis: vascular permeability factor (VPF), vasodilator-stimulated phosphoprotein (VASP), soluble hemopexin (Hpx) receptor-receptor-receptor type (suPAR), cardiotropin-like cytokine-1 (CLCF-1) and anti-CD40 antibodies. It is noted that the role of such factors is not in doubt today, however, from the standpoint of evidence-based medicine, this role needs serious confirmation by specially formulated criteria.

Synchronous inhibition of mTOR and VEGF/NRP1 axis impedes tumor growth and metastasis in renal cancer

Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of mTOR and the disruption of VEGF/NRP1 axis, respectively. We also exploited a liposomal formulation decorated with a proprietary tumor-targeting-peptide (TTP) to simultaneously deliver these two agents in a tumor-targeted manner. The TTP-liposomes encapsulating both Everolimus and EG00229 (EG-L) demonstrated higher in vitro and in vivo growth retardation than the single drug-loaded liposomes (E-L and G-L) in two different ccRCC models and led to a noticeable reduction in lung metastasis in vivo. In addition, EG-L displayed remarkable inhibition of tumor growth in a highly aggressive syngeneic immune-competent mouse model of ccRCC developed in Balb/c mice. Taken together, this study demonstrates an effective approach to achieve improved therapeutic outcome in ccRCC.

Correlation of VEGF with immune suppression involving MDSC, malnutrition, and prognosis in patients with gastric and colorectal cancer.

582 Background: Although a causal relationship for inflammation and immunity of cancer is more widely accepted today, the precise cell mechanisms mediating this relationship have not been elucidated. Vascular endothelial growth factor (VEGF), previously known as vascular permeability factor. 45 kDa protein, belongs to a family of platelet-derived growth factors. VEGF, inflammation-related protein, could contribute to the accumulation of immunosuppressive cells (MDSC, Treg, TAM, and Tie-2-expressingmonocytes) in tumor-bearing hosts through direct or indirect mechanisms. Methods: We tested the serum levels of VEGF by ELISA in 106 patients including 43 with gastric and 63 with colorectal cancer, and they were increased in advanced stages of gastric and colorectal cancer. Production of IL-17, pro-inflammatory cytokine, with a stimulation of PHA was measured by ELISA and MDSC (myeloid-derived suppressor cells), one of major immunosuppressing cells, was measured by flow cytometry (CD11b+CD14-CD33+). Neutrophil to lymphocyte ratio (NLR) and C-reactive protein (CRP) were used as inflammatory markers. Production of IL-12 and SI (stimulation index) of blastogenic response of lymphocytes were used as markers of cell-mediated immune response. Results: The concentrations of VEGF were positively correlated with levels of MDSC, production of IL-17, NLR and CRP, and were inversely correlated with IL-12 production, SI and nutritional markers including prealbumin and retinol binding protein. The patients were both divided into two groups with a serum level of VEGF (330 pg/ml) and OS (overall survival) of patients with stages III and IV gastric or colorectal cancer were both significantly worse in patients with high levels of VEGF than in those with low VEGF although the differences were not significant in patients with stagesⅠand II. Conclusions: The results of the present study suggested that VEGF may have an impact on advancement and progression involving inflammation and serve as useful markers of the immune suppression involving MDSC, malnutrition and poor prognosis in patients with gastric and colorectal cancer.