VEGF expression disparities in brainstem motor neurons of the SOD1G93A ALS model: Correlations with neuronal vulnerability.

Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease characterized by severe muscle weakness mainly due to degeneration and death of motor neurons. A peculiarity of the neurodegenerative processes is the variable susceptibility among distinct neuronal populations, exemplified by the contrasting resilience of motor neurons innervating the ocular motor system and the more vulnerable facial and hypoglossal motor neurons. The crucial role of vascular endothelial growth factor (VEGF) as a neuroprotective factor in the nervous system is well-established since a deficit of VEGF has been related to motoneuronal degeneration. In this study, we investigated the survival of ocular, facial, and hypoglossal motor neurons utilizing the murine SOD1G93A ALS model at various stages of the disease. Our primary objective was to determine whether the survival of the different brainstem motor neurons was linked to disparate VEGF expression levels in resilient and susceptible motor neurons throughout neurodegeneration. Our findings revealed a selective loss of motor neurons exclusively within the vulnerable nuclei. Furthermore, a significantly higher level of VEGF was detected in the more resistant motor neurons, the extraocular ones. We also examined whether TDP-43 dynamics in the brainstem motor neuron of SOD mice was altered. Our data suggests that the increased VEGF levels observed in extraocular motor neurons may potentially underlie their resistance during the neurodegenerative processes in ALS in a TDP-43-independent manner. Our work might help to better understand the underlying mechanisms of selective vulnerability of motor neurons in ALS.

Chitosan bioactive glass scaffolds for in vivo subcutaneous implantation, toxicity assessment, and diabetic wound healing upon animal model.

This study aims to develop chitosan-bioactive glass (BG) scaffolds for diabetic wound healing, toxicity valuation, and subcutaneous implantation in animals for biocompatibility assessment. The scaffolds were prepared by lyophilization technique. In specific BG without sodium (Na), composited with chitosan for better biological activities. The equipped scaffolds were studied for their physiochemical, biological, in vitro and in vivo performances. The chitosan and chitosan-BG (Na free) scaffolds show reliable biocompatibility, cytocompatibility, anti-oxidant, and tissue regeneration. The biocompatibility, toxicity assessments, and diabetic skin wound healing experiments were examined through in vivo studies using Sprague Dawley rats. The extracted tissue samples were analyzed using hematoxylin-eosin- (H and E) and Masson's trichrome staining. Further, tissue excised after scaffold implantation declared non-toxic, non-allergic, and anti-inflammatory nature of chitosan scaffolds. Moreover, the total ribonucleic acid (RNA) expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) for the scaffolds against vascular endothelial growth factor (VEGF), and collagen type one (Col-1) primers. Admirably, the scaffolds achieved the best level of skin wound healing via tissue regeneration by increasing epithetical cell formation and collagen deposition. Thus, the biocompatibility, non-toxicity, anti-inflammatory, and wound healing efficiency proved that the chitosan-BG (Na free) scaffold can be readily substantial for wound healing.

Computational assessment of pigment epithelium-derived factor as an anti-cancer protein during its interaction with the receptors.

Pigment epithelium-derived factor (PEDF) is a member of the serine proteinase inhibitor (serpin) with antiangiogenic, anti-tumorigenic, antioxidant, anti-atherosclerosis, antithrombotic, anti-inflammatory, and neuroprotective properties. The PEDF can bind to low-density lipoprotein receptor-related protein 6 (LRP6), laminin (LR), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), and ATP synthase ß-subunit receptors. In this study, we aimed to investigate the structural basis of the interaction between PEDF and its receptors using bioinformatics approaches to identify the critical amino acids for designing anticancer peptides. The human ATP synthase ß-subunit was predicted by homology modeling. The molecular docking, molecular dynamics (MD) simulation, and Molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) were used to study this protein-receptor complex. The molecular docking showed PEDF could bind to the Laminin and VEGFR2 much stronger than ATP synthase ß-subunit, VEGFR1, and LRP6. The PEDF could effectively interact with various receptors during the simulation. The N-terminal of PEDF has an important role in the interaction with the receptors. The MM/PBSA showed the electrostatic (ΔEElec) and van der Waals interactions (ΔEVdW) contributed positively to the binding process of the complexes. The critical amino acids in the binding interaction of PEDF to its receptors in the MD simulation were determined. The interaction mode of 34-mer PEDF to laminin, VEGFR2, and LRP6 were different from VEGFR1, ATP synthase ß-subunit. The 34-mer PEDF has an important role in the interaction with different receptors and these critical amino acids can be used for designing peptides for future therapeutic aims.Communicated by Ramaswamy H. Sarma.

Quantitative Assessment of the Apical and Basolateral Membrane Expression of VEGFR2 and NRP2 in VEGF-A-stimulated Cultured Human Umbilical Vein Endothelial Cells.

Endothelial cells (ECs) form a precisely regulated polarized monolayer in capillary walls. Vascular endothelial growth factor-A (VEGF-A) induces endothelial hyperpermeability, and VEGF-A applied to the basolateral side, but not the apical side, has been shown to be a strong barrier disruptor in blood-retinal barrier ECs. We show here that VEGF-A presented to the basolateral side of human umbilical vein ECs (HUVECs) induces higher permeability than apical stimulation, which is similar to results obtained with bovine retinal ECs. We investigated with immunocytochemistry and confocal imaging the distribution of VEGF receptor-2 (VEGFR2) and neuropilin-2 (NRP2) in perinuclear apical and basolateral membrane domains. Orthogonal z-sections of cultured HUVECs were obtained, and the fluorescence intensity at the apical and basolateral membrane compartments was measured. We found that VEGFR2 and NRP2 are evenly distributed throughout perinuclear apical and basolateral membrane compartments in unstimulated HUVECs grown on Transwell inserts, whereas basolateral VEGF-A stimulation induces a shift toward basolateral VEGFR2 and NRP2 localization. When HUVECs were grown on coverslips, the distribution of VEGFR2 and NRP2 across the perinuclear apical and basolateral membrane domains was different. Our findings demonstrate that HUVECs dynamically regulate VEGFR2 and NRP2 localization on membrane microdomains, depending on growth conditions and the polarity of VEGF-A stimulation.

Dabigatran and Wet AMD, Results From Retinal Pigment Epithelial Cell Monolayers, the Mouse Model of Choroidal Neovascularization, and Patients From the Medicare Data Base.

Background: Age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Caucasian populations, includes destruction of the blood-retina barrier (BRB) generated by the retinal pigment epithelium-Bruch's membrane complex (RPE/BrM), and complement activation. Thrombin is likely to get access to those structures upon BRB integrity loss. Here we investigate the potential role of thrombin in AMD by analyzing effects of the thrombin inhibitor dabigatran. Material and Methods: MarketScan data for patients aged ≥65 years on Medicare was used to identify association between AMD and dabigatran use. ARPE-19 cells grown as mature monolayers were analyzed for thrombin effects on barrier function (transepithelial resistance; TER) and downstream signaling (complement activation, expression of connective tissue growth factor (CTGF), and secretion of vascular endothelial growth factor (VEGF)). Laser-induced choroidal neovascularization (CNV) in mouse is used to test the identified downstream signaling. Results: Risk of new wet AMD diagnosis was reduced in dabigatran users. In RPE monolayers, thrombin reduced TER, generated unique complement C3 and C5 cleavage products, led to C3d/MAC deposition on cell surfaces, and increased CTGF expression via PAR1-receptor activation and VEGF secretion. CNV lesion repair was accelerated by dabigatran, and molecular readouts suggest that downstream effects of thrombin include CTGF and VEGF, but not the complement system. Conclusions: This study provides evidence of association between dabigatran use and reduced exudative AMD diagnosis. Based on the cell- and animal-based studies, we suggest that thrombin modulates wound healing and CTGF and VEGF expression, making dabigatran a potential novel treatment option in AMD.

Expression of Long Non-Coding RNA (lncRNA) SNHG5 in Patients with Refractory Diabetic Macular Edema and Its Regulatory Mechanism.

BACKGROUND The aim of this study was to assess use of lncRNAs as biomarkers in serum and aqueous humor of patients with diabetic macular edema (DME). MATERIAL AND METHODS Optical coherence tomography and fundus photography were used to analyze the retinal features of the patients. RT-qPCR was used to analyze the differential expression of lncRNA snhg5 in patients who have idiopathic macular hole (MH), DME, or refractory DME. The relationship between SNHG5 and the clinical characteristics of the patients was analyzed. The effect of SNHG5 on the hyperplasia and apoptosis of human retino-microvascular endothelial cells (HRMECs) and its mechanism were analyzed in vitro. RESULTS Patients with idiopathic MH developed retinal nerve epithelium rupture and retinal fundus thickening, and patients with DME or refractory DME showed significant macular edema with hemorrhaging. The refractory DME patients improved after treatment but still showed significant macular edema and multiple laser scarring. SNHG5 expression was not only low in the atrial fluid and plasma in DME patients, but also lower in the refractory DME group compared to the idiopathic MH patients. SNHG5 expression in the aqueous humor and plasma was negatively correlated with disease duration, body mass index, and levels of fasting blood glucose, glycated hemoglobin, proteinuria, and glycosuria. In the in vitro experiments, SNHG5 expression was significantly downregulated in high glucose-induced HMECs. After SNHG5 overexpression, cell proliferation, angiogenesis, and VEGF-A protein levels were distinctly downregulated. CONCLUSIONS SNHG5 correlates with the development of DME and is a potential target for therapy.

Functional In Vitro Assessment of VEGFA/NOTCH2 Signaling Pathway and pRB Proteasomal Degradation and the Clinical Relevance of Mucolipin TRPML2 Overexpression in Glioblastoma Patients.

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients.

The Assessment of the Combined Treatment of 5-ALA Mediated Photodynamic Therapy and Thalidomide on 4T1 Breast Carcinoma and 2H11 Endothelial Cell Line.

Photodynamic therapy (PDT) is a low-invasive method of treatment of various diseases, mainly neoplastic conditions. PDT has been experimentally combined with multiple treatment methods. In this study, we tested a combination of 5-aminolevulinic acid (5-ALA) mediated PDT with thalidomide (TMD), which is a drug presently used in the treatment of plasma cell myeloma. TMD and PDT share similar modes of action in neoplastic conditions. Using 4T1 murine breast carcinoma and 2H11 murine endothelial cells lines as an experimental tumor model, we tested 5-ALA-PDT and TMD combination in terms of cytotoxicity, apoptosis, Vascular Endothelial Growth Factor (VEGF) expression, and, in 2H11 cells, migration capabilities by wound healing assay. We have found an enhancement of cytotoxicity in 4T1 cells, whereas, in normal 2H11 cells, this effect was not statistically significant. The addition of TMD decreased the production of VEGF after PDT in 2H11 cell line. Our results reveal enhanced effectiveness of 5-ALA-PDT with TMD treatment compared to 5-ALA-PDT or TMD treatment alone. The addition of TMD may be a promising proceeding of the anti-tumor effect of PDT by decreasing the VEGF concentration in the culture medium. Further studies, including testing on different cell lines, are needed to confirm this assumption.

Ethnic disparities in demographic, clinicopathologic and biological behaviours and prognosis of gastric cancer in northwest China.

This retrospective study aimed to investigate ethnic disparities in demographic, clinicopathologic, and biological behaviours of gastric cancer (GC) in a high GC incidence area of China. There were 5022 GC patients, including 3987 Han (79.4%) and 987 Hui (14.4%) patients from Northwest China. All patient data were retrieved from 2009 to 2017. Median survival was estimated using the Kaplan-Meier method and compared using the log-rank test. A Cox proportional hazards model was used to assess the impact of covariates. Similarly, low 5-year OS rates were observed in both the Hui and Han groups (23.8% and 24.2% respectively). Hui patients with stage T1 or N0 or with tumours <5 cm had 2.144-fold, 1.426-fold and 1.305-fold increased risks of poor prognosis compared with Han patients with these characteristics respectively (all p < 0.05). Further, Hui patients had 1.265-fold, 1.364-fold and 1.401-fold increased risks of poor prognosis compared with Han patients among those with high expression of Ki67, EGFR and VEGF respectively (all p < 0.05). There are ethnic disparities in the prognosis of GC patients in Northwest China. Understanding the effects of ethnicity on GC will guide reasonable evaluations of prognosis and future interventions to equalise access to high-quality care for GC patients of different ethnicities in China.

Assessment of the Molecular Mechanism of Action of SB3, a Trastuzumab Biosimilar.

BACKGROUND: SB3, a biosimilar of Herceptin® (trastuzumab, hereinafter referred to as reference product) is currently approved in the EU, Korea, Australia, the USA, and Brazil for the treatment of human epidermal growth factor receptor (HER) 2-positive early and metastatic breast cancer and HER2-positive metastatic gastric cancer. Previously, the biological similarity of SB3 to EU- or US-sourced reference product was assessed using various cell-based and binding assays. OBJECTIVE: In this paper, as a part of its similarity assessment, SB3 was evaluated for additional characteristics related to its molecular mechanism of action (MoA). METHODS: For extracellular effects of SB3, HER2-overexpressing cancer cell lines were used to assess expression of surface HER2, shedding of the extracellular domain of HER2, and antibody-dependent cell-mediated phagocytosis (ADCP) activity. For intracellular effects, Akt phosphorylation and vascular endothelial growth factor (VEGF) release were assessed. Additionally, in vitro docetaxel or pertuzumab combination experiments were performed for further characterization; anti-proliferation, HER2/HER3 dimerization inhibition, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays were used. RESULTS: It was confirmed that SB3 is highly similar to the reference products on quality attributes related to extracellular/intracellular efficacy. This similarity was also confirmed during combination studies with docetaxel and pertuzumab. CONCLUSION: Overall, the equivalence of SB3 with reference product in MoA-related qualities in in vitro mono- and combination therapy experiments may support clinical bioequivalence of the two substances.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass-spectrometry-based approach.

The ongoing shift from small molecule drugs to protein therapeutics in the pharmaceuticals industry presents a considerable challenge to generic drug developers who are increasingly required to demonstrate biosimilarity for biological macromolecules, a task that is decidedly more complex than doing the same for small molecule drugs. In this work, we demonstrate a multipronged mass-spectrometry-based workflow that allows rapid and facile molecular characterization of antibody-based protein therapeutics, applied to biosimilars development. Specifically, we use a combination of native mass spectrometry (MS), ion mobility spectrometry (IMS), and global time-resolved hydrogen deuterium exchange (HDX) to provide an unambiguous assessment of the structural, dynamic, and chemical similarity between Avastin (bevacizumab) and a biosimilar in the late stages of pre-clinical development. Minor structural and dynamic differences between the biosimilar and Avastin, and between lots of the biosimilar, were tested for functional relevance using Surface Plasmon Resonance-derived kinetic and equilibrium binding parameters.

Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs.

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 105 cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 105 cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K+, Cl- and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

Assessment of Anti-carcinogenic Effect of Pomegranate in Oral Squamous Cell Carcinoma (Pre-clinical Study).

BACKGROUND AND OBJECTIVE: The survival portion of patients treated from oral cancer isn't in progress. This is why researchers are continuously looking for new anti-cancer drugs either natural product or natural product derivatives. Studies have shown that pomegranate and its constituents might have an anti-tumorigenic effect. So the aim of this study was to assess the anticarcinogenic roles of pomegranate on oral squamous cell carcinoma as an adjuvant of chemotherapy. MATERIALS AND METHODS: The Hep-2 cells were propagated and maintained under basic culture media. Cells were grouped according to culture media and each group was tested for cell proliferation as well as VEGF expression and caspase-3 expressions using ELISA and RT-PCR, respectively. RESULTS: Regarding cell proliferation and VEGF expression, a higher mean value was recorded in group 1 in comparison to group 3 with a significant difference (p = 0.001) and in group 2 in comparison to group 4, with a significant difference (p = 0.049). While concerning caspase-3 expression, a higher mean value was recorded in group 3 in comparison to group 1 with a significant difference (p = 0.045) and in group 4 in comparison to group 2, with a significant difference (p = 0.00). CONCLUSION: Pomegranate can be an adjuvant, natural product for oral cancer treatment in combination with 5-fluorouracil to reduce its dose and nullify its toxic side effects on normal body organs.

MRI assessment of the bone adjacent to giant cell tumours and its association with local recurrence after intralesional curettage.

AIM: To assess the tumour border surrounding giant cell tumour of the bone (GCTB) using magnetic resonance imaging (MRI) and investigate its association with local recurrence. MATERIALS AND METHODS: Sixty-nine GCTBs in proximal tibiae and distal femurs were studied. The pathological basis of the paintbrush border sign was explored. Expression of Ki-67, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), receptor activator of nuclear factor-κ B (RANK), and RANK ligand (RANKL) in GCTBs were investigated using immunohistochemistry. Patients treated with intralesional curettage were analysed retrospectively to investigate the prognostic role of the paintbrush border sign. The differences between rates were tested using the chi-square test or Fisher's exact test, as appropriate. RESULTS: The paintbrush border sign correlated well with infiltrative margins. The expression of MMP-9 was associated with the paintbrush border sign, and positively correlated with RANKL and VEGF expression. GCTBs with the paintbrush border sign had a higher rate of local recurrence (76.19 versus 20.59%, p<0.05). The paintbrush border sign was more common in proximal tibiae, and positively correlated with cystic change. The paintbrush border signs were detected at T1-weighted imaging, but the sign was only evident in four cases on T2-weighted imaging. CONCLUSION: Pathologically, the paintbrush border sign correlates well with invasion of the bone around GCTB. MMP-9 might play a key role in the formation of penetrating irregular margins. The paintbrush border sign is revealed as a risk factor for local recurrence of GCTB. Sagittal T1-weighted imaging is crucial to diagnose the paintbrush border sign.

Quantifying the phosphorylation timescales of receptor-ligand complexes: a Markovian matrix-analytic approach.

Cells interact with the extracellular environment by means of receptor molecules on their surface. Receptors can bind different ligands, leading to the formation of receptor-ligand complexes. For a subset of receptors, called receptor tyrosine kinases, binding to ligand enables sequential phosphorylation of intra-cellular residues, which initiates a signalling cascade that regulates cellular function and fate. Most mathematical modelling approaches employed to analyse receptor signalling are deterministic, especially when studying scenarios of high ligand concentration or large receptor numbers. There exist, however, biological scenarios where low copy numbers of ligands and/or receptors need to be considered, or where signalling by a few bound receptor-ligand complexes is enough to initiate a cellular response. Under these conditions stochastic approaches are appropriate, and in fact, different attempts have been made in the literature to measure the timescales of receptor signalling initiation in receptor-ligand systems. However, these approaches have made use of numerical simulations or approximations, such as moment-closure techniques. In this paper, we study, from an analytical perspective, the stochastic times to reach a given signalling threshold for two receptor-ligand models. We identify this time as an extinction time for a conveniently defined auxiliary absorbing continuous time Markov process, since receptor-ligand association/dissociation events can be analysed in terms of quasi-birth-and-death processes. We implement algorithmic techniques to compute the different order moments of this time, as well as the steady-state probability distribution of the system. A novel feature of the approach introduced here is that it allows one to quantify the role played by each kinetic rate in the timescales of signal initiation, and in the steady-state probability distribution of the system. Finally, we illustrate our approach by carrying out numerical studies for the vascular endothelial growth factor and one of its receptors, the vascular endothelial growth factor receptor of human endothelial cells.

Transient Overexpression of Vascular Endothelial Growth Factor A in Adipose Tissue Promotes Energy Expenditure via Activation of the Sympathetic Nervous System.

Adipose-derived vascular endothelial growth factor A (VEGF-A) stimulates functional blood vessel formation in obese fat pads, which in turn facilitates healthy expansion of the adipose tissue. However, the detailed mechanism(s) governing the process remains largely unknown. Here, we investigated the role of sympathetic nervous system activation in the process. To this end, we induced overexpression of VEGF-A in an adipose tissue-specific doxycycline (Dox)-inducible transgenic mouse model for a short period of time during high-fat diet (HFD) feeding. We found that local overexpression of VEGF-A in adipose tissue stimulated lipolysis and browning rapidly after Dox induction. Immunofluorescence staining against tyrosine hydroxylase (TH) indicated higher levels of sympathetic innervation in adipose tissue of transgenic mice. In response to an increased norepinephrine (NE) level, expression of ß3-adrenoceptor was significantly upregulated, and the downstream protein kinase A (PKA) pathway was activated, as indicated by enhanced phosphorylation of whole PKA substrates, in particular, the hormone-sensitive lipase (HSL) in adipocytes. As a result, the adipose tissue exhibited increased lipolysis, browning, and energy expenditure. Importantly, all of these effects were abolished upon treatment with the ß3-adrenoceptor antagonist SR59230A. Collectively, these results demonstrate that transient overexpressed VEGF-A activates the sympathetic nervous system, which hence promotes lipolysis and browning in adipose tissue.

Inhibition of VEGF-Flk-1 binding induced profound biochemical alteration in the hippocampus of a rat model of BBB breakdown by spider venom. A preliminary assessment using FT-IR spectroscopy.

Phoneutria nigriventer spider venom (PNV) contains ion channels-acting neuropeptides that in rat induces transitory blood-brain barrier breakdown (BBBb) in hippocampus in parallel with VEGF upregulation. We investigated whether VEGF has a neuroprotective role by inhibiting its binding to receptor Flk-1 by itraconazole (ITZ). FT-IR spectroscopy examined the biochemical status of hippocampus and evaluated BBBb in rats administered PNV or ITZ/PNV at periods with greatest toxicity (1-2h), recovery (5h) and visual absence of symptoms (24h), and compared to saline and ITZ controls. The antifungal treatment before venom intoxication aggravated the venom effects and increased BBB damage. FT-IR spectra of venom, hippocampi of controls, PNV and ITZ-PNV showed a 1400 cm-1 band linked to symmetric stretch of carboxylate and 1467 cm-1 band (CH2 bending: mainly lipids) that were considered biomarker and reference bands, respectively. Inhibition of VEGF/Flk-1 binding produced marked changes in lipid/protein stability at 1-2h. The largest differences were observed in spectra regions assigned to lipids, both symmetric (2852 cm-1) and asymmetric (2924 and 2968 cm-1). Quantitative analyses showed greatest increases in the 1400 cm-1/1467 cm-1 ratio also at 1h. Such changes at period of rats' severe intoxication referred to wavenumber region from 3106 cm-1 to 687 cm-1 assigning for C-H and N-H stretching of protein, Amide I, C=N cytosine, N-H adenine, Amide II, CH2 bending: mainly lipids, C-O stretch: glycogen, polysaccharides, glycolipids, z-type DNA, C-C, C-O and CH out-of-plane bending vibrations. We conclude that VEGF has a neuroprotective role and can be a therapeutic target in PNV envenomation. FT-IR spectroscopy showed to be instrumental for monitoring biochemical changes in this model of P. nigriventer venom-induced BBB disruption.

Meningioma assessment: Kinetic parameters in dynamic contrast-enhanced MRI appear independent from microvascular anatomy and VEGF expression.

BACKGROUND AND PURPOSE: Kinetic parameters of T1-weighted dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) are considered to be influenced by microvessel environment. This study was performed to explore the extent of this association for meningiomas. MATERIALS AND METHODS: DCE-MRI kinetic parameters (contrast agent transfer constants Ktrans and kep, volume fractions vp and ve) were determined in pre-operative 3T MRI of meningioma patients for later biopsy sites (19 patients; 15 WHO Io, no previous radiation, and 4 WHO IIIo pre-radiated recurrent tumors). Sixty-three navigated biopsies were consecutively retrieved. Biopsies were immunohistochemically investigated with endothelial marker CD34 and VEGF antibodies, stratified in a total of 4383 analysis units and computationally assessed for VEGF expression and vascular parameters (vessel density, vessel quantity, vascular fraction within tissue [vascular area ratio], vessel wall thickness). Derivability of kinetic parameters from VEGF expression or microvascularization was determined by mixed linear regression analysis. Tissue kinetic and microvascular parameters were tested for their capacity to identify the radiation status in a subanalysis. RESULTS: Kinetic parameters were neither significantly related to the corresponding microvascular parameters nor to tissue VEGF expression. There was no significant association between microvessel density and its presumed correlate vp (P=0.07). The subgroup analysis of high-grade radiated meningiomas showed a significantly reduced microvascular density (AUC 0.91; P<0.0001) and smaller total vascular fraction (AUC 0.73; P=0.01). CONCLUSIONS: In meningioma, DCE-MRI kinetic parameters neither allow for a reliable prediction of tumor microvascularization, nor for a prediction of VEGF expression. Kinetic parameters seem to be determined from different independent factors.

A nephron model for study of drug-induced acute kidney injury and assessment of drug-induced nephrotoxicity.

In this study, we developed a multilayer microfluidic device to simulate nephron, which was formed by "glomerulus", "Bowman's capsule", "proximal tubular lumen" and "peritubular capillary". In this microdevice, artificial renal blood flow was circulating and glomerular filtrate flow was single passing through, mimicking the behavior of a nephron. In this dynamic artificial nephron, we observed typical renal physiology, including the glomerular size-selective barrier, glomerular basement membrane charge-selective barrier, glucose reabsorption and para-aminohippuric acid secretion. To demonstrate the capability of our microdevice, we used it to investigate the pathophysiology of drug-induced acute kidney injury (AKI) and give assessment of drug-induced nephrotoxicity, with cisplatin and doxorubicin as model drugs. In the experiment, we loaded the doxorubicin or cisplatin in the "renal blood flow", recorded the injury of primary glomerular endothelial cells, podocytes, tubular epithelial cells and peritubular endothelial cells by fluorescence imaging, and identified the time-dependence, dose-dependence and the death order of four types of renal cells. Then by measuring multiple biomarkers, including E-cadherin, VEGF, VCAM-1, Nephrin, and ZO-1, we studied the mechanism of cell injuries caused by doxorubicin or cisplatin. Also, we investigated the effect of BSA in the "renal blood flow" on doxorubicin-or-cisplatin-induced nephrotoxicity, and found that BSA enhanced the tight junctions between cells and eased cisplatin-induced nephrotoxicity. In addition, we compared the nephron model and traditional tubule models for assessment of drug-induced nephrotoxicity. And it can be inferred that our biomimetic microdevice simulated the complex, dynamic microenvironment of nephron, yielded abundant information about drug-induced-AKI at the preclinical stage, boosted the drug safety evaluation, and provided a reliable reference for clinical therapy.