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Regulating VEGF signaling in platelet concentrates via specific VEGF sequestering.

ABSTRACT: Platelets contain an abundance of growth factors that mimic the composition of the wound healing milieu, and platelet-derived VEGF in particular can negatively influence wound healing if unregulated. Here, we sought to capture and regulate the activity of VEGF factor from human platelets using poly(ethylene glycol) microspheres. In this communication, we demonstrate that platelet freeze/thaw produced significantly higher levels of Vascular Endothelial Growth Factor (VEGF) than either calcium chloride treatment, protease activated receptor 1 activating peptide (PAR1AP) treatment, or thrombin treatment. PEG microspheres containing a VEGF-binding peptide (VBP), derived from VEGFR2, sequestered VEGF from platelet concentrate, prepared via freeze/thaw, and reduced the bioactivity of platelet concentrate in HUVEC culture, which suggests that VBP microspheres sequestered and reduced the activity of VEGF from patient-derived platelets. Here, we demonstrate the ability of VEGF sequestering microspheres to regulate the activity of VEGF derived from a growth factor-rich autologous human blood product.

SUBMITTER: Belair DG

PROVIDER: S-EPMC4846557 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Regulating VEGF signaling in platelet concentrates via specific VEGF sequestering.

Belair David G DG Le Ngoc Nhi NN Murphy William L WL

Biomaterials science 20160324 5

Platelets contain an abundance of growth factors that mimic the composition of the wound healing milieu, and platelet-derived VEGF in particular can negatively influence wound healing if unregulated. Here, we sought to capture and regulate the activity of VEGF factor from human platelets using poly(ethylene glycol) microspheres. In this communication, we demonstrate that platelet freeze/thaw produced significantly higher levels of Vascular Endothelial Growth Factor (VEGF) than either calcium chl ...[more]

PMID: 27010034

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Project description:BackgroundPlatelets are anucleated blood cells and contain various RNA species. We investigated the changes in the whole transcriptome expression profile of platelet concentrates (PC) during storage to explore biological functions and biomarkers in platelet storage damage.Materials and methodsPlatelets were collected by apheresis from eight healthy blood donors and stored from day 0 to day 4. Platelet phenotyping and function analysis were used to detect platelet activity during storage. RNA-sequencing was used to detect changes in expression of mRNA, lncRNA and circRNA in the PC during storage. Gene ontology and KEGG analyses were applied to predict the functional distribution of differential expression of mRNA. Gene set enrichment analysis was used to analyze the differential levels of gene pathways. Finally, polymerase chain reaction (PCR) analysis was performed to verify the expression of three mRNA (POLE2, DCUN1D4, DAD1).ResultsIn total, 10,767 mRNA, 2,923 lncRNA and 68,550 circRNA were detected in the PC by RNA-sequencing. The expression levels of 222 mRNA changed significantly from day 0 to day 4 of storage: 58 increased continuously and 145 decreased continuously. Differentially expressed mRNA may be involved in physiological processes such as platelet activation, platelet aggregation, endocytosis, and apoptosis. Expression levels of 1,413 lncRNA were obvious. The levels of 42 species increased and the levels of 28 species decreased. The expression levels of 198 species of circRNA changed significantly, with those of 13 species changing continuously. The differential levels of expression of DAD1, DCUN1D4 and POLE1 mRNA, shown by RNA sequencing, were validated by PCR assay.DiscussionChanges in mRNA, lncRNA and circRNA during platelet storage may be closely related to platelet apoptosis and physiological functions in the platelet storage lesion. The expression levels of DAD1, DCUN1D4 and POLE1 could be biomarkers to monitor platelet status in PC bags.

Project description:Background:Cisplatin is one of the first-line drugs for urothelial bladder cancer (UBC) treatment. However, its considerable side effects and the emergence of drug resistance are becoming major limitations for its application. This study aimed to investigate whether matrine and cisplatin could present a synergistic anti-tumor effect on UBC cells. Methods:Cell viability assay was used to assess the suppressive effect of matrine and cisplatin on the proliferation of the UBC cells. Wound healing assay and transwell assay were applied respectively to determine the migration and invasion ability of the cells. The distribution of cell cycles, the generation of reactive oxygen species (ROS) and the apoptosis rate were detected by flow cytometry (FCM). The expressions of the relative proteins in apoptotic signal pathways and the epithelial-mesenchymal transition (EMT) related genes were surveyed by western blotting. The binding modes of the drugs within the proteins were detected by CDOCKER module in DS 2.5. Results:Both matrine and cisplatin could inhibit the growth of the UBC cells in a time- and dose-dependent manner. When matrine combined with cisplatin at the ratio of 2000:1, they presented a synergistic inhibitory effect on the UBC cells. The combinative treatment could impair cell migration and invasion ability, arrest cell cycle in the G1 and S phases, increase the level of ROS, and induce apoptosis in EJ and T24 cells in a synergistic way. In all the treated groups, the expressions of E-cadherin, ?-catenin, Bax, and Cleaved Caspase-3 were up-regulated, while the expressions of Fibronectin, Vimentin, Bcl-2, Caspase-3, p-Akt, p-PI3K, VEGFR2, and VEGF proteins were down-regulated, and among them, the combination of matrine and cisplatin showed the most significant difference. Molecular docking algorithms predicted that matrine and cisplatin could be docked into the same active sites and interact with different residues within the tested proteins. Conclusions:Our results suggested that the combination of matrine and cisplatin could synergistically inhibit the UBC cells' proliferation through down-regulating VEGF/PI3K/Akt signaling pathway, indicating that matrine may serve as a new option in the combinative therapy in the treatment of UBC.

Project description:BackgroundPrevious studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage.Study design and methodsThe optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference.ResultsBoth PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis.ConclusionPACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor-, time- and cost efficiency.

Project description:Hyperglycaemia-induced retinal microvascular endothelial cell apoptosis is a critical and principle event in diabetic retinopathy (DR), which involves a series of complex processes such as mitochondrial dysfunction and oxidative stress. Ginsenoside Re (Re), a key ingredients of ginseng, is considered to have various pharmacologic functions, such as antioxidative, inhibition of inflammation and anti-apoptotic properties. However, the effects of Re in DR and the related mechanisms of endothelial cell injury induced by high glucose (HG) exposure remain unclear. The present study was designed to investigate and evaluate the ability of Re to ameliorate HG-induced retinal endothelial RF/6A cell injury and the potential mechanisms involved in the hypoxia-inducible factor-1-alpha (HIF-1?)/vascular endothelial growth factor (VEGF) signaling regulated by phosphoinositide 3-kinase (PI3K)/AKT pathway. Our results showed that preincubation with Re exerted cytoprotective effects by reversing the HG-induced decrease in RF/6A cell viability, downregulation of apoptosis rate and inhibition of oxidative-related enzymes, thereby reducing the excess intracellular reactive oxygen species (ROS) and HG-triggered RF/6A cell injury. In addition, Western blot analysis results showed ginsenoside Re significantly increased HIF-1? expression in the cytoplasm but decreased its expression in the nucleus, suggesting that it reduced the translocation of HIF-1? from the cytoplasm to the nucleus, and downregulated VEGF level. Moreover, this effect is involved in the activation of the PI3K/Akt pathway. LY294002, a PI3K inhibitor, was used to block the Akt pathway. Afterwards, the effects of Re on the regulation of apoptotic related proteins, VEGF and HIF-1? nuclear transcription was partially reversed. These findings suggested the exerting protective effects of ginsenoside Re were associated with regulating of PI3K/AKT and HIF-1?/VEGF signaling pathway, which indicates that ginsenoside Re may ameliorates HG-induced retinal angiogenesis and suggests the potential for the development of Re as a therapeutic for DR.

Dataset Information

Regulating VEGF signaling in platelet concentrates via specific VEGF sequestering.

Publications

Regulating VEGF signaling in platelet concentrates via specific VEGF sequestering.

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