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In this study, a simple and sensitive UHPLC-ESI-MS/MS method was established for the determination of LXH254 in rat plasma. The developed method was validated according to the Food and Drug administration guidelines. After extraction using ethyl acetate, the sample was separated on an ACQUITY BEH C18 column. The mobile phase consisted of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. The flow rate was 0.3 mL/min. A TSQ triple quadrupole mass spectrometer operated in positive-ion mode was used for mass detection, with multiple reaction monitoring transitions of m/z 503.3 > 459.1 and m/z 435.3 > 367.1 for LXH254 and olaparib (internal standard), respectively. An excellent linearity was achieved in the concentration range of 0.1-1000 ng/mL, with correlation coefficient >0.998. The mean recovery was more than 78.55%. Inter- and intra-day precision (percentage of relative standard deviation) did not exceed 12.87%, and accuracy was in the range of -2.50 to 13.50%. LXH254 was demonstrated to be stable under the tested storage conditions. The validated UHPLC-MS/MS method was further applied to the pharmacokinetic study of LXH254 in rat plasma after oral (2, 5, and 15 mg/kg) and intravenous (2 mg/kg) administrations. The pharmacokinetic study revealed that LXH254 showed low clearance, moderate bioavailability (~30%), and linear pharmacokinetic profile over the oral dose range of 2-15 mg/kg. To the best of our knowledge, this is the first report on the method development and validation of the determination of LXH254 and its application to pharmacokinetic study.

Background Despite advances in therapeutic strategies and critical care management, septic encephalopathy (SE) is still a leading cause of infection-associated death in intensive care units (ICUs). Vitexin, a flavonoids compound, exerts and anti-inflammatory effect through inhibition of proinflammatory cytokines and signaling pathways. This study aimed to explore the anti-inflammatory effects of vitexin in SE and the underlying mechanisms. Methods An SE-inducedC57BL/6 mouse model was established via cecal ligation and puncture (CLP). Western blotting was performed to evaluate the protein expression levels of Chemokine (C-X-C motif) ligand 1 (CXCL1), fractalkine (CX3CL1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, NF-κB p65, p-NF-κB p65, and tumor necrosis factor-α (TNF-α). Flow cytometry was used to detect the expressions ofCD11a/CD18, CD11b/CD18, ICAM-1, and adherent leukocyte. The expression of ICAM-1 was detected by immunohistochemistry. An enzyme-linked immunosorbent assay was performed to evaluate the expression of monocyte chemotactic protein-1 (MCP-1), Interleukin (IL)-6, IL-8, and IL-10. Results In this study, we found that vitexin significantly downregulated the expression of brain endothelial chemokines CXCL1 and CX3CL1 in CLP mice, exerting a potential anti-inflammatory against SE. Our data also showed that vitexin alleviated SE primarily by relying on reducing leukocyte-endothelial adhesion via the mediation of adhesion molecules. Moreover, vitexin suppressed the expression of proinflammatory cytokines, such as MCP-1, IL-6, IL-8, TNF-α, and NF-κB p65, in the CLP mice, while the expression of the anti-inflammatory cytokine IL-10 was elevated. Conclusions Overall, our study demonstrated the protective effect vitexin exerts in SE by reducing leukocyte-endothelial adhesion and inflammatory response. These findings offer a molecular basis for the potential application of vitexin in the treatment of SE and other inflammatory-mediated and immunemediated disorders.

The transcriptional coactivator with the PDZ-binding motif (TAZ) has been associated with different types of cancer. In this study, we examined the TAZ protein expression and cellular localization in 194 cases of human cervical squamous cell carcinoma (SCC). We observed that a normal cervix is characterized by higher expression levels of both nuclear and cytosolic TAZ compared to cervical SCC. Lower membranous and cytosolic TAZ expression levels are associated with lymph node involvement. We observed that TAZ expression levels are associated with β1 integrin and Src in SCC and cell lines derived from human cervical cancers. Of note, knock down of TAZ increased the expression of β1 integrin and Src in both normal and human cervical cancer cells. Our data indicate that the expression and cellular localization of TAZ are inversely associated with the development and progression of cervical SCC, and TAZ-mediated transcription may be involved in the activation of the integrin-Src signalling pathway.

Background Angiopoietin-2 (Ang-2), a ligand of the Tie-2 receptor, plays an important role in maintaining endothelial cells and in destabilizing blood vessels. Collateral artery growth (arteriogenesis) is a key adaptive response to arterial occlusion. It is unknown whether the destabilization of blood vessels by Ang-2 can affect arteriogenesis and modulate mononuclear cell function. This study aimed to investigate the effects of Ang-2 on collateral artery growth. Methods Hindlimb ischaemia model was produced in C57BL/6 mice by femoral artery ligation. Blood flow perfusion was measured using a laser Doppler perfusion imager quantitative RT-PCR analysis was applied to identify the level of angiogenic factors. Results After the induction of hindlimb ischaemia, blood flow recovery was impaired in mice treated with recombinant Ang-2 protein; this was accompanied by a reduction of peri-collateral macrophage infiltration. In addition, quantitative RT-PCR analysis revealed that Ang-2 treatment decreased monocyte chemotactic protein-1 (MCP-1), platelet-derived growth factor-BB (PDGF-BB) mRNA levels in ischaemic adductor muscles. Ang-2 can lead to macrophage M1/M2 polarization shift inhibition in the ischaemic muscles. Furthermore, Ang-2 reduced the in vitro inflammatory response in macrophages and vascular cells involved in arteriogenesis. Conclusions Our results demonstrate that Ang-2 is essential for efficient arteriogenesis, which controls macrophage infiltration. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1055-x) contains supplementary material, which is available to authorized users.

Background Currently, resistance to tyrosine kinase inhibitors, such as gefitinib, has become a major obstacle in improving the clinical outcome of patients with metastatic and advanced-stage esophageal squamous cell carcinoma (ESCC). While cell behavior can be modulated by long non-coding RNAs (lncRNAs), the roles of lncRNAs within extracellular vesicles (exosomes) are largely unknown. Therefore, we investigated the involvement and regulatory functions of potential lncRNAs enclosed in exosomes during formation of chemoresistance in human ESCC. Methods Gefitinib-resistant cell lines were established by continuously grafting TE1 and KYSE-450 cells into gefitinib-containing culture medium. LncRNA microarray assay followed by RT-qPCR were used to verify the differential expression of lncRNA Prostate Androgen-Regulated Transcript 1 (PART1) between gefitinib resistant and parental cell lines. RNA fluorescence in situ hybridization (FISH) was used to investigate whether extracellular PART1 could be incorporated into exosomes and transmitted to recipient cells. Subsequently, a series of in vitro assays and a xenograft tumor model were used to observe the functions of lncRNA PART1 in ESCC cells. A signal transduction reporter array, bioinformatics analysis, western blotting, and immunofluorescence were carried out to verify the regulation of PART1 and its downstream Bcl-2 signaling pathway. Results lncRNA PART1 was upregulated in gefitinib-resistant cells when compared to parental ESCC cells. It was found that STAT1 can bind to the promoter region of lncRNA PART1, resulting in its activation. Knockdown of lncRNA PART1 potently promoted the gefitinib-induced cell death, while elevated PART1 promoted gefitinib resistance by competitively binding to miR-129 to facilitate Bcl-2 expression in ESCC cells. In addition, extracellular PART1 could be incorporated into exosomes and transmitted to sensitive cells, thus disseminating gefitinib resistance. Clinically, high levels of serum lncRNA PART1 in exosome were associated with poor response to gefitinib treatment in ESCC patients. Conclusions LncRNA PART1 promotes gefitinib resistance by regulating miR-129/Bcl-2 pathway, and may serve as a therapeutic target for ESCC patients.

Table S1. Information of the RT-qPCR primer sequences and siRNA sequences. (DOC 47Â kb)

Figure S1. Knockdown of STAT1 suppressed expression of PART1. a RT-qPCR verification of the silence of STAT1 after transfection of specific small interfere RNAs. b RT-qPCR analysis of lncRNA PART1 expression after STAT1 was silenced. ***P

Table S2. Fold change of deregulated lncRNAs between gefitinib-resistant cells and -parental cells. (DOC 30Â kb)

Objective— Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 downregulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin-dependent cooperative binding interaction between VEGFR-2 and α V β 3 . Approach and Results— We studied effects of PAI-1 on VEGF signaling in human umbilical vein endothelial cells. PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in human umbilical vein endothelial cells grown on vitronectin, but not on fibronectin or collagen. PAI-1 inhibited the binding of VEGFR-2 to β 3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to vitronectin and another requiring binding to very low-density lipoprotein receptor. PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. Conclusions— PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a vitronectin-dependent proangiogenic binding interaction involving α V β 3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, vitronectin, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.

Background Coagulation factor V ( FV ) plays a key role in hemostasis, is present in plasma and platelets, and has both pro‐ and anticoagulant properties; however, the contribution of platelet‐derived FV to arterial thrombosis remains undetermined. Methods and Results Using transgenic mice with various levels of FV gene expression that was restricted to the plasma or platelets, the roles of platelet FV were evaluated in the regulation of arterial thrombosis and platelet activation. Mice with higher levels of platelet FV exhibited faster thrombotic occlusion of the carotid artery after injury compared with mice with lower platelet FV levels. Infusion of platelets with higher levels of FV into transgenic mice with undetectable levels of platelet FV reduced the time to carotid artery occlusion. In contrast, infusion of purified recombinant plasma FV into mice with undetectable platelet FV levels failed to reduce the carotid occlusion times following injury. Evaluation of isolated platelets revealed that platelet‐derived FV was critical for the regulation of platelet activation. These effects were associated with an increased level of expression of P‐selectin and increased cGMP in platelets. Conclusions We established that platelet‐derived FV is a critical mediator of arterial thrombosis that involves platelet activation.

Metformin, an anti-diabetic drug commonly used for type 2 diabetes therapy, is associated with anti-angiogenic effects in conditions beyond diabetes. miR-21 has been reported to be involved in the process of angiogenesis. However, the precise regulatory mechanisms by which the metformin-induced endothelial suppression and its effects on miR-21-dependent pathways are still unclear. Bioinformatic analysis and identification of miR-21 and its targets and their effects on metformin-induced antiangiogenic activity were assessed using luciferase assays, quantitative real-time PCR, western blots, scratch assays, CCK-8 assays and tubule formation assays. In this study, miR-21 was strikingly downregulated by metformin in a time- and dose-dependent manner. miR-21 directly targeted the 3′-UTR of PTEN and SMAD7, and negatively regulated their expression. Overexpression of miR-21 abrogated the metformin-mediated inhibition of endothelial cells proliferation, migration, tubule formation and the TGF-β-induced AKT, SMAD- and ERK-dependent phosphorylations, and conversely, down-regulation of miR-21 aggravated metformin’s action and revealed significant promotion effects. Our study broadens our understanding of the regulatory mechanism of miR-21 mediating metformin-induced anti-angiogenic effects, providing important implications regarding the design of novel miRNA-based therapeutic strategies against angiogenesis.

Methylglyoxal (MGO), an active metabolite of glucose, has been reported to induce vascular cell apoptosis in diabetic complication. Polydatin (PD), a small natural compound from Polygonum cuspidatum, has a number of biological functions, such as antioxidative, anti-inflammatory, and nephroprotective properties. However, the protective effects of PD on MGO-induced apoptosis in endothelial cells remain to be elucidated. In this study, human umbilical vein endothelial cells (HUVECs) were used to explore the effects of PD on MGO-induced cell apoptosis and the possible mechanism involved. HUVECs were pretreated with PD for 2 h, followed by stimulation with MGO. Then cell apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) impairment, mitochondrial morphology alterations, and Akt phosphorylation were assessed. The results demonstrated that PD significantly prevented MGO-induced HUVEC apoptosis. PD pretreatment also significantly inhibited MGO-induced ROS production, MMP impairment, mitochondrial morphology changes, and Akt dephosphorylation. These results and the experiments involving N-acetyl cysteine (antioxidant), Cyclosporin A (mitochondrial protector), and LY294002 (Akt inhibitor) suggest that PD prevents MGO-induced HUVEC apoptosis, at least in part, through inhibiting oxidative stress, maintaining mitochondrial function, and activating Akt pathway. All of these data indicate the potential application of PD for the treatment of diabetic vascular complication.

Background: Vascular endothelial growth factor (VEGF) plays a key role in regulating angiogenesis, and this process is largely dependent on the newly formed extracellular matrix (ECM). The levels of vitronectin (VN) are increased in patients with various cardiovascular diseases. A role for VN in regulating VEGF-induced angiogenesis has not been previously reported. We tested the hypothesis that VN regulates VEGFR-2 activation via effects on αvβ3, thus contributing to angiogenesis. Methods: We used a 3-dimensional angiogenesis assay, and examined the effects of VN on VEGF-mediated angiogenesis in aortic endothelial cells (ECs) isolated from wild-type and VN-deficient mice. Results: The addition of multimeric VN significantly enhanced VEGF-induced increases in EC migration and capillary formation. In vitro, Vn-/- ECs migrated significantly slower than wild-type ECs. The addition of VN to Vn-/- ECs increased EC migration and augmented the promigratory effect of VEGF in a manner that involved VEGFR-2 and Src signaling. Analysis of the mechanisms involved revealed that multimeric VN, but not monomeric VN, binds VEGF and enhances VEGF-induced VEGFR-2/Src activation in ECs. Conclusion: These results underscore the importance of VN in the regulation of angiogenesis induced by VEGF.

Background: The flowers of Carthamus tinctorius L. are widely used in traditional Chinese medicine to treat cerebrovascular and cardiovascular diseases. Hydroxysafflor yellow A (HSYA), the main constituent of C. tinctorius L. flowers, is known for its multiple biological activities. The present study investigated the effects of HSYA on angiogenesis in vitro and in a mouse hindlimb ischemia model. Methods: Using human umbilical vein endothelial cells (HUVEC) in vitro and a mouse hindlimb ischemia model in vivo, the angiogenic role of HSYA was evaluated. Results: HSYA significantly increased the capillary-like tube formation and migration of HUVEC. HSYA not only induced a rise in the expression of angiopoietin 1 and Tie-2 but it also increased phosphorylation of Tie-2, Akt, and extracellular signal-regulated kinase 1/2. Furthermore, an anti-Tie-2 neutralizing antibody significantly inhibited HSYA-induced HUVEC tube formation and migration. In vivo, the recovery of perfusion of ischemic hindlimb tissue after femoral artery interruption was significantly increased in HSYA-treated mice compared to vehicle controls. Consistent with these results, the arteriole and capillary densities in ischemic gastrocnemius muscles were significantly increased in HSYA-treated mice. Conclusions: These results indicate the potential utility of HSYA for the treatment of ischemic diseases.

Additional file 1: Table S1. Sequences of primers used in the study.

Elevated plasma levels of C-reactive protein (CRP) are independently associated with increased risk of atherothrombosis. Several lines of evidence suggest that CRP has prothrombogenic effects on injured vessel wall(s) by enhancing tissue factor (TF) expression. Abnormal fibrin formation is correlated with increased thrombotic risk. However, the impact of localized, cell surface-driven in situ tissue factor generation by CRP on clot dynamics and fibrin architecture has not previously been evaluated. We examined the impact of native CRP and modified or monomeric CRP (mCRP) on the fibrin formation and structure in Human Umbilical Vein Endothelial Cells (HUVECs). Fibrin formation and structure were examined using laser scanning confocal microscopy. Incubation with mCRP on the cell surface had faster fibrin polymerization by the analysis of turbidimetry. Confocal microscopy of fibrin clots showed a significantly increased density in the treatment of mCRP compared with native CRP and control in the proximal versus distal relationship to the cell surface. The increased expression and activity of TF on the cell surface was observed by addition of mCRP. Blockage of tissue factor and lipid rafts significantly reduced the density of fibrin network produced by mCRP-stimulated endothelial cells. mCRP changes clot dynamics and alters fibrin architecture by enhancing TF on the endothelial cell surface. These results support the concept that elevated CRP levels may induce fibrinolytic resistance and endothelial dysfunction by altering fibrin clot structure.

Diabetes mellitus (DM) leads to the development of microvascular diseases and is associated with impaired angiogenesis. The presence of vascular endothelial growth factor (VEGF) can block PDGF-BB dependent regulation of neovascularization and vessel normalization. We tested the hypothesis that the inhibition of VEGF improves blood flow in a mouse hindlimb ischemia model produced by femoral artery ligation. In this study, we examined the effect of bevacizumab, a humanized monoclonal antibody against VEGF-A, on blood perfusion and angiogenesis after hindlimb ischemia. We showed that bevacizumab induces functional blood flow in high fat chow (HFC)-fed diabetic mice. Treatment with bevacizumab increased the expression of platelet derived growth factor-BB (PDGF-BB) in ischemic muscle, and led to vascular normalization. It also blocked vascular leakage by improving the recruitment of pericytes associated with nascent blood vessels, but it did not affect capillary formation. Furthermore, treatment with an anti-PDGF drug significantly inhibited blood flow perfusion in diabetic mice treated with bevacizumab. These results indicate that bevacizumab improves blood flow recovery through the induction of PDGF-BB in a diabetic mouse hindlimb ischemia model, and that vessel normalization may represent a useful strategy for the prevention and treatment of diabetic peripheral arterial disease.

This study is to investigate the effect and underlying mechanism of Zinc (Zn) on hepatic stellate cell collagen synthesis. The proliferation and collagen synthesis ability of LX-2 cells were detected after adding Zn. The collagen synthesis related proteins of MMP-13 and TIMP1 along with TGF-β signaling pathway related proteins were detected by Western blot. The role of TGF-β signaling pathway in collagen synthesis inhibition was identified by TGF-β RI siRNA silencing. Compared with control group, LX-2 cell proliferation ability was significantly inhibited at all Zn concentrations (50 μM, 100 μM and 200 μM). Zn at 50 μM did not affect the protein content of αSMA and type I collagen while 100 μM and 200 μM Zn could significantly inhibit αSMA expression. Compared with control group, gene expression and protein content of MMP-13 in 200 μM Zn group was significantly increased while no difference in gene expression and protein content of TIMP1 was found. TGF-β RI content in 200 μM Zn group was significantly decreased and the protein content of TGF-β RII was not affected. MMP-13 expression was significantly increased after TGF-β RI siRNA silencing. Further results showed that in LX-2 cells those TGF-β RI expression was inhibited, LX-2 cell proliferation ability and the expression of synthesis collagen related proteins of αSMA and type I collagen were greatly decreased. Zn could significantly inhibit the expression of αSMA and type I collagen by inhibiting TGF-β RI expression and promoting MMP-13 expression.

It is known that Toll-like receptor (TLR)-4 plays an important role in myocardial infarction and atherothrombosis. The role of TLR-4 in arterial thrombosis is undefined. Both TLR-4-deficient ( TLR-4−/−) and wild-type (WT) mice were subjected to FeCl3carotid artery injury, and the time required to form an occlusive thrombus was measured. The mean time to occlusion in TLR-4−/−mice was significantly greater than that in WT mice after injury (303 ± 32 vs. 165 ± 34 s, P < 0.05). Furthermore, when we used a WT or TLR-4−/−-derived platelet reinfusion in a platelet depletion/reinfusion procedure, there was no significant change in the occlusion time and tissue factor (TF) activity in injured arteries between WT mice and platelet-depleted WT mice. Similarly, no significant difference was observed between TLR-4−/−mice and platelet-depleted TLR-4−/−mice for the WT or TLR-4−/−-derived platelet reinfusion. However, TF expression and activity were significantly reduced in the vascular wall of TLR-4−/−mice compared with WT mice. In vivo, lipopolysaccharide accelerated the occlusion time in WT mice but not TLR-4−/−mice. In vitro, LPS-induced TF activity was reduced in endothelial cells of TLR-4−/−mice relative to WT mice. The data demonstrate that TLR-4 contributes to arterial thrombosis formation in vivo and causes increased TF expression and activity in vitro. The results further suggest that the stimulation is mainly derived by endothelial cells but is not due to platelet-derived TLR-4.

Objective: Platelets can regulate endothelial cell genes expression through their adhesion to the subendothelium in response to vascular injury. Recently it has been reported that some endothelial cells (ECs)-secreted genes can modulate vascular smooth muscle cells (VSMCs) phenotypic transformation by ECs/VSMCs co-culture. However, little is known about the effects of platelets adhesion to ECs interaction on VSMCs phenotype. In this study, we investigated the role of some genes secreted by platelets adhesion to EC in regulating SMC phenotypic transformation. Methods and Results: By Q-RT-PCR, expression of PAI-1, MMP-2 and MMP-9 were up-regulated in ECs after platelets adhesion to ECs comparing to ECs monoculture alone. In migration (scratch) and proliferation (CCK-8) assays, platelets adhesion to ECs increased EC migration-promoting activity and proliferative activity. After adhesion of platelets to ECs/VSMCs co-culture, expression of VSMCs contractile apparatus SM-MHC, Smoothelin-B, SMA and SM22a were significantly decreased comparing to ECs/VSMCs co-culture by Q-RT-PCR, but decreasing slightly in adhesion with platelets from type 2 diabetes. Conversely, it increased the expression of synthetic marker Smemb, CCND-1, CCND-2. Further analysis found that platelets adhesion to ECs/VSMCs co-culture significantly increased VSMCs migration-promoting activity and proliferative activity. In Gelatin Zymography assays, there were high levels of MMP-2 and MMP-9 after adhesion of platelets to ECs/VSMCs co-culture comparing to ECs/VSMCs co-culture alone. Conclusion: These results offer important insights into the mechanisms controlling phenotypic modulation of VSMCs by platelets adhesion to ECs, and help to explain the effects of platelets-ECs-VSMCs interaction on VSMCs contractile/synthetic, migration, and proliferation, suggesting that the adhesion of platelets to ECs is a key process in regulating phenotypic switch of VSMCs.

OBJECTIVE: Type II diabetes mellitus (DM) leads to the development of microvascular diseases and is associated with impaired angiogenesis. Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with DM. However, the role of PAI-1 in the development of DM-associated microvascular disease is poorly defined. RESEARCH DESIGN AND METHODS: Hind-limb ischemia was generated by femoral artery occlusion in diet-induced diabetic mice and age-matched normal chow (NC)-fed mice. Plasma was analyzed for glucose, non-esterified fatty acids, and PAI-1. PAI-039, a specific, small molecule pharmacological inhibitor of PAI-1, was orally administered to mice to determine if PAI-1 attenuates neovasculature formation in DM mice. RESULTS: PAI-1 activity and antigen were significantly increased within 2 weeks of DM onset and remained elevated throughout the experimental period. PAI-039 normalizes elevated plasma and ischemic muscles PAI-1 levels in the DM group. Pharmacologic inhibition of PAI-1 with PAI-039 rescued the ischemia-mediated impairments in blood flow perfusion and accelerated arterialization of capillaries in ischemic muscles in DM group compared to vehicle-DM group. Immunohistochemical analysis indicated increased expression of smooth muscle α-actin on capillary-sized vessels, and increased infiltration by CD45 leukocytes in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated mice. Furthermore, in these mice, VEGF-A was significantly down-regulated, and conversely, the expression of the PDGF-B and MMP 2, 9 were up-regulated in ischemic muscles in the inhibition of PAI-1 after induced ischemia. CONCLUSIONS: Taken together, these findings illustrate that the pharmacologic inhibition of elevated PAI-1 rescues the impairments in ischemia-mediated flow perfusion and capillary arterializations observed in DM, and may have efficacy as a therapeutic strategy to prevent diabetic microangiopathy.

Background Platelets play a fundamental role in maintaining hemostasis and have been shown to participate in hematogenous dissemination of tumor cells. Abundant platelets were detected in the tumor microenvironment outside of the blood vessel, thus, platelet -tumor cell interaction outside of the bloodstream may play a role in regulating primary tumor growth and metastasis initiation. However, it is unclear that platelet depletion affects tumor vessel structure and dynamics. Methods Using thrombocytopenia induction in two different tumor-bearing mouse models, tumor tissues were performed by Westernblotting and immunohistochemical staining. Vascular permeability was evaluated by determination of intratumoral Evans blue and Miles vascular permeability assay. Furthermore, microdialysis was used to examining the intratumoral extracellular angiogenic growth factors (VEGF, TGF-β) by ELISA. Results Platelet depletion showed no change in tumor growth and reduced lung metastasis. Platelet depletion led to reduced tumor hypoxia and Met receptor activation and was associated with a decreased release of MMP-2, 9, PAI-1, VEGF, and TGF-β. Tumor vessels in platelet-depleted mice showed impaired vessel density and maturation. Conclusions Our findings demonstrate that platelets within the primary tumor microenvironment play a critical role in the induction of vascular permeability and initiation of tumor metastasis.

Objective. PAI-1 mRNA and protein have been detected in human platelets. Recently some miRNAs have been found in human platelets, which are involved in the regulation of genes and the protein synthesis. However, little is known about the physiological roles of individual miRNAs in platelets. In this study, we investigated whether miR30c can regulate platelet-derived plasminogen activator inhibitor-1(PAI-1). Methods and Results. Expression of miR-30c, PAI-1, miR-21 and its targeted gene TIMP1 were found in healthy human leukocyte-depleted platelets (LDPs) by real time PCR. In luciferase reporter gene assay, miR-30c targets the 3’ untranslated region (3’ UTR) of PAI-1 mRNA through a miR-30c binding site. Transfection of miR-30c mimic into MEG-01, a megakaryoblastic cell line, significantly reduced PAI-1 protein level compared with negative control. Inhibition of miR-30c by transfecting miR-30c inhibitor significantly increased PAI-1 protein level. Furthermore, miR-21 expression was significantly down-regulated after transfecting with miR-30c mimic in PAI-1-/- mice LDPs, conversely, the expression of its target gene TIMP1 was significantly up-regulated after transfecting with miR-30c mimic in PAI-1-/- mice LDPs. Conclusion. These results provide a novel regulatory mechanism of miR30c- regulated PAI-1 protein through its influence on the downstream miR21 and its target gene TIMP1 expression in platelet, suggesting that miR-30c might be a potential new strategy for anti-thrombosis.

Objective. Platelets were presented in human atherosclerotic plaque. The function of platelets in the development of atherosclerosis is far poorly defined. We addressed the role of platelets in mediating atherosclerosis formation. Methods and Results. Apolipoprotein E-deficient mice (ApoE-/-) were fed with a high-fat Western diet for 12 weeks. Thrombocytopenia was induced by intraperitoneal (i.p.) injections every 3 days with mouse of the platelet-depleting antibody to ApoE-/- mice from 8 weeks. Until the experimental endpoint, atherosclerotic lesion in the aorta and aortic roots were quantified after staining. Size of atherosclerotic plaque in aortic root did not differ between platelet-depleted mice and controls (134±13.1 μm2 [x103] vs 124±11.4 μm2 [x103]; P>0.5). Plasma cholesterol did not differ between groups. However, atherosclerotic lesions of platelet-depleted mice exhibited thinner fibrous caps (62.5±9.2 μm vs. 323±63 μm; P Conclusion. Our results suggest that platelet depletion promotes atherosclerotic plaque instability in the development of early atherosclerosis, likely though the macrophage infiltration. These results offer important insights into the in vivo function of platelet, and help to explain the association between enhanced platelet deposition and thrombotic complications of atherosclerosis, such as myocardial infarction.

Background: Cross-talk between integrins and cadherins regulates cell function. We tested the hypothesis that vitronectin (VN), a multi-functional adhesion molecule present in the extracellular matrix and plasma, regulates vascular permeability via effects on VE-cadherin, a critical regulator of endothelial cell (EC) adhesion. Methodology/Principal Findings: Addition of multimeric VN (mult VN) significantly increased VE-cadherin internalization in human umbilical vein EC (HUVEC) monolayers. This effect was blocked by the anti-aVb3 antibody, pharmacological inhibition and knockdown of Src kinase. In contrast to mult VN, monomeric VN did not trigger VE-cadherin internalization. In a modified Miles assay, VN deficiency impaired vascular endothelial growth factor-induced permeability. Furthermore, ischemia-induced enhancement of vascular permeability, expressed as the ratio of FITC-dextran leakage from the circulation into the ischemic and non-ischemic hindlimb muscle, was significantly greater in the WT mice than in the Vn 2/2 mice. Similarly, ischemia-mediated macrophage infiltration was significantly reduced in the Vn 2/2 mice vs. the WT controls. We evaluated changes in the multimerization of VN in ischemic tissue in a mouse hindlimb ischemia model. VN plays a previously unrecognized role in regulating endothelial permeability via conformational- and integrin-dependent effects on VE-cadherin trafficking. Conclusion/Significance: These results have important implications for the regulation of endothelial function and angiogenesis by VN under normal and pathological conditions.

Thromboembolism is a major source of morbidity and mortality in patients with cancer. Increased incidence of venous thromboembolism (VTE) was associated with anti-vascular endothelial growth factor (VEGF) treatment in cancer. However, the mechanism of VTE initiation remains elusive. In this study, we addressed the effect of bevacizumab, a humanized monoclonal antibody against VEGF-A on either an inferior vena cava stenosis or a saphenous vein model using a xenograft mouse model. We found that treatment of bevacizumab accelerated thrombosis in a saphenous vein model with an increased thrombus weight in an inferior vena cava stenosis model. Administration of bevacizumab inhibited the growth of lung tumor by suppressing angiogenesis. Both intrathrombotic PAI-1 expression and plasma PAI-1 level was significantly increased by the treatment of bevacizumab. Furthermore, pharmacologic inhibition of PAI-1 with PAI-039 blocked the bevacizumab-induced venous thrombosis, but showed no change in tumor growth. Collectively, these findings indicate that PAI-1 plays a role in VET with anti-angiogenesis therapy, and have efficacy as a therapeutic strategy to prevent VET. Citation Format: Ni Chen, Meiping Ren, Rong Li, Xin Deng, Yongjie Li, Kai Yan, Lamei Xiao, Yan Yang, Liqun Wang, Mao Luo, Jianbo Wu. Anti-angiogenesis promotes venous thromboembolism through inducing PAI-1 in a mouse xenograft model of human lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1367. doi:10.1158/1538-7445.AM2015-1367

Abstract 3321 Platelets play a fundamental role in maintaining hemostasis and have been shown to participate in hemorrhagic metastasis. However, the role of platelets in the tumor growth, angiogenesis, and metastasis initiation remains undefined. The B16/F10 melanoma cancer cells model of metastasis and the Lewis lung carcinoma (LLC) spontaneous pulmonary metastasis model were used for this purpose. Using induction of thrombocytopenia, primary tumor growth was monitored and every 3 days anti-GPIbα or rat IgG injections were initiated when tumor reached ∼500mm3and continued until tumor reached to 3 weeks. We showed that platelet depletion had no change in tumor growth but reduced metastasis. Platelet depletion significantly increased pericyte coverage and reduced vascular density compared with control mice. We evaluated the ratio of fluorescence intensities within the plasma and tumor following injection of mice with FITC-dextran. We found that the FITC-dextran was similarly deposited into the tumor tissue in either platelet-depleted or control mice, indicating that tumor vessel perfusion did not differ in either platelet-depleted or control mice. To further gain insight into the molecular mechanisms associated with reduced metastasis resulting from platelet depletion, we assessed hypoxia levels by examining pimonidazole adduct formation in the tumors of platelet-depleted and control mice and found decreased hypoxic levels in the platelet-depleted tumors. In addition, expression of the hypoxia-inducible transcription factor HIF-1α was also significantly reduced in the tumors of platelet-depleted mice. Tumor hypoxia is strongly associated with deposition of hemoglobin. We measured the intratumor hemoglobin content, reflecting the level of erythrocytes extravasation. The hemoglobin content in the tumors of mice with platelet-depletion was significantly higher than that of control mice (172.11 ± 20.2 g/L/g Vs. 110.28 ± 12.4 g/L/g, p Recent studies demonstrated that abundant platelets were detected in the tumor microenvironment apart from the vasculature. Based on the finding platelets in contact with tumor cells outside the bloodstream, we examined the functional effects of co-implantation of B16/F10 tumor cells with platelets on tumor progression and metastasis. B16/F10 melanoma cancer cells were implanted into back of wild type mice. During a 3-weeks growth, co-implantation of B16/F10 with platelets not only led to promoted tumor volume (3968 ± 296 mm3Vs. 2956 ± 180 mm3, p In conclusion, our findings demonstrate for the first time that platelets play a critical role in the initiation of tumor metastasis. Moreover, our findings suggest that platelets within the primary tumor microenvironment are likely involved in tumor progression and metastasis. Disclosures: No relevant conflicts of interest to declare.

Background An increased incidence of venous thromboembolism (VTE) is associated with anti-vascular endothelial growth factor (VEGF) treatment in cancer. However, the mechanism underlying this effect remains elusive. In this study, we examined the effect of bevacizumab, a humanized monoclonal antibody against VEGF-A, on VTE in a murine xenograft A549 cell tumor model. Methods Inferior vena cava stenosis model and FeCl3-induced saphenous vein thrombosis model were performed in a mouse xenograft models of human lung adenocarcinoma. Results We found that treatment with bevacizumab significantly increased the thrombotic response to inferior vena cava obstruction and femoral vein injury. Plasminogen activator inhibitor (PAI-1) expression in tumors, plasma, and thrombi was significantly increased by bevacizumab. However, bevacizumab did not enhance VTE in PAI-1-deficient mice, suggesting that PAI-1 is a major mediator of bevacizumab’s prothrombotic effect. VEGF inhibited expression of PAI-1 by A549 cells, and this effect was neutralized by bevacizumab, suggesting that bevacizumab increases PAI-1 expression in vivo by blocking the inhibitory effect of VEGF on PAI-1 expression by tumor cells. Pharmacological inhibition of PAI-1 with PAI-039 blocked bevacizumab-induced venous thrombosis. Conclusion Collectively, these findings indicate that PAI-1 plays a role in VTE associated with antiangiogenic therapy and the inhibition of PAI-1 shows efficacy as a therapeutic strategy for the prevention of bevacizumab-associated VTE.