Human Umbilical Cord Vascular Endothelial Cells Research Articles

1,8-Dihydroxy-3-methoxy-anthraquinone inhibits tumor angiogenesis through HIF-1α downregulation

Photorhabdus luminescens is a gram-negative bioluminescent bacterium known as an intestinal bacterium that coexists in the digestive tract of insect-pathogenic nematodes. As part of our ongoing exploration to identify bioactive compounds from diverse natural resources, the chemical analysis of the cultures of P. luminescens KACC 12254 via LC/MS and TLC-based analyses enabled the isolation and identification of a major fluorescent compound. Its chemical structure was elucidated as 1,8-dihydroxy-3-methoxyanthraquinone (DMA) using HR-ESI-MS and NMR analysis. In this study, we conducted comprehensive investigations utilizing human colorectal cancer HCT116 cells, human umbilical cord vascular endothelial cells (HUVECs), and zebrafish embryos to assess the potential benefits of DMA in suppressing tumor angiogenesis. Our results convincingly demonstrate that DMA effectively suppresses the stability of hypoxia-inducible factor-1α (HIF-1α) protein and its target genes without inducing any cytotoxic effects. Furthermore, DMA demonstrates the ability to inhibit HIF-1α transcriptional activation and mitigate the production of reactive oxygen species (ROS). In our in vitro experiments, DMA exhibits notable inhibitory effects on VEGF-mediated tube formation, migration, and invasion in HUVECs. Additionally, in vivo investigations using zebrafish embryos confirm the antiangiogenic properties of DMA. Notably, DMA does not exhibit any adverse developmental or cardiotoxic effects in the in vivo setting. Moreover, we observe DMA's capability to restrain tumor growth through the downregulation of PI3K/AKT and c-RAF/ERK pathway. Collectively, these compelling findings underscore DMA's potential as a promising therapeutic candidate for targeted intervention against HIF-1α and angiogenesis in cancer treatment.

Engineering and Validation of a Peptide-Stabilized Poly(lactic-co-glycolic) Acid Nanoparticle for Targeted Delivery of a Vascular Disruptive Agent in Cancer Therapy.

Developing a biocompatible and biodegradable nanoparticle (NP) carrier that integrates drug-loading capability, active targeting, and imaging modality is extremely challenging. Herein, we report an NP with a core of poly(lactic-co-glycolic) acid (PLGA) chemically modified with the drug combretastatin A4 (CA4), a vascular disrupting agent (VDA) in clinical development for ovarian cancer (OvCA) therapy. The NP is stabilized with a short arginine-glycine-aspartic acid-phenylalanine x3 (RGDFFF) peptide via self-assembly of the peptide on the PLGA surface. Importantly, the use of our RGDFFF coating replaces the commonly used polyethylene glycol (PEG) polymer that itself often induces an unwanted immunogenic response. In addition, the RGD motif of the peptide is well-known to preferentially bind to αvβ3 integrin that is implicated in tumor angiogenesis and is exploited as the NP's targeting component. The NP is enhanced with an optical imaging fluorophore label via chemical modification of the PLGA. The RGDFFF-CA4 NPs are synthesized using a nanoprecipitation method and are ∼75 ± 3.7 nm in diameter, where a peptide coating comprises a 2-3 nm outer layer. The NPs are serum stable for 72 h. In vitro studies using human umbilical cord vascular endothelial cells (HUVEC) confirmed the high uptake and biological activity of the RGDFFF-CA4 NP. NP uptake and viability reduction were demonstrated in OvCA cells grown in culture, and the NPs efficiently accumulated in tumors in a preclinical OvCA mouse model. The RGDFFF NP did not induce an inflammatory response when cultured with immune cells. Finally, the NP was efficiently taken up by patient-derived OvCA cells, suggesting a potential for future clinical applications.

Anti-Angiogenic Treatment in Pseudomyxoma Peritonei—Still a Strong Preclinical Rationale

Simple SummaryPatients with pseudomyxoma peritonei that are not cured by the standard treatment (cytoreductive surgery and hyperthermic intraperitoneal chemotherapy) have no efficacious treatment options. Drugs that inhibit formation of new vessels (anti-angiogenic drugs) could be a therapeutic option for these patients. Using patient samples and animal models we show that angiogenesis is important in pseudomyxoma peritonei and that anti-angiogenic drugs may indeed have an effect. Our results support continued efforts to determine the role of anti-angiogenic treatment in pseudomyxoma peritonei.Pseudomyxoma peritonei (PMP) is a rare, slow-growing cancer characterized by progressive accumulation of intraperitoneal mucinous tumor deposits. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) cures approximately 50% of patients, but in unresectable and recurrent cases, treatment options are limited. Anti-angiogenic treatment is being explored as a potential therapeutic option. Using PMP patient samples, microvessel densities (immunostaining for CD31 and CD105) and pro-angiogenic factors were analyzed, and the proliferative response upon incubation with human umbilical cord vascular endothelial cells (HUVEC) was determined. Growth inhibition by anti-angiogenic drugs was analyzed in patient-derived xenograft models of PMP. PMP tumor tissues were found to be highly vascularized and contained key pro-angiogenic factors, in particular related to vascular endothelial growth factor (VEGF) signaling, but interestingly, high levels of fibroblast growth factor 2 were also detected. HUVEC proliferation was stimulated upon incubation with fresh tumor samples and the observed proliferation could be inhibited by VEGF pathway inhibitor bevacizumab. In xenograft models the two VEGF pathway inhibitors, bevacizumab and aflibercept, inhibited tumor growth. This work reemphasizes the importance of angiogenesis as a major driver in PMP and strengthens the preclinical rationale for continued exploration of angiogenesis inhibition in the hope of providing novel treatment to a group of patients that have few other treatment options.

Mechanistic Insight of Platelet-Rich Plasma (PRP) in Mesenchymal and Vascular Progenitors

Background: Platelet-rich plasma (PRP) is broadly used for disease intervention, pain management and tissue repair without clear elucidation of its mechanisms of action. Clinically, 100% PRP is injected and yields unknown local concentrations when applied in cardiac infarcts, osteoarthritic joints, tendon injuries and numerous other conditions. Methods: Human PRP samples (26 donors) were obtained per conventional clinical methods. Human bone-marrow mesenchymal stem/progenitor cells (MSCs) were assayed for migration, proliferation and differentiation in response to different PRP concentrations. Human umbilical cord vascular endothelial cells (HUVEC) were assayed for endothelial tube formation. Cleaved Caspase 3 and TUNEL were assayed to evaluate apoptosis. Pro-inflammatory cytokines and FasL were assayed by ELISA. Osmotic pressure and pH were measured. All quantitative data were statistically treated. Findings: MSCs treated with 50% PRP detached into Caspase-3 and TUNEL debris, relative to scarce cell death upon exposure to ≤25% PRPs. Upon 50%-PRP treatment, MSCs produced ~180-fold more IL-1β and ~20-fold more FasL, relative to ≤25% PRPs. IL-6 and TNFα surged upon MSC exposure to 50% PRP. Osmotic pressure of 50% PRP was aberrantly high. Medium pH was neutral throughout the tested 7 days. Nonetheless, 1% to 25% PRPs induced dose-dependent MSC migration. Diluted PRPs induced endothelial tube formation directly or by PRP-treated MSC secretome. Diluted PRPs further elicited context-dependent biomineralization. BMP2, PDGF-BB, VEGF and pro-inflammatory cytokines including IL-1β, IL-6, TNFα had significantly lower concentration in bone marrow than in platelets. Interpretation: PRP's clinical use by aggregating growth factors is inevitably coupled with accumulation of excessive pro-inflammatory cytokines and apoptotic ligands. Cell death elicited by ≥50% PRPs suggests that PRP concentration and composition should be tailored for a given clinical indication. Robust biomineralization induced by PRPs may have implications in fracture healing, atherosclerosis and fibrodysplasia dissecans. Certain factors are preferentially stored in platelets, rather than bone marrow, likely owing to evolution and survival. Funding Statement: This work is funded by NIH grants R01AR065023, R01DE025643, R01DE023112, and R01DE026297. Declaration of Interests: We declare no competing interests. Dr. Jeremy Mao has co-founded biotechnology companies but without reference to PRP technologies in the past and presence. Ethics Approval Statement: IRB approval mentioned by authors in acquisition and use of human blood samples.

MZD7349 peptide-conjugated PLGA nanoparticles directed against VCAM-1 for targeted delivery of simvastatin to restore dysfunctional HUVECs

Endothelial dysfunction is initial and critical step of atherosclerosis. Impaired bioavailability of endothelial nitric oxide synthase (eNOS) is one of the main reasons of endothelial dysfunction. Improving bioavailability of eNOS by increasing its expression or activity using statins is an effective therapeutic strategy in restoring endothelial dysfunction.In this study, simvastatin (SIM) as a poorly water-soluble drug was loaded in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (SIM-PLGA-NPs). NPs were then conjugated with mZD7349 peptide (mZD7349-SIM-PLGA-NPs) and directed against vascular cell adhesion molecule 1 (VCAM-1). In vitro evaluation of the NPs for targeted delivery of SIM was performed on activated Human Umbilical Cord Vascular Endothelial Cells (HUVECs) by tumor necrosis factor alpha (TNF-α). Effect of mZD7349-SIM-PLGA-NPs and SIM-PLGA-NPs was compared on eNOS phosphorylation (ser-1177).Results of western blot showed SIM post-treatment increased significantly phosphor-eNOS (Ser1177) expression but no total eNOS expression. The study showed that mZD7349-SIM-PLGA-NPs have particle size, zeta potential value, polydispersity index (PDI) and encapsulation efficacy % of 233±18nm, −9.6±1.1mV, 0.59±0.066 and 69±17.3%, respectively. Also phosphor-eNOS (Ser1177) expression in activated HUVECs treated with mZD7349-SIM-PLGA-NPs was significantly (p<0.05) better than treated cells with SIM-PLGA-NPs.The results suggest that mZD7349-SIM-PLGA-NPs may be usable as an appropriate drug carrier for restoring endothelial dysfunction.

Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels.

Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secret and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes in vitro we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes during neoangiogenic events with a wide range of analyses options.

The Bromodomain BET Inhibitor JQ1 Suppresses Tumor Angiogenesis in Models of Childhood Sarcoma.

The bromodomain and extra-terminal domain inhibitor JQ1 has marked antitumor activity against several hematologic malignancies as well as solid tumor models. Here, we investigated its activity in vitro and in vivo against models of childhood rhabdomyosarcoma and Ewing sarcoma. In vitro, JQ1 (but not the inactive enantiomer JQ1R) inhibited cell proliferation and increased G1 fraction of cells, although there was no correlation between cell line sensitivity and suppression of c-MYC or MYCN. In vivo, xenografts showed significant inhibition of growth during the period of treatment, and rapid regrowth after treatment was stopped, activity typical of antiangiogenic agents. Furthermore, xenografts derived from cell lines intrinsically resistant or sensitive to JQ1 in vitro had similar sensitivity in vivo as xenografts. Further investigation showed that JQ1 reduced tumor vascularization. This was secondary to both drug-induced downregulation of tumor-derived growth factors and direct effects of JQ1 on vascular elements. JQ1 suppressed VEGF-stimulated vascularization of Matrigel plugs in mice, and in vitro suppressed differentiation, proliferation, and invasion of human umbilical cord vascular endothelial cells (HUVEC). In HUVECs, JQ1 partially suppressed c-MYC levels, but dramatically reduced AP-1 levels and activity through suppression of the AP-1-associated protein FOSL1. Our data suggest that the antitumor activity of JQ1 in these sarcoma models is largely a consequence of its antiangiogenic activity. Mol Cancer Ther; 15(5); 1018-28. ©2016 AACR.

Malignant stroma increases luminal breast cancer cell proliferation and angiogenesis through platelet-derived growth factor signaling.

BackgroundLuminal, estrogen receptor-positive breast cancers represent more than 70% of cases. Despite initial good prognoses one third of Luminal cancers eventually recur locally or at distant sites and exhibit hormone resistance. Here we demonstrate that factors elaborated by malignant stromal cells can induce Luminal tumor cells proliferation and promote angiogenesis and hormone independence. We recently isolated a malignant mouse mammary gland stromal cell line named BJ3Z that increases proliferation and angiogenesis in estrogen-free xenografted Luminal MCF-7 breast cancer cells.MethodsBJ3Z and Normal mouse mammary Fibroblasts (NMFs) were expression profiled using microarray assays. Messenger RNA levels were confirmed by RT-PCR and by immunohistochemistry (IHC). Breast cancer MCF-7, BT-474, BT-20 and MDA-MB-231cell lines and stromal BJ3Z and NMFs were grown for in vitro assays: breast cancer cell lines were treated with stromal cells conditioned media, for three-dimensional (3D) mono and co-cultures in Matrigel, proliferation was measured by Bromo-deoxyuridine (BrdU) incorporation using IHC. Tubule formation in vitro, a proxy for angiogenesis, was assessed using 3D cultured Human Umbilical cord Vascular Endothelial Cells (HUVEC).ResultsWe show that under estrogen-free conditions, BJ3Z cells but not NMFs increase proliferation of co-cultured Luminal but not basal-like human breast cancer cells in 2D or as 3D Matrigel colonies. Gene expression profiling, RT-PCR analysis and IHC of colony-derived BJ3Z cells and NMFs shows that Platelet Derived Growth Factor ligands (PDGF-A and -B) are elaborated by BJ3Z cells but not NMFs; while PDGF receptors are present on NMFs but not BJ3Z cells. As a result, in colony co-culture assays, BJ3Z cells but not NMFs increase MCF-7 cell proliferation. This can be mimicked by direct addition of PDGF-BB, and blocked by the PDGF receptor inhibitor Imatinib Mesylate. Both normal and malignant stromal cells enhance angiogenesis in an in vitro model. This effect is also due to PDGF and is suppressed by Imatinib.ConclusionsWe provide evidence that Luminal breast cancer cells can be targeted by the PDGF signaling pathway leading to estrogen-independent proliferation and angiogenesis. We speculate that stroma-directed therapies, including anti-PDGFR agents like Imatinib, may be useful in combination with other therapies for treatment of luminal cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-735) contains supplementary material, which is available to authorized users.

Independent Anti-Angiogenic Capacities of Coagulation Factors X and Xa

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

Dual Targeting of the Type 1 Insulin-like Growth Factor Receptor and Its Ligands as an Effective Antiangiogenic Strategy

In pediatric tumor xenograft models, tumor-derived insulin growth factor (IGF-2) results in intrinsic resistance to IGF-IR-targeted antibodies, maintaining continued tumor angiogenesis. We evaluated the antiangiogenic activity of a ligand-binding antibody (MEDI-573) alone or in combination with IGF-I receptor binding antibodies (MAB391, CP01-B02). IGF-stimulated signaling was monitored by increased Akt phosphorylation in sarcoma and human umbilical cord vascular endothelial cells (HUVEC). Angiogenesis was determined in vitro using capillary tube formation in HUVECs and in vivo using a VEGF-stimulated Matrigel assay. Tumor growth delay was examined in 4 sarcoma xenograft models. The IGF ligand-binding antibody MEDI-573 suppressed Akt phosphorylation induced by exogenous IGF-I and IGF-2 in sarcoma cells. Receptor-binding antibodies suppressed IGF-I stimulation of Akt phosphorylation, but IGF-2 circumvented this effect and maintained HUVEC tube formation. MEDI-573 inhibited HUVEC proliferation and tube formation in vitro, but did not inhibit angiogenesis in vivo, probably because MEDI-573 binds murine IGF-I with low affinity. However, in vitro antiangiogenic activity of MEDI-573 was also circumvented by human recombinant IGF-I. The combination of receptor- and ligand-binding antibodies completely suppressed VEGF-stimulated proliferation of HUVECs in the presence of IGF-I and IGF-2, prevented ligand-induced phosphorylation of IGF-IR/IR receptors, and suppressed VEGF/IGF-2-driven angiogenesis in vivo. The combination of CP1-BO2 plus MEDI-573 was significantly superior to therapy with either antibody alone against IGF-I and IGF-2 secreting pediatric sarcoma xenograft models. These results suggest that combination of antibodies targeting IGF receptor and ligands may be an effective therapeutic strategy to block angiogenesis for IGF-driven tumors.

Abstract 3894: A high phosphate environment induces angiogenesis, which is mediated by FOXC2.

Abstract Inorganic phosphate is a common dietary element that is critical to the human body on many levels however the effects of changes in serum phosphate on cell and tissue behavior are not well understood. Due, in part, to the increased consumption of processed foods, the amount of inorganic phosphate in the American diet continues to rise above levels already considered high by the FDA. There is a growing appreciation that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. Recent studies in mice have demonstrated that a diet high in phosphate, relative to a reduced phosphate diet, increases tumorigenesis in the two-stage skin carcinogenesis model as well as the Kras lung cancer model. Using cell cultures models we have determined that high phosphate results in increased proliferation and transformation as measured by XTT, cell counts, soft agar and focus formation assays. Our in vitro studies have also identified specific signaling cascades induced by elevated extracellular phosphate which result in the temporal and dynamic regulation of hundreds of phosphate responsive genes. A subset of the Pi-responsive genes including the Forkhead box protein C2 (FOXC2), osteopontin (spp1), Cyr61, and Vegf among others, has been previously linked to stimulation of angiogenesis leading to the hypothesis that conditioned medium from phosphate treated cells would increase angiogenesis. We confirmed the phosphate-induced expression of these genes in a number of cancer cells including breast, colon, and lung. The increased expression of these genes was determined to be dependent on phosphate transport as well as FGF receptor and ERK1/2 signaling. Using human umbilical cord vascular endothelial cells (HUVECs) we determined that, in fact, conditioned medium from these phosphate treated cancer cells increased both migration and tube formation in the HUVEC model. Knockdown of FOXC2 inhibited phosphate induced gene expression in cancer cells as well as eliminating the stimulation of HUVEC migration in response to Pi-treated cancer cell conditioned medium. These results suggest that a cellular environment with elevated phosphate promotes multiple aspects of tumorigenesis including angiogenesis. Citation Format: Yiming Lin, Laura M. Garneys, Kelly E. Bennett, George R. Beck. A high phosphate environment induces angiogenesis, which is mediated by FOXC2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3894. doi:10.1158/1538-7445.AM2013-3894

Anti-Angiogenic Activity of a Small Molecule STAT3 Inhibitor LLL12

BackgroundRecent data indicate the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is required for VEGF production and angiogenesis in various types of cancers. STAT3 inhibitors have been shown to reduce tumor microvessel density in tumors but a direct anti-angiogenic activity has not been described.Methodology/Principal FindingsWe investigated the direct action of a small molecule inhibitor of STAT3 (LLL12) in human umbilical cord vascular endothelial cells (HUVECs) in vitro, in a Matrigel model for angiogenesis in vivo, and its antitumor activity in a xenograft model of osteosarcoma. LLL12 (100 nM) significantly inhibited VEGF-stimulated STAT3 phosphorylation in HUVECs, reduced their proliferation/migration and inhibited VEGF-induced tube formation. Morphologic analysis of LLL12 treated HUVECs demonstrated marked changes in actin/tubulin distribution and bundling. In scid mice, LLL12 reduced microvessel invasion into VEGF-infused Matrigel plugs by ∼90% at a dose of 5 mg/kg daily. Following a period of tumor progression (2 weeks), LLL12 completely suppressed further growth of established OS-1 osteosarcoma xenografts. Pharmacodynamic studies showed robust phosphorylated STAT3 in control tumors, whereas phospho-STAT3 was not detected in LLL12-treated OS-1 tumors. Treated tumors demonstrated decreased proliferation (Ki67 staining), and decreased microvessel density (CD34 staining), but no significant increase in apoptosis (TUNEL staining), relative to controls. Assay of angiogenic factors, using an antibody array, showed VEGF, MMP-9, Angiopoietin1/2, Tissue Factor and FGF-1 expression were dramatically reduced in LLL12-treated tumors compared to control tumors.ConclusionsThese findings provide the first evidence that LLL12 effectively inhibits tumor angiogenesis both in vitro and in vivo.

Discovery of a pyrazole derivative promoting angiogenesis through modulating reactive oxygen species and interferon-inducible protein 10 levels

Human umbilical cord vascular endothelial cells (HUVECs) cultured without serum and fibroblast growth factor-2 is an in vitro model of ischemic conditions. Our previous study showed that ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) could inhibit apoptosis of HUVECs in this model. In this study, we investigated the effect of MPD on angiogenesis and the possible mechanisms. Capillary-like tube formation assay on Matrigel and cell migration assay were performed to investigate the effect of MPD on angiogenesis. The reactive oxygen species (ROS) and interferon-inducible protein 10 (IP-10) levels were respectively evaluated by intracellular ROS assay and western blot analysis. MPD at 5 and 10 μM promoted vascular structure formation and HUVEC migration in an in vitro ischemic model. MPD promoted angiogenesis through elevating ROS levels and depressing IP-10 level. ROS seemed to be necessary for angiogenesis, and a high level of IP-10 inhibited angiogenesis in ischemic state. ROS provide clues for seeking new key factors involved in angiogenesis. IP-10 may become a new target for future therapeutic intervention. MPD is a good tool for investigating the mechanism of angiogenesis, and MPD might be useful in the development of new drugs in therapy of ischemic diseases.

Selectins Ligand Decorated Drug Carriers for Activated Endothelial Cell Targeting

New active particulate polymeric vectors based on branched polyester copolymers of hydroxy-acid and allyl glycidyl ether were developed to target drugs to the inflammatory endothelial cell surface. The hydroxyl and carboxyl derivatives of these polymers allow grafting of ligand molecules on the polyester backbones at different densities. A known potent nonselective selectin ligand was selected and synthesized using a new scheme. This synthesis allowed the grafting of the ligand to the polyester polymers, preserving its binding activity as assessed by docking simulations. Selectin expression on human umbilical cord vascular endothelial cells (HUVEC) was induced with the pro-inflammatory bacterial lipopolysaccharide (LPS) or with the nonselective inhibitor of nitric oxide synthase L-NAME. Strong adhesion of the ligand decorated nanoparticles was evidenced in vitro on activated HUVEC. Binding of nanoparticles bearing ligand molecules could be efficiently inhibited by prior incubation of cells with free ligand, demonstrating that adhesion of the nanoparticles is mediated by specific interaction between the ligand and the selectin receptors. These nanoparticles could be used for specific drug delivery to the activated vascular endothelium, suggesting their application in the treatment of diseases with an inflammatory component such as rheumatoid arthritis and cancer.

Using fluorescent micro‐particle image velocimetry to interrogate the surface glycocalyx on cultured endothelial cells in collagen microchannels

Numerous studies of endothelial cells in culture have been undertaken that are intended to elucidate mechanisms of endothelial-cell function spanning a broad range of fields in cardiovascular physiology. Since the surface glycocalyx on vascular endothelial cells is likely to have important implications for endothelial-cell function, it is necessary to determine if a physiologically typical surface glycocalyx can be grown and maintained on cultured endothelial cells and required conditions are. To make this determination, we use fluorescent micro- particle image velocimetry (μ-PIV) in cylindrical collagen lined on their luminal surface with a confluent monolayer of human umbilical-cord vascular endothelial cells (HUVECs). From μ-PIV data of the velocity and positions of microspheres in an optical section through the median plane of the tube, a velocity profile of the local microfluidics in the vicinity of the HUVEC luminal surface is generated. This velocity profile was used to estimate the effective hydrodynamic thickness of glycocalyx. By modulating the composition of the perfusate solution, the μ-PIV data can be used to assess whether molecular adsorption from the perfusate is an important determinant of the hydrodynamic properties of the glycocalyx. Results will be shown for a variety of perfusate compositions before and after treatments to degrade the glycocalyx. Supported by NIH grants R01 HL76499.

The new cardioprotector Monohydroxyethylrutoside protects against doxorubicin-induced inflammatory effects in vitro.

Besides its cardiotoxic effect, doxorubicin also elicits inflammatory effects in vivo. 7-Monohydroxyethylrutoside (monoHER) has recently been used as a protector against doxorubicin-induced cardiotoxicity in vivo. It is not known yet whether monoHER can also protect against doxorubicin-induced inflammatory effects. The aim of the present study was (1) to illustrate the inflammatory effects of doxorubicin in vitro and (2) to evaluate a possibly protective effect of monoHER. In order to demonstrate the inflammatory effects of doxorubicin and the possible protection of monoHER, proliferating human umbilical cord vascular endothelial cells (HUVECs) were incubated with different concentrations of doxorubicin ranging from 12.5 to 600 nM with(out) 200 μM monoHER. Resting (confluent) HUVECs were incubated with (0.5–25 μM) doxorubicin with(out) monoHER (0.2–1.2 mM) and the viability of endothelial cells and their propensity to adhere to neutrophils were measured 24 h after treatment. The localisation of adhered neutrophils was determined with immunofluorescence microscopy. To further characterise the mechanism of doxorubicin-induced neutrophil adhesion, the expression of the HUVECs surface adhesion molecules was determined after doxorubicin treatment. Doxorubicin decreased the viability and proliferation capacity of HUVECs in a concentration-dependent manner. The proliferating HUVECs were much more sensitive to doxorubicin (IC50=60.0±20.8 nM) than resting cells (LC50=4.0±0.3 μM). Doxorubicin also increased the adhesion of neutrophils reaching a plateau value at a doxorubicin concentration of ⩾0.4μM (P=0.0113). The induced neutrophil adhesion was accompanied by overexpression of VCAM and E-selectin but not ICAM. Although monoHER did not reverse the effect of doxorubicin on the proliferation of endothelial cells, it significantly protected resting HUVECs against the cytotoxic effect of doxorubicin (⩽25 μM, P<0.0015). In addition, monoHER completely protected against the stimulatory effect of doxorubicin on neutrophil adhesion, and inhibited the doxorubin-induced expression of VCAM and E-selectin on the surface of treated HUVECs. This study illustrates that monoHER, which protects against doxorubicin's cardiotoxic effect, can also protect against doxorubicin-induced inflammatory effects. These data prompt further investigation about the possible link between doxorubicin-induced inflammatory effects and its cardiotoxicity in vivo.

Serum amyloid A mediates the inhibitory effect of Ganoderma lucidum polysaccharides on tumor cell adhesion to endothelial cells

Ganoderma ludicum polysaccharides (GlPS) are the major bioactive composition of Ganoderma lucidum, a well-recognized oriental medical fungus. The published data have shown a complementary effect of GlPS in cancer therapy. The present study was designed to determine the anti-tumor efficacy of GlPS and the possible mechanism covering this effect. Murine Sarcoma 180 (S180) model was established, and GlPS administered orally for 10 days. On the 10th day, tumors were weighed to assess the inhibitory effect of GlPS and sera were collected for proteomic analysis and in vitro study. The in vivo results demonstrated that 25, 50 and 100 mg/kg GlPS inhibited S180 growth by 32.67, 44.80 and 45.24%, respectively (P<0.01). Proteomic study revealed marked protein changes after the process of treatment. Three significantly changed proteins were identified by ESI-Q-TOF-MS and database search indicated that they were haptoblobin, apolipoprotein A-II and serum amyloid A (SAA), respectively. Additionally, the expression change of SAA was confirmed by both Western blot and RT-PCR. The adhesion assay showed that GlPS-treated sera dramatically inhibited the adhesion ability of human prostate carcinoma (PC-3M) cells to human umbilical cord vascular endothelial cells (HUVECs), and this effect partially recovered after immunodepletion by the antibody against SAA. Collectively, these results suggest that GlPS inhibited the tumor growth and tumor cell adhesion to HUVECs via up-regulation of SAA protein expression.