Microvessel Cells Research Articles

A Model for Oxygen Transport from Blood in Microvessels to Tissue

Oxygen is vital for cellular energetics and metabolism in the human body. Blood transports oxygen to the tissues with hemoglobin in red blood cells playing a key role in the transportation of oxygen. To account for the Fåhraeus and Fåhraeus–Lindqvist effects, we use Haynes marginal zone concept, which subdivides each microvessel into a cell free layer surrounding a core region of uniform red blood cells concentration. The marginal zone concept is used to develop a steady state model for the transport of oxygen from blood to tissue where chemical reaction of oxygen occurs to produce energy. The approach is based on fundamentals of fluid flow and mass transfer in the two zones while accounting for the role of hemoglobin in the transport process and including mass transfer and chemical reaction in the tissue to produce energy using the Krogh cylinder concept. In contrast to transport modeling of solutes such as glucose, the present model includes the key role of hemoglobin in the transport of oxygen from blood to tissue. The model is analytical and provides analytical expressions for the oxygen level profiles in the blood cell free layer, the core zone, and the Krogh cylinder. The results are found to agree with published results in the literature for oxygen transport from blood in capillary size microvessel to its Krogh tissue cylinder. The model is not restricted to transport from capillaries and includes transport of oxygen from microvessels to tissue in general. Extensions of the model include further investigations in the case where changes in the blood microvessel or red blood cells occur due to pathological conditions.

Recent advances in the in vitro and in vivo methods to assess impact of P-glycoprotein and breast cancer resistance protein transporters in central nervous system drug disposition.

One challenge in central nervous system (CNS) drug discovery has been ensuring the blood-brain barrier (BBB) penetration of compounds at an efficacious concentration that provides suitable safety margins for clinical investigation. Research providing for the accurate prediction of brain penetration of compounds during preclinical discovery is important to a CNS program. In the BBB, P-glycoprotein (P-gp) (ABCB1) and breast cancer resistance protein (BCRP) (ABCG2) transporters have been demonstrated to play a major role in the active efflux of endogenous compounds and xenobiotics out of the brain microvessel cells and back to the systemic circulation. In the past 10years, there has been significant technological improvement in the sensitivity of quantitative proteomics methods, in vivo imaging, in vitro methods of organoid and microphysiological systems, as well as in silico quantitative physiological based pharmacokinetic and systems pharmacology models. Scientists continually leverage these advancements to interrogate the distribution of compounds in the CNS which may also show signals of substrate specificity of P-gp and/or BCRP. These methods have shown promise toward predicting and quantifying the unbound concentration(s) within the brain relevant for efficacy or safety. In this review, the authors have summarized the in vivo, in vitro, and proteomics advancements toward understanding the contribution of P-gp and/or BCRP in restricting the entry of compounds to the CNS of either healthy or special populations. Special emphasis has been provided on recent investigations on the application of a proteomics-informed approach to predict steady-state drug concentrations in the brain. Moreover, future perspectives regarding the role of these transporters in newer modalities are discussed.

The palliative effects of folic acid on retinal microvessels in diabetic retinopathy via regulating the metabolism of DNA methylation and hydroxymethylation

ABSTRACT Diabetic retinopathy (DR) is one of the severe microvascular complications of diabetes. The protective effects of FA on retinal vascular endothelial cells against high glucose levels involve in multiple aspects in DR; however, the underlying mechanism is not fully elucidated. In present study, we investigated the transcriptome as well as genome-wide DNA methylation and hydroxymethylation signature in human retinal microvascular endothelial ACBRI 181 cells cultured within high glucose (HG) medium supplemented with or without FA by RNA-seq, MeDIP-seq, and hMeDIP-seq. Total 3308 differential expressed genes (DEGs) were involved in multiple biological processes and molecular functions containing angiogenesis, inflammation, S-adenosyl methionine metabolism, and hypoxia response. Moreover, the global DNA methylation and hydroxymethylation in ACBRI 181 cells with FA treatment were both compromised compared to HG. Combined with transcriptome data, four subclusters of DEGs with hyper- or hypomethylated promoters were further verified. Unexpectedly, promoters of these 487 genes all displayed a pattern of increased DNA hydroxymethylation. Furthermore, hyperglycemia rat model was established and administered with FA. The DNA methylation and hydroxymethylation changes of selected target genes COL1A1, ITGA7, MMP-14, and VEGFB confirmed by MeDIP-qPCR were consistent with the results in human ACBRI 181 cells. Finally, the presence of activated DNMT1 and TET2 induced by FA was determined in ACBRI 181 cells and hyperglycemia rat. Taken together, this research provided a resource of expression and epigenetic profiles in retinal microvascular endothelial cell, emphasizing a pharmacological mechanism of FA on DNA methylation and hydroxymethylation regulation in retinal microvessel cells of DR.

P–361 Endometrium optical coherence tomography (OCT) and histomorphometry in implantation window and their relationships with reproductive failure and implantation outcome

Abstract Study question How do endometrium OCT image characteristics during peri-implantation window correlate with histomorphometry and associate with implantation outcomes in women with reproductive failure? Summary answer Endometrium OCT intensity correlated with stromal cell density and gland size. Endometrium with recurrent implantation failure had low OCT intensity but reversed in successful implantation. What is known already OCT is a non-invasive imaging technique using low energy near-infrared light to capture micrometer-scale resolution images from optical scattering media. An image produced by OCT resembles tissue architecture observed in histology, so OCT imaging has been regarded as “optical biopsy”. Our previous findings demonstrated OCT is better than ultrasound to identify endometrial pathology. We also showed association of OCT signal with microvessel density in peri-implantation endometrium. However, other histomorphometry were not evaluated. It is still unclear whether endometrium OCT image characteristics are different in reproductive failure and can predict implantation outcomes. Study design, size, duration This was a prospective study conducted at teaching hospital of The Chinese University of Hong Kong from Jan 2018 to Dec 2019. 46 infertile women with or without recurrent miscarriage (RM) and implantation failure (RIF) were recruited in this study. Endometrium OCT imaging and subsequent biopsy were performed on the seventh day after luteal hormone surge (LH + 7) in natural cycle prior to the consecutive natural conception or embryo transfer (ET) cycle. Participants/materials, setting, methods At least 5 systematic random endometrium OCT images from uterine fundus, body and lower segment of each subject were included for intensity analysis by two independent observers. OCT intensity of each image was classified as low, moderate, high based on optical range and then average OCT intensity in each uterine region was calculated for group comparison. Endometrium glandular epithelial, stromal, endothelial, uNK cells were defined by standard H&E and specific immunostaining for histomorphometry and correlation. Main results and the role of chance OCT intensity significantly correlated with endometrial cell and gland parameters regardless classifications of reproductive failure and implantation outcome. Higher OCT intensity indicated higher stromal cell density, gland to stromal (G/S) ratio and average gland area, but fewer microvessel and uNK cells. None of the endometrium histomorphometry were significantly different among different reproductive failure types and implantation outcomes, suggesting it may not be sensitive enough to detect the abnormal histological features. However, OCT intensity was significantly lower in the uterine fundus and body of RIF group than in that of infertile and RM groups. There was no significant difference of OCT intensity in the lower part of the endometrium among three groups. It indicates that OCT intensity is a sensitive marker to differentiate endometrium with RIF from the endometrium with other conditions and also endometrium with RIF is characterized with less stromal cells and smaller glands. Compared with infertile group with unsuccessful implantation, OCT intensity was higher in all three parts of the uterus from the infertile group with successful implantation, but the results were not statistically different. The results further implied that endometrial cells and gland size may potentially contribute to the endometrium receptivity for implantation. Limitations, reasons for caution Current endometrium OCT imaging depth is within 3mm, change beyond this thickness is inaccessible but still the most important layer for implantation. This is a pilot and small study with lack of normal fertile control. Endometrium OCT imaging in the same natural conception or ET cycle will be more accurate. Wider implications of the findings: OCT imaging could be used as a potential noninvasive modality to evaluate peri-implantation window endometrium. It enables real-time and in-situ visualization of endometrium structure and pathology with no additional biopsy risk and examination delay. Larger clinical trials are needed to confirm its clinical applications and utilities. Trial registration number CREC 2016.160

Ultrasound-mediated microbubbles destruction for treatment of rabbit VX2 orthotopic hepatic tumors

The aim of this study was to determine whether ultrasound (US) mediated microbubbles (MBs) destruction (UMMD) could inhibit VX2 orthotopic hepatic tumor growth in rabbit models. Twenty-four VX2 orthotopic hepatic tumor rabbit models were randomly divided into four groups (n = 6 each): saline group, SonoVue alone group, US alone group, US + SonoVue group. Tumor volume (TV), peak intensity (PI) of contrast enhanced ultrasound (CEUS) in US + SonoVue group were significantly lower than that in other groups after treatment (P < 0.05, for all). Immunohistochemical analysis of tumor tissue showed that microvascular density (MVD) in US + SonoVue group was significantly lower than that in other groups (P < 0.05, for all). Extensive interstitial hemorrhage, intravascular thrombosis, destroyed nucleus membrane, mitochondria vacuolation and chromatin condensation were observed in US + SonoVue group, whereas these changes were rarely appeared in other groups. Our study indicated that UMMD could inhibit the growth of VX2 hepatic tumors in rabbits by irreversible destroying tumor microvessel and tumor cells.

Abstract 5921: Microenvironmental changes in the progression from adenocarcinoma in situ to early-invasive adenocarcinoma of the lung

Abstract Introduction: Lepidic predominant adenocarcinoma of the lung was classified into adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive lepidic predominant adenocarcinoma (LPA) in current WHO classification, 4th edition. Furthermore, LPA is thought to progress in a stepwise fashion from AIS and MIA. However, those classifications were diagnosed by observation of pathologists based on morphology only. The aim of this study was to investigate the microenvironmental changes in the process of development from AIS, MIA to LPA, and subjective marker of stepwise progression. We have published a part of data in this study in Lung Cancer (100:53-62, 2016), and we believe that this study makes a contribution to elucidate the mechanism of development of LPA of the lung. Materials and Methods: Clinicopathological characteristics of AIS (n=51), MIA (n=59), LPA smaller than 3 cm (S-LPA, n=113), and LPA larger than 3 cm (L-LPA, n=47) were analyzed. We then immunohistochemically evaluated the expression levels of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, S100A4), invasion-related molecules (laminin-5, ezrin), and growth factor receptors (EGFR) in cancer cells of each group (n=20). The number of tumor-promoting stromal cells, including podoplanin-positive cancer-associated fibroblasts (PDPN+ CAFs), CD204-positive tumor-associated macrophages (CD204+ TAMs), and CD34+ microvessel cells, were also analyzed. Results: LPA-S showed significantly frequent vascular and pleural invasion than MIA (p&amp;lt;0.05); however, no significant difference in those factors was found between S-LPA and L-LPA. Laminin-5 expression in the non-invasive component of MIA was significantly higher than that of AIS (p&amp;lt;0.001). During the progression from MIA to S-LPA, the expression level of laminin-5 in the invasive component was significantly elevated (p&amp;lt;0.01). Moreover, tumor-promoting stromal cells were more frequently recruited in the invasive area of LPA-S (PDPN+ CAFs; p&amp;lt;0.05, CD204+ TAMs; p&amp;lt;0.001, CD34+ microvessel; p&amp;lt;0.05). Ezrin expression in the invasive component of L-LPA was significantly increased (p&amp;lt;0.05) compared to S-LPA; however, the number of tumor- promoting stromal cells were not different between these two groups. Conclusion: Our current study indicated that microenvironmental molecular changes occur during the progression from MIA to S-LPA. Thus, it was suggested that this process may play an important role in disease progression from AIS to LPA of the lung. Citation Format: MASAHITO NAITO, Yukitoshi Satoh, Genichiro Ishii. Microenvironmental changes in the progression from adenocarcinoma in situ to early-invasive adenocarcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5921. doi:10.1158/1538-7445.AM2017-5921

Microenvironmental changes in the progression from adenocarcinoma in situ to minimally invasive adenocarcinoma and invasive lepidic predominant adenocarcinoma of the lung

ObjectivesInvasive lepidic predominant adenocarcinoma (LPA) of the lung is thought to progress in a stepwise fashion from adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA). The aim of this study was to investigate the microenvironmental changes during the development from AIS to LPA. Materials and methodsClinicopathological characteristics of AIS (n=51), MIA (n=59), LPA smaller than 3cm (LPA-S, n=113), and LPA larger than 3cm (LPA-L, n=47) were analyzed. We then evaluated the expression levels of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, S100A4), invasion-related molecules (laminin-5, ezrin), stem-cell-related molecules (ALDH-1), and growth factor receptors (c-Met, EGFR) in cancer cells of each group (n=20). The number of tumor-promoting stromal cells, including podoplanin-positive cancer-associated fibroblasts (PDPN+ CAFs), CD204-positive tumor-associated macrophages (CD204+ TAMs), and CD34+ microvessel cells, were also analyzed. ResultsNo significant difference in these characteristics was found between LPA-S and LPA-L. Laminin-5 expression in the non-invasive carcinoma component of MIA was significantly higher than that of AIS (p<0.001). During the progression from MIA to LPA-S, the expression level of laminin-5 in the invasive carcinoma component was significantly elevated (p<0.01). Moreover, tumor-promoting stromal cells were more frequently recruited in the invasive area of LPA-S (PDPN+ CAFs; p<0.05, CD204+ TAMs; p<0.001, CD34+ microvessel; p<0.05). Ezrin expression in the invasive carcinoma component of LPA-L was significantly increased (p<0.05) compared to LPA-S; however, the number of tumor-promoting stromal cells were not different between these two groups. ConclusionOur current results indicated that microenvironmental molecular changes occur during the progression from MIA to LPA-S and suggested that this process may play an important role in disease progression from AIS to LPA.

Coronary Microvascular Smooth Muscle‐Endothelial Cell Communication and Notch 3 Signaling in Type 2 Diabetes

The Notch signaling pathway, via cell‐cell communication, is critical during normal vascular development and homeostasis, and it is known to regulate vascular smooth muscle (VSMC) phenotype, including proliferation. The current study tested the hypothesis that VSMC‐endothelial cell (EC) Notch 3/Jagged 1 signaling is aberrant to account for adverse remodeling and stiffness observed in type 2 diabetic coronary resistance microvessels (CRMs). Low‐passage primary coronary VMSCs were isolated from normal and type 2 diabetic db/db mice and were either mono‐ or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively (n=4–6 per group). In separate experiments, normal (n=3) and diabetic db/db CRMs (n=3) were isolated, mounted, and perfusion pressure fixed on a pressure myograph. The size and frequency of fenestrae in the internal elastic laminas of intact CRMs were assessed using Alexa Fluor 633 hydrazide on a confocal microscope. Coronary VSMCs isolated from db/db mice expressed higher Notch 3 mRNA (2.12±0.10 vs. 1.00±0.20; p&lt;0.01) compared to normal. Co‐culture of coronary VSCMs with hCMECs caused a robust induction of Notch 3 expression in normal VSMCs (3.67±0.60 vs. 1.00, p&lt;0.001), which was exacerbated in coronary VSMCs isolated from db/db mice (5.52±0.51 vs. 3.67±0.60, p&lt;0.05). Alpha‐smooth muscle actin (α‐SMA) trended lower in db/db coronary VSMCs (p=0.07), while co‐culture with hCMECs did not alter α‐SMA expression. Diabetic hCMECs expressed lower Jagged 1 mRNA compared to normal (0.58±0.04 vs. 1.00±0.07; p&lt;0.01); co‐culture of hCMECs and coronary VSMCs caused a robust reduction in Jagged 1 expression in normal cells (0.63±0.06 vs. 1.00±0.07, p&lt;0.001), while no further reduction was observed in co‐cultured diabetic cells. The number of fenestrae in the internal elastic lamina was reduced in diabetic CRMs (0.028±0.005 vs. 0.040±0.001 fen/μm2, p&lt;0.05), while the size of the fenestrae remained unchanged. These data show that Notch3/Jagged 1 expression and VSMC differentiation are altered in diabetic coronary resistance microvessel cells, and VSMC‐EC contact via co‐culture induces differential dysregulation of Notch3/Jagged1 signaling. The data further show a reduction in the number of fenestrae in db/db CRMs, suggesting that the opportunity for Notch3/Jagged1 signaling to occur in vivo may be reduced to contribute to adverse remodeling and stiffness observed in those blood vessels.Support or Funding InformationAHA 15UFEL22760002 to MK; NIH K99HL116769 and NCH to AJT.

Recent advance in molecular angiogenesis in glioblastoma: the challenge and hope for anti-angiogenic therapy.

Glioblastoma (GBM) is the most highly malignant brain tumor in the human central nerve system. In this paper, we review new and significant molecular findings on angiogenesis and possible resistance mechanisms. Expression of a number of genes and regulators has been shown to be upregulated in GBM microvessel cells, such as interleukin-8, signal transducer and activator of transcription 3, Tax-interacting protein-1, hypoxia induced factor-1 and anterior gradient protein 2. The regulator factors that may strongly promote angiogenesis by promoting endothelial cell metastasis, changing the microenvironment, enhancing the ability of resistance to anti-angiogenic therapy, and that inhibit angiogenesis are reviewed. Based on the current knowledge, several potential targets and strategies are proposed for better therapeutic outcomes, such as its mRNA interference of DII4-Notch signaling pathway and depletion of b1 integrin expression. We also discuss possible mechanisms underlying the resistance to anti-angiogenesis and future directions and challenges in developing new targeted therapy for GBM.

Purification and culture of central nervous system pericytes.

Pericytes are found on the abluminal surface of endothelial tubes. The function of these cells is still being elucidated, but they have been shown to be important for development and maintenance of the blood-brain barrier, regulation of angiogenesis and capillary blood flow, and regulation of the neural response to injury and disease. Previously used methods to isolate pericytes have relied on negative selection and prolonged culture of microvessel cells and may lead to populations of pericytes contaminated by other neural cell types. We have developed an immunopanning protocol to specifically purify pericytes from capillaries in the rodent optic nerve. This method relies on a combination of negative and positive selection criteria and allows prospective, acute isolation of pericytes. Use of this method will facilitate studies of pericyte cell biology and function and pericyte-endothelial cell interactions.

Abstract 1287: Anastellin, the fibronectin-derived angiogenesis inhibitor, activates a proangiogenic signature in stromal fibroblasts

Abstract Fibronectin is an extracellular matrix protein that is important in the regulation of angiogenesis, tumor progression and tumor metastasis. Anastellin, a peptide fragment derived from the first Type III repeat in Fibronectin (III1c), has been shown to prevent tumor angiogenesis in mouse models of human cancer and to inhibit serum-dependent growth of endothelial microvessel cells. To determine whether anastellin might have additional roles in the tumor microenvironment, we evaluated the effect of anastellin on gene expression in human dermal fibroblasts. Microarray analysis (Human Cancer PathwayFinder RT2 Profiler PCR Array) indicated that addition of anastellin to fibroblasts induced the expression of several genes including the proangiogenic inflammatory mediators, IL-8 and TNF-α. PCR analysis confirmed that expression of both TNF-α and IL-8 were markedly increased within 2 hrs suggesting a direct effect of anastellin on fibroblast gene expression. ELISA assay of the conditioned medium of anastellin treated cells indicated that the increased gene expression was accompanied by the secretion of IL-8 and TNF-α protein, which was detected between 2-4 hrs of anastellin treatment. Changes in gene expression were preceded by increased phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK as well as the nuclear translocation of NF-κB. PCR and ELISA analysis showed that the NF-κB inhibitor (BAY 11-7082) completely inhibited the induction of IL-8 and TNF-α. The inhibitor of p38 MAPK activity (SB 203580) and the inhibitor of JNK (SP600125) each partially attenuated the induction of IL-8 and TNFα while the combination of inhibitors completely blocked gene expression and the nuclear translocation of NF-κB in response to anastellin. Taken together, these data indicate that treatment of dermal fibroblasts with anastellin causes activation of the p38 and JNK MAPK pathways which converge downstream to promote the NF-κB dependent expression of IL-8 and TNF-α. These data suggest that fibronectin matrix remodeling induces the expression of proangiogenic cytokines by stromal cells present in the tumor microenvironment. This mechanism may represent a novel pathway of tumor resistance to angiogenesis blockade. This work is supported by NIH grant CA69612. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1287.

Astrocyte‐derived glutathione attenuates hemin‐induced cytotoxicity in murine cerebral microvessel

Intracerebral hemorrhage (ICH) induces neurovascular injury following the rupture of a blood vessel within the brain via poorly defined mechanisms. The aim of the present study was to determine whether glio‐vascular communication may restrict hemorrhagic vascular injury. Hemin concentration‐ and time‐dependently increased oxidative cytotoxicity in mouse bEND.3 microvessel cells. Cell death was preceded by a NFκB‐mediated increase in inflammatory gene expression, including up‐regulation of inducible nitric oxide synthase (iNOS) expression and activity. The inhibition of iNOS or the addition of a peroxynitrite decomposition catalyst reduced cell death. Interestingly, co‐treatment with astrocyte conditioned media (ACM) reversed hemin‐induced NF ?ÛB activation, nitrotyrosine formation, and apoptotic cell death via the release of the endogenous antioxidant, reduced glutathione (GSH). Prior treatment of astrocytes with the glutathione‐depleting agent, DL‐buthionine (S,R)‐sulfoximine (BSO) or direct addition of diethyl maleate, a thiol depleting agent, to ACM reversed the observed protection. In contrast, the addition of exogenous GSH or the GSH precursor, N‐acetylcysteine, were protective in bEND.3 cells. These data identify a previously unrecognized vasculoprotective role for astrocyte‐derived GSH and indicate therapeutic targeting of GSH may reduce neurovascular injury following ICH.

Melanotransferrin stimulates t-PA-dependent activation of plasminogen in endothelial cells leading to cell detachment

Tissue plasminogen activator (t-PA) is an extracellular serine protease that converts the proenzyme plasminogen into the broad-spectrum substrate serine protease, plasmin. Plasmin, one of the most potent pro-angiogenic factors, is a key element in fibrinolysis, cell migration, tissue remodeling and tumor invasion. In the present investigation, we assessed the impact of the truncated form of soluble melanotransferrin (sMTf) on plasminogen activation by t-PA and subsequent endothelial cell detachment. Co-treatment of human endothelial microvessel cells with plasminogen, t-PA and sMTf significantly increased plasmin formation and activity in the culture medium. Plasmin generated in the presence of sMTf also led to a 30% reduction in fibronectin detection within cell lysates and to a 9-fold increase within the corresponding cell medium. Moreover, the presence of sMTf increases EC detachment by 6-fold compared to cells treated only with plasminogen and t-PA. Although the addition of α 2-antiplasmin completely prevented plasmin formation and EC detachment, epigallocatechin gallate, GM6001 and a specific antibody directed against MMP-2 prevented cellular detachment without interfering with plasminogen activation. Overall, these data suggest that the anti-angiogenic properties of sMTf may result from local overstimulation of plasminogen activation by t-PA, thus leading to subsequent degradation of the Fn matrix and EC detachment.

Cytotoxic and antiangiogenic activity of AW464 (NSC 706704), a novel thioredoxin inhibitor: an in vitro study

AW464 (NSC 706704) is a novel benzothiazole substituted quinol compound active against colon, renal and certain breast cancer cell lines. NCI COMPARE analysis indicates possible interaction with thioredoxin/thioredoxin reductase, which is upregulated under hypoxia. Through activity on HIF1α, VEGF levels are regulated and angiogenesis controlled. A thioredoxin inhibitor could therefore exhibit enhanced hypoxic toxicity and indirect antiangiogenic effects. In vitro experiments were performed on colorectal and breast cancer cell lines under both normoxic and hypoxic conditions and results compared against those obtained with normal cell lines, fibroblasts and keratinocytes. Antiangiogenic effects were studied using both large and microvessel cells. Indirect antiangiogenic effects (production of angiogenic growth factors) were studied via ELISA. We show that AW464 exerts antiproliferative effects on tumour cell lines as well as endothelial cells with an IC50 of ∼0.5 μM. Fibroblasts are however resistant. Proliferating, rather than quiescent, endothelial cells are sensitive to the drug indicating potential antiangiogenic rather than antivascular action. Endothelial differentiation is also inhibited in vitro. Hypoxia (1% O2 for 48 h) sensitises colorectal cells to lower drug concentrations, and in HT29s greater inhibition of VEGF is observed under such conditions. In contrast, bFGF levels are unaffected, suggesting possible involvement of HIF1α. Thus, AW464 is a promising chemotherapeutic drug that may have enhanced potency under hypoxic conditions and also additional antiangiogenic activity.

Anastellin, a Fragment of the First Type III Repeat of Fibronectin, Inhibits Extracellular Signal-Regulated Kinase and Causes G1 Arrest in Human Microvessel Endothelial Cells

Abstract The formation of a microvascular endothelium plays a critical role in the growth and metastasis of established tumors. The ability of a fragment from the first type III repeat of fibronectin (III1C), anastellin, to suppress tumor growth and metastasis in vivo has been reported to be related to its antiangiogenic properties, however, the mechanism of action of anastellin remains unknown. Utilizing cultures of human dermal microvascular endothelial cells, we provide evidence that anastellin inhibits signaling pathways which regulate the extracellular signal-regulated (ERK) mitogen-activated protein kinase pathway and subsequent expression of cell cycle regulatory proteins. Addition of anastellin to primary microvascular endothelial cells resulted in a complete inhibition of serum-dependent proliferation. Growth inhibition correlated with a decrease in serum-dependent expression of cyclin D1, cyclin A and the cyclin-dependent kinase, cdk4, key regulators of cell cycle progression through G1 phase. Consistent with a block in G1-S transition, anastellin inhibited serum-dependent incorporation of [3H]-thymidine into S-phase nuclei. Addition of anastellin to serum-starved microvessel cells resulted in a time-dependent and dose-dependent decrease in basal levels of phosphorylated MEK/ERK and blocked serum-dependent activation of ERK. Adenoviral infection with Ad.ΔB-Raf:ER, an inducible estrogen receptor-B-Raf fusion protein, restored levels of active ERK in anastellin-treated cells, rescued levels of cyclin D1, cyclin A, and cdk4, and rescued [3H]-thymidine incorporation. These data suggest that the antiangiogenic properties of anastellin observed in mouse models of human cancer may be due to its ability to block endothelial cell proliferation by modulating ERK signaling pathways and down-regulating cell cycle regulatory gene expression required for G1-S phase progression.

Nanogels for oligonucleotide delivery to the brain.

Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood-brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine ("nanogel"). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain.

In vitro demonstration of a saturable transport system for leptin across the blood-brain barrier

A saturable blood-to-brain transport system for leptin across the blood-brain barrier (BBB) has been observed in vivo. Since the main component of the non-fenestrated microvessels of the BBB is the endothelial cell, we established an in vitro culture system of these cerebrovascular cells to study leptin transport and to determine whether the self-inhibition of leptin transport characteristic of a saturable system occurs at this level. The results show that 125I-leptin crossed from the luminal to abluminal side of a monolayer of cerebral microvessel cells significantly faster than the albumin and lactalbumin controls. This transport of 125I-leptin across an in vitro BBB was significantly faster than in the opposite direction and was dose-relatedly inhibited by the addition of unlabeled leptin. Thus, the results establish that the saturable transport system for leptin across the BBB occurs at the level of the endothelial cells of the BBB.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Changes in lipoprotein (a) levels measured by six kit methods during growth hormone treatment of growth hormone deficient adults

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of35SO4into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Endostatin Inhibits Microvessel Formation in the ex Vivo Rat Aortic Ring Angiogenesis Assay

Endostatin has demonstrated potent antiangiogenic and antitumor activity in mouse models. We have investigated the ex vivo rat aortic ring assay and a human vein model to assess the biological activity of murine and human endostatin. Rat aortic rings were exposed to recombinant murine endostatin (Spodoptera frugipera; Calbiochem, San Diego, CA) or recombinant human endostatin (Pichia pastoris; EntreMed, Rockville, MD). After 5 days, murine endostatin (500 μg/ml) demonstrated inhibition of microvessel outgrowth with dose-dependent effects (down to 16 μg/ml). No significant inhibition was observed with human endostatin in the rat assay. Human endostatin at 250 and 500 μg/ml inhibited outgrowths from human saphenous vein rings after a 14-day incubation. Electron microscopy assessed the formation of basal lamina, confirming that the microvessels were progenitors of patent vessels. Immunostaining for Factor VIII or CD34 demonstrated that the microvessel cells were endothelial. BrdU incorporation assays supported the presence of proliferating endothelial cells, correlating with neovascularization from the aortic wall. We conclude that the rat aortic ring assay confirms the antiangiogenic activity of murine but not human endostatin, suggesting that the model may have species specificity. However, the human form shows biological activity against human vascular tissue.