Endothelial Cell-conditioned Medium Research Articles

STAT5 and Prolactin Participate in a Positive Autocrine Feedback Loop That Promotes Angiogenesis

We have shown previously that the murine prolactin/growth hormone family member proliferin plays a pivotal role in angiogenesis induced by the FGF2/STAT5 signaling cascade. To delineate the signaling pathway downstream of STAT5 in the human system, where proliferin does not exist, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in hCMEC/D3 human brain endothelial cells. We found that conditioned medium from CA-STAT5A- but not from DN-STAT5A-overexpressing endothelial cells (EC) is sufficient to induce EC migration and tube formation but not proliferation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. We identified prolactin (PRL) as a candidate autocrine factor. CA-STAT5A expression stimulates PRL production at the RNA and protein level, and STAT5A binds to the PRL promoter region, suggesting direct transcriptional regulation. Medium conditioned by CA-STAT5A-overexpressing EC induces phosphorylation of the PRL receptor and activates MAPK. Knockdown of PRL expression by shRNA or blocking of PRL activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. The addition of recombinant PRL restores this activity. STAT5A-induced PRL in the conditioned medium can activate STAT5, STAT1, and to a lesser extent STAT3 in hCMEC/D3 cells, suggesting the existence of a positive feedback loop between STAT5 and PRL that promotes angiogenesis. Furthermore, we find that VEGF, a potent proangiogenic factor, is induced by activation of STAT5A, and VEGF induction depends on PRL expression. These observations demonstrate a STAT5/PRL/VEGF signaling cascade in human brain EC and implicate PRL and VEGF as autocrine regulators of EC migration, invasion, and tube formation.

Inhibition of apelin expression switches endothelial cells from proliferative to mature state in pathological retinal angiogenesis

The recruitment of mural cells such as pericytes to patent vessels with an endothelial lumen is a key factor for the maturation of blood vessels and the prevention of hemorrhage in pathological angiogenesis. To date, our understanding of the specific trigger underlying the transition from cell growth to the maturation phase remains incomplete. Since rapid endothelial cell growth causes pericyte loss, we hypothesized that suppression of endothelial growth factors would both promote pericyte recruitment, in addition to inhibiting pathological angiogenesis. Here, we demonstrate that targeted knockdown of apelin in endothelial cells using siRNA induced the expression of monocyte chemoattractant protein-1 (MCP-1) through activation of Smad3, via suppression of the PI3K/Akt pathway. The conditioned medium of endothelial cells treated with apelin siRNA enhanced the migration of vascular smooth muscle cells, through MCP-1 and its receptor pathway. Moreover, in vivo delivery of siRNA targeting apelin, which causes exuberant endothelial cell proliferation and pathological angiogenesis through its receptor APJ, led to increased pericyte coverage and suppressed pathological angiogenesis in an oxygen-induced retinopathy model. These data demonstrate that apelin is not only a potent endothelial growth factor, but also restricts pericyte recruitment, establishing a new connection between endothelial cell proliferation signaling and a trigger of mural recruitment.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-013-9349-6) contains supplementary material, which is available to authorized users.

1137 FGF2-MEDIATED RECIPROCAL TUMOR CELL-ENDOTHELIAL CELL INTERPLAY CONTRIBUTES TO THE GROWTH OF CHEMO-RESISTANT BLADDER CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research (III)1 Apr 20131137 FGF2-MEDIATED RECIPROCAL TUMOR CELL-ENDOTHELIAL CELL INTERPLAY CONTRIBUTES TO THE GROWTH OF CHEMO-RESISTANT BLADDER CANCER CELLS Yule Chen, Kaijie Wu, Jin Zeng, Lei Li, and Dalin He Yule ChenYule Chen Xi'an, China, People's Republic of More articles by this author , Kaijie WuKaijie Wu Xi'an, China, People's Republic of More articles by this author , Jin ZengJin Zeng Xi'an, China, People's Republic of More articles by this author , Lei LiLei Li Xi'an, China, People's Republic of More articles by this author , and Dalin HeDalin He Xi'an, China, People's Republic of More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.752AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chemo-resistance is of outstanding importance for the limited results of chemotherapy in bladder cancer, because chemo-resistant cancer cells probably provide the main sources of recurrent tumors. However, the mechanisms by which these chemo-resistant cells develop into recurrent tumors remain unclear. It has been recently reported that the proangiogenic factor FGF2, a molecule associated with the recruitment of endothelial cells, was identified as an independent prognostic factor for bladder cancer recurrence after chemotherapy. Here, we explored the mechanisms of chemo-resistant cells mediated bladder cancer recurrence, focusing on FGF2-associated endothelial cell recruitment mediated by drug-resistant bladder cancer cells as well as the possible effect of tumor cell-endothelial cell interaction on chemo-resistant cancer cell growth. METHODS A novel multidrug resistant subline (253J/DOX) of human bladder cancer 253J cell line was generated by culturing these cells in gradually increasing doses of doxorubicin (DOX) for 12 months. The chemo-resistant phenotype was identified. The expression levels of P-glycoprotein and FGF2 and the interaction between chemo-resistant tumor cells and endothelial cells was assessed. RESULTS As compared with parental 253J cells, 253J/DOX cells expressed higher level of the drug transporter P-glycoprotein, accompanied with significant resistance to doxorubicin, pirarubicin and vincristine. Downregulation of P-glycoprotein expression by specific siRNA reversed this multidrug resistant phenotype. In 253J/DOX cells, the expression and secretion levels of FGF2 were both markedly increased. The conditioned medium of 253J/DOX cells recruited abundant endothelial cells and promoted endothelial cell proliferation and tube formation potential, which was blocked by FGF2 neutralizing antibody. Furthermore, co-culture with endothelial cells or conditioned medium of endothelial cells significantly enhanced the proliferative capacity of 253J/DOX cells. CONCLUSIONS Our findings provided the first evidences that FGF2-mediated tumor cell-endothelial cell interplay facilitated the growth of chemo-resistant bladder cancer, which may possibly contribute to bladder cancer recurrence after chemotherapy. On one hand, the residual chemo-resistant cells after chemotherapy can recruit endothelial cells by secreting proangiogenic factors, while on the other hand the recruited endothelial cells may promote residual cancer cell growth. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e464 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yule Chen Xi'an, China, People's Republic of More articles by this author Kaijie Wu Xi'an, China, People's Republic of More articles by this author Jin Zeng Xi'an, China, People's Republic of More articles by this author Lei Li Xi'an, China, People's Republic of More articles by this author Dalin He Xi'an, China, People's Republic of More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Angiogenesis Induced by Signal Transducer and Activator of Transcription 5A (STAT5A) Is Dependent on Autocrine Activity of Proliferin

Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation.

AnIn VitroStudy of Differentiation of Hematopoietic Cells to Endothelial Cells

Bone-marrow-derived endothelial progenitor cells (BM-EPCs) contribute to postnatal neovascularization and therefore are of great interest for cell therapies to treat ischemic diseases. However, their origin and characteristics are still in controversy. In this paper, we identified the origin/lineage of the BM-EPCs that were isolated from bone marrow mononuclear cells and differentiated with the induction of bone-marrow endothelial-cellconditioned medium (ECCM). BM-EPCs were characterized in terms of phenotype, lineage potential, and their functional properties. Endothelial cell colonies derived from BM-EPC were cultured with ECCM for 3 months. Cultured EPC colony cells expressed endothelial cell markers and formed the capillary-like network in vitro. EPC colony cells expressed differential proliferative capacity; some of the colonies exhibited a high proliferative potential (HPP) capacity up to 20 population doublings. More importantly, these HPP-EPCs expressed hematopoietic marker CD45, exhibited endocytic activities, and preserved some of the myeloid cell activity. In addition, the HPP-EPCs secrete various growth factors including VEGF and GM-CSF into the culture medium. The results demonstrate that these EPCs were primarily derived from hematopoietic origin of early precursor cells and maintained high proliferative potential capacity, a feature with a significant potential in the application of cell therapy in ischemic diseases.

Ancrod and Fibrin Formation

Ancrod, derived from Malayan pit viper venom, has been tested as ischemic stroke treatment in clinical trials with inconsistent results. We studied the actions of ancrod on fibrinolysis pathways in patient plasma samples and endothelial cell culture systems. We analyzed fibrinogen levels during the first 6 hours of ancrod infusion in patients entered in the Stroke Treatment with Ancrod Trial. For the in vitro study, human brain microvascular endothelial cells incubated with plasminogen or with human brain microvascular endothelial cell-conditioned medium were co-incubated with ancrod and fibrinogen under normal or oxygen-glucose deprivation conditions over 6 hours. Fibrinogen levels decreased both in vivo and in vitro. Ancrod generated fibrinopeptide A, caused visible clot formation, and reduced levels of tissue-type plasminogen activator antigen in the human brain microvascular endothelial cell system and in a cell-free system with conditioned media. The in vitro results indicate that ancrod causes local fibrin formation and secondary depletion of tissue-type plasminogen activator by binding to fibrin clot. Ancrod-induced fibrin formation could result in cerebral microvascular occlusion and may explain the suboptimal clinical effects of ancrod in human stroke trials.

MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin− progenitor cells in ischaemia

MicroRNA-126 (miR-126), which is enriched in endothelial cells, plays a role in angiogenesis. Based on the seed sequence, miR-126 can also be predicted to regulate vasculogenesis by modulating the endothelial expression of stromal cell-derived factor-1 (SDF-1). Using miR-reporter constructs, we first validated that miR-126 inhibits SDF-1 expression in endothelial cells in vitro. Next, we investigated the potential relevance of this observation with respect to the mobilization of progenitor cells. For this, we studied the migration of human CD34+ progenitor cells towards chemotactic factors present in endothelial cell-conditioned medium. Antagomir-induced silencing of miR-126 elevated SDF-1 expression by human umbilical vein endothelial cells and enhanced migration of the CD34+ cells. In a murine model of hind limb ischaemia, a striking increase in the number of circulating Sca-1(+)/Lin(-) progenitor cells in antagomir-126-treated mice was observed when compared with scramblemir-treated controls. Immunohistochemical staining of capillaries in the post-ischaemic gastrocnemius muscle of miR-126-silenced mice revealed elevated SDF-1 expressing CD31-positive capillaries, whereas a mobilizing effect of miR-126 inhibition was not detected in healthy control animals. miR-126 can regulate the expression of SDF-1 in endothelial cells. In the context of an ischaemic event, systemic silencing of miR-126 leads to the mobilization of Sca-1(+)/Lin(-) progenitor cells into the peripheral circulation, potentially in response to elevated SDF-1 expression by endothelial cells present in the ischaemic tissue.

Postnatal vasculogenesis: Identification, growth and function of endothelial progenitor cells from peripheral blood mononuclear cells

To identify best growth conditions for the in vitro differentiation of human peripheral blood mono- nuclear cells (PBMNCs) into endothelial progenitor cells (EPCs), PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, and IGF-I; M199 with bovine retina-derived growth sup- plement (RDGS); human umbilical vein endothelial cell (HUVEC) conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in M199 with RDGS (cocul- tures system); PHA burst (10lL/10 6 cells) for 24 h and culture in M199 with RDGF. EPCs were identified by FACS using mAbs for endothelial markers. EPCs mi- gration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. Matrigel was used to assess in vitro angiogenesis capability. Spindle-shaped and attached cells sprouted with growth factors, differentiating in EPCs within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDGS, numerous large cell clusters appeared within 1 week but the number of clusters decreased during culture. After stimulation with PHA, many cells clusters and some adherent EC-like cells were observed within 7 days; their number and size increased within 14 days. With HUVEC conditioned medium, spindle- shaped cells were observed after 10 days. FACS con- firmed the endothelial phenotype. Cocultures induced the appearance of double-labeled EPCs expressing endothelial antigens starting from 7 days. EPCs were able to migrate in response to VEGF and to incorporate in capillary-like structures formed by HUVECs on Matrigel. PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions. The contact with mature endothelial cells (ECs) makes this process quicker.

The Effects of the Endothelium on Adrenal Steroidogenesis and Growth are Mainly Mediated by Proteins Other than Endothelin-1

The endothelium releases factors stimulating the adrenal cortex. It is also known that endothelin-1 (ET-1) promotes generation of cortisol and aldosterone, and proliferation of adrenocortical cells. The aim of the study was to find out whether the effect of the endothelium on adrenocortical cells is dominated by the action of ET-1. The effects of endothelial cell-conditioned medium (ECCM), obtained during growth of human umbilical cord vein endothelial cells, on aldosterone and cortisol release by cells of the adrenocortical cancer cell-line NCI-H295R and the promoter activity of steroidogenic acute-regulatory protein (StAR) were studied. The effect of ECCM on proliferation of human primary normal adrenocortical and NCI-H295R cells was also investigated. Concentration-dependent increases in cortisol release that reached 192.7 ± 62.8 in percent of basal secretion, in aldosterone release that reached 188.2 ± 52.3 in percent of basal secretion, and in proliferation after stimulation with ECCM at concentrations of 10-50% were found. ECCM significantly activated the StAR promoter 3-fold in NCI-H295R cells if the ECCM was not pretreated with pronase. These effects of the endothelium were not reversed after co-incubation with endothelin receptor antagonists and could not be mimicked by incubation with endothelin-1. In conclusion, the cultured endothelial cells secrete a protein that stimulates steroidogenesis in adrenal cells and their growth. It was also shown that the ET-1 does not mediate the effect of ECCM on the NCI-H295R cell line.

Abstract 1281: Endothelial cells promote the cancer stem cell phenotype of human colorectal cancer cells

Abstract Background: Cancer stem cells (CSC), which possess the properties of self-renewal, multi-potential differentiation and tumor initiation, can arise either from normal stem cells or due to the influence of the tumor micro-environment. This study was done to determine the potential role of endothelial cell (EC) derived paracrine factors on promoting the CSC phenotype in human colorectal cancer (CRC) cells. Methods: RFP-labeled human CRC cells HCT116 were co-cultured with RF24 ECs (immortalized HUVECs) under low serum conditions. After FAC-sorting for the CRC cells, the CSC population was analyzed by 1) the Aldefluor assay, 2) sphere forming ability, and 3) Western blot analysis for the CSC markers CD133 and CD44. In addition, low serum condition media obtained from either ECs or parental CRC cells (control), was added to CRC cells to determine its effect on the CSC phenotype. In order to characterize the apoptotic effect of EC derived factors on CRC cells, we performed Annexin V staining by FACS, PARP cleavage, Caspase 3 cleavage, and Bcl2 levels by Western blotting. Using MTT assay, we determined the effect of EC derived factors on chemo-sensitivity of CRC cells exposed to 5-FU. Results: Co-culturing of CRC cells with ECs markedly increased the ALDH-positive population from 5% to 18%, and the sphere forming ability by more than 4-fold in CRC cells. In addition, CRC cells also displayed increased CD133 (6-fold) and CD44 (4-fold) protein levels. Furthermore, this effect could be mimicked simply by co-culturing CRC cells with conditioned media obtained from ECs. Similarly, treatment of CRC cells with conditioned medium from ECs significantly increased the ALDH-positive population from 4% to 8%, sphere forming ability >3-fold, and the expression of CD133 (7-fold) and CD44 (6-fold). Conditioned medium from ECs, concomitantly decreased spontaneous apoptosis in CRC cells as demonstrated by a 3-fold decrease in Annexin V-positive population (21% to 7%), down-regulation of cleaved PARP and Caspase 3, and up-regulation of Bcl2. CRC cells exposed to EC conditioned medium also displayed decreased sensitivity to 5-FU [>10-fold increase of the IC50 from 2.0 μg/ml to 22.3 μg/ml (p<0.05)]. Conclusions: Exposure of CRCs to ECs increased CSC properties, and decreased spontaneous apoptosis. Since, this effect can also be mimicked by the conditioned media, additional experiments to identify and validate the soluble factors that mediate this effect are in progress. 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 1281.

Effects of Activated Protein C on Ventilator-Induced Lung Injury in Rats

Background: Mechanical ventilation with a high tidal volume (VT) increases lung and systemic plasminogen activator inhibitor (PAI)-1 levels and alveolar fibrin deposition. Activated protein C (APC) may decrease PAI activity in endothelial cell-conditioned medium and thus enhance fibrinolysis. Objectives: The aims of this study were to test the hypothesis that APC can neutralize PAI-1 activity and improve lung function in an animal model of ventilator-induced lung injury. Methods: Rats were ventilated with a high-volume zero positive end-expiratory pressure (PEEP; HVZP) protocol by a volume-cycled ventilator for 2 h at a VT of 30 ml/kg, a respiratory rate of 25 breaths/min, and an FiO₂ of 0.21. Fifteen minutes before ventilation, the rats received intravenous APC (250 µg/kg, HVZP+APC group) or normal saline (vehicle; HVZP group). Another group that received no ventilation served as the control group. Results: Levels of arterial blood gas tension were comparable between the two ventilation groups throughout the study period. Rats treated with the HVZP protocol exhibited significantly higher total protein and macrophage inflammatory protein-2 concentrations in bronchoalveolar lavage fluid (BALF) and higher lung PAI-1 mRNA expression and plasma active PAI-1 levels than did the control group. Administration of APC tended to reduce the BALF protein content and systemic PAI-1 activity but did not improve the lung histology in the HVZP+APC group. Plasma levels of D-dimers were comparable among the three study groups. Conclusions: These results suggest that APC administered at a higher dosage might improve lung function by reducing alveolar protein leakage and systemic coagulation.

Effects of an endothelial cell‐conditioned medium on the hematopoietic and endothelial differentiation of embryonic stem cells

We have examined the effect of mouse bone marrow endothelial cell-conditioned medium (mEC-CM) on hematopoietic and endothelial differentiation of mouse embryonic stem cells (mESCs). mEC-CM can efficiently promote the differentiation of mESCs into Flk(+) cells and hematopoietic colony-forming cells. mEC-CM proved to be as potent as a cytokine cocktail comprised of VEGF, bFGF, IGF and EGF. After inducing mESCs with mEC-CM, cobblestone-like cells were mechanically selected and identified which had the ability to incorporate DiI-Ac-LDL. DiI-Ac-LDL-positive cells were endothelial-like cells due to their expression of CD31 and Flk1, ability to bind to UEA1 and capacity to form capillary-like tube structures on matrigel. In conclusion, mEC-CM can efficiently promote the differentiation of mESCs into endothelial cells and hematopoietic colony-forming cells. The differentiated endothelial-like cells can be isolated by using DiI-Ac-LDL labeling and mechanical selection.

Intact and injured endothelial cells differentially modulate postnatal murine forebrain neural stem cells

Neural stem cells (NSCs) persist in the forebrain subventricular zone (SVZ) within a niche containing endothelial cells. Evidence suggests that endothelial cells stimulate NSC expansion and neurogenesis. Experimental stroke increases neurogenesis and angiogenesis, but how endothelial cells influence stroke-induced neurogenesis is unknown. We hypothesized intact or oxygen–glucose deprived (OGD) endothelial cells secrete factors that enhance neurogenesis. We co-cultured mouse SVZ neurospheres (NS) with endothelial cells, or differentiated NS in endothelial cell-conditioned medium (ECCM). NS also were expanded in ECCM from OGD-exposed (OGD-ECCM) endothelial cells to assess injury effects. ECCM significantly increased NS production. NS co-cultured with endothelial cells or ECCM generated more immature-appearing neurons and oligodendrocytes, and astrocytes with radial glial-like/reactive morphology than controls. OGD-ECCM stimulated neuroblast migration and yielded neurons with longer processes and more branching. These data indicate that intact and injured endothelial cells exert differing effects on NSCs, and suggest targets for stimulating regeneration after brain insults.

Cerebral Vasospasm: New Strategies in Research and Treatment

Cerebral Vasospasm: New Strategies in Research and Treatment

Evidence for the involvement of endothelial cell products in adrenal CITED2 expression

The adrenal gland contains a well-organized network of blood vessels, and adrenocortical cells are situated in close proximity to endothelial cells. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, including the involvement of endothelial-cell-derived factors. Interestingly, a CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which is necessary for adrenal development, has been linked to aldosterone synthesis. We have therefore examined the effects of endothelial-cell-conditioned medium (ECCM), as produced during the incubation of human umbilical vein endothelial cells for 24 h, on the promoter activity and mRNA and protein expression of CITED2 in adrenocortical cells as represented by the NCI-H295R cell line. We have found a dose-dependent effect of ECCM on CITED2 promoter activity; this peaks at 480%. Activation of the CITED2 promoter occurs in parallel to an increase in CITED2 messenger RNA (as quantified by real-time polymerase chain reaction) and protein. The stimulatory effect of ECCM can be reversed by blocking mitogen-activated protein kinase activity with the MEK1-inhibitor PD98059. We conclude that products secreted by endothelial cells control not only steroidogenesis, but also factors that are important for adrenocortical development, thereby highlighting the role of cellular interactions within adrenocortical development and physiology.

Amino acid‐mediated heterotypic interaction governs performance of a hepatic tissue model

Tissue-engineered models that mimic in vivo tissue organization offer the potential of capturing complex signaling pathways in vitro. In the liver, hepatocytes and endothelial cells are closely associated but separated by the extracellular matrix of the space of Disse. This unique configuration was mimicked by embedding primary hepatocytes in collagen gel and overlaying the matrix with endothelial cells. We demonstrate that during the first few days of culture, the secretion of albumin and fibrinogen was 2-fold higher in cocultures compared to hepatocytes alone. Hepatocyte function in both cultures stabilized to a similar level during the second week, suggesting that endothelial cells can induce the early recovery of hepatocytes after isolation and seeding. Endothelial cell-conditioned medium reproduced the effect of coculture in a dose-dependent fashion, suggesting a role for endothelial cell-derived soluble factors. Endothelial cell-conditioned medium increased mRNA levels of various acute-phase proteins such as albumin, fibrinogen, transferrin, and alpha-macroglobulin in hepatocytes. Surprisingly, the effect of endothelial cell-conditioned medium was not mediated by growth factors or cytokines, or by secreted extracellular matrix, but by the release of the amino acid proline, which mediates endogenous collagen synthesis by hepatocytes. These findings suggest an important role for proline secretion by endothelial cells as a paracrine factor regulating hepatocyte function.

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.

Endothelial factors mediate aldosterone release via PKA-independent pathways

Aldosterone synthesis is primarily regulated by angiotensin II and potassium ions. In addition, endothelial cell-secreted factors have been shown to regulate mineralocorticoid release. We analyzed the pathways that mediate endothelial cell-factor-induced aldosterone release from adrenocortical cells, NCI-H295R using endothelial cell-conditioned medium (ECM). The cAMP antagonist Rp-cAMP caused a 44% decrease in the ECM-induced aldosterone release but inhibition of cAMP-dependent PKA had no effect on aldosterone release. Interestingly, inhibition of cAMP-regulated guanine nucleotide exchange factor Epac with brefeldin-A decreased the ECM-induced aldosterone release by 45%. Similarly, inhibition of p38 MAP-kinase; PI-3-kinase and PKB significantly reduced the ECM-induced aldosterone release whereas inhibition of ERK1/2 and PKC did not decrease aldosterone release. These results provide evidence for the existence of a cAMP-dependent but PKA-independent pathway in mediating the ECM-induced aldosterone release and the significant influence of more than one signaling mechanism.

The Endothelium Secretes Interleukin-6 (IL-6) and Induces IL-6 and Aldosterone Generation by Adrenocortical Cells

Endothelial cells have been shown to induce adrenal steroidogenesis and to enhance aldosterone secretion via angiotensin II and endothelin 1-independent mechanisms. It has been demonstrated that endothelial cells and adrenocortical cells are capable of producing interleukin-6 (IL-6) and IL-6 is a factor known to stimulate adrenal cortisol secretion. We therefore asked whether endothelial cells have an effect on adrenal IL-6 generation and whether IL-6 mediates biosynthesis of aldosterone as is observed after exposure of adrenocortical cells to endothelial cell-conditioned medium (ECCM). Cells from the adrenocortical cancer cell line NCI-H295R were incubated with ECCM produced from human umbilical vein endothelial cells at increasing concentrations. As detected by an enzyme-linked immunosorbent assay, pure ECCM significantly increased IL-6 protein secretion by cultured adrenocortical cells in a dose-dependent fashion, to a 18.0+/-2.0 pg/mL (mean+/-SEM). This was paralleled by an enhanced IL-6 promoter activity as determined with the transfection of an IL-6-promoter-luciferase reporter gene construct. Pure ECCM also induced aldosterone secretion by adrenocortical cells more than three times that of controls with serum-free medium. ECCM PER SE contains significant amounts of IL-6 protein. However, blockade of IL-6 signal transduction did not interfere with aldosterone synthesis. These data suggest that endothelial cells secrete IL-6 and that endothelial cell-derived factors regulate adrenal IL-6 synthesis which does not alter adrenal aldosterone secretion. Our findings support the hypothesis that the endothelium and the adrenal gland may play a role in the development of some forms of hypertension and - more speculative - inflammation.