Expression of prolactin mRNA and of prolactin-like proteins in endothelial cells: evidence for autocrine effects

Formation of new capillary blood vessels, termed angiogenesis, is essential for the growth and development of tissues and underlies a variety of diseases including tumor growth. Members of the prolactin hormonal family bind to endothelial cell receptors and have direct effects on cell proliferation, migration and tube formation. Because many angiogenic and antiangiogenic factors are produced by endothelial cells, we investigated whether endothelial cells expressed the prolactin gene. Here we show that bovine brain capillary endothelial cells (BBCEC) in culture express the full-length prolactin messenger RNA, in addition to a novel prolactin transcript, lacking the third exon of the gene. In addition cultures of BBCEC synthesize and secrete prolactin-like immunoreactive proteins with apparent molecular masses of 23, 21 and 14 kDa. The prolactin-like nature of these proteins in supported by the observation that Nb2-cells, a prolactin-responsive cell line, were stimulated to proliferate when co-cultured with endothelial cells and this stimulation was neutralized with prolactin-directed antibodies. Finally, consistent with a possible autocrine effect of endothelial-derived prolactins, polyclonal and monoclonal prolactin antibodies specifically inhibited basal and basis fibroblast growth-factor-stimulated growth of endothelial cells. Taken together, the present findings support the hypothesis of the prolactin gene being expressed in endothelial cells as proteins that could act in an autocrine fashion to regulate cell proliferation.

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The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

Journal of Cell Science ◽

10.1242/jcs.112.10.1599 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1599-1609 ◽

Cited By ~ 2

Author(s):

B.M. Kraling ◽

D.G. Wiederschain ◽

T. Boehm ◽

M. Rehn ◽

J.B. Mulliken ◽

...

Keyword(s):

Cell Culture ◽

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Protein Levels ◽

Matrix Deposition ◽

Capillary Endothelial Cells ◽

Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

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P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells

Life Sciences ◽

10.1016/0024-3205(92)90537-y ◽

1992 ◽

Vol 51 (18) ◽

pp. 1427-1437 ◽

Cited By ~ 197

Author(s):

Akira Tsuji ◽

Tetsuya Terasaki ◽

Yasushi Takabatake ◽

Yoshiyuki Tenda ◽

Ikumi Tamai ◽

...

Keyword(s):

Endothelial Cells ◽

Efflux Pump ◽

Bovine Brain ◽

Drug Efflux ◽

Brain Capillary ◽

Brain Capillary Endothelial Cells ◽

P Glycoprotein ◽

Capillary Endothelial Cells ◽

Drug Efflux Pump

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Angiogenin Acts as a Direct Mitogen on Bovine Brain Capillary Endothelial Cells

Vascular Endothelium ◽

10.1007/978-1-4615-3736-6_29 ◽

1991 ◽

pp. 249-249

Author(s):

M. P. Dehouck ◽

M. Chamoux ◽

J. C. Fruchart ◽

G. Spik ◽

J. Montreuil ◽

...

Keyword(s):

Endothelial Cells ◽

Bovine Brain ◽

Brain Capillary ◽

Brain Capillary Endothelial Cells ◽

Capillary Endothelial Cells

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Receptor-mediated transcytosis of transferrin through blood-brain barrier endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.270.4.h1149 ◽

1996 ◽

Vol 270 (4) ◽

pp. H1149-H1158 ◽

Cited By ~ 16

Author(s):

L. Descamps ◽

M. P. Dehouck ◽

G. Torpier ◽

R. Cecchelli

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Bovine Brain ◽

Brain Barrier ◽

Double Labeling ◽

Brain Capillary ◽

Brain Capillary Endothelial Cells ◽

Blood Brain ◽

Capillary Endothelial Cells ◽

A Cell

A cell culture model of the blood-brain barrier consisting of a coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes has been used to examine the mechanism of iron transport to the brain. Binding experiments showed that BBCECs express 35,000 high-affinity (concn at 50% receptor saturation = 11.3 +/- 2.1 nM) transferin (Tf) receptors per cell. In contrast to apo-transferrin (apoTf) we observed a specific transport of holo-transferrin (holoTf) across BBCECs. This transport was inhibited completely at low temperature. Moreover, the anti-Tf receptor antibody (OX-26) competitively inhibited holoTf uptake by BBCECs. Pulse-chase experiments demonstrated that only 10% of Tf was recycled to the luminal side of the cells, whereas the majority of Tf was transcytosed to the abluminal side; double-labeling experiments clearly demonstrated that iron crosses BBCECs bound to Tf. No intraendothelial degradation of Tf was observed, suggesting that the intraendothelial pathway through BBCECs bypasses the lysosomal compartment. These results clearly show that the iron-Tf complex is transcytosed across brain capillary endothelial cells by a receptor-mediated pathway without any degradation.

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Abstract 14820: Sirt7 Deficiency in Blood Vessel Components Impairs Vascular Function by Inhibiting Cell Cycle- and Inflammatory-Related Protein Expression

Circulation ◽

10.1161/circ.132.suppl_3.14820 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Yuichi Kimura ◽

Yasuhiro Izumiya ◽

Satoshi Araki ◽

Satoru Yamamura ◽

Yoshiro Onoue ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Blood Flow ◽

Vascular Function ◽

Vascular Dysfunction ◽

Vascular Diseases ◽

Tube Formation ◽

Hindlimb Ischemia ◽

Proliferation Assay

Introduction: Aging is a well-established cardiovascular risk factor and associated with vascular dysfunction. Sirt7, one of the members of mammalian sirtuin family, is thought to be involved in age-related diseases. However, little is known about the relative contribution of Sirt7 in vascular dysfunction. Hypothesis: Sirt7 maintains vascular cell functions and its deficiency plays a critical role in vascular diseases. Methods: Sirt7 loss- and gain-of-function experiments were performed with human aortic smooth muscle cells (HAoSMCs) and human umbilical vein endothelial cells (HUVECs). In vivo, blood flow recovery was evaluated by hindlimb ischemia model in homozygous Sirt7 deficient (Sirt7-/-) and wild-type (WT) mice. Irradiated WT mice were intravenously received bone marrow (BM) cells from WT or Sirt7 -/- mouse to achieve BM transfer. Results: An RNAi-medicated Sirt7 knockdown resulted in a significant inhibition of HAoSMCs proliferation following serum or Platelet-derived growth factor BB (PDGF-BB) stimulation as determined by cell count, BrdU cell proliferation assay and MTS proliferation assay. Knockdown of endogenous Sirt7 also reduced cell migration as revealed by Boyden chamber migration assay. The Cyclin D1 and Cyclin dependent kinase 2 (CDK2) protein levels were significantly decreased in Sirt7 siRNA-treated HAoSMCs in response to serum or PDGF-BB stimulation. In endothelial cells, knockdown of Sirt7 attenuated tube formation, proliferation and migration. These changes were accompanied by reduced ERK activation and VCAM-1 mRNA and protein expression in Sirt7 siRNA-treated HUVECs. Conversely, overexpression of Sirt7 by adenovirus enhanced tube formation and cell proliferation. In vivo, blood flow recovery in response to hindlimb ischemia was significantly attenuated in Sirt7-/- mice compared with WT mice. There was no difference in blood flow recovery between WT mice transplanted with WT or Sirt7-/- BM cells suggesting that Sirt7 deficiency in vascular cells have a predominant effect on attenuated blood flow recovery in response to hindlimb ischemia. Conclusions: Sirt7 in blood vessel components have an important role in maintenance of vascular function. Sirt7 could be a promising therapeutic target for vascular diseases.

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MicroRNA-126-3p Inhibits Angiogenic Function of Human Lung Microvascular Endothelial Cells via LAT1 (L-Type Amino Acid Transporter 1)-Mediated mTOR (Mammalian Target of Rapamycin) Signaling

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.119.313800 ◽

2020 ◽

Vol 40 (5) ◽

pp. 1195-1206 ◽

Cited By ~ 2

Author(s):

Danting Cao ◽

Andrew M. Mikosz ◽

Alexandra J. Ringsby ◽

Kelsey C. Anderson ◽

Erica L. Beatman ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Amino Acid ◽

Cell Apoptosis ◽

Human Lung ◽

Mammalian Target Of Rapamycin ◽

Tube Formation ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial

Objective: MicroRNA-126-3p (miR-126) is required for angiogenesis during organismal development or the repair of injured arterial vasculature. The role of miR-126 in lung microvascular endothelial cells, which are essential for gas exchange and for lung injury repair and regeneration, remains poorly understood. Considering the significant heterogeneity of endothelial cells from different vascular beds, we aimed to determine the role of miR-126 in regulating lung microvascular endothelial cell function and to elucidate its downstream signaling pathways. Approach and Results: Overexpression and knockdown of miR-126 in primary human lung microvascular endothelial cells (HLMVEC) were achieved via transfections of miR-126 mimics and antisense inhibitors. Increasing miR-126 levels in HLMVEC reduced cell proliferation, weakened tube formation, and increased cell apoptosis, whereas decreased miR-126 levels stimulated cell proliferation and tube formation. Whole-genome RNA sequencing revealed that miR-126 was associated with an antiangiogenic and proapoptotic transcriptomic profile. Using validation assays and knockdown approaches, we identified that the effect of miR-126 on HLMVEC angiogenesis was mediated by the LAT1 (L-type amino acid transporter 1), via regulation of mTOR (mammalian target of rapamycin) signaling. Furthermore, downregulation of miR-126 in HLMVEC inhibited cell apoptosis and improved endothelial tube formation during exposure to environmental insults such as cigarette smoke. Conclusions: miR-126 inhibits HLMVEC angiogenic function by targeting the LAT1-mTOR signaling axis, suggesting that miR-126 inhibition may be useful for conditions associated with microvascular loss, whereas miR-126 augmentation may help control unwanted microvascular angiogenesis.

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A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

Proteome Science ◽

10.1186/1477-5956-8-57 ◽

2010 ◽

Vol 8 (1) ◽

pp. 57 ◽

Cited By ~ 11

Author(s):

Gwënaël Pottiez ◽

Barbara Deracinois ◽

Sophie Duban-Deweer ◽

Roméo Cecchelli ◽

Laurence Fenart ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Large Scale ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Barrier Properties ◽

Bovine Brain ◽

Brain Capillary ◽

Capillary Endothelial Cells

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Roundabout4 Suppresses Glioma-Induced Endothelial Cell Proliferation, Migration and Tube Formation in Vitro by Inhibiting VEGR2-Mediated PI3K/AKT and FAK Signaling Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000373982 ◽

2015 ◽

Vol 35 (5) ◽

pp. 1689-1705 ◽

Cited By ~ 27

Author(s):

Heng Cai ◽

Yixue Xue ◽

Zhen Li ◽

Yi Hu ◽

Zhenhua Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Signaling Pathways ◽

Conditioned Medium ◽

Tube Formation ◽

Endothelial Cell Proliferation ◽

Akt Inhibitor ◽

Over Expression

Background and Aims: Endothelial cell (EC) proliferation, migration, and tube formation are the critical steps for tumor angiogenesis, which is involved in the formation of new tumor blood vessels. Roundabout4 (Robo4), a new member of Robo proteins family, is specifically expressed in endothelial cells. This study aimed to investigate the effects of Robo4 on glioma-induced endothelial cell proliferation, migration and tube formation in vitro. Methods and Results: We found that Robo4 was endogenously expressed in Human Brain Microvascular Endothelial Cells (HBMECs), while Robo4 was significantly down-regulated in endothelial cells cultured in glioma conditioned medium. Robo4 over-expression remarkably suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro. In addition, Robo4 influenced the glioma-induced angiogenesis via binding to its ligand Slit2. Further studies demonstrated that the knockdown of Robo4 up-regulated the phosphorylation of VEGFR2, PI3K, AKT and FAK in EC cultured in glioma conditioned medium. VEGFR2 inhibitor SU-1498, AKT inhibitor LY294002 and FAK inhibitor 14 (FAK inhibitor) blocked the Robo4 knockdown-mediated alteration in glioma angiogenesis in vitro. Conclusion: Our results proved that Robo4 suppressed glioma-induced endothelial cell proliferation, migration and tube formation in vitro by inhibiting VEGR2-mediated activation of PI3K/AKT and FAK signaling pathways.

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