Crossveinless 2 regulates bone morphogenetic protein 9 in human and mouse vascular endothelium

Abstract The importance of morphogenetic proteins (BMPs) and their antagonists in vascular development is increasingly being recognized. BMP-4 is essential for angiogenesis and is antagonized by matrix Gla protein (MGP) and crossveinless 2 (CV2), both induced by the activin receptor like-kinase 1 (ALK1) when stimulated by BMP-9. In this study, however, we show that CV2 preferentially binds and inhibits BMP-9 thereby providing strong feedback inhibition for BMP-9/ALK1 signaling rather than for BMP-4/ALK2 signaling. CV2 disrupts complex formation involving ALK2, ALK1, BMP-4, and BMP-9 required for the induction of both BMP antagonists. It also limits VEGF expression, proliferation, and tube formation in ALK1-expressing endothelial cells. In vivo, CV2 deficiency translates into a dysregulation of vascular BMP signaling, resulting in an abnormal endothelium with increased endothelial cellularity and expression of lineage markers for mature endothelial cells. Thus, mutual regulation by BMP-9 and CV2 is essential in regulating the development of the vascular endothelium.

Download Full-text

SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development

Molecular and Cellular Biology ◽

10.1128/mcb.00239-15 ◽

2015 ◽

Vol 35 (12) ◽

pp. 2165-2172 ◽

Cited By ~ 6

Author(s):

Jonathon Marks-Bluth ◽

Anchit Khanna ◽

Vashe Chandrakanthan ◽

Julie Thoms ◽

Thomas Bee ◽

...

Keyword(s):

Endothelial Cells ◽

Selective Advantage ◽

Regulatory Elements ◽

Bmp Signaling ◽

Tissue Specific ◽

Morphogenetic Protein ◽

Smad Signaling Pathway ◽

Endothelial Development

The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin,in vitroreporter assay, andin vivotransgenic data to identify and validateSmad1+63 and theSmad5promoter as tissue-specificcis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortasin vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cellsin vitro. In contrast, CD31+cKit−endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reducedSmad1but notSmad5transcript levels. This is suggestive of a degree ofin vivoselection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.

Download Full-text

Abstract 17763: Inhibition of Bone Morphogenetic Protein Signaling Reduces Endothelial-Mesenchymal Transition and Vascular Smooth Muscle Cell Calcification Associated with Matrix Gla Protein Deficiency

Circulation ◽

10.1161/circ.130.suppl_2.17763 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Megan F Burke ◽

Caitlin O’Rourke ◽

Trejeeve Martyn ◽

Hannah R Shakartzi ◽

Timothy E Thayer ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Bone Morphogenetic Protein ◽

Vascular Calcification ◽

Bmp Signaling ◽

Matrix Gla Protein ◽

Wild Type ◽

Mesenchymal Transition ◽

Morphogenetic Protein ◽

Endothelial Mesenchymal Transition

Background: Matrix Gla protein (MGP) is an extracellular matrix protein that inhibits bone morphogenetic protein (BMP) signaling in vitro. MGP deficiency induces vascular calcification associated with osteogenic transdifferentiation of endothelial cells (via endothelial-mesenchymal transition, EndMT) and vascular smooth muscle cells (VSMCs). We previously reported that treatment with two pharmacologic inhibitors of BMP signaling reduced aortic calcification in MGP-/- mice. We hypothesized that BMP signaling is essential for EndMT and VSMC osteogenic transdifferentiation induced by MGP deficiency. Methods and Results: Aortic levels of mRNAs encoding markers of osteogenesis (Runx2 and osteopontin) and EndMT (nanog, Sox2, and Oct3/4) were greater in MGP-/- than in wild-type mice (P<0.01 for all). Aortic expression of markers of VSMC differentiation (α-smooth muscle actin, transgelin, and calponin) was less in MGP-/- than in wild-type mice (P<0.001 for all). Treatment of MGP-/- mice with the BMP signaling inhibitor, LDN-193189, reduced expression of both osteogenic and EndMT markers (P<0.05 for all) but did not prevent VSMC de-differentiation. Depletion of MGP in cultured wild-type VSMCs with siRNA specific for MGP (siMGP) was associated with a 30-40% reduction in levels of mRNAs encoding markers of VSMC differentiation (P<0.05 for all), an effect that was not prevented by LDN-193189. Incubation in phosphate-containing media induced greater calcification in siMGP-treated VSMCs than in cells treated with control siRNA (P<0.0001). Treatment with LDN-193189 reduced calcification in siMGP-treated VSMCs (50%, P=0.0003). Conversely, infection of MGP-/- VSMCs with adenovirus specifying MGP increased expression of markers of VSMC differentiation by 60-80% (P<0.01 for all) and decreased calcification by 74% (P=0.03). Conclusions: Inhibition of BMP signaling suppresses osteogenic and EndMT gene programs in MGP-/- mice and reduces calcification of siMGP-treated VSMCs. However, MGP deficiency induces VSMC de-differentiation via a BMP-independent mechanism. These findings suggest that the processes underlying vascular calcification in MGP deficiency are mediated by both BMP signaling-dependent and -independent mechanisms.

Download Full-text

Matrigel induces thymosin beta 4 gene in differentiating endothelial cells

Journal of Cell Science ◽

10.1242/jcs.108.12.3685 ◽

1995 ◽

Vol 108 (12) ◽

pp. 3685-3694 ◽

Cited By ~ 4

Author(s):

D.S. Grant ◽

J.L. Kinsella ◽

M.C. Kibbey ◽

S. LaFlamme ◽

P.D. Burbelo ◽

...

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

Morphological Differentiation ◽

Tube Formation ◽

Vessel Formation ◽

Thymosin Beta 4 ◽

Cdna Hybridization ◽

Matrix Components ◽

Cells Cultured

We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillary-like tubes. The mRNA for one clone, thymosin beta 4, was increased 5-fold. Immunostaining localized thymosin beta 4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin beta 4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin beta 4 inhibited tube formation on Matrigel. The results suggest that thymosin beta 4 is induced and likely involved in differentiating endothelial cells. Thymosin beta 4 may play a role in vessel formation in vivo.

Download Full-text

Radiocontrast agents induce endothelin release in vivo and in vitro.

Journal of the American Society of Nephrology ◽

10.1681/asn.v3158 ◽

1992 ◽

Vol 3 (1) ◽

pp. 58-65 ◽

Cited By ~ 1

Author(s):

S N Heyman ◽

B A Clark ◽

N Kaiser ◽

K Spokes ◽

S Rosen ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Concentration ◽

Plasma Level ◽

Vascular Endothelium ◽

Radiocontrast Agent ◽

Transient Rise ◽

Bovine Endothelial Cells ◽

Radiocontrast Agents

The intravascular administration of the ionic radiocontrast agent sodium iothalamate (2.9 g of iodine/kg body wt) to rats induced an increase in plasma concentration of immunoreactive endothelin from 21.3 +/- 1.2 to 36 +/- 3 fmol/mL, preceded by a transient rise in the plasma level of atrial natriuretic peptide and associated with a fall in RBF. Equi-iodine amounts of the nonionic agents ioxaglate and iohexol elicited similar or more marked changes in plasma endothelin, but hypertonic solutions of NaCl, mannitol, or glucose did not. Comparable levels of endothelin produced by infusions of endothelin-1 induced a reduction of up to 29% in RBF. Iothalamate and iohexol stimulated endothelin release from cultured bovine endothelial cells, suggesting a direct effect of ionic and nonionic agents on vascular endothelium. The data invite speculation that under some circumstances endothelin release might play a role in the circulatory changes caused by these compounds and in the pathogenesis of radiocontrast nephropathy.

Download Full-text

Anticancer Effect of Heparin–Taurocholate Conjugate on Orthotopically Induced Exocrine and Endocrine Pancreatic Cancer

Cancers ◽

10.3390/cancers13225775 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5775

Author(s):

Hae Hyun Hwang ◽

Hee Jeong Jeong ◽

Sangwu Yun ◽

Youngro Byun ◽

Teruo Okano ◽

...

Keyword(s):

Pancreatic Cancer ◽

Endothelial Cells ◽

Cancer Cells ◽

Tumor Model ◽

Pancreatic Neuroendocrine Tumor ◽

Tube Formation ◽

Ductal Adenocarcinoma ◽

Orthotopic Model ◽

Anticancer Efficacy

Pancreatic cancers are classified based on where they occur, and are grouped into those derived from exocrine and those derived from neuroendocrine tumors, thereby experiencing different anticancer effects under medication. Therefore, it is necessary to develop anticancer drugs that can inhibit both types. To this end, we developed a heparin–taurocholate conjugate, i.e., LHT, to suppress tumor growth via its antiangiogenic activity. Here, we conducted a study to determine the anticancer efficacy of LHT on pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET), in an orthotopic animal model. LHT reduced not only proliferation of cancer cells, but also attenuated the production of VEGF through ERK dephosphorylation. LHT effectively reduced the migration, invasion and tube formation of endothelial cells via dephosphorylation of VEGFR, ERK1/2, and FAK protein. Especially, these effects of LHT were much stronger on PNET (RINm cells) than PDAC (PANC1 and MIA PaCa-2 cells). Eventually, LHT reduced ~50% of the tumor weights and tumor volumes of all three cancer cells in the orthotopic model, via antiproliferation of cancer cells and antiangiogenesis of endothelial cells. Interestingly, LHT had a more dominant effect in the PNET-induced tumor model than in PDAC in vivo. Collectively, these findings demonstrated that LHT could be a potential antipancreatic cancer medication, regardless of pancreatic cancer types.

Download Full-text

The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

Blood ◽

10.1182/blood-2011-08-376038 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1302-1313 ◽

Cited By ~ 31

Author(s):

Cheng-Hsiang Kuo ◽

Po-Ku Chen ◽

Bi-Ing Chang ◽

Meng-Chen Sung ◽

Chung-Sheng Shi ◽

...

Keyword(s):

Endothelial Cells ◽

Egf Receptor ◽

Tube Formation ◽

Endothelial Tube Formation ◽

A Cell ◽

Lewis Y ◽

Tm Domain

AbstractLewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

Download Full-text

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.

Download Full-text

Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development

Development ◽

10.1242/dev.126.18.4005 ◽

1999 ◽

Vol 126 (18) ◽

pp. 4005-4015 ◽

Cited By ~ 14

Author(s):

M. Weaver ◽

J.M. Yingling ◽

N.R. Dunn ◽

S. Bellusci ◽

B.L. Hogan

Keyword(s):

Cell Types ◽

Surfactant Protein ◽

Bmp Signaling ◽

Dominant Negative ◽

Clara Cell ◽

Mouse Lung ◽

Marker Genes ◽

Distal Marker ◽

Morphogenetic Protein

In the mature mouse lung, the proximal-distal (P-D) axis is delineated by two distinct epithelial subpopulations: the proximal bronchiolar epithelium and the distal respiratory epithelium. Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is Bone Morphogenetic Protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. Previous studies in which Bmp4 was overexpressed in the lung endoderm (Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. M. (1996) Development 122, 1693–1702) suggested that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homologue 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells, a proximal cell type, in the most peripheral regions of the transgenic lungs. We propose a model in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program.

Download Full-text

Accutin, a New Disintegrin, Inhibits Angiogenesis In Vitro and In Vivo by Acting as Integrin vβ3 Antagonist and Inducing Apoptosis

Blood ◽

10.1182/blood.v92.9.3268 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3268-3276 ◽

Cited By ~ 90

Author(s):

Chia Hsin Yeh ◽

Hui-Chin Peng ◽

Tur-Fu Huang

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Fluorescein Isothiocyanate ◽

Adenosine Diphosphate ◽

Tube Formation ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Antiangiogenic Effect

Abstract Endothelial integrins play an essential role in angiogenesis and cell survival. Accutin, a new member of disintegrin family derived from venom of Agkistrodon acutus, potently inhibited human platelet aggregation caused by various agonists (eg, thrombin, collagen, and, adenosine diphosphate [ADP]) through the blockade of fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin IIbβ3). In this report, we describe that accutin specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin vβ3, to human umbilical vein endothelial cells (HUVECs), but not those of other anti-integrin MoAbs such as 2β1, 3β1, and 5β1. Moreover, accutin, but not the control peptide GRGES, dose-dependently inhibited the 7E3 interaction with HUVECs. Both 7E3 and GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein isothiocyanate-conjugated accutin binding to HUVEC. In functional studies, accutin exhibited inhibitory effects on HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin, and the capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner. In addition, it exhibited an effective antiangiogenic effect in vivo when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as examined by electrophoretic and flow cytometric assays. In conclusion, accutin inhibits angiogenesis in vivo and in vitro by blocking integrin vβ3 of endothelial cells and by inducing apoptosis. The antiangiogenic activity of disintegrins might be explored as the target of developing the potential antimetastatic agents. © 1998 by The American Society of Hematology.

Download Full-text

The Functional Implications of Endothelial Gap Junctions and Cellular Mechanics in Vascular Angiogenesis

Cancers ◽

10.3390/cancers11020237 ◽

2019 ◽

Vol 11 (2) ◽

pp. 237 ◽

Cited By ~ 15

Author(s):

Takayuki Okamoto ◽

Haruki Usuda ◽

Tetsuya Tanaka ◽

Koichiro Wada ◽

Motomu Shimaoka

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Gap Junctions ◽

Gap Junction ◽

Cancer Cells ◽

Tumor Development ◽

Tube Formation ◽

Cellular Mechanics

Angiogenesis—the sprouting and growth of new blood vessels from the existing vasculature—is an important contributor to tumor development, since it facilitates the supply of oxygen and nutrients to cancer cells. Endothelial cells are critically affected during the angiogenic process as their proliferation, motility, and morphology are modulated by pro-angiogenic and environmental factors associated with tumor tissues and cancer cells. Recent in vivo and in vitro studies have revealed that the gap junctions of endothelial cells also participate in the promotion of angiogenesis. Pro-angiogenic factors modulate gap junction function and connexin expression in endothelial cells, whereas endothelial connexins are involved in angiogenic tube formation and in the cell migration of endothelial cells. Several mechanisms, including gap junction function-dependent or -independent pathways, have been proposed. In particular, connexins might have the potential to regulate cell mechanics such as cell morphology, cell migration, and cellular stiffness that are dynamically changed during the angiogenic processes. Here, we review the implication for endothelial gap junctions and cellular mechanics in vascular angiogenesis.

Download Full-text