scholarly journals Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

1995 ◽  
Vol 6 (3) ◽  
pp. 327-343 ◽  
Author(s):  
M L Iruela-Arispe ◽  
T F Lane ◽  
D Redmond ◽  
M Reilly ◽  
R P Bolender ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Chorioallantoic Membrane ◽  
Focal Adhesions ◽  
Protein Accumulation ◽  
Dependent Manner ◽  
Plasmin Activity ◽  
Specific Expression ◽  
Chicken Chorioallantoic Membrane

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.

scholarly journals Production of a heparin-binding angiogenesis factor by the embryonic kidney.

1986 ◽  
Vol 103 (3) ◽  
pp. 1101-1107 ◽  
Author(s):  
W Risau ◽  
P Ekblom
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Blood Vessels ◽  
Gel Filtration ◽  
Chorioallantoic Membrane ◽  
Heparin Binding ◽  
Rabbit Cornea ◽  
Embryonic Mouse ◽  
Angiogenesis Factor

Embryonic mouse kidneys induce angiogenesis when transplanted on the quail chorioallantoic membrane (Ekblom, P., H. Sariola, M. Karkinen, and L. Saxén, 1982, Cell Differ., 11:35-39). In these experiments all blood vessels were derived from the quail host, suggesting that kidney endothelium is derived from outside blood vessels. We have now analyzed whether kidney angiogenesis is regulated by kidney-derived soluble factors that stimulate the growth of new blood vessels. In the rabbit cornea, 11-d embryonic kidneys induced angiogenesis, whereas uninduced 11-d kidney mesenchymes did not. To characterize and purify this activity from an embryonic organ, we dissected between 600 and 1,000 14-17-d-old embryonic mouse kidneys for each purification experiment. Growth factor activity for capillary endothelial cells was found to bind to heparin-Sepharose and eluted at 0.9-1.1 M sodium chloride. Gel filtration revealed a molecular weight of 16,000-20,000 of this factor. A major 18,000-mol-wt band was seen after gel electrophoresis and silver staining of partially purified growth factor material. The chromatographed factor is mitogenic for endothelial cells but not for smooth muscle cells and stimulates angiogenesis in vivo in the rabbit cornea. Adult kidneys contained two heparin-binding endothelial cell growth factors. The differentiation-dependent production of an angiogenesis factor by the embryonic kidney suggests an important role of angiogenesis in organogenesis.

scholarly journals An antagonist of integrin alpha v beta 3 prevents maturation of blood vessels during embryonic neovascularization

1995 ◽  
Vol 108 (7) ◽  
pp. 2655-2661 ◽  
Author(s):  
C.J. Drake ◽  
D.A. Cheresh ◽  
C.D. Little
Keyword(s):  
Experimental Data ◽  
Monoclonal Antibody ◽  
Blood Vessels ◽  
Chorioallantoic Membrane ◽  
Normal Pattern ◽  
Basal Surface ◽  
Lumen Formation ◽  
Vessel Development ◽  
Alpha V Beta 3 ◽  
Chicken Chorioallantoic Membrane

Experimental data in this study demonstrate that integrin alpha v beta 3 is fundamentally involved in the maturation of blood vessels during embryonic neovascularization (vasculogenesis). Integrin alpha v beta 3 was specifically expressed on the surface of angioblasts during vessel development in quail embryos and vitronectin, a ligand for alpha v beta 3, localized to the basal surface of these cells. More importantly, microinjection of the anti-alpha v beta 3 monoclonal antibody, LM609, disrupted the normal pattern of vascular development. After exposure to LM609 the angioblasts in experimental embryos appeared as clusters of rounded cells lacking normal cellular protrusions. This led to disruption of lumen formation and abnormal vessel patterning. These findings demonstrate that during vasculogenesis ligation of integrin alpha v beta 3 on the surface of primordial endothelial cells is critical for the differentiation and maturation of blood vessels. Similar studies on chicken chorioallantoic membrane showed that LM609 blocks angiogenesis. Together the two studies suggest that integrin alpha v beta 3 plays a role in neovascularization of tissues.

scholarly journals Kisspeptin-10 Inhibits Angiogenesis in Human Placental Vessels ex Vivo and Endothelial Cells in Vitro

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5927-5934 ◽  
Author(s):  
Thayalini Ramaesh ◽  
James J. Logie ◽  
Antonia K. Roseweir ◽  
Robert P. Millar ◽  
Brian R. Walker ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Biologically Active ◽  
Trophoblast Invasion ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Inhibition Of Proliferation

Recent studies suggest that kisspeptin (a neuropeptide central to the regulation of gonadotrophin secretion) has diverse roles in human physiology, including a putative role in implantation and placental function. Kisspeptin and its receptor are present in human blood vessels, where they mediate vasoconstriction, and kisspeptin is known to inhibit tumor metastasis and trophoblast invasion, both processes involving angiogenesis. We hypothesized that kisspeptin contributes to the regulation of angiogenesis in the reproductive system. The presence of the kisspeptin receptor was confirmed in human placental blood vessels and human umbilical vein endothelial cells (HUVEC) using immunochemistry. The ability of kisspeptin-10 (KP-10) (a shorter biologically active processed peptide) to inhibit angiogenesis was tested in explanted human placental arteries and HUVEC using complementary ex vivo and in vitro assays. KP-10 inhibited new vessel sprouting from placental arteries embedded in Matrigel and tube-like structure formation by HUVEC, in a concentration-dependent manner. KP-10 had no effect on HUVEC viability or apoptosis but induced concentration-dependent inhibition of proliferation and migration. In conclusion, KP-10 has antiangiogenic effects and, given its high expression in the placenta, may contribute to the regulation of angiogenesis in this tissue.

Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

2006 ◽  
Vol 19 (1) ◽  
pp. 205873920601900 ◽  
Author(s):  
M. Bongrazio ◽  
L. DA Silva-Azevedo ◽  
E.C. Bergmann ◽  
O. Baum ◽  
B. Hinz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Western Blot ◽  
Dependent Manner ◽  
Vascular Networks ◽  
Thrombospondin 1 ◽  
Matrix Bound ◽  
Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bin Ren ◽  
Arpita Mukhopadhyay* ◽  
Anthony A Lanahan ◽  
Zhen W Zhuang ◽  
Karen L Moodie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Branching Morphogenesis ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Activation ◽  
Arterial Development ◽  
Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

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

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3268-3276 ◽  
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.

High Glucose Alters Endothelial Cell Response to Shear Stress

2009 ◽  
Author(s):  
Steven F. Kemeny ◽  
Alisa Morss Clyne
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Cell Mechanics ◽  
High Glucose ◽  
Fluid Shear Stress ◽  
Cell Function ◽  
Focal Adhesions ◽  
Endothelial Cell Function

Endothelial cells line the walls of all blood vessels, where they maintain homeostasis through control of vascular tone, permeability, inflammation, and the growth and regression of blood vessels. Endothelial cells are mechanosensitive to fluid shear stress, elongating and aligning in the flow direction [1–2]. This shape change is driven by rearrangement of the actin cytoskeleton and focal adhesions [2]. Hyperglycemia, a hallmark of diabetes, affects endothelial cell function. High glucose has been shown to increase protein kinase C, formation of glucose-derived advanced glycation end-products, and glucose flux through the aldose reductase pathway within endothelial cells [3]. These changes are thought to be related to increased reactive oxygen species production [4]. While endothelial cell mechanics have been widely studied in healthy conditions, many disease states have yet to be explored. Biochemical alterations related to high glucose may alter endothelial cell mechanics.

Human glucagon gene promoter sequences regulating tissue-specific versus nutrient-regulated gene expression

2002 ◽  
Vol 282 (1) ◽  
pp. R173-R183 ◽  
Author(s):  
Min Nian ◽  
Jun Gu ◽  
David M. Irwin ◽  
Daniel J. Drucker
Keyword(s):  
Gene Expression ◽  
Gene Promoter ◽  
Regulatory Sequences ◽  
Dependent Manner ◽  
Specific Expression ◽  
Tissue Specific ◽  
High Fiber ◽  
Fiber Diet ◽  
Regulated Gene Expression

The glucagon-like peptides (GLPs) are synthesized and secreted in a nutrient-dependent manner in rodents; however, the factors regulating human GLP-1 and GLP-2 biosynthesis remain unclear. To understand how nutrients regulate human proglucagon gene expression, we studied the expression of a human proglucagon promoter-growth hormone (GH) transgene in 1.6 human glucagon-GH transgenic mice. Fasting-refeeding significantly decreased and increased the levels of circulating mouse insulin and transgene-derived hGH ( P < 0.05 fasting vs. refeeding) and decreased and upregulated, respectively, the levels of endogenous mouse proglucagon RNA in the ileum but not in the jejunum or colon. High-fiber feeding significantly increased the levels of glucose-stimulated circulating hGH and upregulated levels of mouse intestinal proglucagon gene expression in the jejunum, ileum, and colon ( P < 0.05, 0 vs. 30% fiber diet). In contrast, neither fasting-refeeding nor a high-fiber diet upregulated the expression of the human proglucagon promoter-hGH transgene. These findings demonstrate that human proglucagon gene regulatory sequences specifying tissue-specific expression in gut endocrine cells are not sufficient for recognition of energy-derived signals regulating murine glucagon gene expression in enteroendocrine cells in vivo.

scholarly journals Increased gene copy number of DEFA1/DEFA3 worsens sepsis by inducing endothelial pyroptosis

2019 ◽  
Vol 116 (8) ◽  
pp. 3161-3170 ◽  
Author(s):  
QiXing Chen ◽  
Yang Yang ◽  
JinChao Hou ◽  
Qiang Shu ◽  
YiXuan Yin ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Copy Number ◽  
Treatment Options ◽  
Gene Copy Number ◽  
High Copy Number ◽  
Gene Copy ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Specific Expression

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1–3 (HNP1–3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

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