scholarly journals Expression and Function of Laminins in the Embryonic and Mature Vasculature

2005 ◽  
Vol 85 (3) ◽  
pp. 979-1000 ◽  
Author(s):  
Rupert Hallmann ◽  
Nathalie Horn ◽  
Manuel Selg ◽  
Olaf Wendler ◽  
Friederike Pausch ◽  
...  

Endothelial cells of the blood and lymphatic vasculature are polarized cells with luminal surfaces specialized to interact with inflammatory cells upon the appropriate stimulation; they contain specialized transcellular transport systems, and their basal surfaces are attached to an extracellular basement membrane. In adult tissues the basement membrane forms a continuous sleeve around the endothelial tubes, and the interaction of endothelial cells with basement membrane components plays an important role in the maintenance of vessel wall integrity. During development, the basement membrane of endothelium provides distinct spatial and molecular information that influences endothelial cell proliferation, migration, and differentiation/maturation. Microvascular endothelium matures into phenotypically distinct types: continuous, fenestrated, and discontinuous, which also differ in their permeability properties. Development of these morphological and physiological differences is thought to be controlled by both soluble factors in the organ or tissue environment and by cell-cell and cell-matrix interactions. Basement membranes of endothelium, like those of other tissues, are composed of laminins, type IV collagens, heparan sulfate proteoglycans, and nidogens. However, isoforms of all four classes of molecules exist, which combine to form structurally and functionally distinct basement membranes. The endothelial cell basement membranes have been shown to be unique with respect to their laminin isoform composition. Laminins are a family of glycoprotein heterotrimers composed of an α, β, and γ chain. To date, 5α, 4β, and 3γ laminin chains have been identified that can combine to form 15 different isoforms. The laminin α-chains are considered to be the functionally important portion of the heterotrimers, as they exhibit tissue-specific distribution patterns and contain the major cell interaction sites. Vascular endothelium expresses only two laminin isoforms, and their expression varies depending on the developmental stage, vessel type, and the activation state of the endothelium. Laminin 8 (composed of laminin α4, β1, and γ1 chains) is expressed by all endothelial cells regardless of their stage of development, and its expression is strongly upregulated by cytokines and growth factors that play a role in inflammatory events. Laminin 10 (composed of laminin α5, β1, and γ1 chains) is detectable primarily in endothelial cell basement membranes of capillaries and venules commencing 3–4 wk after birth. In contrast to laminin 8, endothelial cell expression of laminin 10 is upregulated only by strong proinflammatory signals and, in addition, angiostatic agents such as progesterone. Other extracellular matrix molecules, such as BM40 (also known as SPARC/osteonectin), thrombospondins 1 and 2, fibronectin, nidogens 1 and 2, and collagen types VIII, XV, and XVIII, are also differentially expressed by endothelium, varying with the endothelium type and/or pathophysiological state. The data argue for a dynamic endothelial cell extracellular matrix that presents different molecular information depending on the type of endothelium and/or physiological situation. This review outlines the unique structural and functional features of vascular basement membranes, with focus on the endothelium and the laminin family of glycoproteins.

1997 ◽  
Vol 78 (05) ◽  
pp. 1392-1398 ◽  
Author(s):  
A Schneider ◽  
M Chandra ◽  
G Lazarovici ◽  
I Vlodavsky ◽  
G Merin ◽  
...  

SummaryPurpose: Successful development of a vascular prosthesis lined with endothelial cells (EC) may depend on the ability of the attached cells to resist shear forces after implantation. The present study was designed to investigate EC detachment from extracellular matrix (ECM) precoated vascular prostheses, caused by shear stress in vitro and to test the performance of these grafts in vivo. Methods: Bovine aortic endothelial cells were seeded inside untreated polytetrafluoro-ethylene (PTFE) vascular graft (10 X 0.6 cm), PTFE graft precoated with fibronectin (FN), or PTFE precoated with FN and a naturally produced ECM (106 cells/graft). Sixteen hours after seeding the medium was replaced and unattached cells counted. The strength of endothelial cell attachment was evaluated by subjecting the grafts to a physiologic shear stress of 15 dynes/cm2 for 1 h. The detached cells were collected and quantitated. PTFE or EC preseeded ECM coated grafts were implanted in the common carotid arteries of dogs. Results: While little or no differences were found in the extent of endothelial cell attachment to the various grafts (79%, 87% and 94% of the cells attached to PTFE, FN precoated PTFE, or FN+ECM precoated PTFE, respectively), the number of cells retained after a shear stress was significanly increased on ECM coated PTFE (20%, 54% and 85% on PTFE, FN coated PTFE, and FN+ECM coated PTFE, respectively, p <0.01). Implantation experiments in dogs revealed a significant increase in EC coverage and a reduced incidence of thrombus formation on ECM coated grafts that were seeded with autologous saphenous vein endothelial cells prior to implantation. Conclusion: ECM coating significantly increased the strength of endothelial cell attachment to vascular prostheses subjected to shear stress. The presence of adhesive macromolecules and potent endothelial cell growth promoting factors may render the ECM a promising substrate for vascular prostheses.


2005 ◽  
Vol 83 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Tara L Haas

The process of sprouting angiogenesis requires that the endothelial cells degrade the basement membrane matrix and migrate into the interstitial matrix. Matrix metalloproteinases are enzymes capable of cleaving numerous extracellular matrix proteins. Increased production and activity of matrix metalloproteinases in any cell type is associated with a more migratory and invasive phenotype. This paper describes results of recent in-vitro studies of the regulation of transcription and activation of MMP-2 and MT1-MMP in endothelial cells, as well as studies that examined roles of matrix metalloproteinases in activity-induced angiogenesis.Key words: proteolysis, extracellular matrix, angiogenesis, mechanotransduction.


Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Petra E Burgisser ◽  
Dennie Tempel ◽  
Caroline Cheng ◽  
Gerard Pasterkamp ◽  
Henricus Duckers

Background: Vascular development is crucial in normal physiology and pathophysiology, and is in part orchestrated by a dynamic balance between extracellular matrix (ECM) and endothelial cells. The molecular mechanisms that govern this balance and as such play a role in the formation of new blood vessels remain largely unknown. In order to identify new genes involved in blood vessel formation, we performed a genome-wide gene expression array analysis in Flk1+ angioblasts during mouse development using a commercial platform (Affymetrix). Multimerin2 (Mmrn2) expression was found to be upregulated in angioblasts and endothelial cells and in the developing vasculature of zebrafish and mice. Methods & Results: Subsequent in vitro studies using an adenoviral construct to induce Mmrn2 overexpression showed a negative effect on angiogenesis. Mmrn2 overexpression resulted in a reduction of 2D tube formation capacity of human aortic endothelial cells (HAECs), as seen in a decrease in the number of junctions and tubules (P<0.05, N=3). Another observed effect after Mmrn2 overexpression was a G0/G1 cell cycle arrest in HAECs (P<0.001, N=8) and decreased migration of the cells in a Boyden chamber assay (P<0.05, N=10). Further in vitro experiments, using immunofluorescence, showed Mmrn2 was detected outside of HAECs, were it co-localized with the extracellular matrix components perlecan, laminin and fibronectin. Direct interaction with these proteins was confirmed using a combination of co-immunoprecipitation followed by proteomics (LC-MS), and western blot to identify possible binding partners. Interestingly, overexpression of Mmrn2 also resulted in enhanced expression of these extracellular components, suggesting that Mmrn2 changes the phenotype from a more proliferative endothelial cell towards a more productive endothelial cell. Conclusions: These findings suggest that Mmrn2 is an abundant and important extracellular matrix protein, secreted by endothelial cells, which influences the expression of other ECM proteins such as fibronectin, laminin and perlecan. We hypothesize that Mmrn2 switches the phenotype of the endothelial cell and subsequently diminishes endothelial cell proliferation, migration and overall tube formation.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas S. Tombor ◽  
David John ◽  
Simone F. Glaser ◽  
Guillermo Luxán ◽  
Elvira Forte ◽  
...  

AbstractEndothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.


2001 ◽  
Vol 168 (3) ◽  
pp. 409-416 ◽  
Author(s):  
SE Dickson ◽  
R Bicknell ◽  
HM Fraser

Vascular endothelial growth factor (VEGF) is essential for the angiogenesis required for the formation of the corpus luteum; however, its role in ongoing luteal angiogenesis and in the maintenance of the established vascular network is unknown. The aim of this study was to determine whether VEGF inhibition could intervene in ongoing luteal angiogenesis using immunoneutralisation of VEGF starting in the mid-luteal phase. In addition, the effects on endothelial cell survival and the recruitment of periendothelial support cells were examined. Treatment with a monoclonal antibody to VEGF, or mouse gamma globulin for control animals, commenced on day 7 after ovulation and continued for 3 days. Bromodeoxyuridine (BrdU), used to label proliferating cells to obtain a proliferation index, was administered one hour before collecting ovaries from control and treated animals. Ovarian sections were stained using antibodies to BrdU, the endothelial cell marker, CD31, the pericyte marker, alpha-smooth muscle actin, and 3' end DNA fragments as a marker for apoptosis. VEGF immunoneutralisation significantly suppressed endothelial cell proliferation and the area occupied by endothelial cells while increasing pericyte coverage and the incidence of endothelial cell apoptosis. Luteal function was markedly compromised by anti-VEGF treatment as judged by a 50% reduction in plasma progesterone concentration. It is concluded that ongoing angiogenesis in the mid-luteal phase is primarily driven by VEGF, and that a proportion of endothelial cells of the mid-luteal phase vasculature are dependent on VEGF support.


2001 ◽  
Vol 12 (1) ◽  
pp. 85-100 ◽  
Author(s):  
Meredith Gonzales ◽  
Babette Weksler ◽  
Daisuke Tsuruta ◽  
Robert D. Goldman ◽  
Kristine J. Yoon ◽  
...  

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.


2006 ◽  
Vol 17 (4) ◽  
pp. 1985-1994 ◽  
Author(s):  
Elena Toschi ◽  
Ilaria Bacigalupo ◽  
Raffaele Strippoli ◽  
Chiara Chiozzini ◽  
Anna Cereseto ◽  
...  

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the α5β1 and αvβ3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.


2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Luke Hoeppner ◽  
Sutapa Sinha ◽  
Ying Wang ◽  
Resham Bhattacharya ◽  
Shamit Dutta ◽  
...  

Vascular permeability factor/vascular endothelial growth factor A (VEGF) is a central regulator of angiogenesis and potently promotes vascular permeability. VEGF plays a key role in the pathologies of heart disease, stroke, and cancer. Therefore, understanding the molecular regulation of VEGF signaling is an important pursuit. Rho GTPase proteins play various roles in vasculogenesis and angiogenesis. While the functions of RhoA and RhoB in these processes have been well defined, little is known about the role of RhoC in VEGF-mediated signaling in endothelial cells and vascular development. Here, we describe how RhoC modulates VEGF signaling to regulate endothelial cell proliferation, migration and permeability. We found VEGF stimulation activates RhoC in human umbilical vein endothelial cells (HUVECs), which was completely blocked after VEGF receptor 2 (VEGFR-2) knockdown indicating that VEGF activates RhoC through VEGFR-2 signaling. Interestingly, RhoC knockdown delayed the degradation of VEGFR-2 compared to control siRNA treated HUVECs, thus implicating RhoC in VEGFR-2 trafficking. In light of our results suggesting VEGF activates RhoC through VEGFR-2, we sought to determine whether RhoC regulates vascular permeability through the VEGFR-2/phospholipase Cγ (PLCγ) /Ca 2+ /eNOS cascade. We found RhoC knockdown in VEGF-stimulated HUVECs significantly increased PLC-γ1 phosphorylation at tyrosine 783, promoted basal and VEGF-stimulated eNOS phophorylation at serine 1177, and increased calcium flux compared with control siRNA transfected HUVECs. Taken together, our findings suggest RhoC negatively regulates VEGF-induced vascular permeability. We confirmed this finding through a VEGF-inducible zebrafish model of vascular permeability by observing significantly greater vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Furthermore, we showed that RhoC promotes endothelial cell proliferation and negatively regulates endothelial cell migration. Our data suggests a scenario in which RhoC promotes proliferation by upregulating -catenin in a Wnt signaling-independent manner, which in turn, promotes Cyclin D1 expression and subsequently drives cell cycle progression.


Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yukio Shimasaki ◽  
Kai Chen ◽  
John F Keaney

Background: Growing evidence suggests that mitochondrial function contributes to cell phenotype. One important component of mitochondrial function is the membrane potential that is controlled, in part, by uncoupling proteins (UCPs). Based on our previous data, the UCP2 is predominantly expressed in cultured endothelial cells. Therefore, we sought to examine the role of UCP2 in endothelial cell growth and angiogenesis. Methods and Results: Murine lung endothelial cells (MLECs) were isolated from UCP2-null and wild-type mice. UCP2-null cells were found less proliferative than wild-type cells (P<0.02, UCP2-null cells vs. wild-type cells, n=4). This defect of UCP2-null cells was rescued by UCP2 adenovirus transfection (19% increase, p<0.02 vs. LacZ adenovirus treated cells, n=3), and also rescued by transfection with manganese superoxide dismutase (MnSOD) adenovirus (53% increase, P<0.002 vs. LacZ adenovirus treated cells, n=3). We found a reciprocal relation such as no UCP2 expression and higher mitochondrial superoxide level in the MLECs (P<0.005, UCP2-null cells vs. wild-type cells, n=3), suggesting that mitochondrial superoxide may regulate endothelial cell growth. Then, we prepared murine aortic rings from UCP2-null and wild-type mice and embedded in rat tail collagen gel. The sprouting angiogenesis of UCP2-null explants was significantly less than wild-type explants (P<0.02, UCP2-null explants vs. wild-type explants, n=3– 4). Furthermore, MLECs from MnSOD-heterozygous mice showed less proliferation with lower expression of UCP2 protein and higher mitochondrial superoxide level compared to the MLECs from wild-type littermates (P<0.02, MnSOD-heterozygous cells vs. wild-type cells, n=4 – 8). We also observed less sprouting angiogenesis in MnSOD-heterozygous aortic explants than wild-type aortic explants (P<0.05, MnSOD-heterozygous explants vs. wild-type explants, n=3– 6). Conclusions: These data indicate that mitochondrial superoxide controls endothelial cell proliferation and angiogenesis, suggesting that mitochondrial metabolism modulates the endothelial cell growth and angiogenesis.


1999 ◽  
Vol 112 (10) ◽  
pp. 1599-1609 ◽  
Author(s):  
B.M. Kraling ◽  
D.G. Wiederschain ◽  
T. Boehm ◽  
M. Rehn ◽  
J.B. Mulliken ◽  
...  

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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