Publications by authors named "Amber N Stratman"

29 Publications

  • Page 1 of 1

DIAPH1 Variants in Non-East Asian Patients With Sporadic Moyamoya Disease.

JAMA Neurol 2021 Jun 14. Epub 2021 Jun 14.

Yale Center for Genome Analysis, West Haven, Connecticut.

Importance: Moyamoya disease (MMD), a progressive vasculopathy leading to narrowing and ultimate occlusion of the intracranial internal carotid arteries, is a cause of childhood stroke. The cause of MMD is poorly understood, but genetic factors play a role. Several familial forms of MMD have been identified, but the cause of most cases remains elusive, especially among non-East Asian individuals.

Objective: To assess whether ultrarare de novo and rare, damaging transmitted variants with large effect sizes are associated with MMD risk.

Design, Setting, And Participants: A genetic association study was conducted using whole-exome sequencing case-parent MMD trios in a small discovery cohort collected over 3.5 years (2016-2019); data were analyzed in 2020. Medical records from US hospitals spanning a range of 1 month to 1.5 years were reviewed for phenotyping. Exomes from a larger validation cohort were analyzed to identify additional rare, large-effect variants in the top candidate gene. Participants included patients with MMD and, when available, their parents. All participants who met criteria and were presented with the option to join the study agreed to do so; none were excluded. Twenty-four probands (22 trios and 2 singletons) composed the discovery cohort, and 84 probands (29 trios and 55 singletons) composed the validation cohort.

Main Outcomes And Measures: Gene variants were identified and filtered using stringent criteria. Enrichment and case-control tests assessed gene-level variant burden. In silico modeling estimated the probability of variant association with protein structure. Integrative genomics assessed expression patterns of MMD risk genes derived from single-cell RNA sequencing data of human and mouse brain tissue.

Results: Of the 24 patients in the discovery cohort, 14 (58.3%) were men and 18 (75.0%) were of European ancestry. Three of 24 discovery cohort probands contained 2 do novo (1-tailed Poisson P = 1.1 × 10-6) and 1 rare, transmitted damaging variant (12.5% of cases) in DIAPH1 (mammalian diaphanous-1), a key regulator of actin remodeling in vascular cells and platelets. Four additional ultrarare damaging heterozygous DIAPH1 variants (3 unphased) were identified in 3 other patients in an 84-proband validation cohort (73.8% female, 77.4% European). All 6 patients were non-East Asian. Compound heterozygous variants were identified in ena/vasodilator-stimulated phosphoproteinlike protein EVL, a mammalian diaphanous-1 interactor that regulates actin polymerization. DIAPH1 and EVL mutant probands had severe, bilateral MMD associated with transfusion-dependent thrombocytopenia. DIAPH1 and other MMD risk genes are enriched in mural cells of midgestational human brain. The DIAPH1 coexpression network converges in vascular cell actin cytoskeleton regulatory pathways.

Conclusions And Relevance: These findings provide the largest collection to date of non-East Asian individuals with sporadic MMD harboring pathogenic variants in the same gene. The results suggest that DIAPH1 is a novel MMD risk gene and impaired vascular cell actin remodeling in MMD pathogenesis, with diagnostic and therapeutic ramifications.
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http://dx.doi.org/10.1001/jamaneurol.2021.1681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204259PMC
June 2021

The SWELL1-LRRC8 complex regulates endothelial AKT-eNOS signaling and vascular function.

Elife 2021 Feb 25;10. Epub 2021 Feb 25.

Department of Internal Medicine, Cardiovascular Division, Washington University School of Medicine, St. Louis, United States.

The endothelium responds to numerous chemical and mechanical factors in regulating vascular tone, blood pressure, and blood flow. The endothelial volume-regulated anion channel (VRAC) has been proposed to be mechanosensitive and thereby sense fluid flow and hydrostatic pressure to regulate vascular function. Here, we show that the leucine-rich repeat-containing protein 8a, LRRC8A (SWELL1), is required for VRAC in human umbilical vein endothelial cells (HUVECs). Endothelial LRRC8A regulates AKT-endothelial nitric oxide synthase (eNOS) signaling under basal, stretch, and shear-flow stimulation, forms a GRB2-Cav1-eNOS signaling complex, and is required for endothelial cell alignment to laminar shear flow. Endothelium-restricted KO mice develop hypertension in response to chronic angiotensin-II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of type 2 diabetes (T2D). These data demonstrate that LRRC8A regulates AKT-eNOS in endothelium and is required for maintaining vascular function, particularly in the setting of T2D.
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http://dx.doi.org/10.7554/eLife.61313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997661PMC
February 2021

Chemokine mediated signalling within arteries promotes vascular smooth muscle cell recruitment.

Commun Biol 2020 12 4;3(1):734. Epub 2020 Dec 4.

Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.

The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.
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http://dx.doi.org/10.1038/s42003-020-01462-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719186PMC
December 2020

Assessment of Vascular Patterning in the Zebrafish.

Methods Mol Biol 2021 ;2206:205-222

Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.

The zebrafish has emerged as a valuable and important model organism for studying vascular development and vascular biology. Here, we discuss some of the approaches used to study vessels in fish, including loss-of-function tools such as morpholinos and genetic mutants, along with methods and considerations for assessing vascular phenotypes. We also provide detailed protocols for methods used for vital imaging of the zebrafish vasculature, including microangiography and long-term time-lapse imaging. The methods we describe, and the considerations we suggest using for assessing phenotypes observed using these methods, will help ensure reliable, valid conclusions when assessing vascular phenotypes following genetic or experimental manipulation of zebrafish.
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http://dx.doi.org/10.1007/978-1-0716-0916-3_15DOI Listing
March 2021

Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling.

Nat Commun 2020 03 5;11(1):1204. Epub 2020 Mar 5.

Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, USA.

Anti-angiogenic therapies have generated significant interest for their potential to combat tumor growth. However, tumor overproduction of pro-angiogenic ligands can overcome these therapies, hampering success of this approach. To circumvent this problem, we target the resynthesis of phosphoinositides consumed during intracellular transduction of pro-angiogenic signals in endothelial cells (EC), thus harnessing the tumor's own production of excess stimulatory ligands to deplete adjacent ECs of the capacity to respond to these signals. Using zebrafish and human endothelial cells in vitro, we show ECs deficient in CDP-diacylglycerol synthase 2 are uniquely sensitive to increased vascular endothelial growth factor (VEGF) stimulation due to a reduced capacity to re-synthesize phosphoinositides, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), resulting in VEGF-exacerbated defects in angiogenesis and angiogenic signaling. Using murine tumor allograft models, we show that systemic or EC specific suppression of phosphoinositide recycling results in reduced tumor growth and tumor angiogenesis. Our results suggest inhibition of phosphoinositide recycling provides a useful anti-angiogenic approach.
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http://dx.doi.org/10.1038/s41467-020-14956-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058007PMC
March 2020

Consensus guidelines for the use and interpretation of angiogenesis assays.

Angiogenesis 2018 08;21(3):425-532

Laboratory of Cellular Metabolism and Metabolic Regulation, VIB Center for Cancer Biology, Leuven, Belgium.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
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http://dx.doi.org/10.1007/s10456-018-9613-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237663PMC
August 2018

Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor Signaling.

Arterioscler Thromb Vasc Biol 2018 02 28;38(2):353-362. Epub 2017 Dec 28.

From the Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD (S.K., A.N.S., C.H.M., J.G., D.C., B.M.W.); Departments of Biochemistry and Cell Biology and of Pathology, Geisel School of Medicine at Dartmouth College, Lebanon, NH (R.V.S.); Vascular Biology Program, IFOM, FIRC Institute of Molecular Oncology Foundation, Milan, Italy (M.M., E.D.); and Department of Immunology, Genetics and Pathology, Uppsala University, Sweden (E.D.).

Objective: The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6's mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization.

Approach And Results: We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell-cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak.

Conclusions: Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans.
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http://dx.doi.org/10.1161/ATVBAHA.117.309571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785447PMC
February 2018

A novel perivascular cell population in the zebrafish brain.

Elife 2017 04 11;6. Epub 2017 Apr 11.

Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.

The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or 'Mato Cells' in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium.
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http://dx.doi.org/10.7554/eLife.24369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423774PMC
April 2017

Interactions between mural cells and endothelial cells stabilize the developing zebrafish dorsal aorta.

Development 2017 01 2;144(1):115-127. Epub 2016 Dec 2.

Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

Mural cells (vascular smooth muscle cells and pericytes) play an essential role in the development of the vasculature, promoting vascular quiescence and long-term vessel stabilization through their interactions with endothelial cells. However, the mechanistic details of how mural cells stabilize vessels are not fully understood. We have examined the emergence and functional role of mural cells investing the dorsal aorta during early development using the zebrafish. Consistent with previous literature, our data suggest that cells ensheathing the dorsal aorta emerge from a sub-population of cells in the adjacent sclerotome. Inhibition of mural cell recruitment to the dorsal aorta through disruption of pdgfr signaling leads to a reduced vascular basement membrane, which in turn results in enhanced dorsal aorta vessel elasticity and failure to restrict aortic diameter. Our results provide direct in vivo evidence for a functional role for mural cells in patterning and stabilization of the early vasculature through production and maintenance of the vascular basement membrane to prevent abnormal aortic expansion and elasticity.
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http://dx.doi.org/10.1242/dev.143131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278630PMC
January 2017

Building the drains: the lymphatic vasculature in health and disease.

Wiley Interdiscip Rev Dev Biol 2016 11 30;5(6):689-710. Epub 2016 Aug 30.

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.

The lymphatic vasculature is comprised of a network of endothelial vessels found in close proximity to but separated from the blood vasculature. An essential tissue component of all vertebrates, lymphatics are responsible for the maintenance of fluid homeostasis, dissemination of immune cells, and lipid reabsorption under healthy conditions. When lymphatic vessels are impaired due to invasive surgery, genetic disorders, or parasitic infections, severe fluid build-up accumulates in the affected tissues causing a condition known as lymphedema. Malignant tumors can also directly activate lymphangiogenesis and use these vessels to promote the spread of metastatic cells. Although their first description goes back to the times of Hippocrates, with subsequent anatomical characterization at the beginning of the 20th-century, the lack of identifying molecular markers and tools to visualize these translucent vessels meant that investigation of lymphatic vessels fell well behind research of blood vessels. However, after years under the shadow of the blood vasculature, recent advances in imaging technologies and new genetic and molecular tools have accelerated the pace of research on lymphatic vessel development. These new tools have facilitated both work in classical mammalian models and the emergence of new powerful vertebrate models like zebrafish, quickly driving the field of lymphatic development back into the spotlight. In this review, we summarize the highlights of recent research on the development and function of the lymphatic vascular network in health and disease. WIREs Dev Biol 2016, 5:689-710. doi: 10.1002/wdev.246 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wdev.246DOI Listing
November 2016

Temporal-specific roles of Rac1 during vascular development and retinal angiogenesis.

Dev Biol 2016 Mar 10;411(2):183-194. Epub 2016 Feb 10.

Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, United States; Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, United States. Electronic address:

Angiogenesis, the formation of new blood vessels by remodeling and growth of pre-existing vessels, is a highly orchestrated process that requires a tight balance between pro-angiogenic and anti-angiogenic factors and the integration of their corresponding signaling networks. The family of Rho GTPases, including RhoA, Rac1, and Cdc42, play a central role in many cell biological processes that involve cytoskeletal changes and cell movement. Specifically for Rac1, we have shown that excision of Rac1 using a Tie2-Cre animal line results in embryonic lethality in midgestation (embryonic day (E) 9.5), with multiple vascular defects. However, Tie2-Cre can be also expressed during vasculogenesis, prior to angiogenesis, and is active in some hematopoietic precursors that can affect vessel formation. To circumvent these limitations, we have now conditionally deleted Rac1 in a temporally controlled and endothelial-restricted fashion using Cdh5(PAC)-iCreERT2 transgenic mice. In this highly controlled experimental in vivo system, we now show that Rac1 is required for embryonic vascular integrity and angiogenesis, and for the formation of superficial and deep vascular networks in the post-natal developing retina, the latter involving a novel specific function for Rac1 in vertical blood vessel sprouting. Aligned with these findings, we show that RAC1 is spatially involved in endothelial cell migration, invasion, and radial sprouting activities in 3D collagen matrix in vitro models. Hence, Rac1 and its downstream molecules may represent potential anti-angiogeneic therapeutic targets for the treatment of many human diseases that involve aberrant neovascularization and blood vessel overgrowth.
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http://dx.doi.org/10.1016/j.ydbio.2016.02.005DOI Listing
March 2016

Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

PLoS One 2013 31;8(12):e85147. Epub 2013 Dec 31.

Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Sciences Center and University of Missouri School of Medicine, Columbia, Missouri, United States of America.

We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085147PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877341PMC
September 2014

Control of vascular tube morphogenesis and maturation in 3D extracellular matrices by endothelial cells and pericytes.

Methods Mol Biol 2013 ;1066:17-28

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine and Dalton Cardiovascular Research Center, Columbia, MO, USA.

An important advance using in vitro EC tube morphogenesis and maturation models has been the development of systems using serum-free defined media. Using this approach, the growth factors and cytokines which are actually necessary for these events can be determined. The first model developed by our laboratory was such a system where we showed that phorbol ester was needed in order to promote survival and tube morphogenesis in 3D collagen matrices. Recently, we have developed a new system in which the hematopoietic stem cell cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and stromal derived factor-1α (SDF-1α) were added in conjunction with FGF-2 to promote human EC tube morphogenesis in 3D collagen matrices under serum-free defined conditions. This new model using SCF, IL-3, SDF-1α, and FGF-2 also works well following the addition of pericytes where EC tube formation occurs, pericytes are recruited to the tubes, and vascular basement membrane matrix assembly occurs following EC-pericyte interactions. In this chapter, we describe several in vitro assay models that we routinely utilize to investigate the molecular requirements that are critical to EC tube formation and maturation events in 3D extracellular matrix environments.
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http://dx.doi.org/10.1007/978-1-62703-604-7_2DOI Listing
March 2014

Dynamic regulation of the cerebral cavernous malformation pathway controls vascular stability and growth.

Dev Cell 2012 Aug;23(2):342-55

Department of Medicine and Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth.
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http://dx.doi.org/10.1016/j.devcel.2012.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743537PMC
August 2012

CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis.

Blood 2012 Jul 30;120(2):489-98. Epub 2012 May 30.

Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA

Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.
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http://dx.doi.org/10.1182/blood-2012-02-408328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398756PMC
July 2012

Endothelial cell-pericyte interactions stimulate basement membrane matrix assembly: influence on vascular tube remodeling, maturation, and stabilization.

Microsc Microanal 2012 Feb 14;18(1):68-80. Epub 2011 Dec 14.

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65212, USA.

Extracellular matrix synthesis and deposition surrounding the developing vasculature are critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review article, we highlight new data demonstrating a corequirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli), and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors--platelet derived growth factor-BB and heparin binding-epidermal growth factor--are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formation in vitro and in vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.
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http://dx.doi.org/10.1017/S1431927611012402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919655PMC
February 2012

Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices.

Cells Tissues Organs 2012 13;195(1-2):122-43. Epub 2011 Oct 13.

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Dalton Cardiovascular Research Center, Columbia, Mo. 65212, USA.

Considerable progress has been made toward a molecular understanding of how cells form lumen and tube structures in three-dimensional (3D) extracellular matrices (ECM). This progress has occurred through work performed with endothelial and epithelial cell models using both in vitro and in vivo approaches. Despite the apparent similarities between endothelial and epithelial cell lumen and tube formation mechanisms, there are clear distinctions that directly relate to their functional differences. This review will focus on endothelial cell (EC) lumen formation mechanisms which control blood vessel formation during development and postnatal life. Of great interest is that an EC lumen signaling complex has been identified which controls human EC lumen and tube formation in 3D matrices and which coordinates integrin-ECM contacts, cell surface proteolysis, cytoskeletal rearrangements, and cell polarity. This complex consists of the collagen-binding integrin α2β1, the collagen-degrading membrane-type 1 matrix metalloproteinase (MT1-MMP), junction adhesion molecule (Jam)C, JamB, polarity proteins Par3 and Par6b, and the Rho GTPase Cdc42-GTP. These interacting proteins are necessary to stimulate 3D matrix-specific signaling events (including activation of protein kinase cascades that regulate the actin and microtubule cytoskeletons) to control the formation of EC lumens and tube networks. Also, EC lumen formation is directly coupled to the generation of vascular guidance tunnels, enzymatically generated ECM conduits that facilitate EC tube remodeling and maturation. Mural cells such as pericytes are recruited along EC tubes within these tunnel spaces to control ECM remodeling events resulting in vascular basement membrane matrix assembly, a key step in tube maturation and stabilization.
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http://dx.doi.org/10.1159/000331410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325603PMC
April 2012

RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG.

Blood 2011 Jul 31;118(4):1145-53. Epub 2011 May 31.

Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.
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http://dx.doi.org/10.1182/blood-2010-10-315275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3148162PMC
July 2011

Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice.

J Clin Invest 2011 May 1;121(5):1871-81. Epub 2011 Apr 1.

Molecular Medicine Program, University of Utah, Salt Lake City, Utah, USA.

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.
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http://dx.doi.org/10.1172/JCI44393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083782PMC
May 2011

Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting.

Int Rev Cell Mol Biol 2011 ;288:101-65

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri, USA.

Many studies reveal a fundamental role for extracellular matrix-mediated signaling through integrins and Rho GTPases as well as matrix metalloproteinases (MMPs) in the molecular control of vascular tube morphogenesis in three-dimensional (3D) tissue environments. Recent work has defined an endothelial cell (EC) lumen signaling complex of proteins that controls these vascular morphogenic events. These findings reveal a signaling interdependence between Cdc42 and MT1-MMP to control the 3D matrix-specific process of EC tubulogenesis. The EC tube formation process results in the creation of a network of proteolytically generated vascular guidance tunnels in 3D matrices that are utilized to remodel EC-lined tubes through EC motility and could facilitate processes such as flow-induced remodeling and arteriovenous EC sorting and differentiation. Within vascular guidance tunnels, key dynamic interactions occur between ECs and pericytes to affect vessel remodeling, diameter, and vascular basement membrane matrix assembly, a fundamental process necessary for endothelial tube maturation and stabilization. Thus, the EC lumen and tube formation mechanism coordinates the concomitant establishment of a network of vascular tubes within tunnel spaces to allow for flow responsiveness, EC-mural cell interactions, and vascular extracellular matrix assembly to control the development of the functional microcirculation.
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http://dx.doi.org/10.1016/B978-0-12-386041-5.00003-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891664PMC
July 2011

VEGF and FGF prime vascular tube morphogenesis and sprouting directed by hematopoietic stem cell cytokines.

Blood 2011 Apr 14;117(14):3709-19. Epub 2011 Jan 14.

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65212, USA.

Here, we demonstrate a novel, direct-acting, and synergistic role for 3 hematopoietic stem cell cytokines: stem cell factor, interleukin-3, and stromal derived factor-1α, in controlling human endothelial cell (EC) tube morphogenesis, sprouting, and pericyte-induced tube maturation under defined serum-free conditions in 3-dimensional matrices. Angiogenic cytokines such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) alone or VEGF/FGF combinations do not support these responses. In contrast, VEGF and FGF prime EC responses to hematopoietic cytokines via up-regulation of c-Kit, IL-3Rα, and C-X-C chemokine receptor type 4 from either human ECs or embryonic quail vessel explants. In support of these findings, EC Runx1 is demonstrated to be critical in coordinating vascular morphogenic responses by controlling hematopoietic cytokine receptor expression. Combined blockade of hematopoietic cytokines or their receptors in vivo leads to blockade of developmental vascularization in quail embryos manifested by vascular hemorrhage and disrupted vascular remodeling events in multiple tissue beds. This work demonstrates a unique role for hematopoietic stem cell cytokines in vascular tube morphogenesis and sprouting and further demonstrates a novel upstream priming role for VEGF and FGF to facilitate the action of promorphogenic hematopoietic cytokines.
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http://dx.doi.org/10.1182/blood-2010-11-316752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083293PMC
April 2011

Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization.

Blood 2010 Nov 25;116(22):4720-30. Epub 2010 Aug 25.

Departments of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Research Center, University of Missouri at Columbia, Columbia, MO, USA.

Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)-lined tubes in 3-dimensional (3D) extracellular matrices to stimulate vascular maturation including basement membrane matrix assembly. Through the use of this serum-free, defined system, we demonstrate that pericyte motility within 3D collagen matrices is dependent on the copresence of ECs. Using either soluble receptor traps consisting of the extracellular ligand-binding domains of platelet-derived growth factor receptor β, epidermal growth factor receptor (EGFR), and ErbB4 receptors or blocking antibodies directed to platelet-derived growth factor (PDGF)-BB, or heparin-binding EGF-like growth factor (HB-EGF), we show that both of these EC-derived ligands are required to control pericyte motility, proliferation, and recruitment along the EC tube ablumenal surface. Blockade of pericyte recruitment causes a lack of basement membrane matrix deposition and, concomitantly, increased vessel widths. Combined inhibition of PDGF-BB and HB-EGF-induced signaling in quail embryos leads to reduced pericyte recruitment to EC tubes, decreased basement membrane matrix deposition, increased vessel widths, and vascular hemorrhage phenotypes in vivo, in support of our findings in vitro. In conclusion, we report a dual role for EC-derived PDGF-BB and HB-EGF in controlling pericyte recruitment to EC-lined tubes during developmental vascularization events.
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http://dx.doi.org/10.1182/blood-2010-05-286872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996127PMC
November 2010

Endothelial lumen signaling complexes control 3D matrix-specific tubulogenesis through interdependent Cdc42- and MT1-MMP-mediated events.

Blood 2010 Jun 9;115(25):5259-69. Epub 2010 Mar 9.

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, MO 85212, USA.

Here, we define an endothelial cell (EC) lumen signaling complex involving Cdc42, Par6b, Par3, junction adhesion molecule (Jam)-B and Jam-C, membrane type 1-matrix metalloproteinase (MT1-MMP), and integrin alpha(2)beta(1), which coassociate to control human EC tubulogenesis in 3D collagen matrices. Blockade of both Jam-B and Jam-C using antibodies, siRNA, or dominant-negative mutants completely interferes with lumen and tube formation resulting from a lack of Cdc42 activation, inhibition of Cdc42-GTP-dependent signal transduction, and blockade of MT1-MMP-dependent proteolysis. This process requires interdependent Cdc42 and MT1-MMP signaling, which involves Par3 binding to the Jam-B and Jam-C cytoplasmic tails, an interaction that is necessary to physically couple the components of the lumen signaling complex. MT1-MMP proteolytic activity is necessary for Cdc42 activation during EC tube formation in 3D collagen matrices but not on 2D collagen surfaces, whereas Cdc42 activation is necessary for MT1-MMP to create vascular guidance tunnels and tube networks in 3D matrices through proteolytic events. This work reveals a novel interdependent role for Cdc42-dependent signaling and MT1-MMP-dependent proteolysis, a process that occurs selectively in 3D collagen matrices and that requires EC lumen signaling complexes, to control human EC tubulogenesis during vascular morphogenesis.
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http://dx.doi.org/10.1182/blood-2009-11-252692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892954PMC
June 2010

MT1-MMP- and Cdc42-dependent signaling co-regulate cell invasion and tunnel formation in 3D collagen matrices.

J Cell Sci 2009 Dec 24;122(Pt 24):4558-69. Epub 2009 Nov 24.

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, MO 65212, USA.

Complex signaling events control tumor invasion in three-dimensional (3D) extracellular matrices. Recent evidence suggests that cells utilize both matrix metalloproteinase (MMP)-dependent and MMP-independent means to traverse 3D matrices. Herein, we demonstrate that lysophosphatidic-acid-induced HT1080 cell invasion requires membrane-type-1 (MT1)-MMP-mediated collagenolysis to generate matrix conduits the width of a cellular nucleus. We define these spaces as single-cell invasion tunnels (SCITs). Once established, cells can migrate within SCITs in an MMP-independent manner. Endothelial cells, smooth muscle cells and fibroblasts also generate SCITs during invasive events, suggesting that SCIT formation represents a fundamental mechanism of cellular motility within 3D matrices. Coordinated cellular signaling events are required during SCIT formation. MT1-MMP, Cdc42 and its associated downstream effectors such as MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) and Pak4 (p21 protein-activated kinase 4), protein kinase Calpha and the Rho-associated coiled-coil-containing protein kinases (ROCK-1 and ROCK-2) coordinate signaling necessary for SCIT formation. Finally, we show that MT1-MMP and Cdc42 are fundamental components of a co-associated invasion-signaling complex that controls directed single-cell invasion of 3D collagen matrices.
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http://dx.doi.org/10.1242/jcs.050724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787465PMC
December 2009

Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation.

Blood 2009 Dec 12;114(24):5091-101. Epub 2009 Oct 12.

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Center, University of Missouri, Columbia, MO 65212, USA.

We show that endothelial cell (EC)-generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, alpha(5)beta(1), alpha(3)beta(1), alpha(6)beta(1), and alpha(1)beta(1), which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.
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http://dx.doi.org/10.1182/blood-2009-05-222364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788982PMC
December 2009

Formation of endothelial lumens requires a coordinated PKCepsilon-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream of Cdc42 activation.

J Cell Sci 2009 Jun 12;122(Pt 11):1812-22. Epub 2009 May 12.

Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.

In this study, we present data showing that Cdc42-dependent lumen formation by endothelial cells (ECs) in three-dimensional (3D) collagen matrices involves coordinated signaling by PKCepsilon in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCepsilon during this process. Src and Yes are differentially expressed during EC lumen formation and siRNA suppression of either kinase, but not Fyn or Lyn, results in significant inhibition of EC lumen formation. Concurrent with Cdc42 activation, PKCepsilon- and SFK-dependent signaling converge to activate p21-activated kinase (Pak)2 and Pak4 in steps that are also required for EC lumen formation. Pak2 and Pak4 further activate two Raf kinases, B-Raf and C-Raf, leading to ERK1 and ERK2 (ERK1/2) activation, which all seem to be necessary for EC lumen formation. This work reveals a multicomponent kinase signaling pathway downstream of integrin-matrix interactions and Cdc42 activation involving PKCepsilon, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices.
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http://dx.doi.org/10.1242/jcs.045799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684834PMC
June 2009

Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices.

Blood 2009 Jul 1;114(2):237-47. Epub 2009 Apr 1.

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Center, University of Missouri-Columbia, MO 65212, USA.

Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.
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http://dx.doi.org/10.1182/blood-2008-12-196451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714200PMC
July 2009

In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.

Methods Enzymol 2008 ;443:83-101

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Center, University of Missouri-Columbia, Columbia, Missouri, USA.

Discovery and comprehension of detailed molecular signaling pathways underlying endothelial vascular morphogenic events including endothelial lumen formation are key steps in understanding their roles during embryonic development, as well as during various disease states. Studies that used in vitro three-dimensional (3D) matrix endothelial cell morphogenic assay models, in conjunction with in vivo studies, have been essential to identifying molecules and explaining their related signaling pathways that regulate endothelial cell morphogenesis. We present methods to study molecular mechanisms controlling EC lumen formation in 3D collagen matrices. In vitro models representing vasculogenesis and angiogenesis, whereby EC lumen formation and tube morphogenesis readily occur, are described. We also detail different methods of gene manipulation in ECs and their application to analyze critical signaling events regulating EC lumen formation.
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http://dx.doi.org/10.1016/S0076-6879(08)02005-3DOI Listing
January 2009

Mechanisms controlling human endothelial lumen formation and tube assembly in three-dimensional extracellular matrices.

Birth Defects Res C Embryo Today 2007 Dec;81(4):270-85

Department of Medical Pharmacology and Physiology, School of Medicine and Dalton Cardiovascular Center, University of Missouri-Columbia, Columbia, Missouri 65212, USA.

Recent data have revealed new mechanisms that underlie endothelial cell (EC) lumen formation during vascular morphogenic events in development, wound repair, and other disease states. It is apparent that EC interactions with extracellular matrices (ECMs) establish signaling cascades downstream of integrin ligation leading to activation of the Rho GTPases, Cdc42 and Rac1, which are required for lumen formation. In large part, this process is driven by intracellular vacuole formation and coalescence, which rapidly leads to the creation of fluid-filled matrix-free spaces that are then interconnected via EC-EC interactions to create multicellular tube structures. EC vacuoles markedly accumulate in a polarized fashion directly adjacent to the centrosome in a region that strongly accumulates Cdc42 protein as indicated by green fluorescent protein (GFP)-Cdc42 during the lumen formation process. Downstream of Cdc42-mediated signaling, key molecules that have been identified to be required for EC lumen formation include Pak2, Pak4, Par3, Par6, and the protein kinase C (PKC) isoforms zeta and epsilon. Together, these molecules coordinately regulate the critical EC lumen formation process in three-dimensional (3D) collagen matrices. These events also require cell surface proteolysis mediated through membrane type 1 matrix metalloproteinase (MT1-MMP), which is necessary to create vascular guidance tunnels within the 3D matrix environment. These tunnels represent physical spaces within the ECM that are necessary to regulate vascular morphogenic events, including the establishment of interconnected vascular tube networks as well as the recruitment of pericytes to initiate vascular tube maturation (via basement membrane matrix assembly) and stabilization. Current research continues to analyze how specific molecules integrate signaling information in concert to catalyze EC lumen formation, pericyte recruitment, and stabilization processes to control vascular morphogenesis in 3D extracellular matrices.
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http://dx.doi.org/10.1002/bdrc.20107DOI Listing
December 2007
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