Identification of distinct vascular mural cell populations during zebrafish embryonic development.

BACKGROUND: Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells. However, the overlapping and distinct expression patterns of these markers in tandem have not been fully described. RESULTS: Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular cell populations on the cranial, axial, and intersegmental vessels between 1 and 5 days postfertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord-a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. CONCLUSION: Together, our findings highlight the variability and versatility of tracking both pdgfrb and tagln expression in mural cells of the developing zebrafish embryo and reveal unexpected embryonic cell populations that express pdgfrb and tagln.

High-throughput methodology to identify CRISPR-generated Danio rerio mutants using fragment analysis with unmodified PCR products.

Targeted mutagenesis in zebrafish, fruit flies, and C. elegans has been significantly improved over the years through CRISPR technology. CRISPR enables researchers to efficiently examine cellular pathways by inducing small, targeted mutations in vivo. Though these mutations are commonly random insertions or deletions (indels), they often result in functionally disrupted alleles of a target gene if the CRISPR components are appropriately designed. However, current protocols used to identify the presence of CRISPR-generated indels are often labor intensive, time-consuming, or expensive. Here, we describe a straightforward, high-throughput method for identifying the presence of mutations by using a fragment analyzer platform which allows for DNA fragment sizing through high-resolution capillary gel-electrophoresis. Following this protocol, small indels-down to 2 base pairs-can be quickly and reliably identified, thus allowing for large-scale genotyping of newly-generated or stable mutant lines.

Peristaltic pumps adapted for laminar flow experiments enhance in vitro modeling of vascular cell behavior.

Endothelial cells (ECs) are the primary cellular constituent of blood vessels that are in direct contact with hemodynamic forces over their lifetime. Throughout the body, vessels experience different blood flow patterns and rates that alter vascular architecture and cellular behavior. Because of the complexities of studying blood flow in an intact organism, particularly during development, the field has increasingly relied on in vitro modeling of blood flow as a powerful technique for studying hemodynamic-dependent signaling mechanisms in ECs. While commercial flow systems that recirculate fluids exist, many commercially available pumps are peristaltic and best model pulsatile flow conditions. However, there are many important situations in which ECs experience laminar flow conditions in vivo, such as along long straight stretches of the vasculature. To understand EC function under these contexts, it is important to be able to reproducibly model laminar flow conditions in vitro. Here, we outline a method to reliably adapt commercially available peristaltic pumps to study laminar flow conditions. Our proof-of-concept study focuses on 2D models but could be further adapted to 3D environments to better model in vivo scenarios, such as organ development. Our studies make significant inroads into solving technical challenges associated with flow modeling and allow us to conduct functional studies toward understanding the mechanistic role of shear forces on vascular architecture, cellular behavior, and remodeling in diverse physiological contexts.

In vivo dissection of Rhoa function in vascular development using zebrafish.

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

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

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.

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

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.

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

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.

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

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.

Hemodynamics regulate spatiotemporal artery muscularization in the developing circle of Willis.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries, playing a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular diseases and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating differentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on the zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that the arterial expression of CoW endothelial cells (ECs) occurs after their migration from the cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors upon recruitment to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity wall shear stress. Furthermore, pulsatile blood flow is required for differentiation of human brain pdgfrb+ mural cells into VSMCs as well as VSMC differentiation on zebrafish CoW arteries. Consistently, the flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight the role of blood flow activation of endothelial klf2a as a mechanism regulating the initial VSMC differentiation on vertebrate brain arteries.

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

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

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.

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

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.

Identification of overlapping and distinct mural cell populations during early embryonic development.

Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells (vSMCs). However, the expression patterns of these markers used in tandem have not been fully described. Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular populations in the cranial, axial, and intersegmental vessels between 1 and 5 days post-fertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord- a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique sclerotome-derived mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. Together, our findings highlight the variability and versatility of tracking pdgfrb and tagln expression in mural cells of the developing zebrafish embryo.

FAM210B is dispensable for erythroid differentiation in adult mice.

Iron plays a central role in cellular redox processes, but its ability to adopt multiple oxidation states also enables it to catalyze deleterious reactions. The requirement for iron in erythropoiesis has necessitated the evolution of mechanisms with which to handle the iron required for hemoglobinization. FAM210B was identified as a regulator of mitochondrial iron import and heme synthesis in erythroid cell culture and zebrafish models. In this manuscript, we demonstrate that while FAM210B is required for erythroid differentiation and heme synthesis under standard cell culture conditions, holotransferrin supplementation was sufficient to chemically complement the iron-deficient phenotype. As the biology of FAM210B is complex and context specific, and whole-organism studies on FAM210 proteins have been limited, we sought to unravel the role of FAM210B in erythropoiesis using knockout mice. We were surprised to discover that Fam210b -/- mice were viable and the adults did not have erythropoietic defects in the bone marrow. In contrast to studies in C. elegans, Fam210b -/- mice were also fertile. There were some modest phenotypes, such as a slight increase in lymphocytes and white cell count in Fam210b -/- females, as well as an increase in body weight in Fam210b -/- males. However, our findings suggest that FAM210B may play a more important role in cellular iron homeostasis under iron deficient conditions. Here, we will discuss the cell culture conditions used in iron metabolism studies that can account for the disparate finding on FAM210B function. Moving forward, resolving these discrepancies will be important in identifying novel iron homeostasis genes.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

Objective: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (K ATP ) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with distinct hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Approach and Results: Whole-cell voltage-clamp of isolated aortic and mesenteric VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no difference in voltage-gated K + (K v ) or Ca 2+ currents. K v and Ca 2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. Pinacidil-sensitive K ATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, and were considerably larger in CS hiPSC-VSMCs. Consistent with lack of any compensatory modulation of other currents, this resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Increased compliance and dilation in isolated CS mouse aortae, was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular K ATP GoF. Conclusions: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. The results further indicate that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by K ATP overactivity within VSMCs.

Peristalsis-Associated Mechanotransduction Drives Malignant Progression of Colorectal Cancer.

Introduction: In the colorectal cancer (CRC) tumor microenvironment, cancerous and precancerous cells continuously experience mechanical forces associated with peristalsis. Given that mechanical forces like shear stress and strain can positively impact cancer progression, we explored the hypothesis that peristalsis may also contribute to malignant progression in CRC. We defined malignant progression as enrichment of cancer stem cells and the acquisition of invasive behaviors, both vital to CRC progression. Methods: We leveraged our peristalsis bioreactor to expose CRC cell lines (HCT116), patient-derived xenograft (PDX1,2) lines, or non-cancerous intestinal cells (HIEC-6) to forces associated with peristalsis in vitro. Cells were maintained in static control conditions or exposed to peristalsis for 24 h prior to assessment of cancer stem cell (CSC) emergence or the acquisition of invasive phenotypes. Results: Exposure of HCT116 cells to peristalsis significantly increased the emergence of LGR5+ CSCs by 1.8-fold compared to static controls. Peristalsis enriched LGR5 positivity in several CRC cell lines, notably significant in KRAS mutant lines. In contrast, peristalsis failed to increase LGR5+ in non-cancerous intestinal cells, HIEC-6. LGR5+ emergence downstream of peristalsis was dependent on ROCK and Wnt activity, and not YAP1 activation. Additionally, HCT116 cells adopted invasive morphologies when exposed to peristalsis, with increased filopodia density and epithelial to mesenchymal gene expression, in a Wnt dependent manner. Conclusions: Peristalsis associated forces drive malignant progression of CRC via ROCK, YAP1, and Wnt-related mechanotransduction. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00776-w.

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

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 ß, 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.

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

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.

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

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 α2ß1, 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.