Wonshill Koh

The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases

Cerebral cavernous malformation (CCM) is a common vascular dysplasia that affects both systemic and central nervous system blood vessels. Loss of function mutations in the CCM2 gene cause CCM. Here we show that targeted disruption of Ccm2 in mice results in failed lumen formation and early embryonic death through an endothelial cell autonomous mechanism. We show that CCM2 regulates endothelial cytoskeletal architecture, cell-to-cell interactions and lumen formation. Heterozygosity at Ccm2, a genotype equivalent to that in human CCM, results in impaired endothelial barrier function. On the basis of our biochemical studies indicating that loss of CCM2 results in activation of RHOA GTPase, we rescued the cellular phenotype and barrier function in heterozygous mice with simvastatin, a drug known to inhibit Rho GTPases. These data offer the prospect for pharmacological treatment of a human vascular dysplasia with a widely available and safe drug.

Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling

Rho GTPases regulate a diverse spectrum of cellular functions involved in vascular morphogenesis. Here, we show that Cdc42 and Rac1 play a key role in endothelial cell (EC) lumen and tube formation as well as in EC invasion in three-dimensional (3D) collagen matrices and that their regulation is mediated by various downstream effectors, including Pak2, Pak4, Par3 and Par6. RNAi-mediated or dominant-negative suppression of Pak2 or Pak4, two major regulators of cytoskeletal signaling downstream of Cdc42 or Rac1, markedly inhibits EC lumen and tube formation. Both Pak2 and Pak4 phosphorylation strongly correlate with the lumen formation process in a manner that depends on protein kinase C (PKC)-mediated signaling. We identify PKCϵ and PKCζ as regulators of EC lumenogenesis in 3D collagen matrices. Two polarity proteins, Par3 and Par6, are also required for EC lumen and tube formation, as they establish EC polarity through their association with Cdc42 and atypical PKC. In our model, disruption of any member in the Cdc42-Par3-Par6-PKCζ polarity complex impairs EC lumen and tube formation in 3D collagen matrices. This work reveals novel regulators that control the signaling events mediating the crucial lumen formation step in vascular morphogenesis.

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

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.

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

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.

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

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.

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

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.

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.

Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

BackgroundLysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT1-MMP in this process.ResultsLPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (~700 μm over 48 hours) in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (~125 μm vs. ~45 μm) and velocity of invasion (~0.09 μm/min vs. ~0.03 μm/min) only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT)-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process.ConclusionLPA is a fundamental regulator of MT1-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, S1P appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MT1-MMP is required only when tumor cells navigate 3D barriers and not when cells migrate on 2D substrata. We demonstrate that tumor cells require coordinate regulation of LPA/S1P receptors and Rho GTPases to migrate, and additionally, require MT1-MMP in order to invade collagen matrices during neoplastic progression.

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

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 PKCϵ in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCϵ 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, PKCϵ- 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 PKCϵ, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices.

Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish

Talin (tln) binds and activates integrins to couple extracellular matrix‐bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1fl02k mutants. The ethylnitrosourea‐induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor ‐2a, was dominantly expressed in the heart and vessels. Unlike tln1 and ‐2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z‐disks and endothelial/endocardial cell integrity, partly through regulating F‐actin networks in zebrafish. The similar expression profiles of tln1 and integrin β1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z‐disks and vessel lumens. Taken together, the results of this work suggest that Tln1‐mediated Itgβ 1b plays a crucial role in maintaining cardiac sarcomeric Z‐disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.—Wu, Q., Zhang, J., Koh, W., Yu, Q., Zhu, X., Amsterdam, A., Davis, G. E., Arnaout, M. A., Xiong, J.‐W. Talin1 is required for cardiac Z‐disk stabilization and endothelial integrity in zebrafish. FASEB J. 29, 4989–5005 (2015). www.fasebj.org