Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction

Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho-logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm-Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm-Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock-Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm-Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.     

Mechanical Cell-Matrix Feedback Explains Pairwise and Collective Endothelial Cell Behavior In Vitro

In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a) the contractile forces that endothelial cells exert on the ECM, (b) the resulting strains in the extracellular matrix, and (c) the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.     

Cell-cell mechanical communication through compliant substrates

The role of matrix mechanics on cell behavior is under intense investigation. Cells exert contractile forces on their matrix and the matrix elasticity can alter these forces and cell migratory behavior. However, little is known about the contribution of matrix mechanics and cell-generated forces to stable cell-cell contact and tissue formation. Using matrices of varying stiffness and measurements of endothelial cell migration and traction stresses, we find that cells can detect and respond to substrate strains created by the traction stresses of a neighboring cell, and that this response is dependent on matrix stiffness. Specifically, pairs of endothelial cells display hindered migration on gels with elasticity below 5500 Pa in comparison to individual cells, suggesting these cells sense each other through the matrix. We believe that these results show for the first time that matrix mechanics can foster tissue formation by altering the relative motion between cells, promoting the formation of cell-cell contacts. Moreover, our data indicate that cells have the ability to communicate mechanically through their matrix. These findings are critical for the understanding of cell-cell adhesion during tissue formation and disease progression, and for the design of biomaterials intended to support both cell-matrix and cell-cell adhesion.     

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.     

Acceleration of Vascular Sprouting from Fabricated Perfusable Vascular-Like Structures

Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA) and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs), adhered to gold-coated needles (ϕ600 μm) via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d) and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC) activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully vascularized tissues and organs.     

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.     

VEGF and endothelial guidance in angiogenic sprouting

The cellular actions of VEGF need to be coordinated to guide vascular patterning during sprouting angiogenesis. Individual endothelial tip cells lead and guide the blood vessel sprout, while neighboring stalk cells proliferate and form the vascular lumen. Recent studies illustrate how endothelial DLL4/NOTCH signalling, stimulated by VEGF, regulates the sprouting response by limiting tip cell formation in the stalk. The spatial distribution of VEGF, in turn, regulates the shape of the ensuing sprout by directing tip cell migration and determining stalk cell proliferation.     

Modulation of VEGF signalling output by the Notch pathway

The formation of blood vessels within the vascular system entails a variety of cellular processes, including proliferation, migration and differentiation. In many cases, these diverse processes need to be finely coordinated among neighbouring endothelial cells in order to establish a functional vascular network. For instance, during angiogenic sprouting specialized endothelial tip cells follow guidance cues and migrate extensively into avascular tissues while trailing stalk cells must stay connected to the patent blood vessel. The vascular endothelial growth factor (VEGF) and Notch signalling pathways have emerged as the major players in governing these different cellular behaviours. In particular, recent work indicates an important role for Notch signalling in determining how an endothelial cell responds to VEGF. In this review, we provide an overview of these biochemically distinct pathways and discuss how they may interact during endothelial cell differentiation and angiogenesis.     

Endothelial cell motility is compatible with junctional integrity

Confluent endothelial cells in culture are generally regarded as a model of resting endothelium in blood vessels (i.e., forming junctions at points of cell-cell contact, losing ability to proliferate in response to growth factors, and remaining stationary). However, incompatibility between junctional integrity and endothelial cell motility remains uncertain. The aim of this study was to determine whether endothelial cells (in colonies generated from differentiating embryonic stem cells in contact with OP9 stromal cell layer) have a resting endothelial phenotype (i.e., lack motility). Time-lapse analyses showed that though endothelial cells were connected to each other through adherens junctions and tight junctions, they were moving continuously within the colonies. Endothelial cell movement was accompanied by formation of lamellipodia, which transiently accumulated green fluorescent protein-tagged beta-actin and p41-Arc (a subunit of the actin-related protein 2/3 complex) at their anterior tips, suggesting that the movement is an active behavior of endothelial cells. Endothelial cell-specific expression of yellow fluorescent protein-tagged vascular endothelial-cadherin and claudin-5 revealed that adherens junctions and tight junctions persisted during endothelial cell migration. Furthermore, intercellular junctions underwent dynamic remodeling at the leading edge of moving endothelial cells. These results suggest that endothelial cells can remain highly motile without losing intercellular junctions.     

A multiscale model of complex endothelial cell dynamics in early angiogenesis

We introduce a hybrid two-dimensional multiscale model of angiogenesis, the process by which endothelial cells (ECs) migrate from a pre-existing vascular bed in response to local environmental cues and cell-cell interactions, to create a new vascular network. Recent experimental studies have highlighted a central role of cell rearrangements in the formation of angiogenic networks. Our model accounts for this phenomenon via the heterogeneous response of ECs to their microenvironment. These cell rearrangements, in turn, dynamically remodel the local environment. The model reproduces characteristic features of angiogenic sprouting that include branching, chemotactic sensitivity, the brush border effect, and cell mixing. These properties, rather than being hardwired into the model, emerge naturally from the gene expression patterns of individual cells. After calibrating and validating our model against experimental data, we use it to predict how the structure of the vascular network changes as the baseline gene expression levels of the VEGF-Delta-Notch pathway, and the composition of the extracellular environment, vary. In order to investigate the impact of cell rearrangements on the vascular network structure, we introduce the mixing measure, a scalar metric that quantifies cell mixing as the vascular network grows. We calculate the mixing measure for the simulated vascular networks generated by ECs of different lineages (wild type cells and mutant cells with impaired expression of a specific receptor). Our results show that the time evolution of the mixing measure is directly correlated to the generic features of the vascular branching pattern, thus, supporting the hypothesis that cell rearrangements play an essential role in sprouting angiogenesis. Furthermore, we predict that lower cell rearrangement leads to an imbalance between branching and sprout elongation. Since the computation of this statistic requires only individual cell trajectories, it can be computed for networks generated in biological experiments, making it a potential biomarker for pathological angiogenesis.     

Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly

New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.     

Domains I and IV of Annexin A2 Affect the Formation and Integrity of In Vitro Capillary-Like Networks

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

A low-Mr chemoattractant for vascular endothelial cells

The formation of new blood vessels occurs by sprouting from previously existing microvasculature. The process involved directed migration of the vascular endothelial cells towards chemical signals released from the target tissue. We have used the Boyden chemotaxis chamber method to identify chemotactic signals for fetal bovine vascular endothelial cells. Human placenta organ cultures produce a high-Mr chemoattractant for the endothelial cells from which a low-Mr factor can be liberated with trichloroacetic acid treatment and ethanol extraction. This activity was isolated from extracts of human placenta using Sephadex LH-20, Amberlite XAD-2, and silica gel thin-layer chromatography. The Mr of the factor is less than 400, it is lipophilic and resistant to proteolytic enzymes. The factor induces chemotactic migration of both aortic endothelial cells and capillary endothelial cells from the retina, but has no effect on fibroblasts or leukocytes suggesting a specific function of the compound for the vascular endothelial cells.     

Endothelial connexin32 enhances angiogenesis by positively regulating tube formation and cell migration

The gap junction proteins connexin32 (Cx32), Cx37, Cx40, and Cx43 are expressed in endothelial cells, and regulate vascular functions involving inflammation, vasculogenesis and vascular remodeling. Aberrant Cxs expression promotes the development of atherosclerosis which is modulated by angiogenesis; however the role played by endothelial Cxs in angiogenesis remains unclear. In this study, we determined the effects of endothelial Cxs, particularly Cx32, on angiogenesis. EA.hy926 cells that had been transfected to overexpress Cx32 significantly increased capillary length and the number on branches compared to Cx-transfectant cells over-expressing Cx37, Cx40, and Cx43 or mock-treated cells. Treatment via intracellular transfer of anti-Cx32 antibody suppressed tube formation of human umbilical vein endothelial cells (HUVECs) compared to controls. In vitro wound healing assays revealed that Cx32-transfectant cells significantly increased the repaired area while anti-Cx32 antibody-treated HUVECs reduced it. Ex vivo aorta ring assays and in vivo matrigel plaque assays showed that Cx32-deficient mice impaired both vascular sprouting from the aorta and cell migration into the implanted matrigel. Therefore endothelial Cx32 facilitates tube formation, wound healing, vascular sprouting, and cell migration. Our results suggest that endothelial Cx32 positively regulates angiogenesis by enhancing endothelial cell tube formation and cell migration.     

Endothelial adhesion molecules in the development of the vascular tree: the garden of forking paths.

In the past, year targeted null mutation studies have further supported the concept that endothelial cell-matrix and cell-cell adhesion is involved in the formation and maintenance of the network of branched tubes within the vascular tree. In addition, recent results derived from the closely related experimental system of branching tubulogenesis in epithelial cells may provide an appealing model for endothelial biology.     

Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos.

Gap junctional proteins (connexins) form aqueous channels that enable direct cell-cell transfer of ions and small molecules. The distribution and conductance of gap junction channels in cardiac muscle determine the pattern and synchrony of cellular activation. However, the capacity for smooth muscle to restrict contractile events temporally and spatially suggests that cell-cell coupling or its regulation may be decidedly different in this tissue. We isolated a cDNA from vascular smooth muscle which encodes a connexin (Mr 43,187) structurally homologous to cardiac connexin43. Vascular smooth muscle connexin43 mRNA was expressed prominently in smooth muscle tissues, cultured vascular myocytes, and arterial endothelial cells. A model for functional expression of connexins was developed in two-cell B6D2 mouse embryos. Microinjection of in vitro transcribed vascular smooth muscle connexin43 mRNA was shown to be sufficient to induce intercellular coupling in previously uncoupled blastomeres. Through the construction of two deletion mutants of connexin43, we also show that the formation of cell-to-cell connections does not depend upon a predicted cytoplasmic region within 98 residues of the carboxyl terminus. Finally, the identification of connexin43 in smooth muscle and endothelial cells provides supporting evidence for the existence of heterocellular coupling between cells of the vascular intima.