Kayla J. Bayless

Endothelial tubes assemble from intracellular vacuoles in vivo

The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described “longitudinal vacuoles” that “appeared to be extruded and connected from one cell to the next”, observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin–extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation.

Regulation of tissue injury responses by the exposure of matricryptic sites within extracellular matrix molecules.

Extracellular matrix (ECM) is known to provide signals controlling cell shape, migration, proliferation, differentiation, morphogenesis, and survival. Recent data shows that some of these signals are derived from biologically active cryptic sites within matrix molecules (matricryptic sites) that are revealed after structural or conformational alteration of these molecules. We propose the name, matricryptins, for enzymatic fragments of ECM containing exposed matricryptic sites. Mechanisms regulating the exposure of matricryptic sites within ECM molecules include the major mechanism of enzymatic breakdown as well as others including ECM protein multimerization, adsorption to other molecules, cell-mediated mechanical forces, and ECM denaturation. Such matrix alterations occur during or as a result of tissue injury, and thus, the appearance of matricryptic sites within an injury site may provide important new signals to regulate the repair process. Here, we review the data supporting this concept and provide insight into why the increased exposure of matricryptic sites may be an important regulatory step in tissue responses to injury.

RGD-Dependent Vacuolation and Lumen Formation Observed during Endothelial Cell Morphogenesis in Three-Dimensional Fibrin Matrices Involves the αvβ3 and α5β1 Integrins

Recent data have revealed the involvement of the αvβ3 integrin in angiogenesis. However, few studies to date have provided a convincing role for this receptor in in vitro assays of endothelial cell morphogenesis where defined steps can be examined. Here, we present data showing that two integrins, αvβ3 and α5β1, regulate human endothelial cell vacuolation and lumen formation in three-dimensional fibrin matrices. Cells resuspended in fibrin formed intracellular vacuoles that coalesced into lumenal structures. These morphogenic events were completely inhibited by the simultaneous addition of anti-αvβ3 and anti-α5 integrin antibodies. Complete blockade was also accomplished with a combination of the cyclic Arg-Gly-Asp (cRGD) peptide and anti-α5 integrin antibodies. No blockade was observed with the control Arg-Gly-Glu (RGE) peptide alone or in combination with control antibodies. Finally, we were able to demonstrate regression of vacuoles and lumens several hours after the addition of cRGD peptides combined with anti-α5 integrin antibodies. These effects were not observed with control peptides alone or in combination with control antibodies. We report here the novel involvement of both the αvβ3 and α5β1 integrins in vacuolation and lumen formation in a fibrin matrix, implicating a role for multiple integrins in endothelial cell morphogenesis.

Molecular basis of endothelial cell morphogenesis in three-dimensional extracellular matrices

Although many studies have focused on blood vessel development and new blood vessel formation associated with disease processes, the question of how endothelial cells (ECs) assemble into tubes in three dimensions (i.e., EC morphogenesis) remains unanswered. EC morphogenesis is particularly dependent on a signaling axis involving the extracellular matrix (ECM), integrins, and the cytoskeleton, which regulates EC shape changes and signals the pathways necessary for tube formation. Recent studies reveal that genes regulating this matrix‐integrin‐cytoskeletal (MIC) signaling axis are differentially expressed during EC morphogenesis. The Rho GTPases represent an important class of molecules involved in these events. Cdc42 and Rac1 are required for the process of EC intracellular vacuole formation and coalescence that regulates EC lumen formation in three‐dimensional (3D) extracellular matrices, while RhoA appears to stabilize capillary tube networks. Once EC tube networks are established, supporting cells, such as pericytes, are recruited to further stabilize these networks, perhaps by regulating EC basement membrane matrix assembly. Furthermore, we consider recent work showing that EC morphogenesis is balanced by a tendency for newly formed tubes to regress. This morphogenesis‐regression balance is controlled by differential gene expression of such molecules as VEGF, angiopoietin‐2, and PAI‐1, as well as a plasmin‐ and matrix metalloproteinase‐dependent mechanism that induces tube regression through degradation of ECM scaffolds that support EC‐lined tubes. It is our hope that this review will stimulate increased interest and effort focused on the basic mechanisms regulating capillary tube formation and regression in 3D extracellular matrices. Anat Rec 268:252–275, 2002.

Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3

The endothelial cell (EC)–derived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following EC–pericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. EC–pericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in EC–pericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)–, MMP-10–, and ADAM-15 (a disintegrin and metalloproteinase-15)–dependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events.

Novel pathways for implantation and establishment and maintenance of pregnancy in mammals

Uterine receptivity to implantation varies among species, and involves changes in expression of genes that are coordinate with attachment of trophectoderm to uterine lumenal and superficial glandular epithelia, modification of phenotype of uterine stromal cells, silencing of receptors for progesterone and estrogen, suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, angiogenesis and vasculogenesis, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine epithelial and stromal cells in response to progesterone, glucocorticoids, prostaglandins and interferons may influence uterine receptivity to implantation in mammals. Uterine receptivity to implantation is progesterone-dependent; however, implantation is preceded by loss of expression of receptors for progesterone (PGR) so that progesterone most likely acts via PGR-positive stromal cells throughout pregnancy. Endogenous retroviruses expressed by the uterus and/or blastocyst also affect implantation and placentation in various species. Understanding the roles of the variety of hormones, growth factors and endogenous retroviral proteins in uterine receptivity for implantation is essential to enhancing reproductive health and fertility in humans and domestic animals.

MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices

Previous work has shown that endothelial cell (EC)-derived matrix metalloproteinases (MMPs) regulate regression of capillary tubes in vitro in a plasmin- and MMP-1 dependent manner. Here we report that a number of serine proteases can activate MMP-1 and cause capillary tube regression; namely plasma kallikrein, trypsin, neutrophil elastase, cathepsin G, tryptase and chymase. Plasma prekallikrein failed to induce regression without coactivators such as high molecular weight kininogen (HMWK) or coagulation Factor XII. The addition of trypsin, the neutrophil serine proteases (neutrophil elastase and cathepsin G) and the mast cell serine proteases (tryptase and chymase) each caused MMP-1 activation and collagen type I proteolysis, capillary tubular network collapse, regression and EC apoptosis. Capillary tube collapse is accompanied by collagen gel contraction, which is strongly related to the wound contraction that occurs during regression of granulation tissue in vivo. We also report that proMMP-10 protein expression is markedly induced in ECs undergoing capillary tube morphogenesis. Addition of each of the serine proteases described above led to activation of proMMP-10, which also correlated with MMP-1 activation and capillary tube regression. Treatment of ECs with MMP-1 or MMP-10 siRNA markedly delayed capillary tube regression, whereas gelatinase A (MMP-2), gelatinase B (MMP-9) and stromelysin-1 (MMP-3) siRNA-treated cells behaved in a similar manner to controls and regressed normally. Increased expression of MMP-1 or MMP-10 in ECs using recombinant adenoviral delivery markedly accelerated serine protease-induced capillary tube regression. ECs expressing increased levels of MMP-10 activated MMP-1 to a greater degree than control ECs. Thus, MMP-10–induced activation of MMP-1 correlated with tube regression and gel contraction. In summary, our work demonstrates that MMP-1 zymogen activation is mediated by multiple serine proteases and MMP-10, and that these events are central to EC-mediated collagen degradation and capillary tube regression in 3D collagen matrices.

An integrin and Rho GTPase-dependent pinocytic vacuole mechanism controls capillary lumen formation in collagen and fibrin matrices.

A major question that remains unanswered concerning endothelial cell (EC) morphogenesis is how lumens are formed in three‐dimensional extracellular matrices (ECMs). Studies from many laboratories have revealed a critical role for an ECM‐integrin‐cytoskeletal signaling axis during EC morphogenesis. We have discovered a mechanism involving intracellular vacuole formation and coalescence that is required for lumen formation in several in vitro models of morphogenesis. In addition, a series of studies have observed vacuoles in vivo during angiogenic events. These vacuoles form through an integrin‐dependent pinocytic mechanism in either collagen or fibrin matrices. In addition, we have shown that the Cdc42 and Rac1 guanosine triphosphatases (GTPases), which control actin and microtubule cytoskeletal networks, are required for vacuole and lumen formation. These GTPases are also known to regulate integrin signaling and are activated after integrin‐matrix interactions. Furthermore, the expression of green fluorescent protein‐Rac1 or ‐Cdc42 chimeric proteins in ECs results in the targeting of these fusion proteins to intracellular vacuole membranes during lumen formation. Thus, a matrix‐integrin‐cytoskeletal signaling axis involving both the Cdc42 and Rac1 GTPases regulates the process of EC lumen formation in three‐dimensional collagen or fibrin matrices.

Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices

SummaryHere, we describe assay systems that utilize serum-free defined media to evaluate capillary morphogenesis during human endothelial cell (EC) invasion of three-dimensional collagen matrices. ECs invade these matrices over a 1–3-d period to form capillary tubes. Blocking antibodies to the α2β1 integrin interfere with invasion and morphogenesis while other integrin blocking antibodies do not. Interestingly, we observed increased invasion of ECs toward a population of underlying ECs undergoing morphogenesis. In addition, we have developed assays on microscope slides that display the invasion process horizontally, thereby enhancing our ability to image these events. Thus far, we have observed intracellular vacuoles that appear to regulate the formation of capillary lumens, and extensive cell processes that facilitate the interconnection of ECs during morphogenic events. These assays should enable further investigation of the morphologic steps and molecular events controlling human capillary tube formation in three-dimensional extracellular matrices.

Secreted Phosphoprotein 1 (SPP1, Osteopontin) Binds to Integrin Alphavbeta6 on Porcine Trophectoderm Cells and Integrin Alphavbeta3 on Uterine Luminal Epithelial Cells, and Promotes Trophectoderm Cell Adhesion and Migration

Conceptus implantation involves pregnancy-specific alterations in extracellular matrix at the conceptus-maternal interface. Secreted phosphoprotein 1 (SPP1, osteopontin) is induced just before implantation and is present at the conceptus-maternal interface in mammals. In the present study, we investigated mechanisms by which SPP1 facilitates porcine conceptus and uterine luminal epithelial cell attachment. Native bovine milk and wild-type rat recombinant SPP1 stimulated trophectoderm cell migration. Bovine milk SPP1, ovine uterine SPP1, and recombinant wild-type, but not mutated, rat SPP1 promoted dose- and cation-dependent attachment of porcine trophectoderm and uterine luminal epithelial cells, which was markedly reduced in the presence of a linear Arg-Gly-Asp integrin-blocking peptide. Affinity chromatography and immunoprecipitation experiments revealed direct binding of alphavbeta6 trophectoderm and alphavbeta3 uterine epithelial cell integrins to SPP1. Immunofluorescence microscopy using SPP1-coated microspheres revealed colocalization of the alphav integrin subunit and talin at focal adhesions as well as at the apical domain of trophectoderm cells. Similarly, immunofluorescence staining of implantation sites in frozen gravid uterine cross sections localized SPP1 and alphav integrin to the apical surfaces of trophectoderm and luminal epithelium and beta3 integrin to the apical surface of luminal epithelium. To our knowledge, the present study is the first to demonstrate functionally that SPP1 directly binds specific integrins to promote trophectoderm cell migration and attachment to luminal epithelium that may be critical to conceptus elongation and implantation.