TGF-ß signaling is required for maintenance of retinal ganglion cell differentiation and survival.

PURPOSE: To determine the role of TGF-ß1 in the maintenance of retinal ganglion cell line (RGC-5) differentiation and integrity. METHODS: RGC-5 cells were differentiated in media conditioned by human non-pigmented ciliary epithelial cells (HNPE) for 4 days before treatment with TGF-ß1 for 24 h. Cells were examined for morphological changes and harvested for western blot and real-time PCR analysis. For study of apoptosis, differentiated RGC-5 cells were grown in serum-free medium for 24 h in the presence or absence of TGF-ß1 and collected for Annexin V/Propidium iodide FACs analysis. The role of MAPK pathways in TGF-ß1-dependent signaling was determined by treatment with specific inhibitors of ERK, JNK and p38. RESULTS: Differentiation of RGC-5 cells in HNPE-conditioned media (CM) increased the neural cell markers, Brn-3c, NF-160, Thy1.2, Tau and PGP9.5. Treatment with TGF-ß1 significantly increased the length of neurites extended by differentiated RGC-5s, concomitant with increased expression of NF-160 and PGP9.5, but not Brn-3c, Thy1.2 or Tau. TGF-ß1 also decreased RGC-5 cell apoptosis in serum-free medium. p38 phosphorylation, but not smad2/3, JNK or ERK phosphorylation, was increased in TGF-ß1 treated cells. Specific inhibition of p38 signaling reversed TGF-ß1 induced neurite growth. CONCLUSIONS: These findings demonstrate the induction of RGC-5 cell differentiation by HNPE-derived CM and illustrate a role for TGF-ß1 in maintaining RGC-5 cell survival and promoting neurite outgrowth through p38 MAPK.

What tangled webs they weave: Rho-GTPase control of angiogenesis.

The members of the Rho family of small GTPases are involved in an array of cellular processes, including regulation of the actin cytoskeleton, cell polarity, microtubule dynamics, membrane transport, and transcription factor activity. Recent findings have implicated the Rho-proteins as key regulators of angiogenesis, modulating a diversity of cellular processes, including vascular permeability, extracellular matrix remodeling, migration, proliferation, morphogenesis, and survival.

Development of the endothelium.

Our understanding of the regulation of vascular development has exploded over the past decade. Prior to this time, our knowledge of vascular development was primarily based on classic descriptive studies. The identification of stem cells, lineage markers, specific growth factors and their receptors, and signalling pathways has facilitated a rapid expansion in information regarding details of the mechanisms that govern development of the vascular system.

Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival.

Pericytes have been suggested to play a role in regulation of vessel stability; one mechanism for this stabilization may be via pericyte-derived vascular endothelial growth factor (VEGF). To test the hypothesis that differentiation of mesenchymal cells to pericytes/smooth muscle cells (SMC) is accompanied by VEGF expression, we used endothelial cell (EC) and mesenchymal cell cocultures to model cell-cell interactions that occur during vessel development. Coculture of EC and 10T1/2 cells, multipotent mesenchymal cells, led to induction of VEGF expression by 10T1/2 cells. Increased VEGF expression was dependent on contact between EC-10T1/2 and was mediated by transforming growth factorbeta (TGFbeta). A majority of VEGF produced in coculture was cell- and/or matrix-associated. Treatment of cells with high salt, protamine, heparin, or suramin released significant VEGF, suggesting that heparan sulfate proteoglycan might be sequestering some of the VEGF. Inhibition of VEGF in cocultures led to a 75% increase in EC apoptosis, indicating that EC survival in cocultures is dependent on 10T1/2-derived VEGF. VEGF gene expression in developing retinal vasculature was observed in pericytes contacting newly formed microvessels. Our observations indicate that differentiated pericytes produce VEGF that may act in a juxtacrine/paracrine manner as a survival and/or stabilizing factor for EC in microvessels.

Wnt signaling in the vasculature.

The Wnt signaling pathway regulates normal development as well as a variety of pathologies. Studies of the Wnt pathway have focused largely on very early development and on tumorigenesis. Recent observations point to a role for Wnt signaling in vessel development and pathology. Although not yet investigated systematically, several Wnt ligands have been demonstrated to be expressed in the cells of blood vessels in vivo and in vitro, including Wnt-2, -5a, -7a and -10b. Mice deficient for Wnt-2 display vascular abnormalities including defective placental vasculature. Wnt receptors, called frizzled (Fz), are also expressed by vascular cells in culture and in situ. Of the 10 murine Fz identified to date, Fz-1, -2, -3, and -5 have been demonstrated in endothelial and vascular smooth muscle cells; mice deficient for Fz-5 display vascular abnormalities and are embryonic lethal. Two soluble, naturally occurring Wnt antagonists, frizzled-related proteins (FRP)-1 and -3, are also expressed by vascular cells. Stabilization of the downstream signaling component beta-catenin in blood vessels has been demonstrated in several developmental and pathologic states, further supporting the idea that Wnt signaling plays an important regulatory role in the vasculature.

Cell-cell interactions in vascular development.

Research into areas as divergent as hemangiopoiesis and cardiogenesis as well as investigations of diseases such as cancer and diabetic retinopathy have converged to form the face of research in vascular development today. This convergence of disparate topics has resulted in rapid advances in many areas of vascular research. The focus of this review has been the role of cell-cell interactions in the development of the vascular system, but we have included discussions of pathology where the mechanism of disease progression may have parallels with developmental processes. A number of intriguing questions remain unanswered. For example, what triggers abnormal angiogenesis in the disease state? Are the mechanisms similar to those that control developmental neovascularization? Perhaps the difference in development in angiogenesis versus in disease is context driven, that is, an adult versus an embryonic organism. If this is the case, can the controls that curtail developmental vessel formation be applied in pathologies? Can cell-cell interactions be targeted as a control point for new vessel formation? For instance, can perivascular cells be stimulated or eliminated to result in increased vessel stability or instability, respectively? If the hypothesis that mural cell association is required for vessel stabilization is accurate, are there mechanisms to promote or inhibit mural cell recruitment and differentiation as needed? These and other questions lie in wait for the next generation of approaches to discern the mechanisms and the nature of the cell-cell interactions and the influence of the microenvironment on vascular development.

Cellular interactions in vascular growth and differentiation.

In nature, mammalian cells do not exist in isolation, but rather are involved in interactions with other cells and matrix. In this review, several aspects of cellular interactions that are important in vascular growth and development will be highlighted. The cardiovascular system is the earliest to develop in the embryo. A number of growth factors and their receptors mediate the complex stages of migration, assembly, organization, and stabilization of developing vessels. In the adult organism, normal angiogenesis is restricted primarily to tissue growth (such as muscle and fat), the wound healing process and the female reproductive system. However, pathological angiogenesis, such as with tumor growth, diabetic retinopathy, and arthritis, is of great concern. The identification and/or development of exogenous and endogenous angiogenesis inhibitors has added to the understanding of these pathological processes. In addition to cellular interactions via ligands and receptors, cells also interact directly through physical contacts. These interactions facilitate anchorage, communication, and permeability. Since vessels serve as non-leaky conduits for blood flow as well as interfaces for molecular diffusion, the physical interactions between the cells that make up vessels must be specific for the function at hand. Permeability is a specialized function of vessels and is mediated by intracellular mechanisms and intercellular interactions. Cells also interact with the surrounding extracellular matrix. Integrin-matrix interaction is a two-way exchange critical for angiogenesis. Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases play major roles in embryonic remodeling, adult injury, and pathological conditions. Several experimental model systems have been useful in our understanding of cellular interactions. These in vitro models incorporate heterotypic cell-cell interactions and/or allow cell-matrix interactions to occur.

Differential expression of VEGF isoforms in mouse during development and in the adult.

Vascular endothelial growth factor (VEGF), a factor that is critical for development of the vascular system in mouse embryos, exists as at least three isoforms, VEGF120, VEGF164, and VEGF188. The isoforms have different affinities for heparan sulfate as well as for the three known VEGF receptors, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and neuropilin-1, suggesting that different VEGF isoforms may play distinct roles in vascular development. To determine whether there are differences in the organ-specific expression patterns that would support this concept, we used a quantitative RNase protection assay (RPA) to determine the distribution of different VEGF isoform mRNA in developing and adult mouse organs. Results revealed that the ratios of the three VEGF isoforms changed during organ development and that adult organs expressed different levels of the three VEGF isoforms. Because the lung expressed the highest levels of VEGF188 isoform, we used VEGF isoform-specific in situ hybridization in the developing lung and determined that type II alveolar epithelial cells were expressing high levels of VEGF188 mRNA. Finally, targeted exon deletion of the VEGF gene revealed that mice that developed in the absence of the heparan sulfate binding isoforms VEGF164 and VEGF188, displayed a variety of vascular defects, including abnormal pulmonary vascular development. Our results support the concept that different VEGF isoforms have distinct functions in vascular development.

TGF beta is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells.

New vessels form de novo (vasculogenesis) or from pre-existing vessels (angiogenesis) in a process that involves the interaction of endothelial cells (EC) and pericytes/smooth muscle cells (SMC). One basic component of this interaction is the endothelial-induced recruitment, proliferation and subsequent differentiation of pericytes and SMC. We have previously demonstrated that TGF beta induces the differentiation of C3H/10T1/2 (10T1/2) mesenchymal cells toward a SMC/pericyte lineage. The current study tests the hypothesis that TGF beta not only induces SMC differentiation but stabilizes capillary-like structures in a three-dimensional (3D) model of in vitro angiogenesis. 10T1/2 and EC in Matrigel were used to establish cocultures that form cord structures that are reminiscent of new capillaries in vivo. Cord formation is initiated within 2-3 h after plating and continues through 18 h after plating. In longer cocultures the cord structures disassemble and form aggregates. 10T1/2 expression of proteins associated with the SMC/pericyte lineage, such as smooth muscle alpha-actin (SMA) and NG2 proteoglycan, are upregulated in these 3D cocultures. Application of neutralizing reagents specific for TGF beta blocks cord formation and inhibits expression of SMA and NG2 in the 10T1/2 cells. We conclude that TGF beta mediates 10T1/2 differentiation to SMC/pericytes in the 3D cocultures and that association with differentiated mural cells is required for formation of capillary-like structures in Matrigel.

Release of bFGF, an endothelial cell survival factor, by osmotic shock.

PURPOSE: To test the effects of osmotic change on basic fibroblast growth factor (bFGF) release from cultured endothelial cells (ECs). METHODS: Bovine aortic and bovine retinal ECs were exposed to hypoosmotic shock for 2 minutes, were allowed to recover for 15 minutes, and had bFGF release assayed. The role of bFGF in cell recovery was assessed by including neutralizing antibody against bFGF or the addition of exogenous bFGF. Cell number and viability were determined under varying conditions. Apoptosis was assessed by immunoperoxidase detection of digoxigenin-labeled DNA. RESULTS: After shock and recovery, both ECs released significantly greater amounts of bFGF than untreated control. bFGF release after shock for 2 minutes was lower than release after shock and recovery. Bovine retinal endothelial (BRE) cell number was reduced at 48 hours after shock, recovery, and removal of released bFGF compared with cells left in the presence of released bFGF. Cell number was significantly lower when BRE cells were shocked and recovered in the presence of a neutralizing anti-bFGF antibody (P<0.05). Exogenous bFGF reversed this effect. Apoptosis was significantly increased in BRE cells shocked and recovered or in the presence of bFGF antibody (P<0.001). CONCLUSIONS: bFGF is released by cultured ECs in response to osmotically induced cell injury. These results support the concept of bFGF as a "wound" hormone and survival factor for ECs. In further compromised tissue, release of bFGF in this manner may play a role in the pathogenesis of disease.

A secreted frizzled related protein, FrzA, selectively associates with Wnt-1 protein and regulates wnt-1 signaling.

The Wnt gene family encodes proteins that serve key roles in differentiation and development. Wnt proteins interact with seven transmembrane receptors of the Frizzled family and activate a signaling pathway leading to the nucleus. A primary biochemical effect of Wnt-1 signaling is the stabilization of cytoplasmic (beta)-catenin which, in association with transcription factors of the Lef/tcf family, regulates gene expression. The recent identification of a new class of secreted proteins with similarity to the extracellular, ligand-binding domain of Frizzled proteins, soluble Frizzled related proteins (sFRP), suggested that additional mechanisms could regulate Wnt signaling. Here we demonstrate that FrzA, a sFRP that is highly expressed in vascular endothelium and a variety of epithelium, specifically binds to Wnt-1 protein, but not Wnt-5a protein, and modulates Wnt-1 signaling. FrzA associated with Wnt-1 either when expressed in the same cell or when soluble FrzA was incubated with Wnt-1-expressing cells. FrzA efficiently inhibited the Wnt-1 mediated increase in cytoplasmic (beta)-catenin levels as well as the Wnt-1 induction of transcription from a Lef/tcf reporter gene. The effects of FrzA on (beta)-catenin levels could be demonstrated when co-expressed with Wnt-1 or when individual cells expressing FrzA and Wnt-1 were co-cultured. These data demonstrate the existence of a negative regulatory mechanism mediated by the selective binding of FrzA to Wnt-1 protein.

Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro.

Angiogenesis is a complex process that includes recruitment and proliferation of mural cells-smooth muscle cells (SMC) and pericytes. Vascular endothelial growth factor (VEGF) has been shown to play an important role in angiogenesis and is an endothelial cell chemoattractant. In addition, certain VEGF isoforms have been implicated in the normal formation of smooth muscle cell-surrounded arteries. Because VEGF's role as a mural cell chemoattractant had not been explored, we examined the ability of VEGF to influence vascular SMC migration in vitro. A Boyden chamber migration assay demonstrated that VEGF (0-100 ng/ml) caused a dose-dependent migration of SMC. VEGF did not cause proliferation of SMC. Reverse transcriptase-polymerase chain reaction analysis demonstrated the presence of both KDR and flt mRNA, two known VEGF receptors, in SMC cultures. Western blot analysis of SMC lysates confirmed these data, revealing bands migrating at approximately 200 kDa and slightly below 200 kDa consistent with KDR and flt. These observations demonstrate that VEGF receptors are present on SMC, and that VEGF can act as an SMC chemoattractant.

Identification and cloning of a secreted protein related to the cysteine-rich domain of frizzled. Evidence for a role in endothelial cell growth control.

We report the isolation of a cDNA, FrzA (frizzled in aorta; GenBank accession No. U85945), from bovine aortic endothelium. It is the bovine counterpart of the mouse sFRP1, which encodes for a secreted protein that is homologous to the cysteine-rich domain of frizzled. Members of the frizzled family of genes have been shown to be required for tissue polarity and to act as receptors for Wnt. The predicted protein product of this gene includes the cysteine-rich extracellular domain, but not the 7 putative transmembrane domains that are highly conserved among members of the frizzled family. Visualization of FrzA mRNA and protein revealed that it was widely distributed among adult tissues. FrzA is expressed by highly differentiated or polarized cells, eg, neurons, cardiocytes, or various epithelia. Analysis of its expression in endothelium revealed that FrzA mRNA levels were high in endothelial cells scraped from freshly obtained bovine aortas, decreased when cells were placed in culture and began to proliferate, but increased at confluence. Transient transfection assays and an assay using addition of purified protein indicate that FrzA reduces the proliferation of endothelial cells. These data demonstrate the existence of a secreted protein homologous to the extracellular domain of the fz receptor, which we speculate plays a role in controlling cell growth and differentiation, possibly by regulating accessibility to Wnt family members.

Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact.

Embryological data suggest that endothelial cells (ECs) direct the recruitment and differentiation of mural cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal cells (10T1/2 cells) and induce their differentiation toward a smooth muscle cell/pericyte lineage. The present study was undertaken to investigate cell proliferation in these cocultures. ECs and 10T1/2 cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-beta had no effect on EC-stimulated 10T1/2 cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 cells in the absence of contact. The cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated cell sorter analysis revealed that ECs and 10T1/2 cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 cell cocultures inhibited the growth of both cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-beta in coculture-induced mural cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-beta. Furthermore, the decreased proliferation of 10T1/2 cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-beta in the 10T1/2 cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal cells and that heterotypic cell-cell contact and soluble factors play a role in growth control during vessel assembly.

Functional and biochemical interactions of Wnts with FrzA, a secreted Wnt antagonist.

Wnts are highly conserved developmental regulators that mediate inductive signaling between neighboring cells and participate in the determination of embryonic axes. Frizzled proteins constitute a large family of putative transmembrane receptors for Wnt signals. FrzA is a novel protein that shares sequence similarity with the extracellular domain of Frizzled. The Xenopus homologue of FrzA is dynamically regulated during early development. At the neurula stages, XfrzA mRNA is abundant in the somitic mesoderm, but later becomes strongly expressed in developing heart, neural crest derivatives, endoderm, otic vesicle and other sites of organogenesis. To evaluate possible biological functions of FrzA, we analyzed its effect on early Xenopus development. Microinjection of bovine or Xenopus FrzA mRNA into dorsal blastomeres resulted in a shortened body axis, suggesting a block of convergent extension movements. Consistent with this possibility, FrzA blocked elongation of ectodermal explants in response to activin, a potent mesoderm-inducing factor. FrzA inhibited induction of secondary axes by Xwnt8 and human Wnt2, but not by Xdsh, supporting the idea that FrzA interferes with Wnt signaling. Furthermore, FrzA suppressed Wnt-dependent activation of the early response genes in ectodermal explants and in the marginal zone. Finally, immunoprecipitation experiments demonstrate that FrzA binds to the soluble Wingless protein in cell culture supernatants in vitro. Our results indicate that FrzA is a naturally occurring secreted antagonist of Wnt signaling.

PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate.

We aimed to determine if and how endothelial cells (EC) recruit precursors of smooth muscle cells and pericytes and induce their differentiation during vessel formation. Multipotent embryonic 10T1/2 cells were used as presumptive mural cell precursors. In an under-agarose coculture, EC induced migration of 10T1/2 cells via platelet-derived growth factor BB. 10T1/2 cells in coculture with EC changed from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture. Immunohistochemical and Western blot analyses were used to examine the expression of smooth muscle (SM)-specific markers in 10T1/2 cells cultured in the absence and presence of EC. SM-myosin, SM22alpha, and calponin proteins were undetectable in 10T1/2 cells cultured alone; however, expression of all three SM-specific proteins was significantly induced in 10T1/2 cells cocultured with EC. Treatment of 10T1/2 cells with TGF-beta induced phenotypic changes and changes in SM markers similar to those seen in the cocultures. Neutralization of TGF-beta in the cocultures blocked expression of the SM markers and the shape change. To assess the ability of 10T1/2 cells to contribute to the developing vessel wall in vivo, prelabeled 10T1/2 cells were grown in a collagen matrix and implanted subcutaneously into mice. The fluorescently marked cells became incorporated into the medial layer of developing vessels where they expressed SM markers. These in vitro and in vivo observations shed light on the cell-cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.