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.

In vivo adenovirus-mediated delivery of a uPA/uPAR antagonist reduces retinal neovascularization in a mouse model of retinopathy.

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization. We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.

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.

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.

Activin A and follistatin influence expression of somatostatin in the ciliary ganglion in vivo.

An important developmental question concerns whether neurotransmitter phenotype is an inherent property of neurons or is influenced by target tissues. This issue can be addressed in the avian ciliary ganglion (CG) which contains two cholinergic populations, ciliary and choroid neurons, that differentially express the peptide cotransmitter, somatostatin. The present study tests the hypothesis that differences in the level of expression of activin A and its endogenous inhibitor follistatin in CG neuron target tissues are responsible for selective expression of somatostatin in choroid neurons. Intraocular injection of activin A or follistatin (300 ng injected at E10/E11) in cultured embryos resulted in a 39% increase or a 23% decrease, respectively, in somatostatin-positive neurons relative to controls. Chorioallantoic membrane application of follistatin (1 microgram daily from E7 to E13) reduced somatostatin positive neurons by 54%. Neuron number, size, and target tissue morphology were unaffected by these treatments. Together with our previous studies, these data suggest that activin A and follistatin are target-derived molecules that regulate neuropeptide phenotype in the ciliary ganglion.

Activin A and follistatin expression in developing targets of ciliary ganglion neurons suggests a role in regulating neurotransmitter phenotype.

The avian ciliary ganglion contains choroid neurons that innervate choroid vasculature and express somatostatin as well as ciliary neurons that innervate iris/ciliary body but do not express somatostatin. We have previously shown in culture that activin A induces somatostatin immunoreactivity in both neuron populations. We now show in vivo that both targets contain activin A; however, choroid expressed higher levels of activin A mRNA. In contrast, follistatin, an activin A inhibitor, was higher in iris/ciliary body. Iris cell-conditioned medium also contained an activity that inhibited activin A and could be depleted with anti-follistatin antibodies. These results suggest that development of somatostatin is limited to choroid neurons by differential expression of activin A and follistatin in ciliary ganglion targets.