Chitra Suri

Requisite Role of Angiopoietin-1, a Ligand for the TIE2 Receptor, during Embryonic Angiogenesis

Vascular endothelial growth factor (VEGF), which acts via members of a family of endothelial-specific receptor tyrosine kinases, is the only factor that has been shown definitively to play a role in the formation of the embryonic vasculature. Only one other family of receptor tyrosine kinases, comprising TIE1 and TIE2, is largely endothelial cell specific. We have recently cloned a ligand for TIE2, termed Angiopoietin-1. Here we show that mice engineered to lack Angiopoietin-1 display angiogenic deficits reminiscent of those previously seen in mice lacking TIE2, demonstrating that Angiopoietin-1 is a primary physiologic ligand for TIE2 and that it has critical in vivo angiogenic actions that are distinct from VEGF and that are not reflected in the classic in vitro assays used to characterize VEGF. Angiopoietin-1 seems to play a crucial role in mediating reciprocal interactions between the endothelium and surrounding matrix and mesenchyme.

Angiopoietin-2 Is Required for Postnatal Angiogenesis and Lymphatic Patterning, and Only the Latter Role Is Rescued by Angiopoietin-1

VEGF and Angiopoietin-1 requisitely collaborate during blood vessel development. While Angiopoietin-1 obligately activates its Tie2 receptor, Angiopoietin-2 can activate Tie2 on some cells, while it blocks Tie2 activation on others. Our analysis of mice lacking Angiopoietin-2 reveals that Angiopoietin-2 is dispensable for embryonic vascular development but is requisite for subsequent angiogenic remodeling. Unexpectedly, mice lacking Angiopoietin-2 also exhibit major lymphatic vessel defects. Genetic rescue with Angiopoietin-1 corrects the lymphatic, but not the angiogenesis, defects, suggesting that Angiopoietin-2 acts as a Tie2 agonist in the former setting, but as an antagonist in the latter setting. Our studies define a vascular growth factor whose primary role is in postnatal angiogenic remodeling and also demonstrate that members of the VEGF and Angiopoietin families collaborate during development of the lymphatic vasculature.

Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins

NGF and the other neurotrophins all bind to the low affinity NGF receptor (LNGFR). Although early studies suggested that the LNGFR was absolutely required for the formation of a functional neurotrophin receptor, current evidence indicates that the Trk family of receptor tyrosine kinases, in the absence of the LNGFR, can directly bind to and mediate responses to the neurotrophins. Here we describe a functional approach, in fibroblasts, designed to assay for the ability of the LNGFR to potentiate Trk-mediated responses to the neurotrophins. We report that although collaboration between the LNGFR and the Trks could be detected in this system, a truncated form of the LNGFR displayed a much more dramatic ability to interact functionally with each of the Trks, potentiating masked autocrine loops as well as responses to limiting amounts of exogenously provided neurotrophins.

Angiopoietin 2 expression in the retina: upregulation during physiologic and pathologic neovascularization

Vascular development in the embryo requires coordinated signaling through several endothelial cell‐specific receptors; however, it is not known whether this is also required later during retinal vascular development or as part of retinal neovascularization in adults. The Tie2 receptor has been implicated in stabilization and maturation of vessels through action of an agonist ligand, angiopoietin 1 (Ang1) and an antagonistic ligand, Ang2. In this study, we have demonstrated that ang2 mRNA levels are increased in the retina during development of the deep retinal capillaries by angiogenesis and during pathologic angiogenesis in a model of ischemic retinopathy. Mice with hemizygous disruption of the ang2 gene by insertion of a promoterless β‐galactosidase (βgal) gene behind the ang2 promoter, show constitutive βgal staining primarily in cells along the outer border of the inner nuclear layer identified as horizontal cells by colocalization of calbindin. During development of the deep capillary bed or retinal neovascularization, other cells in the inner nuclear layer and ganglion cell layer, in regions of neovascularization, stain for βgal. Thus, there is temporal and spatial correlation of Ang2 expression with developmental and pathologic angiogenesis in the retina, suggesting that it may play a role. J. Cell. Physiol. 184:275–284, 2000.

Absence of hippocampal mossy fiber sprouting in transgenic mice overexpressing brain‐derived neurotrophic factor

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain‐derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain‐derived neurotrophic factor. Despite 2–3‐fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild‐type mice. In addition, no changes of the mRNA expression of two growth‐associated proteins, GAP‐43 and SCG‐10 were found. These data suggest that early and persistent elevations of brain‐derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus. J. Neurosci. Res. 64:268–276, 2001.

Growth Factors in Vascular Morphogenesis: Insights from Gene Knockout Studies in Mice

The basic processes leading to the generation of a mature vascular network are conserved between avians and mammals, however, more is known about the actual players in these processes in mammals due to the relative ease of genetic manipulations in mice.

The Ties That Bind: Emerging Concepts About the Structure and Function of Angiopoietins and Their Receptors in Angiogenesis

The Tie [tyrosine kinase with immunoglobulin and epidermal growth factor (EGF) homology domains] receptors, Tie-1 and Tie-2, have been found to be localized primarily to the endothelial and hematopoietic cells in all organisms (human, mouse, rat, zebrafish) that have been examined (Wilks, 1989;Dumont et al., 1992;Iwama, et al., 1993;Maisonpierre et al., 1993;Sato et al., 1993;Lyons et al., 1998). The only other receptors that share this feature are those that bind and are activated by the (VEGF) family members (discussed in the previous chapter). Tie-1 and Tie-2 are large (-160 kDa) multidomain proteins that are highly homologous. The rat genes share 32% sequence identity in their extracellular regions and 79% sequence identity in their intracellular regions (Maisonpierre et al., 1997). The mouse genes are both on chromosome 4, separated by only 12.2 centimorgans (cM), while the human Tie-1 gene is on 1p33–34, which is a syntenic location (Korhonen et al., 1994). Human Tie-2 is on 9p21. The ligands for Tie-2, the angiopoietins, comprise a unique family of proteins, the first member of which was cloned only a few years ago (Davis et al., 1996;Maisonpierre et al., 1997;Valenzuela et al., 1999). All the angiopoietins are highly homologous to each other; angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are —60% identical in their amino acid sequence; angiopoietin-3 and -4 (Ang3 and 4) show —54% identity to Ang1 (Maisonpierre et al., 1997;Valenzuela et al., 1999). They have diverged enough to serve different functions and to reside on different chromosomes.15 (syntenic); for Ang2, the human gene is on 8p21 and the mouse gene on chromosome 8 (syn-tenic), whereas human Ang4 is on 20p13 and mouse Ang3 is on chromosome 2 (which are also syntenic) (Valenzuela et aI., 1999). So far, there are no definitive ligands for Tie-I.