from leukocytes, tumor cells or the endothelium can degrade HS chains.38 Second, nitric oxide can damage HS chains. Such HSPGs are repaired by undergoing cellular internalization, damaged chain regions are removed, fresh HS chains are re-synthesized on the core proteins, and the repaired HSPG is transported back to the cell surface.39 Third, extracellular sulfatases, which remove specific sulfate moieties, can modify HS motifs. For example, 6-O-sulfate groups can be removed by the Sulf enzymes.40 Fourth, proteoglycans bound to the cell membrane can be shed. Phospholipase activity liberates GPI-linked HSPGs whereas shedding of integral membrane HSPGs involves protease cleavage of the extracellular domain near the transmembrane domain.11 Thus, the HSPG structure can be altered by multiple extracellular factors. The potential influence of such factors on HS-mediated cell signaling is discussed below.

4. Evolution of HSPGs

An evolutionary perspective reveals many fundamental features of HSPGs. Bona fide HS polysaccharide has not been found in plants, unicellular organisms, or prokaryotes. Rigorous structural studies have found that HS occurs in most metazoans, being absent only from the most primitive multicellular animal, the sponges. The two more complex phyla Ctenophora and Cnidaria, whose ancestors represent the earliest forms of Eumetazoa (true metazoans), clearly possess vertebrate-type HS structures (reviewed in Refs. 41 and 42). The long split between Eumetazoans and sponges, 940 million years ago, testifies to the extreme antiquity of HS. On one hand, HS may have arisen by conferring a selection advantage through optimizing pre-existent processes. Potential candidates include processes which were firmly established in single cell organisms, such as cell signaling and adhesion. Additional candidates include totipotent stem cells, innate immunity, chemokine production, and apoptosis, which all occur in sponges. On the other hand, the emergence of the complex HS biosynthetic pathway may have involved selection for a unique process appearing at the divergence of Eumetazoans. In particular, the emergence of an integrated mechanism for whole organ homeostasis enabled this evolutionary

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split.43 Eumetazoa does not include the sponges because they essentially function like a colony of animals. Sponges are comprised of a semiautonomous groups of cells. Each group has an independent inlet to obtain seawater that contains food/oxygen and an independent outlet for removal of waste/carbon dioxide. The earliest Eumetazoans, in contrast, have a single inlet into and a single outlet draining a common gastrovascular cavity. This specialized sealed compartment is surrounded by an evolutionarily novel sheet of cells, the original “epithelium sensu stricto.” This cell layer serves to regulate and separate the fluids in distinct extracellular compartments. Thus, Eumetazoans are characterized by a single circulatory system that enables whole body homeostasis.43

An increase in cellular diversity accompanied this change in body plan. Whereas sponges arise from a single germ layer, early Eumetazoans had two germ layers (endoderm and ectoderm) that enabled the formation of the new epithelium sensu stricto. Neurons were an additional novel early Eumetazoan cell type, which allowed for further coordination of whole body homeostasis. Thus, the emergence of Eumetazoans featured the simultaneous development of primitive circulatory and nervous systems. This common emergence might explain why both systems in higher organisms frequently employ similar signaling components such as FGFs, neuropilins, ephrins and Notch receptors, as elaborated below. From such primordial evolutionary roots, it is not surprising that HSPGs are involved in homeostatic mechanisms, serve to modulate cell type-specific phenotypes, occur in multiple organ systems and regulate embryonic development.

The advent of total genome sequencing has revealed the extent of the lower metazoan HS biosynthetic machinery as known for the roundworm (C. elegans) and the fruit fly (Drosophila melanogaster). These organisms possess genes encoding multiple core proteins and all of the various HS biosynthetic enzymes; thus all the components for HSPG production are present. Numerous studies show that the mammalian and invertebrate gene products are functionally equivalent (reviewed in Ref. 12). However, these invertebrates, in contrast to vertebrates, largely lack multigene families. They exhibit only single genes for NDST, HS6ST, syndecan, and glypican. A notable exception is that Drosophila shows two HS3ST genes which respectively encode gD-type

and AT-type isoforms.35 Thus, these two isoforms stem from the same predecessors of the two major functional groupings of the large mammalian 3-O-sulfortransferase multigene family.

5. HSPGs in Development

The roles of HS in development have been most extensively examined in C. elegans and Drosophila. These invertebrates are not useful for directly studying vascular development as they only have a rudimentary open circulatory system. Nevertheless, fundamental features of the roles of HS in development are revealed by the genetic investigations of these animals. Most importantly, these studies demonstrate a role for HSPGs in several signaling pathways that are known to be operable in vertebrate ECs. Thus, genetic analyses of such lower organisms exemplify potential ways in which HSPGs may function in mammalian ECs.

Studies of mutants lacking various core proteins show that each core is required for signaling events that are specific in time, place, cell type and tissue, and that HSPGs are required for the development of multiple organ systems (reviewed in Refs. 12 and 44). That each protein conveys discrete functions suggests the multitude of core proteins expressed in mammalian ECs should serve to expand endothelial functional diversity. Deletion of a single core protein in lower organisms is not usually lethal, due to the multiplicity of core proteins. However, early embryonic death occurs in Drosophila and C. elegans mutants that have core proteins but are completely lacking in HS (due to mutations in EXT enzymes, see Fig. 2, that prevent polymerization of the HS backbone). Such lethality shows that the HS component of HSPGs plays an essential role in development. The HS-deficient Drosophila mutants show defects in pathways mediated by three HS-binding signaling ligands (wingless, hedgehog, and decapentaplegic, which is a form of tumor growth factor-β (reviewed in Refs. 12 and 44). It is likely that mammalian ECs employ HS to regulate these pathways, as vertebrates have comparable ligands that are known to be involved in vasculogenesis and angiogenesis.

Genetic studies also show distinct roles for HS modification enzymes. Although global HS deficiency is lethal in C. elegans, mutants lacking

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either C5-epimerase, HS2ST or HS6ST are viable. All such mutants have abnormal neuronal guidance; however, each enzyme deficiency affects a unique assortment of specific neurons. Thus, unique properties to HS are conveyed by different HS modifications in a cell type-specific fashion. These deficiencies interfere with developmental processes involving three receptors (Robo, Integrin and Ephrin) (reviewed in Refs. 12 and 44). Signaling through the vertebrates forms of all of these receptors is known to involve HSPGs, with integrins and ephrin receptors controlling key endothelial functions.

In Drosophila, deficiencies of specific HS modification enzymes leads to malformation of key organ systems and lethality. Drosophila mutants lacking a gD-type HS3ST isoform have disrupted signaling through the Notch receptor, which produces multiple developmental defects. The vertebrate Notch pathway conveys an arterial cell phenotype to non-committed ECs;45 thus, a mammalian gD-type HS3ST isoform may participate in this form of EC differentiation. Drosophila mutants lacking HS6ST die from malformation of the tracheal airway system.35,46 Development of the Drosophila trachea is analogous to vertebrate angiogenesis. Both processes control branching morphogenesis of tubular structures through comparable signaling components (including FGFs). The Drosophila HS6ST and FGF receptor (breathless) are co-expressed in tracheal cells; whereas, adjacent inducing cells express branchless (the Drosophila FGF). Mutants lacking branchless or breathless or HS6ST activity have stunted branching of the tracheal system that is phenotypically equivalent. Moreover, FGF-dependent activation of mitogen-activated protein kinase is impaired in HS6STdeficient mutants.46 Thus, 6-O-sulfates of HS are essential for FGFinduced branching morphogenesis in Drosophila. Mammalian FGF signaling also requires these moieties, as elaborated below. Clearly, such genetic investigations into the developmental roles of HS in lower organisms should facilitate the identification and delineation of HS-mediated signaling mechanisms that are operable in vertebrate ECs.

Roles of HS in vertebrate vessel development are now beginning to come to light. HS is critical for the signaling activity of VEGF164 and VEGF188, the splice variants of VEGF-A that contain HS-binding domains. Mice that exclusively express VEGF120 (non-HS-binding)