James D. San Antonio

Heparan sulfate proteoglycans: heavy hitters in the angiogenesis arena

Without new blood vessels, neoplasms cannot expand beyond a few millimeters, the point at which the diffusion of nutrients and the disposal of waste products become rate-limiting. Regulation of angiogenesis thus must be controlled at multiple levels. For instance, the VEGF family of heparin-binding proteins and their primary receptors, VEGFR-1 and VEGFR-2 (KDR), products of the flt-1 and the flk-1 gene, respectively, are required for angioblast differentiation and vasculogenesis, and specific VEGF isoforms play distinct roles in promoting endothelial growth and migration during angiogenesis. In addition, angiogenesis is profoundly affected by several members of the FGF family and their four receptors, and indeed, supplementing the media of endothelial cell cultures with basic FGF (FGF2) and heparin is now well established as a means to obtain optimal growth, migration, and capillary morphogenesis. In addition to producing proangiogenic factors, tumor cells also directly or indirectly generate negative angiogenic stimuli. The ultimate growth rate of the tumors is thus a fine balance between positive and negative angiogenic cues.

Endorepellin, a Novel Inhibitor of Angiogenesis Derived from the C Terminus of Perlecan

Perlecan, a ubiquitous basement membrane heparan sulfate proteoglycan, plays key roles in blood vessel growth and structural integrity. We discovered that the C terminus of perlecan potently inhibited four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan was active at nanomolar concentrations and blocked endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth we have named it “endorepellin.” We also found that endothelial cells possess a significant number of high affinity (K d of 11 nm) binding sites for endorepellin and that endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo.

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates.

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

Decorin Binds Near the C Terminus of Type I Collagen

Decorin belongs to a family of small leucine-rich proteoglycans that are directly involved in the control of matrix organization and cell growth. Genetic evidence indicates that decorin is required for the proper assembly of collagenous matrices. Here, we sought to establish the precise binding site of decorin on type I collagen. Using rotary shadowing electron microscopy and photoaffinity labeling, we mapped the binding site of decorin protein core to a narrow region near the C terminus of type I collagen. This region is located within the cyanogen bromide peptide fragment α1(I) CB6 and is ∼25 nm from the C terminus, in a zone that coincides with the c1 band of the collagen fibrild-period. This location is very close to one of the major intermolecular cross-linking sites of collagen heterotrimers. Thus, decorin protein core possesses a unique binding specificity that could potentially regulate collagen fibril stability.

Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.

Collagen IV is the major protein found in basement membranes. It comprises three heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (“interactomes”) that indicated major structural landmarks, known and predicted ligand‐binding sites, and missense mutations, in order to identify functional and disease‐associated domains, potential interactions between ligands, and genotype–phenotype relationships. The maps documented more than 30 known ligand‐binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin, and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection, and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases, and heparin. The maps indicated that mutations affecting major ligand‐binding sites, for example, for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms, and muscle Cramps [HANAC] syndrome, and early‐onset Alport syndrome, respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. Hum Mutat 32:127–143, 2011.

Heparan Sulfate Proteoglycans: A GAGgle of Skeletal-Hematopoietic Regulators

This review summarizes our current understanding of the presence and function of heparan sulfate proteoglycans (HSPGs) in skeletal development and hematopoiesis. Although proteoglycans (PGs) comprise a large and diverse group of cell surface and matrix molecules, we chose to focus on HSPGs owing to their many proposed functions in skeletogenesis and hematopoiesis. Specifically, we discuss how HSPGs play predominant roles in establishing and regulating niches during skeleto‐hematopoietic development by participating in distinct developmental processes such as patterning, compartmentalization, growth, differentiation, and maintenance of tissues. Special emphasis is placed on our novel hypothesis that mechanistically links endochondral skeletogenesis to the establishment of the hematopoietic stem cell (HSC) niche in the marrow. HSPGs may contribute to these developmental processes through their unique abilities to establish and mediate morphogen, growth factor, and cytokine gradients; facilitate signaling; provide structural stability to tissues; and act as molecular filters and barriers. Developmental Dynamics 237:2622–2642, 2008.

A Key Role for the Integrin α2β1 in Experimental and Developmental Angiogenesis

The alpha2beta1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin alpha2 I or beta1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a beta1 I-like domain inhibitor and by function-blocking anti-alpha2beta1 but not -alpha1beta1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish alpha2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin alpha2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of alpha2beta1 integrin. Collectively, our results provide strong biochemical and genetic evidence of a central role for alpha2beta1 integrin in experimental and developmental angiogenesis.

Non-Enzymatic Glycation of Type I Collagen Diminishes Collagen-Proteoglycan Binding and Weakens Cell Adhesion

Non‐enzymatic glycation of type I collagen occurs in aging and diabetes, and may affect collagen solubility, charge, polymerization, and intermolecular interactions. Proteoglycans1 (PGs) bind type I collagen and are proposed to regulate fibril assembly, function, and cell–collagen interactions. Moreover, on the collagen fibril a keratan sulfate (KS) PG binding region overlaps with preferred collagen glycation sites. Thus, we examined the effect of collagen modified by simple glycation on PG–collagen interactions. By affinity coelectrophoresis (ACE), we found reduced affinities of heparin and KSPGs for glycated but not normal collagen, whereas the dermatan sulfate (DS)PGs decorin and biglycan bound similarly to both, and that the affinity of heparin for normal collagen decreased with increasing pH. Circular dichroism (CD) spectroscopy revealed normal and glycated collagens to assume triple helical conformations, but heparin addition caused precipitation and decreased triple helical content—effects that were more marked with glycated collagen. A spectrophotometric assay revealed slower polymerization of glycated collagen. However, ultrastructural analyses indicated that fibrils assembled from normal and glycated collagen exhibited normal periodicity, and had similar structures and comparable diameter distributions. B‐cells expressing the cell surface heparan sulfate PG syndecan‐1 adhered well to normal but not glycated collagen, and endothelial cell migration was delayed on glycated collagen. We speculate that glycation diminishes the electrostatic interactions between type I collagen and PGs, and may interfere with core protein‐collagen associations for KSPGs but not DSPGs. Therefore in vivo, collagen glycation may weaken PG–collagen interactions, thereby disrupting matrix integrity and cell–collagen interactions, adhesion, and migration. J. Cell. Biochem. 104: 1684–1698, 2008.

Dinosaur peptides suggest mechanisms of protein survival.

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a ‘preservation motif’, and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

Insulin-like growth factor (IGF) binding protein-3 regulation of IGF-I is altered in an acidic extracellular environment

While extracellular acidification within solid tumors is well‐documented, how reduced pH impacts regulation of insulin‐like growth factor‐I (IGF‐I) has not been studied extensively. Because IGF‐I receptor binding is affected by IGF binding proteins (IGFBPs), we examined how pH impacted IGFBP‐3 regulation of IGF‐I. IGF‐I binding in the absence of IGFBP‐3 was diminished at reduced pH. Addition of IGFBP‐3 reduced IGF‐I cell binding at pH 7.4 but increased surface association at pH 5.8. This increase in IGF‐I binding at pH 5.8 corresponded with an increase in IGFBP‐3 cell association. This, however, was not due to an increase in affinity of IGFBP‐3 for heparin at reduced pH although both heparinase III treatment and heparin addition reduced IGFBP‐3 enhancement of IGF‐I binding. An increase in IGF‐I binding to IGFBP‐3, though, was seen at reduced pH using a cell‐free assay. We hypothesize that the enhanced binding of IGF‐I at pH 5.8 is facilitated by increased association of IGFBP‐3 at this pH and that the resulting cell associated IGF‐I is IGFBP‐3 and not IGF‐IR bound. Increased internalization and nuclear association of IGF‐I at pH 5.8 in the presence of IGFBP‐3 was evident, yet cell proliferation was reduced by IGFBP‐3 at both pH 5.8 and 7.4 indicating that IGFBP‐3‐cell associated IGF‐I does not signal the cell to proliferate and that the resulting transfer of bound IGF‐I from IGF‐IR to IGFBP‐3 results in diminished proliferation. Solution binding of IGF‐I by IGFBP‐3 is one means by which IGF‐I‐induced proliferation is inhibited. Our work suggests that an alternative pathway exists by which IGF‐I and IGFBP‐3 both associate with the cell surface and that this association inhibits IGF‐I‐induced proliferation.