Nicole C. Smits

The Diaphragms of Fenestrated Endothelia: Gatekeepers of Vascular Permeability and Blood Composition

Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.

Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus.

The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.

The Heparan Sulfate Motif (GlcNS6S-IdoA2S)3, Common in Heparin, Has a Strict Topography and Is Involved in Cell Behavior and Disease

Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups on HS, which creates distinct binding motifs. Biologically important HS motifs are expected to exhibit regulated expression, yet there is a profound lack of tools to identify such motifs; consequently, little is known of their structures and functions. We have identified a novel phage display-derived antibody (NS4F5) that recognizes a highly regulated HS motif (HSNS4F5), which we have rigorously identified as (GlcNS6S-IdoA2S)3. HSNS4F5 exhibits a restricted expression in healthy adult tissues. Blocking HSNS4F5 on cells in culture resulted in reduced proliferation and enhanced sensitivity to apoptosis. HSNS4F5 is up-regulated in tumor endothelial cells, consistent with a role in endothelial cell activation. Indeed, TNF-α stimulated endothelial expression of HSNS4F5, which contributed to leukocyte adhesion. In a mouse model of severe systemic amyloid protein A amyloidosis, HSNS4F5 was expressed within amyloid deposits, which were successfully detected by microSPECT imaging using NS4F5 as a molecularly targeted probe. Combined, our results demonstrate that NS4F5 is a powerful tool for elucidating the biological function of HSNS4F5 and can be exploited as a probe to detect novel polysaccharide biomarkers of disease processes.

Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.

Phage display-derived human antibodies against specific glycosaminoglycan epitopes.

Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hyaluronic acid, and keratan sulfate). Modification of the backbone by sulfation, deacetylation, and epimerization results in unique sequences within GAG molecules, which are instrumental in the binding of a large number of proteins. Investigating the exact roles of GAGs has long been hampered by the lack of appropriate tools, but we have successfully implemented phage display technology to generate a large panel of antibodies against CS, DS, HS, and heparin epitopes. These antibodies provide unique and highly versatile tools to study the topography, structure, and function of specific GAG domains. In this chapter, we describe the selection, characterization, and application of antibodies against specific GAG epitopes.

Heparan Sulfates in the Lung: Structure, Diversity, and Role in Pulmonary Emphysema

There is an emerging interest in the extracellular matrix (ECM) of the lung, especially in the role it plays in development and disease. There is a rapid change from the classical view of the ECM as a supporting structure towards a view of the ECM as a regulatory entity with profound effects on proliferation, migration, and differentiation of pulmonary cells. In the ECM, a variety of molecules is present in a highly organized pattern. Next to the abundant fiber‐forming molecules such as collagens and elastin, a large number of less abundant molecules are part of the ECM, including proteoglycans. In this review, we will focus on one class of proteoglycans, the heparan sulfate proteoglycans. We will particularly address the structure, biosynthesis, and function of their saccharide moiety, the heparan sulfates, including their role in development and (patho)physiology. Anat Rec, 293:955–967, 2010.

SPECT imaging of peripheral amyloid in mice by targeting hyper-sulfated heparan sulfate proteoglycans with specific scFv antibodies

INTRODUCTION Amyloid deposits are associated with a broad spectrum of disorders including monoclonal gammopathies, chronic inflammation, and Alzheimers disease. In all cases, the amyloid pathology contains, in addition to protein fibrils, a plethora of associated molecules, including high concentrations of heparan sulfate proteoglycans (HSPGs). METHODS We have evaluated radioiodinated scFvs that bind HS for their ability to image amyloid deposits in vivo. scFvs with different binding characteristics were isolated by phage display using HS extracted from bovine kidney or mouse and human skeletal muscle glycosaminoglycans (GAGs). Following purification and radioiodination, the biodistribution of (125)I-scFvs was assessed in mice with inflammation-associated AA amyloidosis or in amyloid-free mice by using SPECT imaging, biodistribution measurements and tissue autoradiography. RESULTS Four different scFvs all showed binding in vivo to amyloid in the spleen, liver and kidney of diseased mice; however, three of the scFvs also bound to sites within these organs in disease free mice. One scFv specific for hypersulfated HSPGs preferentially bound amyloid and did not accumulate in healthy tissues. CONCLUSIONS These data indicate that HS expressed in amyloid deposits has unique qualities that can be distinguished from HS in normal tissues. A scFv specific for rare hypersulfated HS was used to selectively image AA amyloid in mice with minimal retention in normal tissue.

Bispecific T-Cell Engagers (BiTEs) as Treatment of B-Cell Lymphoma

Antibodies are widely used in biochemistry, molecular biology, and medical research, and one of their innovative uses has been as therapeutic agents for the treatment of a variety of diseases, including cancer. At least 45 antibody-based products are currently marketed for therapy or imaging in the United States and Europe with approximately 63 billion US dollars in total worldwide sales in 2013. Important advances have improved the engineering, safety, and efficacy of the first generation of therapeutic antibodies. These developments, along with a greater understanding of the immunomodulatory properties of antibodies, have paved theway for the next generation of novel and improved antibody-based therapeutics, such as bispecific antibodies (BsAbs). BsAbs combine the specificity of two antibodies so that they can simultaneously bind to different antigens. Most BsAbs involve specificities that bind to an antigen on a cancer cell and to T-cell surface glycoprotein CD3 e-chain (CD3) on T cells. As with many of the strategies that are based the on use of monoclonal antibodies, BsAbs have benefited from a steady improvement in technology and lessons learned from earlier clinical and preclinical studies. The engineering of monospecific antibodies to be bispecific opens up a variety of potential therapeutic applications as evidenced by the more than 30 BsAbs currently in clinical development.

Designing multivalent proteins based on natural killer cell receptors and their ligands as immunotherapy for cancer

ABSTRACT Introduction: Natural killer (NK) cells are an important component of the innate immune system that play a key role in host immunity against cancer. NK cell recognition and activation is based on cell surface receptors recognizing specific ligands that are expressed on many types of tumor cells. Some of these receptors are capable of activating NK cell function while other receptors inhibit NK cell function. Therapeutic approaches to treat cancer have been developed based on preventing NK cell inhibition or using NK cell receptors and their ligands to activate NK cells or T cells to destroy tumor cells. Areas covered: This article describes the various strategies for targeting NK cell receptors and NK cell receptor ligands using multivalent proteins to activate immunity against cancer. Expert opinion: NK cell receptors work in synergy to activate NK cell effector responses. Effective anti-cancer strategies will need to not only kill tumor cells but must also lead to the destruction of the tumor microenvironment. Immunotherapy based on NK cells and their receptors has the capacity to accomplish this through triggering lymphocyte cytotoxicity and cytokine production.

Abstract 5607: Lipoprotein lipase binds to the surface of cancer cells and facilitates uptake of lipoproteins.

We examined the hypothesis that some cancer cells have surface-bound lipoprotein lipase (LPL), and we postulate that this membrane-bound LPL facilitates the acquisition of fatty acids (FA) from circulating triglyceride-rich lipoproteins. This deployment of the enzyme links the growth of tumors to dietary fat. Background: Recent studies have explored the association of dietary fat and obesity with increased incidence and aggressiveness of certain cancers. Tumor cells require FA for synthesis of membranes and thus for growth. Cells can acquire lipids through de novo synthesis from glucose and glutamine using fatty acid synthase (FASN) or by acquisition of pre-formed FA using LPL. LPL is a secreted enzyme synthesized by some cancer cell lines and all tumors examined to date (n = 181). It facilitates the uptake of very low density lipoproteins (VLDLs) by extracellular hydrolysis of triglyceride-rich particles such as VLDLs in the circulation or lipoprotein endocytosis followed by intracellular hydrolysis. In previous work, we demonstrated a heparin-releasable pool of LPL, consistent with tumor cell surface-associated LPL binding to a heparan sulfate proteoglycan (HSPG). Methods: We used immunocytochemistry and flow cytometry to demonstrate LPL on the surface of HeLa, BT474 and DU4475 breast cancer, and LiSa-2 liposarcoma cells. Confocal microscopy with fluorophore-labeled VLDLs enabled us to follow the endocytosis of VLDLs. Results: We have demonstrated that cancer cells can acquire lipoprotein particles (VLDLs) from their environment by endocytosis, and that this is mediated by cell-surface LPL bound to a specific HSPG motif. Major findings include: 1) Cell surface LPL is detectable by immunocytochemistry and flow cytometry. 2) The binding of LPL to the cell surface is abrogated by heparin. 3) LPL binding is likewise disrupted by NS4F5, a novel antibody to the specific proteoglycan motif which binds LPL to the surface of vascular endothelial cells. 4) Cancer cells endocytose VLDL particles, and this is abrogated by heparin or NS4F5. 5) VLDL particles accelerate the growth of LPL-expressing cancer cells. Conclusions: This work demonstrates of the use of endocytosis for the acquisition of diet-derived FA by cancer cells, and that this is mediated by cell-surface LPL bound to a specific HSPG motif. Thus endocytosis is a new mechanistic link between dietary lipoproteins and tumor cell growth. Further, these findings suggest that abrogation of LPL binding to the cell surface presents an opportunity for non-cytotoxic, therapeutic intervention. This work was supported by a grant from the Sarcoma Foundation of America (NBK) and a Prouty grant from Norris Cotton Cancer Center (WBK) and NIH Grant RO1CA126618 (WBK). Citation Format: Nancy Benton Kuemmerle, Leslie E. Lupien, Nicole C. Smits, Wilson L. Davis, William B. Kinlaw. Lipoprotein lipase binds to the surface of cancer cells and facilitates uptake of lipoproteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5607. doi:10.1158/1538-7445.AM2013-5607