Nicholas W. Shworak

Normal levels of anticoagulant heparan sulfate are not essential for normal hemostasis

Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.

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.

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.

Mice deficient in heparan sulfate 3-O-sulfotransferase-1: Normal hemostasis with unexpected perinatal phenotypes

Heparan sulfate that contains antithrombin binding sites is designated as anticoagulant heparan sulfate (HSact) since, in vitro, it dramatically enhances the neutralization of coagulation proteases by antithrombin. Endothelial cell production of HSact is controlled by the Hs3st1 gene, which encodes the rate limiting enzyme—heparan sulfate 3-O-sulfotransferase-1 (Hs3st1). It has long been proposed that levels of endothelial HSact may tightly regulate hemostatic tone. This potential in vivo role of HSact was assessed by generating Hs3st1−/− knockout mice. Hs3st1−/− and Hs3st1+/+ mice were evaluated with a variety of methods, capable of detecting altered hemostatic tone. However, both genotypes were indistinguishable. Instead, Hs3st1−/− mice exhibited lethality on a specific genetic background and also showed intrauterine growth retardation. Neither phenotypes result from a gross coagulopathy. So although this enzyme produces the majority of tissue HSact, Hs3st1−/− mice do not show an obvious procoagulant phenotype. These results suggest that the bulk of HSact is not essential for normal hemostasis and that hemostatic tone is not tightly regulated by total levels of HSact. Moreover, the unanticipated non-thrombotic phenotypes suggest structure(s) derived from this enzyme might serve additional/alternative biologic roles. Published in 2003.

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.

Human Follicular Fluid Heparan Sulfate Contains Abundant 3-O-Sulfated Chains with Anticoagulant Activity

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.

Inherent and Benzo[a]pyrene-Induced Differential Aryl Hydrocarbon Receptor Signaling Greatly Affects Life Span, Atherosclerosis, Cardiac Gene Expression, and Body and Heart Growth in Mice

Little is known of the environmental factors that initiate and promote disease. The aryl hydrocarbon receptor (AHR) is a key regulator of xenobiotic metabolism and plays a major role in gene/environment interactions. The AHR has also been demonstrated to carry out critical functions in development and disease. A qualitative investigation into the contribution by the AHR when stimulated to different levels of activity was undertaken to determine whether AHR-regulated gene/environment interactions are an underlying cause of cardiovascular disease. We used two congenic mouse models differing at the Ahr gene, which encodes AHRs with a 10-fold difference in signaling potencies. Benzo[a]pyrene (BaP), a pervasive environmental toxicant, atherogen, and potent agonist for the AHR, was used as the environmental agent for AHR activation. We tested the hypothesis that activation of the AHR of different signaling potencies by BaP would have differential effects on the physiology and pathology of the mouse cardiovascular system. We found that differential AHR signaling from an exposure to BaP caused lethality in mice with the low-affinity AHR, altered the growth rates of the body and several organs, induced atherosclerosis to a greater extent in mice with the high-affinity AHR, and had a huge impact on gene expression of the aorta. Our studies also demonstrated an endogenous role for AHR signaling in regulating heart size. We report a gene/environment interaction linking differential AHR signaling in the mouse to altered aorta gene expression profiles, changes in body and organ growth rates, and atherosclerosis.

Hs3st3-Modified Heparan Sulfate Controls KIT+ Progenitor Expansion by Regulating 3-O-Sulfotransferases

The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.

Obesity Is Mediated by Differential Aryl Hydrocarbon Receptor Signaling in Mice Fed a Western Diet

Background: Obesity is a growing worldwide problem with genetic and environmental causes, and it is an underlying basis for many diseases. Studies have shown that the toxicant-activated aryl hydrocarbon receptor (AHR) may disrupt fat metabolism and contribute to obesity. The AHR is a nuclear receptor/transcription factor that is best known for responding to environmental toxicant exposures to induce a battery of xenobiotic-metabolizing genes. Objectives: The intent of the work reported here was to test more directly the role of the AHR in obesity and fat metabolism in lieu of exogenous toxicants. Methods: We used two congenic mouse models that differ at the Ahr gene and encode AHRs with a 10-fold difference in signaling activity. The two mouse strains were fed either a low-fat (regular) diet or a high-fat (Western) diet. Results: The Western diet differentially affected body size, body fat:body mass ratios, liver size and liver metabolism, and liver mRNA and miRNA profiles. The regular diet had no significant differential effects. Conclusions: The results suggest that the AHR plays a large and broad role in obesity and associated complications, and importantly, may provide a simple and effective therapeutic strategy to combat obesity, heart disease, and other obesity-associated illnesses.