Vikas Prabhakar

Possible Role for Vascular Cell Proliferation in Cerebral Vasospasm After Subarachnoid Hemorrhage

Background and Purpose— During vasospasm after subarachnoid hemorrhage (SAH), cerebral blood vessels show structural changes consistent with the actions of vascular mitogens. We measured platelet-derived vascular growth factors (PDGFs) in the cerebrospinal fluid (CSF) of patients after SAH and tested the effect of these factors on cerebral arteries in vivo and in vitro. Methods— CSF was sampled from 14 patients after SAH, 6 patients not suffering SAH, and 8 normal controls. ELISA was performed for PDGF-AB, transforming growth factor-&bgr;1, and vascular endothelial growth factor. A mouse model was used to compare cerebral vascular cell proliferation and PDGF staining in SAH compared with sham-operated controls. Normal human pial arteries were incubated for 7 days in vitro, 2 groups with human blood clot and 1 with and 1 without PDGF antibodies. Results— PDGF-AB concentrations in CSF from SAH patients were significantly higher than those from non-SAH patients and normal controls, both during the first week after SAH and for all time points measured. Smooth muscle and fibroblast proliferation was observed after SAH in the mouse model, and this cellular replication was observed in conjunction with PDGF protein at the sites of thrombus. In human pial arteries, localized thrombus stimulated vessel wall proliferation, and proliferation was blocked by neutralizing antibodies directed against PDGFs. Conclusions— Vascular mitogens are increased in the CSF of patients after SAH. Proliferation of cells in the vascular wall is associated with perivascular thrombus. Cellular proliferation and subsequent vessel wall thickening may contribute to the syndrome of delayed cerebral vasospasm.

Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification

GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC Is active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

Effect of Contact Time and Force on Monocyte Adhesion to Vascular Endothelium

In this study we examined whether monocytic cell attachment to vascular endothelium was affected by elevating shear stress at a constant shear rate. Contact time, which is inversely related to the shear rate, was fixed and viscosity elevated with dextran to increase the shear stress (and hence the net force on the cell) independently of shear rate. At a fixed contact time, tethering frequencies increased, rolling velocities decreased, and median arrest durations increased with increasing shear stress. Rolling and short arrests (< 0.2 s) were well fit by a single exponential consistent with adhesion via the formation of a single additional bond. The cell dissociation constant, k(off), increased when the shear stress was elevated at constant shear rate. Firmly adherent cells arresting for at least 0.2 s were well fit by a stochastic model involving dissociation from multiple bonds. Therefore, at a fixed contact time and increasing shear stress, bonds formed more frequently for rolling cells resulting in more short arrests, and more bonds formed for firmly arresting cells resulting in longer arrest durations. Possible mechanisms for this increased adhesion include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers.

Biochemical characterization of the chondroitinase ABC I active site

cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via beta-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme-substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme-substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.

The biosynthesis and catabolism of galactosaminoglycans.

Publisher Summary This chapter discusses the structure of galactosaminoglycans (GalAGs) , their roles in biology and medicine, GalAG biosynthesis, their in vivo cellular degradation, and analytical biotechnologies pertinent to the study of GalAGs. The study of biological phenomena, from the molecular to the tissue level, has evolved substantially as scientists have focused on the emerging paradigm of extracellular regulation of cell function. GAG polysaccharides are attached to a protein core and are impelled to the cell surface and into the extracellular space. The cell surface machinery at the cell–extracellular matrix (ECM) interface acts as a complex battery of regulators that dynamically modulates outside cues from diverse signaling molecules. Specifically, GAGs govern signaling molecules by controlling their effective concentration and state at the cell surface. The extensive variation in GalAG composition based on core protein or tissue localization is so pronounced that GalAG production processes must be closely examined to further develop a context for their bioregulatory roles.

Recombinant Expression, Purification, and Biochemical Characterization of Chondroitinase ABC II from Proteus vulgaris

Chondroitin lyases (or chondroitinases) are a family of enzymes that depolymerize chondroitin sulfate (CS) and dermatan sulfate (DS) galactosaminoglycans, which have gained prominence as important players in central nervous system biology. Two distinct chondroitinase ABC enzymes, cABCI and cABCII, were identified in Proteus vulgaris. Recently, cABCI was cloned, recombinantly expressed, and extensively characterized structurally and biochemically. This study focuses on recombinant expression, purification, biochemical characterization, and understanding the structure-function relationship of cABCII. The biochemical parameters for optimal activity and kinetic parameters associated with processing of various CS and DS substrates were determined. The profile of products formed by action of cABCII on different substrates was compared with product profile of cABCI. A homology-based structural model of cABCII and its complexes with CS oligosaccharides was constructed. This structural model provided molecular insights into the experimentally observed differences in the product profile of cABCII as compared with that of cABCI. The critical active site residues involved in the catalytic activity of cABCII identified based on the structural model were validated using site-directed mutagenesis and kinetic characterization of the mutants. The development of such a contaminant-free cABCII enzyme provides additional tools to decode the biologically important structure-function relationship of CS and DS galactosaminoglycans and offers novel therapeutic strategies for recovery after central nervous system injury.

The structural elucidation of glycosaminoglycans.

There is accumulating evidence of the importance of linear polysaccharides in modulating biological phenomena in both the normal and the diseased states. This layer of regulation results from interactions between polysaccharides and other biomolecules, such as proteins, at the cell-extracellular matrix interface. The specific sequence of chemical modifications within the polymer backbone imparts a potential for interaction with other molecular species, and thus there exists important information within the various sulfation, acetylation, and epimerization states of such complex carbohydrates. A variety of factors have made the deciphering of this chemical code elusive. To this end, this report describes several techniques to elucidate the structural information inherent in glycosaminoglycan species. First, the use of depolymerizing enzymes that cleave polysaccharides at specific sites is described. Then, capillary electrophoretic (CE) techniques are employed to characterize the disaccharide species present in an enzymatically-cleaved polysaccharide sample. Mass spectrometry (MS) procedures can further be used to establish the length of an oligosaccharide chain and the presence of specific functional groups.

Glycosaminoglycan characterization methodologies: probing biomolecular interactions.

Interactions between glycans and proteins are central to many of the regulatory processes within biology. The development of analytical methodologies that enable structural characterization of glycosaminoglycan oligosaccharides has fostered improved understanding of the specificity of these biomolecular interactions. This facilitates an appreciation in understanding how changes in GAG structure can regulate physiology as well as pathology. While there are various techniques for studying the interaction of GAGs with proteins, in this chapter we focus on two approaches. First, an integrated analytical methodology, surface non-covalent affinity mass spectrometry (SNA-MS), is described to isolate, enrich, and sequence tissue-derived GAGs that bind to specific proteins. The broad applicability of this powerful platform offers an insight into how changes in cell-surface and extracellular GAG composition and sequence influences the ability of cells and tissues to dynamically alter responses to signaling molecules. Thus, this approach provides a window into understanding how changes at a molecular level manifest with respect to cellular phenotype. Second, surface plasmon resonance, or SPR, represents an additional platform for the study of protein-polysaccharide interaction, specifically for measuring the binding between GAG chains and proteins.

Effect of fluid viscosity and erythrocytes on monocyte adhesion

Monocyte recruitment to vascular endothelium is a key event linked to the development of atherosclerosis. Adhesion is influenced by many parameters including local fluid dynamics, intracellular collisions, and adhesion receptor expression. Elevated medium viscosity as well as the presence of erythrocytes in monocyte-containing media was found to increase monocyte adhesion to vascular endothelial cells. By comparing monocyte rolling velocities and arrest durations in fluids of differing viscosities at the same shear rates, monocyte adhesion was found to be shear stress dependent. Supplementation of the monocyte-containing medium with red blood cells increased tethering frequencies to a greater extent than the effect of elevated viscosity alone.