Methods are provided for inhibiting stenosis following vascular trauma or disease in a mammalian host, comprising administering to the host a therapeutically effective dosage of a therapeutic conjugate containing a vascular smooth muscle binding protein that associates in a specific manner with a cell surface of the vascular smooth muscle cell, coupled to a therapeutic agent dosage form that inhibits a cellular activity of the muscle cell. Methods are also provided for the direct and/or targeted delivery of therapeutic agents to vascular smooth muscle cells that cause a dilation and fixation of the vascular lumen by inhibiting smooth muscle cell contraction, thereby constituting a biological stent.

Therapeutic inhibitor of vascular smooth muscle cells

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

/pdf/endothelial-cell-derived-heparan-sulfate-binds-basic-3pqqpev82k.pdf

Endothelial Cell-derived Heparan Sulfate Binds Basic Fibroblast Growth Factor and Protects It from Proteolytic Degradation

D uring recent years, it has become increasingly evident that arterial smooth muscle cells occur in at least two distinct states, usually referred to as a synthetic and a contractile phenotype. Synthetic-state cells have a fibroblast-like appearance, and their main functions are to proliferate and to produce extracellular matrix components. They are found in the embryo and the young growing organism, where they take part in the formation of the vessel wall. Contractile-state cells have a musclelike appearance and contract in response to chemical and mechanical stimuli. They predominate in the vessels of adults and are primarily involved in the control of blood pressure and flow. However, these cells are able to return to a synthetic phenotype, and this appears to be an important early event in atherogenesis. This review briefly summarizes the present knowledge concerning the regulation of differentiated properties and proliferation of arterial smooth muscle cells. Most of the discussion will deal with studies on cultured cells, which so far are the most abundant. Particular attention will be paid to the role of extracellular matrix components like fibronectin and laminin and of polypeptide mitogens like platelet-derived growth factor (PDGF). It is believed that future studies in this area will help to widen our understanding of vasculogenesis and the initial stages in the pathogenesis of atherosclerosis.

Regulation of differentiated properties and proliferation of arterial smooth muscle cells.

Clinical trials of restenosis after coronary angioplasty.

The objective of this chapter is to discuss the roles of hyaluronan (HA) and proteoglycans (PGs) in developmental processes and tissue remodeling. In the first section I have summarized recent progress on the molecular basis of HA and PG interactions with the surface of cells, since this is the most likely means whereby HA and PGs would influence cell behavior during development. This is followed by a review of cellular studies, mainly performed in vitro, that implicate HA and PGs in specific aspects of cell behavior that would be relevant to development in vivo. Finally, I have chosen three developmental systems to illustrate ways in which HA or PGs are involved in actual morphogenetic and differentiative processes.

Proteoglycans and Hyaluronan in Morphogenesis and Differentiation

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action.

Heparin and related glycosaminoglycans are potent inhibitors of both in vivo and in vitro smooth muscle cell (SMC) proliferation. We have found that epidermal growth factor (EGF) reverses the antiproliferative effects of heparin. Other known SMC mitogens, including platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and thrombin, were unable to prevent heparin action. The EGF specificity was further demonstrated by developing a biological growth assay in which EGF or PDGF, at concentrations as low as 1 ng/ml, stimulated SMC growth in the absence of other serum components. Under these conditions, EGF, but not PDGF, suppressed heparin inhibition as well. The ability of EGF to reverse heparin inhibition was only observed when mitogen and glycosaminoglycan were added to SMC at similar times. If SMC were pretreated with heparin for 48 hours prior to EGF addition, the protective effects of EGF were lost. Heparin did not directly prevent 125I-EGF or platelet-derived EGF-like peptides from binding to the EGF receptor on SMC. However, cultures that were pretreated with heparin for 48 hours bound 49% less 125I-EGF than cultures that had been pretreated with the mucopolysaccharide for only 2 hours or that had not been preexposed to heparin. In previous studies, we have established that heparin exerts its maximal inhibitory activity after a 48-hour treatment of SMC (Reilly et al. 1986). Taken together, these data suggest that heparin may exert its antiproliferative potential by slowly and specifically altering SMC response to EGF-like mitogens of platelet origin.

Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor

of activation. The presenceof a sulphateester inposition3 of the central glucosamineunit in the pentasaccharide is the mostremarkable structuralcharacteristicof this sequence.In spite of its constantoccurrencein fragmentswith a high affinity for ATIII, definitive evidencecouldnot beobtainedwith regard to its essential role inbindingto theprotein.We have synthesized the pentasaccharidewithout the 3-sulphategroup (Figure2). This compoundis devoidof affinity for ATIII and of antifactor X, activity. These findings definitely establishthe fundamentalrole of the 3-sulphatedglucosamine unit in binding andactivatingthe proteaseinhibitor. ATill binding experimentswere performedon all these syntheticoligosaccharides. The results,combinedwith others that wereobtainedon heparinfragments,give a precise estimation of the role that is played by the different monosaccharides in the pentasaccharidesequence.\" Moreover, careful physicochemicalanalysisby nuclear magneticresonancespectroscopyof thesepurecompounds is possible.\" It has revealed unusual conformational properties of L-iduronic acid (a major component of heparin)that are currently being investigatedthoroughly. Thesepropertiesmight explainthe very peculiarbiological behaviourof iduronic-containingglycosaminoglycans. Thus,syntheticheparinfragmentsare themostpowerful tools for investigationat the molecular level. However, becauseof thedifficulties thatare presentlyassociatedwith synthesis,their use is restrictedto interactionsthat only involve a short part of the heparinmolecule.

Christopher F. Reilly

Papers

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action.

Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor

Inhibition of smooth muscle cell proliferation by heparin‐like molecules