Β-cyclodextrin Sulfate Research Articles

903–11 Sustained Inhibition of Intimal Thickening: Beneficial In Vitro and In Vivo Effects of Polymeric β-Cyclodextrin Sulfate

Intimal thickening after vascular injury is thought to be modulated in part by heparin binding growth factors. We hypothesized that placement of a therapeutic polymer in the periadventitial space capable of tightly binding growth factors might alter the vascular response to injury. We first tested the effects of polymeric β -cyclodextrin sulfate (P-CDS) on smooth muscle cell (SMC) proliferation using a quantitative protein accumulation assay. Incubation of rat aortic SMC (2500 cells/microwell in 10% FCS, n = 96) with P-CDS was associated with a dose dependent inhibition of proliferation, with maximum inhibition ≈50% vs. control at 1 mg/ml (p < 0.05). The proliferation assays were repeated using media containing only recombinant basic FGF (bFGF, 5 μg/ml) which had been exposed to P-CDS (1 mg/ml) for varying periods of time (1 min to 3 days). We demonstrated a time dependent reduction of the bFGF mitogenic activity, with complete removal of all activity within 60 minutes. Using radiolabeled bFGF (0.25 μg/ml), we observed rapid association (0.23 min -1 ) and dissociation (7.3 μ 10–5 min -1 ) rates, suggesting a high affinity interaction. We then tested the effects of P-CDS on SMC migration (using a linear under-agarose assay) and demonstrated treatment of rat aortic SMC with P-CDS (1 mg/ml) in the agar resulted in ≈50% reduction in SMC migration vs. control at 72 hours (p < 0.05). Finally, the effects of P-CDS in vivo were studied in the rat carotid balloon injury model. SD rats (< 350 g) were randomized to periadventitial P-CDS (10 mg) or no treatment, additional rats received treatment with unsulfated β-cyclodextrin polymer (PCD). Morphometric analyses at 4 (n = 20), 14 (n = 59), and 88 days (n = 14) demonstrated >40% reduction of intimal area and intima/media area ratios only in the P-CDS rats. No inhibition was seen with P-CD treated rats. Decreased bromodeoxyuridine labeled SMC (79 ± 11 vs 144 ± 23, p = 0.01) were noted in the intima of P-CDS rats at 14 days. At all time points, there were reduced total SMC counts and no reduction in % proliferation in the intima of P-CDS rats, suggesting greater effects on SMC migration than proliferation in vivo . Endothelial cell regrowth (by Evans blue staining) was increased in the P-CDS rats at 14 (4.4 vs 2.2 mm, p = 0.09) and 88 days (13.0 vs 9.2 mm, p < 0.01). Periadventitially applied polymeric β-cyclodextrin sulfate produces sustained inhibition of intimal thickening following vascular injury, possibly by modulating cellular processes mediated by heparin binding growth factors.

Divalent cation-dependent interaction of sulfated polysaccharides with phosphatidylcholine and mixed phosphatidylcholine/phosphatidylglycerol liposomes

The Ca 2+-dependent interaction of various polyanionic polysaccharides (chondroitin sulfate, heparin, dextran sulfate, β-cyclodextrin sulfate, hyaluronic acid and carboxymethyldextran) with multilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes was investigated by calorimetric and fluorescence spectroscopic measurements. It was found that an observed polysaccharide-induced phospholipid phase separation depends on the density of the sulfate groups along the polysaccharide chain independent of the presence of additional carboxyl groups. The phase separation resulting from the drastic dehydration of the covered membrane regions is monitored by the upward shift of the lipid phase transition and by the blue shift of the emission spectrum of a headgroup-dansylated phosphatidylethanolamine (DPE). This shift is only observable if the required polysaccharide chain length contains at least three glycosyl units. The Ca 2+-mediated interaction of dextran sulfate with various phosphatidylcholines, differing in their compressibility, showed the maximal difference between the phase transition temperatures of the lipid phase covered by the polysaccharide and the uneffected lipid domains for dielaidinoyl phosphatidylcholine (DEPC), the most compressible phospholipid investigated here. Mixed negatively charged DMPC/dimyristoyl phosphatidylglycerol (DMPG) liposomes were found to compete with the likewise negatively charged dextran sulfate for the binding of Ca 2+. At excess Ca 2+ concentrations, the binding of the polysaccharide was strengthened, compared to pure DMPC liposomes. The monovalent cation sodium, was able to inhibit the interaction between the membrane surface and dextran sulfate. Various divalent cations were found to mediate the interaction, depending on their ionic radii and electron configuration. Within the second group of the periodic system Ca 2+ is the most effective ion. However, within the horizontal forth period the ability to bind sulfated dextran to membrane surfaces decreases from Ca 2+ to Ni 2+, but then increases again if Cu 2+ or Zn 2+ was used as the mediating ion.