Daniel L. Mooradian

Nitric oxide (NO) donor molecules : effect of NO release rate on vascular smooth muscle cell proliferation in vitro

Nitric oxide (NO) inhibits vascular smooth muscle cell (SMC) growth in vitro. To determine the effects of release rate and exposure time on SMC growth inhibition by NO, we compared the activities of five NO donors that generate NO with half-lives of 2 min (DEA/NO, Et2N[N2O2]Na), 15 min (PAPA/NO, CH3(CH2)2N[N2O2]-(CH2)3NH3+), 39 min, (SPER/NO, H2N(CH2)3NH2+(CH2)4N[N2O2]-(CH2)3NH2), 3 h (DPTA/NO, H2N(CH2)3N[N2O2]-(CH2)3NH3+), and 20 h (DETA/NO, H2N(CH2)2N[N2O2]-(CH2)2NH3+). After 22-h treatment, rat aorta SMC (RA-SMC) DNA synthesis was inhibited with IC50 values of 180, 60, and 40 microM for SPER/NO, DPTA/NO, and DETA/NO, respectively. DEA/NO and PAPA/NO did not inhibit DNA synthesis significantly at any concentration tested (20-500 microM). The inhibitory effect of NO on RA-SMC DNA synthesis was thus greatest when a given molar dose of NO was delivered slowly throughout the 22-h period. The antiproliferative effect of DETA/NO was confirmed by measurement of cell numbers for 7 days. When RA-SMC were treated with 500 microM DETA/NO on days 1, 3, and 5, growth was completely suppressed. Cell viability was > 95%, confirming that DETA/NO was not cytotoxic. The results suggest that NO donors may be useful inhibitors of intimal hyperplasia and restenosis after vascular injury such as balloon angioplasty.

Magnetically orientated tissue-equivalent tubes: application to a circumferentially orientated media-equivalent

Circumferential orientation of collagen fibrils in a media-equivalent (ME) is achieved in a simple and effective way using the orientating effects of a strong magnetic field during collagen fibrillogenesis when the ME is first created. Circumferential orientation of the entrapped smooth muscle cells (SMC) is achieved subsequently via cell contact guidance, the induced SMC orientation along orientated fibrils. After describing the methods used, several lines of evidence are provided showing that the magnetically orientated ME is circumferentially orientated, including collagen birefringence, circumferential SMC orientation, accelerated ME compaction and increased ME stiffness with reduced creep in the circumferential direction as compared to control MEs not exposed to a magnetic field during fibrillogenesis. The optimization of these methods is discussed in order to better mimic the circumferential orientation and mechanical properties of a natural medium. Other applications of magnetically orientated tissue-equivalents are indicated.

Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films

The incorporation of lipidated cell adhesion peptides into self-assembled structures such as films provides the opportunity to develop unique biomimetic materials with well-organized interfaces. Synthetic dialkyl tails have been linked to the amino-terminus, carboxyl-terminus, and both termini of the cell recognition sequence Arg-Gly-Asp (RGD) to produce amino-coupled, carboxyl-coupled, and looped RGD peptide amphiphiles. All three amphiphilic RGD versions self-assembled into fairly stable mixed monolayers that deposited well as Langmuir-Blodgett films on surfaces, except for films containing amino-coupled RGD amphiphiles at high peptide concentrations. FT-IR studies showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. Melanoma cells spread on looped RGD amphiphiles in a concentration-dependent manner, spread indiscriminately on carboxyl-coupled RGD amphiphiles, and did not spread on amino-coupled RGD amphiphiles. Looped RGD amphiphiles promoted the adhesion, spreading, and cytoskeletal reorganization of melanoma and endothelial cells while control looped Arg-Gly-Glu (RGE) amphiphiles inhibited them. Antibody inhibition of the integrin receptor alpha3beta1 blocked melanoma cell adhesion to looped RGD amphiphiles. These results confirm that novel biomolecular materials containing synthetic peptide amphiphiles have the potential to control cellular behavior in a specific manner.

Platelet adhesion to novel phospholipid materials: Modified phosphatidylcholine covalently immobilized to silica, polypropylene, and PTFE materials

Based on the premise of achieving blood compatibility through mimicking the chemical constitutents of the biologically insert surface of the unactivated platelet membrane, a process was developed that entails the covalent grafting of modified phosphatidylcholine molecules to materials including silica, polypropylene, and polytetrafluoroethylene (PTFE) polymer films. These materials were characterized using x-ray photoelectron spectroscopy (XPS) and contactangle measurements. The phosphatidylcholine-containing materials (PC materials) were used as substrates in the plateletadhesion assays and were subjected to enzymatic degradation evaluation. Phosphatidylcholine-grafted silica materials do not support platelet adhesion. In addition the number of adherent platelets correlate with the amount of grafted phospholipid present, as indicated by the phosphorus/ carbon ratio obtained by XPS analysis. Platelet adhesion to phosphatidylcholine-grafted polypropylene and PTFE was inhibited 80% and 90%, respectively, when compared with platelet adhesion to unmodified polypropylene and PTFE.

Nitric Oxide Donors: A Continuing Opportunity in Drug Design

Publisher Summary This chapter illustrates the advantageous, much unique, chemical, and pharmacological properties of nitric oxide (NO) donor compound class by showing how the accumulating knowledge are used in this area in attempting to design solutions to two clinical problems for which adequate pharmacological strategies do not currently exist. These are restenosis following angioplasty and other mechanical interventional procedures for vascular repair and the failure of small-caliber synthetic vascular grafts used to replace obstructed arteries. The chapter attempts to show how the versatility of compounds containing the N 2 O 2 − functional group might be harnessed to solve important clinical problems. As such, agents releases NO at a first-order rate with a half-life of approximately one day in physiological buffer, these compounds can be conveniently used to inhibit vascular smooth muscle cell proliferation in culture completely for a prolonged period with no detectable toxic effect. As the half-life of NO release from such agents generally increases with pH, this is used to design an infusion device in which a homolog with a half-life of 1 min at pH 7.4 could be stabilized in a slightly alkaline reservoir and continuously neutralized 1 min before delivery as an NO-rich solution into the bloodstream to inhibit platelet deposition at an otherwise thrombogenic site. Finally, by incorporating the N 2 O 2 − functional group into a polymeric matrix, an NO point source has been prepared that, when used as a coating for commercial vascular implants, greatly reduced thrombogenicity in a preliminary experiment.

Endothelial Cell Interactions With Synthetic Peptides From the Carboxyl-Terminal Heparin-Binding Domains of Fibronectin

Fibronectin (FN) plays an important role in endothelial cell adhesion, spreading, and motility. Within FN, a number of functional domains have been identified, including the 33/66-kD carboxyl-terminal heparin-binding fragments, which support the adhesion of vascular endothelial cells. A number of synthetic peptides representing amino acid sequences within the 33/66-kD fragments have been shown to promote the adhesion, spreading, and migration of a variety of cell types. Our working hypothesis is that one or more of these sequences may also mediate vascular endothelial cell adhesion, spreading, and migration to the 33/66-kD fragments. In support of this hypothesis, we have demonstrated that endothelial cells from various sources adhered in a concentration-dependent manner to surfaces coated with FN, the 33/66-kD fragments, and synthetic peptides derived from the 33/66-kD fragments of FN. FN and the 33/66-kD fragments also promoted endothelial cell spreading and migration. Although each of the six synthetic peptides tested supported endothelial cell adhesion, only one of these peptides within the carboxyl-terminal heparin-binding domain (FN-C/H-V) promoted endothelial cell spreading and migration. Cell spreading on FN-C/H-V, as well as on FN and the 33/66-kD fragments, was associated with the formation of a well-developed actin cytoskeleton and the formation of focal contacts. FN-C/H-V (but not scrambled FN-C/H-V) inhibited cell spreading on FN and the 33/66-kD fragments in a concentration-dependent manner. FN-C/H-V had a modest effect on the adhesion of a clonal population of rat heart endothelial cells (RHE-1A) to the 33/66-kD fragments of FN and no effect on RHE-1A cell adhesion to FN. These findings suggest that peptide FN-C/H-V is unique among this group of peptides derived from the 33/66-kD heparin-binding fragments of FN in its ability to promote the adhesion, spreading, and migration of vascular endothelial cells and further suggest that the sequence defined by this peptide plays an important role in vascular endothelial cell interactions with the 33/66-kD fragments of FN.

Manipulation of remodeling pathways to enhance the mechanical properties of a tissue engineered blood vessel.

There is a current need for a small diameter vascular graft due to the limited supply of autogenous grafts and the failure of synthetic grafts due to thrombosis and/or intimal hyperplasia. The use of living cells and tissues to fabricate a small diameter graft (i.e., tissue engineered blood vessel, TEBV) could be useful given the endothelialization potential and biocompatibility benefits of such a graft. However, while sufficient strength has been attained in a TEBV, coordinate compliance has yet to be fine-tuned. In this study we investigate the effects of biological response modifiers, retinoic acid (RA) and ascorbic acid (AA) on TEBV biomechanics as a function of time and subsequently correlate observed RA/AA induced changes in TEBV mechanics with alterations in smooth muscle cell (SMC) biochemistry. TEBVs were constructed using a fibrillar type I collagen network populated by human aortic smooth muscle cells (AoSMC). Following construction this TEBV was treated with 0.3 mM AA and 0.1 mM RA (concentrations found to induce changes in VSMC phenotype). Ultimate tensile stress (UTS), rate of relaxation (RR) and elastic efficiency (EE) of RA/AA treated and untreated TEBVs were measured following 1, 7, 15, 30, 45, and 60 days of treatment. At corresponding time points, the effect of these treatments on collagen and elastin protein synthesis and mRNA expression was examined. RA/AA treated TEBV strength increased and stiffness decreased compared to controls as a function of time. Relative collagen synthesis in treated TEBVs exceeded control levels by nearly two-fold at 15 and 30 days of incubation. RA/AA treated collagen gene expression followed a similar trend. Relative elastin synthesis was also greater in treated TEBVs as compared to untreated TEBVs at 15 and 30 days of incubation and correspondingly elastin mRNA expression was significantly elevated at 15 days of incubation. These data provide evidence that RA/AA treated TEBVs exhibit mechanical properties which more closely mimic those of a native vessel than their untreated counterparts and that changes in extracellular matrix composition and matrix gene expression in the presence of RA/AA treatment may play an important role in the development of said mechanical properties.

Conversion of a polysaccharide to nitric oxide-releasing form. dual-mechanism anticoagulant activity of diazeniumdiolated heparin

We describe heparin/diazeniumdiolate conjugates that generate nitric oxide (NO) at physiological pH. Like the heparin from which they were prepared, they inhibit thrombin-induced blood coagulation. Unlike heparin, they can also inhibit and reverse ADP-induced platelet aggregation (as expected for an NO-releasing agent), suggesting potential utility as dual-action antithrombotics.

Angiopeptin (BIM23014C) inhibits vascular smooth muscle cell migration in vitro through a G-protein-mediated pathway and is associated with inhibition of adenylyl cyclase and cyclic AMP accumulation.

Angiopeptin (AP: BIM23014C), a cyclic analogue of the peptide hormone somatostatin, inhibits intimal hyperplasia after balloon angioplasty. This inhibition has been attributed to a direct inhibitory effect on smooth muscle cell (SMC) proliferation. However, the SMC that proliferate in the intima and contribute to intimal hyperplasia arrive there by migrating from the injured media, suggesting that SMC migration may also play an important role in this process. Indeed, in the experiments we describe, AP inhibited the migration of rat aortic SMC cells (RA-SMC) in response to type I collagen, the predominant form of collagen in the vessel media, and did so dose dependently. RA-SMC migration was inhibited 70% in the presence of AP 100 nM. RA-SMC adhesion to type I collagen in these conditions was not inhibited, suggesting that AP does not interfere with RA-SMC recognition of type I collagen; instead, it blocks subsequent signaling events that are necessary for RA-SMC migration in response to type I collagen. AP inhibited the forskolin-stimulated accumulation of cyclic AMP by RA-SMC (35% at 30 nM). In addition, pertussis toxin (PT), which blocks Gi-mediated inhibition of adenylyl cyclase, blocked the inhibitory effect of AP on cyclic AMP (cAMP) accumulation and also blocked the inhibitory effect of AP on RA-SMC migration. These findings suggest that the inhibitory effect of AP on intimal hyperplasia is due at least in part to its effects on SMC migration and that these effects are mediated by a Gi-dependent pathway and may involve inhibition of adenylyl cyclase and cAMP accumulation.

Effect of Glow Discharge Surface Modification of Plasma TFE Vascular Graft Material on Fibronectin and Laminin Retention and Endothelial Cell Adhesion

The effects of glow discharge surface modification of plasma TFE vascular graft material (plasma TFE, Atrium Medical Corp., Hollis, NH) on the binding and retention of fibronectin and laminin as well as endothelial adherence were examined. The binding of both fibronectin and laminin to plasma TFE increased as a function of protein concentration in the range of 0.02 to 200 micrograms/ml. Binding to plasma TFE was not saturable in this range of protein concentrations. Fibronectin and laminin binding to plasma TFE was time-dependent, reaching a maximal level (1.8 and 3.2 micrograms/cm2 of bound fibronectin and laminin, respectively) after 30 min (fibronectin) and 4 hr (laminin). Binding was not the result of an accumulation of unbound protein in graft interstices since graft prewetting did not alter the amount of either fibronectin or laminin associated with plasma TFE. In addition, binding was not the result of protein modification during radiolabeling, since unlabeled fibronectin and laminin could compete effectively with their iodinated counterparts for binding to plasma TFE. Dissociation of bound fibronectin and laminin from plasma TFE was rapid, and less than 12% remained bound 60 min after washing. Plasma TFE subjected to glow discharge in O2 and carrying negatively charged functional groups on graft surfaces, exhibited a fivefold increase in fibronectin and laminin binding. In addition, more than 85% of the bound fibronectin and laminin was retained 24 hr after washing. The enhanced retention of fibronectin or laminin by these grafts was associated with an increase in endothelial cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)