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BackgroundHypercholesterolemia (HC) has previously been shown to augment restenotic response in several animal models and humans. However, the mechanistic aspects of in-stent restenosis (ISR) on a hypercholesterolemic background, including potential augmentation of systemic and local inflammation precipitated by HC are not completely understood. CD47 is a transmembrane protein known to abort crucial inflammatory pathways. Our present studies have examined the interrelation between HC, inflammation, and ISR and investigated the therapeutic potential of stents coated with a CD47-derived peptide (pepCD47) in the hypercholesterolemic rabbit model.Methods and ResultsPepCD47 was immobilized on metal foil coupons and stents using polybisphosphonate coordination chemistry and pyridyldithio/thiol conjugation. The relative abundance of the surface-associated cells on bare metal (BM) and pepCD47 foils exposed to whole rabbit blood showed a 40% inhibition of cell attachment on pepCD47-modified surfaces. Likewise, cytokine expression analyzed in buffy coat-derived cells cultured over the BM and pepCD47-derivatized foils demonstrated a M2/M1 increase with pepCD47 coating. Hypercholesterolemic and normocholesterolemic rabbit cohorts underwent bilateral implantation of BM and pepCD47 stents in the iliac location. Hypercholesterolemia increased neointimal growth in comparison with normocholesterolemic animals at 4 weeks post-stenting. These untoward outcomes were mitigated in the arteries of hypercholesterolemic rabbits treated with pepCD47-derivatized stents. Compared to NC animals, inflammatory cytokine immunopositivity and macrophage infiltration of peri-strut areas increased in HC group animals, and was attenuated in the arteries of hypercholesterolemic rabbits treated with pepCD47 stents.ConclusionsAugmented inflammatory responses triggered by HC underlie severe ISR morphology in hypercholesterolemic rabbits. Blockage of initial platelet and leukocyte attachment to stent struts through CD47 functionalization of stents mitigates pro-restenotic effects of HC.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT −/− mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor β1 ( TGF β 1 ) and collagen ( COL1A1 ) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde-fixed heterograft tissue, such as bovine pericardium (BP), are widely used for treating heart valve disease, a group of disorders that affects millions. Structural valve degeneration (SVD) of BHV due to both calcification and the accumulation of advanced glycation end products (AGE) with associated serum proteins limits durability. We hypothesized that BP modified with poly-2-methyl-2-oxazoline (POZ) to inhibit protein entry would demonstrate reduced accumulation of AGE and serum proteins, mitigating SVD. In vitro studies of POZ-modified BP demonstrated reduced accumulation of serum albumin and AGE. BP-POZ in vitro maintained collagen microarchitecture per two-photon microscopy despite AGE incubation, and in cell culture studies was associated with no change in tumor necrosis factor-α after exposure to AGE and activated macrophages. Comparing POZ and polyethylene glycol (PEG)-modified BP in vitro, BP-POZ was minimally affected by oxidative conditions, whereas BP-PEG was susceptible to oxidative deterioration. In juvenile rat subdermal implants, BP-POZ demonstrated reduced AGE formation and serum albumin infiltration, while calcification was not inhibited. However, BP-POZ rat subdermal implants with ethanol pretreatment demonstrated inhibition of both AGE accumulation and calcification. Ex vivo laminar flow studies with human blood demonstrated BP-POZ enhanced thromboresistance with reduced white blood cell accumulation. We conclude that SVD associated with AGE and serum protein accumulation can be mitigated through POZ functionalization that both enhances biocompatibility and facilitates ethanol pretreatment inhibition of BP calcification.

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde treated bovine pericardium (BP) are widely used to treat heart valve disease. BHV dysfunction from structural valve degeneration (SVD) develops over time, often necessitating device replacement. SVD is associated with both calcification and the accumulation of advanced glycation end products (AGE) and serum proteins within BHV leaflets. Here, we introduce a new strategy for mitigating AGE-associated SVD by modifying BP with a poly(2-methyl-2-oxazoline) (POZ). Hypothesis: Attached POZ will prevent BP deterioration by mitigating serum protein and pro-inflammatory AGE uptake. Methods: Covalently modified BP-POZ was studied for its effects on serum albumin and AGE entry in vitro via a mass-uptake assay and immunostaining. Resistance of BP-POZ to oxidation was assessed in comparison to poly(ethylene glycol) (PEG). Macrophage studies assessed the inflammatory properties of BP-POZ. A juvenile rat subdermal model was used to study serum protein and AGE uptake and calcification within BP-POZ, with or without ethanol pretreatment. A heart valve pulse duplicator was used to assess the protective effects of BP-POZ on BHV hydrodynamics following accelerated glycation. Results: POZ mitigated the accumulation of serum albumin and AGE in vitro . After exposure to AGE and macrophages, POZ preserved BP collagen structure while TNFα levels remained at baseline. Under oxidation conditions, POZ retained its protective function, unlike PEG. In the rat model, POZ reduced BP AGE (p Conclusions: SVD due to AGE and serum proteins can be effectively mitigated through POZ modification, that does not interfere with ethanol pretreatment mitigation of BP calcification.

Background: Inflammatory mechanisms, which can be affected by altered metabolic profiles such as hypercholesterolemia (HC), contribute to the pathophysiology of in-stent restenosis (ISR). CD47 is a transmembrane protein that is known to inhibit immune cell and platelet activation. Previously in a rat model, we showed that immobilizing peptide sequences of CD47 (pepCD47) to stainless steel (SS) stent surfaces decreased platelet and leukocyte association, and significantly reduced ISR. In these current studies, we use HC rabbits to test the hypothesis that the pepCD47-modified stents will reduce ISR in a pro-atherogenic phenotype. Methods: PepCD47 was attached to SS using polybisphosphonate coordination chemistry and pyridyldithio/thiol conjugation. Bare metal (BM) and pepCD47 SS foils (n=6) were exposed to anticoagulated blood from normocholesterolemic (NC) or HC rabbits for 1 hr. The relative abundance of the surface-associated cells was determined fluorimetrically. Rabbits (n=4) were fed a HC diet for 4 weeks prior to placing BMS or pepCD47 stents bilaterally in the iliac locations. The diet was continued until sacrifice (4 weeks). After chemical dissolution of the stent struts, the segments were paraffin embedded, sectioned, stained and assessed morphometrically. The prevalence of peri-strut macrophages was determined by immunohistochemistry (IHC) as a percentage of a strut circumference infiltrated with macrophages. Results: An 8% decrease of platelet and leukocyte attachment to a pepCD47-derivatized SS compared to BM foils (p=NS) was observed with NC blood, while a significant 35% decrease of cell attachment (p Conclusion: These results are the first to show the anti-restenotic properties of pepCD47 modified stents in a translational HC rabbit model. The anti-restenotic effects are likely due to CD47-mediated reduction of stent-triggered inflammation.

Background: Hypercholesterolemia (HC) has previously been shown to augment restenotic response in several animal models. However, the mechanistic aspects of in-stent restenosis (ISR) on an HC background are not fully understood. Methods: HC was induced in 5 NZW rabbits by hypercholesterolemic diet (HCD) fed for 4 weeks prior to bilateral implantation of stainless steel stents in the iliac location. The diet was continued until sacrifice. In parallel, stents were deployed in the iliac arteries of 5 normocholesterolemic (NC) rabbits. All animals were euthanized 4 weeks after stenting. Harvested arteries were formalin-fixed. The stent struts were dissolved in a mixture of nitric and hydrofluoric acids. The destented arteries were paraffin-embedded, sectioned, stained according to the Verhoeff-vanGieson method, and the lumen area, neointimal thickness, neointimal area, neointima-to-media ratio, and percent of luminal stenosis were determined morphometrically. A semiquantitative scale was used to assess the intensity and spread of TNFa expression by immunohistochemistry (IHC). The prevalence of peri-strut macrophages (MΦ) was determined by IHC as a percentage of a strut circumference infiltrated with MΦ. Results: HC diet drastically increased severity of ISR (Fig). The corresponding values of the lumen area, neointimal thickness, neointimal area, neointima-to-media ratio and percent of luminal stenosis for the groups of NC and HC animals were 1.86±0.44 vs 1.48±0.54 (p Conclusions: The inflammatory response to stent deployment is intensified in HC metabolic conditions, leading to the augmented neointimal expansion and ISR.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties similar to the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

AimsHeart valve disease attributed to serotonin (5HT) has been observed with 5HT-secreting carcinoid tumors and in association with medications, such as the diet drug, Dexfenfluoramine, a serotonin transporter (SLC6A4) inhibitor and 5HT receptor (HTR) 2B agonist. HTR2B signaling upregulates TGFβ-1 resulting in increased production of extracellular matrix proteins. SLC6A4 internalizes 5HT, limiting HTR signaling. Selective 5HT reuptake inhibitors (SSRI), widely used antidepressants, target SLC6A4, thus enhancing HTR signaling. However, 5HT and SLC6A4 mechanisms have not been previously associated with degenerative mitral regurgitation (MR). The present studies investigated the hypothesis that both dysregulation of SLC6A4 and inhibition of SLC6A4 contribute to the pathophysiology of MR.Methods and ResultsHere we report SLC6A4 related studies of 225 patients with MR requiring surgery. A multivariate analysis showed that SSRI use in MR patients was associated with surgery at a younger age, indicating more rapidly progressive MR (p=0.0183); this was confirmed in a national dataset (pConclusionsDown regulation and inhibition of SLC6A4 influences MR through enhanced HTR signaling. SSRI may further influence MR through inhibition and down regulation of SLC6A4, upregulation of HTR2B, and increased platelet release of 5HT.Translational PerspectiveDegenerative mitral valve regurgitation (MR) affects millions, and there is no medical therapy for this disease. MR becomes progressively worse, and for severe MR, the only option is cardiac surgery. Serotonin (5HT) is best known as a neurotransmitter. However, 5HT secreting carcinoid tumors cause a cardiac valve disorder in many cases, and 5HT related medications, such as the diet drug Fenfluoramine, have been associated with the development of cardiac valve disease. The present paper presents evidence that diminished serotonin transporter (SLC6A4) expression and inhibition, lead to increased 5HT receptor signaling, contributing to the progression of MR.

In-stent restenosis (ISR) and late stent thrombosis are the major complications associated with the use of metal stents and drug eluting stents respectively. Our lab previously investigated the use of peptide CD47 in improving biocompatibility of bare metal stents in a rat carotid stent model and our results demonstrated a significant reduction in platelet deposition and ISR. However, this study did not characterize the stability of the pepCD47 on metal surfaces post storage, sterilization and deployment. Thus, the objective of the present study was 1) to test the stability of the peptide post - storage, sterilization, exposure to shear and mechanical stress and 2) to begin to expand our current knowledge of pepCD47 coated metal surfaces into the preclinical large animal rabbit model. Our results show that the maximum immobilization density of pepCD47 on metal surfaces is approximately 350 ng/cm2. 100% of the pepCD47 was retained on the metal surface post 24 weeks of storage at 4 °C, exposure to physiological shear stress, and mechanical stress of stent expansion. The bioactivity of the pepCD47 was found to be intact post 24 weeks of storage and ethylene oxide sterilization. Finally our ex vivo studies demonstrated that compared to bare metal the rabbit pepCD47 coated surfaces showed - 45% reduced platelet adhesion, a 10-fold decrease in platelet activation, and 93% endothelial cell retention. Thus, our data suggests that pepCD47 coating on metal surfaces is stable and rabbit pepCD47 shows promising preliminary results in preventing thrombosis and not inhibiting the growth of endothelial cells. Statement of significance Biocompatibility of bare metal stents is a major challenge owing to the significantly high rates of in-stent restenosis. Previously we demonstrated that peptide CD47 functionalization improves the biocompatibility of bare metal stents in rat model. A similar trend was observed in our ex vivo studies where rabbit blood was perfused over the rabbit pepCD47 functionalized surfaces. These results provide valuable proof of concept data for future in vivo rabbit model studies. In addition, we investigated stability of the pepCD47 on metal surface and observed that pepCD47 coating is stable over time and resistant to industrially relevant pragmatic challenges.

The blood compatibility of various intravascular (IV) devices (e.g., catheters, sensors, etc.) is compromised by activation of platelets that can cause thrombus formation and device failure. Such devices also carry a high risk of microbial infection. Recently, nitric oxide (NO) releasing polymers/devices have been proposed to reduce these clinical problems. CD47, a ubiquitously expressed transmembrane protein with proven anti-inflammation/anti-platelet properties when immobilized on polymeric surfaces, is a good candidate to complement NO release in both effectiveness and longevity. In this work, we successfully appended CD47 peptides (pepCD47) to the surface of biomedical grade polyurethane (PU) copolymers. SIRPα binding and THP-1 cell attachment experiments strongly suggested that the pepCD47 retains its biological properties when bound to PU films. In spite of the potentially high reactivity of NO toward various amino acid residues in CD47, the efficacy of surface-immobilized pepCD47 to prevent inflammatory cell attachment was not inhibited after being subjected to a high flux of NO for three days, demonstrating excellent compatibility of the two species. We further constructed a CD47 surface immobilized silicone tubing filled with NO releasing S-nitrosoglutathione/ascorbic acid (GSNO/AA) solution for synergistic biocompatibility evaluation. Via an ex vivo Chandler loop model, we demonstrate for the first time that NO release and CD47 modification could function synergistically at the blood/material interface and produce greatly enhanced anti-inflammatory/anti-platelet effects. This concept should be readily implementable to create a new generation of thromboresistant/antimicrobial implantable devices.

Tissue contacting surfaces of medical devices initiate a host inflammatory response, characterized by adsorption of blood proteins and inflammatory cells triggering the release of cytokines, reactive oxygen species (ROS) and reactive nitrogen species (RNS), in an attempt to clear or isolate the foreign object from the body. This normal host response contributes to device-associated pathophysiology and addressing device biocompatibility remains an unmet need. Although widespread attempts have been made to render the device surfaces unreactive, the establishment of a completely bioinert coating has been untenable and demonstrates the need to develop strategies based upon the molecular mechanisms that define the interaction between host cells and synthetic surfaces. In this review, we discuss a family of transmembrane receptors, known as immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors, which show promise as potential targets to address aberrant biocompatibility. These receptors repress the immune response and ensure that the intensity of an immune response is appropriate for the stimuli. Particular emphasis will be placed on the known ITIM-containing receptor, Signal Regulatory Protein Alpha (SIRPhα), and its cognate ligand CD47. In addition, this review will discuss the potential of other ITIM-containing proteins as targets for addressing the aberrant biocompatibility of polymeric biomaterials.

Addressing the aberrant interactions between immune cells and biomaterials represents an unmet need in biomaterial research. Although progress has been made in the development of bioinert coatings, identifying and targeting relevant cellular and molecular pathways can provide additional therapeutic strategies to address this major healthcare concern. To that end, we describe the immune inhibitory motif, receptor–ligand pairing of signal regulatory protein alpha and its cognate ligand CD47 as a potential signaling pathway to enhance biocompatibility. The goals of this article are to detail the known roles of CD47–signal regulatory protein alpha signal transduction pathway and to describe how immobilized CD47 can be used to mitigate the immune response to biomaterials. Current applications of CD47-modified biomaterials will also be discussed herein.

CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils.

The uniform alignment of endothelial cells inside small-diameter synthetic grafts can be directed by surface topographies such as microgrooves and microfibers to recapitulate the flow-induced elongation and alignment of natural endothelium. These surface micropatterns may also promote directional migration and potentially improve anastomotic ingrowth of endothelial cells inside the synthetic grafts. In this paper, we developed electrospinning and spin casting techniques to pattern the luminal surface of small-diameter polyurethane (PU) grafts with microfibers and microgrooves, respectively, and evaluated endothelial cell orientation on these surface micropatterns. Tracks of circumferentially oriented microfibers were generated by electrospinning PU onto a mandrel rotated at high velocity, whereas longitudinal tracks of microgrooves were generated by spin casting PU over a rotating poly(dimethylsiloxane) mold. We found that both PU grafts possessed longitudinal Young’s moduli in the range of 0.43 ± 0.04 to 2.00 ± 0.40 MPa, comparable with values obtained from native artery. Endothelial cells seeded onto the grafts formed confluent monolayers with individual cells exhibiting elongated morphology parallel to the micropatterns. The cells were phenotypically similar to natural endothelium as assessed by the expression of the endothelial cell-specific marker, vascular endothelial cell cadherin. In addition, the cells were also responsive to stimulation with the pro-inflammatory cytokine tumor necrosis factor-α as assessed by the inducible expression of intercellular adhesion molecule-1. These results demonstrate that our micropatterned PU grafts possessed longitudinal Young’s moduli in the same range as native vascular tissue and were capable of promoting the formation of aligned and cytokine-responsive endothelial monolayers.

Background— Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. Methods and Results— We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37°C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial Ad GFP expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. Conclusions— Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature.

The effectiveness of endovascular stents is hindered by in-stent restenosis (ISR), a secondary re-obstruction of treated arteries due to unresolved inflammation and activation of smooth muscle cells in the arterial wall. We previously demonstrated that immobilized CD47, a ubiquitously expressed transmembrane protein with an established role in immune evasion, can confer biocompatibility when appended to polymeric surfaces. In present studies, we test the hypothesis that CD47 immobilized onto metallic surfaces of stents can effectively inhibit the inflammatory response thus mitigating ISR. Recombinant CD47 (recCD47) or a peptide sequence corresponding to the Ig domain of CD47 (pepCD47), were attached to the surfaces of both 316L-grade stainless steel foils and stents using bisphosphonate coordination chemistry and thiol-based conjugation reactions to assess the anti-inflammatory properties of CD47-functionalized surfaces. Initial in vitro and ex vivo analysis demonstrated that both recCD47 and pepCD47 significantly reduced inflammatory cell attachment to steel surfaces without impeding on endothelial cell retention and expansion. Using a rat carotid stent model, we showed that pepCD47-functionalized stents prevented fibrin and platelet thrombus deposition, inhibited inflammatory cell attachment, and reduced restenosis by 30%. It is concluded that CD47-modified stent surfaces mitigate platelet and inflammatory cell attachment, thereby disrupting ISR pathophysiology.

Polyurethane (PU) components of cardiovascular devices are subjected to oxidation-initiated surface degradation, which leads to cracking and ultimately device failure. In the present study, we investigated a novel bromoalkylation chemical strategy to covalently attach the antioxidant, di-tert-butylphenol (DBP), and/or cholesterol (Chol) to the PU urethane nitrogen groups to hypothetically prevent oxidative degradation. These experiments compared PU, PU-DBP, PU-Chol, and PU-Chol-DBP. A series of comparative oxidative degradation studies involved exposing PU samples (modified and unmodified) to H2O2-CoCl2 for 15 days at 37 degrees C, to cause accelerated oxidative degradation. The extent and effects of degradation were assessed by attenuated total reflectance Fourier transformation infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface contact angle measurements, and mechanical testing. Both the Chol and DBP modification conferred significant resistance to oxidation related changes compared to unmodified PU per FTIR and SEM results. SEM demonstrated cavitation only in unmodified PU. However, contact angle analysis showed significant oxidation-induced changes only in the Chol-modified PU formulations. Most importantly, uniaxial stress-strain testing revealed that only PU-DBP demonstrated bulk elastomeric properties that were minimally affected by oxidation; PU, PU-Chol, PU-Chol-DBP showed marked deterioration of their stress-strain properties following oxidation. In conclusion, these results demonstrate that derivatizing PU with DBP confers significant resistance to oxidative degradation compared with unmodified PU.

Previous research from our group has demonstrated that bromoalkylation of polyurethane elastomers via base mediated activation of the urethane-hard segment nitrogen groups can be used to either attach bisphosphonate groups to confer calcification resistance or append cholesterol to promote endothelial cell adhesion. In the present studies we further explore the potential of this chemical approach by investigating bulk carboxylation of polyurethanes via bromoalkylation to enable surface heparinization for thromboresistance. Thus, polyurethane (PU) was modified with pendant 7-carboxy-5-thiaheptyl groups using a polymer-analogous reaction of bromobutylated PU with tetrabutylammonium 3-mercaptopropionate in mild conditions. The grafting of polyallylamine (PAA) onto the surface of carboxylated PU via direct coupling of amino and carboxy groups resulted in high levels of PAA (up to 8 mug/cm(2)). The surface-aminated PU was further covalently modified with unfractionated heparin as confirmed by FTIR. Fluorescence labeling of PAA hydrochloride and heparin with BODIPY-FL was used to quantify the extent of surface modifications. Heparin was covalently bound at a high level (1.11 +/- 0.06 mug/cm(2)) and was shown to be active, with demonstrable Factor Xa inhibition and platelet factor IV binding. It is concluded that surface amination of bulk-carboxylated PU represents a novel approach for heparinizing PU; carboxylation followed by surface amination represents another important dimension of bromo-alkyl activation of polyurethane hard segments, thereby enabling heparinization.

Gene therapy holds great promise for treating both genetic and acquired disorders. However, progress toward effective human gene therapy has been thwarted by a number of problems including vector toxicity, poor targeting of diseased tissues, and host immune and inflammatory activity to name but a few of the challenges. Gene therapy for cardiovascular disease has been the subject of many fewer clinical trials than other disorders such as cancer or cystic fibrosis. Nevertheless, the challenges are comparable. The present paper reports a review of investigations related to our hypothesis that site specific cardiovascular gene therapy represents an approach that can lead to both optimizing efficacy and reducing the impact of gene vector-related systemic adverse effects. We report experimental studies demonstrating proof of principle in three areas: gene therapy for heart valve disease, gene delivery stents, and gene therapy to treat cardiac arrhythmias. Heart valve disease is the second most common indication for open heart surgery and is now only treatable by surgical removal or repair of the diseased heart valve. Our investigations demonstrate that gene vectors can be immobilized on the surface of prosthetic heart valve leaflets thereby enabling a therapeutic genetic modification of host cells around the valve annulus and on the leaflet. Other animal studies have shown that vascular stents used to relieve arterial obstruction can also be used as gene delivery systems to provide therapeutic vector constructs that can both locally prevent post stenting reobstruction, known as in-stent restenosis, and treat the underlying vascular disease. Cardiac arrhythmias are the cause of sudden death due to heart disease and affect millions of others on a chronic basis. Our group has successfully investigated in animal studies localized gene therapy using an ion channel mutation to treat atrial arrhythmias.

Endothelialization of synthetic surfaces has been challenging with limited success thus far. We investigated the hypothesis that covalent attachment of cholesterol to polyurethane via the urethane nitrogen groups would create a high-affinity surface for attachment and adhesion of endothelial cells. Cholesterol was covalently bound to the polyether polyurethane, Tecothane®, by first derivatizing the polyurethane nitrogen groups with bromoalkyl side chains, followed by reacting mercapto-cholesterol to the bromoalkyl sites. Cholesterol-modified polyurethane demonstrated a qualitatively smoother surface per atomic force microscopy than nonmodified and increased surface energy (contact angle measurements) compared with unmodified polyurethane. Cell attachment assays showed a significantly greater number of attached bovine arterial endothelial cells (p = 0.0003) after 45 min of seeding on cholesterol-modified polyurethane versus unmodified polyurethane. Bovine arterial endothelial cells cultivated on cholesterol-modified Tecothane® showed significantly greater levels of cell retention compared with unmodified Tecothane® when exposed to arterial level shear stress for 2 h (25 dynes/cm2) with 90.0 ± 6.23% cells remaining adherent compared with unmodified polyurethane, 41.4 ± 11.7%, p = 0.0070. Furthermore, ovine endothelial precursors, obtained as blood outgrowth endothelial cells, were seeded on cholesterol-modified polyurethane and exposed to 25 dynes/cm2 shear conditions for 2 h, with the retention of 90.30 ± 3.25% of seeded cells versus unmodified polyurethane, which retained only 4.56 ± 0.85% (p < 0.001). It is concluded that covalently linking cholesterol to polyurethane results in improved material properties that permit increased endothelial cell retention compared with unmodified polyurethane. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 72A: 200–212, 2005

Objective: Calcification of polyurethane prosthetic valve leaflets causes a major functional impairment. Previously we showed that polyurethane heart valves modified with covalently linked bisphosphonate groups were resistant to calcification in vivo. However, we also found that the highly polar anionic bisphosphonate groups on the polyurethane surface attracted sodium counter ion adsorption, and thereby increased the elastomer's water absorption to 20% of total weight. In this study we address the increased water absorption by investigating the hypothesis that covalently attaching cationic diethylamino groups to the bisphosphonate-modified polyurethane will reduce water absorption. Thus we evaluated the mechanical and in vivo anticalcification properties of heart-valve leaflets composed of this modified polymer. Methods: Diethylamino and bisphosphonate groups (DBP) were appended to the polyurethane Biospan's hard segment using previously published bromoalkylation methodology. Water absorption and biaxial mechanical and uniaxial failure testing were used to determine the mechanical properties of the DBP-modified polymer. Rat subdermal implants (60 days) and extended (150 days) single pulmonary leaflet replacements in juvenile sheep provided in vivo assessments of the bisphosphonate-modified polyurethane. Results: The water absorption properties of the DBP-modified polymers and unmodified polyurethanes were 1.86 and 2.3 %, respectively. Biaxial mechanical tests showed the DBP-modified polymer was more compliant than the unmodified control material, but all polymeric material had similar uniaxial failure properties. In both rat subdermal and sheep circulatory implants, the DBP-modified polyurethane resisted calcification, as assessed by scanning electron microscopy, with complete calcification inhibition in prosthetic sheep valve leaflet replacements. Conclusion: DBP polyurethane possesses physical (water absorption) and biomechanical properties comparable to unmodified polyurethane and can resist intrinsic heart-valve leaflet calcification in blood-stream implants. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 385–395, 2003

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

We have previously established that recombinant CD47 can ameliorate the inflammatory response to synthetic polymeric surfaces. Here, we begin to profile, at the transcriptional, translational and cell signaling level, the inflammatory cell response when blood interacts with CD47 modified polyvinyl chloride (PVC) (CD47-PVC). We used qPCR arrays to compare transcriptional changes between human whole blood exposed to CD47-PVC or PVC. Transcription of IL1F5, IL1F10, IL17F, CCL3, CCL8, CCL28, CXCL12, and CXCL13 was upregulated in blood exposed to PVC, compared to CD47-PVC. The increase in CCL3 and CCL8 transcription correlated with an increase in the chemokines' presence in the plasma. Exposure of blood to CD47-PVC resulted in an increase, compared to PVC, in transcription of CCL2, CCL4, CCL20, CXCL1, TGFβ3, GDF3, GDF10, CD40LG, and TNFSF10. CD47-PVC exposure resulted in an increase of the following matrix metalloproteinase related genes: MMP1, MMP7, MMP13, and MMP16. Phosflow cytometry, and assays examining transcription factor binding, cell attachment, and genome-wide chromatin association indicated that members of the JAK-STAT signaling pathway, particularly JAK2 and STAT5, mediate inflammatory cell interactions with CD47-PVC. Our data demonstrate that differential molecular responses to CD47 involve downregulation of cytokines, upregulation of MMPs, and JAK/STAT signaling mechanisms.

The morphological and inflammatory responses of adherent macrophages are correlated to evaluate the biocompatibility of surfaces. Monocyte-derived macrophage (MDM), THP-1, and THP-1 cells expressing GFP-actin chimeric protein were seeded onto glass, polyurethane (PU), and glass surface modified with quaternary ammonium salt functionalized chitosan (CH-Q) and hyaluronic acid (HA). Using confocal microscopy, the surface area, volume and 3D shape factor of adherent macrophages was quantified. For comparison, functional consequences of cell-surface interactions that activate macrophages and thereby elicit secretion of a proinflammatory cytokine were evaluated. Using an enzyme linked immune sorbent assay, tumor necrosis factor-alpha (TNF-α) was measured. On glass, macrophages exhibited mainly an amoeboid shape, exhibited the largest surface area, volume, and 3D shape factor and produced the most TNF-α. On PU, macrophages displayed mainly a hemispherical shape, exhibited an intermediate volume, surface area and 3D shape factor, and produced moderate TNF-α. In contrast, on CH-Q and HA surfaces, macrophages were spherical, exhibited the smallest volume, surface area, and 3D shape factor, and produced the least TNF-α. These studies begin to validate the use of GFP-actin-modified MDM as a novel tool to correlate cell morphology with inflammatory cell response.

CD47 is a transmembrane protein that is a marker of "self". CD47 binding to its cognate receptor in leukocytes and macrophages, signal-regulatory protein alpha (SIRPα), causes inhibition of inflammatory cell attachment. We hypothesized that immobilization of recombinant CD47 on polymeric surfaces would reduce inflammation. Recombinant CD47 was appended to polyvinyl chloride (PVC) or polyurethane (PU) surfaces via photoactivation chemistry. Cell culture studies showed that CD47 immobilization significantly reduced human neutrophil (HL-60) and human monocyte derived macrophage (MDM) (THP-1) attachment to PVC and PU respectively. A neutralizing antibody, directed against SIRPα, inhibited THP-1 and HL-60 binding to PU and PVC surfaces respectively. This antibody also increased the level of SIRPα tyrosine phosphorylation, thereby indicating a direct role for SIRPα mediated signaling in preventing inflammatory cell attachment. Studies using human blood in an ex vivo flow-loop showed that CD47 modified PVC tubing significantly reduced cell binding and neutrophil activation compared to unmodified tubing or poly-2-methoxy-ethylacrylate (PMEA) coated tubing. In ten-week rat subdermal implants, CD47 functionalized PU films showed a significant reduction in markers of MDM mediated oxidative degradation compared to unmodified PU. In conclusion, CD47 functionalized surfaces can resist inflammatory cell interactions both in vitro and in vivo.

Despite many advances in designing biocompatible materials, inflammation remains a problem in medical devices and implants. We report two methods, microcontact printing and photodegradation by UV exposure, to pattern dextran and hyaluronic acid on glass, as well as demonstrate their utility for use as an anti-inflammatory biomaterial. The dextran/glass patterned surface can be further modified by grafting hyaluronic acid to glass, creating a binary polysaccharide patterned surface. We used two geometries, 90 µm squares and 22 µm stripes, to study the human macrophage (THP-1) adhesion on the patterned surfaces containing dextran, hyaluronic acid and the binary pattern. The results indicate that a majority of the macrophages are non-adherent on hyaluronic acid for three day culture. The ranking of surfaces according to macrophage adhesion is 3-aminopropyl triethoxysilane-modified glass culture dish, dextranized surfaces, glass, and hyaluronic acid-modified surfaces. On the binary pattern of dextran and hyaluronic acid, macrophages preferentially attach and adhere to the dextranized area. Patterned surfaces provide an excellent platform for mimicking the complexity of the glycocalyx and investigating the interface between this surface and cells. This binary polysaccharide pattern also offers a new route to address anti-inflammatory potential of surface coatings on biomaterials in a high through-put fashion.

We previously showed that blood outgrowth endothelial cells (BOECs) had a high affinity for polyurethane (PU) covalently configured with cholesterol residues (PU-Chol). However, the molecular mechanisms responsible for this enhanced affinity were not determined. CD47, a multifunctional transmembrane glycoprotein involved in cellular attachment, can form a cholesterol-dependent complex with integrin alpha(v)beta(3) and heterotrimeric G proteins. We tested herein the hypothesis that CD47, and the other components of the multi-molecular complex, enhance the attachment of BOECs to PU-Chol. Immunoprecipitation studies, of human and ovine BOECs, demonstrated that CD47 associates with integrin alpha(v) and integrin beta(3) as well as G(alphai-2) protein. The three-fold increase in BOEC attachment to PU-Chol, compared to unmodified PU, was reversed with the addition of blocking antibodies specific for CD47 and integrin alpha(v) and integrin beta(3). Similar results were observed with the addition of methyl-beta-cyclodextrin (MbetaCD), a known disruptor of the CD47 complex as well as of the membrane cholesterol content, to seeded BOEC or PU-Chol films. Reducing CD47 expression, via lentivirus transduced shRNA, decreased BOEC binding to PU-Chol by 50% compared to control groups. These data are the first demonstration of a role for the CD47 cholesterol-dependent signaling complex in BOEC attachment onto synthetic surfaces.

Oxidative degradation of the polyurethane elastomeric (PU) components greatly reduces the efficacy of PU-containing cardiovascular devices. Covalently appending the phenol-based antioxidant, 4-substituted 2,6-di-tert-butylphenol (DBP), to PU hard segments effectively reduced oxidative degradation of the PU in vivo and in vitro in prior studies by our group. In these experiments, we analyze the contribution of the tethering molecule to the antioxidant capabilities of the DBP-modified PU. Bromoalkylation chemistry was used to link DBP to the hard segment of the polyether PU, Tecothane, via our original linker (PU-DBP) or variants containing side chains with one (PU-C-DBP) or three (PU-3C-DBP) carbons. Two additional DBP variants were fabricated in which the DBP group was appended to the alkyl chain via an oxygen atom (PU-O-DBP) or an amide linkage in the middle of the tether (PU-NHCO-DBP). All DBP variant films and unmodified control films were subject to oxidative degradation via 15-day immersion in a solution of 20% H(2)O(2) + 0.1M CoCl(2). At the end of the oxidation protocol, films were analyzed for the presence of oxidation-related endpoints via scanning electron microscopy, contact angle measurements, and Fourier transformation infrared spectroscopy (FTIR). All DBP-containing variants resisted oxidation damage significantly better than the unmodified control PU. SEM analysis of oxidized PU-C-DBP and PU-O-DBP showed evidence of surface cracking, consistent with oxidative degradation of the PU surfaces. Similarly, there was a trend in increased ether crosslinking, a marker for oxidative degradation, in PU-C-DBP and PU-NHCO-DBP films. Consistent with these FTIR results, both PU-C-DBP and PU-NHCO-DBP had significant reductions in measured surface hydrophobicity as a result of oxidation. These data show for the first time that the choice of linker molecule significantly affects the efficiency of the linked phenolic antioxidant.

Polyurethane cardiovascular implants are subject to oxidation initiated surface degradation, which is mediated by monocyte-derived macrophages (MDM); this often leads to surface cracking and device failure. The present studies examined the hypothesis that covalently attaching antioxidant, di-tert-butylphenol (DBP), to the urethane nitrogens of a polyether polyurethane (PU) via bromo-alkylation reactions could prevent this problem. PU was configured with two dosages of DBP, 0.14 mM DBP/g PU of DBP (PU-DBP) and a more highly modified (HM) 0.40 mM DBP/g PU (PU-DBP-HM). THP-1 cells, a human MDM cell line, stimulated with phorbol ester and seeded on PU, PU-DBP, and PU-DBP-HM films were assessed for reactive oxygen species (ROS) production via a fluorescent based dihydrorhodamine-123 assay. Results from these studies showed a significant dose-dependent reduction of ROS levels for THP-1 cells seeded on PU-DBP versus unmodified PU. PU, PU-DBP, or PU-DBP-HM films were implanted into subdermal pouches of Sprague-Dawley rats. Films were explanted after 10 weeks and assessed for oxidative degradation via light and scanning electron microscopy (SEM) and Fourier transformation infrared spectroscopy (FTIR). Light microscopy showed extensive surface cracking, which was confirmed via SEM, on unmodified PU surfaces that was absent in both PU-DBP and PU-DBP-HM explanted films. FTIR analysis showed reduction in oxidation-induced ether crosslinking that was directly related to DBP dosages. It is concluded that modifying PU with the covalent attachment of an antioxidant confers biodegradation resistance in vivo in a dose dependent manner; this effect is likely due to quenching of the ROS generated by the adherent macrophages.

To explore the feasibility of utilizing two implantable devices made from modified polyurethane films with antibody tethered replication-defective adenoviruses encoding for green fluorescent protein (AdGFP) as gene delivery platforms.Intra-aortic button implants of collagen-coated polyurethane films with antibody tethered AdGFP were sutured into the infrarenal aorta of adult pigs and pulmonary valve leaflet in juvenile sheep was replaced by polyurethane pulmonary valve cusp replacement with antibody-tethered AdGFP. After seven days, the buttons, prosthetic leaflets, and their surrounding tissues were explanted and evaluated for biocompatibility and AdGFP-mediated gene transfer by fluorescent microscopy and PCR analysis.In vivo analysis of gene transfer from collagen-coated polyurethane films in pig infrarenal aorta implants, one week explants of the collagen-coated polyurethane films demonstrated (14.2 +/- 2.5)% of neointimal cells on the surface of the implant. In sheep pulmonary valve leaflet replacement studies, polyurethane films with antibody tethered AdGFP vector demonstrated (25.1 +/- 5.7)% of cells attached to polyurethane valve leaflets were transduced in one week. PCR analyses showed that GFP DNA was not detectable in blood or distal tissues.Site-specific intravascular delivery of adenoviral vectors for gene therapy can be achieved with these two kinds of polyurethane implants utilizing the antivector antibody tethering mechanism.

The clinical and experimental use of polyurethane heart valve prostheses has been compromised by thrombosis and calcified thrombus. This is caused in part by the lack of an intact endothelium on these implant surfaces. We hypothesize that endothelial seeding of a polyurethane heart valve leaflet with autologous sheep blood outgrowth endothelial cells (BOECs) could be achieved with cholesterol-modified polyurethane (PU-Chol) to promote BOEC adhesion, thereby resulting in an intact, shear-resistant endothelium that would promote resistance to thrombosis.Cholesterol-derivatized polyurethane was formulated by bromoalkylation of the urethane nitrogens followed by reactive attachment of mercaptocholesterol. In vitro shear flow studies were carried out comparing BOEC retention on control surfaces versus PU-Chol using forces comparable to those observed in vivo with cardiac valves (75 dyne/cm2). Autologous sheep BOECs were seeded onto PU-Chol before pulmonary leaflet replacement surgery under cardiopulmonary bypass. Studies were terminated at 30 and 90 days followed by retrieval analyses.Blood outgrowth endothelial cell seeding of PU-Chol surfaces resulted in an endothelial monolayer that was positive for von Willebrand factor. Polyurethane-cholesterol demonstrated significantly greater BOEC adhesion under 75 dyne/cm2 shear force in vitro than control polyurethane (75.3% +/- 12.3% versus 5.8% +/- 3.9%, respectively; p0.001). Sheep pulmonary cusp replacements demonstrated retention of seeded BOECs on PU-Chol leaflets with no significant differences in the extent of cellular density comparing unimplanted specimens with explants. Control explants (nonseeded PU-Chol and nonseeded polyurethane) demonstrated no evidence of endothelial recruitment.Polyurethane-cholesterol represents a polyurethane formulation with very high adhesive properties for BOECs under heart valve level shear forces both in vitro and in vivo.

The timing of granule cell migration in the developing cerebellum is regulated by thyroid hormone. Granule cell migration depends on the recognition of extracellular neuronal guidance molecule(s), such as laminin, and this, in turn, requires cell surface adhesion molecules (integrins) that are anchored on the cell membrane by the actin cytoskeleton. While many of the actions of thyroid hormone, specifically 3,5,3′-triiodothyronine (T3), are mediated by regulated gene expression, both thyroxine (T4) and 3,3′,5′-triiodothyronine (rT3) also exert direct, positive control of the quantity of polymerized actin in cultured astrocytes without affecting gene expression. T4-dependent actin polymerization has been shown to (i) participate in the immobilization of laminin to the cell surface, (ii) help deposit laminin in the molecular layer of the developing cerebellum, and (iii) anchor integrin(s) that recognize laminin present in the extracellular matrix. In this study, we show that both T4 and rT3, but not T3, directly regulate the F-actin content of elongating neurites of cerebellar neurons. T4 and rT3 also promoted extensive granule cell migration from cerebellar explants, as well as, dense cell clustering and extensive neuronal process formation when granule cells were grown on a laminin-coated surface. Both granule cell migration and neuronal process outgrowth were markedly attenuated by the addition of integrin-blocking antibodies or binding peptides, by the absence of thyroid hormone or the presence of T3. These data suggest that the T4-dependent actin polymerization in developing neurons is necessary for these migrating cells to recognize the laminin guidance molecule, thereby providing a novel molecular mechanism for the profound influence of thyroid hormone on brain development that is independent of regulated gene expression.

The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen-coated polyurethane films investigated an abdominal aorta implant model in pigs using a button configuration that simulated the blood contacting environment of a vascular graft. One week explants of the collagen-coated polyurethane films demonstrated 14.3+/-2.5% of neointimal cells on the surface of the implant transduced with green fluorescent protein - adenovirus (AdGFP) vector loadings of 1 x 10(8) PFU. PCR studies demonstrated no detectable vector DNA in blood or distal organs. Similarly, polyurethane films with direct attachment of antivector antibodies to the surface were used in sheep pulmonary valve leaflet replacement studies, simulating the blood contacting environment of a prosthetic heart valve cusp. Polyurethane films with antibody tethered AdGFP vector (10(8) PFU) demonstrated 25.1+/-5.7% of attached cells transduced in these 1 week studies, with no detectable vector DNA in blood or distal organs. In vivo GFP expression was confirmed with immunohistochemistry. It is concluded that site-specific intravascular delivery of adenoviral vectors for gene therapy can be achieved with polyurethane implants utilizing the antivector antibody tethering mechanism.

LIMITED SUCCESS has been reported in restoring function to spinal cord-injured rodents. Lower limb paralysis caused by complete spinal cord transection in neonatal rats less than 14 days of age may resolve, presumably because of the plasticity of the still-developing central nervous system (CNS). Less success has been achieved in functional repair of the injured adult spinal cord. Implants of immobilized nerve growth factor (NGF) appear to enhance the regrowth of ascending sensory axons across spinal cord gaps in adult rats. Limited but progressive improvement (over a 6-month period) of hind limb function in spinal cord-transected adult rats has been observed over a 6-month period after bridging the transected cord with multiple intercostal nerve grafts. In addition, much has been learned recently about the biology of stem cells. Stem cells isolated from the brain and the spinal cord of both neonatal and adult mice reportedly retain the potential to differentiate into neurons, astrocytes, and oligodendrocytes. Undifferentiated stem cells have been reported to propagate in culture by adding epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to the growth medium. Recent reports suggest that transplantation of immortalized neuronal stem cells into the spinal cord and cerebral cortex led to differentiation of such cells into site-specific neuronal and glial cells and also some functional recovery of the associated defects seen in spinal cord injury, Sly disease, and the myelin degenerative disorder found in the shiverer (shi) mouse. Thus, neuronal stem cells propagated in vitro appear to retain the developmental plasticity necessary to respond to local environmental cues. We postulated that resected segments of the spinal cord could be regenerated in adult rats by implanting spinal cord progenitor cells associated with an appropriate scaffolding or matrix.

Objective: This study focuses on developing a treatment for in-stent restenosis (ISR), the re-obstruction of a stented artery following arterial angioplasty. This work involves creating a system for the delivery of magnetic nanoparticle (NP) loaded endothelial precursor cells to magnetically responsive stents. The goal of this research is to understand NP/cell interactions in response to external magnetic fields as well as characterizing cell viability post incubation with magnetic NPs.

Polyurethane is a durable elastomer capable of serving as a gene delivery platform in the cardiovascular system. Precoating polyurethane cardiovascular devices with a layer of autologous endothelial cells would hypothetically protect the device from thrombus formation. However, biocompatibility issues often hinder attempts to seed vascular devices with endothelial cells. In these studies, blood outgrowth endothelial cells (BOEC's) were used to test the hypothesis that covalent attachment of cholesterol to the polyurethane amide group would create a high affinity surface for attachment and adhesion of autologous BOEC's, prior to implantation as cardiovascular devices. Cholesterol was covalently appended to the polyether polyurethane Tecothane, by activating polyurethane nitrogens and attaching a bromo-alkyl side chain. Expanded ovine BOEC's were cultured on the modified polyurethane to compare viability and cell retention under vascular flow conditions. BOEC susceptibility to lentivirus mediated ectopic gene expression was also analyzed, using a VSV-g pseudotyped, self-inactivating lentivirus with an internal CMV promoter driving an eGFP gene, in conjunction with growth on cholesterol modified polyurethane. Cell adhesion was investigated with BOEC's cultivated until confluence on Tecothane ± appended cholesterol, exposed to physiological shear stress (25 dynes/cm2) for two hours. BOEC's growing on cholesterol modified polyurethane retained 94.1 ± 0.36 % of cells. In contrast cells cultured on unmodified polyurethane retained only 4.56 ± 0.85 % (p = 0.02, n = 3) of the initial cells. Green fluorescent protein (GFP) encoding lentivirus (MOI = 250) vectors transduced 100% of cultured BOEC's. GFP expression was observed in all subcultured BOEC's beyond four cell passages strongly suggesting permanent ectopic gene incorporation. GFP expressing BOEC's continued to grow and express GFP on cholesterol modified polyurethane surfaces. In conclusion, cholesterol modified polyurethane seeded with BOEC's represent a formulation with great potential for intravascular cell based therapies.