Serotonin transporter deficiency in mice results in an increased susceptibility to HTR2B-dependent pro-fibrotic mechanisms in the cardiac valves and left ventricular myocardium.

Increased serotonin (5HT) concentration and signaling, can lead to pathological remodeling of the cardiac valves. We previously showed that a reduction of the 5HT transporter (SERT) expression in the mitral valve (MV) contributes to the progression of degenerative MV regurgitation (MR). We sought to investigate the myocardial and valvular phenotype of SERT-/- mice in order to identify remodeling mechanisms specific to the MV and left ventricular (LV) remodeling. Using 8- and 16-week-old WT and SERT-/- mice we show that male and female animals deficient of SERT have pathological remodeling of the cardiac valves, myocardial fibrosis, diminished ejection fraction and altered left ventricular dimensions. In the MV and intervalvular area of the aortic valve (AV)-MV, gene expression, including Col1a1 mRNA, was progressively altered with age up until 16 weeks of age. In contrast, in the AV and myocardium, most gene expression changes occurred earlier and plateaued by 8 weeks. To explore basal differences in susceptibility to remodeling stimuli among cardiac valves, valve interstitial cells (VIC) were isolated from AV, MV, tricuspid valve (TV), pulmonary valve (PV) and fibroblasts (Fb) from the myocardial apex from 16 weeks old wild type (WT) mice. After 24h stimulation with 10 µM of 5HT, the gene expression of Col1a1 and Acta2 were upregulated in MVIC to a higher degree than in VIC from other valves and Fb. Treatment with TGFß1 similarly upregulated Cola1 and Acta2 in MVIC and AVIC, while the increase was milder in right heart VIC and Fb. Experiments were also carried out with human VIC. In comparison to mice, human left heart VIC were more sensitive to 5HT and TGFß1, upregulating COL1A1 and ACTA2; TGFß1 upregulated HTR2B expression in all VIC. Our results support the hypothesis that a deleterious cardiac effect of SERT downregulation may be mediated by increased susceptibility to HTR2B-dependent pro-fibrotic mechanisms, which are distinct among VIC populations and cardiac fibroblasts, regardless of SERT activity. Given that HTR2B mechanisms involved in VIC and myocardial remodeling response are due to both 5HT and also to downstream related TGFß1 and TNFα activity, targeting HTR2B could be a therapeutic strategy for dual treatment of MR and LV remodeling.

Pediatric Device Clinical Trials Activity: 1999-2022.

OBJECTIVES: This study assessed the state of pediatric medical device (PMD) development by comparing PMD clinical trials to pediatric trials evaluating drugs and biologics, from 1999 to 2022. METHODS: The site www.clinicaltrials.gov was used to identify and quantify both PMD clinical trials and pediatric trials for drugs and biologics. Clinical specialty was also assessed. The institutions included were the 7 children's hospitals primarily affiliated with the Food and Drug Administration (FDA) Pediatric Device Consortia (PDC) grant program between 2018 and 2023. For a national comparison, an additional search assessed PMD trials across all US medical institutions. RESULTS: A total of 243 PMD clinical trials were identified at the FDA-PDC institutions on the basis of the year of initiation; the average number of PMD trials initiated per year per institution was 1.5 from 1999 to 2022. However, PMD trials significantly increased during the period 2014 to 2022 compared with 1999 to 2013 (P < .001); the rate of initiation of drug and biologic pediatric trials demonstrated no significant differences between these time periods. A national survey of all institutions initiating PMD trials, and drugs and biologics trials, identified 1885 PMD trials out of a total 12 943. A comparable trend was noted in the national survey with initiation of PMD trials increasing significantly from 2014 to 2022 (P < .001), compared with 1999 to 2013, whereas the rate of initiation of drug and biologic trials during these periods did not demonstrate a significant change. CONCLUSIONS: Although pediatric clinical trial initiation for drugs and biologics remained stable from 1999 to 2022, the rate of new PMD trials significantly increased during the period 2014 to 2022 at FDA-PDC institutions and nationally.

Nanocarrier Design for Dual-Targeted Therapy of In-Stent Restenosis.

The injury-triggered reocclusion (restenosis) of arteries treated with angioplasty to relieve atherosclerotic obstruction remains a challenge due to limitations of existing therapies. A combination of magnetic guidance and affinity-mediated arterial binding can pave the way to a new approach for treating restenosis by enabling efficient site-specific localization of therapeutic agents formulated in magnetizable nanoparticles (MNPs) and by maintaining their presence at the site of arterial injury throughout the vulnerability period of the disease. In these studies, we investigated a dual-targeted antirestenotic strategy using drug-loaded biodegradable MNPs, surface-modified with a fibrin-avid peptide to provide affinity for the injured arterial wall. The MNPs were characterized with regard to their magnetic properties, efficiency of surface functionalization, disassembly kinetics, and interaction with fibrin-coated substrates. The antiproliferative effects of MNPs formulated with paclitaxel were studied in vitro using a fetal cell line (A10) exhibiting the defining characteristics of neointimal smooth muscle cells. Animal studies examined the efficiency of combined (physical/affinity) MNP targeting to stented arteries in Sprague Dawley rats using fluorimetric analysis and fluorescent in vivo imaging. The antirestenotic effect of the dual-targeted therapy was determined in a rat model of in-stent restenosis 28 days post-treatment. The results showed that MNPs can be efficiently functionalized to exhibit a strong binding affinity using a simple two-step chemical process, without adversely affecting their size distribution, magnetic properties, or antiproliferative potency. Dual-targeted delivery strongly enhanced the localization and retention of MNPs in stented carotid arteries up to 7 days post-treatment, while minimizing redistribution of the carrier particles to peripheral tissues. Of the two targeting elements, the effect of magnetic guidance was shown to dominate arterial localization (p = 0.004 vs. 0.084 for magnetic targeting and peptide modification, respectively), consistent with the magnetically driven MNP accumulation step defining the extent of the ultimate affinity-mediated arterial binding and subsequent retention of the carrier particles. The enhanced arterial uptake and sustained presence of paclitaxel-loaded MNPs at the site of stent deployment were associated with a strong inhibition of restenosis in the rat carotid stenting model, with both the neointima-to-media ratio (N/M) and % stenosis markedly reduced in the dual-targeted treatment group (1.62 ± 0.2 and 21 ± 3 vs. 2.17 ± 0.40 and 29 ± 6 in the control animals; p < 0.05). We conclude that the dual-targeted delivery of antirestenotic agents formulated in fibrin-avid MNPs can provide a new platform for the safe and effective treatment of in-stent restenosis.

Hypercholesterolemia exacerbates in-stent restenosis in rabbits: Studies of the mitigating effect of stent surface modification with a CD47-derived peptide.

BACKGROUND AND AIMS: Hypercholesterolemia (HC) has previously been shown to augment the restenotic response in 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 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: PepCD47 was immobilized on metal foils and stents using polybisphosphonate coordination chemistry and pyridyldithio/thiol conjugation. Cytokine expression in buffy coat-derived cells cultured over bare metal (BM) and pepCD47-derivatized foils demonstrated an M2/M1 macrophage shift with pepCD47 coating. HC and normocholesterolemic (NC) rabbit cohorts underwent bilateral implantation of BM and pepCD47 stents (HC) or BM stents only (NC) in the iliac location. RESULTS: A 40 % inhibition of cell attachment to pepCD47-modified compared to BM surfaces was observed. HC increased neointimal growth at 4 weeks post BM stenting. These untoward outcomes were mitigated in hypercholesterolemic rabbits treated with pepCD47-derivatized stents. Compared to NC animals, inflammatory cytokine immunopositivity and macrophage infiltration of peri-strut areas increased in HC animals and were attenuated in HC rabbits treated with pepCD47 stents. CONCLUSIONS: Augmented inflammatory responses underlie severe ISR morphology in hypercholesterolemic rabbits. Blockage of initial platelet and leukocyte attachment to stent struts through CD47 functionalization of stents mitigates the pro-restenotic effects of hypercholesterolemia.

Valved Conduits for Right Ventricular Outflow Tract Reconstruction: A Review of Current Technologies and Future Directions.

The need for right ventricular outflow tract reconstruction is common and growing in congenital heart surgery given expanding indications for the repair of congenital as well as acquired heart disease. Various valved conduit options currently exist including homografts, xenograft pulmonary valved conduits (Contegra™), and porcine valved conduits. The major limitation for all conduits is implant durability, which requires reoperation. Currently, cryopreserved homografts are often used given their superiority shown in long-term data. Significant limitations remain in the cost and availability of the graft, particularly for smaller sizes. Contegra conduits are available in a variety of sizes. Nonetheless, the data regarding long-term durability are less robust and studies comparing durability with homografts have been conflicting. Additionally, there is concern for increased rates of late endocarditis in this conduit. Porcine valved conduits offer a reliable option but are limited by structural valve degeneration associated with all types of bioprosthetic heart valve replacements. New developments in the field of tissue engineering have produced promising bio-restorative valved conduits that may overcome many of the limitations of previous conduit technologies. These remain in the early stages of clinical testing. This review summarizes the clinical data surrounding the conduits used most commonly in clinical practice today and explores emerging technologies that may bring us closer to developing the ideal conduit.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

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 ß1 (TGFß1) 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 valve structural degeneration associated with metabolic syndrome: Mitigation with polyoxazoline modification.

Bioprosthetic heart valves (BHV), made from glutaraldehyde-fixed xenografts, are widely used for surgical and transcatheter valve interventions but suffer from limited durability due to structural valve degeneration (SVD). We focused on metabolic syndrome (MetS), a risk factor for SVD and a highly prevalent phenotype in patients affected by valvular heart disease with a well-recognized cluster of comorbidities. Multicenter patient data (N = 251) revealed that patients with MetS were at significantly higher risk of accelerated SVD and required BHV replacement sooner. Using a next-generation proteomics approach, we identified significantly differential proteomes from leaflets of explanted BHV from MetS and non-MetS patients (N = 24). Given the significance of protein infiltration in MetS-induced SVD, we then demonstrated the protective effects of polyoxazoline modification of BHV leaflets to mitigate MetS-induced BHV biomaterial degeneration (calcification, tissue cross-linking, and microstructural changes) in an ex vivo serum model and an in vivo with MetS rat subcutaneous implants.

Age-related enhanced degeneration of bioprosthetic valves due to leaflet calcification, tissue crosslinking, and structural changes.

AIMS: Bioprosthetic heart valves (BHVs), made from glutaraldehyde-fixed heterograft materials, are subject to more rapid structural valve degeneration (SVD) in paediatric and young adult patients. Differences in blood biochemistries and propensity for disease accelerate SVD in these patients, which results in multiple re-operations with compounding risks. The goal of this study is to investigate the mechanisms of BHV biomaterial degeneration and present models for studying SVD in young patients and juvenile animal models. METHODS AND RESULTS: We studied SVD in clinical BHV explants from paediatric and young adult patients, juvenile sheep implantation model, rat subcutaneous implants, and an ex vivo serum incubation model. BHV biomaterials were analysed for calcification, collagen microstructure (alignment and crimp), and crosslinking density. Serum markers of calcification and tissue crosslinking were compared between young and adult subjects. We demonstrated that immature subjects were more susceptible to calcification, microstructural changes, and advanced glycation end products formation. In vivo and ex vivo studies comparing immature and mature subjects mirrored SVD in clinical observations. The interaction between host serum and BHV biomaterials leads to significant structural and biochemical changes which impact their functions. CONCLUSIONS: There is an increased risk for accelerated SVD in younger subjects, both experimental animals and patients. Increased calcification, altered collagen microstructure with loss of alignment and increased crimp periods, and increased crosslinking are three main characteristics in BHV explants from young subjects leading to SVD. Together, our studies establish a basis for assessing the increased susceptibility of BHV biomaterials to accelerated SVD in young patients.

Intraprocedural endothelial cell seeding of arterial stents via biotin/avidin targeting mitigates in-stent restenosis.

Impaired endothelialization of endovascular stents has been established as a major cause of in-stent restenosis and late stent thrombosis. Attempts to enhance endothelialization of inner stent surfaces by pre-seeding the stents with endothelial cells in vitro prior to implantation are compromised by cell destruction during high-pressure stent deployment. Herein, we report on the novel stent endothelialization strategy of post-deployment seeding of biotin-modified endothelial cells to avidin-functionalized stents. Acquisition of an avidin monolayer on the stent surface was achieved by consecutive treatments of bare metal stents (BMS) with polyallylamine bisphosphonate, an amine-reactive biotinylation reagent and avidin. Biotin-modified endothelial cells retain growth characteristics of normal endothelium and can express reporter transgenes. Under physiological shear conditions, a 50-fold higher number of recirculating biotinylated cells attached to the avidin-modified metal surfaces compared to bare metal counterparts. Delivery of biotinylated endothelial cells to the carotid arterial segment containing the implanted avidin-modified stent in rats results in immediate cell binding to the stent struts and is associated with a 30% reduction of in-stent restenosis in comparison with BMS.

Inhibition of advanced glycation end product formation and serum protein infiltration in bioprosthetic heart valve leaflets: Investigations of anti-glycation agents and anticalcification interactions with ethanol pretreatment.

Bioprosthetic heart valves (BHV) fabricated from heterograft tissue, such as glutaraldehyde pretreated bovine pericardium (BP), are the most frequently used heart valve replacements. BHV durability is limited by structural valve degeneration (SVD), mechanistically associated with calcification, advanced glycation end products (AGE), and serum protein infiltration. We investigated the hypothesis that anti-AGE agents, Aminoguanidine, Pyridoxamine [PYR], and N-Acetylcysteine could mitigate AGE-serum protein SVD mechanisms in vitro and in vivo, and that these agents could mitigate calcification or demonstrate anti-calcification interactions with BP pretreatment with ethanol. In vitro, each of these agents significantly inhibited AGE-serum protein infiltration in BP. However, in 28-day rat subdermal BP implants only orally administered PYR demonstrated significant inhibition of AGE and serum protein uptake. Furthermore, BP PYR preincubation of BP mitigated AGE-serum protein SVD mechanisms in vitro, and demonstrated mitigation of both AGE-serum protein uptake and reduced calcification in vivo in 28-day rat subdermal BP explants. Inhibition of BP calcification as well as inhibition of AGE-serum protein infiltration was observed in 28-day rat subdermal BP explants pretreated with ethanol followed by PYR preincubation. In conclusion, AGE-serum protein and calcification SVD pathophysiology are significantly mitigated by both PYR oral therapy and PYR and ethanol pretreatment of BP.

Robust Chemical Strategy for Stably Labeling Polyester-Based Nanoparticles with BODIPY Fluorophores.

Aliphatic polyesters are among materials most extensively used for producing biodegradable polymeric nanoparticles currently in development as delivery carriers and imaging agents for a range of biomedical applications. Their clinical translation requires robust particle labeling methodologies that allow reliably monitoring the fate of these formulations in complex biological environments. In the present study, a practical and versatile synthetic strategy providing conjugates of poly(D,L-lactide) representative of this class of polymers with BODIPY fluorophores varying in functional groups and excitation/emission maxima was investigated as a tool for making traceable nanoparticles. Polymer-probe conjugation was accomplished by carbodiimide-induced and 4-(dimethylamino)pyridinium 4-toluenesulfonate-catalyzed esterification of the polymer's terminal hydroxyl group, either directly with a carboxy-functionalized fluorophore or with amine-protected amino acids (Boc-glycine or Boc-6-aminohexanoic acid). In the latter case, the amino acid-derivatized polymeric precursors were reacted with amine-reactive BODIPY dyes after the removal of the protective group. Unlike nanoparticles encapsulating a strongly hydrophobic BODIPY505/515 (logPo/w = 4.3), nanoparticles labeled covalently with its carboxy-functionalized analogue (BODIPY FL) demonstrated stable particle-tracer association under perfect sink conditions. Furthermore, in contrast to the encapsulated dye rapidly partitioning from particles onto cell membranes but not stably retained by cultured cells, the internalization of the covalently attached probe was an irreversible process requiring the presence of serum, consistent with active nanoparticle uptake by endocytosis. In conclusion, the conjugation of particle-forming polymers with BODIPY fluorophores offers an effective and accessible labeling strategy for making traceable polyester-based biodegradable nanoparticles and is expected to facilitate their development and optimization as therapeutic carriers and diagnostic agents.

Stent-Based Gene Delivery for Coronary Disease.

Percutaneous coronary interventions (PCI) are the mainstay for treatment of advanced coronary disease. A majority of PCI involve deployment of a stent in the affected vascular segment. This chapter introduces the concept of using stents as a platform for delivering gene therapies to the vasculature with the overarching aim of mitigating in-stent restenosis (ISR), late stent thrombosis (LST), and neoatherosclerosis (NA), a triad of delayed complications that reduce the overall success rate of PCI. The chapter provides a detailed methodology for coatless reversible attachment of adenoviral (Ad) and adeno-associated viral (AAV) vectors to the metal stent struts along with representative in vitro and in vivo results.

Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1.

In-stent restenosis (ISR) complicates revascularization in the coronary and peripheral arteries. Apolipoprotein A1 (apoA1), the principal protein component of HDL possesses inherent anti-atherosclerotic and anti-restenotic properties. These beneficial traits are lost when wild type apoA1(WT) is subjected to oxidative modifications. We investigated whether local delivery of adeno-associated viral (AAV) vectors expressing oxidation-resistant apoA1(4WF) preserves apoA1 functionality. The efflux of 3H-cholesterol from macrophages to the media conditioned by endogenously produced apoA1(4WF) was 2.1-fold higher than for apoA1(WT) conditioned media in the presence of hypochlorous acid emulating conditions of oxidative stress. The proliferation of apoA1(WT)- and apoA1(4FW)-transduced rat aortic smooth muscle cells (SMC) was inhibited by 66% ± 10% and 65% ± 11%, respectively, in comparison with non-transduced SMC (p < 0.001). Conversely, the proliferation of apoA1(4FW)-transduced, but not apoA1(WT)-transduced rat blood outgrowth endothelial cells (BOEC) was increased 41% ± 5% (p < 0.001). Both apoA1 transduction conditions similarly inhibited basal and TNFα-induced reactive oxygen species in rat aortic endothelial cells (RAEC) and resulted in the reduced rat monocyte attachment to the TNFα-activated endothelium. AAV2-eGFP vectors immobilized reversibly on stainless steel mesh surfaces through the protein G/anti-AAV2 antibody coupling, efficiently transduced cells in culture modeling stent-based delivery. In vivo studies in normal pigs, deploying AAV2 gene delivery stents (GDS) preloaded with AAV2-eGFP in the coronary arteries demonstrated transduction of the stented arteries. However, implantation of GDS formulated with AAV2-apoA1(4WF) failed to prevent in-stent restenosis in the atherosclerotic vasculature of hypercholesterolemic diabetic pigs. It is concluded that stent delivery of AAV2-4WF while feasible, is not effective for mitigation of restenosis in the presence of severe atherosclerotic disease.

Poly-2-methyl-2-oxazoline-modified bioprosthetic heart valve leaflets have enhanced biocompatibility and resist structural degeneration.

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.

Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces.

Spatially and temporally controlled delivery of biologicals, including gene vectors, represents an unmet need for regenerative medicine and gene therapy applications. Here we describe a method of reversible attachment of serotype 2 adeno-associated viral vectors (AAV2) to metal surfaces. This technique enables localized delivery of the vector to the target cell population in vitro and in vivo with the subsequent effective transduction of cells adjacent to the metal substrate. The underlying bioengineering approach employs coordination chemistry between the bisphosphonic groups of polyallylamine bisphosphonates and the metal atoms on the surface of metallic samples. Formation of a stable polybisphosphonate monolayer with plentiful allyl-derived amines allows for further chemical modification to consecutively append thiol-modified protein G, an anti-AAV2 antibody, and AAV2 particles. Herein we present a detailed protocols for the metal substrate modification, for the visualization of the metal surface-immobilized vector using direct and indirect fluorescent AAV2 labeling and scanning electron microscopy, for quantification of the surface-immobilized vector load with RT-PCR, and for the localized vector transduction in vitro and in vivo.

Altered Responsiveness to TGFß and BMP and Increased CD45+ Cell Presence in Mitral Valves Are Unique Features of Ischemic Mitral Regurgitation.

[Figure: see text].

Noncalcific Mechanisms of Bioprosthetic Structural Valve Degeneration.

Bioprosthetic heart valves (BHVs) largely circumvent the need for long-term anticoagulation compared with mechanical valves but are increasingly susceptible to deterioration and reduced durability with reoperation rates of ≈10% and 30% at 10 and 15 years, respectively. Structural valve degeneration is a common, unpreventable, and untreatable consequence of BHV implantation and is frequently characterized by leaflet calcification. However, 25% of BHV reoperations attributed to structural valve degeneration occur with minimal leaflet mineralization. This review discusses the noncalcific mechanisms of BHV structural valve degeneration, highlighting the putative roles and pathophysiological relationships between protein infiltration, glycation, oxidative and mechanical stress, and inflammation and the structural consequences for surgical and transcatheter BHVs.

Model studies of advanced glycation end product modification of heterograft biomaterials: The effects of in vitro glucose, glyoxal, and serum albumin on collagen structure and mechanical properties.

Glutaraldehyde cross-linked heterograft tissues, bovine pericardium (BP) or porcine aortic valves, are the leaflet materials in bioprosthetic heart valves (BHV) used in cardiac surgery for heart valve disease. BHV fail due to structural valve degeneration (SVD), often with calcification. Advanced glycation end products (AGE) are post-translational, non-enzymatic reaction products from sugars reducing proteins. AGE are present in SVD-BHV clinical explants and are not detectable in un-implanted BHV. Prior studies modeled BP-AGE formation in vitro with glyoxal, a glucose breakdown product, and serum albumin. However, glucose is the most abundant AGE precursor. Thus, the present studies investigated the hypothesis that BHV susceptibility to glucose related AGE, together with serum proteins, results in deterioration of collagen structure and mechanical properties. In vitro experiments studied AGE formation in BP and porcine collagen sponges (CS) comparing 14C-glucose and 14C-glyoxal with and without bovine serum albumin (BSA). Glucose incorporation occurred at a significantly lower level than glyoxal (p<0.02). BSA co-incubations demonstrated reduced glyoxal and glucose uptake by both BP and CS. BSA incubation caused a significant increase in BP mass, enhanced by glyoxal co-incubation. Two-photon microscopy of BP showed BSA induced disruption of collagen structure that was more severe with glucose or glyoxal co-incubation. Uniaxial testing of CS demonstrated that glucose or glyoxal together with BSA compared to controls, caused accelerated deterioration of viscoelastic relaxation, and increased stiffness over a 28-day time course. In conclusion, glucose, glyoxal and BSA uniquely contribute to AGE-mediated disruption of heterograft collagen structure and deterioration of mechanical properties.

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization.

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 like 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.

Experimental Single-Platform Approach to Enhance the Functionalization of Magnetically Targetable Cells.

Magnetic guidance shows promise as a strategy for improving the delivery and performance of cell therapeutics. However, clinical translation of magnetically guided cell therapy requires cell functionalization protocols that provide adequate magnetic properties in balance with unaltered cell viability and biological function. Existing methodologies for characterizing cells functionalized with magnetic nanoparticles (MNP) produce aggregate results, both distorted and unable to reflect variability in either magnetic or biological properties within a preparation. In the present study, we developed an inverted-plate assay allowing determination of these characteristics using a single-platform approach, and applied this method for a comparative analysis of two loading protocols providing highly uniform vs. uneven MNP distribution across cells. MNP uptake patterns remarkably different between the two protocols were first shown by fluorimetry carried out in a well-scan mode on endothelial cells (EC) loaded with BODIPY558/568-labeled MNP. Using the inverted-plate assay we next demonstrated that, in stark contrast to unevenly loaded cells, more than 50% of uniformly functionalized EC were captured within 5 min over a broad range of MNP doses. Furthermore, magnetically captured cells exhibited unaltered viability, substrate attachment, and proliferation rates. Conducted in parallel, magnetophoretic mobility studies corroborated the markedly superior guidance capacity of uniformly functionalized cells, confirming substantially faster cell capture kinetics on a clinically relevant time scale. Taken together, these results emphasize the importance of optimizing cell preparation protocols with regard to loading uniformity as key to efficient site-specific delivery, engraftment, and expansion of the functionalized cells, essential for both improving performance and facilitating translation of targeted cell therapeutics.