Biology subjects

Levy, R. J.

Publications and source records attributed to Levy, R. J..

6 recordsLinked to original sources

bioRxiv2023

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

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.

bioengineering↗

bioRxiv2022

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[1]. 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 proximal to 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.

bioengineering↗

bioRxiv2020

Deterioration of Glutaraldehyde Crosslinked Heterograft Biomaterials due to Advanced Glycation End Product Formation and Serum Albumin Infiltration

Bioprosthetic heart valves (BHV) are fabricated from glutaraldehyde cross-linked heterograft tissue, such as bovine pericardium (BP) or porcine aortic valves. BHV develop structural valve degeneration (SVD), often with calcification, requiring BHV replacement. Advanced glycation end products (AGE) are post-translational, non-enzymatic carbohydrate protein modifications. AGE are present in SVD-BHV clinical explants and not detectable in unimplanted BHV. Here, we studied the hypothesis that BHV susceptibility to AGE formation and serum protein infiltration results in deterioration of both leaflet collagen structure and mechanical properties. In vitro experiments studied BP and porcine collagen sponges (CS) for susceptibility to AGE formation using 14C-glucose and 14C-glyoxal with and without bovine serum albumin (BSA), as a model serum protein. The results showed AGE formation is a rapid and progressive process. BSA co-incubations reduced glyoxal and glucose uptake by BP and CS. Incubating BP in BSA caused a substantial increase in BP mass, enhanced by glyoxal co-incubation. Per two-photon microscopy, BP with AGE formation and BSA infiltration each induced significant disruption in collagen microarchitecture, with loss of collagen alignment and crimp. These effects are cumulative with the greatest disruption occurring when there was both AGE formation and BSA infiltration. Uniaxial testing of CS demonstrated that AGE formation, together with BSA uptake compared to controls, caused a significant deterioration in mechanical properties with a loss of viscoelastic relaxation and increased stiffness. It is concluded that AGE-BSA associated collagen structural disruption and deterioration of mechanical properties contribute to SVD.

biophysics↗

bioRxiv2020

Inhibition and down regulation of the serotonin transporter contribute to the progression of degenerative mitral regurgitation

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 TGF{beta}-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 (p<0.001). Aspirin use by MR patients was associated with surgery at an older age (p=0.0055). Quantitative reverse transcriptase PCR of MR leaflet RNA from 44 patients, and 20 normal mitral leaflets from heart transplant recipients, demonstrated down regulation in MR of both SLC6A4 and vesicular monoamine transporter-2 (SLC18A2), that packages 5HT (p<0.001). Human mitral valve interstitial cells cultivated with Fluoxetine, a SSRI, demonstrated down regulation of SLC6A4 and upregulation of HTR2B, compared to untreated, in cells from both normal and MR leaflets. Platelet 5HT studies in healthy subjects without heart disease used ADP-induced activation to model MR-associated activation. Fluoxetine significantly increased platelet activation and plasma 5HT levels, while Aspirin inhibited ADP platelet activation. ConclusionsDown 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.

physiology↗

bioRxiv2020

Glycation and Serum Albumin Infiltration Contribute to the Structural Degeneration of Bioprosthetic Heart Valves

BackgroundBioprosthetic heart valves (BHV) are widely used to treat heart valve disease but are fundamentally limited by structural valve degeneration (SVD). Non-calcific mechanisms of SVD entirely account for approximately 30% of SVD cases and contribute to calcific SVD but remain understudied. Glycation mechanisms have not been previously associated with SVD, despite being established as degenerative in collagenous native tissues. ObjectivesTo determine whether blood component infiltration-based glycation and concomitant human serum albumin (HSA) deposition contribute mechanistically to SVD. MethodsImmunohistochemistry (IHC) was used to identify advanced glycation end-products (AGEs) and serum albumin accumulation in 45 aortic valve BHV explanted due to SVD, glutaraldehyde-treated bovine pericardium (BP) incubated in vitro in glyoxal and HSA, and rat subcutaneous BP implants. Structural impacts of glycation-related mechanisms were evaluated by second harmonic generation (SHG) collagen imaging. Hydrodynamic effects of valve glycation and concomitant HSA exposure were studied with an ISO-5840-compliant pulse duplicator system using surgical grade BHV. ResultsAll 45 clinical explants and in vitro-incubated BP demonstrated accumulated AGE and HSA compared to un-implanted, un-exposed BHV. SHG revealed instigation of collagen malalignment similar to that in SVD explants by glycation and HSA infiltration. Rat subdermal explants also showed AGE and serum albumin accumulation. Pulse duplication demonstrated significantly reduced orifice area and increased pressure gradient and peak fluid velocity following glyoxal and HSA incubations. ConclusionsGlycation and concomitant HSA infiltration occur in clinical BHV and contribute to structural and functional degeneration of leaflet tissue, thus representing novel, interacting mechanisms of BHV SVD.

bioengineering↗

bioRxiv2020

Novel software for automated morphometric analysis of stented arteries

Morphometric analysis of explanted arteries remains the gold standard in assessing pathological mechanisms of arterial disease, and the efficacy of therapeutic interventions. Software currently used for morphometric analysis (ImageJ, ImagePro, etc.) requires manual tracing of each region of interest by the user to obtain direct and derived data needed for analysis. Manual segmentation of borders between differently stained arterial compartments is time-consuming and prone to bias, even when the operator is blind to the treatment type. Here, we report newly designed morphometric software, floodSeg, which greatly aids analysis through semiautomated segmentation, followed by automated computation and data entry. The program includes tools for both rapid component selection, and precision user-controlled contour correction. In practice, segmentation occurs following the selection of seed points and corresponding threshold values, for each desired component. The flood fill algorithm is used to map out components, followed by Sklanskys convex-hull algorithm to obtain the outer contours. If necessary, convexity defects can be overcome through manual point placement on top of existing points, and regeneration of the contour. floodSeg was tested using a set of non-uniformly stained stented rat arteries, and compared against manually obtained results. The accuracy of the resulting measurements was within the expected limit based on repeated manual measurements by the same operator, and did not exceed 3%. Most notably, the duration for data acquisition using floodSeg was less than 20% of the time required for manual measurements by an experienced operator. Thus, our contribution is an improvement on widely used software, with significant potential for application in a multitude of areas of pathology practice.

bioinformatics↗