Novel Payloads to Mitigate Maladaptive Inward Arterial Remodeling in Drug-Coated Balloon Therapy.

Drug-coated balloon therapy is a minimally invasive endovascular approach to treat obstructive arterial disease, with increasing utilization in the peripheral circulation due to improved outcomes as compared to alternative interventional modalities. Broader clinical use of drug-coated balloons is limited by an incomplete understanding of device- and patient-specific determinants of treatment efficacy, including late outcomes that are mediated by postinterventional maladaptive inward arterial remodeling. To address this knowledge gap, we propose a predictive mathematical model of pressure-mediated femoral artery remodeling following drug-coated balloon deployment, with account of drug-based modulation of resident vascular cell phenotype and common patient comorbidities, namely, hypertension and endothelial cell dysfunction. Our results elucidate how postinterventional arterial remodeling outcomes are altered by the delivery of a traditional anti-proliferative drug, as well as by codelivery with an anti-contractile drug. Our findings suggest that codelivery of anti-proliferative and anti-contractile drugs could improve patient outcomes following drug-coated balloon therapy, motivating further consideration of novel payloads in next-generation devices.

Reduced Smooth Muscle Contractile Capacity Facilitates Maladaptive Arterial Remodeling.

Albeit seldom considered explicitly, the vasoactive state of a central artery can contribute to luminal control and thereby affect the in vivo values of flow-induced wall shear stress and pressure-induced intramural stress, which in turn are strong determinants of wall growth and remodeling. Here, we test the hypothesis that diminished vasoactive capacity compromises effective mechano-adaptations of central arteries. Toward this end, we use consistent methods to re-interpret published data on common carotid artery remodeling in a nonpharmacologic mouse model of induced hypertension and a model of connective tissue disorder that results in Marfan syndrome. The mice have identical genetic backgrounds and, in both cases, the data are consistent with the hypothesis considered. In particular, carotid arteries with strong (normal) vasoactive capacity tend to maintain wall thickness and in vivo axial stretch closer to homeostatic, thus resulting in passive circumferential wall stress and energy storage close to normal. We conclude that effective vasoactivity helps to control the biomechanical state in which the cells and matrix turnover, thus helping to delineate mechano-adaptive from maladaptive remodeling. Future analyses of experimental data and computational models of growth and remodeling should account for this strong coupling between smooth muscle contractile capacity and central arterial remodeling.

Diet alters age-related remodeling of aortic collagen in mice susceptible to atherosclerosis.

Vascular cells restructure extracellular matrix in response to aging or changes in mechanical loading. Here, we characterized collagen architecture during age-related aortic remodeling in atherosclerosis-prone mice. We hypothesized that changes in collagen fiber orientation reflect an altered balance between passive and active forces acting on the arterial wall. We examined two factors that can alter this balance, endothelial dysfunction and reduced smooth muscle cell (SMC) contractility. Collagen fiber organization was visualized by second-harmonic generation microscopy in aortic adventitia of apolipoprotein E (apoE) knockout (KO) mice at 6 wk and 6 mo of age on a chow diet and at 7.5 mo of age on a Western diet (WD), using image analysis to yield mean fiber orientation. Adventitial collagen fibers became significantly more longitudinally oriented with aging in apoE knockout mice on chow diet. Conversely, fibers became more circumferentially oriented with aging in mice on WD. Total collagen content increased significantly with age in mice fed WD. We compared expression of endothelial nitric oxide synthase and acetylcholine-mediated nitric oxide release but found no evidence of endothelial dysfunction in older mice. Time-averaged volumetric blood flow in all groups showed no significant changes. Wire myography of aortic rings revealed decreases in active stress generation with age that were significantly exacerbated in WD mice. We conclude that the aorta displays a distinct remodeling response to atherogenic stimuli, indicated by altered collagen organization. Collagen reorganization can occur in the absence of altered hemodynamics and may represent an adaptive response to reduced active stress generation by vascular SMCs.NEW & NOTEWORTHY The following major observations were made in this study: 1) aortic adventitial collagen fibers become more longitudinally oriented with aging in apolipoprotein E knockout mice fed a chow diet; 2) conversely, adventitial collagen fibers become more circumferentially oriented with aging in apoE knockout mice fed a high-fat diet; 3) adventitial collagen content increases significantly with age in mice on a high-fat diet; 4) these alterations in collagen organization occur largely in the absence of hemodynamic changes; and 5) circumferential reorientation of collagen is associated with decreased active force generation (contractility) in aged mice on a high-fat diet.

Brief communication: Maximum ingested bite size in captive western lowland gorillas (Gorilla gorilla gorilla).

OBJECTIVES: Previously, we found that maximum ingested bite size (Vb ), the largest piece of food an animal can consume without biting it into smaller pieces first, isometrically scales relative to body size in strepsirrhines and with negative allometry in anthropoids. In the current study, we rectify the omission of great apes from the earlier sample to now characterize the Vb of the entire size-range of the order. MATERIALS AND METHODS: Five gorillas (Gorilla gorilla gorilla-G. g. gorilla) were studied to ascertain Vb in relation to the mechanical properties of five foods. RESULTS: Gorilla Vb ranged from 166.38 cm3 (for the least obdurate food: watermelon) to 8 cm3 (for the most obdurate food: turnip), with an average Vb of 33.50 cm3 across all food types. CONCLUSIONS: When these data were compared to those from our previous studies, we found that gorillas consumed relatively slightly smaller volumes of food compared to the trend found across primates. However, because the more frugivorous gorillas consumed relatively larger pieces of food than the large folivorous monkeys previously studied, including the gorilla data increased the slope of the linear regression between body mass and Vb in anthropoids. Thus, the addition of the largest living primate brings the anthropoid Vb trend closer to the Vb trend of the order. Notwithstanding, there is still negative allometry in anthropoid Vb , in contrast with the isometry in strepsirrhine Vb . Future research should include species with body masses between the smaller anthropoids and gorillas by studying the Vb of large papionids and the other great apes.

Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10-70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.

Removing vessel constriction on the embryonic heart results in changes in valve gene expression, morphology, and hemodynamics.

BACKGROUND: The formation of healthy heart valves throughout embryonic development is dependent on both genetic and epigenetic factors. Hemodynamic stimuli are important epigenetic regulators of valvulogenesis, but the resultant molecular pathways that control valve development are poorly understood. Here we describe how the heart and valves recover from the removal of a partial constriction (banding) of the OFT/ventricle junction (OVJ) that temporarily alters blood flow velocity through the embryonic chicken heart (HH stage 16/17). Recovery is described in terms of 24- and 48-hr gene expression, morphology, and OVJ hemodynamics. RESULTS: Collectively, these studies show that after 24 hr of recovery, important epithelial-mesenchymal transformation (EMT) genes TGFßRIII and Cadherin 11 (CDH11) transcript levels normalize return to control levels, in contrast to Periostin and TGFß,3 which remain altered. In addition, after 48 hr of recovery, TGFß3 and CDH11 transcript levels remain normalized, whereas TGFßRIII and Periostin are down-regulated. Analyses of OFT cushion volumes in the hearts show significant changes, as does the ratio of cushion to cell volume at 24 hr post band removal (PBR). Morphologically, the hearts show visible alteration following band removal when compared to their control age-matched counterparts. CONCLUSIONS: Although some aspects of the genetic/cellular profiles affected by altered hemodynamics seem to be reversed, not all gene expression and cardiac growth normalize following 48 hr of band removal. Developmental Dynamics 247:531-541, 2018. © 2017 Wiley Periodicals, Inc.

Perfusion Tissue Culture Initiates Differential Remodeling of Internal Thoracic Arteries, Radial Arteries, and Saphenous Veins.

Adaptive remodeling processes are essential to the maintenance and viability of coronary artery bypass grafts where clinical outcomes depend strongly on the tissue source. In this investigation, we utilized an ex vivo perfusion bioreactor to culture porcine analogs of common human bypass grafts: the internal thoracic artery (ITA), the radial artery (RA), and the great saphenous vein (GSV), and then evaluated samples acutely (6 h) and chronically (7 days) under in situ or coronary-like perfusion conditions. Although morphologically similar, primary cells harvested from the ITA illustrated lower intimal and medial, but not adventitial, cell proliferation rates than those from the RA or GSV. Basal gene expression levels were similar in all vessels, with only COL3A1, SERPINE1, FN1, and TGFB1 being differentially expressed prior to culture; however, over half of all genes were affected nominally by the culturing process. When exposed to coronary-like conditions, RAs and GSVs experienced pathological remodeling not present in ITAs or when vessels were studied in situ. Many of the remodeling genes perturbed at 6 h were restored after 7 days (COL3A1, FN1, MMP2, and TIMP1) while others (SERPINE1, TGFB1, and VCAM1) were not. The findings elucidate the potential mechanisms of graft failure and highlight strategies to encourage healthy ex vivo pregraft conditioning.

Contractile Smooth Muscle and Active Stress Generation in Porcine Common Carotids.

The mechanical response of intact blood vessels to applied loads can be delineated into passive and active components using an isometric decomposition approach. Whereas the passive response is due predominantly to the extracellular matrix (ECM) proteins and amorphous ground substance, the active response depends on the presence of smooth muscle cells (SMCs) and the contractile machinery activated within those cells. To better understand determinants of active stress generation within the vascular wall, we subjected porcine common carotid arteries (CCAs) to biaxial inflation-extension testing under maximally contracted or passive SMC conditions and semiquantitatively measured two known markers of the contractile SMC phenotype: smoothelin and smooth muscle-myosin heavy chain (SM-MHC). Using isometric decomposition and established constitutive models, an intuitive but novel correlation between the magnitude of active stress generation and the relative abundance of smoothelin and SM-MHC emerged. Our results reiterate the importance of stretch-dependent active stress generation to the total mechanical response. Overall these findings can be used to decouple the mechanical contribution of SMCs from the ECM and is therefore a powerful tool in the analysis of disease states and potential therapies where both constituent are altered.

Therapeutic Engineered Hydrogel Coatings Attenuate the Foreign Body Response in Submuscular Implants.

BACKGROUND: Biomedical devices are implanted into mammalian soft tissues to improve, monitor, or restore form or function. The utility of these implants is limited by the subsequent foreign body response (FBR), beginning with inflammation and terminating in a collagen envelope around the device, known as the capsule. This capsule then can contract and distort the shape of the device or limit its effectiveness in interacting with the surrounding host tissues. In the current study, we investigated the effect of therapeutic collagen-coated silicone discs in a rat model of the FBR. METHODS: A 3-dimensional printed mold was used to fabricate collagen-coated silicone discs incorporating 3 therapeutic agents: colchicine, a function-blocking antibody against interleukin 8 (IL-8) receptor B, and a powerful anti-inflammatory steroid, dexamethasone. Discs were implanted submuscularly into a well-characterized rat model of the FBR and evaluated for inflammatory response, fibrotic development, and cytokine release. RESULTS: Coated silicone discs exhibited reduced collagen deposition and little to no foreign body giant cells at the host-silicone interface when compared with the silicone-only group. Therapeutic hydrogels demonstrate a significant decrease in cellular infiltration into the coatings over the 2-week time point in contrast to therapeutic-free hydrogel coatings. Cytokine analysis revealed significant differences between therapeutic-free and therapeutic-containing coatings when compared with silicone-only controls. Levels of IL-1ß, IL-6, monocyte chemotactic protein 1, and macrophage inflammatory protein 3α were affected 48 hours after implantation, while differences in IL-18, growth-regulated oncogene/keratinocyte chemoattractant, and macrophage inflammatory protein 3α were observed 1 week after implantation. CONCLUSIONS: By utilizing the host's innate immune response, our engineered hydrogel coatings delivered therapeutic moieties directly to the implant microenvironment, thus delaying the FBR up to 2 weeks.

Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor.

UNLABELLED: Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from <3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14(+) monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus. IMPORTANCE: Outbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a critical role in EVA pathogenesis. To this point, however, the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of the cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV-susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

Design and Fabrication of a Three-Dimensional In Vitro System for Modeling Vascular Stenosis.

Vascular stenosis, the abnormal narrowing of blood vessels, arises from defective developmental processes or atherosclerosis-related adult pathologies. Stenosis triggers a series of adaptive cellular responses that induces adverse remodeling, which can progress to partial or complete vessel occlusion with numerous fatal outcomes. Despite its severity, the cellular interactions and biophysical cues that regulate this pathological progression are poorly understood. Here, we report the design and fabrication of a three-dimensional (3D) in vitro system to model vascular stenosis so that specific cellular interactions and responses to hemodynamic stimuli can be investigated. Tubular cellularized constructs (cytotubes) were produced, using a collagen casting system, to generate a stenotic arterial model. Fabrication methods were developed to create cytotubes containing co-cultured vascular cells, where cell viability, distribution, morphology, and contraction were examined. Fibroblasts, bone marrow primary cells, smooth muscle cells (SMCs), and endothelial cells (ECs) remained viable during culture and developed location- and time-dependent morphologies. We found cytotube contraction to depend on cellular composition, where SMC-EC co-cultures adopted intermediate contractile phenotypes between SMC- and EC-only cytotubes. Our fabrication approach and the resulting artery model can serve as an in vitro 3D culture system to investigate vascular pathogenesis and promote the tissue engineering field.

Comparison of Aortic Collagen Fiber Angle Distribution in Mouse Models of Atherosclerosis Using Second-Harmonic Generation (SHG) Microscopy.

Characterization of collagen fiber angle distribution throughout the blood vessel wall provides insight into the mechanical behavior of healthy and diseased arteries and their capacity to remodel. Atherosclerotic plaque contributes to the overall mechanical behavior, yet little is known experimentally about how collagen fiber orientation is influenced by atherogenesis. We hypothesized that atherosclerotic lesion development, and the factors contributing to lesion development, leads to a shift in collagen fiber angles within the aorta. Second-harmonic generation microscopy was used to visualize the three-dimensional organization of collagen throughout the aortic wall and to examine structural differences in mice maintained on high-fat Western diet versus age-matched chow diet mice in a model of atherosclerosis. Image analysis was performed on thoracic and abdominal sections of the aorta from each mouse to determine fiber orientation, with the circumferential (0°) and blood flow directions (axial ±90°) as the two reference points. All measurements were used in a multiple regression analysis to determine the factors having a significant influence on mean collagen fiber angle. We found that mean absolute angle of collagen fibers is 43° lower in Western diet mice compared with chow diet mice. Mice on a chow diet have a mean collagen fiber angle of ±63°, whereas mice on a Western diet have a more circumferential fiber orientation (~20°). This apparent shift in absolute angle coincides with the development of extensive aortic atherosclerosis, suggesting that atherosclerotic factors contribute to collagen fiber angle orientation.

Impact of cryopreservation on elastomuscular artery mechanics.

Low temperatures slow or halt undesired biological and chemical processes, protecting cells, tissues, and organs during storage. Cryopreservation techniques, including controlled media exchange and regulated freezing conditions, aim to mitigate the physical consequences of freezing. Dimethyl sulfoxide (DMSO), for example, is a penetrating cryoprotecting agent (CPA) that minimizes ice crystal growth by replacing intracellular water, while polyvinyl alcohol (PVA) is a nonpenetrating CPA that prevents recrystallization during thawing. Since proteins and ground substance dominate the passive properties of soft biological tissues, we studied how different freezing rates, storage temperatures, storage durations, and the presence of cryoprotecting agents (5% [v/v] DMSO + 1 mg/mL PVA) impact the histomechanical properties of the internal thoracic artery (ITA), a clinically relevant blood vessel with both elastic and muscular characteristics. Remarkably, biaxial mechanical analyses failed to reveal significant differences among the ten groups tested, suggesting that mechanical properties are virtually independent of the cryopreservation technique. Scanning electron microscopy revealed minor CPA-independent delamination in rapidly frozen samples, while cryoprotected ITAs had better post-thaw viability than their unprotected counterparts using methyl thiazole-tetrazolium (MTT) metabolic assays, especially when frozen at a controlled rate. These results can be used to inform ongoing and future studies in vascular engineering, physiology, and mechanics.

Hydrophilic Coating Microstructure Mediates Acute Drug Transfer in Drug-Coated Balloon Therapy.

Drug-coated balloon (DCB) therapy is a promising endovascular treatment for obstructive arterial disease. The goal of DCB therapy is restoration of lumen patency in a stenotic vessel, whereby balloon deployment both mechanically compresses the offending lesion and locally delivers an antiproliferative drug, most commonly paclitaxel (PTX) or derivative compounds, to the arterial wall. Favorable long-term outcomes of DCB therapy thus require predictable and adequate PTX delivery, a process facilitated by coating excipients that promotes rapid drug transfer during the inflation period. While a variety of excipients have been considered in DCB design, there is a lack of understanding about the coating-specific biophysical determinants of essential device function, namely, acute drug transfer. We consider two hydrophilic excipients for PTX delivery, urea (UR) and poly(ethylene glycol) (PEG), and examine how compositional and preparational variables in the balloon surface spray-coating process impact resultant coating microstructure and in turn acute PTX transfer to the arterial wall. Specifically, we use scanning electron image analyses to quantify how coating microstructure is altered by excipient solid content and balloon-to-nozzle spray distance during the coating procedure and correlate obtained microstructural descriptors of coating aggregation to the efficiency of acute PTX transfer in a one-dimensional ex vivo model of DCB deployment. Experimental results suggest that despite the qualitatively different coating surface microstructures and apparent PTX transfer mechanisms exhibited with these excipients, the drug delivery efficiency is generally enhanced by coating aggregation on the balloon surface. We illustrate this microstructure-function relation with a finite element-based computational model of DCB deployment, which along with our experimental findings suggests a general design principle to increase drug delivery efficiency across a broad range of DCB designs.

Acute Mechanical Consequences of Vessel-Specific Coronary Bypass Combinations.

PURPOSE: Premature coronary artery bypass graft (CABG) failure has been linked to geometric, mechanical, and compositional discrepancies between host and graft tissues. Acute hemodynamic disturbances and the introduction of wall stress gradients trigger a myriad of mechanobiological processes at the anastomosis that can be associated with restenosis and graft failure. Although the origins of coronary artery disease dictate the anastomotic target, an opportunity exists for graft-vessel optimization through rationale graft selection. METHODS: Here we explored the four distinct regions of the left (L) and right (R) ITA (1 = proximal, 2 = submuscular, 3 = middle, 4 = distal), and four common target vessels in the coronary circulation including the proximal and distal left anterior descending (PLAD & DLAD), right coronary (RCA), and left circumflex (LCX) arteries. Benchtop biaxial mechanical data was used to acquire constitutive model parameters of these tissues and enable vessel-specific computational models to elucidate the mechanical consequences of 32 unique graft-target combinations. RESULTS: Simulations revealed the maximum principal wall stresses for the PLAD, RCA, and LCX occurred when anastomosed with LITA1, and the maximum flow-induced shear stress occurred with LITA4. The DLAD, on the other hand, reached stress maximums when anastomosed to LITA4. Using a normalized objective function of simulation output variables, we found LITA2 to be the best graft choice for both LADs, RITA3 for the RCA, and LITA3 for the LCX. CONCLUSION: Although mechanical compatibility is just one of many factors determining bypass graft outcomes, our data suggests improvements can be made to the grafting process through vessel-specific regional optimization.

Full-field strain mapping of healthy and pathological mouse aortas using stereo digital image correlation.

The murine aorta is a complex, heterogeneous structure that undergoes large and sometimes asymmetrical deformations under loading. For analytical convenience, mechanical behavior is predominantly described using global quantities that fail to capture critical local information essential to elucidating aortopathic processes. Here, in our methodological study, we used stereo digital image correlation (StereoDIC) to measure the strain profiles of speckle-patterned healthy and elastase-infused, pathological mouse aortas submerged in a temperature-controlled liquid medium. Our unique device rotates two 15-degree stereo-angle cameras that gather sequential digital images while simultaneously performing conventional biaxial pressure-diameter and force-length testing. A StereoDIC Variable Ray Origin (VRO) camera system model is employed to correct for high-magnification image refraction through hydrating physiological media. The resultant Green-Lagrange surface strain tensor was quantified at different blood vessel inflation pressures, axial extension ratios, and after aneurysm-initiating elastase exposure. Quantified results capture large, heterogeneous, inflation-related, circumferential strains that are drastically reduced in elastase-infused tissues. Shear strains, however, were very small on the tissue's surface. Spatially averaged StereoDIC-based strains were generally more detailed than those determined using conventional edge detection techniques.

Small-diameter artery decellularization: Effects of anionic detergent concentration and treatment duration on porcine internal thoracic arteries.

Engineered replacement materials have tremendous potential for vascular applications where over 400,000 damaged and diseased blood vessels are replaced annually in the United States alone. Unlike large diameter blood vessels, which are effectively replaced by synthetic materials, prosthetic small-diameter vessels are prone to early failure, restenosis, and reintervention surgery. We investigated the differential response of varying 0%-6% sodium dodecyl sulfate and sodium deoxycholate anionic detergent concentrations after 24 and 72 h in the presence of DNase using biochemical, histological, and biaxial mechanical analyses to optimize the decellularization process for xenogeneic vascular tissue sources, specifically the porcine internal thoracic artery (ITA). Detergent concentrations greater than 1% were successful at removing cytoplasmic and cell surface proteins but not DNA content after 24 h. A progressive increase in porosity and decrease in glycosaminoglycan (GAG) content was observed with detergent concentration. Augmented porosity was likely due to the removal of both cells and GAGs and could influence recellularization strategies. The treatment duration on the other hand, significantly improved decellularization by reducing DNA content to trace amounts after 72 h. Prolonged treatment times reduced laminin content and influenced the vessel's mechanical behavior in terms of altered circumferential stress and stretch while further increasing porosity. Collectively, DNase with 1% detergent for 72 h provided an effective and efficient decellularization strategy to be employed in the preparation of porcine ITAs as bypass graft scaffolding materials with minor biomechanical and histological penalties.

Development of a TaqMan® Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion's reproductive tract. Two alleles (CXCL16 S and CXCL16 r ) were identified in the equine population and correlated with the susceptibility or resistance of a CD3+ T cell subpopulation in peripheral blood to in vitro EAV infection, respectively. Interestingly, CXCL16 S has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on CXCL16 S/r would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan® allelic discrimination qPCR assay for the genotyping of the equine CXCL16 gene based on the identification of a single nucleotide polymorphism in position 1,073 based on NCBI gene ID: 100061442 (or position 527 based on Ensembl: ENSECAG00000018406.2) located in exon 2. One hundred and sixty horses from four breeds were screened for the CD3+ T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine CXCL16 allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3+ T cell phenotype were homozygous for CXCL16 r while horses with the susceptible CD3+ T cell phenotype carried at least one CXCL16 S allele or homozygous for CXCL16 S . In addition, genotypification with the TaqMan® allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan® allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the CXCL16 genotype. It is highly recommended that colts that carry the susceptible genotype (CXCL16  S/S or CXCL16 S/r ) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.

Longitudinal histomechanical heterogeneity of the internal thoracic artery.

The internal thoracic artery (ITA) is the principal choice for coronary artery bypass grafting (CABG) due to its mechanical compatibility, histological composition, anti-thrombogenic lumen, and single anastomotic junction. Originating at the subclavian artery, traversing the thoracic cavity, and terminating at the superior epigastric and musculophrenic bifurcation, bilateral ITAs follow a protracted circuitous pathway. The physiological hemodynamics, anatomical configuration, and perivascular changes that occur throughout this length influence the tissue's microstructure and gross mechanical properties. Since histomechanics play a major role in premature graft failure we used inflation-extension testing to quantify the regional material and biaxial mechanical properties at four distinct locations along the left (L) and right (R) ITA and fit the results to a structurally-motivated constitutive model. Our comparative analysis of 44 vessel segments revealed a significant increase in the amount of collagen but not smooth muscle and a significant decrease in elastin and elastic lamellae present with distance from the heart. A subsequent decrease in the total deformation energy and isotropic contribution to the strain energy was present in the LITA but not RITA. Circumferential stress and compliance generally decreased along the length of the LITA while axial stress increased in the RITA. When comparing RITAs to LITAs, some morphological and histological differences were found in proximal sections while distal sections revealed differences predominantly in compliance and axial stress. Overall, this information can be used to better guide graft selection, graft preparation, and xenograft-based tissue-engineering strategies for CABG.

Mechanics of ascending aortas from TGFß-1, -2, -3 haploinsufficient mice and elastase-induced aortopathy.

Transforming growth factor-beta (TGFß-1, -2, -3) ligands act through a common receptor complex yet each is expressed in a unique and overlapping fashion throughout development. TGFß plays a role in extra-cellular matrix composition with mutations to genes encoding TGFß and TGFß signaling molecules contributing to diverse and deadly thoracic aortopathies common in Loeys-Dietz syndrome (LDS). In this investigation, we studied the TGFß ligand-specific mechanical phenotype of ascending thoracic aortas (ATA) taken from 4-to-6 months-old Tgfb1+/-, Tgfb2+/-, and Tgfb3+/- mice, their wild-type (WT) controls, and an elastase infusion model representative of severe elastolysis. Heterozygous mice were studied at an age without dilation to elucidate potential pre-aortopathic mechanical cues. Our findings indicate that ATAs from Tgfb2+/- mice demonstrated significant wall thickening, a corresponding decrease in biaxial stress, decreased biaxial stiffness, and a decrease in stored energy. These results were unlike the pathological elastase model where decreases in biaxial stretch were found along with increases in diameter, biaxial stress, and biaxial stiffness. ATAs from Tgfb1+/- and Tgfb3+/-, on the other hand, had few mechanical differences when compared to wild-type controls. Although aortopathy generally occurs later in development, our findings reveal that in 4-to-6 month-old animals, only Tgfb2+/- mice demonstrate a significant phenotype that fails to model ubiquitous elastolysis.