Porcine Aortic Valve Leaflets Research Articles

BFGF attenuates aortic valvular interstitial cell calcification by inhibiting endoplasmic reticulum stress-mediated apoptosis

The potential protective effect of basic fibroblast growth factor (BFGF) on the cardiovascular system has been proposed previously, however, its effect on calcific aortic valve disease (CAVD) and underlying mechanisms have not been elucidated. The valvular interstitial cell (VIC) were isolated from porcine aortic valve leaflets. To investigate the effect of BFGF on osteogenic differentiation of VIC, the osteogenic induced medium (OIM) and BFGF were added. The protein expression level was detected by Western blot, and apoptosis was determined by flow cytometry. The effect of BFGF on CAVD process in vivo was assessed by a rat CAVD model, which was identified by echocardiography and Alizarin red staining. The expression level of BFGF in the aortic valve and serum were significantly upregulated in CAVD patients compared to control group. In addition, exogenous BFGF injection attenuates CAVD process in vivo. The protein markers of osteogenic differentiation, endoplasmic reticulum stress (ERS), and apoptosis were significantly upregulated by culture with OIM. On the contrary, the aforementioned proteins were suppressed after adding 100 ng/mL of BFGF. Inhibition of PI3K/Akt and ERK1/2 pathways by specific inhibitors abolished the protective effect of BFGF. In conclusion, BFGF could alleviate the VIC calcification by inhibiting ERS-mediated apoptosis, which is partly regulated by activation of the PI3K/Akt and ERK1/2 signaling pathways. BFGF may provide a potential avenue for CAVD therapy.

Ultrastructural and Immunohistochemical Detection of Hydroxyapatite Nucleating Role by rRNA and Nuclear Chromatin Derivatives in Aortic Valve Calcification: In Vitro and In Vivo Pro-Calcific Animal Models and Actual Calcific Disease in Humans.

Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death.

Prevention by the CXCR2 antagonist SCH527123 of the calcification of porcine heart valve cusps implanted subcutaneously in rats.

Calcification is a main cause of bioprosthetic heart valves failure. It may be promoted by the inflammation developed in the glutaraldehyde (GA)-fixed cusps of the bioprosthesis. We tested the hypothesis that antagonizing the C-X-C chemokines receptor 2 (CXCR2) may prevent the calcification of GA-fixed porcine aortic valves. Four-week-old Sprague Dawley males were transplanted with 2 aortic valve cusps isolated from independent pigs and implanted into the dorsal wall. Four groups of 6 rats were compared: rats transplanted with GA-free or GA-fixed cusps and rats transplanted with GA-fixed cusps and treated with 1 mg/kg/day SCH5217123 (a CXCR2 antagonist) intraperitoneally (IP) or subcutaneously (SC) around the xenograft, for 14 days. Then, rats underwent blood count before xenografts have been explanted for histology and biochemistry analyses. A strong calcification of the xenografts was induced by GA pre-incubation. However, we observed a significant decrease in this effect in rats treated with SCH527123 IP or SC. Implantation of GA-fixed cusps was associated with a significant increase in the white blood cell count, an effect that was significantly prevented by SCH527123. In addition, the expression of the CD3, CD68 and CXCR2 markers was reduced in the GA-fixed cusps explanted from rats treated with SCH527123 as compared to those explanted from non-treated rats. The calcification of GA-fixed porcine aortic valve cusps implanted subcutaneously in rats was significantly prevented by antagonizing CXCR2 with SCH527123. This effect may partly result from an inhibition of the GA-induced infiltration of T-cells and macrophages into the xenograft.

Strain energy density as a Gaussian process and its utilization in stochastic finite element analysis: Application to planar soft tissues

Data-based approaches are promising alternatives to the traditional analytical constitutive models for solid mechanics. Herein, we propose a Gaussian process (GP) based constitutive modeling framework, focusing on planar, hyperelastic and incompressible soft tissues. Specifically, the strain energy density of soft tissues is modeled as a GP, which can be regressed to experimental stress–strain data obtained from biaxial stretching experiments. Moreover, the GP model can be weakly constrained to be convex. A key advantage of a GP-based model is that, in addition to the mean value, it provides a probability density (i.e. associated uncertainty) for the strain energy density. To simulate the effect of this uncertainty, a non-intrusive stochastic finite element analysis (SFEA) framework is proposed. The proposed framework is verified against an artificial dataset based on the Gasser–Ogden–Holzapfel model and applied to a real experimental dataset of a porcine aortic valve leaflet tissue. The results show that the proposed framework can be trained with limited experimental data and fits the data better than several existing models. The SFEA framework provides a straightforward way of using the experimental data and quantifying the resulting uncertainty in simulation-based predictions.

HIF1A inhibitor PX-478 reduces pathological stretch-induced calcification and collagen turnover in aortic valve.

HIF1A is significantly upregulated in calcified human aortic valves (AVs). Furthermore, HIF1A inhibitor PX-478 was shown to inhibit AV calcification under static and disturbed flow conditions. Since elevated stretch is one of the major mechanical stimuli for AV calcification, we investigated the effect of PX-478 on AV calcification and collagen turnover under a pathophysiological cyclic stretch (15%) condition. Porcine aortic valve (PAV) leaflets were cyclically (1 Hz) stretched at 15% for 24 days in osteogenic medium with or without PX-478. In addition, PAV leaflets were cyclically stretched at a physiological (10%) and 15% for 3 days in regular medium to assess its effect of on HIF1A mRNA expression. It was found that 100 μM (high concentration) PX-478 could significantly inhibit PAV calcification under 15% stretch, whereas 50 μM (moderate concentration) PX-478 showed a modest inhibitory effect on PAV calcification. Nonetheless, 50 μM PX-478 significantly reduced PAV collagen turnover under 15% stretch. Surprisingly, it was observed that cyclic stretch (15% vs. 10%) did not have any significant effect on HIF1A mRNA expression in PAV leaflets. These results suggest that HIF1A inhibitor PX-478 may impart its anti-calcific and anti-matrix remodeling effect in a stretch-independent manner.

IMMUNE ANALYSIS OF TISSUE ENGINEERED PORCINE AORTIC VALVE LEAFLETS AFTER ALPHA-GALACTOSE CLEAVAGE

Bioprosthetic heart valves are prone to structural valve deterioration (SVD) due to an inflammatory immune response resulting in calcification, stenosis, and ultimately valve failure. The antigenicity of xenografts is thought to underlie the immune response, with galactose-α-1,3-galactose (alpha-gal) the principal antigen of investigation. The objective of this study is to further characterize the role of alpha-gal in SVD and determine whether the addition of alpha-gal cleavage to tissue engineered porcine aortic valve (PAV) leaflets will attenuate the xenoreactive humoral immune response.

Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview.

Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo, in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.

Calcium-Dependent Cytosolic Phospholipase A2α as Key Factor in Calcification of Subdermally Implanted Aortic Valve Leaflets.

Calcium-dependent cytosolic phospholipase A2α (cPLA2α) had been previously found to be overexpressed by aortic valve interstitial cells (AVICs) subjected to in vitro calcific induction. Here, cPLA2α expression was immunohistochemically assayed in porcine aortic valve leaflets (iAVLs) that had undergone accelerated calcification subsequent to 2- to 28-day-long implantation in rat subcutis. A time-dependent increase in cPLA2α-positive AVICs paralleled mineralization progression depending on dramatic cell membrane degeneration with the release of hydroxyapatite-nucleating acidic lipid material, as revealed by immunogold particles decorating organelle membranes in 2d-iAVLs, as well as membrane-derived lipid byproducts in 7d- to 28d-iAVLs. Additional positivity was detected for (i) pro-inflammatory IL-6, mostly exhibited by rat peri-implant cells surrounding 14d- and 28d-iAVLs; (ii) calcium-binding osteopontin, with time-dependent increase and no ossification occurrence; (iii) anti-calcific fetuin-A, mostly restricted to blood plasma within vessels irrorating the connective envelopes of 28d-iAVLs; (iv) early apoptosis marker annexin-V, limited to sporadic AVICs in all iAVLs. No positivity was found for either apoptosis executioner cleaved caspase-3 or autophagy marker MAP1. In conclusion, cPLA2α appears to be a factor characterizing AVL calcification concurrently with a distinct still uncoded cell death form also in an animal model, as well as a putative target for the prevention and treatment of calcific valve diseases.

Rac1 mediates cadherin-11 induced cellular pathogenic processes in aortic valve calcification

Background: Calcific aortic valve disease (CAVD), a major cause for surgical aortic valve replacement, currently lacks available pharmacological treatments. Cadherin-11 (Cad11), a promising therapeutic target, promotes aortic valve calcification in vivo, but direct Cad11 inhibition in clinical trials has been unsuccessful. Targeting of downstream Cad11 effectors instead may be clinically useful; however, the downstream effectors that mediate Cad11-induced aortic valve cellular pathogenesis have not been investigated. Approach and Results: Immunofluorescence of calcified human aortic valves revealed that GTP-Rac1 is highly upregulated in calcified leaflets and is 2.15 times more co-localized with Cad11 in calcified valves than GTP-RhoA. Using dominant negative mutants in porcine aortic valve interstitial cells (PAVICs), we show that Cad11 predominantly regulates Runx2 nuclear localization via Rac1. Rac1-GEF inhibition via NSC23766 effectively reduces calcification in ex vivo porcine aortic valve leaflets treated with osteogenic media by 2.8-fold and also prevents Cad11-induced cell migration, compaction, and calcification in PAVICs. GTP-Rac1 and Trio, a known Cad11 binding partner and Rac1-GEF, are significantly upregulated in Nfatc1Cre; R26-Cad11Tg/Tg (Cad11 OX) mice that conditionally overexpress Cad11 in the heart valves by 3.1-fold and 6.3-fold, respectively. Finally, we found that the Trio-specific Rac1-GEF inhibitor, ITX3, effectively prevents Cad11-induced calcification and Runx2 induction in osteogenic conditions. Conclusion: Here we show that Cad11 induces many cellular pathogenic processes via Rac1 and that Rac1 inhibition effectively prevents many Cad11-induced aortic disease phenotypes. These findings highlight the therapeutic potential of blocking Rac1-GEFs in CAVD.

Prevention of the calcification of porcine heart valve leaflets implanted subcutaneously in rats by an interleukin-8 receptors antagonist (SCH527123)

Bioprosthetic heart valve calcification may result from interactions between the host's immune system and the glutaraldehyde (GA)-fixed tissue that makes up the leaflets. We hypothesized that antagonizing interleukin-8 (IL-8) receptors, CXCR-1 and CXCR-2, may prevent the calcification of GA-fixed porcine aortic valves implanted subcutaneously in rats. Three-week-old Sprague Dawlay males were transplanted with 2 aortic valve leaflets isolated from pigs and implanted into the dorsal wall. Four groups of 3–4 rats (6–8 independent leaflets each time) were compared: glutaraldehyde (GA)-free leaflets, GA-fixed leaflets (0.6% GA for 24 h before implantation), GA-fixed leaflets and treatment of rats with 1 mg/kg SCH5217123 (a CXCR1/2 antagonist) injected once a day locally subcutaneously (SC) or intraperitoneally (IP). Fourteen days later, rats underwent blood leukocyte count and CT-scan, before porcine leaflets explantation and histology and biochemistry analyses. As expected, strong calcification of ectopic valves was induced by the GA. However, we observed a significant about 40% decrease in this effect when rats were treated with the SCH527123. These effects were confirmed by CT-scan acquisitions and Alizarin red staining of valves. Besides, GA pre-incubation of valves significantly increased leukocytes count in rat blood, an effect that is significantly prevented by IP SCH527123 (6.9, 16.6 and 6.8 × 10 9 /L in the GA-free, GA and GA + IP SCH527123 groups respectively, P = 0.0129). Immunohistochemistry of ectopic valves highlighted higher infiltration of CD3 (lymphocytes) and CD68 (macrophages) cells in the GA group compared to the GA-free and GA + SCH527123 groups. Therefore, the calcification of GA-fixed porcine aortic valve leaflets, implanted subcutaneously in rats, is significantly prevented by antagonizing IL-8 receptors with SCH527123. This effect might partly result from an inhibition of the GA-induced leukocytes activation and recruitment into the graft tissue.

The Comparison of Different Constitutive Laws and Fiber Architectures for the Aortic Valve on Fluid-Structure Interaction Simulation.

Built on the hybrid immersed boundary/finite element (IB/FE) method, fluid–structure interaction (FSI) simulations of aortic valve (AV) dynamics are performed with three different constitutive laws and two different fiber architectures for the AV leaflets. An idealized AV model is used and mounted in a straight tube, and a three-element Windkessel model is further attached to the aorta. After obtaining ex vivo biaxial tensile testing of porcine AV leaflets, we first determine the constitutive parameters of the selected three constitutive laws by matching the analytical stretch–stress relations derived from constitutive laws to the experimentally measured data. Both the average error and relevant R-squared value reveal that the anisotropic non-linear constitutive law with exponential terms for both the fiber and cross-fiber directions could be more suitable for characterizing the mechanical behaviors of the AV leaflets. We then thoroughly compare the simulation results from both structural mechanics and hemodynamics. Compared to the other two constitutive laws, the anisotropic non-linear constitutive law with exponential terms for both the fiber and cross-fiber directions shows the larger leaflet displacements at the opened state, the largest forward jet flow, the smaller regurgitant flow. We further analyze hemodynamic parameters of the six different cases, including the regurgitant fraction, the mean transvalvular pressure gradient, the effective orifice area, and the energy loss of the left ventricle. We find that the fiber architecture with body-fitted orientation shows better dynamic behaviors in the leaflets, especially with the constitutive law using exponential terms for both the fiber and cross-fiber directions. In conclusion, both constitutive laws and fiber architectures can affect AV dynamics. Our results further suggest that the strain energy function with exponential terms for both the fiber and cross-fiber directions could be more suitable for describing the AV leaflet mechanical behaviors. Future experimental studies are needed to identify competent constitutive laws for the AV leaflets and their associated fiber orientations with controlled experiments. Although limitations exist in the present AV model, our results provide important information for selecting appropriate constitutive laws and fiber architectures when modeling AV dynamics.

Brillouin Spectroscopy as an Innovative Tool to Investigate Biomechanical Properties of Native Human Aortic Valve and Bioprostheses Tissue

Objective: Proper aortic valve (AV) function is enabled by a complex multi-layered extracellular matrix structure and embedded valvular interstitial and endothelial cells (VICs/VECs). In a highly dynamic environment, VICs and VECs respond to biomechanical stimuli thereby influencing valvular matrix remodelling and pathological changes. Thus, it is of cardinal importance to understand the mechanobiology of AV tissue. It is hypothesized that biomechanical properties of native human AV as well as biological AV prostheses tissue can be determined using Brillouin spectroscopy. Methods: Native AV tissue (n=4) and explanted AV prosthesis (n=1) were obtained from operating theatre after AV replacement, 1mm-thick vibratome cuts were prepared (cross section in radial direction) and directly analysed by means of a confocal Brillouin microscope (10x/0.25NA). Here, the inelastically back-scattered laser light (780nm) is analysed by a custom-build Brillouin spectrometer based on a two-stage virtually imaged phased array (VIPA) set-up. Brillouin maps have been acquired pixel-by-pixel (acquisition time 1s/pixel). Tissue slides adjacent to the vibratome sections were fixed and examined histologically. Results: In stenotic AV tissue mineralized regions in the fibrosa exhibit higher Brillouin shift values compared to non-mineralized spongiosa (Figure 1A). The matrix of the examined explanted AV bioprosthesis (glutaraldehyde-fixed porcine AV) showed higher Brillouin shift values than the massive endocarditic vegetation covering both surfaces of the cusp. Calcified regions at the coaptation area were correlated with higher Brillouin shift values (Figure 1B). Conclusions: Brillouin spectroscopy enables detection of differentiated degrees of tissue stiffness within native stenotic and fibrotic human AV. It represents a promising tool to analyze biomechanical properties of human AV. Figure 1. Procedure of Brillouin spectroscopy on (a) stenotic human aortic valve and (b) aortic valve prothesis (from porcine AV cusps) with florid endocarditis and subsequent histological analysis, high Brillouin shift values indicate enhanced stiffness

The focal mechanical properties of normal and diseased porcine aortic valve tissue measured by a novel microindentation device

Cells sense and respond to the heterogeneous mechanical properties of their tissue microenvironment, with implications for the development of many diseases, including cancer, fibrosis, and aortic valve disease. Characterization of tissue mechanical heterogeneity on cellular length scales of tens of micrometers is thus important for understanding disease mechanobiology. In this study, we developed a low-cost bench-top microindentation system to readily map focal microscale soft tissue mechanical properties. The device was validated by comparison with atomic force microscopy nanoindentation of polyacrylamide gels. To demonstrate its utility, the device was used to measure the focal microscale elastic moduli of normal and diseased porcine aortic valve leaflet tissue. Consistent with previous studies, the fibrosa layer of intact leaflets was found to be 1.91-fold stiffer than the ventricularis layer, with both layers exhibiting significant heterogeneity in focal elastic moduli. For the first time, the microscale compressive moduli of focal proteoglycan-rich lesions in the fibrosa of early diseased porcine aortic valve leaflets were measured and found to be 2.44-fold softer than those of normal tissue. These data provide new insights into the tissue micromechanical environment in valvular disease and demonstrate the utility of the microindentation device for facile measurement of the focal mechanical properties of soft tissues.

Mechanical characterization and identification of material parameters of porcine aortic valve leaflets

The ideal artificial heart valve does not exist yet. Understanding of mechanical and structural properties of natural tissues is necessary to improve the design of biomimetic aortic valve. Besides these properties are needed for the finite element modeling as input parameters. In this study we propose a new method combining biaxial tests and digital image correlation. These tests are carried out on porcine aortic valves. In this work, we use a modified version of the HGO (Holzapfel-Gasser-Ogden) model which is classically used for hyper-elastic and anisotropic soft tissues. This model can include fiber orientation. The identification of HGO model parameters can be determined using experimental data and two different protocols. One protocol is based on the identification of collagen fibers orientation as well as the mechanical parameters. The second one, is based on a complementary experiment to determine orientation (confocal laser scanning microscope). Both lead to determine different sets of material parameters. We show that the model is more likely to reproduce the actual mechanical behavior of the heart valves in the second case and that a minimum of three different loading conditions for the biaxial tensile tests is required to obtain a relevant set of parameters.

Investigation of air plasma generated by surface microdischarge for decellularized porcine aortic valve leaflets modification

AbstractIn this study, the surface microdischarge (SMD) device was used to modify the mechanical properties of decellularized porcine aortic valve leaflets (DPAVL). It was found that SMD treatment under NOx mode considerably improved the ultimate tensile strength and the Young's modulus of DPAVL, whereas treatment under O3 mode had no effect. The absorbance band peak at ~1,375 cm−1, measured by Fourier‐transform infrared spectrometer, increased with treatment time in the freeze‐dried DPAVL group under NOx mode treatment. The microstructure was not destroyed and blood compatibility test showed no toxicity. The reaction between plasma‐activated species and the DPAVL and the pH change, other than temperature/UV irradiation, plays a major role. All of the results indicate that cold atmospheric plasma treatment seems to be a potential alternative for DPAVL modification.

Disturbed Flow Increases UBE2C (Ubiquitin E2 Ligase C) via Loss of miR-483-3p, Inducing Aortic Valve Calcification by the pVHL (von Hippel-Lindau Protein) and HIF-1α (Hypoxia-Inducible Factor-1α) Pathway in Endothelial Cells.

Objective- Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results- Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483-dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions- These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.

Coupled OPG-Fc on Decellularized Aortic Valves by EDC/NHS Attenuates Rat MSCs Calcification In Vitro.

Valve calcification commonly damages natural human heart valves and tissue-engineered heart valves (TEHVs), and no ideal intervention is available in clinical practice. It is increasingly considered that osteoprotegerin (OPG) inhibits vascular calcification. Herein we aimed to explore whether free OPG-Fc fusion protein or coupled OPG-Fc on decellularized aortic valves attenuates calcification. Calcification of rat bone marrow-derived mesenchymal stromal cells (MSCs) was induced by osteogenic differentiation media, and the effects of free OPG-Fc or OPG-Fc coupled on the decellularized porcine aortic heart valve leaflet scaffolds by coupling agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) on calcification were observed. Mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers were assessed to determine the calcification kinetics. Our results indicated that the matrix calcium content and the ALP activity, as well as the mRNA expression levels of a bone morphogenetic protein-2 (BMP-2), osteopontin (OPN), and osteocalcin (OC), of the MSCs seeded on plates with free OPG-Fc or on the OPG-Fc-coupled scaffolds decreased compared with their control MSCs without coupled OPG-Fc. The results suggest that both free and immobilized OPG-Fc on the decellularized aortic valve scaffolds by EDC/NHS can attenuate the calcification of MSCs induced by osteogenic differentiation media, implying that OPG-Fc might be a new treatment or prevention strategy for the calcification of natural human heart valves and TEHVs in the future.

In Vivo Response of Acellular Porcine Pericardial for Tissue Engineered Transcatheter Aortic Valves

Current heart valve prostheses have limitations that include durability, inability to grow in pediatric patients, and lifelong anticoagulation. Transcatheter aortic valve replacements are minimally invasive procedures, and therefore have emerged as an alternative to traditional valve prostheses. In this experiment, the regenerative capacity of potential tissue engineered transcatheter valve scaffolds (1) acellular porcine pericardium and (2) mesenchymal stem cell-seeded acellular porcine pericardium were compared to native porcine aortic valve cusps in a rat subcutaneous model for up to 8 weeks. Immunohistochemistry, extracellular matrix evaluation, and tissue biomechanics were evaluated on the explanted tissue. Acellular valve scaffolds expressed CD163, CD31, alpha smooth muscle actin, and vimentin at each time point indicating host cell recellularization; however, MSC-seeded tissue showed greater recellularization. Inflammatory cells were observed with CD3 biomarker in native porcine pericardial tissue throughout the study. No inflammation was observed in either acellular or MSC-seeded scaffolds. There was no mechanical advantage observed in MSC-seeded tissue; however after the first week post-explant, there was a decrease in mechanical properties in all groups (p < 0.05). MSC-seeded and acellular porcine pericardium expressed decreased inflammatory response and better host-cell recellularization compared to the native porcine aortic valve cusps.

Strain effects on collagen proteolysis in heart valve tissues

Collagen is at the heart of any and all questions concerning semilunar valvular leaflet composition, structure, and function. Whether during development, physiological homeostasis, or pathological degeneration, it is the structural-mechanical state of the heart valve leaflet collagen network that ultimately confers valvular function, and the difference between health and disease. In the current study, the effects of physiologically relevant strain states on collagen catabolism are investigated in porcine aortic and pulmonary valve leaflets. Application of bacterial collagenase to the tissues which acts to simulate collagen degradation by endogenous matrix metalloproteinases, biaxial stress relaxation, and histology are all used to serve as measures of functional and compositional collagen catabolism. Current stress-relaxation results are used in conjunction with previous equibiaxial testing to confirm that a mechanism exists to prevent collagen catabolism when stretched at physiologically relevant strain states. Collectively, these in vitro results indicate that biaxial strain states are capable of impacting the susceptibility of valvular collagens to catabolism, and that at physiological strain states, a protective mechanism exists to effectively block collagen catabolism. The results of the study will be broadly applicable to clarify the roles of tissue microarchitecture and load transmission in a variety of other developmental, homeostatic, or pathogenic tissue processes such as tumor growth, embryogenesis, thrombi formation, and atherogenesis.

MiR-214 is Stretch-Sensitive in Aortic Valve and Inhibits Aortic Valve Calcification.

miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1). Porcine aortic valve (PAV) leaflets were cyclically stretched at 15% for 48h in regular medium and for 1week in osteogenic medium to simulate the early remodeling and late calcification stages of stretch-induced AV disease, respectively. For both stages, 10% cyclic stretch served as the physiological counterpart. RT-qPCR revealed that miR-214 expression was significantly downregulated during the late calcification stage, whereas the mRNA expression of ATF4 and BCL2L1 was upregulated and downregulated, respectively, during both early remodeling and late calcification stages. When PAV leaflets were statically transfected with miR-214 mimic in osteogenic medium for 2weeks, calcification was significantly reduced compared to the control mimic case. This implies that miR-214 may have a protective role in stretch-induced calcific AV disease.