Naren Vyavahare

Aluminum chloride pretreatment of elastin inhibits elastolysis by matrix metalloproteinases and leads to inhibition of elastin-oriented calcification.

Calcification of elastin occurs in many pathological cardiovascular diseases including atherosclerosis. We have previously shown that purified elastin when subdermally implanted in rats undergoes severe calcification and aluminum chloride (AlCl(3)) pretreatment of elastin inhibits calcification. In the present study we investigated whether matrix metalloproteinase (MMP) binding to elastin and elastin degradation is prevented by AlCl(3) pretreatment. Subdermal implantation of AlCl(3)-pretreated elastin showed significantly lower MMP-9 and MMP-2 activity surrounding the implant as compared to the control implants. AlCl(3) pretreatment also significantly inhibited elastin implant calcification at the seven-day implant period (AlCl(3)-pretreated 4.07 +/- 1.27, control 23.82 +/- 2.24 microg/mg; p<0.0001). Moreover, elastin gel zymography studies showed that gel pretreatment with AlCl(3) inhibited elastolysis by MMP-9. We also demonstrate significant suppression of MMP-2 activity in aortic wall segments of AlCl(3)-pretreated porcine bioprosthetic heart valve implants as compared to control valve implants in sheep mitral valve replacement studies. AlCl(3) pretreatment also significantly inhibited calcification of elastin in this model. Thus, we conclude that aluminum ion binding to elastin prevents MMP-mediated elastolysis and thus prevents elastin calcification.

The Effect of Glycosaminoglycan Stabilization on Tissue Buckling in Bioprosthetic Heart Valves

Bioprosthetic valves are used in thousands of heart valve replacement surgeries. Existing glutaraldehyde-crosslinked bioprosthetic valves fail due to either calcification or degeneration. Glutaraldehyde crosslinking does not stabilize valvular glycosaminoglycans (GAGs). GAGs, predominantly present in the medial spongiosa layer of native heart valve cusps, play an important role in regulating physico-mechanical behavior of the native cuspal tissue during dynamic motion. The primary objective of this study was to identify the role of cuspal GAGs in valve tissue buckling. Glutaraldehyde-crosslinked cusps showed extensive buckling compared to fresh, native cusps. Removal of GAGs by treatment with GAG-degrading enzymes led to a marked increase in buckling behavior in glutaraldehyde-crosslinked cusps. We demonstrate that the retention of valvular GAGs by carbodiimide crosslinking together with chemical attachment of neomycin trisulfate (a hyaluronidase inhibitor), prior to glutaraldehyde crosslinking, reduces the extent of buckling in bioprosthetic heart valves. Furthermore, following exposure to GAG-digestive enzymes, neomycin-trisulfate-bound cusps experienced no alterations in buckling behavior. Such moderate buckling patterns mimicked that of fresh, untreated cusps subjected to similar bending curvatures. Thus, GAG stabilization may subsequently improve the durability of these bioprostheses.

On the biomechanical role of glycosaminoglycans in the aortic heart valve leaflet

While the role of collagen and elastin fibrous components in heart valve valvular biomechanics has been extensively investigated, the biomechanical role of the glycosaminoglycan (GAG) gelatinous-like material phase remains unclear. In the present study, we investigated the biomechanical role of GAGs in porcine aortic valve (AV) leaflets under tension utilizing enzymatic removal. Tissue specimens were removed from the belly region of porcine AVs and subsequently treated with either an enzyme solution for GAG removal or a control (buffer with no enzyme) solution. A dual stress level test methodology was used to determine the effects at low and high (physiological) stress levels. In addition, planar biaxial tests were conducted both on-axis (i.e. aligned to the circumferential and radial axes) and at 45° off-axis to induce maximum shear, to explore the effects of augmented fiber rotations on the fiber-fiber interactions. Changes in hysteresis were used as the primary metric of GAG functional assessment. A simulation of the low-force experimental setup was also conducted to clarify the internal stress system and provide viscoelastic model parameters for this loading range. Results indicated that under planar tension the removal of GAGs had no measureable affect extensional mechanical properties (either on- or 45° off-axis), including peak stretch, hysteresis and creep. Interestingly, in the low-force range, hysteresis was markedly reduced, from 35.96±2.65% in control group to 25.00±1.64% (p<0.001) as a result of GAG removal. Collectively, these results suggest that GAGs do not play a direct role in modulating the time-dependent tensile properties of valvular tissues. Rather, they appear to be strongly connected with fiber-fiber and fiber-matrix interactions at low force levels. Thus, we speculate that GAGs may be important in providing a damping mechanism to reduce leaflet flutter when the leaflet is not under high tensile stress.

A novel synthetic route for the preparation of hydrolytically degradable synthetic hydrogels

A variety of approaches have been described for the modification of synthetic, water soluble polymers with hydrolytically degradable bonds and terminal vinyl groups that can be crosslinked in situ by photo- or redox-initiated free radical polymerization. However, changes in macromer concentration, functionality, and molecular weight commonly used to achieve variable degradation rates simultaneously alter hydrogel mechanical properties. Herein, we describe a novel, two-step synthetic route for the preparation of hydrolytically degradable, crosslinkable PEG-based macromers based on chemical intermediaries that form ester linkages with variable alkyl chain length. Changes in the concentration of a single macromer were shown to provide effective variation of degradation, but with corresponding significant changes in tensile properties. Through variation in the alkyl chain length of the chemical intermediary, variable degradation times ranging from weeks to months are achieved, without significantly affecting initial gelation efficiency, swelling, or tensile properties. When modified with adhesive ligands, hydrogels supported viability of encapsulated and adherent cells. Controlled release of a model protein (Immunoglobulin G) was attained as a function of hydrogel degradation rate. Independent control of hydrogel degradation and mechanical properties will offer improved flexibility for studying the effect of these material characteristics on cellular function and may be useful in the design of matrices for tissue engineering and controlled release of bioactive molecules.

High-glucose levels and elastin degradation products accelerate osteogenesis in vascular smooth muscle cells

Diabetes mellitus (DM) is a chronic disease in which the body either does not use or produce the glucose metabolising hormone insulin efficiently. Calcification of elastin in the arteries of diabetics is a major predictor of cardiovascular diseases. It has been previously shown that elastin degradation products work synergistically with transforming growth factor-beta 1 (TGF-β1) to induce osteogenesis in vascular smooth muscle cells. In this study, we tested the hypothesis that high concentration of glucose coupled with elastin degradation products and TGF-β1 (a cytokine commonly associated with diabetes) will cause a greater degree of osteogenesis compared to normal vascular cells. Thus, the goal of this study was to analyse the effects of high concentration of glucose, elastin peptides and TGF-β1 on bone-specific markers like alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2). We demonstrated using relative gene expression and specific protein assays that elastin degradation products in the presence of high glucose cause the increase in expression of the specific elastin–laminin receptor-1 (ELR-1) and activin receptor-like kinase-5 (ALK-5) present on the surface of the vascular cells, in turn leading to overexpression of typical osteogenic markers like ALP, OCN and RUNX2. Conversely, blocking of ELR-1 and ALK-5 strongly suppressed the expression of the osteogenic proteins. In conclusion, our results indicate that glucose plays an important role in amplifying the osteogenesis induced by elastin peptides and TGF-β1, possibly by activating the ELR-1 and ALK-5 signalling pathways.

Efficacy of Reversal of Aortic Calcification by Chelating Agents

Elastin-specific medial vascular calcification, termed “Monckeberg’s sclerosis,” has been recognized as a major risk factor for various cardiovascular events. We hypothesize that chelating agents, such as disodium ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and sodium thiosulfate (STS) might reverse elastin calcification by directly removing calcium from calcified tissues into soluble calcium complexes. We assessed the chelating ability of EDTA, DTPA, and STS on removal of calcium from hydroxyapatite (HA) powder, calcified porcine aortic elastin, and calcified human aorta in vitro. We show that both EDTA and DTPA could effectively remove calcium from HA and calcified tissues, while STS was not effective. The tissue architecture was not altered during chelation. In the animal model of aortic elastin-specific calcification, we further show that local periadventitial delivery of EDTA loaded in to poly(lactic-co-glycolic acid) nanoparticles regressed elastin-specific calcification in the aorta. Collectively, the data indicate that elastin-specific medial vascular calcification could be reversed by chelating agents.

In vivo vascular tissue engineering: influence of cytokine and implant location on tissue specific cellular recruitment

In vivo tissue engineering has been explored as a means to create autologous vascular replacements. Elastin is necessary to sustain continual pulsatile flow and to prevent the dilatation of vascular tissues. Unfortunately, elastogenesis in tissue‐engineered constructs has been very limited. To overcome this limitation, we have created tubular elastin scaffolds from porcine carotid arteries. Elastin would provide the necessary elasticity to the graft on implanting these scaffolds as vascular grafts. In this study, elastin tubes with agarose gel containing either stromal‐derived factor‐1 α [SDF; for homing of endothelial cells (ECs)] or basic fibroblast growth factor (bFGF; for homing of myofibroblasts) were implanted into adipose tissue, as it is a known source of stem/progenitor cells. We also implanted these tubes into subdermal pouches (as a control location). We observed a difference in the types of cells recruited—ECs were recruited in large numbers by SDF in the adipose tissue, whereas the adipose‐FGF group had a vascularized (smooth muscle and EC‐positive), collagenous capsule (adventitia) with many smooth muscle α‐actin (SMA)‐positive cells in the elastin scaffold layer (media). These results were in contrast to the subdermal group, which only recruited fibroblasts and some SMA‐positive cells. Also, more cell infiltration and neo‐collagen formation was seen in adipose implants. This study provides novel results by the use of specific cytokines and implant locations to recruit tissue‐specific cells to create autologous vascular grafts. Copyright

Neomycin and pentagalloyl glucose enhanced cross-linking for elastin and glycosaminoglycans preservation in bioprosthetic heart valves

Glutaraldehyde cross-linked bioprosthetic heart valves fail within 12–15 years of implantation due to limited durability. Glutaraldehyde does not adequately stabilize extracellular matrix components such as glycosaminoglycans and elastin, and loss of these components could be a major cause of degeneration of valve after implantation. We have shown earlier that neomycin-based cross-linking stabilizes glycosaminoglycans in the tissue but fails to stabilize elastin component. Here, we report a new treatment where neomycin and pentagalloyl glucose (PGG) were incorporated into glutaraldehyde cross-linking neomycin-PGG-Glutaraldehyde (NPG) to stabilize both glycosaminoglycans and elastin in porcine aortic valves. In vitro studies demonstrated a marked increase in extracellular matrix stability against enzymatic degradation after cross-linking and 10 month storage in NPG group when compared to glutaraldehyde controls. Tensile properties showed increased lower elastic modulus in both radial and circumferential directions in NPG group as compared to glutaraldehyde, probably due to increased elastin stabilization with no changes in upper elastic modulus and extensibility. The enhanced extracellular matrix stability was further maintained in NPG-treated tissues after rat subdermal implantation for three weeks. NPG group also showed reduced calcification when compared to glutaraldehyde controls. We conclude that NPG cross-linking would be an excellent alternative to glutaraldehyde cross-linking of bioprosthetic heart valves to improve its durability.

Neomycin and carbodiimide crosslinking as an alternative to glutaraldehyde for enhanced durability of bioprosthetic heart valves

Glutaraldehyde cross-linked porcine aortic valves, referred to as bioprosthetic heart valves (BHVs), are often used in heart valve replacements. Glutaraldehyde does not stabilize glycosaminoglycans (GAGs) and they are lost during preparation, in vivo implantation, cyclic fatigue, and storage. We report that binding of neomycin, a hyaluronidase inhibitor, to the tissues with carbodiimide cross-linking improves GAG retention without reducing collagen and elastin stability. It also led to improved biomechanical properties. Neomycin carbodiimide cross-linking did not significantly reduce calcification in a rat subdermal implantation model when they were stored in formaldehyde after cross-linking. Removal of formaldehyde storage significantly reduced calcification.

PORCINE VENA CAVA AS AN ALTERNATIVE TO BOVINE PERICARDIUM IN BIOPROSTHETIC PERCUTANEOUS HEART VALVES

Percutaneous heart valves are revolutionizing valve replacement surgery by offering a less invasive treatment option for high-risk patient populations who have previously been denied the traditional open chest procedure. Percutaneous valves need to be crimped to accommodate a small-diameter catheter during deployment, and they must then open to the size of heart valve. Thus the material used must be strong and possess elastic recoil for this application. Most percutaneous valves utilize bovine pericardium as a material of choice. One possible method to reduce the device delivery diameter is to utilize a thin, highly elastic tissue. Here we investigated porcine vena cava as an alternative to bovine pericardium for percutaneous valve application. We compared the structural, mechanical, and in vivo properties of porcine vena cava to those of bovine pericardium. While the extracellular matrix fibers of pericardium are randomly oriented, the vena cava contains highly aligned collagen and elastin fibers that impart strength to the vessel in the circumferential direction and elasticity in the longitudinal direction. Moreover, the vena cava contains a greater proportion of elastin, whereas the pericardium matrix is mainly composed of collagen. Due to its high elastin content, the vena cava is significantly less stiff than the pericardium, even after crosslinking with glutaraldehyde. Furthermore, the vena cavas mechanical compliance is preserved after compression under forces similar to those exerted by a stent, whereas pericardium is significantly stiffened by this process. Bovine pericardium also showed surface cracks observed by scanning electron microscopy after crimping that were not seen in vena cava tissue. Additionally, the vena cava exhibited reduced calcification (46.64 ± 8.15 μg Ca/mg tissue) as compared to the pericardium (86.79 ± 10.34 μg/mg). These results suggest that the vena cava may provide enhanced leaflet flexibility, tissue resilience, and tissue integrity in percutaneous heart valves, ultimately reducing the device profile while improving the durability of these valves.