Prevention of abdominal aortic aneurysm progression by targeted inhibition of matrix metalloproteinase activity with batimastat-loaded nanoparticles.

RATIONALE: Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms. Systemic treatments of MMP inhibitors have shown effectiveness in animal models, but it did not translate to clinical success either because of low doses used or systemic side effects of MMP inhibitors. We propose a targeted nanoparticle (NP)-based delivery of MMP inhibitor at low doses to the abdominal aortic aneurysms site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects. OBJECTIVE: Our previous study showed that poly(d,l-lactide) NPs conjugated with an antielastin antibody could be targeted to the site of an aneurysm in a rat model of abdominal aortic aneurysms. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth. METHODS AND RESULTS: Poly(d,l-lactide) NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced abdominal aortic aneurysms in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control versus 40% elastin antibody-conjugated BB-94-loaded NPs) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition. CONCLUSIONS: Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression.

Pentagalloyl Glucose (PGG) Prevents and Restores Mechanical Changes Caused by Elastic Fiber Fragmentation in the Mouse Ascending Aorta.

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aorta that can lead to dissection or rupture. Degradation of elastic fibers is a consistent histopathological feature of TAA that likely contributes to disease progression. Pentagalloyl glucose (PGG) shows promise for stabilizing elastic fibers in abdominal aortic aneurysms, but its efficacy and mechanical effects in the thoracic aorta are unknown. We simulated TAAs using elastase (ELA) to degrade elastic fibers in the mouse ascending aorta and determined the preventative and restorative potential of PGG. Biaxial mechanical tests, constitutive model fitting, and multiphoton imaging were performed on untreated (UNT), PGG, ELA, PGG + ELA, and ELA + PGG treated aortas. PGG treatment alone does not significantly alter mechanical properties or wall structure compared to UNT. ELA treatment alone causes an increase in the unloaded diameter and length, decreased compliance, significant changes in the material constants, and separation of the outer layers of the aortic wall compared to UNT. PGG treatment before or after ELA ameliorates the mechanical and structural changes associated with elastic fiber degradation, with preventative PGG treatment being most effective. These results suggest that PGG is a potential pharmaceutical option to stabilize elastic fibers in TAA.

Elastin stabilization prevents impaired biomechanics in human pulmonary arteries and pulmonary hypertension in rats with left heart disease.

Pulmonary hypertension worsens outcome in left heart disease. Stiffening of the pulmonary artery may drive this pathology by increasing right ventricular dysfunction and lung vascular remodeling. Here we show increased stiffness of pulmonary arteries from patients with left heart disease that correlates with impaired pulmonary hemodynamics. Extracellular matrix remodeling in the pulmonary arterial wall, manifested by dysregulated genes implicated in elastin degradation, precedes the onset of pulmonary hypertension. The resulting degradation of elastic fibers is paralleled by an accumulation of fibrillar collagens. Pentagalloyl glucose preserves arterial elastic fibers from elastolysis, reduces inflammation and collagen accumulation, improves pulmonary artery biomechanics, and normalizes right ventricular and pulmonary hemodynamics in a rat model of pulmonary hypertension due to left heart disease. Thus, targeting extracellular matrix remodeling may present a therapeutic approach for pulmonary hypertension due to left heart disease.

Reversal of elastase-induced abdominal aortic aneurysm following the delivery of nanoparticle-based pentagalloyl glucose (PGG) is associated with reduced inflammatory and immune markers.

OBJECTIVE: An Abdominal aortic aneurysm (AAA), a deadly disease in elderly population, is featured by expansion of aortic diameter, degradation and weakening of vasculature. Its common and significant characteristics are disarray and inflammation in vasculature. We tested the hypothesis that the reversal of abdominal aortic aneurysm by pentagalloyl glucose-loaded nanoparticles (PGG-NPs) therapy that targets degraded elastin suppresses inflammatory and immune markers to ameliorate the pathophysiology of the disease in advance stage aneurysm in a porcine pancreatic elastase (PPE)-induced mouse model of AAA. METHODS AND RESULTS: After induction of aneurysm in pathogen-free C57BL/6 male mice by applying PPE peri-adventitially to the abdominal aorta, once a week for two doses of intravenous injections of pentagalloyl glucose-loaded nanoparticles (PGG-NPs) conjugated with elastin targeted antibody were used to reverse the aneurysms. We showed that PGG-NPs therapy could suppress infiltration of macrophages, CD8 and CD4 subsets of T cells, matrix metalloproteinases (MMPs), inflammatory cytokines interferon (IFN-γ) and interleukin (IL)-6 at the local and systemic level. Moreover, such PGG-NPs therapy increases the induction of anti-inflammatory cytokines IL-13, IL-27 and IL-10 at the local and systemic level. The therapy also led to remodeling of elastic lamina at the aneurysm site. CONCLUSION: Nanoparticles-loaded pentagalloyl glucose therapy can be an effective treatment option against advanced stage aneurysms to reverse the disease by ameliorating inflammation and restoring arterial homeostasis.

Systemic delivery of targeted nanotherapeutic reverses angiotensin II-induced abdominal aortic aneurysms in mice.

Abdominal aortic aneurysm (AAA) disease causes dilation of the aorta, leading to aortic rupture and death if not treated early. It is the 14th leading cause of death in the U.S. and 10th leading cause of death in men over age 55, affecting thousands of patients. Despite the prevalence of AAA, no safe and efficient pharmacotherapies exist for patients. The deterioration of the elastic lamina in the aneurysmal wall is a consistent feature of AAAs, making it an ideal target for delivering drugs to the AAA site. In this research, we conjugated nanoparticles with an elastin antibody that only targets degraded elastin while sparing healthy elastin. After induction of aneurysm by 4-week infusion of angiotensin II (Ang II), two biweekly intravenous injections of pentagalloyl glucose (PGG)-loaded nanoparticles conjugated with elastin antibody delivered the drug to the aneurysm site. We show that targeted delivery of PGG could reverse the aortic dilation, ameliorate the inflammation, restore the elastic lamina, and improve the mechanical properties of the aorta at the AAA site. Therefore, simple iv therapy of PGG loaded nanoparticles can be an effective treatment option for early to middle stage aneurysms to reverse disease progression and return the aorta to normal homeostasis.

Nanoparticle-based targeted delivery of pentagalloyl glucose reverses elastase-induced abdominal aortic aneurysm and restores aorta to the healthy state in mice.

AIM: Abdominal aortic aneurysms (AAA) is a life-threatening weakening and expansion of the abdominal aorta due to inflammatory cell infiltration and gradual degeneration of extracellular matrix (ECM). There are no pharmacological therapies to treat AAA. We tested the hypothesis that nanoparticle (NP) therapy that targets degraded elastin and delivers anti-inflammatory, anti-oxidative, and ECM stabilizing agent, pentagalloyl glucose (PGG) will reverse advance stage aneurysm in an elastase-induced mouse model of AAA. METHOD AND RESULTS: Porcine pancreatic elastase (PPE) was applied periadventitially to the infrarenal aorta in mice and AAA was allowed to develop for 14 days. Nanoparticles loaded with PGG (EL-PGG-NPs) were then delivered via IV route at 14-day and 21-day (10 mg/kg of body weight). A control group of mice received no therapy. The targeting of NPs to the AAA site was confirmed with fluorescent dye marked NPs and gold NPs. Animals were sacrificed at 28-d. We found that targeted PGG therapy reversed the AAA by decreasing matrix metalloproteinases MMP-9 and MMP-2, and the infiltration of macrophages in the medial layer. The increase in diameter of the aorta was reversed to healthy controls. Moreover, PGG treatment restored degraded elastic lamina and increased the circumferential strain of aneurysmal aorta to the healthy levels. CONCLUSION: Our results support that site-specific delivery of PGG with targeted nanoparticles can be used to treat already developed AAA. Such therapy can reverse inflammatory markers and restore arterial homeostasis.

Targeted Gold Nanoparticles as an Indicator of Mechanical Damage in an Elastase Model of Aortic Aneurysm.

Elastin is a key structural protein and its pathological degradation deterministic in aortic aneurysm (AA) outcomes. Unfortunately, using current diagnostic and clinical surveillance techniques the integrity of the elastic fiber network can only be assessed invasively. To address this, we employed fragmented elastin-targeting gold nanoparticles (EL-AuNPs) as a diagnostic tool for the evaluation of unruptured AAs. Electron dense EL-AuNPs were visualized within AAs using micro-computed tomography (micro-CT) and the corresponding Gold-to-Tissue volume ratios quantified. The Gold-to-Tissue volume ratios correlated strongly with the concentration (0, 0.5, or 10 U/mL) of infused porcine pancreatic elastase and therefore the degree of elastin damage. Hyperspectral mapping confirmed the spatial targeting of the EL-AuNPs to the sites of damaged elastin. Nonparametric Spearman's rank correlation indicated that the micro-CT-based Gold-to-Tissue volume ratios had a strong correlation with loaded (ρ = 0.867, p-val = 0.015) and unloaded (ρ = 0.830, p-val = 0.005) vessel diameter, percent dilation (ρ = 0.976, p-val = 0.015), circumferential stress (ρ = 0.673, p-val = 0.007), loaded (ρ = - 0.673, p-val = 0.017) and unloaded (ρ = - 0.697, p-val = 0.031) wall thicknesses, circumferential stretch (ρ = - 0.7234, p-val = 0.018), and lumen area compliance (ρ = - 0.831, p-val = 0.003). Likewise, in terms of axial force and axial stress vs. stretch, the post-elastase vessels were stiffer. Collectively, these findings suggest that, when combined with CT imaging, EL-AuNPs can be used as a powerful tool in the non-destructive estimation of mechanical and geometric features of AAs.

Gold nanoparticles that target degraded elastin improve imaging and rupture prediction in an AngII mediated mouse model of abdominal aortic aneurysm.

Background: Abdominal aortic aneurysms (AAA) are characterized by a progressive disruption and weakening of the extracellular matrix (ECM) leading to dilation of the aorta which can be fatal if not treated. Current diagnostic imaging modalities provides little insight on the varying degree of ECM degeneration that precedes rupture in AAAs. Targeted delivery of contrast agents such as gold nanoparticles (GNPs) that bind to degraded matrix could prove useful when combined with computed tomography (CT) to provide a non-invasive surrogate marker of AAA rupture potential. Methods: AAAs were induced by chronic infusion of angiotensin II (AngII) into low density-lipoprotein receptor-deficient (LDLr -/-) mice in combination with a high-fat diet. Abdominal ultrasound was used to monitor disease progression and to assess the circumferential strain throughout the cardiac cycle. At six weeks, GNPs conjugated with an elastin antibody (EL-GNP) were injected retro-orbitally. Mice were euthanized 24 hours after EL-GNP injection, and aortas were explanted and scanned ex-vivo with a micro-CT system. Histological assessment and 3D models of the aneurysms with micro-CT were used to determine the EL-GNPs distribution. Isolated vessel burst pressure testing was performed on each aneurysmal aorta to quantify rupture strength and to assess rupture location. Results: Aneurysms were found along the suprarenal aorta in AngII infused mice. Darkfield microscopy indicated EL-GNPs accumulation around the site of degraded elastin while avoiding the healthy and intact elastin fibers. Using nonlinear regression, the micro-CT signal intensity of EL-GNPs along the suprarenal aortas correlated strongly with burst pressures (R2=0.9415) but not the dilation as assessed by ultrasound measurements. Conclusions: Using an established mouse model of AAA, we successfully demonstrated in vivo targeting of EL-GNPs to damaged aortic elastin and correlated micro-CT-based signal intensities with burst pressures. Thus, we show that this novel targeting technique can be used as a diagnostic tool to predict the degree of elastin damage and therefore rupture potential in AAAs better than the extent of dilation.

Site-specific chelation therapy with EDTA-loaded albumin nanoparticles reverses arterial calcification in a rat model of chronic kidney disease.

Medial arterial calcification (MAC) is a common outcome in diabetes and chronic kidney disease (CKD). It occurs as linear mineral deposits along the degraded elastin lamellae and is responsible for increased aortic stiffness and subsequent cardiovascular events. Current treatments for calcification, particularly in CKD, are predominantly focused on regulating the mineral disturbance and other risk factors. Ethylene diamine tetraacetic acid (EDTA), a chelating agent, can resorb mineral deposits, but the systemic delivery of EDTA may cause side effects such as hypocalcemia and bone resorption. We have developed elastin antibody conjugated albumin nanoparticles that target only degraded elastin in vasculature while sparing healthy tissues. In this study, we tested a targeted nanoparticle-based EDTA chelation therapy to reverse CKD-associated MAC. Renal failure was induced in Sprague-Dawley rats by a high adenine diet supplemented by high P and Ca for 28 days that led to MAC. Intravenous delivery of DiR dye-loaded nanoparticles confirmed targeting to vascular degraded elastin and calcification sites within 24 hours. Next, EDTA-loaded albumin nanoparticles conjugated with an anti-elastin antibody were intravenously injected twice a week for two weeks. The targeted nanoparticles delivered EDTA at the site of vascular calcification and reversed mineral deposits without any untoward effects. Systemic EDTA injections or blank nanoparticles were ineffective in reversing MAC. Reversal of calcification seems to be stable as it did not return after the treatment was stopped for an additional four weeks. Targeted EDTA chelation therapy successfully reversed calcification in this adenine rat model of CKD. We consider that targeted NP therapy will provide an attractive option to reverse calcification and has a high potential for clinical translation.

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.

Synthesis and characterization of biodegradable elastomeric polyurethane scaffolds fabricated by the inkjet technique.

Biodegradable polyurethanes (PUs) were synthesized from methylene di-p-phenyl-diisocyanate (MDI), polycaprolactone diol (PCL-diol) and N,N-bis (2-hydorxyethyl)-2-aminoethane-sulfonic acid (BES), serving as a hard segment, soft segment and chain extender, respectively. MDI was chosen due to its reactivity and wide application in synthesis of biomedical polyurethanes due to its reactivity; PCL-diol was chosen because of its biodegradability; and BES was chosen because it allowed the introduction sulfonic acid groups onto the polymer chains. We evaluated the polyurethanes' degradation rate, mechanical properties, hydrophilicity, antithrombogenecity, and ability to support fibroblast cell attachment and growth by comparing with polymers having a 2,2-(methylimino)diethanol (MIDE) chain extender. Mechanical testing demonstrated that the PU containing BES has tensile strengths of about 17 MPa and elongations up to 400%, about three times the strength and four times the elongation than the MIDE based PUs. The polymers showed decreased in vitro degradation rates, lower glass transition temperature (T(g)) and hydrophilicity possibly due to enhanced microphase separation. Preliminary cytocompatibility studies showed that all the PUs are non-toxic, but PU containing BES exhibited much lower cell attachment and proliferation than the MIDE chain extended polymers. An in vitro platelet adhesion assay showed lower platelet attachment on BES containing PU. Additionally, due to the existence of sulfonic acid groups, the BES extended PU became water soluble in basic condition and insoluble in acidic condition, a phenomenon that is reversible at pH value of 8.7, making this a pH sensitive polymer attractive for bioprinting applications. By adding acetic acid into an inkjet cartridge and printing it onto a PU solution with pH above 8.7, precision fabricated scaffolds can be obtained, suggesting that BES based PUs are promising candidates as synthetic inks used for customizable fabrication of tissue engineering scaffolds.

Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves.

Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.

The effect of hyaluronic acid incorporation on fibroblast spreading and proliferation within PEG-diacrylate based semi-interpenetrating networks.

The nanometer-scale mesh size of many synthetic crosslinked hydrogel networks restricts encapsulated cells to a rounded morphology that can inhibit cellular processes such as proliferation and migration that are essential for the early stages of remodeling and tissue formation. The objective of these studies was to investigate an approach for accelerating cellular remodeling based on the creation of semi-interpenetrating networks (IPNs) composed of hydrolytically degradable poly(ethylene glycol) (PEG) diacrylate macromers and native, enzymatically degradable extracellular matrix (ECM) components (collagen, gelatin and hyaluronic acid (HA)). Among the three ECM components investigated, addition of HA at concentrations of 0.12% w/v and greater supported fibroblast spreading throughout the three-dimensional network and significantly increased proliferation relative to control hydrogels without HA. Incorporation of HA resulted in relatively small changes in hydrogel physical/chemical properties such as swelling, degradation rate, and elastic modulus. Fibroblast spreading was eliminated by the addition of hyaluronidase inhibitors, demonstrating that cell-mediated enzymatic degradation of HA is a necessary mechanism responsible for the observed increases in fibroblast activity. By accelerating early cellular remodeling and growth, these semi-IPNs may be useful vehicles for cell transplantation in a variety of tissue engineering applications.

Biocompatibility and remodeling potential of pure arterial elastin and collagen scaffolds.

Surgical therapy of cardiovascular disorders frequently requires replacement of diseased tissues with prosthetic devices or grafts. In typical tissue engineering approaches, scaffolds are utilized to serve as templates to support cell growth and remodeling. Decellularized vascular matrices have been previously investigated as scaffolds for tissue engineering. However, cell migration into these scaffolds was inadequate due to the very tight matrix organization specific to the aortic structure. To address this problem, we prepared two types of decellularized scaffolds from porcine vascular tissues. Pure elastin scaffolds and pure collagen scaffolds were prepared by selectively removing the collagen component or elastin, respectively. In the current study, we use a subdermal implantation model to demonstrate that arterial elastin and collagen scaffolds exhibit enhanced potential for repopulation by host cells in vivo. Notably, numerous new collagen fibers and bundles were found within the remodeled elastin scaffolds and new elastin fibers within collagen scaffolds, respectively, clearly indicating their ability to support de novo extracellular matrix synthesis. We also show that biological cues such as growth factors are required for efficient repopulation of elastin and collagen scaffolds. Finally, we bring evidence that these scaffolds can be endothelialized in vitro for thrombosis resistance and thus can serve as promising candidates for cardiovascular tissue engineering.

Tannic acid treatment enhances biostability and reduces calcification of glutaraldehyde fixed aortic wall.

Progressive degeneration and calcification of glutaraldehyde (Glut) fixed tissues used in cardiovascular surgery restrict their long-term clinical performance. This limited biological stability may be attributable to the inability of Glut to adequately protect certain tissue components such as elastin from enzymatic attack. The aim of our studies was to develop novel tissue-processing techniques targeted specifically at elastin stabilization by using tannic acid (TA), a plant polyphenol capable of protecting elastin from digestion by specific enzymes. In present studies we demonstrated that Glut does not adequately protect porcine aorta from elastase-mediated degradation in vitro. The addition of TA to the Glut fixation process increased the stability of Glut-fixed aorta to elastase digestion by 15-fold and also decreased calcification in the rat subdermal model by 66%. TA was found to be chemically compatible with Glut fixation and did not hinder collagen crosslinking as shown by minor changes in thermal denaturation temperatures, resistance to collagenase and mechanical properties. In vitro and in vivo studies also revealed that TA binding to aortic wall was stable over an extended period of time. TA-mediated elastin stabilization in Glut-fixed cardiovascular implants may significantly extend the clinical durability of these tissue replacements.

Elastin stabilization in cardiovascular implants: improved resistance to enzymatic degradation by treatment with tannic acid.

The long-term performance of tissue-derived, glutaraldehyde (Glut)-treated cardiovascular implants such as prosthetic heart valves and vascular grafts is limited by the bio-degeneration of tissue components. While collagen is satisfactorily preserved by Glut, elastin is not stabilized and is highly vulnerable to degradation. The aim of our studies was to develop methods for efficient stabilization of elastin and subsequently reduce its vulnerability towards enzymatic degradation. More specifically, we investigated the use of tannic acid (TA)1 as a novel agent that specifically targets elastin stabilization. Basic investigations on in vitro interactions between Glut, TA and pure aortic elastin provided clear evidence that Glut treatment does not protect elastin from enzymatic degradation. TA bound to elastin in a time-dependent pattern and this binding increased the resistance of elastin to enzymatic degradation. In addition, when TA was used in mixture with Glut, the kinetic of TA binding to elastin was enhanced and this was translated into improved elastin stabilization. Our results clearly documented the superiority of TA as an elastin-stabilizing agent by comparison with the commonly utilized Glut-based tissue crosslinking techniques.

Role of elastin in pathologic calcification of xenograft heart valves.

Bioprosthetic heart valves fabricated from glutaraldehyde crosslinked porcine aortic valves often fail because of calcific degeneration. Calcification occurs in both cusp and aortic wall portions of bioprosthetic heart valves. The purpose of this study was to discern the role of different aortic wall components in the calcification process. Thus, we selectively extracted cells and other extracellular matrix proteins from porcine aorta using trypsin/DNase/RNase, cyanogen bromide (CNBr), and sodium hydroxide (NaOH) treatments and subdermally implanted these pretreated aortas in young rats. Total DNA and phospholipid data showed complete removal of cells by CNBr and NaOH treatments, whereas trypsin/DNase/RNase treatment was effective in removing DNA but not phospholipids. As shown by amino acid data and Masson's trichrome staining, collagen was removed in CNBr and NaOH treatments. Control fresh porcine aorta calcified significantly after 21 days of implantation (Ca 26.4 +/- 2.4 microg/mg). Removal of cells and collagen from the aorta by CNBr treatment did not lead to a statistically significant reduction in aortic calcification (Ca 20.8 +/- 3.0 microg/mg). Moreover, partial degradation of elastin fibers caused by NaOH (during extraction) and trypsin treatment (after implantation) of the aorta significantly increased elastin-oriented calcification (Ca 94.4 +/- 9.3 and 58.4 +/- 4.6 microg/mg, respectively). Our results indicate that the elastin component of the aorta may undergo independent calcification irrespective of devitalized cell-mediated calcification observed in glutaraldehyde crosslinked aortas. Our results also demonstrate the importance of studying elastin-oriented calcification in decellularized elastin-rich aortic matrices currently used in tissue-engineering applications.

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.

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-ß1) 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-ß1 (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-ß1 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-ß1, possibly by activating the ELR-1 and ALK-5 signalling pathways.