Biomedical subjects

N Vyavahare

Publications and source records attributed to N Vyavahare.

10 recordsLinked to original sources

PubMed2001

Periodate-mediated glycosaminoglycan stabilization in bioprosthetic heart valves.

Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 +/- 5.97 microg/mg, glutaraldehyde-fixed tissue Ca 107.25 +/- 6.56 microg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices.

Animals↗

PubMed2001

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.

Aluminum Chloride↗

PubMed2001

Bisphosphonate derivatized polyurethanes resist calcification.

Calcification of polyurethane cardiovascular implants is an important disease process that has the potential to compromise the long-term function of devices such as polymer heart valves and ventricular assist systems. In this study we report the successful formulation and characterization of bisphosphonate-derivatized polyurethanes, hypothesized to resist implant calcification based on the pharmacologic activity of the immobilized bisphosphonate. Fully polymerized polyurethanes (a polyurea-polyurethane and a polycarbonate polyurethane) were modified (post-polymerization) with bromoalkylation of the hard segments followed by attachment of a bisphosphonate group at the bromine site. These bisphosphonate-polyurethanes resisted calcification in rat 60 day subdermal implants compared to nonmodified control polyurethane implants, that calcify. Bisphosphonates-modified polyurethanes were also studied in circulatory implants using a pulmonary valve cusp replacement model in sheep. Polyurethane cusps modified with bisphosphonate did not calcify in 90 day implants. compared to control polyurethane cusps implants, that demonstrated nodular surface oriented calcific deposits. It is concluded that bisphosphonate modified polyurethanes resist calcification both in subdermal implants and in the circulation. This novel biomaterial approach offers great promise for long-term blood stream implantation with calcification resistance.

Animals↗

PubMed2001

Localized adenovirus gene delivery using antiviral IgG complexation.

Gene therapy with viral vectors has progressed to clinical trials. However, the localization of viral vector delivery to diseased target sites remains a challenge. We tested the hypothesis that an adenoviral vector could be successfully delivered by complexation with a specific antibody that is bound to a biodegradable matrix designed for achieving localized gene transduction. We report the first successful delivery system based upon antibody immobilization of virions in a type I collagen-avidin gel using a polyclonal biotinylated IgG specific for the adenovirus hexon. In vitro stability studies demonstrated retention of viral vector activity with antibody-complexed adenovirus collagen gel preparations, in comparison to loss of vector activity from collagen gels prepared with nonspecific biotinylated IgG. Cell culture investigations using this antibody-controlled release system for adenoviral vector transduction of rat aortic smooth muscle cells (A10) demonstrated a significantly more localized reporter expression (beta-galactosidase) compared with non-antibody-complexed controls. Herpes simplex thymidine kinase (HSVtk) adenoviral vectors were immobilized on avidin-collagen gels via this antibody-complexation approach, and ganciclovir was added to rat smooth muscle cells (A10) in culture with the gels. With complexed HSVtk adenovirus, only cells either in contact with the virus-containing gel or within 50 microm were killed. By comparison, at the same adenovirus and ganciclovir dose, non-antibody-complexed HSVtk adenoviral delivery with ganciclovir resulted in the death of virtually all cells. Myocardial gene transfer studies in pigs demonstrated significantly more efficient right ventricular adenoviral GFP expression with anti-hexon antibody-complexed matrix injections, compared with direct vector injections. Thus, our results show that matrix formulations based on antibody-complexation delivery of adenovirus resulted in site-specific localization of transgene expression that enhances the efficiency of therapeutic vector strategies and provides a potent means for localization, to avoid distal side-effects. This approach has therapeutic potential as an implantable preparation that through the means of antibody-complexation, can localize and optimize viral vector gene therapy.

Adenoviridae↗

PubMed2000

Inhibition of matrix metalloproteinase activity attenuates tenascin-C production and calcification of implanted purified elastin in rats.

Elastin, a major extracellular matrix protein present in arterial walls provides elastic recoil and resilience to arteries. Elastin is prone to calcification in a number of cardiovascular diseases including atherosclerosis and bioprosthetic heart valve mineralization. We have recently shown that purified elastin when implanted subdermally in rats undergoes severe calcification. In the present study, we used this elastin implant model to investigate the molecular mechanisms underlying elastin calcification. Intense matrix metalloproteinase (MMP-2) and tenascin-C (TN-C) expression were seen in the proximity of the initial cal-cific deposits at 7 days. Gelatin zymography studies showed both MMP-2 (latent and active form) and MMP-9 expression within the implants. To investigate the role of MMPs in calcification, rats were administered a MMP inhibitor, (2S:-allyl-N:-hydroxy-3R:-isobutyl-N:-(1S:-methylcarbamoyl-2-ph enylet hyl)-succinamide (BB-1101) by daily injection, either systemically or at the implant site. The site-specific BB-1101 administration almost completely suppressed TN-C expression, as shown by immunohistochemical staining, within the implants. The systemic BB-1101 injections also significantly reduced TN-C expression within the elastin implants. Moreover, calcification of elastin implants was significantly reduced in the site-specific administration group (5.43 +/- 1.03 microg/mg Ca for BB-1101 group versus 21.71 +/- 1.19 for control group, P: < 0.001). Alizarin Red staining clearly showed that the elastin fibers were heavily calcified in the control group, whereas in BB-1101 group the calcification was scarce with few fibers showing initial calcification deposits. The systemic administration of BB-1101 also significantly reduced elastin calcification (28.07 +/- 5.81 control versus 16.92 +/- 2.56 in the BB-1101 group, P: < 0.05), although less than the site-specific administration. Thus, the present studies indicate that MMPs and TN-C play a role in elastin-oriented calcification.

Alkaline Phosphatase↗

PubMed1999

Mechanisms of bioprosthetic heart valve failure: fatigue causes collagen denaturation and glycosaminoglycan loss.

Bioprosthetic heart valve (BPHV) degeneration, characterized by extracellular matrix deterioration, remodeling, and calcification, is an important clinical problem accounting for thousands of surgeries annually. Here we report for the first time, in a series of in vitro accelerated fatigue studies (5-500 million cycles) with glutaraldehyde fixed porcine aortic valve bioprostheses, that the mechanical function of cardiac valve cusps caused progressive damage to the molecular structure of type I collagen as assessed by Fourier transform IR spectroscopy (FTIR). The cyclic fatigue caused a progressive loss of helicity of the bioprosthetic cuspal collagen, which was evident from FTIR spectral changes in the amide I carbonyl stretching region. Furthermore, cardiac valve fatigue in these studies also led to loss of glycosaminoglycans (GAGs) from the cuspal extracellular matrix. The GAG levels in glutaraldehyde crosslinked porcine aortic valve cusps were 65.2 +/- 8.66 microg uronic acid/10 mg of dry weight for control and 7.91 +/- 1.1 microg uronic acid/10 mg of dry weight for 10-300 million cycled cusps. Together, these molecular changes contribute to a significant gradual decrease in cuspal bending strength as documented in a biomechanical bending assay measuring three point deformation. We conclude that fatigue-induced damage to type I collagen and loss of GAGs are major contributing factors to material degeneration in bioprosthetic cardiac valve deterioration.

Animals↗

PubMed1999

Elastin calcification and its prevention with aluminum chloride pretreatment.

Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3).

Aluminum Chloride↗

PubMed1999

Identification and characterization of calcifying valve cells from human and canine aortic valves.

BACKGROUND AND AIM OF THE STUDY: Cardiac valve calcification is the predominant pathology in patients needing valve replacement. The aim of this study was to determine if aortic valve cells calcify spontaneously and, if so, to characterize the nodular complex and response to growth factors. METHODS: Aortic valves were obtained from humans undergoing surgical valve replacement, and from female dogs. The valvular endothelium was removed and explants cultured in medium. RESULTS: A population of valvular interstitial cells spontaneously formed distinct calcified nodules containing hydroxyapatite within two to three weeks in canine and within six weeks in human aortic valves. The nodules contained an inner ring of dead cells surrounded by an outer ring of living cells. Cells associated with nodules had osteoblast-like characteristics and stained positively for extracellular bone matrix proteins. Incubating canine cells with potential calcifying stimuli tested the stimulus for calcification. The rate of nodule formation was increased with transforming growth factor beta-1 (+25 nodules), 25-hydroxycholesterol (+9 nodules) and bone morphogenetic protein 2 (+4 nodules) as compared with vehicle control (+3 nodules) over 25 days. CONCLUSIONS: We identified a population of valvular interstitial cells with osteoblast-like characteristics that spontaneously form calcific nodules in cell culture. In addition, the rate of calcific nodule formation was increased with transforming growth factor beta-1 and 25-hydroxycholesterol. Further study of these 'calcifying valve cells' may yield a new in vitro model for testing therapy aimed at preventing calcific valve stenosis.

Animals↗

PubMed1998

Inhibition of aortic wall calcification in bioprosthetic heart valves by ethanol pretreatment: biochemical and biophysical mechanisms.

The effectiveness of ethanol pretreatment on preventing calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific collagen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calcification of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80+/-8.45 microg/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90+/-7.24 microg/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circulatory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02+/-4.42 microg/mg compared to the control calcium level of 56.35+/-6.14 microg/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) studies of ethanol-pretreated BPHV aortic wall (vs. control) demonstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T1) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The optimum condition of 80% ethanol pretreatment almost completely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification occurred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effective for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bioprosthetic aortic wall calcification only in synergistic combination with another agent.

Animals↗

PubMed1997

Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms.

BACKGROUND: Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction. METHODS AND RESULTS: Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid. CONCLUSIONS: Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Animals↗