Background-Elastin calcification is a widespread feature of vascular pathology, and circumstantial evidence exists for a correlation between elastin degradation and calcification. We hypothesized that matrix metalloproteinase (MMP)-mediated vascular remodeling plays a significant role in elastin calcification. Methods and Results-In the present studies, we determined that short-term periadventitial treatment of the rat abdominal aorta with low concentrations of calcium chloride (CaCl 2 ) induced chronic degeneration and calcification of vascular elastic fibers in the absence of aneurysm formation and inflammatory reactions. Furthermore, the rate of progression of calcification depended on the application method and concentration of CaCl 2 applied periarterially. Initial calcium deposits, associated mainly with elastic fibers, were persistently accompanied by elastin degradation, disorganization of aortic extracellular matrix, and moderate levels of vascular cell apoptosis. Application of aluminum ions (known inhibitors of elastin degradation) before the CaCl 2 -mediated injury significantly reduced elastin calcification and abolished both extracellular matrix degradation and apoptosis. We also found that MMP-knockout mice were resistant to CaCl 2 -mediated aortic injury and did not develop elastin degeneration and calcification. Conclusion-Collectively, these data strongly indicate a correlation between MMP-mediated elastin degradation and vascular calcification.

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.

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Glycosaminoglycans (GAGs) are important structural and functional components in native aortic heart valves and in glutaraldehyde (Glut)-fixed bioprosthetic heart valves (BHVs). However, very little is known about the fate of GAGs within the extracellular matrix of BHVs and their contribution to BHV longevity. BHVs used in heart valve replacement surgery have limited durability due to mechanical failure and pathologic calcification. In the present study we bring evidence for the dramatic loss of GAGs from within the BHV cusp structure during storage in saline and both shortand long-term Glut fixation. In order to gain insight into role of GAGs, we compared properties of fresh and Glut-fixed porcine heart valve cusps before and after complete GAG removal. GAG removal resulted in significant morphological and functional tissue alterations, including decreases in cuspal thickness, reduction of water content and diminution of rehydration capacity. By virtue of this diminished hydration, loss of GAGs also greatly increased the ''with-curvature'' flexural rigidity of cuspal tissue. However, removal of GAGs did not alter calcification potential of BHV cups when implanted in the rat subdermal model. Controlling the extent of pre-implantation GAG degradation in BHVs and development of improved GAG crosslinking techniques are expected to improve the mechanical durability of future cardiovascular bioprostheses.

Background-Maintaining the integrity of arterial elastin is vital for the prevention of abdominal aortic aneurysm (AAA) development. We hypothesized that in vivo stabilization of aortic elastin with pentagalloyl glucose (PGG), an elastin-binding polyphenol, would interfere with AAA development. Methods and Results-Safety and efficacy of PGG treatment were first tested in vitro using cytotoxicity, elastin stability, and PGG-elastin interaction assays. For in vivo studies, the efficacy of PGG was evaluated within a well-established AAA model in rats on the basis of CaCl 2 -mediated aortic injury. With this model, PGG was delivered periadventitially at 2 separate time points during the course of AAA development; aortic diameter, elastin integrity, and other pathological aspects were monitored and evaluated in PGG-treated aortas compared with saline-treated control aortas. Our results show that a one-time periadventitial delivery of noncytotoxic levels of PGG inhibits elastin degeneration, attenuates aneurysmal expansion, and hinders AAA development in rats without interfering with the pathogenic mechanisms typical of this model, namely inflammation, calcification, and high metalloproteinase activities. PGG binds specifically to arterial elastin and, in doing so, preserves the integrity of elastic lamellae despite the presence of high levels of proteinases derived from inflammatory cells. Conclusions-Periadventitial administration of PGG hinders the development of AAA in a clinically relevant animal model. Stabilization of aortic elastin in aneurysm-prone arterial segments offers great potential toward the development of safe and effective therapies for AAAs.

Over 300,000 heart valve replacements are performed annually to replace stenotic and regurgitant heart valves. Bioprosthetic heart valves (BHVs), derived from glutaraldehyde crosslinked (GLUT) porcine aortic valve leaflets or bovine pericardium are often used. However, valve failure can occur within 12–15 years due to calcification and/or progressive degeneration. In this study, we have developed a novel fabrication method that utilizes carbodiimide, neomycin trisulfate, and pentagalloyl glucose crosslinking chemistry (TRI) to better stabilize the extracellular matrix of porcine aortic valve leaflets. We demonstrate that TRI treated leaflets show similar biomechanics to GLUT crosslinked leaflets. TRI treated leaflets had better resistance to enzymatic degradation in vitro and demonstrated better tearing toughness after challenged with enzymatic degradation. When implanted subcutaneously in rats for up to 90 days, GLUT control leaflets calcified heavily while TRI treated leaflets resisted calcification, retained more ECM components, and showed better biocompatibility.

We have developed a method for fabricating bacterial colony arrays and complex patterns using commercially available ink-jet printers. Bacterial colony arrays with a density of 100 colonies/cm(2) were obtained by directly ejecting Escherichia coli (E. coli) onto agar-coated substrates at a rapid arraying speed of 880 spots per second. Adjusting the concentration of bacterial suspensions allowed single colonies of viable bacteria to be obtained. In addition, complex patterns of viable bacteria as well as bacteria density gradients were constructed using desktop printers controlled by a simple software program.