Operads for complex system design specification, analysis and synthesis.

As the complexity and heterogeneity of a system grows, the challenge of specifying, documenting and synthesizing correct, machine-readable designs increases dramatically. Separation of the system into manageable parts is needed to support analysis at various levels of granularity so that the system is maintainable and adaptable over its life cycle. In this paper, we argue that operads provide an effective knowledge representation to address these challenges. Formal documentation of a syntactically correct design is built up during design synthesis, guided by semantic reasoning about design effectiveness. Throughout, the ability to decompose the system into parts and reconstitute the whole is maintained. We describe recent progress in effective modelling under this paradigm and directions for future work to systematically address scalability challenges for complex system design.

Arterial pharmacokinetics of red wine polyphenols: implications for novel endovascular therapies targeting restenosis.

Drug-eluting stents (DESs) are endovascular devices that provide controlled release of compounds to interfere with restenosis, an adverse outcome of angioplasty characterized by thickening of the arterial wall. Accumulating evidence suggests that arterial pharmacokinetics determine the biological effect and potential toxicity of stent-based therapeutics. The aim of this study was to examine how drug polarity, drug load, and protein binding influence release from a polymer film and distribution within arterial tissue. The transport and safety profile of resveratrol (RESV) and quercetin (QUER), two red wine polyphenols known to interfere with events in the pathogenesis of restenosis, were compared with paclitaxel (Taxol), a lipophilic drug used in DES. In bovine arteries, RESV showed considerable protein binding and arterial kinetics that were found to mimick Taxol. In contrast, the less lipophilic QUER showed limited tissue distribution. Measured diffusivity of RESV and QUER was coupled with a novel computational method for assessment of biphasic drug release kinetics and arterial drug retention profiles. Modeling revealed that drugs associated with high- and low-protein-binding affinity result in markedly distinct arterial drug profiles. These data underscore the importance of arterial partitioning and propagation of drug within arterial tissue in the rational design of DES coatings.

Novel nanocomposite stent coating releasing resveratrol and quercetin reduces neointimal hyperplasia and promotes re-endothelialization.

Late-term thrombosis associated with drug-eluting stents may be due to the non-selective actions of antimitogenic drugs on endothelial cells, leading to delayed vascular healing after stenting angioplasty. Currently, there is a need for stent-based therapies that can both attenuate neointimal hyperplasia and promote re-endothelialization. The aim of this study was to compare the effects of a resveratrol (R)- and quercetin (Q)-eluting stent with that of a bare metal stent (BMS) on neointimal hyperplasia and re-endothelialization in a rat model of arterial angioplasty and stenting. Miniature stents (2.5×1.25mm) were sprayed with nanocomposite coatings containing two concentrations of R:Q (50:25µg/cm(2) (RQ1) or 150:75µg/cm(2) (RQ2)). The stents were deployed into the common carotid artery of rats and their impact on vascular remodeling was compared to that of BMS. Luminal stenosis in arteries stented with RQ2-eluting stents was reduced by 64.6% (p<0.05) compared to arteries stented with BMS. Accompanying this effect was a 59.8% reduction in macrophage infiltration (p<0.05). There were no differences found between RQ1 and BMS. Finally, the RQ2-coated stent accelerated re-endothelialization by 50% compared with BMS (p<0.05). Thus, compared with BMS, local delivery of R and Q from a stent platform significantly reduced in-stent stenosis, while promoting re-endothelialization. These data suggest that R and Q may be favorable candidates for novel stent coatings, potentially reducing the risk of late thrombosis associated with drug-eluting stents.

A dietary approach to increase in-stent stenosis and face validity of a rat model for arterial angioplasty and stenting.

OBJECTIVE: To expedite the investigation of new devices for inhibiting restenosis, we aimed to develop a modified model of arterial angioplasty and stenting in rats that showed greater face validity than the traditional rat model. METHODS: Carotid arteries from Sprague-Dawley rats fed a normal or an atherogenic diet containing a low dose of cholate underwent balloon pre-dilation followed by placement of a bare metal stent. Vessel patency was followed for 28d using ultrasound. Stented vessels were then harvested and were subjected to histologic analysis. Plasma lipid profiles and biomarkers of endothelial dysfunction, inflammation and thrombosis were assessed. RESULTS: There was significant interaction between stenting injury and the atherogenic diet, leading to higher levels of markers for inflammation, platelet activation, and endothelial dysfunction, as well as neointimal hyperplasia, compared with stented rats on normal chow. There was a significant correlation between plasma IL-6 and TXB(2) in stented rats, a relationship which may have contributed to exaggerated vessel remodeling with increased platelet sensitivity. Compared to normal chow, the atherogenic diet also increased fibrin and proteoglycan deposition near stent struts. CONCLUSIONS: Arterial stenting, in combination with the atherogenic diet, led to exacerbated endothelial dysfunction, inflammation, platelet activation, and vascular remodeling compared with stented rats on normal chow. By reproducing key features of clinical restenosis that are lacking in other rat models, this modified rat model may serve as a valuable screening tool to rapidly evaluate new coatings and devices before moving candidates into expensive, more time-consuming rabbit or porcine models.

Synergistic effect of resveratrol and quercetin released from drug-eluting polymer coatings for endovascular devices.

This study describes the development and evaluation of novel polymer films that provide controlled release of two vascular-protective polyphenols for endovascular devices. Resveratrol (RESV) and quercetin (QUER) have antimigratory and antiproliferative actions on vascular smooth muscle cells (VSMCs), inhibit both platelet and inflammatory cell activation, and promote endothelial cell function. Our aim is to develop and characterize coatings that release these drugs within a therapeutic range. The most synergistic drug combination, as determined by isobolographic analysis, was incorporated into an arborescent poly(styrene-isobutylene-styrene) tri-block polymer (arbIBS) and applied to stainless steel coupons using an electrospray process. Physical characterization of the resulting coating revealed a film featuring micro-scale architecture consisting of drug-containing domains. To determine drug-mediated effects, vascular cells were exposed to coatings incorporating several loadings of RESV and QUER. Results from this study indicate that arbIBS exhibits no cytotoxicity, and that the films release RESV and QUER at therapeutic levels, dose-dependently inhibiting macrophage activation, VSMC proliferation, and platelet stimulation. We conclude that RESV and QUER released from arbIBS interfere with key processes responsible for in-stent stenosis, suggesting that RESV and QUER may have utility as therapeutics in a novel coating for device-based interventions.

Effect of the coating morphology on the drug release from engineered drug-polymer nanocomposites.

The ElectroNanospray process (Nanocopoeia, Inc) transforms drugs and polymers into many nanoscale material states including powders, liquids, encapsulated particles, and coatings. This enabling technology platform allows application of polymers and drugs to the surface of medical devices such as coronary stents in a single-stage process. Modification of ElectroNanospray process parameters resulted in surface coatings with rich morphologies ranging in appearance from smooth and heterogeneous to highly porous and rough (open matrix). The traditional approach of measuring percent release over time by HPLC shows that the drug release profiles change significantly with coating morphology. In this study, we employed high resolution imaging techniques such as SEM, Atomic Force Microscopy (AFM) and Confocal Raman Microscopy to elucidate the drug release process on these coatings in situ, indicating a correlation of release kinetics with coating morphology.

Multimodal dynamic imaging of therapeutic biomedical coatings in aqueous medium.

Drug release from therapeutic biomedical films such as drug-polymer composite coatings on drug eluting stents is a highly complex and poorly understood process. The dynamics of drug release and the evolution of surface morphology during release have direct impact on the performance of the device. This information is not easily accessible, and there have been few systematic studies to investigate drug release from biomedical coatings in real time. In this study, the complementary analytical techniques of confocal Raman microscopy, in-liquid atomic force microscopy, scanning electron microscopy, and high performance liquid chromatography were used to examine real-time mobilization and release of the drug rapamycin from polyisobutylene-block-polystyrene thin films, during immersion in buffered saline for 12 h. Each technique was found to have distinct limitations in either temporal or spatial resolution; in combination, however, the overlapping techniques provided a level of detail that is not available using any single approach.

Prevention of cold ischemia/rewarming-induced ERK 1/2, p38 kinase and HO-1 activation by trophic factor supplementation of UW solution.

We have previously shown that trophic factor supplementation (TFS) of University of Wisconsin (UW) solution reduced early apoptotic changes in vascular endothelial cells. Here, we examine the effect of TFS on cell signaling pathways related to cell growth, differentiation, and apoptosis after cold ischemic storage. In this study, the effect of TFS on the phosphorylation of signaling molecules ERK (extracellular regulated-signaling kinase) 1/2 and p38 MAPK (mitogen activated protein kinases) and of HO-1 (hemeoxygenase-1), relative to changes seen in unmodified UW solution, were determined by Western blot in cells stored under cold ischemic conditions. Primary cultures of canine kidney proximal tubule cells (CKPTC) and human umbilical vein endothelial cells (HUVEC) were used in this study. There was a significant decrease, relative to UW solution, after 1 min rewarming in ERK 1 and 2 activity in CKPTCs. For p38 MAPK, a significant decrease after 5 min rewarming was seen in CKPTC (p<0.05) while significant reductions relative to UW solution were seen in HUVECs after both 1 and 5 min rewarming (p<0.05). Phosphorylated HO-1 was also decreased by 43% and 50% in HUVECs, relative to UW solution, after 1 and 5 min rewarming (p<0.05 at each time point). Collectively, TFS not only limits ERK 1/2 and p38 MAPK activity induced by cold ischemic injury and subsequent rewarming, but also substantially restricted increases in HO-1 phosphorylation.

The effect of trophic factor supplementation on cold ischemia-induced early apoptotic changes.

We have previously shown that trophic factor supplementation (TFS) of University of Wisconsin (UW) solution enhanced kidney viability after cold storage. Here, we use an in vitro model to study the effect of TFS on early apoptotic changes after cold ischemic storage. Mitochondrial membrane potential was determined by fluorescence intensity in primary canine kidney tubule cells, Madin-Darby canine kidney cells, and human umbilical vein endothelial cells. In addition, caspase 3 enzyme activity assay and immunofluorescence staining were performed to evaluate apoptosis. There was a 15% increase in mitochondrial membrane potential in human umbilical vein endothelial cells stored in trophic factor supplemented University of Wisconsin solution after four-hour rewarming (P<0.05). TFS suppressed caspase 3 enzyme activity and activation in human umbilical vein endothelial cells. We confirmed that the presence of TFS in UW solution has a beneficial effect by protecting mitochondrial function and reducing early apoptotic changes in vascular endothelial cells.

Adolescent use and misuse of marijuana.

Substance use by adolescents and young adults continues to be a serious problem. Marijuana remains the most commonly used illicit substance with close to 50% of high school seniors admitting use at some time. Each year 2.6 million individuals in this country become new users and most are under 19 years old. Individuals who provide health care services to this age group must have an understanding of the drug, its pharmacokinetics, and the many short- and long-term adverse effects. Familiarity with risk factors associated with initiating use can be helpful in screening older children and targeting anticipatory guidance toward those most likely to benefit. This article reviews these issues and includes commentary on a recently published review of treatment programs.

The effect of environmental factors on the response of human corneal epithelial cells to nanoscale substrate topography.

We have previously shown that human corneal epithelial cells sense and react to nanoscale substrate topographic stimuli [Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci 2003;116(10):1881-92; Karuri NW, Liliensiek S, Teixeira AI, Abrams G, Campbell S, Nealey PF, et al. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells. J Cell Sci 2004;117(15):3153-64]. Here we demonstrate that cellular responses to nanoscale substrate topographies are modulated by the context in which these stimuli are presented to cells. In Epilife medium, cells aligned preferentially in the direction perpendicular to nanoscale grooves and ridges. This is in contrast to a previous study where cells cultured in DMEM/F12 medium aligned in the direction parallel to nanoscale topographic features [Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci 2003;116(10):1881-92]. Additionally, cell alignment in Epilife medium was dependent on pattern pitch. Cells switched from perpendicular to parallel alignment when the pitch was increased from 400 to 4,000 nm. There was a transition region (between 800 and 1,600 nm pitch) where both parallel and perpendicular alignments were favored compared to all other cellular orientations. Cells formed focal adhesions parallel to the substrate topographies in this transition region. On the nano- and microscale patterns, 400 and 4,000 nm pitch, focal adhesions were almost exclusively oriented obliquely to the topographic patterns.

Cooperative modulation of neuritogenesis by PC12 cells by topography and nerve growth factor.

Understanding axonal formation and contact guidance are of critical importance for the design of materials that interface with neuronal tissue. Contact guidance of neurites by topographic features is well known, but the role that topography plays in the modulation of neuritogenesis has not been addressed. To test this, we cultured PC12 cells with a range of nerve growth factor (NGF) concentrations on surfaces with ridge widths ranging from 70 to 1900 nm. We find that neuritogenesis by PC12 cells cultured with sub-optimal concentrations of NGF (25 and 5 ng/ml) is modulated by topographic feature size. The threshold for induction of neuritogenesis was markedly reduced when cells were cultured on ridges of 70 and 250 nm. In contrast, contact guidance of neurites was independent of feature size. These results suggest that the scale of topographic features can act cooperatively with NGF signaling to regulate the formation of neurites. These findings may have general relevance to differentiation processes in neurons as well as in other cell types.

Suppression of cold ischemic injury in stored kidneys by the antimicrobial peptide bactenecin.

BACKGROUND: Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys. METHODS: Bactenecin (L- and D-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro. RESULTS: Bactenecin (both L- and D- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin-Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics. CONCLUSIONS: These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage.

Temporal regulation of VEID-7-amino-4-trifluoromethylcoumarin cleavage activity and caspase-6 correlates with organelle loss during lens development.

Lens fiber cell differentiation involves extensive reconstruction of the cell's architecture, including the degradation and elimination of all membrane-bound organelles via a process that has been likened to apoptosis. Using caspase reporter assays under conditions in which nonspecific cleavage of the reporter peptides by the proteasome has been inhibited, we investigated whether any specific caspase activities are temporally correlated with this process of organelle loss. Extracts from neonatal mouse lenses contained strong VEID-7-amino-4-trifluoromethylcoumarin (AFC) and minor IETD-AFC and LEVD-AFC cleavage activities, but no DEVD-AFC cleavage activity. Further testing suggested that the VEID-AFC and IETD-AFC cleavage activities were likely due to the same enzyme. In lens extracts from rat embryos, VEID-AFC cleavage activity increased during the period when organelles are eliminated, between embryonic days 15.5 and 18.5, whereas procaspase-6 protein levels decreased, suggesting that this enzyme is responsible for VEID-AFC cleavage. By contrast, in extracts from alpha AE7 transgenic mouse lenses in which apoptosis was induced, strong DEVD-AFC cleavage activity and activated caspase-3 protein were detected. Thus, within the same tissue, different caspase activities can predominate depending on the context, normal differentiation versus apoptosis. These results highlight the difference between normal fiber cell differentiation and apoptosis and the capacity of the lens to differentially regulate these two processes.