Molecular basis of cell-biomaterial interaction: insights gained from transcriptomic and proteomic studies.

With the growing interest in clinical interventions that involve medical devices, the role for new biomaterials in modern medicine is currently expanding at a phenomenal rate. Failure of most implant materials stems from an inability to predict and control biological phenomena, such as protein adsorption and cell interaction, resulting in an inappropriate host response to the materials. Contemporary advances in biological investigation are starting to shift focus in the biomaterials field, in particular with the advent of high-throughput methodologies for gene and protein expression profiling. Here, we examine the role that emerging transcriptomic and proteomic technologies could play in relation to biomaterial development and usage. Moreover, a number of studies are highlighted which have utilized such approaches in order to try to create a deeper understanding of cell-biomaterial interactions and, hence, improve our ability to predict and control the biocompatibility of new materials.

TGF-beta1-induced thrombospondin-1 expression through the p38 MAPK pathway is abolished by fluvastatin in human coronary artery smooth muscle cells.

Thrombospondin-1 (TSP-1) and transforming growth factor-beta1 (TGF-beta1) are both implicated in the pathogenesis of in-stent restenosis. This study evaluated the hypothesis that the HMG-CoA reductase inhibitor fluvastatin inhibits TGF-beta1 induced TSP-1 expression via inhibition of p38 mitogen activated protein kinase (MAPK) phosphorylation in human coronary artery smooth muscle cells (HCASMC) and may therefore have anti-restenosis potential. Fluvastatin significantly reduced TSP-1 mRNA and protein expression in HCASMC in a concentration-dependent manner with a significant reduction in expression observed after treatment with 0.25 microM fluvastatin. TGF-beta1 (5 ng/ml) induced phosphorylation of p38 MAPK and induced TSP-1 mRNA and protein expression in HCASMC. Fluvastatin abolished TGF-beta1-induced phosphorylation of p38 MAPK and TGF-beta1-induced TSP-1 expression. Blockade of the p38 MAPK pathway with the upstream inhibitor SB-203580 also abolished TGF-beta1-induced TSP-1 expression. We conclude that fluvastatin decreases expression of TSP-1 and abolishes the ability of TGF-beta1 to induce TSP-1 expression in HCASMC; this may be achieved by preventing signalling through the p38 MAPK pathway. Targeted delivery of fluvastatin may therefore be a useful therapeutic objective for prevention of the intimal hyperplasia associated with in-stent restenosis.

Surface-induced changes in protein adsorption and implications for cellular phenotypic responses to surface interaction.

Understanding external factors that determine cellular phenotypic responses is of key interest in the field of biomaterials. Currently, material surface characteristics, protein adsorption and cellular phenotypic responses are all considered to be interrelated and ultimately determine the biocompatibility of materials. The exact nature of the relationship between these distinct, yet related, phenomena still remains to be elucidated. Through the use of a series of thermoresponsive N-isopropylacrylamide-based co-polymer films, we aimed to shed light on the relationship between surface hydrophobicity, protein adsorption and subsequent cellular response. Despite changes in co-polymer hydrophobicity mediated by altered ratios of constituent monomers, differential cellular response was only apparent in the presence of serum. Co-polymer films displayed alterations with respect to the amount of protein adsorbed on the surface, with individual serum proteins (albumin and fibronectin) displaying contrasting adsorption characteristics. Changes in protein adsorption corresponded to changes in cell adhesion, cytoskeletal organisation and cell morphology, as well as to changes in cell movement and intracellular signalling events. Examination of focal adhesion kinase (FAK), and extracellular signal-regulated kinase (ERK 1/2), important mediators of adhesion and growth factor-related signalling events, revealed a comparative reduction in phosphorylation of these signalling proteins in cells grown on co-polymers in comparison to those cultured on tissue culture polystyrene (TCP; used as a control surface). We also associated surface-mediated phenotypic alterations of cells grown on TCP and co-polymer films with particular changes in gene expression. These results indicate that cellular response to interaction with our series of co-polymer films is determined by the surface-adsorbed protein layer, which in turn is determined by the changing surface chemistry as the ratio of the co-monomers is altered.

Local drug delivery in restenosis injury: thermoresponsive co-polymers as potential drug delivery systems.

The success of percutaneous transluminal coronary angioplasty in treatment of acute coronary syndromes has been compromised by the incidence of restenosis. The physical insult of balloon insertion can damage or remove the endothelial monolayer, thereby generating a prothrombotic surface. The resulting inappropriate response to injury can also lead to penetration of inflammatory cells, conversion of the underlying media to a synthetic phenotype, deposition of extracellular matrix, constrictive remodeling, and neointimal hyperplasia. While stent implantation at the time of balloon insertion has offset some of these events, inflammatory responses to the implanted biomaterial (stent) and intimal hyperplasia are still prominent features of the procedure, leading in 20-30% of cases to in-stent restenosis within a year. Systemic delivery of drugs designed to offset in-stent restenosis injury has been largely unsuccessful, which has led to the development of strategies for coating stents with drugs for local delivery. Drug-eluting stents constitute an innovative means of further reducing the incidence of restenosis injury and clinical trials have shown encouraging results. This review focuses on properties of a class of environment-sensitive hydrogels, the N-isopropylacrylamide-based thermoresponsive co-polymers, on their potential roles as stent coatings, on their demonstrated ability to incorporate and release drugs that modify vascular endothelial and smooth muscle cell functions, and on issues that still await clarification, prior to their adoption in a clinical setting.

Interaction of soft condensed materials with living cells: phenotype/transcriptome correlations for the hydrophobic effect.

The assessment of biomaterial compatibility relies heavily on the analysis of macroscopic cellular responses to material interaction. However, new technologies have become available that permit a more profound understanding of the molecular basis of cell-biomaterial interaction. Here, both conventional phenotypic and contemporary transcriptomic (DNA microarray-based) analysis techniques were combined to examine the interaction of cells with a homologous series of copolymer films that subtly vary in terms of surface hydrophobicity. More specifically, we used differing combinations of N-isopropylacrylamide, which is presently used as an adaptive cell culture substrate, and the more hydrophobic, yet structurally similar, monomer N-tert-butylacrylamide. We show here that even discrete modifications with respect to the physiochemistry of soft amorphous materials can lead to significant impacts on the phenotype of interacting cells. Furthermore, we have elucidated putative links between phenotypic responses to cell-biomaterial interaction and global gene expression profile alterations. This case study indicates that high-throughput analysis of gene expression not only can greatly refine our knowledge of cell-biomaterial interaction, but also can yield novel biomarkers for potential use in biocompatibility assessment.

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells.

INTRODUCTION: Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS: A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS: The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS: This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.

Role of hemin in the modulation of H2O2-mediated endothelial cell injury.

Heme oxygenase (HO) has been primarily regarded as the rate-limiting enzyme in the degradation of heme. However it has recently been proposed that the inducible isoform, HO-1 (EC 1.14.99.3), functions as a stress-responsive antioxidant enzyme, with the capacity to protect against oxidant-mediated vascular injury. This study used an in vitro model of endothelial permeability to determine the effects of the HO-1-inducing agent hemin on noncytotoxic endothelial injury mediated by acute oxidant stress. Effects of hemin on oxidant-mediated cytotoxicity in a number of endothelial cell types were also investigated. A 20-min exposure of human umbilical vein endothelial cell (HUVEC) monolayers to H(2)O(2) resulted in a significant concentration-dependent increase in permeability, which was reversible 48 h later. Pretreatment of monolayers with hemin for 2 h followed by 18 h in complete medium resulted in HO-1 induction and the attenuation of H(2)O(2)-mediated increases in endothelial permeability, and significantly improved the restoration of endothelial barrier function 48 h later. In HUVEC and in the human microvascular endothelial cell line HMEC-1, hemin treatment as above resulted in protection against cytotoxicity, but not in bovine aortic endothelial cells (BAECs), where such toxicity was potentiated. This potentiation was inhibited by incubation with the HO inhibitor tin protoporphyrin IX, supporting a role for HO-1 in the potentiation of the cytotoxic response. When the exposure time of BAEC to hemin was extended to 24 h, H(2)O(2)-mediated cytotoxicity was attenuated. We conclude that hemin treatment is cytoprotective against noncytotoxic endothelial injury in vitro, under conditions that may not offer global protection against cytotoxic injury to vascular endothelium. This would indicate that HO-1 induction associated with cytotoxic injury in vivo is not always beneficial and therefore that the use of hemin as a therapeutic agent to offset oxidant injury in vascular endothelium should be undertaken with caution.

In vitro neuronal and vascular responses to 5-hydroxytryptamine: modulation by 4-methylthioamphetamine, 4-methylthiomethamphetamine and 3,4-methylenedioxymethamphetamine.

4-Methylthioamphetamine and 4-methylthiomethamphetamine are thioarylethylamines structurally related to 3,4-methylenedioxymethamphetamine (MDMA, 'Ecstasy'). This study compared effects of these agents and MDMA on 5-hydroxytryptamine (5-HT) signalling systems in the brain and vasculature in vitro. Both 4-methylthioamphetamine and 4-methylthiomethamphetamine (100 micro M) reduced the rate of specific high affinity [3H]5-HT reuptake in isolated rat brain synaptosomes to 14% and 10% of control, respectively. The concentration required for half-maximal inhibition (IC(50)) of [3H]5-HT reuptake by 4-methylthioamphetamine (0.27 micro M) was significantly lower (P<0.005) than that for inhibition by MDMA (1.28 micro M) and that for inhibition by 4-methylthiomethamphetamine (0.89 micro M). Both 4-MTA and 4-MTMA caused a significant release of preloaded [3H]5-HT from synaptosomes, but were significantly less effective than MDMA at the concentrations tested (1-100 micro M). In isolated rat aorta, a 15-min preincubation with 4-methylthioamphetamine or 4-methylthiomethamphetamine significantly reduced the maximal contraction (E(max)) induced by 5-HT to 71% or 91% of control (P<0.05 in each case), respectively. In addition, 4-methylthiomethamphetamine (100 micro M), but not 4-methylthioamphetamine, significantly increased the concentration of 5-HT required for half-maximal contraction (EC(50)) from 4.13 to 20.08 micro M (P<0.0001). In contrast, MDMA did not significantly alter the E(max) or the EC(50) of 5-HT-induced aortic contraction. It is concluded that both 4-methylthioamphetamine and 4-methylthiomethamphetamine are potent inhibitors of [3H]5-HT reuptake in the brain. Furthermore, unlike MDMA, they both directly inhibit 5-HT-mediated vascular contraction. These results suggest that these compounds may be potentially more harmful than MDMA in the context of human misuse.