Restoring cigarette smoke-induced impairment of efferocytosis in alveolar macrophages.

Cigarette smoke has been associated with susceptibility to different pulmonary and airway diseases. Impaired alveolar macrophages (AMs) that are major phagocytes in the lung have been associated with patients with airway diseases and active smokers. In the current report, we show that exposure to second-hand cigarette smoke (SHS) significantly reduced efferocytosis in vivo. More importantly, delivery of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to the alveolar space restored and refurbished the efferocytosis capability of AMs. Exposure to SHS significantly reduced expression of CD16/32 on AMs, and treatment with GM-CSF not only restored but also significantly increased the expression of CD16/32 on AMs. GM-CSF treatment increased uptake and digestion/removal of apoptotic cells by AMs. The latter was attributed to increased expression of Rab5 and Rab7. Increased efferocytosis of AMs was also tested in a disease condition. AMs from GM-CSF-treated, influenza-infected, SHS-exposed mice showed significantly better efferocytosis activity, and mice had significantly less morbidity compared with phosphate-buffered saline-treated group. GM-CSF-treated mice had increased amphiregulin levels in the lungs, which in addition to efferocytosis of AMs may have attributed to their protection against influenza. These results will have great implications for developing therapeutic approaches by harnessing mucosal innate immunity to treat lung and airway diseases and protect against pneumonia.

Correlation of factor IXa subsite modulations with effects on substrate discrimination.

BACKGROUND: A key feature of factor IXa is its allosteric transformation from an enzymatically latent form into a potent procoagulant. Although several small molecules have been found to be capable of partially affecting FIXa function (i.e. ethylene glycol, Ca(2+), and low molecular weight heparin [LMWH]), the resulting modest changes in peptidolytic activity have made the study of their mechanisms of action challenging. As these effects provide hints about potential regulatory forces that may be operational in the full expression of FIXa coagulant activity, their description remains of great interest. Studies of crystal structures have yielded insights into the structural changes induced by these effectors, but there remains a paucity of information to correlate any given structural change with specific consequences for FIXa function. OBJECTIVES: To correlate structural changes induced by these modulators with defined consequences for FIXa substrate discrimination and function. METHODS: A peptidomics-based mass spectrometry (MS) approach was used to examine the patterns of hydrolysis of four combinatorial chemistry-derived pentapeptide libraries by FIXa under various conditions in a soluble, active enzyme system. RESULTS: Ethylene glycol specifically altered the S3 subsite of FIXa to render it more tolerant to side chains at the P3 substrate position, whereas Ca(2+) enhanced tolerance at the S2 subsite. In contrast, LMWH altered both the S2 and S1' subsites. CONCLUSIONS: These results demonstrate the role of plasticity in regulating FIXa function with respect to discrimination of extended substrate sequences, as well as providing crucial insights into active site modulations that may be capitalized on by various physiologic cofactors of FIXa and in future drug design.

Tuning electrospinning parameters for production of 3D-fiber-fleeces with increased porosity for soft tissue engineering applications.

Degrapol® and PLGA electrospun fiber fleeces were characterized with regard to fiber diameter, alignment, mechanical properties as well as scaffold porosity. The study showed that electrospinning parameters affect fiber diameter and alignment in an inverse relation: fiber diameter was increased with increased flow rate, with decrease in working distance and collector velocity, whereas fiber alignment increased with the working distance and collector velocity but decreased with increased flow rate. When Degrapol® or PLGA-polymers were co-spun with increasing ratios of a water-soluble polymer that was subsequently removed; fibrous scaffolds with increased porosities were obtained. Mechanical properties correlated with fiber alignment rather than fiber diameter as aligned fiber scaffolds demonstrated strong mechanical anisotropy. For co-spun fibers the Young's modulus correlated inversely with the amount of co-spun polymer. Cell proliferation was independent of the porosity of the scaffold, but different between the two polymers. Furthermore, fibrous scaffolds with different porosities were analyzed for cell infiltration suggesting that cell infiltration was enhanced with increased porosity and increasing time. These experiments indicate that 3D-fiber fleeces can be produced with controlled properties, being prerequisites for successful scaffolds in tissue engineering applications.

Skeletal myogenesis on highly orientated microfibrous polyesterurethane scaffolds.

Skeletal myogenesis is a complex process, which is known to be intimately depending on an optimal outside-in substrate-cell signaling. Current attempts to reproduce skeletal muscle tissue in vitro using traditional scaffolds mainly suffer from poor directionality of the myofibers, resulting in an ineffective vectorial power generation. In this study, we aimed at investigating skeletal myogenesis on novel biodegradable microfibrous scaffolds made of DegraPol, a block polyesterurethane previously demonstrated to be suitable for this application. DegraPol was processed by electrospinning in the form of highly orientated ("O") and nonorientated ("N/O") microfibrous meshes and by solvent-casting in the form of nonporous films ("F"). The effect of the fiber orientation at the scaffold surface was evaluated by investigating C2C12 and L6 proliferation (via SEM analysis and alamarBlue test) and differentiation (via RT-PCR analysis and MHC immunostaining). We demonstrated that highly orientated elastomeric microfibrous DegraPol scaffolds enable skeletal myogenesis in vitro by aiding in (a) myoblast adhesion, (b) myotube alignment, and (c) noncoplanar arrangement of cells, by providing the necessary directional cues along with architectural and mechanical support.

In-vitro engineering of implantable human urinary tract tissue matrices.

Due to lack or destruction of functional tissues, congenital and acquired diseases of the genitourinary tract may need, for the engineering of these tissues, biomaterials as cell carriers. Cell cultures of human urothelial and bladder smooth muscle cells were established and growth of the latter, also under mechanical stimulation was analysed. Collagen and polyesterurethane foam scaffolds were evaluated for their aptitude as cell carriers. Scaffolds made of collagens, naturally occurring extracellular matrix proteins, are biocompatible and can induce tissue regeneration and are therefore apt for tissue engineering. The attachment of serum proteins to their surfaces does further improve these characteristics, mimicking an almost natural cell environment. We investigated the aptitude of equine collagen scaffolds (Tissue Fleece) modified by coating foetal bovine serum proteins, for human bladder smooth muscle cell attachment and growth. Furthermore we evaluated a highly porous biodegradable polyesterurethane foam (DegraPol), as scaffold for smooth muscle tissue engineering. These cell-polymer constructs were characterised by histology, scanning electron microscopy, immunohistochemistry and proliferation assays. Cell attachment and growth on collagen scaffolds improved when pre-coated with serum proteins. Cell penetration assessed by histology showed similar results on modified and native scaffolds. Further these cell-scaffold constructs were implanted in the dorsal subcutaneous space of athymic mice. In vivo studies showed the presence of the transplanted smooth muscle cells until day 3. Thereafter angiogenesis was induced and infiltration of mouse fibroblasts and polymorphonuclear cells was observed. Smooth muscle cells grown on DegraPol showed the same morphology as when grown on a control polystyrene surface. Positive immunostaining with anti-alpha smooth muscle actin indicated the preservation of the specific cell phenotype. Micrographs from scanning electron microscopy showed that the cells grew on the foam surface as well as inside the pores. The smooth muscle cells proliferated well on DegraPol, however, proliferation rate decreased due to apoptosis with time in culture. Although the above scaffolds provide an adequate milieu for cell attachment, their ability for cell penetration and growth is reduced. For this reason we are now evaluating a biodegradable poly (ethylene glycol) (PEG) hydrogel, which allows integration of cells within the matrix and also provides an excellent scaffold for the controlled incorporation of biological signals.

Diversion of prescription drugs to the black market: what the states are doing to curb the tide.

The diversion of legitimate controlled substances to the black market is a major cause of medical emergencies, fatalities, and drug-related dependencies. The effects harm not only the illegal user, but also the legitimate patient who may be getting shorted on treatments and innocent medical providers who may be charged with false claim offenses or other professional failures because of diversions that take place during their watch. The dollar magnitude of this crime is estimated to rival the black markets for both crack cocaine and heroine combined. This article addresses the various ways prescription drugs are diverted to the black market, some monitoring programs employed by the states, and guidelines that doctors, pharmacists, and other providers can use to protect themselves against possible liabilities arising from the diversion of prescription drugs. We will also address some of the oppositions to monitoring programs that have been asserted and replies to these oppositions.

DegraPol-foam: a degradable and highly porous polyesterurethane foam as a new substrate for bone formation.

Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. To evaluate the suitability of DegraPol-foam, a degradable, elastic, and highly porous polyesterurethane foam as carrier for BMP-induced bone formation, a fraction containing all the active BMPs (BMP cocktail) was combined with DegraPol-foam and implanted subcutaneously into rats. DegraPol-BMP scaffolds were found to induce osteogenesis 2 weeks after implantation as evidenced by morphological and biochemical observations. In addition, the osteoblast-compatibility of DegraPol-foam was examined here. In vitro, primary rat osteoblasts and osteoblasts from the human cell line (HFO1) attached and proliferated preferentially on the surface of the DegraPol-foam. Both cell types exhibited relatively high attachment and low doubling time that resulted in a confluent cell multilayer with spindle-shaped morphology on the surface of the foam. Osteoblasts produced high concentrations of collagen type I and osteocalcin, and expressed increasing levels of alkaline phosphatase (ALP) activity. Taken collectively, both osteoblasts from rat tibia and from the human cell line HFO1 showed high cell attachment and growth, and preserved their phenotype. The geometrical structure of DegraPol is a suitable carrier for BMP for the induction of bone formation.

Hepatic artery embolisation with a novel radiopaque polymer causes extended liver necrosis in pigs due to occlusion of the concomitant portal vein.

BACKGROUND/AIM: In an attempt to overcome some of the problems encountered with the materials available for liver embolisation, we investigated a novel radiopaque polymer of the polyurethane family (Degra-Bloc). METHODS: Hepatic artery embolisation of one liver lobe using polyurethane was performed in 19 healthy pigs. Microcirculatory changes were assessed by laser Doppler flowmetry. Radiological and pathological examinations of the livers, hearts and lungs removed provided information about the extent and effect of the embolisation. RESULTS: None of the pigs died due to hepatic failure or toxicity of polyurethane. Microcirculation of embolised liver lobes significantly decreased from 106 (+/-15) perfusion units (PU) to 45 (+/-6) PU immediately after embolisation and further to 28 (+/-7) PU before euthanasia. At this time conventional and angiographic X-ray controls demonstrated the radiopaque casts extending up to the peripheral arteries with signs of degradation over time but without formation of collateral vessels. The main pathological findings consisted of destruction of the portal tract structures and also of large areas of liver necrosis. Polyurethane was encountered in arterioles as small as 10-20 microm, but not in liver sinusoids, hearts or lungs. CONCLUSIONS: The novel polymer called DegraBloc is a biocompatible, slowly degradable, radiopaque embolic agent. The occlusion of the arterial tree up to the smallest arteriolar diameter combined with concomitant portal vein occlusion leads to sharp segmental necrosis in pig livers without formation of significant collaterals and without systemic embolism. In the treatment of liver tumours polyurethane might provide a promising alternative to conventional embolic materials, provided that it is used with care in patients with advanced liver cirrhosis.

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function.

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.

In vitro evaluation of the biofunctionality of osteoblasts cultured on DegraPol-foam.

The biofunctionality of osteoblasts cultured on DegraPol-foam, a biodegradable, elastic, and highly porous polyesterurethane-foam, was determined here to examine the possible use of this structure as bone repair material. Osteoblasts from rat tibia and from the cell line (MC3T3-E1) exhibited relatively high attachment and low doubling time that result in a confluent cell multilayer on the surface of the foam. They produced high concentrations of collagen type I and osteocalcin, and expressed increasing alkaline phosphatase activity. Exposure to 1,25-dihydroxy vitamin D (Vit. D) increased dose- and time-dependent alkaline phosphatase activity and osteocalcin concentration, and decreased the level of collagen type I and cell density. Maximal effects of Vit. D on alkaline phosphatase activity (2.2 fold), osteocalcin (1.5 fold), collagen type I (50% reduction), and on cell density (35% reduction) were found at 100 ng Vit. D ml(-1). Osteoblasts cultured on DegraPol-foam in the presence of Vit. D exhibited more spreading and less spindle-like morphology than cells cultured in the absence of Vit. D. Cell ingrowth into the pores of the foam was not affected by Vit. D treatment. Taken collectively, the osteoblasts, capability of responding to Vit. D confirms the osteoblast compatibility of DegraPol-foam and the possible use of this scaffold in the bone healing process.

Chondrocyte-biocompatibility of DegraPol-foam: in vitro evaluations.

Histological and biochemical investigations were carried out in order to evaluate the chondrocyte compatibility of a recently developed biodegradable polyesterurethane-foam (DegraPol-foam). Therefore, cell adhesion, cell growth, and the preservation of chondrocyte phenotype was measured in rat xyphoid chondrocytes seeded on DegraPol-foam. Chondrocytes, isolated from xyphoids of adult male rats, exhibited relatively high cell adhesion on DegraPol-foam (about 60% of that found on TCPS). Scanning electron microscopy (SEM) showed that chondrocytes grew on the surface and into the open cell pores of the foam. Morphologically, cells found on the surface of the foam exhibited a flat cell appearance and built a confluent cell multilayer. In contrast, the interior of the foam cells showed rounded morphology in cell aggregates and cell islets. In addition, chondrocytes proliferated on the DegraPol-foam (doubling-time of about 12.5 days) and preserved their phenotype for up to 14 days. Compared to freshly isolated chondrocytes, cells seeded on the foam produced high concentrations of collagen type II for up to 2 weeks: the ratio of type II/I collagen was 1.2-1.4 fold higher than the ratio found in freshly isolated cells. No significant difference was observed in chondroitin sulfate levels produced by freshly isolated cells and cells cultured on DegraPol-foam for up to 14 days. To sum up, our results indicate that DegraPol-foam is a compatible substrate for chondrocytes.

New versatile, elastomeric, degradable polymeric materials for medicine.

The present investigation was focused on the cell compatibility of recently developed biodegradable polyesterurethane-foam (DegraPol-foam) to chondrocytes and osteoblasts. Both chondrocytes and osteoblasts, isolated from adult male rats, exhibited relatively high cell adhesion on DegraPol-foam. Scanning electron microscopy (SEM) showed that cells grew on the surface and into the open cell pores of the foam. Morphologically, cells found on the surface of the foam exhibited a flat cell appearance and built a confluent cell multilayer. In contrast, inside the foams cell showed rounded morphology building cell aggregates and cell islets. In addition, chondrocytes and osteoblasts proliferated on the DegraPol-foam and preserved their phenotype for up to 2 weeks. During degradation of these polymers, small crystalline particles of short-chain poly[(R)-3-hydroxybutyric acid] (Mn approximately 2300) (PHB-P) and lysine methyl ester are released. Therefore, lysine methyl ester and PHB-P, as possible degradation products of the polymer, are investigated here for their effects on macrophages and osteoblasts. Results obtained in the present study clearly indicate that macrophages and, to a lesser degree, osteoblasts have the ability to take up (phagocytose) PHB-P. At low concentrations, particles of PHB failed to induce cytotoxic effects or to activate macrophages. Osteoblasts showed only limited PHB-P phagocytosis and no signs of any cellular damage. At high concentrations of PHB-P, the cell viability of macrophages and to a lesser extent of osteoblasts was affected.

Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: generation of an inactive factor VIII cofactor.

Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.

Degradable and highly porous polyesterurethane foam as biomaterial: effects and phagocytosis of degradation products in osteoblasts.

Recently, a new class of biodegradable PHB-based polyesterurethane (DegraPol/btc) has been prepared and found to exhibit favorable cell and tissue compatibility. The present study has been designed to evaluate the response of primary isolated rat tibia osteoblasts to small crystalline particles of short-chain poly[(R)-3-hydroxybutyric acid] (PHB-P diameter: 2-20 microm), of fluorescent-labeled analogs (DPHP-P), and of lysine methyl ester as possible degradation products of DegraPol/btc. Observations made using confocal microscopy clearly indicate that osteoblasts have the capability of taking up PHB-P particles. Although in single-cell analysis the number of DPHB-P-positive osteoblasts gradually increased up to 16 days, the fluorescence intensity per osteoblast increased only during the first 4 h after DPHB-P incubation, and then it retained the 4 h level up to 16 days. No significant change in the production levels of collagen type I and osteocalcin was detectable after treatment with low concentrations of PHB-P for up to 32 days. In contrast, a time- and dose-dependent alteration of the alkaline phosphatase (ALP) activity was found. Maximal activity was measured after 4 days of treatment with 2 microg of PHB-P/mL (170% of control cells). Rat peritoneal macrophages co-cultured with osteoblasts in a transwell culture system mimicked the observed PHB-P induced ALP elevation. Therefore, the PHB-P-induced ALP increase could be the result of direct or indirect stimulation of osteoblasts, possibly via soluble factors produced by contaminating osteoclasts. Taken collectively, the data demonstrate that osteoblasts are capable of phagocytosing PHB-P and that this process is accompanied at low PHB-P concentrations by dose- and time-dependent alteration of alkaline phosphatase activity but not of collagen type I or osteocalcin.

In vivo performance of a new biodegradable polyester urethane system used as a nerve guidance channel.

Biodegradable nerve guidance channels (NGCs) represent a promising alternative to current clinical nerve repair procedures. To be suitable as a NGC material, the polymer system should possess elastomeric properties and degrade at a defined rate without interfering with the regenerating environment. Polymers made of non-crystallizable blocks of poly[glycolide-co-(epsilon-caprolactone)]-diol and crystallizable blocks of poly[(R)-3-hydroxybutyric acid-co-(R)-3-hydroxyvaleric acid]-diol (PHB) can be modulated so as to respond to those criteria. Tubular structures were fabricated from three different types of materials containing either 41, 17 or 8 wt% PHB. Nerve regeneration through a 10 mm long NGC using a transected sciatic nerve model with an 8 mm gap was studied in rats at 4, 12 and 24 weeks. Out of 26 implanted NGCs, 23 contained regenerated tissue cables centrally located within the channel lumen and composed of numerous myelinated axons and Schwann cells. No significant difference in the degree of regeneration was observed between the various channel types. The inflammatory reaction associated with the polymer degradation had not interfered with the nerve regeneration process. Macrophages and giant cells surrounded polymer material remnants. A weight loss of 33, 74 and 88% for polymers containing 41, 17 and 8 wt% PHB was observed after 24 weeks by nuclear magnetic resonance (NMR) anaylsis, respectively. In all cases, the polymer fragments had a porous appearance with multiple surface cracks as evidenced by scanning electron microscopical analysis. Guidance channels made of 8 wt% PHB containing polymer displayed the highest degree of degradation at 24 weeks with only small polymer fragments remaining. The present study suggests that this new biodegradable elastomeric polymeric material holds promises for its utilization as nerve guidance channels.

Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain. A fluorescence resonance energy transfer study.

Coagulation factor VIIa (fVIIa), a soluble serine protease, exhibits full proteolytic activity only when bound to its cofactor, tissue factor (TF). Both proteins interact with membranes; TF is an integral membrane protein, while fVIIa binds reversibly to phospholipid surfaces via its Gla domain. In this study, we examine the extent to which the location of the fVIIa active site in the fVIIa.TF complex is determined by the fVIIa Gla domain. A fluorescein dye was covalently attached to the active site of fVIIa lacking the Gla domain (Gla domainless fVIIa, GD-fVIIa) via a tripeptide tether to yield fluorescein-D-Phe-Pro-Arg-GD-fVIIa (Fl-FPR-GD-fVIIa). The location of the active site of GD-fVIIa relative to the membrane surface was determined using fluorescence resonance energy transfer between the fluorescein dye in the active site of GD-fVIIa and octadecylrhodamine (OR) at the surface of phospholipid vesicles. As expected, no energy transfer was observed between Fl-FPR-GD-fVIIa and OR in vesicles composed of phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) because the Gla domain is required for the binding of fVIIa to phospholipid. However, when Fl-FPR-GD-fVIIa was titrated with PC or PC/PS vesicles into which purified TF had been reconstituted, energy transfer was observed. Based on the dependence of fluorescence resonance energy transfer on OR density, the average distance of closest approach between fluorescein in the active site of Fl-FPR-GD-fVIIa.TF and OR at the vesicle surface was determined to be 78 A (kappa2 = (2)/(3)). Since this value is nearly the same as that obtained with intact Fl-FPR-fVIIa bound to TF, the presence or absence of the fVIIa Gla domain has only a small effect on the location of the active site in the fVIIa.TF complex. The extracellular domain of tissue factor therefore must be fairly rigid and fixed relative to the surface to position and maintain the fVIIa active site far above the membrane even in the absence of the fVIIa Gla domain.

Multiblock copolyesters as biomaterials: in vitro biocompatibility testing.

Cell adhesion, cell growth and cell activities of macrophages and fibroblasts, cultured on newly developed degradable multiblock-copolyesters were studied to examine the biocompatibility and the possible use of these polymers for medical applications. The biocompatibility and the biodegradability of the polymers were confirmed by subcutaneous implantation of polymer foils in rats. The newly developed polymers, two polyesters (DegraPol/bsc43 and DegraPol/bsd43) and a polyesterether (DegraPol/bst41), were found to exhibit good cell compatibility; the cell-to-substrate interactions induced neither cytotoxic effects nor activation of macrophages. The adhesion and growth of fibroblasts and macrophages were different among the substrate. Fibroblasts adhered on the polyesters to about 60% of control cell cultured on tissue culture polystyrene (TCPS) and proliferated in the same doubling time as on TCPS. On the polyetherester cells exhibited weak adhesion; however, they proliferated up to day 4 after plating at the same doubling time as on TCPS (of about 42 h), and then decreased their doubling time to 27 h. Macrophages attached to the polyesters to about 40-60% of TCPS but no significant change was seen in the doubling time of cells cultured on TCPS and the polyesters. Again on the polyetherester, macrophages exhibited relatively low adhesion (25% of TCPS) and high doubling time (about 100 h). Fibroblasts produced high amounts (up to 500% of control cells) of collagen type I and type IV, and fibronectin. Macrophages responded to lipopolysaccharide treatment by the production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha), indicating that the cell-to-polymer interactions allow fibroblasts and macrophages to maintain their phenotype. All three test polymers exhibit favourable tissue compatibility. The formed capsule was just a few cell layers thick (<30 microm). After 2 months implanted subcutaneously in rats, the molecular weight of the test polymers was reduced by >20% depending on their chemical structure. Taken collectively, the present data demonstrate that the newly developed multiblock copolyesters are biocompatible and biodegradable.

Development of degradable polyesterurethanes for medical applications: in vitro and in vivo evaluations.

To evaluate the biocompatibility of a newly developed degradable class of polyesterurethanes and their possible use as biomaterials, we investigated the cell and tissue interactions with these polymers using a small number of chemical base entities. The polymers were prepared by chain extension with diisocyanates of PHB/HV-diol and either PCL-diol or Diorez, another aliphatic polyester-diol. Regardless of the chemical composition of the four tested polyesterurethanes used as substrates, no morphological difference was observed either in the macrophages (macrophage cell line J774) or in the fibroblasts (fibroblast cell line 3T3) cultured on the polymers. In contrast, however, cell adhesion and growth of macrophages and fibroblasts were affected by the polymer properties. Compared to macrophages cultured on tissue culture polystyrene (TCPS), cells cultured on the test polymers exhibited levels of cell adhesion that varied from 65-100% of TCPS, and the doubling time was 25-43% higher on the polymers than on TCPS. Likewise, fibroblasts adhered to the polymers at lower rates (50-85% of TCPS) and grew at higher doubling times (125-140% of TCPS). Furthermore, cells cultured on the test polymers preserved their phenotypes: fibroblasts produced high amounts (up to 280% of control cells) of collagens Type I and Type IV and fibronectin; and macrophages produced nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) in the same concentrations as control cells and responded to lipopolysaccharide treatment by the elevation of the production of NO and TNF-alpha, indicating that the cell-to-polymer interactions allow fibroblasts and macrophages to maintain their phenotypes. In vivo investigations showed that all four test polymers exhibit favorable tissue compatibility. The formed capsule was 60-250 microns thick. In addition, the polymers are degradable. After one year's subcutaneous implantation in rats, the molecular weight of the test polymers were reduced to about 50%, depending on the composition. Taken collectively, the present data demonstrate that the newly developed polyesterurethanes are cell and tissue compatible and biodegradable.

Factor VIIa-tissue factor: functional importance of protein-membrane interactions.

The first enzyme in the blood clotting cascade consists of two distinct protein subunits: a catalytic subunit (factor VIIa; FVIIa) and an essential regulatory subunit (tissue factor; TF). FVIIa is a soluble plasma protease, while TF is a cell-surface, integral-membrane protein. The recently reported X-ray crystal structure of the complex of FVIIa and the isolated extracellular domain of TF has provided important insights into the protein-protein interactions that bind these two subunits together (1). Equally important in the functioning of the TF-FVIIa complex, but much less well understood, are a series of protein-phospholipid interactions involving TF, FVIIa, and the natural substrates of this enzyme, as well as protein-protein interactions important in substrate recognition by TF-FVIIa. Here we review recent studies on the membrane organization and role of protein-phospholipid interactions in the function of TF-FVIIa, the enzyme that triggers blood clotting in hemostasis and thrombosis.

The location of the active site of blood coagulation factor VIIa above the membrane surface and its reorientation upon association with tissue factor. A fluorescence energy transfer study.

The topography of membrane-bound blood coagulation factor VIIa (fVIIa) was examined by positioning a fluorescein dye in the active site of fVIIa via a tripeptide tether to yield fluorescein-D-phenylalanyl-L-prolyl-L-arginyl-fVIIa (Fl-FPR-fVIIa). The location of the active-site probe relative to the membrane surface was determined, both in the presence and absence of tissue factor (TF), using fluorescence energy transfer between the fluorescein dye and octadecylrhodamine (OR) at the phospholipid vesicle surface. When Fl-FPR-fVIIa was titrated with phospholipid vesicles containing OR, the magnitude of OR-, calcium ion-, and phosphatidylserine-dependent fluorescence energy transfer revealed that the average distance of closest approach between fluorescein in the active site of fVIIa and OR at the vesicle surface is 82 A assuming a random orientation of donor and acceptor dyes (kappa2 = 2/3; the orientational uncertainty totals approximately 10%). The active site of fVIIa is therefore located far above the membrane surface, and the elongated fVIIa molecule must bind at one end to the membrane and project approximately perpendicularly out of the membrane. When Fl-FPR-fVIIa was titrated with vesicles that contained TF, the efficiency of energy transfer was increased by a TF-dependent translational and/or rotational movement of the fVIIa protease domain relative to the membrane surface. If this movement was solely translational, the height of the active site of fVIIa was lowered by an average of 6 A after binding to TF. The association of fVIIa with TF on the membrane surface therefore causes a significant reorientation of the active site relative to the membrane surface. This cofactor-dependent realignment of the active-site groove presumably facilitates and optimizes fVIIa cleavage of its membrane-bound substrates.