Interaction of a block-co-polymeric biomaterial with immunoglobulin G modulates human monocytes towards a non-inflammatory phenotype.

Monocyte interactions with implanted biomaterials can contribute significantly to the ability of a biomaterial to support tissue integration and wound healing, as opposed to a chronic pro-inflammatory foreign body reaction, provided the materials are designed to do so. However, there are few biomaterials available designed to regulate immune cell response with the intention of reducing the pro-inflammatory activation state. Material chemistry is a powerful tool for regulating protein and cell interactions that can be incorporated into surfaces while maintaining desired mechanical properties. The aspects of material chemistry that can support monocyte activation away from a pro-inflammatory state are still poorly understood. Protein adsorption is a key initial event that transforms the surface of a biomedical device into a biological substrate that will govern subsequent cellular interactions. In this study, the chemistry of degradable block polyurethanes, termed degradable polar hydrophobic ionic (D-PHI) polyurethanes, were studied for their unique interactions with bound immunoglobulin G (IgG), a pro-inflammatory protein that supports monocyte-biomaterial interactions. The specific immunological active sites of the polyurethane-adsorbed protein were compared with IgG's adsorbed state on a homopolymeric material with surface chemistry conducive to cell interactions, e.g. tissue culture polystyrene (TCPS). IgG-coated TCPS supported sustained monocyte adhesion and enhanced monocyte spreading, effects not observed with IgG-coated PU. The degradable PU was subsequently shown to reduce the number of exposed IgG-Fab sites following pre-adsorption vs. IgG adsorbed to TCPS, with antibody inhibition experiments demonstrating that Fab-site exposure appears to dominate monocyte-biomaterial interactions. Minor changes in chemical segments within the PU molecular chains were subsequently investigated for their influence on directing IgG interactions towards reducing pro-inflammatory activity. A reduction in chemical heterogeneity within the PU, without significant differences in other material properties known to regulate monocyte response, was shown to increase Fab exposure and subsequently led to monocyte interactions similar to those observed for IgG-coated TCPS. These results infer that reduced IgG-Fab site exposure can be directed by material chemistry to attenuate pro-inflammatory monocyte interactions with biomaterial surfaces, and identify the chemical features of polymeric biomaterial design responsible for this process. STATEMENT OF SIGNIFICANCE: There is currently limited understanding of material design features that can regulate protein-material interactions in order to prevent adverse inflammatory responses to implanted biomaterials. In this paper, monocyte interactions with biomaterials (specifically a block co-polymeric degradable polyurethane [D-PHI] and tissue culture polystyrene [TCPS]) were investigated as a function of their interactions with adsorbed immunoglobulin G (IgG). D-PHI was shown to attenuate IgG-induced monocyte retention and spreading by reducing IgG-Fab site exposure upon adsorption relative to TCPS. Aspects of D-PHI chemistry important in regulating Fab site exposure were determined. This study thus identifies features of biomaterials, using D-PHI as a case study, which can contribute to the development of new immunomodulatory biomaterial design.

Immunomodulatory polymeric scaffold enhances extracellular matrix production in cell co-cultures under dynamic mechanical stimulation.

Despite the importance of immune cells in regulating the wound healing process following injury, there are few examples of synthetic biomaterials that have the capacity to push the body's immune cells toward pro-regeneration phenotypes, and fewer still that are designed with the intention of achieving this immunomodulatory character. While monocytes and their derived macrophages have been recognized as important contributors to tissue remodeling in vivo, this is primarily believed to be due to their ability to regulate other cell types. The ability of monocytes and macrophages to generate tissue products themselves, however, is currently not well appreciated within the field of tissue regeneration. Furthermore, while monocytes/macrophages are found in remodeling tissue that is subjected to mechanical loading, the effect this biomechanical strain on monocytes/macrophages and their ability to regulate tissue-specific cellular activity has not been understood due to the complexity of the many factors involved in the in vivo setting, hence necessitating the use of controlled in vitro culture platforms to investigate this phenomenon. In this study, human monocytes were co-cultured with human coronary artery smooth muscle cells (VSMCs) on a tubular (3mm ID) degradable polyurethane scaffold, with a unique combination of non-ionic polar, hydrophobic and ionic chemistry (D-PHI). The goal was to determine if such a synthetic matrix could be used in a co-culture system along with dynamic biomechanical stimulus (10% circumferential strain, 1Hz) conditions in order to direct monocytes to enhance tissue generation, and to better comprehend the different ways in which monocytes/macrophages may contribute to new tissue production. Mechanical strain and monocyte co-culture had a complementary and non-mitigating effect on VSMC growth. Co-culture samples demonstrated increased deposition of sulphated glycosaminoglycans (GAGs) and elastin, as well as increases in the release of FGF-2, a growth factor that can stimulate VSMC growth, while dynamic culture supported increases in collagen I and III as well as increased mechanical properties (elastic modulus, tensile strength) vs. static controls. Macrophage polarization toward an M1 state was not promoted by the biomaterial or culture conditions tested. Monocytes/macrophages cultured on D-PHI were also shown to produce vascular extracellular matrix components, including collagen I, collagen III, elastin, and GAGs. This study highlights the use of synthetic biomaterials having immunomodulatory character in order to promote cell and tissue growth when used in tissue engineering strategies, and identifies ECM deposition by monocytes/macrophages as an unexpected source of this new tissue. STATEMENT OF SIGNIFICANCE: The ability of biomaterials to regulate macrophage activation towards a wound healing phenotype has recently been shown to support positive tissue regeneration. However, the ability of immunomodulatory biomaterials to harness monocyte/macrophage activity to support tissue engineering strategies in vitro holds enormous potential that has yet to be investigated. This study used a monocyte co-culture on a degradable polyurethane (D-PHI) to regulate the response of VSMCs in combination with biomechanical strain in a vascular tissue engineering context. Results demonstrate that immunomodulatory biomaterials, such as D-PHI, that support a desirable macrophage activation state can be combined with biomechanical strain to augment vascular tissue production in vitro, in part due to the novel and unexpected contribution of monocytes/macrophages themselves producing vascular ECM proteins.

Monocyte/macrophage cytokine activity regulates vascular smooth muscle cell function within a degradable polyurethane scaffold.

Tissue engineering strategies rely on the ability to promote cell proliferation and migration into porous biomaterial constructs, as well as to support specific phenotypic states of the cells in vitro. The present study investigated the use of released factors from monocytes and their derived macrophages (MDM) and the mechanism by which they regulate vascular smooth muscle cell (VSMC) response in a VSMC-monocyte co-culture system within a porous degradable polyurethane (D-PHI) scaffold. VSMCs cultured in monocyte/MDM-conditioned medium (MCM), generated from the culture of monocytes/MDM on D-PHI scaffolds for up to 28 days, similarly affected VSMC contractile marker expression, growth and three-dimensional migration when compared to direct VSMC-monocyte co-culture. Monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) were identified as two cytokines present in MCM, at concentrations that have previously been shown to influence VSMC phenotype. VSMCs cultured alone on D-PHI scaffolds and exposed to MCP-1 (5 ng ml(-1)) or IL-6 (1 ng ml(-1)) for 7 days experienced a suppression in contractile marker expression (with MCP-1 or IL-6) and increased growth (with MCP-1) compared to no cytokine medium supplementation. These effects were also observed in VSMC-monocyte co-culture on D-PHI. Neutralization of IL-6, but not MCP-1, was subsequently shown to decrease VSMC growth and enhance calponin expression for VSMC-monocyte co-cultures on D-PHI scaffolds for 7 days, implying that IL-6 mediates VSMC response in monocyte-VSMC co-cultures. This study highlights the use of monocytes and their derived macrophages in conjunction with immunomodulatory biomaterials, such as D-PHI, as agents for regulating VSMC response, and demonstrates the importance of monocyte/MDM-released factors, such as IL-6 in particular, in this process.

Development of a three-dimensional in vitro model system to study orthodontic tooth movement.

Few three-dimensional (3-D) models exist to study the cellular aspects and molecular regulation of orthodontic tooth movement (OTM). The aim of this study was to develop a 3-D in vitro model to study mechanical loading of human periodontal ligament (PDL) fibroblasts (hPDLF). hPDLF were seeded within collagen gels to form a PDLF analogue. Characterisation of the seeded collagen gels revealed that the gels supported cell proliferation, viability and the emergence of a possible contractile phenotype, replicating the constrained condition of the human PDL in vivo. We next developed a 3-D model that incorporated a seeded collagen gel interlocked mechanically at two ends to movable end plates. The movable end plates allowed for static tensile or compressive loading of the hPDLF-seeded collagen gels. Preliminary testings showed that this 3-D model mimicked PDL strains similar to those observed during OTM. Our 3-D model of OTM therefore offers promise for use as a model system in future studies to improve our understanding of the effects of OTM on PDLF.

Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves.

The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.69 +/- 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 +/- 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated approximately 85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.

Bone regeneration via a mineral substrate and induced angiogenesis.

Angiogenesis and biomineral substrates play major roles in bone development and regeneration. We hypothesized that macroporous scaffolds of biomineralized 85:15 poly(lactide-co-glycolide), which locally release vascular endothelial growth factor-165 (VEGF), would direct simultaneous regeneration of bone and vascular tissue. The presence of a bone-like biomineral substrate significantly increased regeneration of osteoid matrix (32 +/- 7% of total tissue area; mean +/- SD; p < 0.05) and mineralized tissue (14 +/- 2%; P < 0.05) within a rat cranium critical defect compared with a non-mineralized polymer scaffold (19 +/- 8% osteoid and 10 +/- 2% mineralized tissue). Further, the addition of VEGF to a mineralized substrate significantly increased the generation of mineralized tissue (19 +/- 4%; P < 0.05) compared with mineralized substrate alone. This appeared to be due to a significant increase in vascularization throughout VEGF-releasing scaffolds (52 +/- 9 vessels/mm(2); P < 0.05) compared with mineralized scaffolds without VEGF (34 +/- 4 vessels/mm(2)). Surprisingly, there was no significant difference in total osteoid between the two samples, suggesting that increased vascularization enhances mineralized tissue generation, but not necessarily osteoid formation. These results indicate that induced angiogenesis can enhance tissue regeneration, supporting the concept of therapeutic angiogenesis in tissue-engineering strategies.

Identifying substrates for endothelium-specific Tie-2 receptor tyrosine kinase from phage-displayed peptide libraries for high throughput screening.

The peptide substrate specificity of Tie-2 was probed using the phage display method in order to identify efficient substrate for high throughput screening. Two random peptide libraries, pGWX3YX4 and pGWX4YX4, were constructed, in which all twenty amino acid residues were represented at the X positions flanking the fixed tyrosine residue Y. A fusion protein of GST and the catalytic domain of human Tie-2 was used to perform the phage phosphorylation. The phosphorylated phage particles were enriched by panning over immobilized anti-phosphotyrosine antibody pY20 for a total of 5 rounds. Four phage clones (3T61, 3T68, C1-90 and D1-15) that express a peptide sequence that can be phosphorylated by the recombinant catalytic domain of human Tie-2 were identified. Synthetic peptides made according to the sequences of the 4 selected clones from the two libraries, which had widely different sequences, were active substrates of Tie-2. Kinetic analysis revealed that D1-15 had the best catalytic efficiency with a k(cat)/K(m) of 5.9x10(4) M(-1) s(-1). Three high throughput screening assay formats, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radioactive plate binding (RPB) and time-resolved fluorescent resonance energy transfer (TR-FRET) were developed to assess the suitability of these phage display selected peptides in screening Tie-2 inhibitors. Three out of four peptides were functional in the DELFIA assay and D1-15 was functional in the TR-FRET assay.

Mechanical regulation of localized and appositional bone formation around bone-interfacing implants.

The local mechanical environment around bone-interfacing implants determines, in large part, whether bone formation leading to functional osseointegration will occur. Previous attempts to relate local peri-implant tissue strains to tissue formation have not accounted for implant surface geometry, which has been shown to influence early tissue healing in vivo. Furthermore, the process by which mechanically regulated peri-implant bone formation occurs has not been considered previously. In the current study, we used a unit cell approach and the finite element method to predict the local tissue strains around porous-surfaced and plasma-sprayed implants, and compared the predictions to patterns of bone formation reported in earlier in vivo experiments. Based on the finite element predictions, we determined that appositional bone formation occurred when the magnitudes of the strain components at the tissue-host bone interface were <8%. Localized, de novo bone formation occurred when the distortional tissue strains were less than approximately 3%. Based on these threshold tissue strains, we propose a mechanoregulatory model to relate local tissue strains to the process of peri-implant bone formation. The mechanoregulatory model is novel in that it predicts both appositional and localized bone formation and its predictions are dependent on implant surface geometry. The model provides initial criteria with which the osseointegration potential of bone-interfacing implants may be evaluated, particularly under conditions of immediate or early loading.

Differences in osseointegration rate due to implant surface geometry can be explained by local tissue strains.

Experimental evidence indicates that the surface geometry of bone-interfacing implants influences the nature and rate of tissues formed around implants. In a previously reported animal model study, we showed that non-functional, press-fitted porous-surfaced implants placed in rabbit femoral condyle sites osseointegrated more rapidly than plasma-sprayed implants. We hypothesized that the accelerated osseointegration observed with the porous-surfaced design was the result of this design providing a local mechanical environment that was more favourable for bone formation. In the present study, we tested this hypothesis using finite element analysis and homogenization methods to predict the local strains in the pre-mineralized tissues formed around porous-surfaced and plasma-sprayed implants. We found that, for loading perpendicular to the implant interface, the porous surface structure provided a large region that experienced low distortional and volumetric strains, whereas the plasma-sprayed implant provided little local strain protection to the healing tissue. The strain protected region, which was within the pores of the sintered porous surface layer. corresponded to the region where the difference in the amount of mineralization between the two implant designs was the greatest. Low distortional and volumetric strains are believed to favour osteogenesis, and therefore the model results provide initial support for the hypothesis that the porous-surfaced geometry provides a local mechanical environment that favours more rapid bone formation in certain situations.

Osseointegration of sintered porous-surfaced and plasma spray-coated implants: An animal model study of early postimplantation healing response and mechanical stability.

The osseointegration and long-term success of bone-interfacing implants are dependent on mechanical stability of the implant relative to host bone during the early healing period. The geometric design of an implant surface may play an important role in affecting early implant stabilization, possibly by influencing tissue healing dynamics. In this study, we compared the early tissue healing response and resulting implant stability for two surface designs by characterizing the histological and mechanical properties of the healing tissue around Ti6Al4V sintered porous-surfaced and Ti plasma-sprayed implants. The implants were inserted transversely in rabbit femoral condyles and evaluated at 0, 4, 8, and 16 days postimplantation. At 4 and 8 days after implantation, the early healing tissue (fibrin and collagenous matrix) was more extensively integrated with the three-dimensional interconnected structure of the sintered porous surface than with the irregular geometry of the plasma-sprayed coating. In addition, histological examination indicated that initial matrix mineralization leading to osseointegration occurred more rapidly with the porous-surfaced implants. The more extensive tissue integration and more rapid matrix mineralization with the porous-surfaced implants were reflected in the mechanical test data, which demonstrated greater attachment strength and interfacial stiffness for the porous-surfaced implants 4 and 8 days postimplantation (p <.05). Sixteen days after implantation, both implant designs were osseointegrated and had comparable attachment characteristics. These data demonstrate that appropriate surface design selection can improve early implant stability and induce an accelerated healing response, thereby improving the potential for implant osseointegration.

Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice.

The mechanism of liver cell injury induced by an overdose of the analgesic acetaminophen (AAP) remains controversial. Recently, it was hypothesized that a significant number of hepatocytes die by apoptosis. Since caspases have been implicated as critical signal and effector proteases in apoptosis, we investigated their potential role in the pathophysiology of AAP-induced liver injury. Male C3Heb/FeJ mice were fasted overnight and then treated with 500 mg/kg AAP. Liver injury became apparent at 4 h and was more severe at 6 h (plasma ALT activities: 4110 +/- 320 U/liter; centrilobular necrosis). DNA fragmentation increased parallel to the increase of plasma ALT values. At 6 h there was a 420% increase of DNA fragmentation and a 74-fold increase of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells located predominantly around central veins. However, the activity of the proapoptotic caspase-3 was not increased at any time after AAP. In contrast, injection of the anti-Fas antibody Jo-2 (positive control) caused a 28-fold increase of caspase-3 activity and severe DNA fragmentation before significant ALT release. Treatment with the caspase inhibitor ZVAD-CHF2 had no effect on AAP toxicity but completely prevented Jo-mediated apoptosis. In contrast, Jo-induced caspase activation and apoptosis could be inhibited by AAP treatment in a time- and dose-dependent manner. We conclude that AAP-induced DNA fragmentation does not involve caspases, suggesting a direct activation of endonucleases through elevated Ca2+ levels. In addition, electrophilic metabolites of AAP may inactivate caspases or their activation pathway. This indicates that AAP metabolism has the potential to inhibit signal transduction mechanisms of receptor-mediated apoptosis.

Automation of yeast two-hybrid screening.

We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.

Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and Fas-antibody-induced liver injury.

Endotoxin (ET) induces neutrophil sequestration in hepatic sinusoids, the activation of proinflammatory transcription factors (nuclear factor KB [NF-kappaB]) with up-regulation of adhesion molecules on sinusoidal endothelial cells and hepatocytes. However, if galactosamine (Gal) is co-administered with ET, neutrophils transmigrate and attack parenchymal cells. This suggests that a signal from parenchymal cells triggers neutrophil transmigration. In this study, we tested the hypothesis that parenchymal cell apoptosis may induce neutrophil transendothelial migration in the Gal/ET model. Treatment of C3Heb/FeJ mice with 700 mg/kg Gal and 100 microg/kg ET induced tumor necrosis factor alpha (TNF-alpha) formation (13.25 +/- 0.75 ng/mL) and hepatic NF-kappaB activation at 90 minutes; the generation of the C-X-C chemokine KC (2.86 +/- 0.30 ng/mL at 5 hours); sinusoidal neutrophil sequestration (380 +/- 21 polymorphonuclear leukocytes/50 high-power fields) and apoptosis (925% +/- 29% increase of DNA fragmentation; and a 45-fold increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells) at 6 hours, followed by transmigration of neutrophils and development of substantial necrosis (38% +/- 3% of hepatocytes; alanine transaminase [ALT]: 1,500 +/- 300 U/L) at 7 hours. Administration of uridine (1,000 mg/kg) did not reduce plasma levels of TNF-alpha and KC, NF-kappaB activation, or polymorphonuclear leukocyte sequestration, but attenuated apoptosis by 90% to 94%. In these livers, neutrophils did not transmigrate and liver injury was prevented (necrosis: < 5%; ALT: 40 +/- 3 U/L). However, massive apoptosis and liver injury initiated by the anti-Fas antibody, Jo2, did not recruit neutrophils into the liver. We conclude that excessive parenchymal cell apoptosis represents an important signal for transmigration of primed neutrophils sequestered in sinusoids during endotoxemia in vivo. However, apoptosis per se does not cause neutrophil sequestration in the liver vasculature.

Activation of caspase 3 (CPP32)-like proteases is essential for TNF-alpha-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model.

Endotoxin (ET)-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. Members of the caspase family have been implicated in the signal transduction pathway of apoptosis. The aim of this study was to characterize ET-induced hepatic caspase activation and apoptosis and to investigate their effect on neutrophil-mediated liver injury. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine (Gal) and 100 microg/kg Salmonella abortus equi ET increased caspase 3-like protease activity (Asp-Val-Glu-Asp-substrate) by 1730 +/- 140% at 6 h. There was a parallel enhancement of apoptosis (assessed by DNA fragmentation ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). In contrast, activity of caspase 1 (IL-1beta-converting enzyme)-like proteases (Tyr-Val-Ala-Asp-substrate) did not change throughout the experiment. Caspase 3-like protease activity and apoptosis was not induced by Gal/ET in ET-resistant mice (C3H/HeJ). Furthermore, only murine TNF-alpha but not IL-1alphabeta increased caspase activity and apoptosis. Gal/ET caused neutrophil-dependent hepatocellular necrosis at 7 h (area of necrosis, 45 +/- 3%). Delayed treatment with the caspase 3-like protease inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD) (10 mg/kg at 3 h) attenuated apoptosis by 81 to 88% and prevented liver cell necrosis (< or = 5%). Z-VAD had no effect on the initial inflammatory response, including the sequestration of neutrophils in sinusoids. However, Z-VAD prevented neutrophil transmigration and necrosis. Our data indicate that activation of the caspase 3 subfamily of cysteine proteases is critical for the development of parenchymal cell apoptosis. In addition, excessive hepatocellular apoptosis can be an important signal for transmigration of primed neutrophils sequestered in sinusoids.

Increased P-selectin gene expression in the liver vasculature and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock.

We studied the role of P-selectin, an adhesion molecule known to be important for neutrophil localization to sites of inflammation, in a model of inflammatory liver injury. Male C3Heb/FeJ (ET-sensitive) mice treated with 700 mg/kg galactosamine and 100 microg/kg Salmonella abortus equi endotoxin (Gal/ET), murine tumor necrosis factor alpha (TNF-alpha, 15 microg/kg), or interleukin-1 (IL-1, 13-23 microg/kg), showed increased P-selectin mRNA levels in the liver. In contrast, C3H/HeJ (ET-resistant) mice responded only to cytokines with P-selectin mRNA formation. Whereas no P-selectin expression was detectable in control livers, there was temporary staining of endothelium in large blood vessels but not in sinusoids between 3 and 5 h after ET, TNF-alpha, or IL-1 treatment. Severe liver injury induced by Gal/ET at 7 h was not inhibited by an anti-P-selectin antibody in C3Heb/FeJ mice or in P-selectin-deficient animals. Sequestration of neutrophils in sinusoids, i.e. those neutrophils that have been identified as critical for the injury, was not affected by the antibody or in P-selectin-deficient mice. However, the temporary margination in portal and post-sinusoidal venules was reduced by 75% in anti-P-selectin antibody-treated animals and by 51% in P-selectin-deficient mice. We conclude that hepatic P-selectin gene transcription in vivo involves cytokines. However, blocking P-selectin neither attenuated sinusoidal neutrophil sequestration nor prevented neutrophil-induced liver injury during endotoxin shock but attenuated neutrophil margination in larger vessels. Thus, our data demonstrate similarities and fundamental differences in the requirement for adhesion molecules to localize neutrophils in the liver vasculature compared to other organs during an inflammatory response.

Method-based differences in the automated analysis of the three-dimensional morphology of trabecular bone.

The three-dimensional (3D) morphology of trabecular bone is frequently quantified using computer programs. However, there are no standardized implementations of morphology programs and many variations are possible. Even though programs may use the same basic method, results can be significantly different because of differences in implementation. Morphology data from different laboratories therefore may not be comparable. The method of directed secants, with parallel plate assumptions, is commonly used to quantify 3D morphology. We examined the effect of several variations in the implementation of this method on measurements of trabecular plate number (Tb.N), trabecular thickness, and trabecular spacing. Three-dimensional micromagnetic resonance images of 10 bovine trabecular bone specimens were analyzed using several variations of the directed secant method. An analysis of covariance with repeated measures suggested that variations in the algorithm used to count test line intersections, variations in the criteria used to classify a test coordinate as bone or marrow, and variations in the number of test grid rotations had significant effects on Tb.N (p < 0.0001). The largest difference in Tb.N (52%) was due to the method used to count test line intersections with the bone-marrow interface. Variations in the classification algorithm and variations in the number of test line grid rotations resulted in a 6% difference in Tb.N. The spacing of the test line grids did not affect Tb.N (p = 0.28), and all differences were independent of volume fraction (p = 0.67). These data show that there can be significant differences in trabecular bone morphology measurements due only to the method used for the measurements. To facilitate comparisons between laboratories, we have made validated computer programs to measure trabecular bone morphology available over the Internet.

Remodeling and neointimal formation in the carotid artery of normal and P-selectin-deficient mice.

BACKGROUND: Inflammatory reactions such as leukocyte activation with platelet adherence and release of inflammatory mediators occur after percutaneous transluminal coronary angioplasty and may play a role in restenosis. Vascular remodeling with neointimal formation was studied in normal C57Bl/J6 and P-selectin-deficient mice. METHODS AND RESULTS: The left common carotid artery was ligated just proximal to the carotid bifurcation. Four weeks later, left carotids and contralateral controls were snap-frozen. Computer-aided morphometry was performed to measure ratios of neointimal to medial area (NI/M) in 10 sections per animal as a measure of the thickness of the neointimal lesion. For normal mice, NI/M was 1.13+/-0.2 (n=20), whereas NI/M was reduced by 76% to 0.27+/-0.1 (n= 19) in P-selectin knockout mice. Vascular constriction (as measured by the length of external elastic lamina) was the same in both groups, but the circumference of the lumen in knockout mice was 26% larger. Also, normal and P-selectin-deficient mice were killed at 3 and 7 days after ligation (n=6 for each group per time point). Histological staining and immunostaining for CD45 showed no inflammatory cell presence in P-selectin knockout mice. However, in normal mice, leukocyte infiltration was observed in the adventitia, media, and developing neointima. Also, P-selectin immunostaining was observed in media and developing neointima of normal mice. CONCLUSIONS: These data suggest that P-selectin is involved in processes leading to cell migration and proliferation associated with vascular remodeling, presumably by mediating leukocyte recruitment and the interaction between platelets and leukocytes.

Temporal expression of c-fos mRNA following balloon injury in the rat common carotid artery.

OBJECTIVE: Restenosis is a common problem which limits the effectiveness of percutaneous transluminal coronary angioplasty (PTCA). The cellular mechanisms of restenosis appear to involve smooth muscle cell (SMC) migration to the neointima in response to mitogens and growth factors, resulting in proliferation and deposition of cells in the lumen of the vessel. An antibody directed against PDGF attenuates this response in the rat. Thus, signaling cascades induced by growth factors including PDGF may be important targets for therapeutic intervention. METHODS: Since a number of growth factors activate c-fos via the p21-ras signaling pathway, we examined c-fos expression in a time course experiment involving restenotic lesions in rat carotid arteries. Sections of arteries collected at 1, 3, 7, 14 and 28 days following balloon injury were hybridized using a fluorescein-labeled RNA probe to c-fos. Immunohistochemistry was performed with antibodies to proliferating cell nuclear antigen (PCNA) and alpha-smc actin to characterize cellular constituents of the neointima, and detect any correlation between fos expression and PCNA localization. RESULTS: Expression of c-fos was low at day 1. By day 3, the media and adventitia were positively stained. At days 7 and 14, most cells in the neointima were labeled. By day 28, c-fos was expressed mainly in scattered cells along the luminal surface. Control sections revealed little labeling and confirmed specific staining by the antisense strand, PCNA localization and c-fos expression were similar at days 1, 3, 7 and 28, but at day 14 c-fos was expressed throughout the lesion, with PCNA localized mainly along the luminal edge. The majority of the cells making up the neointima stained rather intensely for alpha-smc actin, identifying them as SMCs. CONCLUSIONS: Results of these experiments indicate that, while c-fos expression correlates with lesion formation, it may be associated with a cellular process distinct from proliferation in this model.

Trabecular bone morphology from micro-magnetic resonance imaging.

Micro-magnetic resonance imaging (micro-MRI) is potentially a widely available tool to image and quantify the three-dimensional structure of trabecular bone. However, it has not been demonstrated that the same quantitative measurements can be obtained using micro-MRI as would be obtained from conventional light microscope images. Bovine trabecular bone from several anatomic sites was imaged with both optical and micro-MRI methods. The six faces of approximately cubic trabecular bone specimens were examined with the light microscope, and the volume of bone internal to these faces was then imaged using an 8.6 T 25 mm bore magnet. Three-dimensional measures of bone morphology were calculated from both the optical and micro-MR images using the method of directed secants. Quantitative measures from the two imaging methods were compared by paired t-tests. Volume fractions (BV/TV) measured by micro-MRI were linearly related to (r2 = 0.81) and did not differ statistically from (p = 0.96) similar measurements from optical images. The trabecular plate number (Tb.N) measured by micro-MRI also was linearly related to (r2 = 0.53) and did not differ statistically from (p = 0.17) similar measurements from optical images. The orientation of trabeculae predicted from micro-MRI was within 6 degrees of that calculated from optical images in 10 out of 16 specimens. The micro-MRI morphology measurements are relatively easy to perform, and since several hundred small-bore high-field strength MRI systems are available, this technique could be used widely to quantify the morphology of trabecular bone.

Estimation of ozone with total ozone portable spectroradiometer instruments. II. Practical operation and comparisons.

We used a microcomputer-controlled total ozone portable spectroradiometer instrument #21 (MTOPS21) to measure solar radiation at 298, 304 and 310 nm in Greenbelt, Md., during 1995. One day's ozone measurements from a Brewer instrument (B105) were used to calibrate the 304- and 310-nm channel ratios to a theoretical model. Total ozone estimates were then determined for the entire MTOPS21 data set. Differences between individual B105 and MTOPS21 ozone estimates show a 1% drop as solar zenith angles increase and depend on atmospheric attenuation and SO(2) variation at the ±2% level. Daily average values agree well (<0.5% average offset, 2% standard deviation).