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.

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.

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.

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.

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.

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.

Medial smooth muscle cell proliferation in the balloon injured rabbit aorta: effect of a thiazole compound with platelet inhibitory activity.

Twenty-seven adult male New Zealand rabbits (3-4 kgs) were used in this study. Six rabbits received vehicle, 3 groups of 6 each received doses of 4,5-bis(p-methoxyphenyl)-2-(trifluoromethyl)-thiazole, (U-53,059), at 0.3 mg/kg, 3.0 mg/kg and 30.0 mg/kg/day respectively. Drug and vehicle doses were given orally each day starting 3 days before balloon injury and continuing for the entire 2 week time period. Three rabbits were used as nontreated sham controls. In the vehicle and U-53,059 treated groups aortae were denuded of endothelial cells by balloon catheter injury. Two weeks after injury platelet aggregation to collagen was measured and the aortae removed for analysis of surface characteristics by scanning electron microscopy and lesion size by morphometry. All doses of U-53,059 inhibited platelet aggregation. The 3.0 and 30.0 mg/kg groups had the greatest inhibitory effect. All balloon injured aortae had the same morphologic characteristics. All vessels had similar extent and intensity of Evan's blue staining, similar areas of leukocyte/platelet adhesion, and a myointimal cell cover of transformed smooth muscle cells. The myointimal proliferative response was not inhibited at any of the drug doses studied.

Smooth muscle proliferation in chronically injured canine pulmonary arteries is reduced by a potent platelet aggregation inhibitor U-53,059.

Dirofilaria immitis (DI) infection chronically injures canine pulmonary arteries. This injury produces endothelial cell loss, platelet/leukocyte adhesion, and smooth muscle proliferation. In the present study we assessed the effect of the cyclooxygenase inhibitor, U-53,059, on platelet function, platelet kinetics, coagulation, and smooth muscle proliferation in DI infected dogs. Platelet aggregation to the combination of arachidonic acid/ADP was significantly inhibited by U-53,059. Coagulation and hematologic parameters were not effected by either DI infection or U-53,059 treatment. Platelet survival and the number of platelet dense granules were reduced in DI infection. Quantification of the lesions demonstrated that U-53,059 reduced both severity and density compared to non-treated dogs. U-53,059 is a potent and effective inhibitor of platelet aggregation which modifies smooth muscle proliferation produced by chronic vascular injury.

NF-kappa B is activated during acute inflammation in vivo in association with elevated endothelial cell adhesion molecule gene expression and leukocyte recruitment.

Leukocytes accumulate at sites of inflammation in response to the induced expression of endothelial cell adhesion molecules. The nuclear transcription factor kappa B (NF-kappa B) plays a critical role in the cytokine-induced expression of these genes in cultured endothelium. We examined the relationship between NF-kappa B activation and endothelial cell adhesion molecule gene expression in vivo during the initiation of acute inflammation. Nuclear NF-kappa B DNA-binding activity was rapidly increased within lung and heart tissues of rats administered endotoxin, consistent with the translocation of NF-kappa B complexes from the cytoplasm to the nucleus. This NF-kappa B was composed of p50 and p65 subunits, and could bind NF-kappa B elements in the E-selectin promoter. NF-kappa B activation was maximal within 30 min and persisted for at least 3 hr after endotoxin treatment. NF-kappa B activation preceded the transcriptional activation of the P-selectin, E-selectin, VCAM-1, and ICAM-1 genes. In the lung, increased expression of P-selectin and ICAM-1 protein was detected immunohistochemically. These molecular events were temporally associated with the sequestration of leukocytes and the development of pulmonary inflammation. NF-kappa B activation is therefore an early event in the initiation of acute inflammation in vivo. This molecular pathway may be of consequence in the pathogenesis of acute inflammatory disease.

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.

1,4-Dihydronaphthoquinones as water-soluble inhibitors of 5-lipoxygenase.

The acetate derivatives of 1,4-dihydronaphthoquinones showed significant inhibition of 5-lipoxygenase. Among them, 1-acetyl-2-n-butyl-4-methoxy-naphthalene and 1-acetyl-2, 3-diethyl- 4- methoxy-naphthalene were found to be the best inhibitors. A series of HCl salts of the amino acid esters and other derivatives of the two parent molecules, 1-hydroxy-2-n-butyl-4-methoxy-naphthalene and 1-hydroxy-2, 3-diethyl-4-methoxynaphthalene, were synthesized as water-soluble potential inhibitors of 5-lipoxygenase to improve the formulation characteristics of this class of compounds. The derivatives were evaluated for leukotriene (LT) C4/D4 and LTB4 inhibitory activity. The HCl salts of the L-valine esters from the two parent molecules exhibited the best potency for inhibition of LTC4/D4 (IC50 0.11-0.90 microM) in ionophore A23187-stimulated rat mononuclear cells and of LTB4 in A23187-stimulated rat blood (55.5-79.2% inhibition) following a single oral dose of 50 mg/kg.

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.

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.

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.

Prevalence of uveitis: a retrospective study.

BACKGROUND: Uveitis is commonly seen in optometric practice, and anterior uveitis accounts for more than 75% of all uveitis. Uveitis may be the initial manifestation of an associated systemic disease process; and, with proper testing and diagnosis, the patient's ocular and systemic condition may be more effectively treated. METHODS: Retrospectively, 194 charts from a referral practice with a general diagnosis of uveitis, retinitis, or chorioretinal scar were reviewed to determine medical history, clinical findings, tests performed, and specific diagnoses obtained. RESULTS: The majority of cases, 87.6%, were anterior uveitis with more than half, 54.7%, presenting with undetermined etiology. Trauma was the cause of the next most common group of anterior uveitis patients, 21.8%, and accounted for 19.1% of all uveitis cases. Glaucomatocyclitic crisis was seen in 8 cases, 4.1%, while there were 7 patients with sarcoid disease, and 5 with ankylosing spondylitis or systemic lupus erythematosus. Posterior uveitis was the principle finding in 12.4% of cases with ocular histoplasmosis being most common. There were 4 cases of toxoplasmosis and 3 cases of pars planitis. CONCLUSIONS: Utilization of laboratory testing proved to be most valuable in the treatment and management of uveitis. Although there were fewer cases of pars planitis reported in our study in comparison to previous reports, the mix of cases of uveitis in our study was comparable to the work of others in the field. Accurate diagnosis of these cases is quite important since the treatment and prognosis for recovery is variable and different with its associated diseases.

Heparin inhibits fibrin, but not leukocytes, in a model of deep-vein thrombosis.

Previous studies with models of deep-vein thrombosis (DVT) have demonstrated that leukocyte (PMN)-mediated vein injury may be an initiating event in DVT (14, 17). Since heparin (H) can prevent DVT, we studied its effect on vascular injury and thrombosis in our model. Three groups of rabbits were treated with H either sc (73 and 147 U/kg) or iv (662 U/kg). Scanning electron microscopy revealed that the 73 U/kg sc dose was ineffective. All veins had PMN accumulation, fibrin deposition and complex thrombus formation. There was no increase in anti-Xa activity; activated partial thromboplastin times (APTT) and whole blood clotting times were normal. The 147 U/kg sc and the intravenous dose did not inhibit PMN-mediated vein injury. The endothelium was sloughed by migrating PMNs, basement membrane was exposed, and platelets adhered to it. Thrombosis was completely absent in the iv dose group. This correlated with increased anti-Xa activity and prolonged APTT and whole blood clotting times. Our results indicate that heparin does not inhibit the PMN adhesion and migration which produces vascular injury. However, the anticoagulant activity of heparin effectively reduces fibrin deposition and complex thrombus formation.

Eicosanoid regulation of oosporogenesis by Lagenidium giganteum.

Cell proliferation is inhibited in many biological systems by lipid peroxides and related products derived from polyunsaturated fatty acids. Using developmentally synchronized cultures of Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, it has been documented that oxidative lipid metabolism is necessary for the induction and subsequent maturation of its sexual stage, the oospore. Addition of lipoxygenase inhibitors to liquid cultures of the fungus results in the stage-specific disruption of antheridial induction, gametangial fusion, induction of meiosis and spore cell wall formation. Oosporogenesis is inhibited by these compounds at concentrations which have no discernible effect on mycelial viability or asexual reproduction. Cyclooxygenase inhibition had comparable effects using ibuprofen and to a lesser extent with indomethacin. Phenylbutazone and the salicylates affected oosporogenesis only at concentrations which decreased asexual reproduction or mycelial viability. The inhibitory effects of NDGA on oosporogenesis could be reversed using partly purified eicosanoid extracts from growth media.

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.