Rat hepatic stellate cells become retinoid unresponsive during activation.

Hepatic stellate cells (HSC) play an essential role in fibrogenesis. Many stimuli cause HSC to activate, lose their Vitamin A and produce collagen. It is unclear whether Vitamin A loss causes activation, potentiates it or is simply an event in the cascade of activation changes. We determine if exogenous retinoids prevent the activation of freshly isolated rat HSC activated by plating on plastic. We also determine if retinoids: (1) reverse HSC activation; (2) maintain/restore HSC intracellular retinoid levels; (3) maintain expression of HSC nuclear receptors for retinoic acid (RAR) in HSC that are becoming activated or are chronically activated. Markers of activation in freshly isolated HSC were decreased by either retinol or retinoic acid without increases in HSC retinoid concentration. mRNA levels for RAR-alpha, RAR-beta and RAR-gamma, the nuclear receptors for retinoic acid, decreased during activation of freshly isolated HSC even with retinoid supplementation. RAR-alpha, RAR-beta and RAR-gamma mRNA and RAR-beta protein was undetectable in chronically activated HSC and remained absent after retinoic acid supplementation. Activation markers in chronically activated HSC were only slightly decreased after retinoid exposure. We conclude that exposure of HSC to extracellular retinoids diminishes some markers of activation but does not prevent HSC activation.

Initial signaling of the fibronectin receptor (alpha5beta1 integrin) in hepatic stellate cells is independent of tyrosine phosphorylation.

BACKGROUND/AIMS: Activation of hepatic stellate cells (HSC) plays an integral role in hepatic fibrosis. HSC activation increases fibronectin (alpha(5)beta(1)) receptor expression and interactions between alpha(5)beta(1) and the extracellular matrix increase collagen synthesis. It is unclear how signaling by the alpha(5)beta(1) receptor initiates these changes. We aimed to determine the signaling cascade after alpha(5)beta(1) stimulation in activated HSC. METHODS: HSC were isolated from male Sprague-Dawley rats. Activated HSC were exposed to beads coated with fibronectin (ligand for alpha(5)beta(1)) or D-polylysine (inert control). HSC were stained with FTC-labeled antibodies against classes of signaling molecules. Tyrosine phosphorylation was blocked using genistein or herbimycin A. The fraction of beads with localized immunostaining (indicating accumulation of signaling protein) was determined. RESULTS: The majority of cytoskeletal proteins, Src substrates, Src kinases and members of the ERK and JNK signaling molecule families require actin cytoskeletal organization and tyrosine-kinase-mediated phosphorylation to accumulate. Several proteins (e.g. tensin, FAK) accumulated in the absence of tyrosine phosphorylation. CONCLUSIONS: The alpha(5)beta(1) integrin-ligand interaction induces accumulation of cytoskeletal molecules, activating multiple kinase pathways. Initial integrin signaling by alpha(5)beta(1) are associated with cytoskeletal proteins and are independent of tyrosine phosphorylation. We suggest that there may be cytoskeletal changes that may be targeted to diminish HSC activation.

The peroxisomal proliferator clofibrate enhances the hepatic cytoplasmic movement of fatty acids in rats.

The role of cytosolic fatty acid binding protein (FABP) in cellular fatty acid metabolism remains poorly defined. The intracellular movement of fatty acids is thought to be facilitated through codiffusion with FABP. Peroxisomal proliferators like clofibrate induce FABP and may stimulate fatty acid use by increasing cytoplasmic diffusion rates. Our aim was to determine if induction of FABP by clofibrate increases the cytoplasmic transport of a fluorescent fatty acid NBD-stearate. Male rats were fed clofibrate for 9 days to increase the hepatic concentration of FABP. Hepatocytes were isolated using collagenase perfusion. Cytosolic FABP was measured by enzyme-linked immunosorbent assay (ELISA) using a specific antibody to hepatic FABP. Two-dimensional laser photobleaching (FRAP) was used to measure the cytoplasmic movement of NBD-stearate in hepatocytes. Cytoplasmic transport of NBD-stearate occurred by isotropic diffusion with no evidence for convective or directed transport. Treatment with clofibrate increased FABP levels, the fraction of NBD-stearate found in cytosol, and the observed cytoplasmic diffusion rate. The increase in cytoplasmic movement exceeded that predicted from FABP binding changes suggesting that clofibrate also enhances fatty acid diffusion within intracellular membranes. Nonspecific effects of clofibrate on cytoplasmic viscosity or pore size were not observed. These data suggest that clofibrate treatment induces cytosolic FABP and stimulates the intracellular movement of fatty acids by 2 distinct mechanisms. Soluble proteins like FABP promote the cytoplasmic transport of fatty acids by increasing diffusion. These data further support a role for intracellular binding proteins in facilitating the intracellular movements of fatty acids.

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate.

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.

Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP.

Sex differences in multiple steps in hepatic transport of palmitate support a balanced uptake mechanism.

Hepatic clearance of long-chain fatty acids is substantially faster in females than in males, a fact that may underlie known gender-related differences in lipoprotein metabolism and associated disease states. To further investigate the transport steps responsible for this difference, we used a novel method combining multiple-indicator dilution and steady-state measurements of palmitate extraction from albumin solutions. We found that cytoplasmic transport of palmitate is sufficiently slow (diffusion constants 9.0 and 5.9 x 10(-9) cm2/s for male and female liver, respectively) that the steady-state concentration of palmitate in the center of the cell should be approximately 0.5 of that found in the cytoplasm just beneath the plasma membrane. Previous studies in cultured liver cells using nonphysiological fatty acids have shown more rapid cytoplasmic transport in females. This sex difference reflects higher concentrations of cytosolic fatty acid-binding protein, which acts as a carrier system to transport fatty acids across cell water layers. The current study confirmed slow cytoplasmic diffusion rates in intact perfused rat liver using a physiological fatty acid and found a similar female-to-male ratio. Female liver also had a greater influx rate constant and a larger vascular volume than male liver but had a similar rate of metabolism. Rapid cytoplasmic diffusion enhances movement of palmitate into deeper layers of the cell cytoplasm, thus reducing efflux. The larger sinusoidal volume in females not only permits more dissociation of palmitate from albumin within the sinusoids but also may generate a greater permeability-surface area product. These multiple sex-related differences combine to produce a nearly twofold greater steady-state uptake rate by female liver.

Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid binding protein (FABP). Previous work suggested that FABP decreases fatty acid binding to immobile membranes, causing faster cytoplasmic diffusion. However, the specificity for binding to FABP has not been addressed. The aim of the current study was to determine whether specific FABP binding is required or whether binding to other proteins will produce the same effect. A model cytoplasm consisted of a fatty acid, proteins, and liposomes to simulate intracellular membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the movement of the fluorescent fatty acid 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazoaminostearate (NBD-stearate) in model cytoplasm, in normal and permeabilized Hep G2 cells, and after incubation of permeabilized cells with bovine serum albumin (BSA) or FABP. Increasing protein in the model cytoplasm increased the diffusion rate in proportion to the extent of protein binding. Cell permeabilization reduced diffusion of NBD-stearate to < 5% of controls. Incubation of permeabilized cells with FABP or BSA resulted in a concentration-dependent increase in the NBD-stearate diffusion rate. BSA was more effective than FABP in binding NBD-stearate and increasing its diffusion rate after permeabilization. Proteins like FABP promote the diffusion of fatty acids. Removal of these proteins drastically reduces cytoplasmic diffusion. Substitution with BSA reestablishes the diffusive flux, suggesting that specific binding to FABP is not required. These data support a role for intracellular binding proteins in facilitating the cytoplasmic movement of fatty acids.

The effects of axial rotational alignment of the femoral component on knee stability and patellar tracking in total knee arthroplasty demonstrated on autopsy specimens.

Four fresh-frozen anatomic knee specimens were tested for knee stability, patellar tracking, and patellofemoral contact points with the femoral component positioned in 5 degrees internal, 5 degrees external, or neutral axial rotational alignment of the femoral component referenced on the posterior femoral condyles. The externally rotated specimens had varus-valgus stability of the knee that was closest to the normal control. The internally rotated specimens shifted into valgus alignment with flexion. Patellar tracking also was closest to normal in the externally rotated specimens. Patellofemoral contact was more evenly distributed between the medial and lateral contact areas in the externally rotated specimens than in the internally rotated or in the neutral specimens. Internal rotation of the femoral component in the knee with perpendicular resection of the tibia causes undesirable changes in knee stability, patellar tracking, and patellofemoral contact points. Neutral positioning produces similar but less negative effects on knee stability and patellar kinematics. External rotation improves both patellar tracking and knee stability characteristics.

Evaluation of the effect of the femoral articular surface material on the wear of a metal-backed patellar component.

Wear characteristics of metal-backed, polyethylene patellar components were tested using cobalt-chromium, titanium alloy (Ti), and ion-implanted titanium alloy (IITi) articular surfaces. Patellar components were cycled in a bovine serum bath at 3 Hz for 1 million cycles, under a compressive load that varied from 343 N at 0 degree flexion to 2255 N at 120 degrees flexion. After testing, the polyethylene articular surfaces of the patellar components were evaluated for wear and graded using a subjective numbering system. Overall wear damage to the polyethylene surface was much worse with both Ti and IITi than with cobalt-chromium. Differences in mean wear scores were statistically significant when cobalt-chromium was compared with either Ti or IITi, but there were no statistically significant differences between Ti and IITi. Polyethylene surfaces that articulated against Ti femoral surfaces had more severe scratching. The IITi test group had areas of delamination not observed in the other test groups. Subjective evaluation of the metal surfaces showed evidence of wear damage as well. The metal articular surface of IITi resisted scratching as long as the treated surface was intact. In the high-stress areas, however, such as the edges of the intercondylar notch, the ion-implanted surface quickly wore away, exposing the untreated titanium alloy. The cobalt-chromium femoral articular surface had the least amount of scratching and no evidence of loss of metal.

Postoperative blood retrieval and transfusion in cementless total knee arthroplasty.

Postoperative wound drainage was aspirated, collected, and transfused in 197 patients undergoing unilateral or bilateral cementless knee arthroplasty using the Solcotrans or the Constavac Blood Conservation blood-retrieval devices. Operative technique, method of hemostasis, and postoperative management were identical in all patients. Drainage-tube suction pressure was minimized, and the duration of drainage collection was less than 8 hours in all patients. In the 153 patients who underwent unilateral total knee arthroplasty (TKA), the amount of blood retrieved and transfused averaged 829 cm3 (59% of total blood lost). For the 44 patients who underwent bilateral procedures, 1,131 cm3 of blood was salvaged (56% of total blood lost). Transfused banked blood averaged 1.7 units (25% homologous) in unilateral cases, while bilateral procedures required 3.0 units (35% homologous). Complications (4%) included wound hematomata in five patients and deep venous thrombosis in two. Transient chills, fever, or tachycardia were seen in four patients at the time of transfusion. Transfusion requirements of banked blood appeared to be significantly reduced, especially in simultaneous bilateral knee arthroplasty, when compared to previous experience in patients who did not undergo postoperative blood retrieval.

The effect of central stem and stem length on micromovement of the tibial tray.

The effects of a central stem and its length on cementless tibial tray micromovement were investigated using preserved cadaver tibial specimens. For axial loading tests, cyclical compressive loads ranging 50-1000 N were applied to the anterolateral portion of the implanted tray. With subsidence on the loaded side and lift-off on the contralateral side, micromotions on both sides and bending of the tray were measured. The three groups consisted of a stemless group, a 7.5 cm (short) central stem group, and a 15 cm (long) central stem group. For shear loading tests, shear loads ranging 20-250 N were applied to the central portion of the posterior rim of the tray anteriorly for 1,000 cycles. Displacement values for subsidence and micromotion were measured on the medial and lateral side of the trays and compared for each group. For axial tests, the long stem minimized subsidence and lift-off (P < .05) when compared to the stemless group. Although the short stem also tended to prevent contralateral lift-off, no significant difference was shown when compared to the stemless group. However for shear loading, both central stem lengths significantly reduced subsidence and micromotion (P < .05). The authors conclude that the tibial tray with a long stem can achieve better initiation fixation of the implant to bone when compared to the short stem and no stem groups.

Articular surface material effect on metal-backed patellar components. A microscopic evaluation.

This study evaluated patellar wear with three commonly used metallic surfaces against a polyethylene surface. Wear characteristics of metal-backed patellar components were tested against cobalt chromium, titanium alloy, and ion-implanted titanium alloy (IITi) articular surfaces. Adult bovine serum was used as a lubricant. The specimens were cycled at 2.9 Hz for 1 X 10(6) cycles. The axial load was 343.2 N at 0 degrees flexion and increased to 2255.6 N at 110 degrees flexion. After testing, the polyethylene and metal surfaces were examined for wear using scanning electron microscopy. Patellae tested against the titanium-alloy surfaces had severe abrasion, surface cold flow, and pitting. The patellae tested against IITi were similar to the titanium-alloy group at the edges of the components, and delamination of the surface layers occurred in the high stress areas. The patellae tested against cobalt-chromium alloy had the least wear. Among the three metal surfaces, the titanium surface had the most severe surface abrasion and the cobalt-chromium surface had the least. At higher power magnification (X500), the cobalt-chromium surface had minimal surface abrasion even in the high stress areas. The IITi and titanium surfaces had severe scratching and surface abrasion, but the titanium surfaces were clearly more severely affected. The ion-implanted surface appeared to have been worn away in the high stress areas. Ion implantation does not produce a major improvement in the effect the titanium-alloy surface has on the polyethylene but does improve the wear on the metal surface itself. The cobalt-chromium surface was less damaged than the titanium and IITi and also caused less damage to the polyethylene. This experiment suggests that titanium and ion-implanted titanium are inferior materials to cobalt chromium when used as an articular surface in total knee replacements.

Influence of prosthetic joint line position on knee kinematics and patellar position.

Prosthetic joint line position after total knee arthroplasty (TKA) was investigated using sagittal roentgenograms obtained from six fresh frozen cadaver knees. A specially designed knee testing device was developed that allowed for a controlled flexion angle while maintaining a constant quadriceps force. Pre- and postoperative roentgenograms were obtained from 30 degrees to 120 degrees in 15 degrees intervals. Steinman pins inserted into the medial femoral condyle and patella were used as reference points in the roentgenograms. A displacement vector between the medial femoral condyle and tibial plateau was used to analyze the tibiofemoral joint relationship. The functional patellar length (Insall-Salvati ratio), was used to determine correct patellar height. Another displacement vector was used to measure the patellofemoral joint relationship, and the angle between the patellar cut surface and femoral long axis was also calculated. Bone resection thickness from the femoral, tibial, and patellar surfaces was equal to the prosthetic thickness. This reconstructive scheme produced correct ligament balance and flexibility of the knee without the aid of tensioning devices or special measurements. Patellar tracking appeared to be identical before and after surgery. This accurate but simple surgical technique also reproduced normal knee extensor mechanisms that may influence longevity and long-term results of TKA.