Membrane-like Interface Research Articles

Lipoprotein-associated phospholipase A2: A paradigm for allosteric regulation by membranes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2's specificity toward oxidized phospholipids.

Structure Dependence of Pyridine and Benzene Derivatives on Interactions with Model Membranes.

Pyridine-based small-molecule drugs, vitamins, and cofactors are vital for many cellular processes, but little is known about their interactions with membrane interfaces. These specific membrane interactions of these small molecules or ions can assist in diffusion across membranes or reach a membrane-bound target. This study explores how minor differences in small molecules (isoniazid, benzhydrazide, isonicotinamide, nicotinamide, picolinamide, and benzamide) can affect their interactions with model membranes. Langmuir monolayer studies of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE), in the presence of the molecules listed, show that isoniazid and isonicotinamide affect the DPPE monolayer at lower concentrations than the DPPC monolayer, demonstrating a preference for one phospholipid over the other. The Langmuir monolayer studies also suggest that nitrogen content and stereochemistry of the small molecule can affect the phospholipid monolayers differently. To determine the molecular interactions of the simple N-containing aromatic pyridines with a membrane-like interface, 1H one-dimensional NMR and 1H-1H two-dimensional NMR techniques were utilized to obtain information about the position and orientation of the molecules of interest within aerosol-OT (AOT) reverse micelles. These studies show that all six of the molecules reside near the AOT sulfonate headgroups and ester linkages in similar positions, but nicotinamide and picolinamide tilt at the water-AOT interface to varying degrees. Combined, these studies demonstrate that small structural changes of small N-containing molecules can affect their specific interactions with membrane-like interfaces and specificity toward different membrane components.

Role and regulation of VEGF and its receptors 1 and 2 in the aseptic loosening of total hip implants

It was hypothesized that vascular endothelial growth factor (VEGF) in fibroblasts participates in aseptic loosening of total hip replacement (THR) implants. Therefore, osteoarthritic (OA) samples (n = 11) were compared with synovial membrane-like interface tissues from revision THR (n = 10). VEGF-A and its receptors were stained using streptavidin-immunoperoxidase method. Their regulation by hypoxia and cytokines were studied in cultured fibroblasts using quantitative real-time polymerase chain reaction (qRT-PCR). VEGFR1(+) lining cells (p < 0.01), stromal fibroblast-like cells (p = 0.001) and stromal macrophage-like cells (p < 0.05) were more numerous in rTHR than in OA. As to VEGFR2(+), only stromal fibroblast-like cells in rTHR outnumbered those found in OA (p < 0.05). VEGFRs in synovial fibroblasts were not affected by hypoxia, but VEGF increased 2.4-fold (p < 0.05). Interleukin-4 up-regulated VEGFR1 expression 23-fold. This is the first study to describe a difference between rTHR and OA in VEGF receptors, particularly VEGFR1. Hypoxia increased VEGF, but the VEGFR1 increase in the lining and stroma is probably IL-4 driven, in accordance with the M2-type macrophage dominance in interface tissues. VEGF/VEGFR system is also affected by hypoxia and may play a role in angiogenesis and bone pathology in aseptic loosening of total hip implants.

BMPs in periprosthetic tissues around aseptically loosened total hip implants

Background and purpose Primary and dynamically maintained periprosthetic bone formation is essential for osseointegration of hip implants to host bone. Bone morphogenetic proteins (BMPs) play a role in osteoinductive bone formation. We hypothesized that there is an increased local synthesis of BMPs in the synovial membrane-like interface around aseptically loosened total hip replacement (THR) implants, as body attempts to generate or maintain implant fixation.Patients and methods We compared synovial membrane-like interface tissue from revised total hip replacements (rTHR, n = 9) to osteoarthritic control synovial membrane samples (OA, n = 11. Avidin-biotin-peroxidase complex staining and grading of BMP-2, BMP-4, BMP-6, and BMP-7 was done. Immunofluorescence staining was used to study BMP proteins produced by mesenchymal stromal/stem cells (MSCs) and osteoblasts.Results and interpretation All BMPs studied were present in the synovial lining or lining-like layer, fibroblast-like stromal cells, interstitial macrophage-like cells, and endothelial cells. In OA and rTHR samples, BMP-6 positivity in cells, inducible by the proinflammatory cytokines tumor necrosis factor−α and interleukin-1β, predominated over expression of other BMPs. Macrophage-like cells positive for BMP-4, inducible in macrophages by stimulation with particles, were more frequent around loosened implants than in control OA samples, but apparently not enough to prevent loosening. MSCs contained BMP-2, BMP-4, BMP-6, and BMP-7, but this staining diminished during osteogenesis, suggesting that BMPs are produced by progenitor cells in particular, probably for storage in the bone matrix.

Toll‐like receptors and aseptic loosening of hip endoprosthesis—a potential to respond against danger signals?

Bacterial remnants and subclinical biofilms residing on prosthesis surfaces have been speculated to play a role in hip implant loosening by opsonizing otherwise relatively inert wear particles. The innate immune system recognizes these microbial pathogen-associated molecular patterns (PAMPs) using Toll-like receptors (TLRs). Our objective was to evaluate the possible presence of TLRs in aseptic synovial membrane-like interface tissue. Bacterial culture-negative, aseptic (n = 4) periprosthetic synovial membrane-like tissue was compared to osteoarthritis synovial membrane (n = 5) for the presence of cells positive for all known human functional TLRs, stained using specific antibodies by immunohistochemistry, and evaluated using morphometry. In comparison to osteoarthtritic synovium, the number of TLR-positive cells was found to be increased in the aseptic setting, reflecting the considerable macrophage infiltration to the tissues investigated. Thus aseptic periprosthetic tissue seems to be very reactive to PAMPs. It has been recently recognized that TLR do not only respond to traditional PAMPs, but also to endogenous alarmings or danger signals released from necrotic and activated cells. Alarming-TLR interaction in the periprosthetic tissue might be a novel mechanism of aseptic loosening of endoprosthesis.

Seppo Santavirta: The Life and Work of an Orthopaedic Surgeon and Scientist. A Tribute From His Friends

Seppo Santavirta: The Life and Work of an Orthopaedic Surgeon and Scientist. A Tribute From His Friends

Elastic waves guided by a material interface

The propagation of interfacial small-amplitude waves along a rectilinear thin film separating two pre-stressed, incompressible, elastic media is addressed. The film is modelled as a material surface possessing its own mass density and normal and flexural stiffnesses. It is shown that these features induce dispersion as the obtained secular equations are polynomials of the second degree in the wavenumber when bending stiffness is absent (membrane-like interface), and of the fourth degree otherwise (plate-like interface). In both case, beyond the modified Stoneley mode, a bending mode for the interface, an additional propagating wave can exist, with amplitude polarized along the interface (extensional mode). The associated bifurcation problem is analyzed with focus on the effects of compressive residual forces at the interface. The buckling strain of a compressed metal layer embedded in an elastomeric medium is computed also with an exact approach, to provide the range of validity of the proposed simplified model of material interface.

Toll‐like receptors in the interface membrane around loosening total hip replacement implants

Toll-like receptors (TLRs) have been known to act as sensors of innate immunity and respond to ligands of microbial and endogenous components. Tissues and cells typical for interface membrane of foreign body reaction were analyzed to evaluate potential role of TLRs in the pathogenesis of the so called "aseptic loosening of total hip replacement." Fourteen cases of interface membrane around aseptic loose total hip replacement implants were stained by single and double immunohistochemical methods to examine cellular localization of toll-like receptor (TLR)-4 and TLR-9. Osteoarthritic synovium was used as control tissues. Cultured macrophages were used to study TLR-4 and TLR-9 mRNA levels by quantitative reverse transcriptase-polymerase chain reaction. The effect of titanium particle stimulation on macrophages was also examined in the culture. Extensive immunolocalization of TLR-4 and TLR-9 positive cells was observed in the synovial membrane-like interface membrane of foreign body granulomas compared with control synovial membranes. TLR and CD68 double staining demonstrated that the TLR positive cells in aseptic loosening were mostly monocyte/macrophages and foreign body giant cells. TLR-4 and TLR-9 mRNA expression was also found in macrophage-colony stimulating factor treated rat macrophages, but this expression decreased (p < 0.05 or less) upon stimulation with titanium particles although matrix metalloproteinase (MMP)-9 mRNA levels used as macrophage activation marker were increased (p = 0.01). The interface membrane around loosening total hip replacement implants is apparently well equipped with TLRs and, thus, probably very sensitive to various structural components of microbes and to endogenous TLR ligands. This seems to be due to recruitment of monocyte/macrophages as particles per se seemed to down-regulate some of the key TLRs. This suppression after particle phagocytosis might prevent excessive and harmful host responses, and injury to innocent bystander cells/tissues.

Increased collagen degradation around loosened total hip replacement implants

To assess collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. The medical indication for the primary total hip replacement was osteoarthritis in all study patients. Samples from the study patients were compared with control synovial membranes obtained from trauma (hip fracture) patients. Proteoglycans were extracted with 4M guanidinium chloride. Denatured collagen in the remaining matrix was solubilized with alpha-chymotrypsin. Nonsoluble matrix and supernatant fractions were acid hydrolyzed before measurement of hydroxyproline. The proportion of soluble (in vivo-degraded) collagen of the total sample collagen content was calculated. Proteinases were stained using the avidin-biotin-peroxidase complex method. Collagen in the interface membrane from the implants was highly degraded (mean +/- SEM 20 +/- 3%) compared with that in the control synovial membranes (12 +/- 1%; P = 0.007). In controls, the degree of collagen degradation did not correlate with levels of matrix metalloproteinase 1 (MMP-1), MMP-13, or cathepsin K, although MMP-1 approached statistical significance. In interface membranes, the correlations were r = 0.88 (P = 0.002), r = 0.92 (P = 0.001), and r = 0.98 (P < 0.0001) for MMP-1, MMP-13, and cathepsin K, respectively. In normal synovial membrane, collagen matrix remodeling may be mainly an intracellular process. In contrast, pathologic tissue destruction in the interface membrane from prosthetic hip joints is associated with a shift toward MMP-13 and cathepsin K, which become activated and overcome their endogenous inhibitors (tissue inhibitors of metalloproteinases and cystatin C). The highly significant correlation between collagen degradation and cathepsin K indicates an extracellular role of this acidic endoproteinase, consistent with previous observations concerning the acidity of the interface membrane.

Significance and role of increased expression of extracellular matrix metalloproteinase induced in the aseptic loosening of prostheses

To investigate the expression of extracellular matrix metalloproteinase induced (EMMPRIN) in the interface tissue, and explore the role of EMMPRIN in the aseptic loosening of prostheses. Immunohistochemistry was performed to characterize the EMMPRIN-expressing cells at sites of interface tissue around aseptic loosened hip prostheses in 16 cases. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to study the existence of EMMPRIN mRNA in interface tissue samples. And it was followed up by computer assisted image analysis in order to detect the A values of their expression. Synovium of hip joint of 8 femoral neck fracture were in control group. Strong immunostaining of EMMPRIN was found in the macrophages and fibroblasts of lining-like layers and vascular endothelium of synovial membrane-like interface tissue around loosened prostheses. Expression of EMMPRIN was significantly higher in interface tissue than the control synovium (z=-3.252, P=0.001). RT-PCR of interface tissue samples disclosed the presence of EMMPRIN mRNA of 14 cases. In interface tissue, the A value of EMMPRIN increased significantly compared to control synovium (P<0.01). Over-expression of EMMPRIN up-regulates the production of matrix metalloproteinase (MMPs) in the interface tissue. And it can promote the bone destruction around prostheses. Thereby it may be one of methods to prevent and treat aseptic loosening of prostheses by repression the biology activity of EMMPRIN.

Expression of caspase-1 in synovial membrane-like interface tissue around loosened hip prostheses.

Caspase-1 expression in synovial membrane-like interface tissue (SMLIT) around loosened hip prostheses and osteoarthritic synovial samples was studied. Caspase-1 mRNA was found in SMLIT and synovial tissue. There is no difference in the copy numbers of caspase-1 mRNA between these samples. Both precursor and active forms of caspase-1 proteins appeared in these samples, but the number of positive cells was higher in SMLIT than in synovial tissue. Double labeling revealed that most caspase-1-positive cells were macrophages and fibroblasts. In the lining-like layers and deep stroma of SMLIT, many cells were double positive for active caspase-1 and interleukin-1 beta (IL-1beta). In contrast, the number of active caspase-1/IL-18 double-positive cells was very low. We conclude that caspase-1 synthesis is increased in SMLIT. Caspase-1 can be involved in implant loosening by processing IL-1beta precursor into its mature form, which is a potent osteoclast-activating factor and a major proinflammatory mediator.

Hyaluronan synthases, hyaluronan, and its CD44 receptor in tissue around loosened total hip prostheses.

Aseptic loosening of prosthetic components, the most common long-term complication after total hip replacement (THR), is characterized by the formation of a synovial membrane-like interface tissue (SMLIT). It was hypothesized that the hyaluronan synthase (HAS)/hyaluronan (HA)/HA receptor CD44 signalling system is responsible for the synovial-like differentiation of the interface membrane. SMLIT was therefore compared with osteoarthritis (OA) synovial membrane by using reverse transcriptase polymerase chain reaction (RT-PCR) of HAS 1, 2 and 3, histochemical HA assay, and immunohistochemistry of CD44 and its non-HA ligands. All three isoforms of HAS were found in these samples. HA and CD44 were most abundant in the lining, but the signal was actually stronger in aseptic loosening than in OA (p<0.01). The non-HA CD44 ligands, collagen type VI, fibronectin, osteopontin, and MCP-1, had a similar distribution pattern in both tissues. These results confirm the synovial-like structure of the interface tissue lining. The pressure waves and movement of the HA-rich pseudosynovial fluid seem to drive HA into the implant-to-host interface, which itself also produces HA. HA may be responsible for the induction of a synovial-like lining at the interface through HA-CD44 signalling.

No lymphokines in T-cells around loosened hip prostheses.

Research results have been contradictory about the role of lymphocytes and immune response in aseptic loosening of total hip replacement (THR). Conclusive evidence is still lacking in spite of extensive in vivo and in vitro studies. Our study was designed to check whether T-cells were activated and if they produced lymphokines in synovial membrane-like interface tissue around loosened THRs. Tissue sections were stabilized and permeabilized to allow the cytokine-specific antibodies to penetrate through the cell membrane and the membranes of intracellular organelles. This technique, combined with computer-assisted image analysis, permits the detection and quantitation of lymphokine-producing cells. We found that the number of T-cells was low, and none of the T-cells was activated, as shown by the absence of interleukin-2 receptor (IL-2R) immunoreactivity. There was no cell producing lymphokines, such as interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and tumor necrosis factor-beta (TNF-beta). Our results suggest that T-cell-mediated immune response is not actively involved in aseptic loosening of THR.

Distribution of tenascin-X in different synovial samples and synovial membrane-like interface tissue from aseptic loosening of total hip replacement.

The distribution of tenascin-X (Tn-X) was investigated in synovial samples from rheumatoid arthritis (RA), osteoarthritis (OA) and knee injuries, and in synovial membrane-like interface tissue (SMLIT) from aseptic loosening of total hip replacement (THR). An affinity purified rabbit antiserum against Tn-X was applied in avidin-biotin-peroxidase complex method. Double immunofluorescence labeling was used to assess the spatial relationship of Tn-X and Tn-C. All samples showed Tn-X immunoreactivity. Strong staining appeared in the lining and lining-like layers of RA and SMLIT samples, respectively. An intensive immunoreactivity was also found in pannus tissue in RA, and around multinucleate giant cells and polyethylene wear debris in SMLIT. Staining intensity/extent varied significantly in different samples in the following rank order: SMLIT, RA, OA, knee synovium membrane. Double labeling revealed two patterns of Tn-X/Tn-C distribution, reciprocal and co-localization. Our results suggest that Tn-X is an essential component of normal synovial membrane, and that inflammatory mediators may increase local Tn-X production. Tn-X distribution is not always reciprocal to that of Tn-C.

Increased expression of EMMPRIN in the tissue around loosened hip prostheses.

Matrix metalloproteinases (MMPs) have been shown to play a role in aseptic loosening of total hip replacement (THR). Extracellular matrix metalloproteinase inducer (EMMPRIN) can upregulate expression of several MMPs but has little effect on their tissue inhibitor (TIMP). Using the avidin-biotin-peroxidase complex immunostaining method, we detected strong immunoreactivity of EMMPRIN in the lining-like layers, sublining area and vascular endothelium of synovial membrane-like interface tissue around loosened prostheses. In contrast, EMMPRIN staining was very weak in the synovial samples from patients with hip arthrosis. Double immunofluorescence labeling revealed EMMPRIN/MMP-1 double-positive cells in lining-like layers and the sublining area of interface tissue. Our findings indicate that EMMPRIN expression is upregulated in interface tissue, and that locally accumulated EMMPRIN may modulate MMP-1 expression. An imbalance in the activity of MMPs and TIMP may lead to tissue destruction and periprosthetic osteolysis. These biological responses, combined with mechanical stress caused by micromotion and oscillating fluid pressure, may eventually cause aseptic loosening of THR.

Ligand binding at membrane mimetic interfaces

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels.