Impact of type-1 collagen hydrogel density on integrin-linked morphogenic response of SH-SY5Y neuronal cells.

Cellular metabolism and behaviour is closely linked to cytoskeletal tension and scaffold mechanics. In the developing nervous system functional connectivity is controlled by the interplay between chemical and mechanical cues that initiate programs of cell behaviour. Replication of functional connectivity in neuronal populations in vitro has proven a technical challenge due to the absence of many systems of biomechanical regulation that control directional outgrowth in vivo. Here, a 3D culture system is explored by dilution of a type I collagen hydrogel to produce variation in gel stiffness. Hydrogel scaffold remodelling was found to be linked to gel collagen concentration, with a greater degree of gel contraction occurring at lower concentrations. Gel mechanics were found to evolve over the culture period according to collagen concentration. Less concentrated gels reduced in stiffness, whilst a biphasic pattern of increasing and then decreasing stiffness was observed at higher concentrations. Analysis of these cultures by PCR revealed a program of shifting integrin expression and highly variable activity in key morphogenic signal pathways, such as mitogen-associated protein kinase, indicating genetic impact of biomaterial interactions via mechano-regulation. Gel contraction at lower concentrations was also found to be accompanied by an increase in average collagen fibre diameter. Minor changes in biomaterial mechanics result in significant changes in programmed cell behaviour, resulting in adoption of markedly different cell morphologies and ability to remodel the scaffold. Advanced understanding of cell-biomaterial interactions, over short and long-term culture, is of critical importance in the development of novel tissue engineering strategies for the fabrication of biomimetic 3D neuro-tissue constructs. Simple methods of tailoring the initial mechanical environment presented to SH-SY5Y populations in 3D can lead to significantly different programs of network development over time.

The five-year radiological results of the uncemented Oxford medial compartment knee arthroplasty.

This study reports on the first 150 consecutive Oxford cementless unicompartmental knee arthroplasties (UKA) performed in an independent centre (126 patients). All eligible patients had functional scores (Oxford knee score and high activity arthroplasty score) recorded pre-operatively and at two- and five-years of follow-up. Fluoroscopically aligned radiographs were taken at five years and analysed for any evidence of radiolucent lines (RLLs), subsidence or loosening. The mean age of the cohort was 63.6 years (39 to 86) with 81 (53.1%) males. Excellent functional scores were maintained at five years and there were no progressive RLLs demonstrated on radiographs. Two patients underwent revision to a total knee arthroplasty giving a revision rate of 0.23/100 (95% confidence interval 0.03 to 0.84) component years with overall component survivorship of 98.7% at five years. There were a further four patients who underwent further surgery on the same knee, two underwent bearing exchanges for dislocation and two underwent lateral UKAs for disease progression. This was a marked improvement from other UKAs reported in New Zealand Joint Registry data and supports the designing centre's early results.

Heme oxygenase-1 protects against Alzheimer's amyloid-ß(1-42)-induced toxicity via carbon monoxide production.

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer's disease, catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediates cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid ß peptide (Aß) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aß(1-42) toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrP(c), and rat primary hippocampal neurons. Aß(1-42) (containing protofibrils) caused a concentration-dependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrP(c)-expressing cells showing greater susceptibility to Aß(1-42) toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aß(1-42). The CO-donor CORM-2 protected cells against Aß(1-42) toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aß(1-42) treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aß toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aß also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aß toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aß. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.

Should a Patients BMI Status be Used to Restrict Access to Total Hip and Knee Arthroplasty? Functional Outcomes of Arthroplasty Relative to BMI - Single Centre Retrospective Review.

We reviewed the experience of a dedicated orthopaedic elective service to determine whether we could establish a BMI group where arthroplasty was no longer effective as assessed by the patient's functional outcome. This was a prospective observational study with retrospective analysis of data collected on 1439 total hip arthroplasty, 934 total knee arthroplasty and 326 unicompartment knee arthroplasty patients. Functional scores (WOMAC, Oxford hip and knee scores and HAAS) were obtained preoperatively and at 12 months post op. Patients had their BMI recorded at the preoperative assessment and were divided into BMI groups (BMI<25, BMI 25-30, BMI 30-35 and BMI > 35). Patients with a BMI of ≤ 30 had significantly better functional scores at 12 months post op compared to those with a BMI of > 35. The absolute gain in functional scores from pre op to 12 months post op did not differ significantly between BMI groups, the only significant difference we found for absolute gain showed patients with a BMI of > 35 have a greater increase in HAAS scores following total hip arthroplasty compared to patients with a BMI of 30 or less (p = 0.0435). Our patients with higher BMI's had worse preoperative and post operative functional scores but their benefit from surgery measured by the change in functional scores showed no difference compared to patients with lower BMI. We could find no reason on the basis of the 12-month results to limit surgery to obese patients because of an expected poorer functional outcome.

The outcome of bone substitute wedges in medial opening high tibial osteotomy.

BACKGROUND: Opening wedge high tibial osteotomy often requires bone grafting to improve the union rate and avoid instability at the osteotomy site. Autograft and allograft have both been associated with complications and the use of bone substitute wedges has been advocated to improve the outcome. This study investigated the clinical, radiological and histological outcomes of using biphasic calcium phosphate ceramic (Triosite) wedges in opening wedge high tibial osteotomy and determined whether the presence of the graft would compromise the satisfactory conversion to a total knee replacement. METHODS: A consecutive cohort underwent radiological review to determine whether the osteotomy healed and the correction was maintained. Biopsies were performed on those undergoing second procedures. All patients converted to total knee arthroplasty were assessed separately as to any surgical complications attributed to the Triosite wedge. RESULTS: There were 36 osteotomies in 33 patients with a minimum of 4 years follow up. All osteotomies healed. There was an average 90 (5-14) of correction, which was maintained. Histological assessment of 17 cases confirmed adequate bone replacement of the Triosite although some areas of tricalcium phosphate remained visible. Conversion to a total knee arthroplasty occurred in 11 cases with no complications. CONCLUSION: Biphasic calcium phosphate ceramic wedges (Triosite) can be reliably used in opening wedge high tibial osteotomy with a low incidence of complications and satisfactory conversion to total knee arthroplasty.

Neprilysin, obesity and the metabolic syndrome.

OBJECTIVE: Neprilysin (NEP), a zinc metalloendopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity, and investigated NEP production by adipocytes in-vitro. METHODS AND RESULTS: In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homoeostasis model assessment and body mass index (BMI) in all subjects (P<0.01). Quantitative reverse transcriptase PCR (RT-PCR) and western blotting showed that in human pre-adipocytes NEP expression is upregulated 25- to 30-fold during differentiation into adipocytes. Microarray analysis of mRNA from differentiated human adipocytes confirmed high-NEP expression comparable with adiponectin and plasminogen activator inhibitor-1. In a murine model of diet-induced insulin resistance, plasma NEP levels were significantly higher in high-fat diet (HFD)-fed compared with normal chow diet (NCD)-fed animals (1642 ± 529 and 820 ± 487 pg µl(-1), respectively; P<0.01). Tissue NEP was increased in mesenteric fat in HFD compared with NCD-fed mice (P<0.05). NEP knockout mice did not display any changes in insulin resistance, glucose tolerance, or body and epididymal fat pad weight compared with wild-type mice. CONCLUSION: In humans, NEP activity correlated with BMI and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors. NEP protein production in human adipocytes increased during cell differentiation and plasma and adipose tissue levels of NEP were increased in obese insulin-resistant mice. Our results indicate that NEP associates with cardiometabolic risk in the presence of insulin resistance and increases with obesity.

The emerging role of ACE2 in physiology and disease.

The renin-angiotensin-aldosterone system (RAAS) is a key regulator of systemic blood pressure and renal function and a key player in renal and cardiovascular disease. However, its (patho)physiological roles and its architecture are more complex than initially anticipated. Novel RAAS components that may add to our understanding have been discovered in recent years. In particular, the human homologue of ACE (ACE2) has added a higher level of complexity to the RAAS. In a short period of time, ACE2 has been cloned, purified, knocked-out, knocked-in; inhibitors have been developed; its 3D structure determined; and new functions have been identified. ACE2 is now implicated in cardiovascular and renal (patho)physiology, diabetes, pregnancy, lung disease and, remarkably, ACE2 serves as a receptor for SARS and NL63 coronaviruses. This review covers available information on the genetic, structural and functional properties of ACE2. Its role in a variety of (patho)physiological conditions and therapeutic options of modulation are discussed.

Reactive oxygen species (ROS)-mediated beta-cleavage of the prion protein in the mechanism of the cellular response to oxidative stress.

The PrP(C) [cellular isoform of PrP (prion protein)] can undergo a conformational conversion to produce a proteinase-resistant form PrP(Sc) (scrapie isoform of PrP), a step critical for the development of prion disease. Although essential for disease progression, the normal cellular function of PrP(C) remains unknown. Suggestions to date have centred on a protective role against oxidative stress. We have demonstrated that ROS (reactive oxygen species)-mediated beta-cleavage of PrP(C) occurs at the cell surface, can be inhibited following hydroxyl radical quenching and has a prerequisite for the octarepeat region in the N-terminus of the protein. Significantly, two disease-associated mutants of PrP, namely PG14 and A116V (Ala(116)-->Val), were unable to undergo beta-cleavage and this lack of proteolysis was accompanied by functional consequences in cells expressing these mutant proteins. The cells were found to be less viable following exposure to copper and H2O2, had reduced levels of glutathione peroxidase and increased amounts of intracellular oxygen radicals. These results suggest that beta-cleavage of PrP(C) is an initial consequence following exposure to ROS in the extracellular environment contributing to a pathway involved in antioxidant protection of neuronal cells.

Membrane-associated zinc peptidase families: comparing ACE and ACE2.

In contrast to the relatively ubiquitous angiotensin-converting enzyme (ACE), expression of the mammalian ACE homologue, ACE2, was initially described in the heart, kidney and testis. ACE2 is a type I integral membrane protein with its active site domain exposed to the extracellular surface of endothelial cells and the renal tubular epithelium. Here ACE2 is poised to metabolise circulating peptides which may include angiotensin II, a potent vasoconstrictor and the product of angiotensin I cleavage by ACE. To this end, ACE2 may counterbalance the effects of ACE within the renin-angiotensin system (RAS). Indeed, ACE2 has been implicated in the regulation of heart and renal function where it is proposed to control the levels of angiotensin II relative to its hypotensive metabolite, angiotensin-(1-7). The recent solution of the structure of ACE2, and ACE, has provided new insight into the substrate and inhibitor profiles of these two key regulators of the RAS. As the complexity of this crucial pathway is unravelled, there is a growing interest in the therapeutic potential of agents that modulate the activity of ACE2.

Roles of proteolysis and lipid rafts in the processing of the amyloid precursor protein and prion protein.

In the amyloidogenic pathway, the APP (amyloid precursor protein) is proteolytically processed by the beta- and gamma-secretases to release the Abeta (amyloid-beta) peptide that is neurotoxic and aggregates in the brains of patients suffering from Alzheimer's disease. In the non-amyloidogenic pathway, APP is cleaved by alpha-secretase within the Abeta domain, precluding deposition of intact Abeta peptide. The cellular form of the PrP(C) (prion protein) undergoes reactive oxygen species-mediated beta-cleavage within the copper-binding octapeptide repeats or, alternatively, alpha-cleavage within the central hydrophobic neurotoxic domain. In addition, PrP(C) is shed from the membrane by the action of a zinc metalloprotease. Members of the ADAM (a disintegrin and metalloproteinase) family of zinc metalloproteases, notably ADAM10 and TACE (ADAM17) display alpha-secretase activity towards APP and appear to be responsible for the alpha-cleavage of PrP(C). The amyloidogenic cleavage of APP by the beta- and gamma-secretases appears to occur preferentially in cholesterol-rich lipid rafts, while the conversion of PrP(C) into the infectious form PrP(Sc) also appears to occur in these membrane domains.

Angiotensin-converting enzyme-2: a molecular and cellular perspective.

Angiotensin-converting enzyme-2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein which functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the C-terminus of its substrates. ACE2 efficiently hydrolyses the potent vasoconstrictor angiotensin II to angiotensin (1-7). It is a consequence of this action that ACE2 participates in the renin-angiotensin system. However, ACE2 also hydrolyses dynorphin A (1-13), apelin-13 and des-Arg(9) bradykinin. The role of ACE2 in these peptide systems has yet to be revealed. A physiological role for ACE2 has been implicated in hypertension, cardiac function, heart function and diabetes, and as a receptor of the severe acute respiratory syndrome coronavirus. This paper reviews the biochemistry of ACE2 and discusses key findings such as the elucidation of crystal structures for ACE2 and testicular ACE and the development of ACE2 inhibitors that have now provided a basis for future research on this enzyme.

The search for alpha-secretase and its potential as a therapeutic approach to Alzheimer s disease.

In the nonamyloidogenic processing pathway the Alzheimer s amyloid precursor protein (APP) is proteolytically cleaved by alpha-secretase. As this cleavage occurs at the Lys16-Leu17 bond within the amyloid beta domain, it prevents deposition of intact amyloidogenic peptide. In addition, the large ectodomain (sAPP(alpha)) released by the action of alpha-secretase has several neuroprotective properties. Studies with a range of hydroxamic acid-based compounds, such as batimastat, indicate that alpha-secretase is a zinc metalloproteinase, and members of the adamalysin family of proteins, TACE, ADAM10 and ADAM9, all fulfil some of the criteria required of alpha-secretase. APP is constitutively cleaved by alpha-secretase in most cell lines. However, on stimulation with muscarinic agonists or activators of protein kinase C, such as phorbol esters, the alpha-secretase cleavage of APP is up-regulated. The constitutive alpha-secretase activity is primarily at the cell surface, while the regulated activity is predominantly located within the Golgi. The beneficial action of cholinesterase inhibitors may in part be due to activation of muscarinic receptors, resulting in an up-regulation of alpha-secretase. Other agents can also increase the nonamyloidogenic cleavage of APP including estrogen, testosterone, various neurotransmitters and growth factors. As the alpha-secretase cleavage of APP both precludes the deposition of the amyloid beta peptide and releases the neuroprotective sAPP(alpha), pharmacological up-regulation of alpha-secretase may provide alternative therapeutic approaches for Alzheimer s disease.

Ectodomain shedding of cystinyl aminopeptidase from human placental membranes.

Cystinyl aminopeptidase (CAP; EC 3.4.11.3) is an integral protein of the placental membrane that is also found in a soluble form in maternal serum during pregnancy. CAP was found to be shed from human placental membranes in a temperature- and time-dependent process. The released form of CAP was hydrophilic as assessed by phase separation in Triton X-114 and high-speed centrifugation. This ectodomain shedding of CAP was inhibited by the hydroxamic acid-based compounds marimastat and BB3103. The inhibition profile for the shedding of CAP was distinct to that for the release of angiotensin converting enzyme, implicating the involvement of distinct zinc metallosecretases in the shedding of these two proteins. These results have implications for our understanding of the mechanism underlying the reduction in serum levels of CAP observed in certain pregnancy-related disorders, such as pre-eclampsia.

Determination of glycosyl-phosphatidylinositol membrane protein anchorage.

A diverse range of proteins are modified by the post-translational covalent attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Hydropathy plots and other computer algorithms can be used to predict the presence of a GPI anchor attachment signal in the nascent polypeptide chain. However, the presence of a GPI anchor on the mature protein requires experimental evidence, including sensitivity of the protein to release from cells or membranes with bacterial phosphatidylinositol-specific phospholipase C, recognition by anti-cross-reacting determinant antibodies, or metabolic labelling with components of the anchor. GPI-anchored proteins are resistant to solubilisation with detergents such as Triton X-100 due to their association with cholesterol and glycosphingolipids in membrane domains known as lipid rafts. This detergent insolubility can be used to provide evidence for the presence of a GPI anchor on a protein and to isolate lipid rafts.

Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes: direct evidence for a GPI anchor and ligand binding of the released form.

Transforming growth factor-beta (TGF-beta) is a key modulator of epidermal development and homeostasis, and has been shown to potently regulate keratinocyte migration and function during wound repair. There are three cloned TGF-beta receptors termed type I, type II, and type III that are found on most cell types. The types I and II are the signaling receptors, while the type III is believed to facilitate TGF-beta binding to the types I and II receptors. Recently, we reported that in addition to these receptors, human keratinocytes express a 150 kDa TGF-beta 1 binding protein (r150) which forms a heteromeric complex with the TGF-beta signaling receptors. This accessory receptor was described as glycosyl phosphatidylinositol-specific anchored based on its sensitivity to phosphatidylinositol phospholipase C (PIPLC). In the present study, we demonstrate that the GPI-anchor is contained in r150 itself and not on a tightly associated protein and that it binds TGF-beta 1 with an affinity similar to those of the types I and II TGF-beta signaling receptors. Furthermore, the PIPLC released (soluble) form of this protein is capable of binding TGF-beta 1 independently from the signaling receptors. In addition, we provide evidence that r150 is released from the cell surface by an endogenous phospholipase C. Our observation that r150 interacts with the TGF-beta signaling receptors, together with the finding that the soluble r150 binds TGF-beta 1 suggest that r150 in either its membrane anchored or soluble form may potentiate or antagonize TGF-beta signaling. Elucidating the mechanism by which r150 functions as an accessory molecule in TGF-beta signaling may be critical to understanding the molecular mechanisms underlying the regulation of TGF-beta action in keratinocytes.

Epidemiologic usefulness of spoligotyping for secondary typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110.

Restriction fragment length polymorphism (RFLP) analysis of IS6110 is commonly used to DNA fingerprint Mycobacterium tuberculosis. However, low-copy (< or =5) IS6110 M. tuberculosis strains are poorly differentiated, requiring secondary typing. When spoligotyping was used as the secondary method, only 13% of Maryland culture-positive tuberculosis (TB) patients with low-copy IS6110-spoligotyped clustered strains had epidemiologic linkages to another patient, compared to 48% of those with high-copy strains clustered by IS6110 alone (P < 0.01). Spoligotyping did not improve a population-based molecular epidemiologic study of recent TB transmission.

Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding.

Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.

Differential effects of glycosphingolipids on the detergent-insolubility of the glycosylphosphatidylinositol-anchored membrane dipeptidase.

The insolubility of glycosylphosphatidylinositol (GPI)-anchored proteins in certain detergents appears to be an intrinsic property of their association with sphingolipids and cholesterol in lipid rafts. We show that the GPI-anchored protein membrane dipeptidase is localized in detergent-insoluble lipid rafts isolated from porcine kidney microvillar membranes, and that these rafts, which lack caveolin, are enriched not only in sphingomyelin and cholesterol, but also in the glycosphingolipid lactosylceramide (LacCer). Dipeptidase purified from porcine kidney was reconstituted into artificial liposomes in order to investigate the relationship between glycosphingolipids and GPI-anchored protein detergent-insolubility. Dipeptidase was insoluble in liposomes containing extremely low concentrations of LacCer. In contrast, identical concentrations of glucosylceramide or galactosylceramide failed to promote significant detergent-insolubility. Cholesterol was shown to enhance the detergent-insoluble effect of LacCer. GC-MS analysis revealed dramatic differences between the fatty acyl compositions of LacCer and those of the other glycosphingolipids. However, despite these differences, we show that the unusually marked effect of LacCer to promote the detergent-insolubility of dipeptidase cannot be singularly attributed to the fatty acyl composition of this glycosphingolipid molecule. Instead, we suggest that the ability of LacCer to confer detergent-insolubility on this GPI-anchored protein is dependent on the structure of the lipid molecule in its entirety, and that this glycosphingolipid may have an important role to play in the stabilization of lipid rafts, particularly the caveolin-free glycosphingolipid signalling domains.