Inhibition of sphingosine 1-phosphate protects mice against chondrocyte catabolism and osteoarthritis.

OBJECTIVE: Cartilage loss observed in osteoarthritis (OA) is prevented when osteoclasts in the subchondral bone are inhibited in mice. Here, we investigated the role of the osteoclast secretome and of the lipid mediator sphingosine 1-phosphate (S1P) in chondrocyte metabolism and OA. MATERIALS AND METHODS: We used SphK1LysMCre and wild type mice to assess the effect of murine osteoclast secretome in chondrocyte metabolism. Gene and protein expressions of matrix metalloproteinase (Mmp) were quantified in chondrocytes and explants by RT-qPCR and Western blots. SphK1LysMCre mice or wild type mice treated with S1P2 receptor inhibitor JTE013 or anti-S1P neutralizing antibody sphingomab are analyzed by OA score and immunohistochemistry. RESULTS: The osteoclast secretome increased the expression of Mmp3 and Mmp13 in murine chondrocytes and cartilage explants and activated the JNK signaling pathway, which led to matrix degradation. JTE013 reversed the osteoclast-mediated chondrocyte catabolism and protected mice against OA, suggesting that osteoclastic S1P contributes to cartilage damage in OA via S1P/S1P2 signaling. The activity of sphingosine kinase 1 (SphK1) increased with osteoclast differentiation, and its expression was enhanced in subchondral bone of mice with OA. The expression of Mmp3 and Mmp13 in chondrocytes was low upon stimulation with the secretome of Sphk1-lacking osteoclasts. Cartilage damage was significantly reduced in SphK1LysMCre mice, but not the synovial inflammation. Finally, intra-articular administration of sphingomab inhibited the cartilage damage and synovial inflammation. CONCLUSIONS: Lack of S1P in myeloid cells and local S1P neutralization alleviates from osteoarthritis in mice. These data identify S1P as a therapeutic target in OA.

The Matrilin-3 T298M mutation predisposes for post-traumatic osteoarthritis in a knock-in mouse model.

OBJECTIVE: The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. METHODS: A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. RESULTS: The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. CONCLUSIONS: The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

Identification of TGFß signatures in six murine models mimicking different osteoarthritis clinical phenotypes.

OBJECTIVE: TGFß is a key player in cartilage homeostasis and OA pathology. However, few data are available on the role of TGFß signalling in the different OA phenotypes. Here, we analysed the TGFß pathway by transcriptomic analysis in six mouse models of OA. METHOD: We have brought together seven expert laboratories in OA pathophysiology and, used inter-laboratories standard operating procedures and quality controls to increase experimental reproducibility and decrease bias. As none of the available OA models covers the complexity and heterogeneity of the human disease, we used six different murine models of knee OA: from post-traumatic/mechanical models (meniscectomy (MNX), MNX and hypergravity (HG-MNX), MNX and high fat diet (HF-MNX), MNX and seipin knock-out (SP-MNX)) to aging-related OA and inflammatory OA (collagenase-induced OA (CIOA)). Four controls (MNX-sham, young, SP-sham, CIOA-sham) were added. OsteoArthritis Research Society International (OARSI)-based scoring of femoral condyles and ribonucleic acid (RNA) extraction from tibial plateau samples were done by single operators as well as the transcriptomic analysis of the TGFß family pathway by Custom TaqMan® Array Microfluidic Cards. RESULTS: The transcriptomic analysis revealed specific gene signatures in each of the six models; however, no gene was deregulated in all six OA models. Of interest, we found that the combinatorial Gdf5-Cd36-Ltbp4 signature might discriminate distinct subgroups of OA: Cd36 upregulation is a hallmark of MNX-related OA while Gdf5 and Ltbp4 upregulation is related to MNX-induced OA and CIOA. CONCLUSION: These findings stress the OA animal model heterogeneity and the need of caution when extrapolating results from one model to another.

Dynamics of genomic architecture during composite breed development in cattle.

Some livestock breeds face the challenge of reduced genetic variation, increased inbreeding depression owing to genetic drift and selection. Hybridization can reverse these processes and increase levels of productivity and adaptation to various environmental stressors. Samples from American Brangus were used to evaluate the indicine/taurine composition through nine generations (~45 years) after the hybridization process was completed. The purpose was to determine how hybridization alters allelic combinations of a breed over time when genetic factors such as selection and drift are operating. Furthermore, we explored genomic regions with deviations from the expected composition from the progenitor breeds and related these regions to traits under selection. The Brangus composition deviated from the theoretical expectation, defined by the breed association, of 62.5% taurine, showing taurine composition to be 70.4 ± 0.6%. Taurine and indicine proportion were not consistent across chromosomes. Furthermore, these non-uniform areas were found to be associated with traits that were probably under selection such as intermuscular fat and average daily gain. Interestingly, the sex chromosomes were predominantly taurine, which could be due to the composite being formed particularly in the final cross that resulted in progeny designated as purebred Brangus. This work demonstrated the process of new breed formation on a genomic level. It suggests that factors like genetic drift, selection and complementarity shift the genetic architecture into a uniquely different population. These findings are important to better understand how hybridization and crossbreeding systems shape the genetic architecture of composite populations.

Stratified primary care versus non-stratified care for musculoskeletal pain: findings from the STarT MSK feasibility and pilot cluster randomized controlled trial.

BACKGROUND: Musculoskeletal (MSK) pain from the five most common presentations to primary care (back, neck, shoulder, knee or multi-site pain), where the majority of patients are managed, is a costly global health challenge. At present, first-line decision-making is based on clinical reasoning and stratified models of care have only been tested in patients with low back pain. We therefore, examined the feasibility of; a) a future definitive cluster randomised controlled trial (RCT), and b) General Practitioners (GPs) providing stratified care at the point-of-consultation for these five most common MSK pain presentations. METHODS: The design was a pragmatic pilot, two parallel-arm (stratified versus non-stratified care), cluster RCT and the setting was 8 UK GP practices (4 intervention, 4 control) with randomisation (stratified by practice size) and blinding of trial statistician and outcome data-collectors. Participants were adult consulters with MSK pain without indicators of serious pathologies, urgent medical needs, or vulnerabilities. Potential participant records were tagged and individuals sent postal invitations using a GP point-of-consultation electronic medical record (EMR) template. The intervention was supported by the EMR template housing the Keele STarT MSK Tool (to stratify into low, medium and high-risk prognostic subgroups of persistent pain and disability) and recommended matched treatment options. Feasibility outcomes included exploration of recruitment and follow-up rates, selection bias, and GP intervention fidelity. To capture recommended outcomes including pain and function, participants completed an initial questionnaire, brief monthly questionnaire (postal or SMS), and 6-month follow-up questionnaire. An anonymised EMR audit described GP decision-making. RESULTS: GPs screened 3063 patients (intervention = 1591, control = 1472), completed the EMR template with 1237 eligible patients (intervention = 513, control = 724) and 524 participants (42%) consented to data collection (intervention = 231, control = 293). Recruitment took 28 weeks (target 12 weeks) with > 90% follow-up retention (target > 75%). We detected no selection bias of concern and no harms identified. GP stratification tool fidelity failed to achieve a-priori success criteria, whilst fidelity to the matched treatments achieved "complete success". CONCLUSIONS: A future definitive cluster RCT of stratified care for MSK pain is feasible and is underway, following key amendments including a clinician-completed version of the stratification tool and refinements to recommended matched treatments. TRIAL REGISTRATION: Name of the registry: ISRCTN. TRIAL REGISTRATION NUMBER: 15366334. Date of registration: 06/04/2016.

Implementing core NICE guidelines for osteoarthritis in primary care with a model consultation (MOSAICS): a cluster randomised controlled trial.

OBJECTIVE: To determine the effectiveness of a model osteoarthritis consultation, compared with usual care, on physical function and uptake of National Institute for Health and Care Excellence (NICE) osteoarthritis recommendations, in adults ≥45 years consulting with peripheral joint pain in UK general practice. METHOD: Two-arm cluster-randomised controlled trial with baseline health survey. Eight general practices in England. PARTICIPANTS: 525 adults ≥45 years consulting for peripheral joint pain, amongst 28,443 population survey recipients. Four intervention practices delivered the model osteoarthritis consultation to patients consulting with peripheral joint pain; four control practices continued usual care. The primary clinical outcome of the trial was the SF-12 physical component score (PCS) at 6 months; the main secondary outcome was uptake of NICE core recommendations by 6 months, measured by osteoarthritis quality indicators. A Linear Mixed Model was used to analyse clinical outcome data (SF-12 PCS). Differences in quality indicator outcomes were assessed using logistic regression. RESULTS: 525 eligible participants were enrolled (mean age 67.3 years, SD 10.5; 59.6% female): 288 from intervention and 237 from control practices. There were no statistically significant differences in SF-12 PCS: mean difference at the 6-month primary endpoint was -0.37 (95% CI -2.32, 1.57). Uptake of core NICE recommendations by 6 months was statistically significantly higher in the intervention arm compared with control: e.g., increased written exercise information, 20.5% (7.9, 28.3). CONCLUSION: Whilst uptake of core NICE recommendations was increased, there was no evidence of benefit of this intervention, as delivered in this pragmatic randomised trial, on the primary outcome of physical functioning at 6 months. TRIAL REGISTRATION: ISRCTN06984617.

Effect of a model consultation informed by guidelines on recorded quality of care of osteoarthritis (MOSAICS): a cluster randomised controlled trial in primary care.

OBJECTIVE: To determine the effect of a model osteoarthritis (OA) consultation (MOAC) informed by National Institute for Health and Care Excellence (NICE) recommendations compared with usual care on recorded quality of care of clinical OA in general practice. DESIGN: Two-arm cluster randomised controlled trial. SETTING: Eight general practices in Cheshire, Shropshire, or Staffordshire UK. PARTICIPANTS: General practitioners and nurses with patients consulting with clinical OA. INTERVENTION: Following six-month baseline period practices were randomised to intervention (n = 4) or usual care (n = 4). Intervention practices delivered MOAC (enhanced initial GP consultation, nurse-led clinic, OA guidebook) to patients aged ≥45 years consulting with clinical OA. An electronic (e-)template for consultations was used in all practices to record OA quality care indicators. OUTCOMES: Quality of OA care over six months recorded in the medical record. RESULTS: 1851 patients consulted in baseline period (1015 intervention; 836 control); 1960 consulted following randomisation (1118 intervention; 842 control). At baseline wide variations in quality of care were noted. Post-randomisation increases were found for written advice on OA (4-28%), exercise (4-22%) and weight loss (1-15%) in intervention practices but not controls (1-3%). Intervention practices were more likely to refer to physiotherapy (10% vs 2%, odds ratio 5.30; 95% CI 2.11, 13.34), and prescribe paracetamol (22% vs 14%, 1.74; 95% CI 1.27, 2.38). CONCLUSIONS: The intervention did not improve all aspects of care but increased core NICE recommendations of written advice on OA, exercise and weight management. There remains a need to reduce variation and uniformly enhance improvement in recorded OA care. TRIAL REGISTRATION NUMBER: ISRCTN06984617.

Rearrangement reactions of 1,1-divinyl-2-phenylcyclopropanes.

1,1-Divinyl-2-phenylcyclopropanes are entry points to a rich area of rearrangement chemistry. With N,N-diallyl amide substrates, tandem radical cyclizations can be initiated at room temperature. Warming provides products of pure thermal rearrangements with acids, ester, and amides. These isomerizations give vinylcyclopentenes resulting from divinylcyclopropane rearrangements and more deeply rearranged tricyclic spirolactams resulting from aromatic Cope rearrangements followed by ene reactions. Conversion of the carbonyl group to an alcohol or ether opens retro-ene pathways followed by either tautomerization or Claisen rearrangement.

Should General Practitioners Issue a Sick Certificate to Employees Who Consult for Low Back Pain in Primary Care?

PURPOSE: Back pain is a common problem and has significant societal impact. Sickness certification is commonly issued to patients consulting their general practitioner with low back pain. The aim of this study was to investigate the association of certification for low back pain with clinical outcomes and cost consequences. METHODS: A prospective cohort study using linked questionnaire and medical record data from 806 low back pain patients in 8 UK general practices: comparison of 116 (14.4%) who received a sickness certificate versus 690 who did not receive certification. The primary clinical measure was the Roland and Morris Disability Questionnaire (RMDQ). Data on back pain consultation and work absenteeism were used to calculate healthcare and societal costs. RESULTS: Participants issued a sickness certificate had higher back-related disability at baseline consultation and 6-month follow-up [mean difference 3.1 (95% CI 1.8, 4.4) on the RMDQ], indicating worse health status. After fully adjusting for baseline differences, most changes in clinical outcomes at 6 months were not significantly different between study groups. Productivity losses were significantly higher for the certification group, with most absence occurring after the expected end of certification; mean difference in costs due to absenteeism over 6 months was £1,956 (95% CI £941, £3040). CONCLUSIONS: There was no clear evidence of a difference in clinical outcomes between individuals issued a sickness certificate and those not issued a certification for their back pain. With little overall contrast in clinical outcomes, policy makers and care providers may wish to draw on the likely difference in societal costs alongside issues in ethical and moral care in their consideration of patient care for low back pain.

Fates of imine intermediates in radical cyclizations of N-sulfonylindoles and ene-sulfonamides.

Two new fates of imine intermediates formed on radical cyclizations of ene-sulfonamides have been identified, reduction and hydration/fragmentation. Tin hydride-mediated cyclizations of 2-halo-N-(3-methyl-N-sulfonylindole)anilines provide spiro[indoline-3,3'-indolones] or spiro-3,3'-biindolines (derived from imine reduction), depending on the indole C2 substituent. Cyclizations of 2-haloanilide derivatives of 3-carboxy-N-sulfonyl-2,3-dihydropyrroles also presumably form spiro-imines as primary products. However, the lactam carbonyl group facilitates the ring-opening of these cyclic imines by a new pathway of hydration and retro-Claisen-type reaction, providing rearranged 2-(2'-formamidoethyl)oxindoles.

Radical cyclizations of cyclic ene sulfonamides occur with ß-elimination of sulfonyl radicals to form polycyclic imines.

Radical cyclizations of cyclic ene sulfonamides provide stable bicyclic and tricyclic aldimines and ketimines in good yields. Depending on the structure of the precursor, the cyclizations occur to provide fused and spirocyclic imines with five-, six-, and seven-membered rings. The initial radical cyclization produces an α-sulfonamidoyl radical that undergoes elimination to form the imine and a phenylsulfonyl radical. In a related method, 3,4-dihydroquinolines can also be produced by radical translocation reactions of N-(2-iodophenylsulfonyl)tetrahydroiso-quinolines. In either case, very stable sulfonamides are cleaved to form imines (rather than amines) under mild reductive conditions.

Rotational isomers of N-methyl-N-arylacetamides and their derived enolates: implications for asymmetric Hartwig oxindole cyclizations.

The rotational preferences of N-(2-bromo-4,6-dimethylphenyl)-N-methyl 2-phenylpropanamide were studied as a model of precursors for Hartwig asymmetric oxindole cyclizations. The atropisomers of this compound were separated by flash chromatography, and then the enantiomers were resolved and the interconversions of the stereocenter and the N-Ar axis were studied. Under thermal conditions, the axis is very stable. Under the basic conditions of the Hartwig cyclization, both the stereocenter and the chiral axis equilibrate via enolate formation. The N-Ar rotation barrier of a 2-phenylacetamide analogue was reduced from 31 kcal mol(-1) in the precursor to 17 kcal mol(-1) in the enolate. Reasons for this dramatic barrier reduction and implications of both N-Ar and amide C-N rotations for Hartwig cyclizations are discussed.

Animal models in OA: a means to explore bone.

Cartilage damage which characterizes osteoarthritis is accompanied with bone lesions. Joint integrity results from the balance in the physiological interactions between bone and cartilage. Several local factors regulate physiological remodeling of cartilage, the disequilibrium of these leading to a higher cartilage catabolism. Several cytokines secreted by bone cells can induce chondrocyte differentiation which suggests their role in the dialogue between both cells. Several animal models of osteoarthritis have been developed in order to assess the mechanism of cartilage loss and chondrocyte functions that encompassed surgical, chemical, or genetic approaches. Indeed, the animal models are helpful to investigate the cartilage changes in relation to changes in bone remodeling. Accumulative in vivo evidence show that increased bone resorption occurs at early stage of the development of osteoarthritis. Inhibition of bone resorbing molecules prevents cartilage damage, confirming the role of bone factors in the cross talk between both tissues. Among these numerous molecules, some participate to the imbalance in cartilage homeostasis and in the pathophysiology of osteoarthritis. These local factors are potential candidates for new drug targets.

Incidence, progression and sequence of development of radiographic knee osteoarthritis in a symptomatic population.

OBJECTIVES: Symptomatic knee osteoarthritis (OA) is a common disabling condition. Attention has tended to focus on the tibiofemoral joint (TFJ). However, there is evidence that the patellofemoral joint (PFJ) is involved in many cases, but its place in the sequence of development and progression of knee OA is unclear. This study estimates the cumulative incidence, progression and inter-relationship of radiographic changes of OA in the TFJ and the PFJ in symptomatic adults. METHODS: A population-based observational cohort of 414 adults aged ≥ 50 years with knee pain who had knee x-rays (weight-bearing posteroanterior semiflexed, skyline and lateral views) in 2002-3 and again in 2005-6 (mean interval 36.7 months) was studied. The outcome measure was the development of incident or progressive radiographic OA. RESULTS: The 3-year cumulative incidences of patellofemoral joint osteoarthritis (PFJOA) and tibiofemoral joint osteoarthritis (TFJOA) were 28.8% and 21.7%, respectively. Corresponding estimates of 3-year cumulative progression were 18.9% and 25.3%. PFJOA at baseline was common and increased the risk of incident TFJOA (adjusted OR 2.2, 95% CI 1.1 to 4.1) but less clearly progression of TFJOA (adjusted OR 1.7, 95% CI 0.3 to 9.0). TFJOA at baseline increased the risk of PFJOA incidence and progression (adjusted OR 3.1, 95% CI 1.2 to 8.4 and OR 4.5, 95% CI 1.8 to 11.2, respectively). CONCLUSIONS: These results suggest a common sequence in the development of radiographic knee OA in symptomatic adults beginning in the PFJ, with subsequent addition and progression of TFJOA. It is proposed that isolated symptomatic PFJOA may be one marker for the future development of TFJOA and a target for the early management of knee OA.

Extracellular matrix alters epithelial differentiation.

Extracellular matrix (ECM) induces and maintains the differentiation of epithelial cells, not by totally altering their state of differentiation, but by activating overt differentiation. Recent studies of cultured mammary cells provide an elegant molecular analysis of this kind of progressive cell differentiation. Other studies show that ECM can not only activate and enhance a differentiated state, but can even alter it in bringing about transformation of epithelium to mesenchyme.

Genotype by environment interaction in response to cold stress in a composite beef cattle breed.

Extreme weather conditions such as cold stress influence the productivity and survivability of beef cattle raised on pasture. The objective of this study was to identify and evaluate the extent of the impact of genotype by environment interaction due to cold stress on birth weight (BW) and weaning weight (WW) in a composite beef cattle population. The effect of cold stress was modelled as the accumulation of total cold load (TCL) calculated using the Comprehensive Climate Index units, considering three TCL classes defined based on temperature: less than -5°C (TCL5), -15°C (TCL15) and -25°C (TCL25). A total of 4221 and 4217 records for BW and WW, respectively, were used from a composite beef cattle population (50% Red Angus, 25% Charolais and 25% Tarentaise) between 2002 and 2015. For both BW and WW, a univariate model (ignoring cold stress) and a reaction norm model were implemented. As cold load increased, the direct heritability slightly increased in both BW and WW for TCL5 class; however, this heritability remained consistent across the cold load of TCL25 class. In contrast, the maternal heritability of BW was constant with cold load increase in all TCL classes, although a slight increase of maternal heritability was observed for TCL5 and TCL15. The direct and maternal genetic correlation for BW and maternal genetic correlation for WW across different cold loads between all TCL classes were high (r > 0.99), whereas the lowest direct genetic correlations observed for WW were 0.88 for TCL5 and 0.85 for TCL15. The Spearman rank correlation between the estimated breeding value of top bulls (n = 79) using univariate and reaction norm models across TCL classes showed some re-ranking in direct and maternal effects for both BW and WW particularly for TCL5 and TCL15. In general, cold stress did not have a big impact on direct and maternal genetic effects of BW and WW.

Does isolated patellofemoral osteoarthritis matter?

OBJECTIVES: To describe the structure-pain and structure-function associations in isolated patellofemoral osteoarthritis (PF OA). DESIGN: Population-based study of 819 adults aged > or =50 years with knee pain. The severity of knee pain, stiffness and disability were measured using the Western Ontario and McMaster Osteoarthritis Index (WOMAC). Three radiographic views of the knee were obtained. RESULTS: Isolated PF OA was mild in 142 participants and moderate/severe in 44. Mean WOMAC scores for pain, stiffness and function were associated with radiographic severity of PF OA (F(2,389)=4.7, P=0.01; F(2,392)=4.5, P=0.012 and F(2,392)=6.1, P=0.002, respectively, adjusted for age, gender, and body mass index (BMI)). Post-hoc tests demonstrated statistically significant differences for mean pain, stiffness and function score between those with mild PF OA and those with normal X-rays. In task-specific items there was evidence of a stepped response, the proportion of participants with moderate/severe/extreme pain or difficulty in performing everyday tasks increasing with the severity of PF OA. The strongest association was observed for pain going up and down stairs (age-gender-BMI adjusted odds ratio (OR) 3.0; 95% confidence interval (CI) 1.4,6.6. Functional tasks most strongly related to radiographic severity were: descending stairs (OR 3.2; (CI 1.5,6.5)), getting in/out of the bath (3.2; 1.5,6.6), getting in/out of a car (3.0; 1.4,6.1). CONCLUSIONS: Mild isolated PF OA is significantly associated with symptoms of pain, stiffness and functional limitation. Further research on its recognition in clinical practice and the development of targeted treatments to prevent or slow progression are warranted.

Radiographic hand osteoarthritis: patterns and associations with hand pain and function in a community-dwelling sample.

OBJECTIVE: Patterns of radiographic osteoarthritis (ROA) of the hand are often examined by row, with the four joints of the thumb studied inconsistently. The objectives of this study were to determine relationships of ROA at different hand joints, use the findings to define radiographic sub-groups and investigate their associations with pain and function. METHODS: Sixteen joints in each hand were scored for the presence of ROA in a community-dwelling cohort of adults, 50-years-and-over, with self-reported hand pain or problems. Principal components analysis (PCA) with varimax rotation was used to study patterns of ROA in the hand joints and identify distinct sub-groups. Differences in pain and function between these sub-groups were assessed using Australian/Canadian Osteoarthritis Index (AUSCAN), Grip Ability Test (GAT) and grip and pinch strength. RESULTS: PCA was undertaken on data from 592 participants and identified four components: distal interphalangeal joints (DIPs), proximal interphalangeal joints (PIPs), metacarpophalangeal joints (MCPs), thumb joints. However, the left thumb interphalangeal (IP) joint cross-loaded with the PIP and thumb groups. On this basis, participants were categorised into four radiographic sub-groups: no osteoarthritis (OA), finger only OA, thumb only OA and combined thumb and finger OA. Statistically significant differences were found between the sub-groups for AUSCAN function, and in women alone for grip and pinch strength. Participants with combined thumb and finger OA had the worst scores. CONCLUSION: Individual thumb joints can be clustered together as a joint group in ROA. Four radiographic sub-groups of hand OA can be distinguished. Pain and functional difficulties were highest in participants with both thumb and finger OA.

Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix. I. Morphometric analysis of transfilter "induction".

The present study was undertaken to determine whether or not physical contact with the substratum is essential for the stimulatory effect of extracellular matrix (ECM) on corneal epithelial collagen synthesis. Previous studies showed that collagenous substrata stimulate isolated epithelia to produce three times as much collagen as they produce on noncollagenous substrate; killed collagenous substrata (e.g., lens capsule) are just as effective as living substrata (e.g., living lens) in promoting the production of new corneal stroma in vitro. In the experiments to be reported here, corneal epithelia were placed on one side of Nucleopore filters of different pore sizes and killed lens capsule on the other, with the expectation that contact of the reacting cells with the lens ECM should be limited by the number and size of the cell processes that can tranverse the pores. Transfilter cultures were grown for 24 h in [3H]proline-containing median and incorporation of isotope into hot trichloroacetic acid-soluble protein was used to measure corneal epithelial collagen production. Epithelial collagen synthesis increases directly as the size of the pores in the interposed filter increases and decreases as the thickness of the filter layer increases. Cell processes within Nucleopore filters were identified with the transmission electron microscope with difficulty; with the scanning electron microscope, however, the processes could easily be seen emerging from the undersurface of even 0.1-mum pore size filters. Morphometric techniques were used to show that cell surface area thus exposed to the underlying ECM is linearly correlated with enhancement of collagen synthesis. Epithelial cell processes did not pass through ultrathin (25-mum thick) 0.45-mum pore size Millipore filters nor did "induction" occur across them. The results are discussed in relation to current theories of embryonic tissue interaction.

Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.

This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium.