Chondrocyte mechanotransduction: effects of compression on deformation of intracellular organelles and relevance to cellular biosynthesis

OBJECTIVE: The effects of mechanical deformation of intact cartilage tissue on chondrocyte biosynthesis in situ have been well documented, but the mechanotransduction pathways that regulate such phenomena have not been elucidated completely. The goal of this study was to examine the effects of tissue deformation on the morphology of a range of intracellular organelles which play a major role in cell biosynthesis and metabolism. DESIGN: Using chemical fixation, high pressure freezing, and electron microscopy, we imaged chondrocytes within mechanically compressed cartilage explants at high magnification and quantitatively and qualitatively assessed changes in organelle volume and shape caused by graded levels of loading. RESULTS: Compression of the tissue caused a concomitant reduction in the volume of the extracellular matrix (ECM), chondrocyte, nucleus, rough endoplasmic reticulum, and mitochondria. Interestingly, however, the Golgi apparatus was able to resist loss of intraorganelle water and retain a portion of its volume relative to the remainder of the cell. These combined results suggest that a balance between intracellular mechanical and osmotic gradients govern the changes in shape and volume of the organelles as the tissue is compressed. CONCLUSIONS: Our results lead to the interpretive hypothesis that organelle volume changes appear to be driven mainly by osmotic interactions while shape changes are mediated by structural factors, such as cytoskeletal interactions that may be linked to extracellular matrix deformations. The observed volume and shape changes of the chondrocyte organelles and the differential behavior between organelles during tissue compression provide evidence for an important mechanotransduction pathway linking translational and post-translational events (e.g., elongation and sulfation of glycosaminoglycans (GAGs) in the Golgi) to cell deformation.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis

Matrix metalloproteinases (MMPs) are a family of Zn2+-dependent endopeptidases targeting extracellular matrix (ECM) compounds as well as a number of other proteins. Their proteolytic activity acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. In the context of neuro-inflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system. This review focuses on the role of MMPs in multiple sclerosis (MS) and bacterial meningitis (BM), two neuro-inflammatory diseases where current therapeutic approaches are insufficient to prevent severe disability in the majority of patients. Inhibition of enzymatic activity may prevent MMP-mediated neuronal damage due to an overactive or deviated immune response in both diseases. Downregulation of MMP release may be the molecular basis for the beneficial effect of IFN-beta and steroids in MS. Instead, synthetic MMP inhibitors offer the possibility to shut off enzymatic activity of already activated MMPs. In animal models of MS and BM, they efficiently attenuated clinical disease symptoms and prevented brain damage due to excessive metalloproteinase activity. However, the required target profile for the therapeutic use of this novel group of compounds in human disease is not yet sufficiently defined and may be different depending on the type and stage of disease. Currently available MMP inhibitors show little target-specificity within the MMP family and may lead to side-effects due to interference with physiological functions of MMPs. Results from human MS and BM indicate that only a restricted number of MMPs specific for each disease is up-regulated. MMP inhibitors with selective target profiles offer the possibility of a more efficient therapy of MS and BM and may enter clinical trials in the near future.

Meta-analyses qualify metzincins and related genes as acute rejection markers in renal transplant patients

Definition of acute renal allograft rejection (AR) markers remains clinically relevant. Features of T-cell-mediated AR are tubulointerstitial and vascular inflammation associated with excessive extracellular matrix (ECM) remodeling, regulated by metzincins, including matrix metalloproteases (MMP). Our study focused on expression of metzincins (METS), and metzincins and related genes (MARGS) in renal allograft biopsies using four independent microarray data sets. Our own cases included normal histology (N, n = 20), borderline changes (BL, n = 4), AR (n = 10) and AR + IF/TA (n = 7). MARGS enriched in all data sets were further examined on mRNA and/or protein level in additional patients. METS and MARGS differentiated AR from BL, AR + IF/TA and N in a principal component analysis. Their expression changes correlated to Banff t- and i-scores. Two AR classifiers, based on METS (including MMP7, TIMP1), or on MARGS were established in our own and validated in the three additional data sets. Thirteen MARGS were significantly enriched in AR patients of all data sets comprising MMP7, -9, TIMP1, -2, thrombospondin2 (THBS2) and fibrillin1. RT-PCR using microdissected glomeruli/tubuli confirmed MMP7, -9 and THBS2 microarray results; immunohistochemistry showed augmentation of MMP2, -9 and TIMP1 in AR. TIMP1 and THBS2 were enriched in AR patient serum. Therefore, differentially expressed METS and MARGS especially TIMP1, MMP7/-9 represent potential molecular AR markers.

Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells

Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro, HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL).

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.

Single application of low-dose mycophenolate mofetil-OX7-immunoliposomes ameliorates experimental mesangial proliferative glomerulonephritis

IgA nephropathy, one of the most frequent forms of glomerulonephritis, characterized by mesangial hypercellularity and glomerular extracellular matrix (ECM) expansion, often leads to end-stage renal disease over a prolonged period. We investigated whether antiproliferative treatment in a single low dose specifically targeted to the glomerular mesangium by immunoliposomes (ILs) results in an amelioration of mesangial proliferative glomerulonephritis in rats (anti-Thy1.1 nephritis). Mycophenolate mofetil (MMF) containing ILs was generated that targets the Thy1.1 antigen (OX-7) in rat mesangial cells. Treatment benefit of a single intravenous dose of these ILs given 2 days after disease induction was investigated by stereology, immunohistochemistry, and functional analyses (creatinine, albuminuria) until day +9 and was compared among untreated and free MMF-treated rats using six male Wistar rats per group. MMF-loaded OX7-IL prevented creatinine increase and albuminuria. Stereological analyses of MMF OX7-IL-treated animals yielded 30% reduction of mesangial cells on day +9 and a 40% reduction of glomerular ECM volume on day +5, compared with all of the other nephritic animals. Furthermore, at days +5 and +9 we observed decreased ECM content and decreased glomerular volume (day +5) in the MMF-OX7-IL-treated group compared with the nephritic group treated with free MMF. In conclusion, MMF-OX7-IL-based directed drug delivery represents a novel approach for treating mesangial cell-mediated forms of glomerulonephritis.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)- and heparin (KRSR)-binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis

BACKGROUND/AIMS: Mammalian target of rapamycin (mTOR) signalling is central in the activation of hepatic stellate cells (HSCs), the key source of extracellular matrix (ECM) in fibrotic liver. We tested the therapeutic potential of the mTOR inhibitor rapamycin in advanced cirrhosis. METHODS: Cirrhosis was induced by bile duct-ligation (BDL) or thioacetamide injections (TAA). Rats received oral rapamycin (0.5 mg/kg/day) for either 14 or 28 days. Untreated BDL and TAA-rats served as controls. Liver function was quantified by aminopyrine breath test. ECM and ECM-producing cells were quantified by morphometry. MMP-2 activity was measured by zymography. mRNA expression of procollagen-alpha1, transforming growth factor-beta1 (TGF-beta1) and beta2 was quantified by RT-PCR. RESULTS: Fourteen days of rapamycin improved liver function. Accumulation of ECM was decreased together with numbers of activated HSCs and MMP-2 activity in both animal models. TGF-beta1 mRNA was downregulated in TAA, TGF-beta2 mRNA was downregulated in BDL. 28 days of rapamycin treatment entailed a survival advantage of long-term treated BDL-rats. CONCLUSIONS: Low-dose rapamycin treatment is effectively antifibrotic and attenuates disease progression in advanced fibrosis. Our results warrant the clinical evaluation of rapamycin as an antifibrotic drug.

Meprins, membrane-bound and secreted astacin metalloproteinases

The astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the 'hatching' subfamily comprising alveolin, ovastacin, LCE, HCE ('low' and 'high' choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six astacin family genes (two meprins, three BMP1/tolloid-like, one ovastacin), but in Caenorhabditis elegans there are 40. Meprins are the only astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions.

Induction of tenascin-C by cyclic tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK signaling pathways

Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-beta1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-beta1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

Renal allografts with IF/TA display distinct expression profiles of metzincins and related genes

Chronic renal allograft injury is often reflected by interstitial fibrosis (IF) and tubular atrophy (TA) without evidence of specific etiology. In most instances, IF/TA remains an irreversible disorder, representing a major cause of long-term allograft loss. As members of the protease family metzincins and functionally related genes are involved in fibrotic and sclerotic processes of the extracellular matrix (ECM), we hypothesized their deregulation in IF/TA. Gene expression and protein level analyses using allograft biopsies with and without Banff'05 classified IF/TA illustrated their deregulation. Expression profiles of these genes differentiated IF/TA from Banff'05 classified Normal biopsies in three independent microarray studies and demonstrated histological progression of IF/TA I to III. Significant upregulation of matrix metalloprotease-7 (MMP-7) and thrombospondin-2 (THBS-2) in IF/TA biopsies and sera was revealed in two independent patient sets. Furthermore, elevated THBS-2, osteopontin (SPP1) and beta-catenin may play regulatory roles on MMP. Our findings further suggest that deregulated ECM remodeling and possibly epithelial to mesenchymal transition (EMT) are implicated in IF/TA of kidney transplants, and that metzincins and related genes play an important role in these processes. Profiling of these genes may be used to complement IF/TA diagnosis and to disclose IF/TA progression in kidney transplant recipients.

Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors

BACKGROUND Bone-to-tendon healing after rotator cuff repairs is mainly impaired by poor tissue quality. The tenocytes of chronic rotator cuff tendon tears are not able to synthesize normal fibrocartilaginous extracellular matrix (ECM). We hypothesized that in the presence of platelet-released growth factors (PRGF), tenocytes from chronically retracted rotator cuff tendons proliferate and synthesize the appropriate ECM proteins. MATERIALS AND METHODS Tenocytes from 8 patients with chronic rotator cuff tears were cultured for 4 weeks in 2 different media: standard medium (Iscove's Modified Dulbecco's Media + 10% fetal calf serum + 1% nonessential amino acids + 0.5 μg/mL ascorbic acid) and media with an additional 10% PRGF. Cell proliferation was assessed at 7, 14, 21, and 28 days. Messenger (m)RNA levels of collagens I, II, and X, decorin, biglycan, and aggrecan were analyzed using real time reverse-transcription polymerase chain reaction. Immunocytochemistry was also performed. RESULTS The proliferation rate of tenocytes was significantly higher at all time points when cultured with PRGF. At 21 days, the mRNA levels for collagens I, II, and X, decorin, aggrecan, and biglycan were significantly higher in the PRGF group. The mRNA data were confirmed at protein level by immunocytochemistry. CONCLUSIONS PRGFs enhance tenocyte proliferation in vitro and promote synthesis of ECM to levels similar to those found with insertion of the normal human rotator cuffs. CLINICAL RELEVANCE Biologic augmentation of repaired rotator cuffs with PRGF may enhance the properties of the repair tissue. However, further studies are needed to determine if application of PRGF remains safe and effective in long-term clinical studies. LEVEL OF EVIDENCE Basic Science Study, Cell Biology.