Focal Experimental Injury Leads to Widespread Gene Expression and Histologic Changes in Equine Flexor Tendons

It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3cm regions) for histologic (scoring and immunohistochemistry) and gene expression (real time PCR) analysis of extracellular matrix changes. The histopathology score was significantly higher in transected tendons compared to control tendons in all regions except for the most distal (P ≤ 0.03) with no differences between overstressed (medial) and stress-deprived (lateral) tendon halves. Proteoglycan scores were increased by transection in all but the most proximal region (P < 0.02), with increased immunostaining for aggrecan, biglycan and versican. After correcting for location within the tendon, gene expression for aggrecan, versican, biglycan, lumican, collagen types I, II and III, MMP14 and TIMP1 was increased in transected tendons compared with control tendons (P < 0.02) and decreased for ADAMTS4, MMP3 and TIMP3 (P < 0.001). Aggrecan, biglycan, fibromodulin, and collagen types I and III expression positively correlated with all histopathology scores (P < 0.001), whereas lumican, ADAMTS4 and MMP14 expression positively correlated only with collagen fiber malalignment (P < 0.001). In summary, histologic and associated gene expression changes were significant and widespread six weeks after injury to the equine SDFT, suggesting rapid and active development of tendinopathy throughout the entire length of the tendon. These extensive changes distant to the focal injury may contribute to poor functional outcomes and re-injury in clinical cases. Our data suggest that successful treatments of focal injuries will need to address pathology in the entire tendon, and that better methods to monitor the development and resolution of tendinopathy are required.     

Reduction of Tendon Adhesions following Administration of Adaprev,a Hypertonic Solution of Mannose-6-Phosphate: Mechanism of Action Studies

Repaired tendons may be complicated by progressive fibrosis, causing adhesion formation or tendon softening leading to tendon rupture and subsequent reduced range of motion. There are few therapies available which improve the gliding of damaged tendons in the hand. We investigate the role of Mannose 6-phosphate (M6P) in a 600 mM hypertonic solution (Adaprev) on tendon adhesion formation in vivo using a mouse model of severed tendon in conjunction with analysis of collagen synthesis, cellular proliferation and receptors involved in TGF beta signalling. Cytotoxicity was assessed by measuring tissue residency, mechanical strength and cell viability of tendons after treatment with Adaprev. To elicit potential modes of action, in vitro and ex vivo studies were performed investigating phosphorylation of p38, cell migration and proliferation. Adaprev treatment significantly (p<0.05) reduced the development of adhesions and improved collagen organisation without reducing overall collagen synthesis following tendon injury in vivo. The bioavailability of Adaprev saw a 40% reduction at the site of administration over 45 minutes and tendon fibroblasts tolerated up to 120 minutes of exposure without significant loss of cell viability or tensile strength. These favourable effects were independent of CI-MPR and TGF-β signalling and possibly highlight a novel mechanism of action related to cellular stress demonstrated by phosphorylation of p38. The effect of treatment reduced tendon fibroblast migration and transiently halted tendon fibroblast proliferation in vitro and ex vivo. Our studies demonstrate that the primary mode of action for Adaprev is potentially via a physical, non-chemical, hyperosmotic effect.     

Loss of tenomodulin expression is a risk factor for age‐related intervertebral disc degeneration

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti‐angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament‐specific marker and anti‐angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age‐dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd^?/?) mice exhibited more rapid progression of age‐related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd^?/?Chm1^?/?) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF‐derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age‐related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age‐related IVD degeneration.     

Differential expression of Tenomodulin and Chondromodulin-1 at the insertion site of the tendon reflects a phenotypic transition of the resident cells

The insertion site of the tendon to the skeletal element is hypovascular and is one of the most common sites of dysfunction in the musculoskeletal system. However, the resident cells have been poorly defined due to a lack of a specific marker for tenocytes. We previously reported that Tenomodulin (Tnmd) and Chondromodulin-1 (Chm1) are homologous angiogenesis inhibitors and predominantly expressed in the avascular region of tendons and cartilage, respectively. In this study, we analyzed the expression of Tnmd, Chm1, alpha 1 chain of the type I collagen (Col1a1) and alpha 1 chain of the type II collagen (Col2a1) at the insertion site of the Achilles, patellar, or rotator cuff tendons of 1-week-old rabbits by in situ hybridization analysis. Tnmd was co-expressed with Col1a1 in tenocytes of these tendons, while Chm1 and Col2a1 were detected in chondrocytes of the hyaline cartilage. Interestingly, the cell population between Tnmd/Col1a1 positive tenocytes and Chm1/Col2a1 positive chondrocytes expressed Col1a1 but none of the other markers (Tnmd, Chm1, and Col2a1). Red blood cells were exclusively present at the interface between the tendon substance and cartilage in the insertion site of the Achilles tendon. Lack of Tnmd and Chm1 in this newly characterized cell population may allow the transitional zone between the poorly vascularized tendon and cartilage to establish the unique vascular pattern for blood supply.     

Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1−/− tendon. In Col6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm²) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.     

Capture of linear fragments at a double-strand break in yeast

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed ‘filler DNA’) can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10⁻⁶ to 10⁻⁴ per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture.     

Procollagen C proteinase enhancer 1 genes are important determinants of the mechanical properties and geometry of bone and the ultrastructure of connective tissues

Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs ~10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce^−/− mice are viable and fertile, Pcolce^−/− male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce^−/− male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce^−/− vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce^−/− mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce^−/− tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.     

Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala^*Gly-Pro-Ile-Gly (k_cat, 5.41 s⁻¹; K_m, 32 μM) and Met-Gly-Pro-Arg^*Gly-Phe-Pro-Gly-Ser (k_cat, 351 s⁻¹; K_m, 214 μM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.     

Macrophage autophagy protects against liver fibrosis in mice

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5^fl/fl LysM-Cre mice, referred to as atg5^−/−) and their wild-type (Atg5^fl/fl, referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5^−/− mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5^−/− mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5^−/− mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5^−/− mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5^−/− macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5^−/− mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

α2β1 Integrin,GPVI Receptor,and Common FcRγ Chain on Mouse Platelets Mediate Distinct Responses to Collagen in Models of Thrombosis

Objective

Platelets express the α2β1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ^−/−) or the complex was depleted. The development of α2β1^−/− and GPVI^−/− mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro.

Approach and Results

To understand the different roles played by the α2β1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2β1^−/−, FcRγ^−/−, and GPVI^−/− mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2β1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2β1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ^−/− platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI^−/− and wild type platelets. The difference between FcRγ^−/− and GPVI^−/− platelet phosphotyrosine levels correlated with the in vivo thrombosis findings.

Conclusion

Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2β1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings suggest a more complex signaling network downstream of the platelet collagen receptors than previously appreciated.     

Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E₂, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE₂ had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.     

Functions of lumican and fibromodulin: lessons from knockout mice

Lumican and fibromodulin are collagen-binding leucine-rich proteoglycans widely distributed in interstitial connective tissues. The phenotypes of lumican-null (Lum ^–/–), Fibromodulin-null (Fmod ^–/–) and compound double-null (Lum ^–/– Fmod ^–/–) mice identify a broad range of tissues where these two proteoglycans have overlapping and unique roles in modulating the extracellular matrix and cellular behavior. The lumican-deficient mice have reduced corneal transparency and skin fragility. The Lum ^–/– Fmod ^–/– mice are smaller than their wildtype littermates, display gait abnormality, joint laxity and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis and extreme tendon weakness are the likely cause for joint-laxity. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum ^+/+ Fmod ^–/– mice, with further loss in stiffness in a lumican gene dose-dependent way. At the level of ultrastructure, the Lum ^–/– cornea, skin and tendon show irregular collagen fibril contours and increased fibril diameter. The Fmod ^–/– tendon contains irregular contoured collagen fibrils, with increased frequency of small diameter fibrils. The tendons of Lum ^–/– Fmod ^–/– have an abnormally high frequency of small and large diameter fibrils indicating a de-regulation of collagen fibril formation and maturation. In tissues like the tendon, where both proteoglycans are present, fibromodulin may be required early in collagen fibrillogenesis to stabilize small-diameter fibril-intermediates and lumican may be needed at a later stage, primarily to limit lateral growth of fibrils Published in 2003.     

The Effect of Lubricin on the Gliding Resistance of Mouse Intrasynovial Tendon

The purpose of this study was to investigate the role of lubricin on the gliding resistance of intrasynovial tendons by comparing lubricin knockout, heterozygous, and wild type mice. A total of thirty-six deep digital flexor (DDF) tendons in the third digits of each hind paw from eighteen adult mice were used, including six lubricin knockout mice (Prg4 –/–), six heterozygous mice (Prg4 +/–), and six wild type mice (Prg4 +/+). The tendon gliding resistance was measured using a custom-made device. Tendon structural changes were evaluated by scanning electron and light microscopy. The gliding resistance of intrasynovial tendons from lubricin knockout mice was significantly higher than the gliding resistance of either wild type or heterozygous mice. The surface of the lubricin knockout tendons appeared to be rougher, compared to the wild type and heterozygous tendons. Synovial hyperplasia was found in the lubricin knockout mice. Cartilage-like tissue was found in the tendon and pulley of the lubricin knockout mice. Our findings confirm the importance of lubricin in intrasynovial tendon lubrication. This knockout model may be useful in determining the effect of lubricin on tendon healing and the response to injury.     

HGF Mediates the Anti-inflammatory Effects of PRP on Injured Tendons

Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE₂ and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE₂ production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE₂ levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE₂.     

Absence of Endochondral Ossification and Craniosynostosis in Posterior Frontal Cranial Sutures of Axin2?/? Mice

During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2^−/− mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2^−/− PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2^−/− PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2^−/− PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2^−/− PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2^−/− PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.     

The Tumor Suppressor Gene,RASSF1A,Is Essential for Protection against Inflammation -Induced Injury

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a^+/−, Rassf1a^−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a ^IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a^−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a^−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a^−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.     

Changes in Morphological and Elastic Properties of Patellar Tendon in Athletes with Unilateral Patellar Tendinopathy and Their Relationships with Pain and Functional Disability

Background

Patellar tendinopathy (PT) is one of the most common knee disorders among athletes. Changes in morphology and elasticity of the painful tendon and how these relate to the self-perceived pain and dysfunction remain unclear.

Objectives

To compare the morphology and elastic properties of patellar tendons between athlete with and without unilateral PT and to examine its association with self-perceived pain and dysfunction.

Methods

In this cross-sectional study, 33 male athletes (20 healthy and 13 with unilateral PT) were enrolled. The morphology and elastic properties of the patellar tendon were assessed by the grey and elastography mode of supersonic shear imaging (SSI) technique while the intensity of pressure pain, self-perceived pain and dysfunction were quantified with a 10-lb force to the most painful site and the Victorian Institute of Sport Assessment-patella (VISA-P) questionnaire, respectively.

Results

In athletes with unilateral PT, the painful tendons had higher shear elastic modulus (SEM) and larger tendon than the non-painful side (p<0.05) or the dominant side of the healthy athletes (p<0.05). Significant correlations were found between tendon SEM ratio (SEM of painful over non-painful tendon) and the intensity of pressure pain (rho  = 0.62; p = 0.024), VISA-P scores (rho  = −0.61; p = 0.026), and the sub-scores of the VISA-P scores on going down stairs, lunge, single leg hopping and squatting (rho ranged from −0.63 to −0.67; p<0.05).

Conclusions

Athletes with unilateral PT had stiffer and larger tendon on the painful side than the non-painful side and the dominant side of healthy athletes. No significant differences on the patellar tendon morphology and elastic properties were detected between the dominant and non-dominant knees of the healthy control. The ratio of the SEM of painful to non-painful sides was associated with pain and dysfunction among athletes with unilateral PT.