Involvement of small leucine-rich proteoglycans and telocytes in thin and thick human endometrium: immunohistochemical and ultrastructural examination.

Thin endometrium, defined as an endometrial thickness of less than 7 mm during the late follicular phase, is a common cause of frequent cancelation of embryo transfers or recurrent implantation failure during assisted reproductive treatment. Small proteoglycans regulate intracellular signaling cascades by bridging other matrix molecules and tissue elements, affecting cell proliferation, adhesion, migration, and cytokine concentration. The aim of the study is to investigate the role of small leucine-rich proteoglycans in the pathogenesis of thin and thick human endometrium and their differences from normal endometrium in terms of fine structure properties. Normal, thin, and thick endometrial samples were collected, and small leucine-rich proteoglycans (SLRPs), decorin, lumican, biglycan, and fibromodulin immunoreactivities were comparatively analyzed immunohistochemically. The data were compared statistically. Moreover, ultrastructural differences among the groups were evaluated by transmission electron microscopy. The immunoreactivities of decorin, lumican, and biglycan were higher in the thin endometrial glandular epithelium and stroma compared to the normal and thick endometrium (p < .001). Fibromodulin immunoreactivity was also higher in the thin endometrial glandular epithelium than in the normal and thick endometrium (p < .001). However, there was no statistical difference in the stroma among the groups. Ultrastructural features were not profoundly different among cases. Telocytes, however, were not seen in the thin endometrium in contrast to normal and thin endometrial tissues. These findings suggest a possible role of changes in proteoglycan levels in the pathogenesis of thin endometrium.

Loss of hepatic SMLR1 causes hepatosteatosis and protects against atherosclerosis due to decreased hepatic VLDL secretion.

BACKGROUND AND AIMS: The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 ( SMLR1 ), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. APPROACH AND RESULTS: To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes ( Smlr1 -LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1 -LKO mice versus controls. CONCLUSIONS: We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

The role of SLRPs and large aggregating proteoglycans in collagen fibrillogenesis, extracellular matrix assembly, and mechanical function of fibrocartilage.

PURPOSE: Proteoglycans, especially small leucine rich proteoglycans (SLRPs), play major roles in facilitating the development and regulation of collagen fibers and other extracellular matrix components. However, their roles in fibrocartilage have not been widely reviewed. Here, we discuss both SLRP and large aggregating proteoglycan's roles in collagen fibrillogenesis and extracellular matrix assembly in fibrocartilage tissues such as the meniscus, annulus fibrosus (AF), and TMJ disc. We also discuss their expression levels throughout development, aging and degeneration, as well as repair. METHODS: A review of literature discussing proteoglycans and collagen fibrillogenesis in fibrocartilage was conducted and data from these manuscripts were analyzed and grouped to discuss trends throughout the tissue's architectural zones and developmental stage. RESULTS: The spatial collagen architecture of these fibrocartilaginous tissues is reflected in the distribution of proteoglycans expressed, suggesting that each proteoglycan plays an important role in the type of architecture presented and associated mechanical function. CONCLUSION: The unique structure-function relationship of fibrocartilage makes the varied architectures throughout the tissues imperative for their success and understanding the functions of these proteoglycans in developing and maintaining the fiber structure could inform future work in fibrocartilage replacement using tissue engineered constructs.

The Genes Encoding Small Leucine-Rich Proteoglycans Undergo Differential Expression Alterations in Colorectal Cancer, Depending on Tumor Location.

Small leucine-rich proteoglycans (SLRPs) regulate different processes and undergo significant alterations in various diseases. Colon carcinomas (CCs) are heterogeneous pathologies with important clinical and molecular differences depending on their location, which makes it interesting to analyze the alterations in SLRPs in right- and left-sided tumors (RS- and LSCCs). SLRP transcription levels were studied in 32 CCs using qPCR compared to healthy colon mucosae samples from the same patients, 20 of them from LSCCs and the remaining 12 from RSCCs. Protein expression of genes with significant differences in their transcriptions was analyzed by immunohistochemistry. The alterations observed were related to survival data. The arrangement of transcription of SLRPs was quite similar in ascending and descending colon, but RS- and LSCCs displayed different patterns of alteration, with a greater number of deregulations occurring in the latter. The analysis of protein expression also indicated changes in the location of these molecules, largely moving to the cell interior. While podocan underexpression showed a trend toward better outcomes, no differences were observed in terms of overall survival. In vitro studies using the HT29 tumor cell line suggest that deregulation of SLRPs could affect cell proliferation. SLRPs constitute new differential markers of RS- and LSCCs, showing differences dependent on the anatomical location of the tumor.

Novel corneal targeting cell penetrating peptide as an efficient nanocarrier with an effective antimicrobial activity.

Delivery of therapeutics to the ocular tissues is challenging due to various anatomical and physiological barriers imposed. Cell penetrating peptides (CPPs) have emerged as potent drug nanocarriers that have been shown to overcome these barriers and enhance bioavailability of therapeutic macromolecules in deep ocular tissues. In the present study, an ocular targeting CPP has been designed by exploring potential targets of anterior ocular tissues in particular receptors, transporters and glycosaminoglycans (GAGs). The novel 11 mer peptide sequence, Corneal Targeting Sequence 1 (CorTS 1), has been developed by modifying leucine rich repeat (LRR) motif ensuring that it interacts with small leucine rich proteoglycans and collagen present in the corneal stroma. CorTS 1 exhibited dose dependent cellular translocation from 5 µM in Human Corneal Epithelial cell line (HCE) with no cytotoxicity. CorTS 1 was also found to deliver protein cargo inside HCE cells. Ex vivo tissue penetration study of CorTS 1 demonstrated in goat eyes revealed an augmented accumulation of peptide in the stromal region of cornea than in aqueous humor. Interestingly, CorTS 1 showed an antimicrobial activity against MRSA and Fusarium dimerum. Therefore, CorTS 1 can be a promising candidate with dual traits of antimicrobial agent and nanocarrier for ocular drugs.

Small Leucine-Rich Proteoglycans (SLRPs) in the Retina.

Retinal diseases such as age-related macular degeneration (AMD), retinopathy of prematurity (ROP), and diabetic retinopathy (DR) are the leading causes of visual impairment worldwide. There is a critical need to understand the structural and cellular components that play a vital role in the pathophysiology of retinal diseases. One potential component is the family of structural proteins called small leucine-rich proteoglycans (SLRPs). SLRPs are crucial in many fundamental biological processes involved in the maintenance of retinal homeostasis. They are present within the extracellular matrix (ECM) of connective and vascular tissues and contribute to tissue organization and modulation of cell growth. They play a vital role in cell-matrix interactions in many upstream signaling pathways involved in fibrillogenesis and angiogenesis. In this comprehensive review, we describe the expression patterns and function of SLRPs in the retina, including Biglycan and Decorin from class I; Fibromodulin, Lumican, and a Proline/arginine-rich end leucine-rich repeat protein (PRELP) from class II; Opticin and Osteoglycin/Mimecan from class III; and Chondroadherin (CHAD), Tsukushi and Nyctalopin from class IV.

Biglycan: an emerging small leucine-rich proteoglycan (SLRP) marker and its clinicopathological significance.

Extracellular matrix (ECM) plays an important role in the structural organization of tissue and delivery of external cues to the cell. Biglycan, a class I small leucine-rich proteoglycans (SLRP), is a key component of the ECM that participates in scaffolding the collagen fibrils and mediates cell signaling. Dysregulation of biglycan expression can result in wide range of clinical conditions such as metabolic disorder, inflammatory disorder, musculoskeletal defects and malignancies. In this review, we aim to update our current understanding regarding the link between altered expression of biglycan and different clinicopathological states. Biglycan interacts with toll like receptors (TLR)-2 and TLR-4 on the immune cells which initiates inflammation and aggravates inflammatory disorders. ECM unbound soluble biglycan acts as a DAMP (danger associated molecular pattern) resulting in sterile inflammation. Dysregulation of biglycan expression is also observed in inflammatory metabolic conditions such as atherosclerosis and obesity. In cancer, high-biglycan expression facilitates tumor growth, invasion and metastasis which is associated with poor clinical outcome. As a pivotal structural component of the ECM, biglycan strengthens the musculoskeletal system and its absence is associated with musculoskeletal defects. Thus, SLRP biglycan is a potential marker which is significantly altered in different clinicopathological states.

Right Ventricle Has Normal Myofilament Function But Shows Perturbations in the Expression of Extracellular Matrix Genes in Patients With Tetralogy of Fallot Undergoing Pulmonary Valve Replacement.

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

Immunolocalization of Keratan Sulfate in Rat Spinal Tissues Using the Keratanase Generated BKS-1(+) Neoepitope: Correlation of Expression Patterns with the Class II SLRPs, Lumican and Keratocan.

This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development.

Expression, localization and synthesis of small leucine-rich proteoglycans in developing mouse molar tooth germ.

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

From Translation to Protein Degradation as Mechanisms for Regulating Biological Functions: A Review on the SLRP Family in Skeletal Tissues.

The extracellular matrix can trigger cellular responses through its composition and structure. Major extracellular matrix components are the proteoglycans, which are composed of a core protein associated with glycosaminoglycans, among which the small leucine-rich proteoglycans (SLRPs) are the largest family. This review highlights how the codon usage pattern can be used to modulate cellular response and discusses the biological impact of post-translational events on SLRPs, including the substitution of glycosaminoglycan moieties, glycosylation, and degradation. These modifications are listed, and their impacts on the biological activities and structural properties of SLRPs are described. We narrowed the topic to skeletal tissues undergoing dynamic remodeling.

Expression of minor cartilage collagens and small leucine rich proteoglycans may be relatively reduced in osteoarthritic cartilage.

BACKGROUND: In osteoarthritis (OA), cartilage matrix is lost despite vigorous chondrocyte anabolism. In this study, we attempted to determine whether altered matrix synthesis is involved in this paradox in disease progression through gene expression analysis and ultrastructural analysis of collagen fibrils within the cartilage matrix. METHODS: Cartilage tissues were obtained from 29 end-stage OA knees and 11 control knees. First, cDNA microarray analysis was performed and the expression of 9 genes involved in collagen fibrillogenesis was compared between OA and control cartilages. Then their expression was investigated in further detail by a quantitative polymerase chain reaction (qPCR) analysis combined with laser capture microdissection. Finally, collagen fibril formation was compared between OA and control cartilage by transmission electron microscopy. RESULTS: The result of the microarray analysis suggested that the expression of type IX and type XI collagens and fibrillogenesis-related small leucine-rich proteoglycans (SLRPs) may be reduced in OA cartilage relative to the type II collagen expression. The qPCR analysis confirmed these results and further indicated that the relative reduction in the minor collagen and SLRP expression may be more obvious in degenerated areas of OA cartilage. An ultrastructural analysis suggested that thicker collagen fibrils may be formed by OA chondrocytes possibly through reduction in the minor collagen and SLRP expression. CONCLUSIONS: This may be the first study to report the possibility of altered collagen fibrillogenesis in OA cartilage. Disturbance in collagen fibril formation may be a previously unidentified mechanism underlying the loss of cartilage matrix in OA.

Vertebrate SLRP family evolution and the subfunctionalization of osteoglycin gene duplicates in teleost fish.

BACKGROUND: Osteoglycin (OGN, a.k.a. mimecan) belongs to cluster III of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). In vertebrates OGN is a characteristic ECM protein of bone. In the present study we explore the evolution of SLRP III and OGN in teleosts that have a skeleton adapted to an aquatic environment. RESULTS: The SLRP gene family has been conserved since the separation of chondrichthyes and osteichthyes. Few gene duplicates of the SLRP III family exist even in the teleosts that experienced a specific whole genome duplication. One exception is ogn for which duplicate copies were identified in fish genomes. The ogn promoter sequence and in vitro mesenchymal stem cell (MSC) cultures suggest the duplicate ogn genes acquired divergent functions. In gilthead sea bream (Sparus aurata) ogn1 was up-regulated during osteoblast and myocyte differentiation in vitro, while ogn2 was severely down-regulated during bone-derived MSCs differentiation into adipocytes in vitro. CONCLUSIONS: Overall, the phylogenetic analysis indicates that the SLRP III family in vertebrates has been under conservative evolutionary pressure. The retention of the ogn gene duplicates in teleosts was linked with the acquisition of different functions. The acquisition by OGN of functions other than that of a bone ECM protein occurred early in the vertebrate lineage.

[Differential expression of small leucine-rich proteoglycans gene in Lumican transgenic mouse cornea].

Objective: To investigate the differential expression of small leucine-rich proteoglycans at mRNA level in Lumican transgenic mouse cornea with Real-time Quantitative PCR Detecting System. Methods: Experimental research. Ten Lumican transgenic mice (5 male and 5 female) were chosen as experimental group and 10 wild mice (5 male and 5 female) were chosen as control group. All the mice were killed and enucleated both eyes at eight weeks of age. Gene expression levels of Lumican, Decorin, Biglycan, Keratocan, Fibromodulin in the excised corneas were analyzed by real-time quantitative polymerase chain reaction (RT-Q-PCR) using Real-time Quantitative PCR Detecting System. Differential expression within each group were analysed by fold changes and independent t-test. Results: There were statistic different expression level of Lumican, Decorin, Biglycan and Keratocan mRNA between experimental and control group. The expression level of Lumican RNA was found to be 1.497-fold increased relative to the control (t=4.34, P<0.05) , while Decorin, Biglycan, Keratocan were 0.648-fold (t=-9.98, P<0.05) , 0.522-fold (t=-7.74,P<0.05), 0.323-fold (t=-95.94, P<0.05)decreased in transgenic mice. Fibromodulin mRNA up regulated 1.193-fold in transgenic mice without statistic difference (t=1.66, P>0.05). Conclusions: Lumican gene mutation(cDNA 569T>C) results in abnormal SLRP expression in transgenic mouse cornea at mRNA level, which may indicate that this mutation changes the structure of Lumican and impairs the function of regulating SLRP expression. Also, Lumican gene mutation leads to amio acid exchanging(L199P), which may hinder Lumican from binding to collagens and result in abnormal expression of SLRP at mRNA level. (Chin J Ophthalmol, 2018, 54:911-917).

Deep Sequencing of Complex Proteoglycans: A Novel Strategy for High Coverage and Site-specific Identification of Glycosaminoglycan-linked Peptides.

Proteoglycans are distributed in all animal tissues and play critical, multifaceted, physiological roles. Expressed in a spatially and temporally regulated manner, these molecules regulate interactions among growth factors and cell surface receptors and play key roles in basement membranes and other extracellular matrices. Because of the high degree of glycosylation by glycosaminoglycan (GAG), N-glycan and mucin-type O-glycan classes, the peptide sequence coverage of complex proteoglycans is revealed poorly by standard mass spectrometry-based proteomics methods. As a result, there is little information concerning how proteoglycan site specific glycosylation changes during normal and pathological processes. Here, we developed a workflow to improve sequence coverage and identification of glycosylated peptides in proteoglycans. We applied this workflow to the small leucine-rich proteoglycan decorin and three hyalectan proteoglycans: neurocan, brevican, and aggrecan.We characterized glycosylation of these proteoglycans using LC-MS methods easily implemented on instruments widely used in proteomics laboratories. For decorin, we assigned the linker-glycosite and three N-glycosylation sites. For neurocan and brevican, we identified densely glycosylated mucin-like regions in the extended domains. For aggrecan, we identified 50 linker-glycosites and mucin-type O-glycosites in the extended region and N-glycosites in the globular domains, many of which are novel and have not been observed previously. Most importantly, we demonstrate an LC-MS and bioinformatics approach that will enable routine analysis of proteoglycan glycosylation from biological samples to assess their role in pathophysiology.

Effects of mechanical stretching on the morphology of extracellular polymers and the mRNA expression of collagens and small leucine-rich repeat proteoglycans in vaginal fibroblasts from women with pelvic organ prolapse.

To determine the effect of mechanical stretching load and the efficacy of postmenopausal estrogen therapy (ET) on pelvic organ prolapse (POP), vaginal fibroblasts isolated from postmenopausal women with or without POP were subjected to 0.1-Hz uniaxial cyclic mechanical stretching (CS) with 10% elongation and 10-8 M 17-ß-estradiol (E2) treatment. We investigated the morphological characteristics of extracellular polymers using scanning electron microscopy (SEM) and monitored the mRNA expression of type I collagen (COL I) and type III collagen (COL III) as well as the small leucine-rich proteoglycan (SLRP) family members decorin (DCN), biglycan (BGN), fibromodulin (FMO), and lumican (LUM), using real-time quantitative polymerase chain reaction (RT-PCR). Using SEM, certain viscoelastic polymers were found to be randomly distributed among fibroblasts, which for normal fibroblasts formed clusters of plum flower-like patterns under static-culture conditions and resembled stretched strips when stretched in culture, whereas polymers among POP fibroblasts resembled stretched strips under static-cultured conditions and presented broken networks when stretched in culture. RT-PCR revealed that COL I, DCN, BGN, FMO, and LUM mRNA expression was significantly higher in POP than in normal fibroblasts under static-culture condition. Following CS, COL I and BGN mRNA expression was significantly up-regulated in normal fibroblasts, and DCN and FMO mRNA expression was down-regulated in POP fibroblasts. Following concomitant CS and E2 treatment, significantly elevated COL I and DCN mRNA expression was observed in normal fibroblasts, and significantly elevated COL I and BGN mRNA expression was observed in POP fibroblasts. COL III mRNA expression was not significantly different between the POP and normal group, and CS did not significantly affect expression in either group, though COL III was down-regulated in normal fibroblasts concomitantly treated with E2 and CS. We conclude that the morphological distribution of extracellular polymers in POP fibroblasts exhibited higher sensitivity and lower tolerance to stretching loads than do normal fibroblasts. These mechanical properties were further reflected in the transcription of COL I. Defects in the compensatory function of BGN for DCN and LUM for FMO exist in POP fibroblasts, which further affect the structure and function of COL I in response to stretching load, ultimately resulting in abnormal reconstruction of pelvic supportive connective tissues and the occurrence of POP. ET can maintain stretching-induced elevations in COL I and DCN transcription in healthy women and improve stretching-induced COL I, DCN, BGN, and FMO transcriptional changes in POP women to prevent and improve POP. Only down-regulated COL III transcription was observed upon concomitant CS and E2 treatment in normal fibroblasts, which suggests that the tensile strength, not the elasticity, of the supportive connective tissues is damaged in POP and that the higher tensile strength induced by ET in healthy fibroblasts prevents POP. These findings confirm the role of higher sensitivity and lower tolerance to mechanical stretching in the pathogenesis of POP and further provide evidence supporting the use of ET to prevent and inhibit POP in postmenopausal women.

Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.

Hyaluronan (HA), a high molecular weight non-sulfated glycosaminoglycan, is an integral component of the extracellular matrix of developing and mature connective tissues including tendon. There are few published reports quantifying HA content during tendon growth and maturation, or detailing its effects on the mechanical properties of the tendon extracellular matrix. Therefore, the goal of the current study was to examine the role of HA synthesis during post-natal skeletal growth and maturation, and its influence on tendon structure and biomechanical function. For this purpose, the morphological, biochemical, and mechanical properties of Achilles tendons from wild type (WT) and hyaluronan synthase 1 and 3 deficient mouse strains (Has1-/- (Has1KO), Has3-/- (Has3KO), and Has1-/- 3-/- (Has1/3KO)) were determined at 4, 8, and 12 weeks of age. Overall, HAS-deficient mice did not show any marked differences from WT mice in Achilles tendon morphology or in the HA and chondroitin/dermatan sulfate (CS/DS) contents. However, HAS1-deficiency (in the single or Has1/3 double KO) impeded post-natal formation of the retrocalcaneal bursa, implicating HAS1 in regulating HA metabolism by cells lining the bursal cavity. Together, these data suggest that HA metabolism via HAS1 and HAS3 does not markedly influence the extracellular matrix structure or function of the tendon body, but plays a role in the formation/maintenance of peritendinous bursa. Additional studies are warranted to elucidate the relationship of HA and CS/DS metabolism to tendon healing and repair in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2622-2632, 2018.

Effects of aging and resistance training in rat tendon remodeling.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.