Pregnant biglycan knockout mice have altered cardiorenal adaptations and a shorter gestational length, but do not develop a pre-eclamptic phenotype.

INTRODUCTION: Pre-eclampsia complicates 4.6% of pregnancies and is linked to impaired placentation; likely due to dysregulated vasculogenesis/angiogenesis. Proteoglycans, such as biglycan, are located on the endothelial surface of fetal capillaries. Biglycan is reduced in the placenta of pregnancies complicated by fetal growth restriction and pre-eclampsia. Importantly, biglycan stimulates angiogenesis in numerous tissues. Therefore, this study investigated whether biglycan knockdown in mice results in a pre-eclamptic phenotype. METHODS: Wild-type (WT) and Bgn-/- mice underwent cardiorenal measurements prior to and during pregnancy. One cohort of mice underwent post-mortem on gestational day 18 (E18) and another cohort underwent post-mortem on postnatal day 1 (PN1), with maternal and offspring tissues of relevance collected. RESULTS: Bgn-/- dams had increased heart rate (+9%, p < 0.037) and reduced systolic (-11%, p < 0.001), diastolic (-15%, p < 0.001), and mean arterial (-12%, p < 0.001) pressures at all ages investigated compared to WT. Additionally, Bgn-/- dams had reduced urine flow rate (-64%, p < 0.001) as well as reduced urinary excretions (-49%, p < 0.004) during late gestation compared to WT. Bgn-/- pups had higher body weight (+8%, p = 0.004; E18 only) and a higher liver-to-brain weight ratio (+43%, p < 0.001). Placental weight was unaltered with only minor changes in vasculogenic and angiogenic gene abundances detected, which did not correlate to changes in protein expression. DISCUSSION: This study demonstrated that total knockdown of biglycan is not associated with features of pre-eclampsia.

Regulation of Macrophage and Dendritic Cell Function by Chondroitin Sulfate in Innate to Antigen-Specific Adaptive Immunity.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a linear acidic polysaccharide comprised of repeating disaccharides, modified with sulfate groups at various positions. Except for hyaluronan (HA), GAGs are covalently bound to core proteins, forming proteoglycans (PGs). With highly negative charges, GAGs interact with a variety of physiologically active molecules, including cytokines, chemokines, and growth factors, and control cell behavior during development and in the progression of diseases, including cancer, infections, and inflammation. Heparan sulfate (HS), another type of GAG, and HA are well reported as regulators for leukocyte migration at sites of inflammation. There have been many reports on the regulation of immune cell function by HS and HA; however, regulation of immune cells by CS has not yet been fully understood. This article focuses on the regulatory function of CS in antigen-presenting cells, including macrophages and dendritic cells, and refers to CSPGs, such as versican and biglycan, and the cell surface proteoglycan, syndecan.

Biglycan enhances the ability of migration and invasion in endometrial cancer.

OBJECTIVE: This study aimed to confirm that biglycan (BGN) can promote the migration and invasion in endometrial cancer both in vitro and in vivo and the possible therapeutic value of BGN in endometrial cancer. METHODS: Western blot was used to screen out the higher protein level of BGN in human endometrial cancer cells; BGN knocked down cells were constructed by lentiviral transfection; The effect of BGN in endometrial cancer detected by wound healing, transwell migration, and invasion, endothelial tube formation assay in vitro, and xenograft model in vivo. RESULTS: (1) We found that BGN expression level is higher in the Ishikawa (ISK, high differentiation) and AN3CA (poor differentiation) cells than other endometrial cancer cells. (2) BGN enhances endometrial cancer cell wound healing, invasion, and migration ability and formation ability of endothelial cells in vitro. Xenograft model has confirmed the outcome in vivo. CONCLUSIONS: BGN might play an important role on metastasis in human endometrial cancer and it might be a target marker for the molecular therapy of advanced and recurrence endometrial cancer.

Biglycan mediates suture expansion osteogenesis via potentiation of Wnt/ß-catenin signaling.

Mechanical force across sutures is known to modulate suture osteogenesis. However, the underlying mechanisms still remain poorly understood. Biglycan is a component of extracellular matrix (ECM) that is postulated to release from ECM and function as a signaling molecule. Biglycan stimulates the bone formation through Wnt/ß-catenin signaling. To investigate the involvement of biglycan and Wnt/ß-catenin signaling in suture expansion osteogenesis, we observed the expansion force-induced response in mouse midpalatal suture expansion model in vivo, and the mechanical strain-induced response of Wnt/ß-catenin signaling in biglycan-deficient calvarial osteoblasts in vitro. Our data showed that expansion force significantly enhanced new bone formation at the edge of midpalatal sutures. Stronger biglycan positive staining was visible at the edge of expanding midpalatal sutures. The spatio-temporal expression of biglycan was highly consistent with ALP and COL-1, which also coincided with new bone formation throughout the midpalatal suture expansion process. Both protein and mRNA levels of biglycan, ß-catenin, and osteogenic markers including Runx2, ALP and COL-1 were increased together. In addition, mechanical strain sufficiently induced upregulation of osteoblastic biglycan, which was paralleled with the strain-induced potentiation of Wnt/ß-catenin signaling and Runx2 transcriptional activity. However, silencing osteoblastic biglycan resulted in an attenuated increase in the expression of nuclear active ß-catenin and Runx2 in response to mechanical strain. Our data demonstrated that biglycan as a component of ECM mediates suture expansion osteogenesis through the activation of Wnt/ß-catenin signaling.

Fibromodulin and Biglycan Modulate Periodontium through TGFß/BMP Signaling.

A full understanding of the key regulators controlling periodontal development and homeostasis is necessary for the design of improved periodontal regenerative therapies. Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules suggested to regulate collagen organization and cell signaling. Mice with double-deficiency of 2 SLRPs, fibromodulin and biglycan (dKO), acquire skeletal abnormalities, but their roles in regulating the periodontium remain undefined and were the focus of our studies. Transmission electron microscopy studies showed abnormal collagen fibrils in the periodontal ligament (PDL) and altered remodeling of alveolar bone in dKO mice. Immunohistochemistry (IHC) revealed increased staining of SLRPs (asporin, lumican, and decorin) and dentin matrix protein-1 (DMP1, a mechanosensory/osteocyte marker), while osteoblast markers, bone sialoprotein and osteopontin, remained unchanged. Disruption of homeostasis was further evidenced by increased expression of receptor-activator of nuclear factor-κB ligand (RANKL) and elevated numbers of osteoclasts, especially noted around the alveolar bone of molars (buccal side) and incisors. Polymerase chain reaction (PCR) array revealed hyperactive transforming growth factors beta/bone morphogenetic protein (TGFß/BMP) signaling in dKO PDL tissues, which was further confirmed by elevated expression of phosphorylated Smad5 (p-Smad5) by IHC in dKO PDL. These studies highlight the importance of SLRPs in maintaining periodontal homeostasis through regulation of TGFß/BMP signaling, matrix turnover, and collagen organization.

Biglycan enhances gastric cancer invasion by activating FAK signaling pathway.

Biglycan (BGN) is an important member of small leucine-rich proteoglycans family, and has been implicated in oncogenesis and development of various human cancer types. Here we report that BGN promotes tumor invasion and metastasis of gastric cancer both in vitro and in vivo. BGN expression is significantly higher in gastric cancer tissues and associated with lymph node metastasis, depth of tumor invasion and TNM stage. BGN enhances gastric cancer cell wound healing, migration and invasion ability as well as the tube formation ability of endothelial cells in vitro. Animal experiments results in vivo are consistent with outcomes in vitro. BGN induces increased phosphorylation of FAK (Tyr576/577, Tyr925 and Tyr397) and Paxillin. These results indicate that BGN is upregulated, and plays an oncogenic role, in gastric cancer metastasis by activating the FAK signaling pathway.

Role of Toll-like receptor-4 in renal graft ischemia-reperfusion injury.

Toll-like receptor-4 (TLR-4) has been increasingly recognized as playing a critical role in the pathogenesis of ischemia-reperfusion injury (IRI) of renal grafts. This review provides a detailed overview of the new understanding of the involvement of TLR-4 in ischemia-reperfusion injury of renal grafts and its clinical significance in renal transplantation. TLR-4 not only responds to exogenous microbial motifs but can also recognize molecules which are released by stressed and necrotic cells, as well as degraded products of endogenous macromolecules. Upregulation of TLR-4 is found in tubular epithelial cells, vascular endothelial cells, and infiltrating leukocytes during renal ischemia-reperfusion injury, which is induced by massive release of endogenous damage-associated molecular pattern molecules such as high-mobility group box chromosomal protein 1. Activation of TLR-4 promotes the release of proinflammatory mediators, facilitates leukocyte migration and infiltration, activates the innate and adaptive immune system, and potentiates renal fibrosis. TLR-4 inhibition serves as the target of pharmacological agents, which could attenuate ischemia-reperfusion injury and associated delayed graft function and allograft rejection. There is evidence in the literature showing that targeting TLR-4 could improve long-term transplantation outcomes. Given the pivotal role of TLR-4 in ischemia-reperfusion injury and associated delayed graft function and allograft rejection, inhibition of TLR-4 using pharmacological agents could be beneficial for long-term graft survival.

Glycosaminoglycan chains of biglycan promote bone morphogenetic protein-4-induced osteoblast differentiation.

Biglycan (BGN) has been reported to promote bone morphogenetic protein-4 (BMP-4) stimulated osteoblastic differentiation. However, the underlying mechanism has yet to be fully elucidated. The glycosaminoglycan (GAG) chains of BGN have a variety of biological functions. In the present study, we explored the potential role of the GAG chains of BGN in promoting BMP-4-induced osteoblast differentiation. BGN knockout (KO) murine calvarial cells were transfected with adenovirus overexpressing wild-type BGN (Adv-BGN), adenovirus expressing GAG-mutant BGN (Adv-BGNm) and adenovirus without BGN (Adv-Emp). Transfected cells were treated with or without BMP-4. Subsequently, BMP-4 signaling and function were assessed by evaluating the expression of the osteoblast differentiation-related proteins, Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the binding specificity of the transfected cells to BMP-4 was also investigated using immunofluorescence staining. Our study demonstrated that a mutant BGN lacking GAG chains decreased BGN-assisted BMP-4 signaling and osteoblast differentiation and that the expression of this mutant BGN in biglycan knockout (BGN­KO) calvarial osteoblasts could not rescue its differentiation deficiency as efficiently as wild-type (WT) BGN. These results strongly suggest that the GAG chains of BGN promote BGN-assisted BMP-4 function.

The proteoglycan biglycan enhances antigen-specific T cell activation potentially via MyD88 and TRIF pathways and triggers autoimmune perimyocarditis.

Biglycan is a proteoglycan ubiquitously present in extracellular matrix of a variety of organs, including heart, and it was reported to be overexpressed in myocardial infarction. Myocardial infarction may be complicated by perimyocarditis through unknown mechanisms. Our aim was to investigate the capacity of TLR2/TLR4 ligand biglycan to enhance the presentation of specific Ags released upon cardiomyocyte necrosis. In vitro, OVA-pulsed bone marrow-derived dendritic cells from wild-type (WT; C57BL/6) and TLR2-, TLR4-, MyD88-, or TRIF-deficient mice were cotreated with LPS, biglycan, or vehicle and incubated with OVA-recognizing MHC I- or MHC II-restricted T cells. Biglycan enhanced OVA-specific cross-priming by >80% to MHC I-restricted T cells in both TLR2- and TLR4-pathway-dependent manners. Accordingly, biglycan-induced cross-priming by both MyD88- and TRIF-deficient dendritic cells (DCs) was strongly diminished. OVA-specific activation of MHC II-restricted T cells was predominantly TLR4 dependent. Our first in vivo correlate was a model of experimental autoimmune perimyocarditis triggered by injection of cardiac Ag-pulsed DCs (BALB/c). Biglycan-treated DCs triggered perimyocarditis to a comparable extent and intensity as LPS-treated DCs (mean scores 1.3 ± 0.3 and 1.5 ± 0.4, respectively). Substitution with TLR4-deficient DCs abolished this effect. In a second in vivo approach, WT and biglycan-deficient mice were followed 2 wk after induction of myocardial infarction. WT mice demonstrated significantly greater myocardial T lymphocyte infiltration in comparison with biglycan-deficient animals. We concluded that the TLR2/4 ligand biglycan, a component of the myocardial matrix, may enhance Ag-specific T cell priming, potentially via MyD88 and TRIF, and stimulate autoimmune perimyocarditis.

Role of subchondral bone during early-stage experimental TMJ osteoarthritis.

Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease that affects both cartilage and subchondral bone. We used microarray to identify changes in gene expression levels in the TMJ during early stages of the disease, using an established TMJ OA genetic mouse model deficient in 2 extracellular matrix proteins, biglycan and fibromodulin (bgn(-/0)fmod(-/-)). Differential gene expression analysis was performed with RNA extracted from 3-week-old WT and bgn(-/0)fmod(-/-) TMJs with an intact cartilage/subchondral bone interface. In total, 22 genes were differentially expressed in bgn(-/0)fmod(-/-) TMJs, including 5 genes involved in osteoclast activity/differentiation. The number of TRAP-positive cells were three-fold higher in bgn(-/0)fmod(-/-) TMJs than in WT. Quantitative RT-PCR showed up-regulation of RANKL and OPG, with a 128% increase in RANKL/OPG ratio in bgn(-/0)fmod(-/-) TMJs. Histology and immunohistochemistry revealed tissue disorganization and reduced type I collagen in bgn(-/0)fmod(-/-) TMJ subchondral bone. Early changes in gene expression and tissue defects in young bgn(-/0)fmod(-/-) TMJ subchondral bone are likely attributed to increased osteoclast activity. Analysis of these data shows that biglycan and fibromodulin are critical for TMJ subchondral bone integrity and reveal a potential role for TMJ subchondral bone turnover during the initial early stages of TMJ OA disease in this model.

[Signaling of biglycan and cytokines in bovine intervertebral disc cells].

OBJECTIVE: To evaluate the effect of biglycan on the signaling of cytokines (epidermal growth factor, osteogenic protein-1, and interleukin-1) in bovine intervertebral disc cells. METHODS: Nucleoplasty (NP) and annulus fibrosus (AF) cells of the intervertebral disc tissues were isolated from the tails of young adult bovine. First, the cells were treated in 3 ways: Biglycan alone, cytokines alone (epidermal growth factor, osteogenic protein-1, or interleukin-1), and biglycan combined with cytonkines. Western blot was used to observe the singling of biglycan and cytokines in bovine intervertebral disc cells, and to identify the effect of biglycan on cytokines mentioned above. RESULTS: Biglycan upregulated the signaling (3- 4 folds) with the optimal effect at 10 min and 20 µmol/L both in the AF cells and NP cells. Epidermal growth factor, osteogenic protein-1, or interleukin-1 also upregulated the protein expression in the extracellular matrix of intervertebral disc cells. When combined different biglycan concentrations with epidermal growth factor, osteogenic protein-1, or interleukin-1 to treat the intervertebral disc cells, the concentration of biglycan rose, whereas the cytokine signal decreased both in the bovine AF and NP cells (P<0.01). There was no significant difference between the AF and NP cells. CONCLUSION: Biglycan can adhere to the intervertebral disc cells to activate the extracellular signal-regulated kinase (ERK) pathway and this effect is time and concentration dependent. Byglycan can decrease not only the anabolism effect of epidermal growth factor and osteogenic protein-1, but also the catabolism effect of interleukin-1. This regulatory role of biglycan may be very important to maintain the metabolism balance. Biglycan may be good for the repair of intervertebral disc.

Regulation of pre-adipocyte proliferation and apoptosis by the small leucine-rich proteoglycans, biglycan and decorin.

Evidence for a functional role for extracellular matrix (ECM) proteins in adipose tissue is demonstrated in dynamic changes in expression of ECM genes during adipocyte differentiation and in obesity. Components of the ECM may regulate adipose cell number expansion by restricting pre-adipocyte proliferation, regulating apoptosis and inhibiting adipogenesis. Although pre-adipocytes express multiple proteoglycans, their role in pre-adipocyte proliferation up to now has remained unknown. The study described here was conducted to characterize roles of small leucine-rich proteoglycans (SLRPs) in adipocyte proliferation. Pre-adipocytes were seeded on plates coated with biglycan and decorin and were allowed to differentiate. In addition, pre-adipocytes were incubated on plates coated with biglycan, decorin, or fibronectin and measurements were made of cell proliferation and apoptosis. We are able to report that SLRPs decorin and biglycan did not affect differentiation of our 3T3-L1 cells; however, biglycan and decorin did reduce proliferation of pre-adipocytes, partly by induction of apoptosis. Furthermore, anti-proliferative capabilities of decorin and biglycan were nullified with removal of GAG side-chains suggesting that the chains played key roles in anti-proliferative effects of the SLRPs. We also found that co-treatment of decorin or biglycan with the proteoglycan fibronectin restored normal proliferation, an indication that multiple ECM proteins may act in concert to regulate overall proliferation rates of pre-adipocytes. These studies indicate that SLRPs may compose a regulatory factor in adipose tissue expansion, through hyperplasia.

A mouse model of spontaneous preterm birth based on the genetic ablation of biglycan and decorin.

Preterm premature rupture of membranes is responsible for one-third of preterm births. Ehlers-Danlos syndrome (EDS) is associated with preterm premature rupture of membranes in humans. In particular, an EDS variant is caused by a genetic mutation resulting in abnormal secretion of biglycan and decorin, two small leucine-rich proteoglycans highly expressed in reproductive tissues. Because biglycan/decorin null mutant (Bgn(-/-)Dcn(-/-)) mice demonstrate phenotypic changes similar to EDS, we used this model to test whether either biglycan or decorin or both play a role in the attainment of successful term gestation. Wild-type biglycan null mutant, decorin null mutant, and biglycan/decorin null mutant pregnancies were assessed for the length of gestation, pup and placenta weight, and litter size. Quantitative real-time PCR was performed to measure biglycan and decorin gene expression, and immunohistochemistry was performed to assess protein expression in placenta and fetal membranes at embryonic days E12, E15, and E18. Bgn(-/-)Dcn(-/-) dams displayed preterm birth, whereas the possession of at least two biglycan or decorin wild-type alleles was protective of preterm birth. The number of Bgn(-/-)Dcn(-/-) pups was decreased at postnatal day P1 but not at E18. Biglycan and decorin were upregulated in the placenta in the absence of each other and were developmentally regulated in fetal membranes, suggesting that these two proteoglycans demonstrate genetic complementation and contribute to gestational success in a dose-dependent manner. Thus, the biglycan/decorin null mutant mouse is a model of genetically induced preterm birth and perinatal loss. This model presents novel targets for preventive or therapeutic manipulation of preterm birth.

Role of glycosaminoglycans of biglycan in BMP-2 signaling.

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, µCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function.

Biglycan regulates the expression of EGF receptors through EGF signaling pathways in human articular chondrocytes.

Biglycan is a member of the family of small leucine-rich proteoglycans. It is an important structural component of articular cartilage and participates in the assembly of the chondrocyte extracellular matrix through formation of protein interactions with collagen type VI and large proteoglycan aggregates. Biglycan also possesses signaling properties. In articular chondrocytes, short-term activation of epidermal growth factor receptors (EGFR) with biglycan initiated mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3K) signaling events, similar to the effect of epidermal growth factor (EGF) observed in other cell types. The extent and duration of intracellular signaling resolves biological effects initiated by EGFR stimulation, thus, establishing cell fate. In this study, we elucidate a novel regulatory mechanism of EGFR expression in human articular chondrocytes that is modulated by prolonged biglycan treatment and is in contrast to changes detected in the expression of EGFR following EGF stimulation. Treatment of chondrocytes for 24 hr with biglycan upregulated EGFR mRNA and protein expression, whereas treatment with EGF downregulated EGFR message and protein levels. Biglycan and EGF treatment protracted extracellular signal-regulated kinases (ERK1/2) and Akt phosphorylation, albeit to different extents. Mechanistic studies with mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathway-specific inhibitors revealed that biglycan and EGF distinctly modulate the expression of EGFR in chondrocytes. Biglycan promoted the coactivation of ERK1/2 and Akt, however, phosphorylated Akt induced a prolonged inhibition of ERK1/2. Consequently, total EGFR mRNA and protein expression was increased. This regulatory mechanism contrasts the modulation of EGFR expression by exogenous EGF, which strongly protracts ERK1/2 activation, therefore, inducing a decrease of EGFR message and protein levels. Thus, biglycan might impinge on the expression of total EGFR and possibly, on the cell-surface expression of the receptors. These observations suggest that biglycan might play a critical role in the regulation of chondrocyte and pericellular matrix homeostasis.