Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

A comprehensive map of proteoglycan expression and deposition in the pulmonary arterial wall in health and pulmonary hypertension.

Changes in the extracellular matrix of pulmonary arteries (PAs) are a key aspect of vascular remodeling in pulmonary hypertension (PH). Yet, our understanding of the alterations affecting the proteoglycan (PG) family remains limited. We sought to investigate the expression and spatial distribution of major vascular PGs in PAs from healthy individuals and various PH groups (chronic obstructive pulmonary disease: PH-COPD, pulmonary fibrosis: PH-PF, idiopathic: IPAH). PG regulation, deposition, and synthesis were notably heightened in IPAH, followed by PH-PF, with minor alterations in PH-COPD. Single-cell analysis unveiled cell-type and disease-specific PG regulation. Agrin expression, a basement membrane PG, was increased in IPAH, with PA endothelial cells (PAECs) identified as a major source. PA smooth muscle cells (PASMCs) mainly produced large-PGs, aggrecan and versican, and small-leucine-like proteoglycan (SLRP) biglycan, whereas the major PGs produced by adventitial fibroblasts were SLRP decorin and lumican. In IPAH and PF-PH, the neointima-forming PASMC population increased the expression of all investigated large-PGs and SLRPs, except fibroblast-predominant decorin (DCN). Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs in patients with IPAH and PH-PF. We demonstrated that transforming growth factor-beta (TGF-ß) regulates versican and biglycan expression, indicating their contribution to vessel fibrosis in IPAH and PF-PH. We furthermore show that certain circulating PG levels display a disease-dependent pattern, with increased decorin and lumican across all patient groups, while versican was elevated in PH-COPD and IPAH and biglycan reduced in IPAH. These findings suggest unique compartment-specific PG regulation in different forms of PH, indicating distinct pathological processes.NEW & NOTEWORTHY Idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries (PAs) displayed the greatest proteoglycan (PG) changes, with PH associated with pulmonary fibrosis (PH-PF) and PH associated with chronic obstructive pulmonary disease (PH-COPD) following. Agrin, an endothelial cell-specific PG, was solely upregulated in IPAH. Among all cells, neo-intima-forming smooth muscle cells (SMCs) displayed the most significant PG increase. Increased levels of circulating decorin, lumican, and versican, mainly derived from SMCs, and adventitial fibroblasts, may serve as systemic indicators of pulmonary remodeling, reflecting perivascular fibrosis and neointima formation.

Dermal papilla cell-secreted biglycan regulates hair follicle phase transit and regeneration by activating Wnt/ß-catenin.

Alopecia is a prevalent problem of cutaneous appendages and lacks effective therapy. Recently, researchers have been focusing on mesenchymal components of the hair follicle, i.e. dermal papilla cells, and we previously identified biglycan secreted by dermal papilla cells as the key factor responsible for hair follicle-inducing ability. In this research, we hypothesized biglycan played an important role in hair follicle cycle and regeneration through regulating the Wnt signalling pathway. To characterize the hair follicle cycle and the expression pattern of biglycan, we observed hair follicle morphology in C57BL/6 mice on Days 0, 3, 5, 12 and 18 post-depilation and found that biglycan is highly expressed at both mRNA and protein levels throughout anagen in HFs. To explore the role of biglycan during the phase transit process and regeneration, local injections were administered in C57BL/6 and nude mice. Results showed that local injection of biglycan in anagen HFs delayed catagen progression and involve activating the Wnt/ß-catenin signalling pathway. Furthermore, local injection of biglycan induced HF regeneration and up-regulated expression of key Wnt factors in nude mice. In addition, cell analyses exhibited biglycan knockdown inactivated the Wnt signalling pathway in early-passage dermal papilla cell, whereas biglycan overexpression or incubation activated the Wnt signalling pathway in late-passage dermal papilla cells. These results indicate that biglycan plays a critical role in regulating HF cycle transit and regeneration in a paracrine and autocrine fashion by activating the Wnt/ß-catenin signalling pathway and could be a potential treatment target for hair loss diseases.

Clonal Proliferation Within Smooth Muscle Cells in Unstable Human Atherosclerotic Lesions.

BACKGROUND: Studies in humans and mice using the expression of an X-linked gene or lineage tracing, respectively, have suggested that clones of smooth muscle cells (SMCs) exist in human atherosclerotic lesions but are limited by either spatial resolution or translatability of the model. METHODS: Phenotypic clonality can be detected by X-chromosome inactivation patterns. We investigated whether clones of SMCs exist in unstable human atheroma using RNA in situ hybridization (BaseScope) to identify a naturally occurring 24-nucleotide deletion in the 3'UTR of the X-linked BGN (biglycan) gene, a proteoglycan highly expressed by SMCs. BGN-specific BaseScope probes were designed to target the wild-type or deletion mRNA. Three different coronary artery plaque types (erosion, rupture, and adaptive intimal thickening) were selected from heterozygous females for the deletion BGN. Hybridization of target RNA-specific probes was used to visualize the spatial distribution of mutants. A clonality index was calculated from the percentage of each probe in each region of interest. Spatial transcriptomics were used to identify differentially expressed transcripts within clonal and nonclonal regions. RESULTS: Less than one-half of regions of interest in the intimal plaque were considered clonal with the mean percent regions of interest with clonality higher in the intimal plaque than in the media. This was consistent for all plaque types. The relationship of the dominant clone in the intimal plaque and media showed significant concordance. In comparison with the nonclonal lesions, the regions with SMC clonality had lower expression of genes encoding cell growth suppressors such as CD74, SERF-2 (small EDRK-rich factor 2), CTSB (cathepsin B), and HLA-DPA1 (major histocompatibility complex, class II, DP alpha 1), among others. CONCLUSIONS: Our novel approach to examine clonality suggests atherosclerosis is primarily a disease of polyclonally and to a lesser extent clonally expanded SMCs and may have implications for the development of antiatherosclerotic therapies.

Proteomic analysis of extracellular vesicles derived from canine mammary tumour cell lines identifies protein signatures specific for disease state.

BACKGROUND: Canine mammary tumours (CMT) are among the most common types of tumours in female dogs. Diagnosis currently requires invasive tissue biopsies and histological analysis. Tumour cells shed extracellular vesicles (EVs) containing RNAs and proteins with potential for liquid biopsy diagnostics. We aimed to identify CMT subtype-specific proteome profiles by comparing the proteomes of EVs isolated from epithelial cell lines derived from morphologically normal canine mammary tissue, adenomas, and carcinomas. METHODS: Whole-cell protein lysates (WCLs) and EV-lysates were obtained from five canine mammary cell lines: MTH53A (non-neoplastic); ZMTH3 (adenoma); MTH52C (simple carcinoma); 1305, DT1406TB (complex carcinoma); and their proteins identified by LC-MS/MS analyses. Gene Ontology analysis was performed on differentially abundant proteins from each group to identify up- and down-regulated biological processes. To establish CMT subtype-specific proteomic profiles, weighted gene correlation network analysis (WGCNA) was carried out. RESULTS: WCL and EVs displayed distinct protein abundance signatures while still showing the same increase in adhesion, migration, and motility-related proteins in carcinoma-derived cell lines, and of RNA processing and RNA splicing factors in the adenoma cell line. WGCNA identified CMT stage-specific co-abundant EV proteins, allowing the identification of adenoma and carcinoma EV signatures not seen in WCLs. CONCLUSIONS: EVs from CMT cell lines exhibit distinct protein profiles reflecting malignancy state, allowing us to identify potential biomarkers for canine mammary carcinomas, such as biglycan. Our dataset could therefore potentially serve as a basis for the development of a less invasive clinical diagnostic tool for the characterisation of CMT.

Youthful small extracellular vesicles restore the function and reparative capacity of inflammatory-impaired periodontal ligament stem cells via delivering protein biglycan for bone regeneration.

Regenerating inflamed bone defects represents a severe clinical challenge due to the undesirable inflammatory microenvironment. The inflammatory stimulus poses a weighty threat to the regenerative capacity of endogenously derived mesenchymal stem cells (MSCs), which are mainly responsible for osteogenic differentiation, thereby resulting in compromised endogenous bone formation. Consequently, alleviating the biological characteristics of inflammatory-impaired MSCs is crucial for promoting inflamed bone regeneration. Nano-sized small extracellular vesicles (sEVs) have emerged as promising therapeutic tools to orchestrate MSCs fate due to their intrinsic biocompatibility and encapsulated bioactive contents. In the present study, we extracted sEVs from youthful and adult dental pulp MSCs and explored their ability to recover inflammation-compromised periodontal ligament stem cells (IPDLSCs). The results indicated that both types of sEVs were capable of facilitating IPDLSCs osteogenesis. However, young sEVs exhibited a more robust potential at a lower concentration compared to adult sEVs. Mechanically, young sEVs enhanced the expression of bone morphogenetic protein 4 (BMP4) via delivering the protein Biglycan, which correspondingly promoted the osteogenic capability of IPDLSCs. Collectively, our findings emphasized that young sEVs hold enormous potential to rescue the inherent function and regenerative competence of inflammation-impaired MSCs, shedding light on their promising therapeutic prospects for infected bone regeneration.

Bioinformatics Analysis and Experimental Validation of Differential Genes and Pathways in Bone Nonunions.

Non-union fractures pose a significant clinical challenge, often leading to prolonged pain and disability. Understanding the molecular mechanisms underlying non-union fractures is crucial for developing effective therapeutic interventions. This study integrates bioinformatics analysis and experimental validation to unravel key genes and pathways associated with non-union fractures. We identified differentially expressed genes (DEGs) between non-union and fracture healing tissues using bioinformatics techniques. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to elucidate the biological processes and pathways involved. Common DEGs were identified, and a protein-protein interaction (PPI) network was constructed. Fibronectin-1 (FN1), Thrombospondin-1 (THBS1), and Biglycan (BGN) were pinpointed as critical target genes for non-union fracture treatment. Experimental validation involved alkaline phosphatase (ALP) and Alizarin Red staining to confirm osteogenic differentiation. Our analysis revealed significant alterations in pathways related to cell behavior, tissue regeneration, wound healing, infection, and immune responses in non-union fracture tissues. FN1, THBS1, and BGN were identified as key genes, with their upregulation indicating potential disruptions in the bone remodeling process. Experimental validation confirmed the induction of osteogenic differentiation. The study provides comprehensive insights into the molecular mechanisms of non-union fractures, emphasizing the pivotal roles of FN1, THBS1, and BGN in extracellular matrix dynamics and bone regeneration. The findings highlight potential therapeutic targets and pathways for further investigation. Future research should explore interactions between these genes, validate results using in vivo fracture models, and develop tailored treatment strategies for non-union fractures, promising significant advances in clinical management.

Differential Proteoglycan Expression in Atherosclerosis Alters Platelet Adhesion and Activation.

Proteoglycans are differentially expressed in different atherosclerotic plaque phenotypes, with biglycan and decorin characteristic of ruptured plaques and versican and hyaluronan more prominent in eroded plaques. Following plaque disruption, the exposure of extracellular matrix (ECM) proteins triggers platelet adhesion and thrombus formation. In this study, the impact of differential plaque composition on platelet function and thrombus formation was investigated. Platelet adhesion, activation and thrombus formation under different shear stress conditions were assessed in response to individual proteoglycans and composites representing different plaque phenotypes. The results demonstrated that all the proteoglycans tested mediated platelet adhesion but not platelet activation, and the extent of adhesion observed was significantly lower than that observed with type I and type III collagens. Thrombus formation upon the rupture and erosion ECM composites was significantly reduced (p < 0.05) compared to relevant collagen alone, indicating that proteoglycans negatively regulate platelet collagen responses. This was supported by results demonstrating that the addition of soluble biglycan or decorin to whole blood markedly reduced thrombus formation on type I collagen (p < 0.05). Interestingly, thrombus formation upon the erosion composite displayed aspirin sensitivity, whereas the rupture composite was intensive to aspirin, having implications for current antiplatelet therapy regimes. In conclusion, differential platelet responses and antiplatelet efficacy are observed on ECM composites phenotypic of plaque rupture and erosion. Proteoglycans inhibit thrombus formation and may offer a novel plaque-specific approach to limit arterial thrombosis.

Biglycan promotes hepatic fibrosis through activating heat shock protein 47.

BACKGROUND: Biglycan (BGN) is a small leucine-rich proteoglycan that participates in the production of excess extracellular matrix (ECM) and is related to fibrosis in many organs. However, the role of BGN in liver fibrosis remains poorly understood. This study aimed to investigate the role and mechanism of BGN in liver fibrosis. METHODS: Human liver samples, Bgn-/0 (BGN KO) mice and a human LX-2 hepatic stellate cells (HSCs) model were applied for the study of experimental fibrosis. GEO data and single-cell RNA-seq data of human liver tissue were analysed as a bioinformatic approach. Coimmunoprecipitation, immunofluorescence staining, western blotting and qRT-PCR were conducted to identify the regulatory effects of BGN on heat shock protein 47 (HSP47) expression and liver fibrosis. RESULTS: We observed that hepatic BGN expression was significantly increased in patients with fibrosis and in a mouse model of liver fibrosis. Genetic deletion of BGN disrupted TGF-ß1 pathway signalling and alleviated liver fibrosis in mice administered carbon tetrachloride (CCl4 ). siRNA-mediated knockdown of BGN significantly reduced TGF-ß1-induced ECM deposition and fibroblastic activation in LX-2 cells. Mechanistically, BGN directly interacted with and positively regulated the collagen synthesis chaperon protein HSP47. Rescue experiments showed that BGN promoted hepatic fibrosis by regulating ECM deposition and HSC activation by positively regulating HSP47. CONCLUSION: Our data indicate that BGN promotes hepatic fibrosis by regulating ECM deposition and HSC activation through an HSP47-dependent mechanism. BGN may be a new biomarker of hepatic fibrosis and a novel target for disease prevention and treatment.

Relationship of biglycan and decorin expression with clinicopathological features and prognosis in patients with oral squamous cell carcinoma.

OBJECTIVE: This study focused on investigating relation between biglycan (BGN) and decorin (DCN) expression and prognostic outcome for oral squamous cell carcinoma (OSCC) cases. MATERIAL AND METHODS: BGN and DCN mRNA and protein expression was detected by qRT-PCR and Western-blotting (WB) assays from 31 OSCC samples as well as healthy samples. This work harvested 101 paraffin-embedded OSCC together with 30 healthy samples, and conducted immunohistochemical (IHC) staining for assessing pathological changes. Association of DCN with BGN within OSCC was explored by Spearman's analysis. Survival rate was explored by Kaplan-Meier (KM) approach. Multivariate analysis was conducted by Cox regression. RESULTS: WB and qRT-PCR results showed BGN up-regulation (p < 0.001, p < 0.0001) whereas DCN down-regulation (p < 0.0001, p < 0.0001) with fresh OSCC tissues; the expression of BGN and DCN associated with the OSCC histopathological grade. IHC results suggested elevated BGN level (p < 0.0001) whereas DCN down-regulation (p < 0.0001) with paraffin embedded OSCC tissues. The expression of BGN and DCN associated with histopathologic grades and tumor stage of OSCC. The result of Spearman's analysis demonstrated significant association between the expression of BGN and DCN in OSCC. Survival analysis revealed that patients with higher BGN/lower DCN level showed poor overall survival (OS) as well as tumor-specific survival (TSS). Multivariate analysis proved that BGN and DCN independently predicted the prognosis of OS and TSS. CONCLUSION: BGN and DCN expression levels can be adopted for predicting OSCC prognostic outcome.

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.

Stroma biglycan expression can be a prognostic factor in prostate cancers.

OBJECTIVES: This study analyzes the relationship between biglycan expression in prostate cancer and clinicopathological parameters to clarify the potential link between biglycan and prognosis and progression to castration-resistant prostate cancer (CRPC). METHODS: We retrospectively analyzed 60 cases of prostate cancer patients who underwent robot-assisted laparoscopic radical prostatectomy in Hokkaido University Hospital. RESULTS: Biglycan was expressed in the tumor stroma but not in tumor cells. There was no significant relationship with biochemical recurrence (p = 0.5237), but the expression of biglycan was 36.1% in the group with progression to CRPC. This indicates a significant relationship with progression to CRPC (p = 0.0182). Furthermore, the expression of biglycan-positive blood vessels was significantly higher (15.9%) in the group with biochemical recurrence than in the group without biochemical recurrence (8.5%) (p = 0.0169). The biglycan-positive vessels were 28.6% in the group with progression to CRPC, which was significantly higher than that in the group without progression to CRPC (p < 0.0001). CONCLUSION: This is the first study to show that stroma biglycan is a useful prognostic factor for prostate cancer.

Biglycan Involvement in Heart Fibrosis: Modulation of Adenosine 2A Receptor Improves Damage in Immortalized Cardiac Fibroblasts.

Cardiac fibrosis is a common pathological feature of different cardiovascular diseases, characterized by the aberrant deposition of extracellular matrix (ECM) proteins in the cardiac interstitium, myofibroblast differentiation and increased fibrillar collagen deposition stimulated by transforming growth factor (TGF)-ß activation. Biglycan (BGN), a small leucine-rich proteoglycan (SLRPG) integrated within the ECM, plays a key role in matrix assembly and the phenotypic control of cardiac fibroblasts. Moreover, BGN is critically involved in pathological cardiac remodeling through TGF-ß binding, thus causing myofibroblast differentiation and proliferation. Adenosine receptors (ARs), and in particular A2AR, may play a key role in stimulating fibrotic damage through collagen production/deposition, as a consequence of cyclic AMP (cAMP) and AKT activation. For this reason, A2AR modulation could be a useful tool to manage cardiac fibrosis in order to reduce fibrotic scar deposition in heart tissue. Therefore, the aim of the present study was to investigate the possible crosstalk between A2AR and BGN modulation in an in vitro model of TGF-ß-induced fibrosis. Immortalized human cardiac fibroblasts (IM-HCF) were stimulated with TGF-ß at the concentration of 10 ng/mL for 24 h to induce a fibrotic phenotype. After applying the TGF-ß stimulus, cells were treated with two different A2AR antagonists, Istradefylline and ZM241385, for an additional 24 h, at the concentration of 10 µM and 1 µM, respectively. Both A2AR antagonists were able to regulate the oxidative stress induced by TGF-ß through intracellular reactive oxygen species (ROS) reduction in IM-HCFs. Moreover, collagen1a1, MMPs 3/9, BGN, caspase-1 and IL-1ß gene expression was markedly decreased following A2AR antagonist treatment in TGF-ß-challenged human fibroblasts. The results obtained for collagen1a1, SMAD3, α-SMA and BGN were also confirmed when protein expression was evaluated; phospho-Akt protein levels were also reduced following Istradefylline and ZM241385 use, thus suggesting that collagen production involves AKT recruited by the A2AR. These results suggest that A2AR modulation might be an effective therapeutic option to reduce the fibrotic processes involved in heart pathological remodeling.

Microscopic and Biochemical Hallmarks of BICD2-Associated Muscle Pathology toward the Evaluation of Novel Variants.

BICD2 variants have been linked to neurodegenerative disorders like spinal muscular atrophy with lower extremity predominance (SMALED2) or hereditary spastic paraplegia (HSP). Recently, mutations in BICD2 were implicated in myopathies. Here, we present one patient with a known and six patients with novel BICD2 missense variants, further characterizing the molecular landscape of this heterogenous neurological disorder. A total of seven patients were genotyped and phenotyped. Skeletal muscle biopsies were analyzed by histology, electron microscopy, and protein profiling to define pathological hallmarks and pathogenicity markers with consecutive validation using fluorescence microscopy. Clinical and MRI-features revealed a typical pattern of distal paresis of the lower extremities as characteristic features of a BICD2-associated disorder. Histological evaluation showed myopathic features of varying severity including fiber size variation, lipofibromatosis, and fiber splittings. Proteomic analysis with subsequent fluorescence analysis revealed an altered abundance and localization of thrombospondin-4 and biglycan. Our combined clinical, histopathological, and proteomic approaches provide new insights into the pathophysiology of BICD2-associated disorders, confirming a primary muscle cell vulnerability. In this context, biglycan and thrombospondin-4 have been identified, may serve as tissue pathogenicity markers, and might be linked to perturbed protein secretion based on an impaired vesicular transportation.

Analyses of damage-associated molecular patterns, particularly biglycan, in cisplatin-induced rat progressive renal fibrosis.

Damage-associated molecular patterns (DAMPs) and their receptors (TLR-2 and -4) may play important roles in renal fibrosis, of which the pathogenesis is complicated. We used rat renal lesions induced by a single intraperitoneal injection of cisplatin at 6 mg/kg body weight; consisting of tissue damage of renal tubules on days 1 and 3, further damage and regeneration with inflammation mainly on days 5 and 7, and interstitial fibrosis on days 9, 12, 15, and 20. Microarray analyses on days 5 (the commencement of inflammation) and 9 (the commencement of interstitial fibrosis) showed that DAMPs increased by more than two-fold relative to control included common extra-cellular matrix (ECM) components such as laminin (Lamc2) and fibronectin, and heat shock protein family, as well as fibrinogen, although it was limited analysis; Lamc2, an element of basement membrane, may be regarded as an indicator for damaged renal tubules. In the real-time RT-PCR analyses, TLR-2 significantly increased transiently on day 1, whereas TLR-4 significantly increased on days 9 and 15, almost in agreement with the increased biglycan (a small leucine-rich proteoglycan as ubiquitous ECM component). As M1/M2 macrophages participated in renal lesions, such as inflammation and fibrosis, presumably, TLR-4, which may be expressed in immune cells, could play crucial roles in the formation of renal lesions in association with biglycan.

Decorin evokes reversible mitochondrial depolarization in carcinoma and vascular endothelial cells.

Decorin, a small leucine-rich proteoglycan with multiple biological functions, is known to evoke autophagy and mitophagy in both endothelial and cancer cells. Here, we investigated the effects of soluble decorin on mitochondrial homeostasis using live cell imaging and ex vivo angiogenic assays. We discovered that decorin triggers mitochondrial depolarization in triple-negative breast carcinoma, HeLa, and endothelial cells. This bioactivity was mediated by the protein core in a time- and dose-dependent manner and was specific for decorin insofar as biglycan, the closest homolog, failed to trigger depolarization. Mechanistically, we found that the bioactivity of decorin to promote depolarization required the MET receptor and its tyrosine kinase. Moreover, two mitochondrial interacting proteins, mitostatin and mitofusin 2, were essential for downstream decorin effects. Finally, we found that decorin relied on the canonical mitochondrial permeability transition pore to trigger tumor cell mitochondrial depolarization. Collectively, our study implicates decorin as a soluble outside-in regulator of mitochondrial dynamics.

The role of biglycan in the healthy and thoracic aneurysmal aorta.

The class I small leucine-rich proteoglycan biglycan is a crucial structural extracellular matrix component that interacts with a wide range of extracellular matrix molecules. In addition, biglycan is involved in sequestering growth factors such as transforming growth factor-ß and bone morphogenetic proteins and thereby regulating pathway activity. Biglycan consists of a 42-kDa core protein linked to two glycosaminoglycan side chains and both are involved in protein interactions. Biglycan is encoded by the BGN gene located on the X-chromosome and is expressed in various tissues, including vascular tissue, skin, brain, kidney, lung, the immune system, and the musculoskeletal system. Although an increasing amount of data on the biological function of biglycan in the vasculature has been produced, its role in thoracic aortic aneurysms is still not fully elucidated. This review focuses on the role of biglycan in the healthy thoracic aorta and the development of thoracic aortic aneurysm and dissections in both mice and humans.

Rapamycin-induced autophagy in osteosarcoma cells is mediated via the biglycan/Wnt/ß-catenin signaling axis.

Biglycan is a class I secreted small leucine-rich proteoglycan (SLRP), which regulates signaling pathways connected to bone pathologies. Autophagy is a vital catabolic process with a dual role in cancer progression. Here, we show that biglycan inhibits autophagy in two osteosarcoma cell lines (P ≤ 0.001), while rapamycin-induced autophagy decreases biglycan expression in MG63 osteosarcoma cells and abrogates the biglycan-induced cell growth increase (P ≤ 0.001). Rapamycin also inhibits ß-catenin translocation to the nucleus, inhibiting the Wnt pathway (P ≤ 0.001) and reducing biglycan's colocalization with the Wnt coreceptor LRP6 (P ≤ 0.05). Furthermore, biglycan exhibits protective effects against the chemotherapeutic drug doxorubicin in MG63 OS cells through an autophagy-dependent manner (P ≤ 0.05). Cotreatment of these cells with rapamycin and doxorubicin enhances cells response to doxorubicin by decreasing biglycan (P ≤ 0.001) and ß-catenin (P ≤ 0.05) expression. Biglycan deficiency leads to increased caspase-3 activation (P ≤ 0.05), suggesting increased apoptosis of biglycan-deficient cells treated with doxorubicin. Computational models of LRP6 and biglycan complexes suggest that biglycan changes the receptor's ability to interact with other signaling molecules by affecting the interdomain bending angles in the receptor structure. Biglycan binding to LRP6 activates the Wnt pathway and ß-catenin nuclear translocation by disrupting ß-catenin degradation complex (P ≤ 0.01 and P ≤ 0.05). Interestingly, this mechanism is not followed in moderately differentiated, biglycan-nonexpressing U-2OS OS cells. To sum up, biglycan exhibits protective effects against the doxorubicin in MG63 OS cells by activating the Wnt signaling pathway and inhibiting autophagy.

Novel antiangiogenic therapy targeting biglycan using tumor endothelial cell-specific liposomal siRNA delivery system.

Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) into MEND because αV ß3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.

Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells.

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.