Increased atherosclerosis in mice with increased vascular biglycan content

Objective

The response to retention hypothesis of atherogenesis proposes that atherosclerosis is initiated via the retention of atherogenic lipoproteins by vascular proteoglycans. Co-localization studies suggest that of all the vascular proteoglycans, biglycan is the one most closely co-localized with LDL. The goal of this study was to determine if over-expression of biglycan in hyperlipidemic mice would increase atherosclerosis development.

Methods

Transgenic mice were developed by expressing biglycan under control of the smooth muscle actin promoter, and were crossed to the LDL receptor deficient (C57BL/6 background) atherosclerotic mouse model. Biglycan transgenic and non-transgenic control mice were fed an atherogenic Western diet for 4–12 weeks.

Results

LDL receptor deficient mice overexpressing biglycan under control of the smooth muscle alpha actin promoter had increased atherosclerosis development that correlated with vascular biglycan content.

Conclusion

Increased vascular biglycan content predisposes to increased lipid retention and increased atherosclerosis development.     

Targeted Disruption of Two Small Leucine-rich Proteoglycans, Biglycan and Decorin, Excerpts Divergent Effects on Enamel and Dentin Formation

Small leucine-rich proteoglycans have been suggested to affect mineralization of dental hard tissues. To determine the functions of two of these small proteoglycans during the early stages of tooth formation, we characterized the dental phenotypes of biglycan (BGN KO) and decorin deficient (DCN KO) mice and compared them to that of wild type mice. Each targeted gene disruption resulted in specific effects on dentin and enamel formation. Dentin was hypomineralized in both knock out mice, although the effect was more prominent in the absence of decorin. Enamel formation was dramatically increased in newborn biglycan knockout mice but delayed in absence of decorin. Increased enamel formation in the former case resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the later case was most probably an indirect consequence of the high porosity of the underlying dentin. Enamelin expression was unchanged in BGN KO, and reduced in DCN KO. Dentin sialoprotein (DSP), a member of the family of phosphorylated extracellular matrix proteins that play a role in dentinogenesis, was overexpressed in BGN-KO odontoblasts and in the sub-odontoblastic layer. In contrast, a decreased expression of DSP was detected in DCN KO. Dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and osteopontin (OPN) were upregulated in BGN KO and downregulated in the DCN KO. Despite the strong effects induced by these deficiencies in newborn mice, no significant difference was detected between the three genotypes in adult mice, suggesting that the effects reported here in newborn mice are transient and subjected to self-repair.     

TGF-β/Extracellular Matrix Interactions in Dentin Matrix: A Role in Regulating Sequestration and Protection of Bioactivity

TGF-β isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin–pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore^TM 3000 system to investigate the binding of TGF-β isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-β isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-β isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-β isoforms. However, the association with TGF-β3 was much weaker than that with TGF-β1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-β was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-β isoforms 1 and 3, which may explain why TGF-β3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-β1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-β/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.     

Biglycan protects cardiomyocytes against hypoxia/reoxygenation injury: Role of nitric oxide

Biglycan, a proteoglycan component of extracellular matrix, has been suspected to contribute to the development of atherosclerosis, but overexpression of biglycan in transgenic mice has been shown to induce cardioprotective genes including nitric oxide (NO) synthases in the heart. Therefore, here we hypothesized if exogenous administration of biglycan exerts cytoprotection. Primary cardiomyocytes from neonatal rats were subjected to 150 min hypoxia and 2 h reoxygenation. Mortality of cardiomyocytes was dose-dependently attenuated by pretreatment with 1-100 nM biglycan. Biglycan enhanced eNOS mRNA and protein, and significantly increased NO content of cardiomyocytes. The NO synthase inhibitor l-nitro-arginine-methyl-ester significantly attenuated the cytoprotective effect of biglycan. This is the first demonstration that biglycan leads to cytoprotection against hypoxia/reoxygenation injury, and that this phenomenon is partially mediated by an NO-dependent mechanism.     

Biglycan及血管内皮生长因子对结肠癌细胞迁移、侵袭能力的影响及机制

目的:观察biglycan及血管内皮生长因子(vascular endothelial growth factor,VEGF)对结肠癌细胞系HCT116迁移、侵袭能力的影响并探讨其可能的机制。方法:在稳定转染biglycan的结肠癌细胞系HCT116中转染VEGF siRNA,实验设置未转染对照组(mock组)、空载质粒和非特异性干扰片段转染对照组(vector+siRNA-NC组)、biglycan cDNA和非特异性干扰片段转染组(biglycan+siRNA-NC组)以及biglycan cDNA和VEGF siRNA共转染组(biglycan+siRNA-VEGF组)。采用实时荧光定量逆转录聚合酶链反应(RT-PCR)法检测biglycan和VEGF的mRNA表达;采用划痕实验检测细胞的迁移能力;采用Transwell法检测细胞的侵袭能力;采用蛋白质印迹(Western blotting)法检测SNAIL、E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶-9(MMP-9)的表达;采用明胶酶谱法检测MMP-2、MMP-9的活性。结果:与mock组及vector+siRNA-NC组比较,biglycan+siRNA-NC组细胞的迁移、侵袭能力均显著提高(P0.05),vimentin、MMP-2、MMP-9、SNAIL蛋白的表达水平显著提高,E-cadherin蛋白的表达水平则显著下降(P0.05),MMP-2和MMP-9的活性显著提高(P0.05)。与biglycan+siRNA-NC组比较,biglycan+siRNA-VEGF组细胞的迁移、侵袭能力均显著降低(P0.05),SNAIL、vimentin、MMP-2、MMP-9蛋白的表达水平均显著下降,E-cadherin蛋白的表达水平显著提高(P0.05),MMP-2、MMP-9的活性显著下降(P0.05)。结论:biglycan通过促进VEGF的表达来促进结肠癌细胞系HCT116的迁移和侵袭,下调结肠癌细胞中VEGF的表达可逆转biglycan对HCT116迁移和侵袭的促进作用。     

Biglycan-Deficient Mice Have Delayed Osteogenesis after Marrow Ablation

Biglycan (bgn) is a small proteoglycan in skeletal tissue that binds and regulates collagen and TGF-beta activities. Mice deficient in bgn (bgn-KO) develop age-dependent osteopenia and have multiple metabolic defects in their bone marrow stromal cells including increased apoptosis, reduced numbers of colony-forming units-fibroblastic (CFU-F) and decreased collagen production. In the present study we tested the hypothesis that bone formation capability in response to a physiological stress is compromised in bgn deficiency. We tested this theory using an in vivo bone marrow ablation assay. Ablation was performed on 6-week-old wild type (wt) and bgn-KO mice and bones were analyzed at days 7, 10, and 17 postsurgery. X-ray analysis showed that bone marrow ablation in femora induced vigorous new bone formation within 10 days in both genotypes but appeared greater in the wt compared to the bgn-KO. In order to quantitate the changes in bone formation in the ablated animals, bone densities of the proximal, midshaft, and distal femora were assessed using peripheral quantitative computed tomography (pQCT). The ratio of cancellous bone density at the midshaft (ablated limb/control limb) was significantly higher in wt compared to bgn-KO at day 10 postsurgery. Wt and bgn-KO femora had similar total and cancellous bone densities at days 7 and 17 postsurgery at all three locations indicating that the ablation effects were temporal and limited to the cancellous bone of the mid-shaft region. These data indicate that the absence of bgn directly impeded bone formation. Our results support the concept that bgn is important in controlling osteogenesis following marrow ablation.     

Proteoglycans in Predentin: The Last 15 Micrometers Before Mineralization

Small leucine-rich proteoglycans (SLRPs) regulate extracellular matrix organization. In order to investigate the distribution and potential functions of decorin, biglycan (BGN), and fibromodulin (3 SLRPs, potentially related to dentinogenesis), we performed light and electron immunochemistry on teeth from rats, and on wild-type and biglycan knockout mice (BGN KO). Immunohistochemical data demonstrate that chondroitin sulfate/dermatan sulfate (CS/DS) and keratan sulfate (KS) distributions displayed reverse gradients in predentin. The decrease of CS/DS labeling from the proximal to the distal predentin contrasted with the sharp decorin increase observed in the distal predentin near the predentin/dentin transition, an effect possibly attributable to the deglycosylation action of stromelysin-1. In contrast, BGN concentration was apparently constant throughout the whole predentin. Additional immunolabelings showed, for the first time, the presence of fibromodulin in predentin. Compared with the wild-type mouse, the mean diameter of collagen fibrils in the BGN KO was smaller in the proximal predentin but larger in the central and distal predentin, the metadentin was broader, and the dentin mineralization appeared altered and heterogeneous. Altogether, our data suggest an important role for BGN in dentin formation and mineralization.     

青少年特发性脊柱侧凸患者髂骨中双糖链蛋白多糖和核心蛋白聚糖的表达

目的:观察非胶原骨基质蛋白中双糖链蛋白多糖(Biglycan)和核心蛋白聚糖(Decorin)在青少年特发性脊柱侧凸(adolescentidiopathicscoliosis,AIS)患者髂骨中的表达情况。方法:从10例行后路自体骨融合的AIS女性患者(AIS组)和6例无骨代谢性疾病需行髂骨自体骨移植的其它骨科疾病女性患者(对照组)获取髂骨标本。AIS组患者年龄11.9￣19.1岁,平均14.9岁,Cobb角55°(45°￣61°);对照组平均年龄40.8岁。标本于10%缓冲福尔马林液中固定6h,在福尔马林酸中脱钙3周。切片厚度为5!m,行免疫组化染色。用半定量方法分析双糖链蛋白多糖和核心蛋白聚糖在单位视野骨细胞与骨基质中的表达。结果:AIS组髂骨中骨细胞与骨基质表达核心蛋白聚糖各6例,表达双糖链蛋白多糖各6例。对照组核心蛋白聚糖在骨细胞与骨基质中均为阳性表达;双糖链蛋白多糖在骨基质中均为阳性表达,在骨细胞中阳性表达5例。!2检验显示AIS组上述两种非胶原骨基质蛋白多糖的阳性表达率明显低于对照组(P<0.05)。结论:双糖链蛋白多糖和核心蛋白聚糖在AIS患者髂骨中的表达较对照组低,研究其在AIS患者骨骼中的表达和分布,有助于研究AIS的骨质密度和骨生长发育异常的分子基础,进一步认识该病的病因与发病机理。