XXIX Italian Society for the Study of Connective Tissues (SISC) Meeting,October 29–30, 2009, Alghero,Italy

Soluble substances in the bovine nuchal ligament were removed by preliminary extractions with a buffer and dilute alkali solution. The insoluble residue was then extracted with 6 M guanidine HCl and with 6 M guanidine HCl containing a reducing agent (20 mM dithiothreitol) successively. Each of the two extracts contained a glycoprotein; that in the first extract was designated Glycoprotein A and that in the second Glycoprotein B. They were purified by ion-exchange chromatography and gel filtration till essentially homogeneous. Both proteins had similar molecular weights of 35,000 in SDS-polyacrylamide gel electrophoresis and by gel filtration, and their chemical compositions resembled each other closely. It is suggested that Glycoprotein B was present in the native state as a disulfide-bonded, aggregated form of Glycoprotein A. The compounds also showed similarity with the microfibrillar glycoprotein(s) previously reported in bovine nuchal ligament extracts.     

N-Terminal Sequence of a Core Protein from a Biglycan Isolated from Bovine Aorta

A biglycan was isolated from bovine aorta intima media by 4M guanidine HCl extraction of the tissue; the material was fractionated and purified by using isopycnic ultracentrifugation and DEAE Sephacel ion-exchange chromatography. Core proteins, resulting from digestion of the proteoglycan preparation with chondroitinase ABC, were resolved by SDS-polyacrylamide gel electrophoresis into three bands. The apparent molecular weight of the fast migrating major protein band was 47 kDa and the other slow-moving minor protein bands were 90 and 105 kDa. These proteins were recognized by a monoclonal anti-proteoglycan ΔDi-6S (MAb 3-B-3/Cl). The amino acid composition of 47 kDa core protein revealed a high content of aspartic acid, glutamic acid and leucine, similar to those found for biglycans isolated from bovine cartilage, rat vascular smooth muscle cell culture and human bone. The N-terminal sequence of 47 kDa core protein was determined as Asp-Glu-Glu-Ala-X-Gly-Ala-Glu-Thr-Thr-X-Gly-Ile-Pro-Asp which is identical to the sequence of bovine articular cartilage biglycan. The proteoglycan had two glycosaminoglycan chains.     

Differences in mRNA and protein expression of small proteoglycans in vaginal wall tissue from women with and without stress urinary incontinence

BACKGROUND: To investigate changes in mRNA and protein levels of biglycan (BGN), decorin (DCN) and fibromodulin (FMOD) in vaginal wall tissue from women with stress urinary incontinence (SUI) compared to menstrual-cycle matched continent women. METHODS: We determined mRNA expressions of BGN, DCN and FMOD by quantitative real-time PCR. They were localized in vaginal wall tissue by immunohistochemistry. We performed western blot analysis to examine protein expression. RESULTS: BGN, DCN and FMOD co-localized with collagen and elastin in the extracellular matrix (ECM) of vaginal wall tissue from both groups. The mRNA expression of FMOD was significantly lower in cases versus controls in the proliferative phase (P = 0.03). DCN mRNA expression in cases was higher in the proliferative (P = 0.05) and secretory phases (P = 0.02) versus controls. BGN mRNA expression showed no significant differences in either phase. Protein expression of FMOD in cases was lower in the proliferative phase versus controls (six out of nine pairs), whereas DCN and BGN protein expression in the secretory phase in cases was higher (seven out of nine pairs). CONCLUSION: BGN, DCN and FMOD expressions in vaginal wall tissue differ in women with SUI and are hormonally modulated. Differences in small proteoglycans may contribute to the altered pelvic floor connective tissues found in these women.     

DCN与BGN在发育期小鼠牙髓牙本质复合体形成中的作用研究

目的:研究富含亮氨酸的蛋白多糖类物质:核心蛋白聚糖(DCN)及双糖蛋白聚糖(BGN)在小鼠牙髓牙本质复合体不同发育时期的分布特点,探讨其在牙髓牙本质复合体形成过程中的作用.方法:取出生后第5 d、10 d、15 d、25 d的BALB/℃小鼠36只,引颈处死,解剖分离含下颌第一磨牙区的下颌骨,新鲜配制4%多聚甲醛固定24 h,甲酸-甲酸钠复合脱钙液脱钙1～3周,石蜡包埋,近远中向5μm连续切片.免疫组化PV两步法,观察各发育时期第一磨牙牙本质、成牙本质细胞及牙髓细胞中DCN、BGN的组织学分布特点.结果:牙本质形成初期,BGN在前期牙本质与成牙本质细胞强阳性表达.随后,前期牙本质与成牙本质细胞染色开始减弱,而牙髓细胞阳性表达呈逐渐增强趋势.第5 d,DCN在前期牙本质中强阳性表达,而在牙本质、成牙本质细胞中一直为阴性;第13 d,牙髓细胞开始呈弱阳性表达,随着牙齿发育而逐渐增强.在观察期间,两者在前期牙本质中持续阳性表达,并且随着牙本质的发育成熟,其染色逐渐减低.结论:DCN与BGN在牙体组织和牙发育过程中特定阶段聚集在特定的部位,二者可能在细胞与细胞、细胞与基质相互作用中发挥特异作用并参与牙本质的矿化过程.     

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.The passage of leukocytes from the circulation toward the surrounding extravascular space is a critical event in the inflammatory response (1–3). The multistep adhesion cascade defines the increasingly adhesive steps leukocytes make with the endothelium to exit the circulation and enter the surrounding microenvironment (4). Each step of the cascade [tethering, rolling, slow rolling, firm adhesion, and transendothelial migration (TEM)] critically depends on cell adhesion molecules expressed on both leukocytes and endothelial cells. Such cell adhesion molecules have been identified and characterized to act in a sequential and interdependent manner. The molecular mechanism driving leukocyte integrin function during the multistep adhesion cascade is relatively well defined (4–6). In contrast, mechanisms underpinning the regulation of nonintegrin receptors are only beginning to emerge. One such example, L-selectin, is known to promote the initial tethering and subsequent rolling of leukocytes along activated endothelial cells (7). Knocking out L-selectin in mice has provided compelling evidence for this cell adhesion molecule in directing neutrophils toward sites of inflammation (8). Intriguingly, knocking out L-selectin has a dramatic impact on neutrophil chemotaxis in vivo, but the molecular basis for this observation remains elusive (9). Following firm adhesion, L-selectin is broadly considered to be proteolytically cleaved (or shed) at a defined extracellular membrane/proximal domain by membrane-associated matrix metalloproteinases (10). The best-characterized “sheddase” is a disintegrin and metalloproteinase (ADAM17; or TNF-α concerting enzyme) (11). Shedding of L-selectin hinges on its interaction with calmodulin (CaM); binding of CaM to L-selectin protects from shedding, and leukocyte activation leads to CaM dissociation from the L-selectin tail to drive shedding (12). What promotes CaM dissociation is not known, but mutating the L-selectin tail can have a profound impact on the shedding response (13–15). L-selectin shedding is a rapid event, and the biological significance of its outcome may be numerous (10). In respect to leukocyte recruitment, L-selectin shedding would rapidly halt any further contribution of this molecule toward cell adhesion, signaling, or migration. The timing of L-selectin shedding is therefore critical in relation to input signals derived from other surface receptors contributing to the same cellular event (7). Our understanding of coordinated receptor signaling during TEM is extremely poor, mainly because insight into the molecular regulation of each individual cell adhesion molecule is still lacking.The contribution of L-selectin to the multistep adhesion cascade has been defined through two major experimental approaches: function-blocking studies (using soluble ligand or monoclonal antibody) and gene KO/knock-in mice. Such studies have built the foundation of our knowledge of the adhesion cascade. However, these approaches limit our understanding of where the targeted molecule’s first nonredundant step is required. Direct imaging of cell adhesion molecules in space and time, during recruitment under flow conditions, can provide additional clues beyond the first nonredundant point of execution. Here, we have used a series of advanced imaging techniques to pinpoint where and when L-selectin is cleaved during the multistep adhesion cascade. Using primary human monocytes, we reveal the shedding event was occurring specifically during TEM and not before. Through stable expression of WT and mutant forms of L-selectin tagged to GFP or red fluorescent protein (RFP), we could define the shedding event on a mechanistic level in THP-1 cells. Fluorescence lifetime imaging microscopy (FLIM) enabled quantitative measurement of the fluorescence resonance energy transfer (FRET) efficiency between L-selectin–GFP and CaM-RFP, allowing us to monitor the subcellular distribution of their interaction during TEM. We show, for the first time to our knowledge, that L-selectin shedding can regulate the invasive behavior of monocytes crossing activated endothelial monolayers under flow. Furthermore, we show that polarity in transmigrated monocytes is disrupted if shedding of L-selectin is blocked. Taken together, these results reveal previously unidentified roles for L-selectin that extend beyond tethering and rolling.     

Effects of the angiotensin II receptor blocker losartan on the monocyte expression of biglycan in hypertensive patients

1. Recently, we demonstrated that biglycan (BGN) is increased in circulating monocyte cells from hypertensive patients and that angiotensin (Ang) II is able to increase BGN expression. The present study was designed to investigate the effects of treatment with the angiotensin AT₁ receptor antagonist losartan on monocyte BGN mRNA and protein expression in essential hypertension. 2. One hundred and twenty‐six newly diagnosed hypertensive patients without additional risk factors for atherosclerosis and cardiovascular disease were treated with 100 mg losartan once daily for 6 months. Biglycan mRNA and protein expression was determined in monocytes isolated from peripheral blood before (T₀) and after (T₁) therapy. Plasma levels of interleukin (IL)‐6, tumour necrosis factor (TNF)‐α and high sensitivity C‐reactive protein (hs‐CRP) were also determined. In addition, BGN mRNA and protein expression was determined after the ex vivo addition of 1 μmol/L AngII to monocytes isolated from 20 randomly selected hypertensive patients. 3. Biglycan mRNA and protein expression, blood pressure and plasma levels of fibrinogen, IL‐6, TNF‐α and CRP were significantly lower at T₁ than at T₀. Variations in BGN expression were associated with inflammatory markers, but not directly with blood pressure. In AngII‐stimulated monocytes, BGN mRNA and protein expression was significantly lower at T₁ that at T₀. Moreover, mean BGN mRNA expression in AngII‐stimulated monocytes isolated from losartan‐treated patients was similar to baseline expression in unstimulated monocytes from untreated patients. 4. The results of the present study show that losartan can reduce BGN expression in monocytes from hypertensive patients, without any linear association with blood pressure, suggesting that the effects of AngII on BGN expression in monocytes may be modulated, in part, by an AT₁ receptor blocker.     

The small leucine‐rich repeat proteoglycans in tissue repair and atherosclerosis

Proteoglycans consist of a protein core with one or more covalently attached glycosaminoglycan (GAG) side chains and have multiple roles in the initiation and progression of atherosclerosis. Here we discuss the potential and known functions of a group of small leucine‐rich repeat proteoglycans (SLRPs) in atherosclerosis. We focus on five SLRPs, decorin, biglycan, lumican, fibromodulin and PRELP, because these have been detected in atherosclerotic plaques or demonstrated to have a role in animal models of atherosclerosis. Decorin and biglycan are modified post‐translationally by substitution with chondroitin/dermatan sulphate GAGs, whereas lumican, fibromodulin and PRELP have keratan sulphate side chains, and the core proteins have leucine‐rich repeat (LRR) motifs that are characteristic of the LRR superfamily. The chondroitin/dermatan sulphate GAG side chains have been implicated in lipid retention in atherosclerosis. The core proteins are discussed here in the context of (i) interactions with collagens and their implications in tissue integrity, fibrosis and wound repair and (ii) interactions with growth factors, cytokines, pathogen‐associated molecular patterns and cell surface receptors that impact normal physiology and disease processes such as inflammation, innate immune responses and wound healing (i.e. processes that are all important in plaque development and progression). Thus, studies of these SLRPs in the context of wound healing are providing clues about their functions in early stages of atherosclerosis to plaque vulnerability and cardiovascular disease at later stages. Understanding of signal transduction pathways regulated by the core protein interactions is leading to novel roles and therapeutic potential for these proteins in wound repair and atherosclerosis.     

A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc

This study provides a comparative analysis of the temporal and spatial distribution of 5 intervertebral disc (IVD) proteoglycans (PGs) in sheep. The main PGs in the 2 and 10 y old sheep groups were polydisperse chondroitin sulphate and keratan sulphate substituted species. Their proportions did not differ markedly either with spinal level or disc zone. In contrast, the fetal discs contained 2 slow migrating (by composite agarose polyacrylamide gel electrophoresis, CAPAGE), relatively monodisperse chondroitin sulphate-rich aggrecan species which were also identified by monoclonal antibody 7-D-4 to an atypical chondroitin sulphate isomer presentation previously found in chick limb bud, and shark cartilage. The main small PG detectable in the fetal discs was biglycan, whereas decorin predominated in the 2 and 10 y old IVD samples; its levels were highest in the outer annulus fibrosus (AF). Versican was most abundant in the AF of the fetal sheep group; it was significantly less abundant in the 2 and 10 y old groups. Furthermore, versican was immunolocalised between adjacent layers of annular lamellae suggesting that it may have some role in the provision of the viscoelastic properties to this tissue. Versican was also diffusely distributed throughout the nucleus pulposus of fetal IVDs, and its levels were significantly lower in adult IVD specimens. This is the first study to identify versican in ovine IVD tissue sections and confirmed an earlier study which demonstrated that ovine IVD cells synthesised versican in culture (Melrose et al. 2000). The variable distribution of the PGs identified in this study provides further evidence of differences in phenotypic expression of IVD cell populations during growth and development and further demonstrates the complexity of the PGs in this heterogeneous but intricately organised connective tissue.