Lumican mRNA Research Articles

Lumican accumulates with fibrillar collagen in fibrosis in hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in-depth assessment of lumican in clinical and experimental HCM. Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n=15), and controls from hearts deemed unsuitable for transplantation (n=8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild-type littermates (n=8-10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid-ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n=3) and examined using confocal microscopy. Lumican mRNA was increased threefold in HCM patients (P<0.05) and correlated strongly with expression of collagen I (R2 =0.60, P<0.01) and III (R2 =0.58, P<0.01). Lumican protein was increased by 40% in patients with HCM (P<0.01) and correlated with total (R2 =0.28, P=0.05) and interstitial (R2 =0.30, P<0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P<0.001), and lumican protein increased 20-fold (P<0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co-localization measured in patients and mice with HCM (patients: +19%, P<0.01; mice: +13%, P<0.01). dSTORM super-resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to (-15nm, P<0.05), overlapped more frequently with (+7.3%, P<0.05) and to a larger degree with (+5.6%, P<0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8nm, P<0.001), longer (+345.9nm, P<0.001), and fewer (-8.9%, P<0.001) collagen fibres. The ECM proteoglycan lumican is increased in HCM and co-localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM.

Lumican, an Exerkine, Protects against Skeletal Muscle Loss.

Exerkines are soluble factors secreted by exercised muscles, mimicking the effects of exercise in various organs, including the muscle itself. Lumican is reportedly secreted from muscles; however, its roles in skeletal muscle remain unknown. Herein, we found that lumican mRNA expression in the extensor digitorum longus was significantly higher in exercised mice than in unloading mice, and lumican stimulated myogenesis in vitro. Additionally, lumican knockdown significantly decreased muscle mass and cross-sectional area (CSA) of the muscle fiber in the gastrocnemius muscle of exercised mice. Lumican upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) and a p38 inhibitor near completely blocked lumican-stimulated myogenesis. Inhibitors for integrin α2β1 and integrin ανβ3 also prevented lumican-stimulated myogenesis. Systemic lumican treatment, administered via the tail vein for 4 weeks, significantly increased relative muscle masses by 36.1% in ovariectomized mice. In addition, intramuscular lumican injection into unloaded muscles for 2 weeks significantly increased muscle mass by 8.5%. Both intravenous and intramuscular lumican treatment significantly increased muscle CSA. Our in vitro and in vivo experiments indicate that lumican is a muscle-secreted exerkine that affords protection against muscle loss by activating p38 MAPK via integrin receptors.

The Involvement of Lumican in Human Ovulatory Processes.

Based on a previous global transcriptome sequencing project, we hypothesized that Lumican (LUM) might play a role in ovulatory processes. We sought to determine LUM gene expression under various conditions in human preovulatory follicles. The in vitro expression of LUM mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to identify human LUM expression in follicles at different developmental stages. Cell signaling studies were performed by treating human MGCs with human chorionic gonadotropin (hCG) and both, different stimulators and inhibitors to determine their effect on LUM expression by using qRT-PCR. Cell confluence studies were carried out to study the correlation between LUM expression and follicle cell proliferation. Follicular MGCs and CGCs of women undergoing in vitro fertilization (IVF) procedures due to endometriosis were analyzed for differences in LUM expression patterns by qRT-PCR. LUM mRNA expression was significantly higher in MGCs as compared to CGCs. In CGCs, LUM mRNA was higher in mature metaphase II (MII) oocytes than in germinal vesicle (GV) and metaphase I (MI) oocytes. LUM expression was significantly upregulated in response to hCG in cultured MGCs. Immunohistochemistry of human ovaries revealed LUM was mostly present in MGCs of large preovulatory and postovulatory follicles and absent from primordial follicles. Using pharmacological activators and inhibitors, we demonstrated that LUM induction by luteinizing hormone (LH)/hCG is carried through the mitogen-activated protein kinase (MEK) pathway. LUM expression was induced in high-density cell cultures in a confluence-dependent manner. MGCs from follicles of subjects with endometriosis exhibited reduced mRNA transcription levels compared to control subjects. Our study confirms that LUM is a newly discovered ovulatory gene. LUM might play an important role during the preovulatory period up until ovulation as well as in endometriosis infertility. A better understanding of LUM's role might provide potential new treatment paradigms for some types of female infertility.

Lumican inhibits immune escape and carcinogenic pathways in colorectal adenocarcinoma.

Lumican (LUM), a small leucine-rich proteoglycan, is a component of the extracellular matrix. Abnormal LUM expression is potentially associated with cancer progression. In the present study, we confirmed high LUM mRNA expression in colorectal adenocarcinoma (COAD) through the UALCAN database. The Kaplan-Meier method, univariate, and multivariate COX analysis showed that high LUM expression is an independent determinant of poor prognosis in COAD. A COX regression model was constructed based on clinical information and LUM expression. The receiver operating characteristic (ROC) curve indicated that this model was highly accurate in monitoring COAD prognosis. The co-expression network of LUM was determined by LinkedOmics, which showed that LUM expression was closely related to immune escape and the miR200 family. Furthermore, we studied the co-expression network of LUM and found that LUM could promote tumor metastasis and invasion. The Tumor Immune Estimation Resource website showed that LUM was closely related to immune infiltration and correlated with regulatory T cells, tumour-associated macrophages, and dendritic cells. We found that LUM cultivated cancer progression by targeting the miR200 family to promote epithelial-to-mesenchymal transition. These findings suggest that LUM is a potential target for inhibiting immune escape and carcinogenic pathways.

Autologous Cell Therapy for Aged Human Skin: A Randomized, Placebo-Controlled, Phase-I Study

Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 10⁶ cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis.

Hypoxia-induced autophagy of stellate cells inhibits expression and secretion of lumican into microenvironment of pancreatic ductal adenocarcinoma.

Lumican is secreted by pancreatic stellate cells and inhibits cancer progression. Extracellular lumican inhibits cancer cell replication and restrains growth of early-stage pancreatic adenocarcinoma (PDAC) such that patients with tumors containing stromal lumican experience a three-fold longer survival after treatment. In the present study, patient tumor tissues, ex-vivo cultures of patient-derived xenografts (PDX), PDAC stellate and tumor cells were used to investigate whether hypoxia (1% O2) within the tumor microenvironment influences stromal lumican expression and secretion. We observed that hypoxia significantly reduced lumican expression and secretion from pancreatic stellate cells, but not cancer cells. Although hypoxia enhanced lactate dehydrogenase A (LDHA) expression and lactate secretion from all cells, neither hypoxia-induced nor exogenous lactate influenced lumican expression. Autophagy was induced by hypoxia in ex vivo cultures of PDX and pancreatic stellate cells, but not cancer cells cultured in 2D. Autophagic flux inhibitors, bafilomycin A1, chloroquine diphosphate salt, and ammonium chloride prevented hypoxia-mediated reduction in lumican expression in stellate cells. Furthermore, inhibition of AMP-regulated protein kinase (AMPK) phosphorylation or hypoxia-inducible factor (HIF)-1α expression within hypoxic stellate cells restored lumican expression levels. Hypoxia did not affect lumican mRNA expression, indicating that hypoxia-induced reduction of lumican occurs post-transcriptionally; in addition, AMPK inhibition prevented hypoxia-reduced phosphorylation of the mTOR/p70S6K/4EBP signaling pathway, a key contributor to protein synthesis. Taken together, these findings demonstrate that hypoxia reduces stromal lumican in PDAC through autophagy-mediated degradation and reduction in protein synthesis within pancreatic cancer stellate cells.

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-β-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.

Effects of low-dose of TGF-β1 on maintaining bovine corneal stromal cell growth and retarding extra cellular matrix fibrosis in a three-dimensional culture model

Background Transforming growth factor-β1 (TGF-β1) plays an important role in corneal wound healing.The effects of TGF-β1 on the synthesis of extra cellular matrix (ECM) vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β1 on keratocytes under the two-dimensional culture condition, and the effect of low concentration of TGF-β1 on the synthesis of ECM in keratocytes remains unclear. Objective This study was to investigate the growth of Pellet, a three-dimensional model of corneal stroma cells in vitro, and its ECM synthesis under a low concentration of TGF-β1. Methods Bovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10% fetal bovine serum (FBS). Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β1+ 5% FBS and 0.50 ng/ml TGF-β1+ 5% FBS were established, respectively.The morphology of Pellets was observed under the natural light at 48 hours, 1 week, 2 weeks and 3 weeks after culture.In 3 weeks after culture, the cell structures was observed by hematoxylin-eosin staining, and Calcein-AM/propidium (Calcein-AM/PI) staining was used to assay the cell viability.Real-time fluorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin (α-SMA), fibronectin (FN), type Ⅰ collagen (ColⅠ) and type Ⅲ collagen (Col Ⅲ) mRNA and proteins.RT-PCR was employed to detect the expressions of lumican (LUM) mRNA and keratocan (KERA) mRNA in the cells. Results Cells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β1+ 5% FBS group and 0.50 ng/ml TGF-β1+ 5% FBS group, and most cells possessed complete structures.The death rate of the cells was (33.60±1.65)% in the 0.25 ng/ml TGF-β1+ 5% FBS group and (30.90±0.78)% in the 0.50 ng/ml TGF-β1+ 5% FBS group, showing an insignificant difference between them (t=0.144, P=0.887). The expressions of α-SMA, FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β1+ 5% FBS group were lower than those in the 0.50 ng/ml TGF-β1+ 5% FBS group (tα-SMA=4.622, P=0.010; tFN =2.973, P=0.040; t Col Ⅲ=7.845, P<0.001), but the expression of Col Ⅰ in 0.25 ng/ml TGF-β1+ 5% FBS group was higher than that in 0.50 ng/ml TGF-β1+ 5% FBS group (tColⅠ=4.022, P=0.016). The ratio of Col Ⅲ/ColⅠ in 0.25 ng/ml TGF-β1+ 5% FBS group was lower than that in the 0.50 ng/ml TGF-β1+ 5% FBS group in both mRNA and protein level (tmRNA =-3.039, P=0.038; tprotein=3.215, P=0.032). The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β1+ 5% FBS group increased over time.While in 0.50 ng/ml TGF-β1+ 5% FBS group, the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks. Conclusions Low-dose TGF-β1 in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β1, implying a scarless expression. Key words: Corneal keratocyte; Transforming growth factor-β1; Three-dimensionl model; Bovine

Changes of ocular biological parameters and Lumican expression in the monocularly deprivation myopic model of mutant Lumican transgenic mice

Objective: To investigate ocular changes in the monocularly deprivation myopic model of mutant Lumican transgenic mice. Comparing influences on biological parameters and sclera development between Lumican transgenic and form deprivation mice, and to prepare for further study of pathogenesis of pathological myopia (PM). Methods: Experimental research. Lumican transgenic mice and wild mice were monocularly lid-sutured at ten days after birth. All eyes were divided into 6 groups, group A(32 eyes): control eyes in transgenic mice; group B(34 eyes): sutured eyes in transgenic mice; group C(34 eyes): fellow eyes in transgenic mice; group D(28 eyes): control eyes in wild mice; group E(32 eyes): sutured eyes in wild mice; group F(32 eyes): fellow eyes in wild mice. Refraction was measured by streak retinoscopye and axial length was measured by vernier caliper at 8 weeks (56 days) after birth. Lumican expression was detected by quantitative real-time PCR in all groups. Results: The refraction in group B and group E were (-0.38±1.10) D and (0.14±1.26)D respectively, which were significantly different compared with contralateral groups and normal control groups (F=9.525, 10.067; P<0.01). The mean axial length were also increased in group B ((3.28 ± 0.07)mm and group E (3.24 ± 0.09)mm, (F=7.183, 6.671; P<0.05). Expression level of Lumican mRNA in sclera was increased in group B, which was significantly different from group A and group C (F= 6.262; P<0.05). The expression of Lumican mRNA was increased in group B and C when compared with group E and F (t=4.772, 2.218, P<0.05). Conclusions: Form-deprivation in mutant Lumican transgenic mice causes myopic changes in deprived eyes. The gene expression level of Lumican in sclera of transgenic mice is significantly increased compared with contralateral eyes or that of wild group. Lumican mutation may effect the development of PM, and the interaction of genetic and environmental factors may lead to development of PM. (Chin J Ophthalmol, 2016, 52: 850-855).

Effect of interleukin-1beta and dehydroepiandrosterone on the expression of lumican and fibromodulin in fibroblast-like synovial cells of the human temporomandibular joint.

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican differs from that of fibromodulin in synovial tissue and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.

Expression of mRNA of Apolipoprotein E, Apolipoprotein A-IV, and Matricellular Proteins in the Myocardium and Intensity of Fibroplastic Processes during Experimental Hypercholesterolemia

The expression of mRNA of matricellular proteins (osteopontin, and lumican), apolipoproteins E and A-IV, and microsomal triglyceride transfer protein, and the intensity of fibroplastic processes were studied in the myocardium of rats during experimental chronic hypercholesterolemia. We have found that the development of chronic hypercholesterolemia was followed by an increase in volume density of interstitial connective tissue in the myocardium reflecting the activation of fibroplastic processes. A slight positive correlation was observed between the connective tissue density in the myocardium and expression of osteopontin mRNA (r=0.408) and lumican mRNA (r=0.470). Myocardium remodeling during hypercholesterolemia is realized against the background of increased expression of apolipoproteins E and A-IV mRNA and microsomal triglyceride transfer protein mRNA involved in transport and metabolism of lipoproteins in several tissues and probably play a pivotal role in the regulation of lipoprotein transport and metabolism in the myocardium. We concluded that the increase in the expression of apolipoproteins (E and A-IV) and microsomal triglyceride transfer protein play adaptive and compensatory role and is related to the increase in lipoprotein utilization by macrophages.

Small leucine-rich proteoglycans in the vertebrae of Atlantic salmon Salmo salar

We analysed the distribution and expression of the small leucine-rich proteoglycans (SLRPs) decorin, biglycan and lumican in vertebral columns of Atlantic salmon Salmo salar L. with and without radiographically detectable deformities. Vertebral deformities are a reoccurring problem in salmon and other intensively farmed species, and an understanding of the components involved in the pathologic development of the vertebrae is important in order to find adequate solutions to this problem. Using immunohistology and light microscopy, we found that in non-deformed vertebrae biglycan, lumican and decorin were all expressed in osteoblasts at the vertebral growth zones and at the ossification front of the chondrocytic arches. Hence, the SLRPs are expressed in regions where intramembranous and endochondral ossification take place. In addition, mRNA expression of biglycan, decorin and lumican was demonstrated in a primary osteoblast culture established from Atlantic salmon, supporting the in vivo findings. Transcription of the SLRPs increased during differentiation of the osteoblasts in vitro and where lumican mRNA expression increased later in the differentiation compared with decorin and biglycan. Intriguingly, in vertebral fusions, biglycan, decorin and lumican protein expression was extended to trans-differentiating cells at the border between arch centra and osteoblast growth zones. In addition, mRNA expression of biglycan, decorin and lumican differed between non-deformed and fused vertebrae, as shown by quantitative PCR (qPCR). Western blotting revealed an additional band of biglycan in fused vertebrae which had a higher molecular weight than in non-deformed vertebrae. Fourier-transform infrared (FTIR) spectroscopy revealed more spectral focality in the endplates of vertebral fusions and significantly more non-reducible collagen crosslinks compared with non-deformed vertebrae, thus identifying differences in bone structure.

Lumican is increased in experimental and clinical heart failure, and its production by cardiac fibroblasts is induced by mechanical and proinflammatory stimuli

During progression to heart failure (HF), myocardial extracellular matrix (ECM) alterations and tissue inflammation are central. Lumican is an ECM-localized proteoglycan associated with inflammatory conditions and known to bind collagens. We hypothesized that lumican plays a role in the dynamic alterations in cardiac ECM during development of HF. Thus, we examined left ventricular cardiac lumican in a mouse model of pressure overload and in HF patients, and investigated expression, regulation and effects of increased lumican in cardiac fibroblasts. After 4weeks of aortic banding, mice were divided into groups of hypertrophy (AB) and HF (ABHF) based on lung weight and left atrial diameter. Sham-operated mice were used as controls. Accordingly, cardiac lumican mRNA and protein levels were increased in mice with ABHF. Similarly, cardiac biopsies from patients with end-stage HF revealed increased lumican mRNA and protein levels compared with control hearts. Invitro, mechanical stretch and the proinflammatory cytokine interleukin-1β increased lumican mRNA as well as secreted lumican protein from cardiac fibroblasts. Stimulation with recombinant glycosylated lumican increased collagen typeI alpha2, lysyl oxidase and transforming growth factor-β1 mRNA, which was attenuated by costimulation with an inhibitor of the proinflammatory transcription factor NFκB. Furthermore, lumican increased the levels of the dimeric form of collagen type I, decreased the activity of the collagen-degrading enzyme matrix metalloproteinase-9 and increased the phosphorylation of fibrosis-inducing SMAD3. In conclusion, cardiac lumican is increased in experimental and clinical HF. Inflammation and mechanical stimuli induce lumican production by cardiac fibroblasts and increased lumican altered molecules important for cardiac remodeling and fibrosis in cardiac fibroblasts, indicating a role in HF development.

Lumican expression in pancreatic ductal adenocarcinoma.

The present study was aimed to investigate lumican expression in and correlation with severity of pancreatic ductal adenocarcinoma (PDA). We assessed mRNA expression and protein localization (using immunohistochemistry) in PDA samples collected from 260 patients. Additionally, we compared lumican expression with expression of Ki-67, VEGF and mutated p53 proteins, which are markers of cancer progression. Expression levels of lumican mRNA and protein in cancer tissue were significantly higher than those in tumor-adjacent tissue (t=5.69, p<0.05). The stromal expression of lumican in poorly differentiated cases was significantly higher at stage T4 than stage T2-3 (χ²=21.06, p<0.05); similarly, the stromal expression of lumican was significantly higher in TNM stage III-IV than in stage I-Il (χ²=17.01, p<0.05). Additionally, expression of Ki67 was higher in poorly differentiated cases than in highly-moderately differentiated cases (χ²=13.06, p<0.05). Finally, in highly-moderately differentiated samples, stromal expression of lumican was negatively correlated with expression of Ki-67, VEGF and mutated P53 (p<0.05). Lumican expression is higher in pancreatic ductal adenocarcinoma than in tumor-adjacent tissue, and the correlation of lumican expression with TNM stage in poorly differentiated samples, in contrast with its negative correlation with expression of Ki-67, VEGF and mutated P53 mutation in highly-moderately differentiated samples.

Abstract 5531: Lumican as a novel differential diagnostic maker for Bowen's disease and actinic keratosis

Abstract BACKGROUND: Lumican is a member of the small leucine-rich proteoglycan (SLRP) family and regulates the assembly and diameter of collagen fibrils in the extracellular matrix in human tissue. Mice homozygous for a null mutation in lumican display corneal opacification and skin laxity. Recent studies have shown that lumican is expressed in various types of malignant tumors including breast, colorectal, and pancreatic cancer, and osteosarcoma and malignant melanoma, and that it plays an important role in the growth of tumors. However, little has been studied about the expression pattern and role of lumican in non-melanoma skin cancer. In this study, we examined the expression levels of lumican in in situ lesions of squamous cell carcinoma (SCC) of the skin, actinic keraotsis and Bowen's disease, to discover whether lumican can be used as a novel differential diagnostic marker for such skin tumors, particularly for bowenoid actinic keratosis and Bowen's disease in sun-exposed areas of the body. Bowenoid actinic keratosis and Bowen's disease are often difficult to distinguish in both the clinical setting and commonly prepared pathological specimens. Furthermore, invasive SCC arising in actinic keratosis is considered low-risk clinically, while Bowen's disease with invasion is considered high-risk. Thus, it is important to distinguish between actinic keratosis and Bowen's disease, and reliable differential markers for these diseases are required.METHOD: Twenty-nine cases of actinic keratosis, including 4 of the bowenoid sub-type, and 37 cases of Bowen's disease, including 6 in sun-exposed areas of the body, were selected from among patients undergoing surgical resection at the Department of Dermatology at the Nippon Medical School from 2006 to 2010. We evaluated lumican expression and mRNA in these cases using immunohistochemistry and in situ hybridization analysis, respectively. RESULTS: Immunohistochemical analysis indicated that Lumican was expressed in 34 of 37 patients with Bowen's disease (91.8%), but was not detected in any of the cases of actinic keratosis. In situ hybridization analysis revealed that lumican mRNA was expressed in the cytoplasm of immunohistochemically lumican-positive tumor cells in Bowen's disease. CONCLUSION: These findings suggest that lumican plays an important role in the pathogenesis of Bowen's disease and will be a useful marker for the differential diagnosis between Bowen's disease and actinic keratosis, particularly for bowenoid actinic keratosis and Bowen's disease occurring in sun-exposed areas of the body. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5531. doi:1538-7445.AM2012-5531

Self-assembled matrix by umbilical cord stem cells.

Corneal integrity is critical for vision. Corneal wounds frequently heal with scarring that impairs vision. Recently, human umbilical cord mesenchymal stem cells (cord stem cells) have been investigated for tissue engineering and therapy due to their availability and differentiation potential. In this study, we used cord stem cells in a 3-dimensional (3D) stroma-like model to observe extracellular matrix organization, with human corneal fibroblasts acting as a control. For 4 weeks, the cells were stimulated with a stable Vitamin C (VitC) derivative ±TGF-β1. After 4 weeks, the mean thickness of the constructs was ∼30 μm; however, cord stem cell constructs had 50% less cells per unit volume, indicating the formation of a dense matrix. We found minimal change in decorin and lumican mRNA, and a significant increase in perlecan mRNA in the presence of TGF-β1. Keratocan on the other hand decreased with TGF-β1 in both cell lineages. With both cell types, the constructs possessed aligned collagen fibrils and associated glycosaminoglycans. Fibril diameters did not change with TGF-β1 stimulation or cell lineage; however, highly sulfated glycosaminoglycans associated with the collagen fibrils significantly increased with TGF-β1. Overall, we have shown that cord stem cells can secrete their own extracellular matrix and promote the deposition and sulfation of various proteoglycans. Furthermore, these cells are at least comparable to commonly used corneal fibroblasts and present an alternative for the 3D in vitro tissue engineered model.

Expression of lumican and fibromodulin following interleukin-1 beta stimulation of disc cells of the human temporomandibular joint

Small leucine-rich repeat proteoglycans (SLRP) are present in the extracellular matrix of the temporomandibular joint (TMJ) disc. Lumican and fibromodulin, classified as class 2 SLRPs, play important roles in TMJ assembly, proliferation and inflammation. Degenerative change in the TMJ disc gives rise to the process of internal derangement (ID). In this study, we immunohistochemically examined the expression of lumican and fibromodulin in nine human TMJ specimens and examined the gene expression of both proteoglycans in cultured human TMJ disc cells under interleukin-1 beta (IL-1 β)-stimulated conditions. An articular disc cell line was established by collagenase treatment of a TMJ disc. The subcultured cells were then incubated for 1, 3, 6, 12, 24 or 48 h under both normal and IL-1 β (1 ng/mL) conditions. The gene expression of lumican and fibromodulin was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We demonstrated that the expression of lumican significantly differs from that of fibromodulin in the deformed disc and that IL-1 β induces a significant increase in lumican mRNA, but not in fibromodulin mRNA, after 24∼48 h culture compared to cells cultured in the absence of IL-1 β (P<0.05). These results indicate that lumican and fibromodulin display different behaviors and that lumican may promote regeneration of the TMJ after degeneration and deformation induced by IL-1 β.

Abstract 2264: Lumican as a new target to develop molecularly-targeted drugs for pancreatic cancer

Abstract Background: Lumican is a member of a small leucine-rich proteoglycan family and its overexpression has been reported in breast, colorectal, neuroendocrine cell, uterine cervical and pancreatic cancers. The expression of lumican in stromal tissues in breast cancer is associated with a high tumor grade, a low estrogen receptor expression level, and young age. Lumican expression in the cytoplasm in advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic cancer, but the role of lumican in pancreatic cancer is still not well understood. In this study, we clarified the role of lumican in pancreatic cancer. Methods: Immunohistochemical analysis using anti-lumican antibody was performed in 65 pancreatic cancer patients. Lumican mRNA and protein were examined in pancreatic cancer cell lines (i.e. PANC-1, MIA PaCa-2, KLM-1) by real-time PCR and western blot. Lumican stably transfected PANC-1 cells were prepared. Then, the growth rates in vitro and in vivo were compared with mock cells. The lumican expression of PANC-1 cell was knocked down using siRNA. Then, the growth rate in vitro was compared with the control cell. Results: Immunohistochemically, lumican was localized in the cytoplasm of cancer cells in 33 of 65 patients (51%). Lumican expression in cancer cells did not correlated with clinicopathological factors. However, the survival rate of patients with lumican-positive cancer cells was significantly lower than those with lumican-negative cancer cells in the surgery-only group. Lumican mRNA and the protein were detected in all pancreatic cancer cell lines and PANC-1 cells expressed median level of lumican in the cancer cell lines examined. The growth rate of lumican-transfected PANC-1 cells was significantly higher than that of mock cells both in vitro and in vivo. Moreover, the growth rate of lumican-knocked down PANC-1 cell was significantly lower than that of the control cell. Conclusion: Lumican might regulate pancreatic cancer cell growth and become a new target to develop anti-cancer drug for pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2264.

Equine lumican (LUM) cDNA sequence and spatio‐temporal expression in an experimental model of normal and pathological wound healing

The development of exuberant granulation tissue, a situation that in some ways resembles the human keloid, compromises both the aesthetic and functional outcomes of wound repair in horses. To help elucidate the underlying molecular mechanisms the spatio-temporal expression of lumican (LUM) mRNA and protein for their potential contributions to tissue remodelling of body and limb wounds, was examined in an established experimental model. Expression was studied in intact skin and in samples of 1-, 2-, 3-, 4- and 6-week-old wounds of the body and forelimb. Temporal gene expression was determined by reverse transcriptase polymerase chain reaction, and protein expression was mapped immunohistochemically. A significant increase in LUM mRNA expression was observed in response to wounding at both anatomical locations, and a significantly higher mRNA level was recorded in thoracic than in limb wounds at weeks 1, 3 and 6 of repair. The immunohistochemical observations partially corroborated the mRNA data. To the authors' knowledge this study is the first to document that the cDNA for LUM is expressed over the different phases of wound repair in horses and suggests that LUM might be involved in both inflammation and remodelling in response to dermal injury. Further studies are now required to verify and quantify the temporal expression of this protein to provide the basis for targeted therapies that might prevent the development of exuberant granulation tissue in horse wound repair.

M1747 Knockout of KLF11, a Tumor Suppressor for Pancreatic Cancer, Displays An Abnormal Glucose Metabolism

negative cancer cells. Lumican mRNA and the protein were detected in all cancer cell lines. The phosphorylation of AKT, known as an important factor in cellular behaviors such as growth, survival, and drug resistance, was up-regulated in lumican knocked down cancer cells. Conclusion: Lumican in cancer cells might inhibit AKT signal pathway and play important roles in effectiveness of anti-cancer drugs in pancreatic cancer patients. It may be a novel candidate for anti-cancer drug via AKT signal pathway.