Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly.

The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the ß and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease.

Interclass small leucine-rich repeat proteoglycan interactions regulate collagen fibrillogenesis and corneal stromal assembly.

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn(-/0)/Lum(-/-) mouse model and single Lum(-/-) or Bgn(-/0) mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn(-/0)/Lum(-/-) corneas demonstrated gross opacity that was not seen in the Bgn(-/0) or wild type corneas and greater than that in the Lum(-/-) mice. The Bgn(-/0)/Lum(-/-) corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum(-/-) and no opacity in Bgn(-/0) or wild type corneas. In the Bgn(-/0)/Lum(-/-) corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn(-/0) or wild type corneas and was restricted to the posterior stroma in Lum(-/-) mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.

Ablation of keratan sulfate accelerates early phase pathogenesis of ALS.

Biopolymers consist of three major classes, i.e., polynucleotides (DNA, RNA), polypeptides (proteins) and polysaccharides (sugar chains). It is widely accepted that polynucleotides and polypeptides play fundamental roles in the pathogenesis of neurodegenerative diseases. But, sugar chains have been poorly studied in this process, and their biological/clinical significance remains largely unexplored. Amyotrophic lateral sclerosis (ALS) is a motoneuron-degenerative disease, the pathogenesis of which requires both cell autonomous and non-cell autonomous processes. Here, we investigated the role of keratan sulfate (KS), a sulfated long sugar chain of proteoglycan, in ALS pathogenesis. We employed ALS model SOD1(G93A) mice and GlcNAc6ST-1(-/-) mice, which are KS-deficient in the central nervous system. Unexpectedly, SOD1(G93A)GlcNAc6ST-1(-/-) mice exhibited a significantly shorter lifespan than SOD1(G93A) mice and an accelerated appearance of clinical symptoms (body weight loss and decreased rotarod performance). KS expression was induced exclusively in a subpopulation of microglia in SOD1(G93A) mice, and became detectable around motoneurons in the ventral horn during the early disease phase before body weight loss. During this phase, the expression of M2 microglia markers was transiently enhanced in SOD1(G93A) mice, while this enhancement was attenuated in SOD1(G93A)GlcNAc6ST-1(-/-) mice. Consistent with this, M2 microglia were markedly less during the early disease phase in SOD1(G93A)GlcNAc6ST-1(-/-) mice. Moreover, KS expression in microglia was also detected in some human ALS cases. This study suggests that KS plays an indispensable, suppressive role in the early phase pathogenesis of ALS and may represent a new target for therapeutic intervention.

Lumican expression, localization and antitumor activity in prostate cancer.

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Lumican regulates ventilation-induced epithelial-mesenchymal transition through extracelluar signal-regulated kinase pathway.

BACKGROUND: Mechanical ventilation used in patients with acute lung injury can damage pulmonary epithelial cells through production of inflammatory cytokines and excess deposition of the extracellular matrix protein lumican. Lumican participates in macrophage inflammatory protein (MIP)-2 and transforming growth factor-ß1 (TGF-ß1) signaling during the fibroproliferative phase of acute lung injury, which involves a process of epithelial-mesenchymal transition (EMT). The mechanisms regulating interactions between mechanical ventilation and lung injury are unclear. We hypothesized that lung damage and EMT by high tidal volume (Vt) mechanical stretch causes upregulation of lumican that modulates MIP-2 and TGF-ß1 through the extracellular signal-regulated kinase (ERK) 1/2 pathway. METHODS: Male C57BL/6 mice (either wild type or lumican null) aged 3 months and weighing between 25 and 30 g were exposed to low Vt (6 mL/kg) or high Vt (30 mL/kg) mechanical ventilation with room air for 2 to 8 h. Nonventilated mice were used as control subjects. RESULTS: We found that high Vt mechanical ventilation increased microvascular permeability, neutrophil influx, production of free radicals, MIP-2 and TGF-ß1 proteins, positive staining of α-smooth muscle actin and S100A4/fibroblast-specific protein-1, Masson trichrome staining and extracellular collagen, and activation of lumican and ERK1/2 in wild-type mice. Decreased staining of the epithelial marker E-cadherin was also observed. Mechanical stretch-augmented EMT was attenuated with lumican-deficient mice and pharmacologic inhibition of ERK1/2 activity by PD98059. CONCLUSIONS: The data suggest that lumican promotes high Vt mechanical ventilation-induced lung injury and EMT through the activation of the ERK1/2 pathway.

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo.

Keratan sulfate (KS) proteoglycan side chains are abundant in the human cartilage matrix, but these chains have been said to be absent in murine skeletal tissues. We previously showed that KS suppresses cartilage damage and ameliorates inflammation in mice arthritis model. Because mice deficient of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) (KS biosynthesis enzyme) are now available, we decided to do further examinations. We examined, in culture, the difference between GlcNAc6ST-1(-/-) and wild-type (WT) mice for interleukin (IL)-1α-induced glycosaminoglycan (GAG) release from the articular cartilage. Arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail and subsequent intraperitoneal injection of lipopolysaccharide. We examined the differences in arthritis severities in the two genotypes. After intraperitoneal KS administration in phosphate-buffered saline (PBS) or PBS alone, we evaluated the potential of KS in ameliorating arthritis and protecting against cartilage damage in deficient mice. GAG release induced by IL-1α in the explants, and severity of arthritis were greater in GlcNAc6ST-1(-/-) mice than their WT littermates. Intraperitoneal KS administration effectively suppressed arthritis induction in GlcNAc6ST-1(-/-) mice. Thus, GlcNAc6ST-1(-/-) mice cartilage is more fragile than WT mice cartilage, and exogenous KS can suppress arthritis induction in GlcNAc6ST-1(-/-) mice. Vestigial KS chain or altered glycosylation in articular cartilage in GlcNAc6ST-1(-/-) mice may be protective against arthritis and associated cartilage damage as well as cartilage damage in culture. KS may offer therapeutic opportunities for chondroprotection and suppression of joint damage in inflammatory arthritis and may become a therapeutic agent for treating rheumatoid arthritis.

Proteome profiling of wild type and lumican-deficient mouse corneas.

To elucidate how the deficiency of a major corneal proteoglycan, lumican, affects corneal homeostasis, we used mass spectrometry to derive the proteome profile of the lumican-deficient and the heterozygous mouse corneas and compared these to the wild type corneal proteome. 2108 proteins were quantified in the mouse cornea. Selected proteins and transcripts were investigated by Western blot and quantitative RT-PCR, respectively. We observed major changes in the composition of the stromal extracellular matrix (ECM) proteins in the lumican-deficient mice. Lumican deficiency altered cellular proteins in the stroma and the corneal epithelium. The ECM changes included increases in fibril forming collagen type I, Collagen type VI, fibromodulin, perlecan, laminin ß2, collagen type IV, nidogen/entactin and anchoring collagen type VII in the Lum⁺/⁻ and the Lum⁻/⁻ mouse corneas, while the stromal proteoglycans decorin, biglycan and keratocan were decreased in the Lum⁻/⁻( corneas. Cellular protein changes included increases in alcohol dehydrogenase, superoxide dismutase and decreases in epithelial cytokeratins 8 and 14. We also detected proteins that are novel to the cornea. The proteomes will provide an insight into the lumican-deficient corneal phenotype of stromal thinning and loss of transparency and a better understanding of pathogenic changes in corneal and ocular dystrophies.

Impaired skin wound healing in lumican-null mice.

BACKGROUND: Previous studies have demonstrated that the lack of lumican delayed corneal wound healing in lumican-null (Lum(-/-) ) mice. This defect is rescued by the addition of glycosylated lumican core protein to the injured corneas. OBJECTIVES: We examined the hypothesis that lumican is also required for the healing of cutaneous wounds using Lum(-/-) mice. METHODS: We demonstrated the basic thinner skin phenotypes in Lum(-/-) mice at different time points and the changes in arrangement of collagen fibres by transmission electron microscopy (TEM). A full skin thickness wound was generated by punch biopsy (6 mm diameter) in experimental Lum(-/-) and wild-type mice. The closure of injured skin was measured after various periods of time (3, 6, 12, 18 days). Specimens of injured and uninjured skin (serving as control) were then subjected to morphological examination with haematoxylin and eosin and Masson trichrome stains, and by TEM. Immunohistochemical staining with anti-CD68 antibody was used to assess the presence of macrophages in injured skin healing for various periods of time. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to elucidate the transforming growth factor (TGF)-ß1-induced myofibroblast phenotypic genes. RESULTS: Skin of adult Lum(-/-) mice (3 months and older) was much thinner (40% less) than that of age-matched wild-type mice. This phenomenon was aggravated in older mice. TEM revealed disoriented and irregular collagen fibrils in the dermis of Lum(-/-) mice. Delayed wound healing with an increase in inflammatory macrophages was compatible with the delayed response of the expression of TGF-ß1, type I collagen α1 and fibronectin at the mRNA level by semiquantitative RT-PCR in the Lum(-/-) mice. CONCLUSIONS: Our data demonstrate that lumican plays pivotal roles in skin collagen fibrillogenesis and wound healing.

Knockdown of zebrafish lumican gene (zlum) causes scleral thinning and increased size of scleral coats.

The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans approximately 4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5'-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5'-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.

Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.

BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Targeted expression of a lumican transgene rescues corneal deficiencies in lumican-null mice.

PURPOSE: To investigate whether targeted expression of lumican in the mouse cornea rescued the Lum(-/-) phenotype. METHODS: Lum(-/-)/Kera-Lum mice were generated by crossing Lum(-/-) mice with Kera-Lum transgenic mice that overexpressed lumican under the control of the keratocan promoter. Mouse eyes were analyzed in vivo by confocal microscopy through focusing (CMTF) to determine corneal sublayer thickness and haze. Subsequently, one cornea from each mouse was processed for SDS-PAGE/western blotting while the other was used for either electron microscopy (EM) or real-time polymerase chain reaction (RT-PCR). RESULTS: Overall, corneas of Lum(-/-)/Kera-Lum mice showed significant improvement over Lum(-/-) but were still deficient when compared to wildtype (WT) mice. Specifically, analysis of Lum(-/-)/Kera-Lum mouse eyes by CMTF showed a similar stromal but slightly increased epithelial thickness compared to matching Lum(-/-) mice. Analysis of the CMTF scans for light backscattering revealed a small yet significant reduction in corneal haze in Lum(-/-)/Kera-Lum mice as compared to Lum(-/-) mice. At the EM level, the pronounced disarray of the posterior fibrillar matrix seen in Lum(-/-) mice was not observed in Lum(-/-)/Kera-Lum mice. Moreover, analyses of collagen fibril diameter distributions showed a significant reduction in the number of large-diameter (>40 nm) fibrils in Lum(-/-)/Kera-Lum mice as compared to Lum(-/-) mice. No significant differences in keratocan expression were found at the mRNA level, but western blot analysis detected an approximately twofold increase in keratocan protein levels in Lum(-/-)/Kera-Lum over Lum(-/-) mice. CONCLUSIONS: Together these data suggest that despite the low keratocan promoter activity driving the transgene in Lum(-/-) cornea, transgenic lumican expression was sufficient to partially rescue corneal phenotypic deficiencies.

A novel role of the lumican core protein in bacterial lipopolysaccharide-induced innate immune response.

Lumican is an extracellular matrix protein modified as a proteoglycan in some tissues. The core protein with leucine-rich repeats, characteristic of the leucine-rich-repeat superfamily, binds collagen fibrils and regulates its structure. In addition, we believe that lumican sequestered in the pericellular matrix interacts with cell surface proteins for specific cellular functions. Here we show that bacterial lipopolysaccharide sensing by the Toll-like receptor 4 signaling pathway and innate immune response is regulated by lumican. Primary cultures of lumican-deficient (Lum(-/-)) macrophages show impaired innate immune response to lipopolysaccharides with lower induction of tumor necrosis factor alpha (TNFalpha) and interleukin-6. Macrophage response to other pathogen-associated molecular patterns is not adversely affected by lumican deficiency, suggesting a specific role for the lumican core protein in the Toll-like receptor 4 pathway. An exogenous recombinant lumican core protein increases lipopolysaccharide-mediated TNFalpha induction and partially rescues innate immune response in Lum(-/-) macrophages. We further show that the core protein binds lipopolysaccharide. Immunoprecipitation of lumican from peritoneal lavage co-precipitates CD14, a cell surface lipopolysaccharide-binding protein that is involved in its presentation to Toll-like receptor 4. The Lum(-/-) mice are hypo-responsive to lipopolysaccharide-induced septic shock, with poor induction of pro-inflammatory cytokines, TNFalpha, and interleukins 1beta and 6 in the serum. Taken together, the data indicates a novel role for lumican in the presentation of bacterial lipopolysaccharide to CD14 and host response to this bacterial endotoxin.

Collagen fibril assembly during postnatal development and dysfunctional regulation in the lumican-deficient murine cornea.

The transparent cornea is the outer barrier of the eye and is its major refractive surface. Development of a functional cornea requires a postnatal maturation phase involving development, growth and organization of the stromal extracellular matrix. Lumican, a leucine-rich proteoglycan, is implicated in regulating assembly of collagen fibrils and the highly organized extracellular matrix essential for corneal transparency. We investigated the regulatory role(s) of lumican in fibril assembly during postnatal corneal development using wild type (Lum+/+) and lumican-null (Lum-/-) mice. In Lum+/+ mice, a regular architecture of small-diameter fibrils is achieved in the anterior stroma by postnatal day 10 (P10), while the posterior stroma takes longer to reach this developmental maturity. Thus, the anterior and the posterior stroma follow distinct developmental timelines and may be under different regulatory mechanisms. In Lum-/- mice, it is the posterior stroma where abnormal lateral associations of fibrils and thicker fibrils with irregular contours are evident as early as P10. In contrast, the anterior stroma is minimally perturbed by the absence of lumican. In Lum+/+ mice, lumican is expressed throughout the developing stroma at P10, with strong expression limited to the posterior stroma in the adult. Therefore, the posterior stroma, which is most vulnerable to lumican-deficiency, demonstrates an early developmental defect in fibril structure and architecture in the Lum-/- mouse. These defects underlie the reported increased light scattering and opacity detectable in the adult. Our findings emphasize the early regulation of collagen structure by lumican during postnatal development of the cornea.

Neonatal development of the corneal stroma in wild-type and lumican-null mice.

PURPOSE: Between days 8 and 14 of neonatal development, the corneal stroma of the mouse undergoes critical changes in tissue thickness, cell density, and light scattering. The authors investigate the stromal matrix structure in wild-type and lumican-deficient corneas in this developmental phase. METHODS: Wild-type (n = 44) and lumican-deficient (n = 42) mouse corneas at neonatal days 8, 10, 12, and 14 were investigated by synchrotron x-ray diffraction to establish the average collagen fibril spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal matrix. RESULTS: Collagen interfibrillar spacing in the normal mouse cornea became more closely packed between days 8 and 14, though not significantly so. In lumican-null mice, interfibrillar spacing was significantly elevated at days 8, 10, and 12, but not day 14, compared with that in wild-type mice. At all stages investigated, collagen fibrils were, on average, marginally thinner than normal in lumican-null mutants, and the spatial distribution of the fibrils was less well organized. CONCLUSIONS: Transient thickening of the corneal stroma of the normal mouse at eye opening is probably not caused by widespread, homogeneous rearrangement of collagen fibrils but more likely by a temporary increase in cell or stromal "lake" volume. Lumican, structurally influential in adult mouse corneas, is also a key molecule in the neonatal development of the stromal matrix.

Neonatal corneal stromal development in the normal and lumican-deficient mouse.

PURPOSE: The purpose of this study was to characterize temporally stromal growth and transparency in lumican-deficient and normal neonatal mice. METHODS: Lumican-deficient mice and CD1 wild-type mice were evaluated by in vivo confocal microscopy through-focusing (CMTF) to quantify stromal and epithelial thickness and corneal light-scattering and by laser scanning CM to determine density of keratocytes from 1 day to 12 weeks after birth. RESULTS: CD1 corneas showed a rapid loss of light-scattering, decreasing by 50% from day 1 to day 12, that paralleled a 60% decrease in density of keratocytes. By contrast, the stroma demonstrated a marked swelling from day 8 to day 12, followed by thinning at day 14. Compared to corneas from CD1 mice, lumican-deficient corneas showed significantly increased (P < 0.05) light-scattering beginning at week 3 that remained elevated above wild-type levels for the duration of the study. Stromal development was also markedly altered, with thinning detected at week 3, followed by no detectable stromal growth for the duration of the study. Density of keratocytes was significantly increased, but the total cell number was similar compared with that in the wild-type cornea, suggesting no effect on keratocyte differentiation. CONCLUSIONS: Development of normal neonatal corneal transparency appears related to changes in density of keratocytes. The stroma, however, undergoes a marked swelling and thinning at the time of eyelid opening (days 8-14). In the lumican-deficient mouse, stromal swelling is abolished, indicating that this critical phase in stromal development is lumican dependent and essential for normal stromal growth and maintenance of stromal transparency.

Altered collagen fibril formation in the sclera of lumican-deficient mice.

PURPOSE: To better understand the role of lumican (corneal keratan sulfate proteoglycan) in the scleral extracellular matrix, collagen fibril size, shape, and organization were evaluated in the sclera of wild-type mice and in mice homozygous or heterozygous for a null mutation in the lumican gene. METHODS. Anterior and posterior sclera from 6-month-old wild-type (lum+/lum+) and lumican-deficient mice (lum+/lum- and lum-/lum-) were analyzed by transmission electron microscopy. In addition, lumican was characterized in the sclera of wild-type and lumican-deficient mice by Western blot analyses. RESULTS: Lumican was present in the mouse sclera as an approximately 48-kDa core protein containing short glycosaminoglycan side chains consisting of moderate- to low-sulfated keratan sulfate. The wild-type mouse sclera consisted of irregularly arranged lamellae of collagen fibrils with an average diameter of 47.37 +/- 0.648 nm in the anterior sclera and 54.68 +/- 0.342 nm the posterior sclera. Collagen fibrils in the sclera of lumican mutant mice (lum+/lum- and lum-/lum-) were significantly larger in diameter in anterior (72.61 +/- 0.445 and 84.47 +/- 0.394 nm, respectively) and posterior (75.92 +/- 0.361 and 80.90 +/- 0.490 nm, respectively) scleral regions compared with wild-type mice (P < 0.001). CONCLUSIONS: The results of the present study indicate that null mutations in one or both alleles of the lumican gene result in significant defects in scleral collagen fibril formation that could lead to alterations in ocular shape and size and severely affect vision.

Functions of lumican and fibromodulin: lessons from knockout mice.

Lumican and fibromodulin are collagen-binding leucine-rich proteoglycans widely distributed in interstitial connective tissues. The phenotypes of lumican-null (Lum(-/-)), Fibromodulin-null (Fmod(-/-)) and compound double-null (Lum(-/-)Fmod(-/-)) mice identify a broad range of tissues where these two proteoglycans have overlapping and unique roles in modulating the extracellular matrix and cellular behavior. The lumican-deficient mice have reduced corneal transparency and skin fragility. The Lum(-/-)Fmod(-/-) mice are smaller than their wildtype littermates, display gait abnormality, joint laxity and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis and extreme tendon weakness are the likely cause for joint-laxity. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum(+/+)Fmod(-/-) mice, with further loss in stiffness in a lumican gene dose-dependent way. At the level of ultrastructure, the Lum(-/-) cornea, skin and tendon show irregular collagen fibril contours and increased fibril diameter. The Fmod(-/-) tendon contains irregular contoured collagen fibrils, with increased frequency of small diameter fibrils. The tendons of Lum(-/-)Fmod(-/-) have an abnormally high frequency of small and large diameter fibrils indicating a de-regulation of collagen fibril formation and maturation. In tissues like the tendon, where both proteoglycans are present, fibromodulin may be required early in collagen fibrillogenesis to stabilize small-diameter fibril-intermediates and lumican may be needed at a later stage, primarily to limit lateral growth of fibrils

An x-ray diffraction investigation of corneal structure in lumican-deficient mice.

PURPOSE: The corneas of mice homozygous for a null mutation in lumican, a keratan sulfate-containing proteoglycan, are not as clear as normal. In the present study, mutant corneas were examined by synchrotron x-ray diffraction to see what structural changes might lie behind the loss of transparency. METHODS: X-ray diffraction patterns were obtained from the corneas of 6-month-old and 2-month-old lumican-null and wild-type mice. Measured in each cornea were the average collagen fibril diameter, average collagen fibril spacing, and the level of order in the collagen array. RESULTS: The x-ray reflection arising from regularly packed collagen was well-defined on all x-ray patterns from 6-month-old wild-type corneas. Patterns from 6-month-old lumican-deficient corneas, however, contained interfibrillar reflections that were measurably more diffuse, a fact that points to a widespread alteration in the way the collagen fibrils are configured. The same distinction between mutant and wild-type corneas was also noted at 2-months of age. Average collagen fibril spacing was marginally higher in corneas of 6-month-old lumican-null mice than in corneas of normal animals. Unlike x-ray patterns from wild-type corneas, patterns from lumican-deficient corneas of both ages registered no measurable subsidiary x-ray reflection, evidence of a wider than normal range of fibril diameters. CONCLUSIONS: The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice is in disarray. There is also a considerable variation in the diameter of the hydrated collagen fibrils. These abnormalities, seen at 2 months as well as 6 months of age, probably contribute to the reduced transparency.

Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons.

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma.

PURPOSE: Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects. METHODS: Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice. CONCLUSIONS: The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.