The synergistic effect of NELL1 and adipose-derived stem cells on promoting bone formation in osteogenesis imperfecta treatment.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by bone fragility and deformity. Mesenchymal stem cells (MSCs) infusion can improve bone performance mainly due to their differentiation into osteoblasts in OI therapy. The osteoinductive activity of NELL1 have benefited various bone defect and osteoporotic models by promoting bone formation. The present study investigated the efficacy of combined use of NELL1 and adipose-derived mesenchymal stem cells (ADSCs) in OI treatment. METHODS: Lentiviral vector carrying mouse Nell1 gene was constructed and lentivirus were used to infect ADSCs. The osteogenic capacity of MC3T3-E1 and ADSCs stimulated by recombinant mouse NELL1 protein (rmNELL1) and Nell1 gene genetically modified ADSCs (lenti-Nell1-ADSCs) were estimated by real-time quantitative PCR. Thirty adult male OI type I mice with single Col1a1 gene knockout were randomly divided into five groups and received intravenously injected PBS, rmNELL1 (1.25 mg/Kg), ADSCs (2 × 105 cells per mice), rmNELL1 (1.25 mg/Kg) combined with ADSCs (2 × 105 cells per mice), or lenti-Nell1-ADSCs (2 × 105 cells per mice) respectively. Six wildtype (WT) mice served as positive control. Bone formation was examined after 4 weeks using micro-CT, histological and immunohistochemical methods. RESULTS: Three osteoblast related genes of MC3T3-E1 and ADSCs were significantly up-regulated by rmNELL1 in vitro. Lenti-Nell1-ADSCs showed greatly enhanced osteogenic differentiation capacity. The infused lenti-Nell1-ADSCs could migrate to femur and differentiate into ALPL-positive cells. Systemic administration of rmNELL1 combined with ADSCs or lenti-Nell1-ADSCs markedly improved the femoral microstructure and promoted bone formation through increasing the ALPL and osteocalcin (OCN) expression, much better than mice that received single rmNELL1 or ADSCs. And Nell1 gene engineered ADSCs achieved slightly better outcomes than that of combinative use of rmNELL1 and ADSCs. CONCLUSIONS: NELL1 and ADSCs exhibited synergistic effect on stimulating bone formation of OI mice, which might provide an alternative strategy in OI treatment. Compared with dose escalation or multiple administration of rmNELL1, lentivirus-mediated long term expression of NELL1 might be more feasible and convenient. However, further studies are needed to confirm the safety and optimize the therapeutic regime.

Notch signaling inhibition protects against LPS mediated osteolysis.

Chronic inflammatory responses have profound effects on the differentiation and activity of both the bone-forming osteoblasts and bone-resorbing osteoclasts. Importantly, inflammatory bone diseases characterized by clinical osteolysis promote bone resorption and decrease bone formation by uncoupling the process in favor of excess resorption. Notch signaling regulates osteoclast development and thus its manipulation has the potential to suppress resorptive potential. Here, we have utilized a genetic model of Notch inhibition in osteoclasts by expression of dnMAML to prevent formation of transcriptional complex essential for downstream Notch signaling. Using this model and LPS as a tool for experimental inflammatory osteolysis, we have demonstrated that dnMAML-expressing osteoclasts exhibited significantly lower maturation and resorption/functional potential ex vivo using TRAP staining and calcium phosphate coated surfaces. Moreover, we observed that while LPS stimulated the formation of wildtype osteoclasts pre-treated with RANKL, dnMAML expression produced resistance to osteoclast maturation after LPS stimulation. Genetically, Notch-inhibited animals showed a significantly lower TRAP and CTX-1 levels in serum after LPS treatment compared to the control groups in addition to a marked reduction in osteoclast surfaces in calvaria sections. This report provides evidence for modulation of Notch signaling activity to protect against inflammatory osteolysis. Taken together, the findings of this study will help guide the development of Notch signaling-based therapeutic approaches to prevent bone loss.

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

Oligosaccharide modification by N-acetylglucosaminyltransferase-V in macrophages are involved in pathogenesis of bleomycin-induced scleroderma.

Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), which catalyses the formation of ß1,6 GlcNAc (N-acetylglucosamine) branches on N-glycans, is associated with various pathologies, such as cancer metastasis, multiple sclerosis and liver fibrosis. In this study, we demonstrated the involvement of GnT-V in the pathophysiology of scleroderma. High expression of GnT-V was observed in infiltrating cells in skin section samples from systemic and localized patients with scleroderma. Most of the infiltrating cells were T cells and macrophages, most of which were CD163(+) M2 macrophages. To determine the role of GnT-V in scleroderma, we next investigated skin sclerosis in GnT-V knockout (MGAT5(-/-) ) mice. Expression of GnT-V was also elevated in bleomycin (BLM)-injected sclerotic skin, and MGAT5(-/-) mice were resistant to BLM-induced skin sclerosis with reduced collagen type 1 α1 content, suggesting the biological significance of GnT-V in skin sclerosis. Furthermore, the number of CD163(+) M2 macrophages and CD3-positive T cells in BLM-induced skin sclerosis was significantly fewer in MGAT5(-/-) mice. In bone marrow-derived macrophages (BMDMs), IL-4-induced expressions of Fizz1 and Ym1 were significantly reduced in MGAT5(-/-) mice-derived BMDMs. Taken together, these results suggest the induction of GnT-V in skin sclerosis progression is possibly dependent on increased numbers of M2 macrophages in the skin, which are important for tissue fibrosis and remodelling.

Fibrocytes are not an essential source of type I collagen during lung fibrosis.

Progressive fibrosis involves accumulation of activated collagen-producing mesenchymal cells. Fibrocytes are hematopoietic-derived cells with mesenchymal features that potentially have a unique and critical function during fibrosis. Fibrocytes have been proposed as an important direct contributor of type I collagen deposition during fibrosis based largely on fate-mapping studies. To determine the functional contribution of hematopoietic cell-derived type I collagen to fibrogenesis, we use a double-transgenic system to specifically delete the type I collagen gene across a broad population of hematopoietic cells. These mice develop a robust fibrotic response similar to littermate genotype control mice injured with bleomycin indicating that fibrocytes are not a necessary source of type I collagen. Using collagen-promoter GFP mice, we find that fibrocytes express type I collagen. However, fibrocytes with confirmed deletion of the type I collagen gene have readily detectable intracellular type I collagen indicating that uptake of collagen from neighboring cells account for much of the fibrocyte collagen. Collectively, these results clarify several seemingly conflicting reports regarding the direct contribution of fibrocytes to collagen deposition.

Upregulating CXCR4 in human fetal mesenchymal stem cells enhances engraftment and bone mechanics in a mouse model of osteogenesis imperfecta.

Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4(+) cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.

High- and low-dose OPG-Fc cause osteopetrosis-like changes in infant mice.

BACKGROUND: Receptor activator of nuclear factor-κB ligand (RANKL) inhibitors are being considered for use in children with osteogenesis imperfecta (OI). We sought to assess efficacy of two doses of a RANKL inhibitor, osteoprotegerin-immunoglobulin Fc segment complex (OPG-Fc), in a growing animal model of OI, the col1α2-deficient mouse (oim/oim) and its wild-type controls (+/+). METHODS: Treated mice showed runting and radiographic evidence of osteopetrosis with either high- (20 mg/kg twice weekly) or low-dose (1 mg/kg/week) OPG-Fc. Because of this adverse event, OPG-Fc treatment was halted, and the mice were killed or monitored for recovery with monthly radiographs and assessment of serum osteoclast activity (tartrate-resistant acid phosphatase 5b, TRACP-5b) until 25 wk of age. RESULTS: Twelve weeks of OPG-Fc treatment resulted in radiographic and histologic osteopetrosis with no evidence of bone modeling and negative tartrate-resistant acid phosphatase staining, root dentin abnormalities, and TRACP-5b activity suppression. Signs of recovery appeared 4-8 wk post-treatment. CONCLUSION: Both high- and low-dose OPG-Fc treatment resulted in osteopetrotic changes in infant mice, an outcome that was not seen in studies with the RANKL inhibitor RANK-immunoglobulin Fc segment complex (RANK-Fc) or in studies with older animals. Further investigations of RANKL inhibitors are necessary before their consideration for use in children.

A collagen α2(I) mutation impairs healing after experimental myocardial infarction.

Collagen breakdown and de novo synthesis are important processes during early wound healing after myocardial infarction (MI). We tested the hypothesis that collagen I, the main constituent of the extracellular matrix, affects wound healing after MI. The osteogenesis imperfecta mouse (OIM), lacking procollagen-α2(I) expression, represents a model of the type III form of the disease in humans. Homozygous (OIM/OIM), heterozygous (OIM/WT), and wild-type (WT/WT) mice were subjected to a permanent myocardial infarction protocol or sham surgery. Baseline functional and geometrical parameters determined by echocardiography did not differ between genotypes. After MI but not after sham surgery, OIM/OIM animals exhibited significantly increased mortality, due to early ventricular rupture between day 3 and 7. Echocardiography at day 1 demonstrated increased left ventricular dilation in OIM/OIM animals. Less collagen I mRNA within the infarct area was found in OIM/OIM animals. At 2 days after MI, MMP-9 expression in the infarct border zone was higher in OIM/OIM than in WT/WT animals. Increased granulocyte infiltration into the infarct border zone occurred in OIM/OIM animals. Neither granulocyte depletion nor MMP inhibition reduced mortality in OIM/OIM animals. In this murine model, deficiency of collagen I leads to a myocardial wound-healing defect. Both structural alterations within pre-existing collagen matrix and impaired collagen de novo expression contribute to a high rate of early myocardial rupture after MI.

Deficient degradation of homotrimeric type I collagen, α1(I)3 glomerulopathy in oim mice.

Col1a2-deficient (oim) mice synthesize homotrimeric type I collagen due to nonfunctional proα2(I) collagen chains. Our previous studies revealed a postnatal, progressive type I collagen glomerulopathy in this mouse model, but the mechanism of the sclerotic collagen accumulation within the renal mesangium remains unclear. The recent demonstration of the resistance of homotrimeric type I collagen to cleavage by matrix metalloproteinases (MMPs), led us to investigate the role of MMP-resistance in the glomerulosclerosis of Col1a2-deficient mice. We measured the pre- and post-translational expression of type I collagen and MMPs in glomeruli from heterozygous and homozygous animals. Both the heterotrimeric and homotrimeric isotypes of type I collagen were equally present in whole kidneys of heterozygous mice by immunohistochemistry and biochemical analysis, but the sclerotic glomerular collagen was at least 95-98% homotrimeric, suggesting homotrimeric type I collagen is the pathogenic isotype of type I collagen in glomerular disease. Although steady-state MMP and Col1a1 mRNA levels increased with the disease progression, we found these changes to be a secondary response to the deficient clearance of MMP-resistant homotrimers. Increased renal MMP expression was not sufficient to prevent homotrimeric type I collagen accumulation.

Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type I alpha 2-deficient mouse.

Fetal mesenchymal stem cell (fetal MSC) therapy has potential to treat genetic diseases with early onset, including those affecting the kidney and urinary tract. A collagen type I alpha 2-deficient mouse has a deletion in the alpha2 chain of the procollagen type I gene, resulting in the synthesis of abnormal alpha1(I)(3) homotrimers, which replace normal alpha 1(I)2 alpha 2(I)1 heterotrimers and a glomerulopathy. We first confirmed that col1 alpha 2-deficient homozygous mice show abnormal collagen deposition in the glomeruli, which increases in frequency and severity with postnatal age. Intrauterine transplantation of human MSCs from first trimester fetal blood led postnatally to a reduction of abnormal homotrimeric collagen type I deposition in the glomeruli of 4-12 week-old col1 alpha 2-deficient mice. Using bioluminescence imaging, in situ hybridization and immunohistochemistry in transplanted col1 alpha 2-deficient mice, we showed that the damaged kidneys preferentially recruited donor cells in glomeruli, around mesangial cells. Real-time RT-PCR demonstrated that this effect was seen at an engraftment level of 1% of total cells in the kidney, albeit higher in glomeruli. We conclude that intrauterine transplantation of human fetal MSCs improves renal glomerulopathy in a collagen type I-deficient mouse model. These data support the feasibility of prenatal treatment for hereditary renal diseases.

Periodontal Ehlers-Danlos syndrome associated with type III and I collagen deficiencies.

In 1997, Ehlers-Danlos syndrome type VIII (EDS-VIII) was excluded from the diagnostic categories as there were insufficient data regarding the clinical features and the precise underlying molecular basis. However, a recent review of published cases shows that EDS-VIII has distinctive clinical features, which would suggest that it should be considered as a separate entity in future nosological classifications. The underlying molecular defect in EDS-VIII is unknown. A reduction of collagen type III was reported in a single case, but no consistent biochemical or structural changes are detectable. We report a patient with EDS-VIII who showed a reduction in the collagen type I and type III synthesis rates. Type I and type III procollagen and collagen synthesis and secretion rates were investigated in cultured fibroblasts and compared with five healthy controls and three patients with EDS type IV whose fibroblasts were cultured in parallel.

Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.

BACKGROUND/AIMS: Studies in animal models and humans suggest a link between endotoxemia and non-alcoholic steatohepatitis. Since Kupffer cells are responsible for clearing endotoxin and are activated via endotoxin interaction with Toll-like receptor 4 (TLR-4), we examined the relationship between hepatic TLR-4 expression and Kupffer cell content during the genesis of steatohepatitis. METHODS: Male C57BL/6, C3H/HouJ and TLR-4 mutant C3H/HeJ mice were fed control or methionine/choline-deficient diet (MCDD). In one group of C57BL/6 mice, Kupffer cells were depleted by weekly intraperitoneal injections of clodronate liposomes. After 3 weeks, serum ALT activity and portal endotoxin levels were measured. Real-time PCR was used to examine mRNA expression of TLR-4, TLR-2, CD14, MD-2, TGFbeta, TNFalpha, CD36, PPAR-alpha, liver fatty acid binding protein (L-FABP) and collagen alpha1. RESULTS: We observed histological evidence typical of steatohepatitis, portal endotoxemia and enhanced TLR-4 expression in wild type mice fed MCDD. In contrast, injury and lipid accumulation markers were significantly lower in TLR-4 mutant mice. Destruction of Kupffer cells with clodronate liposomes blunted histological evidence of steatohepatitis and prevented increases in TLR-4 expression. CONCLUSIONS: These findings demonstrate the importance of TLR-4 signaling and underscore a direct link between TLR-4 and Kupffer cells in the pathogenesis of steatohepatitis.

Alpha 2(I) collagen deficient oim mice have altered biomechanical integrity, collagen content, and collagen crosslinking of their thoracic aorta.

Collagen and elastin are the primary determinants of vascular integrity, with elastin hypothesized to be the major contributor to aortic compliance and type I collagen the major contributor to aortic strength and stiffness. Type I collagen is normally heterotrimeric composed of two alpha1(I) and one alpha2(I) collagen chains, alpha1(I)(2)alpha2(I). Recent investigations have reported that patients with recessively inherited forms of Ehlers Danlos syndrome that fail to synthesize proalpha2(I) chains have increased risks of cardiovascular complications. To assess the role of alpha2(I) collagen in aortic integrity, we used the osteogenesis imperfecta model (oim) mouse. Oim mice, homozygous for a COL1A2 mutation, synthesize only homotrimeric type I collagen, alpha1(I)3. We evaluated thoracic aortas from 3-month-old oim, heterozygote, and wildtype mice biomechanically for circumferential breaking strength (Fmax) and stiffness (IEM), histologically for morphological differences, and biochemically for collagen content and crosslinking. Circumferential biomechanics of oim and heterozygote descending thoracic aortas demonstrated the anticipated reduced Fmax and IEM relative to wildtype mice. Histological analyses of oim descending aortas demonstrated reduced collagen staining relative to wildtype aortas suggesting decreased collagen content, which hydroxyproline analyses of ascending and descending oim aortas confirmed. These findings suggest the reduced oim thoracic aortic integrity correlates with the absence of the alpha2(I)collagen chains and in part with reduced collagen content. However, oim ascending aortas also demonstrated a significant increase in pyridinoline crosslinks/collagen molecule as compared to wildtype ascending aortas. The role of increased collagen crosslinks is uncertain; increased crosslinking may represent a compensatory mechanism for the decreased integrity.

Altered disc mechanics in mice genetically engineered for reduced type I collagen.

STUDY DESIGN: Mechanically test lumbar discs of transgenic mice in compression-tension and torsion. OBJECTIVES: Determine if a reduction in type I collagen results in decreased disc mechanics. SUMMARY OF BACKGROUND DATA: Quantitative relationships between disc structure and function would improve the understanding of disc generation and are essential relationships for functional tissue engineering. The reduced type I collagen transgenic mouse has been used in structure-function studies of bone and tendon, but not intervertebral discs. Methods for testing mouse discs have recently been developed, making disc structure-function studies possible. METHODS: Microradiographed and mechanically tested lumbar discs from control and collagen-reduced mice in both compression-tension and torsion were used. Disc area and polar moment of inertia were determined from radiographic data, stiffness from mechanical data, and apparent modulus from geometric and mechanical data. RESULTS: Collagen-reduced discs had a larger area and polar moment of inertia compared to controls. The linear and torsional stiffness of collagen-reduced and control discs were not significantly different. Finally, the apparent modulus of collagen-reduced discs was significantly less than controls in compression (73% of control) and torsion (50%). CONCLUSIONS: Compared to controls, collagen-reduced discs had reduced apparent modulus in both loading directions, suggesting that the transgenic disc tissue was mechanically inferior to controls. These results are consistent with the widely accepted functional role of type I collagen in disc mechanics, and therefore supports the use of transgenic mice to study structure-function relationships of the disc. Future work will focus on quantifying structure-function relationships related to degeneration, as well as those relevant to the design of tissue-engineered disc replacements.

Effect of altered matrix proteins on quasilinear viscoelastic properties in transgenic mouse tail tendons.

Tendons have complex mechanical behaviors that are viscoelastic, nonlinear, and anisotropic. It is widely held that these behaviors are provided for by the tissue's composition and structure. However, little data are available to quantify such structure-function relationships. This study quantified tendon mechanical behaviors, including viscoelasticity and nonlinearity, for groups of mice that were genetically engineered for altered extracellular matrix proteins. Uniaxial tensile stress-relaxation experiments were performed on tail tendon fascicles from the following groups: eight week old decorin knockout, eight week old reduced type I collagen, three week old control, and eight week old control. Data were fit using Fung's quasilinear viscoelastic model, where the model parameters represent the linear viscoelastic and nonlinear elastic response. The viscoelastic properties demonstrated a larger and faster stress relaxation for the decorin knockout and a smaller and slower stress relaxation for the three week control. The elastic parameter, A, in the eight week control group was significantly greater than in the collagen reduction and three week control groups. This study provides quantitative evidence for structure-function relationships in tendon, including the role of proteoglycan in viscoelasticity. Future studies should directly correlate composition and structure with tendon mechanics for the design and evaluation of tissue-engineered constructs or tendon repairs.

Alendronate treatment for infants with osteogenesis imperfecta: demonstration of efficacy in a mouse model.

Recent non-placebo-controlled studies of the bisphosphonate pamidronate have shown it to be effective in reducing fractures and improving bone density in infants and children with osteogenesis imperfecta (OI). To evaluate the effects of bisphosphonate treatment in a controlled study, the oim/oim mouse model of OI was studied. Nursing infant mouse pups (approximately 2 wk old) with moderate to severe OI (oim/oim mouse) and age- and background-matched control mice (+/+) were treated either with the third-generation bisphosphonate alendronate (ALN), or with saline. Fracture risk, bone quality, and growth were evaluated over a 12-wk treatment period. ALN at a dose of 0.03 mg/kg/d or saline was administered via s.c. injection to infant oim/oim and wild-type (+/+) mice from 2 to 14 wk of age (n = 20 per subgroup). The average number of fractures sustained by the ALN-treated oim/oim mice was reduced significantly compared with the untreated oim/oim mice (0.7 +/- 0.7 fractures/mouse versus 2.0 +/- 0.2 fractures/mouse). Bone density increased significantly in the femur and the spine with treatment (2.0 +/- 0.5 versus 1.2 +/- 0.5 in femur and 2.1 +/- 0.5 versus1.6 +/- 0.5 in spine). Histologic evaluation revealed the percentage of metaphyseal tibial bone increased significantly with treatment in both +/+ and oim/oim mice. Mechanical testing revealed an increase in structural stiffness for both treated +/+ and oim/oim mice compared with untreated animals. None of the material properties examined were significantly altered with treatment, nor was spinal curvature affected. Weight gain and long bone growth were comparable in the treated and untreated oim/oim mice. In wild-type mice, femur lengths were significantly shorter in the treated mice compared with untreated counterparts. This animal study demonstrates that treatment of OI in mice as early as 2 wk of age with ALN appears to be effective in reducing fractures and increasing bone properties. Based on the data from this study, ALN therapy in infants with OI should prove to be effective.

OIM and related animal models of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by fragile bones, skeletal deformity, and growth retardation. This heritable disorder of connective tissue is the result of mutations affecting the COL1A1 and COL1A2 genes of type I collagen. Progress in OI research has been limited because of dependence on human fibroblast and osteoblast specimens and the absence of a naturally occurring animal model for this genetic disorder. Recent technology in molecular biology has led to the development of transgenic models of OI based on site directed mutagenesis of type I collagen genes. OIM is a naturally occurring model which incorporates both the phenotypic and biochemical defects of moderate to severe osteogenesis imperfecta. This powerful tool permits the development of models based on different type I collagen mutations. The collagen type I mutation in OIM is a C propeptide deletion which impairs the production of normal pro-alpha2(I). Tissues in OIM contain only [pro-alpha1(I)]3 homotrimer. Thus, although several animal models are now available for research in osteogenesis imperfecta few are viable or fully mimic human disease disorders. OIM duplicates the phenotype and biochemistry of human disease and has a normal life span.