Search

Rationaleαv integrins, key regulators of transforming growth factor-β activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvβ6) and fibroblasts (αvβ1) in fibrotic lungs.ObjectivesWe evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis.MethodsSelective αvβ6 and αvβ1, dual αvβ6/αvβ1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-β cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-β signaling. Bleomycin-challenged mice treated with dual αvβ6/αvβ1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation.Measurements and main resultsInhibition of integrins αvβ6 and αvβ1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvβ6/αvβ1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone.ConclusionsIn the fibrotic lung, dual inhibition of integrins αvβ6 and αvβ1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-β.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Nonalcoholic fatty liver disease is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of nonalcoholic fatty liver disease and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease and may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants are orally administered polymers that bind bile acids in the intestine, forming nonabsorbable complexes. Bile acid sequestrants interrupt intestinal reabsorption of bile acids, decreasing their circulating levels. We determined that treatment with the bile acid sequestrant sevelamer reversed the liver injury and prevented the progression of NASH, including steatosis, inflammation, and fibrosis in a Western diet-induced NASH mouse model. Metabolomics and microbiome analysis revealed that this beneficial effect is associated with changes in the microbiota population and bile acid composition, including reversing microbiota complexity in cecum by increasing Lactobacillus and decreased Desulfovibrio The net effect of these changes was improvement in liver function and markers of liver injury and the positive effects of reversal of insulin resistance.

BackgroundBone is one of the most frequent metastatic sites of advanced breast cancer. Current therapeutic agents aim to inhibit osteoclast-mediated bone resorption but only have palliative effects. During normal bone remodeling, the balance between bone resorption and osteoblast-mediated bone formation is essential for bone homeostasis. One major function of osteoblast during bone formation is to secrete type I procollagen, which will then be processed before being crosslinked and deposited into the bone matrix.MethodsSmall RNA sequencing and quantitative real-time PCR were used to detect miRNA levels in patient blood samples and in the cell lysates as well as extracellular vesicles of parental and bone-tropic MDA-MB-231 breast cancer cells. The effects of cancer cell-derived extracellular vesicles isolated by ultracentrifugation and carrying varying levels of miR-218 were examined in osteoblasts by quantitative real-time PCR, Western blot analysis, and P1NP bone formation marker analysis. Cancer cells overexpressing miR-218 were examined by transcriptome profiling through RNA sequencing to identify intrinsic genes and pathways influenced by miR-218.ResultsWe show that circulating miR-218 is associated with breast cancer bone metastasis. Cancer-secreted miR-218 directly downregulates type I collagen in osteoblasts, whereas intracellular miR-218 in breast cancer cells regulates the expression of inhibin β subunits. Increased cancer secretion of inhibin βA results in elevated Timp3 expression in osteoblasts and the subsequent repression of procollagen processing during osteoblast differentiation.ConclusionsHere we identify a twofold function of cancer-derived miR-218, whose levels in the blood are associated with breast cancer metastasis to the bone, in the regulation of type I collagen deposition by osteoblasts. The adaptation of the bone niche mediated by miR-218 might further tilt the balance towards osteolysis, thereby facilitating other mechanisms to promote bone metastasis.

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.

Exposure to the halogenated hydrocarbon carbon tetrachloride (CCl4) leads to hepatic lipid peroxidation, inflammation and fibrosis. Dietary supplementation of ω-3 fatty acids has been increasingly advocated as being generally anti-inflammatory, though its effect in models of liver fibrosis is mixed. This raises the question of whether diets high in ω-3 fatty acids will result in a greater sensitivity or resistance to liver fibrosis as a result of environmental toxicants like CCl4. In this study, we fed CCl4-treated mice a high ω-3 diet (using a mix of docosahexaenoic acid and eicosapentaenoic acid ethyl esters). We also co-administered an inhibitor of soluble epoxide hydrolase, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which has been shown to boost anti-inflammatory epoxy fatty acids that are produced from both ω-3 and ω-6 dietary lipids. We showed that soluble epoxide inhibitors reduced CCl4-induced liver fibrosis. Three major results were obtained. First, the ω-3-enriched diet did not attenuate CCl4-induced liver fibrosis as judged by collagen deposition and collagen mRNA expression. Second, the ω-3-enriched diet raised hepatic tissue levels of several inflammatory lipoxygenase metabolites and prostaglandins, including PGE2. Third, treatment with TPPU in drinking water in conjunction with the ω-3-enriched diet resulted in a reduction in liver fibrosis compared to all other groups. Taken together, these results indicate that dietary ω-3 supplementation alone did not attenuate CCl4-induced liver fibrosis. Additionally, oxylipin signaling molecules may play role in the CCl4-induced liver fibrosis in the high ω-3 diet groups.

Background & aimsCigarette smoke has been identified as an independent risk factor for chronic pancreatitis (CP). Little is known about the mechanisms by which smoking promotes development of CP. We assessed the effects of aryl hydrocarbon receptor (AhR) ligands found in cigarette smoke on immune cell activation in humans and pancreatic fibrosis in animal models of CP.MethodsWe obtained serum samples from patients with CP treated at Stanford University hospital and healthy individuals (controls) and isolated CD4+ T cells. Levels of interleukin-22 (IL22) were measured by enzyme-linked immunosorbent assay and smoking histories were collected. T cells from healthy nonsmokers and smokers were stimulated and incubated with AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene) or antagonists and analyzed by flow cytometry. Mice were given intraperitoneal injections of caerulein or saline, with or without lipopolysaccharide, to induce CP. Some mice were given intraperitoneal injections of AhR agonists at the start of caerulein injection, with or without an antibody against IL22 (anti-IL22) starting 2 weeks after the first caerulein injection, or recombinant mouse IL22 or vehicle (control) intraperitoneally 4 weeks after the first caerulein injection. Mice were exposed to normal air or cigarette smoke for 6 h/d for 7 weeks and expression of AhR gene targets was measured. Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. Pancreatic stellate cells and T cells were isolated and studied using immunoblot, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent analyses.ResultsMice given AhR agonists developed more severe pancreatic fibrosis (based on decreased pancreas size, histology, and increased expression of fibrosis-associated genes) than mice not given agonists after caerulein injection. In mice given saline instead of caerulein, AhR ligands did not induce fibrosis. Pancreatic T cells from mice given AhR agonists and caerulein were activated and expressed IL22, but not IL17 or interferon gamma. Human T cells exposed to AhR agonists up-regulated expression of IL22. In mice given anti-IL22, pancreatic fibrosis did not progress, whereas mice given recombinant IL22 had a smaller pancreas and increased fibrosis. Pancreatic stellate cells isolated from mouse and human pancreata expressed the IL22 receptor IL22RA1. Incubation of the pancreatic stellate cells with IL22 induced their expression of the extracellular matrix genes fibronectin 1 and collagen type I α1 chain, but not α2 smooth muscle actin or transforming growth factor-β. Serum samples from smokers had significantly higher levels of IL22 than those from nonsmokers.ConclusionsAhR ligands found in cigarette smoke increase the severity of pancreatic fibrosis in mouse models of pancreatitis via up-regulation of IL22. This pathway might be targeted for treatment of CP and serve as a biomarker of disease.

BackgroundPrimary congenital glaucoma (PCG) and early onset glaucomas are one of the major causes of children and young adult blindness worldwide. Both autosomal recessive and dominant inheritance have been described with involvement of several genes including CYP1B1, FOXC1, PITX2, MYOC and PAX6. However, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes.MethodsTo elucidate further genetic causes of these conditions whole exome sequencing (WES) was performed in an Italian patient, diagnosed with PCG and retinal detachment, and his unaffected parents. Sanger sequencing of the complete coding region of COL1A1 was performed in a total of 26 further patients diagnosed with PCG or early onset glaucoma. Exclusion of pathogenic variations in known glaucoma genes as CYP1B1, MYOC, FOXC1, PITX2 and PAX6 was additionally done per Sanger sequencing and Multiple Ligation-dependent Probe Amplification (MLPA) analysis.ResultsIn the patient diagnosed with PCG and retinal detachment, analysis of WES data identified compound heterozygous variants in COL1A1 (p.Met264Leu; p.Ala1083Thr). Targeted COL1A1 screening of 26 additional patients detected three further heterozygous variants (p.Arg253*, p.Gly767Ser and p.Gly154Val) in three distinct subjects: two of them diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (OI), one patient with a diagnosis of PCG at age 4 years. All five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. Molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main COL1A1 binding proteins: Hsp47 and fibronectin.ConclusionsDominant inherited mutations in COL1A1 are known causes of connective tissues disorders such as OI. These disorders are also associated with different ocular abnormalities, although recognition of the common pathology for both features is seldom being recognized. Our results expand the role of COL1A1 mutations in different forms of early-onset glaucoma with and without signs of OI. Thus, we suggest including COL1A1 mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with OI.

Although cancer-associated fibroblasts (CAFs) are viewed as a promising therapeutic target, the design of rational therapy has been hampered by two key obstacles. First, attempts to ablate CAFs have resulted in significant toxicity because currently used biomarkers cannot effectively distinguish activated CAFs from non-cancer associated fibroblasts and mesenchymal progenitor cells. Second, it is unclear whether CAFs in different organs have different molecular and functional properties that necessitate organ-specific therapeutic designs. Our analyses uncovered COL11A1 as a highly specific biomarker of activated CAFs. Using COL11A1 as a 'seed', we identified co-expressed genes in 13 types of primary carcinoma in The Cancer Genome Atlas. We demonstrated that a molecular signature of activated CAFs is conserved in epithelial cancers regardless of organ site and transforming events within cancer cells, suggesting that targeting fibroblast activation should be effective in multiple cancers. We prioritized several potential pan-cancer therapeutic targets that are likely to have high specificity for activated CAFs and minimal toxicity in normal tissues.

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.

During the progression of pulmonary hypertension (PH), proximal pulmonary arteries (PAs) increase in both thickness and stiffness. Collagen, a component of the extracellular matrix, is mainly responsible for these changes via increased collagen fiber amount (or content) and crosslinking. We sought to differentiate the effects of collagen content and cross-linking on mouse PA mechanical changes using a constitutive model with parameters derived from experiments in which collagen content and cross-linking were decoupled during hypoxic pulmonary hypertension (HPH). We employed an eight-chain orthotropic element model to characterize collagen's mechanical behavior and an isotropic neo-Hookean form to represent elastin. Our results showed a strong correlation between the material parameter related to collagen content and measured collagen content (R(2)=0.82, P

BackgroundAlthough cardiovascular disease (CVD) and type 2 diabetes mellitus (T2D) share many common risk factors, potential molecular mechanisms that may also be shared for these 2 disorders remain unknown.Methods and resultsUsing an integrative pathway and network analysis, we performed genome-wide association studies in 8155 blacks, 3494 Hispanic American, and 3697 Caucasian American women who participated in the national Women's Health Initiative single-nucleotide polymorphism (SNP) Health Association Resource and the Genomics and Randomized Trials Network. Eight top pathways and gene networks related to cardiomyopathy, calcium signaling, axon guidance, cell adhesion, and extracellular matrix seemed to be commonly shared between CVD and T2D across all 3 ethnic groups. We also identified ethnicity-specific pathways, such as cell cycle (specific for Hispanic American and Caucasian American) and tight junction (CVD and combined CVD and T2D in Hispanic American). In network analysis of gene-gene or protein-protein interactions, we identified key drivers that included COL1A1, COL3A1, and ELN in the shared pathways for both CVD and T2D. These key driver genes were cross-validated in multiple mouse models of diabetes mellitus and atherosclerosis.ConclusionsOur integrative analysis of American women of 3 ethnicities identified multiple shared biological pathways and key regulatory genes for the development of CVD and T2D. These prospective findings also support the notion that ethnicity-specific susceptibility genes and process are involved in the pathogenesis of CVD and T2D.

NLR inflammasomes, caspase 1 activation platforms critical for processing key pro-inflammatory cytokines, have been implicated in the development of nonalcoholic fatty liver disease (NAFLD). As the direct role of the NLRP3 inflammasome remains unclear, we tested effects of persistent NLRP3 activation as a contributor to NAFLD development and, in particular, as a modulator of progression from benign hepatic steatosis to steatohepatitis during diet-induced NAFLD. Gain of function tamoxifen-inducible Nlrp3 knock-in mice allowing for in vivo temporal control of NLRP3 activation and loss of function Nlrp3 knockout mice were placed on short-term choline-deficient amino acid-defined (CDAA) diet, to induce isolated hepatic steatosis or long-term CDAA exposure, to induce severe steatohepatitis and fibrosis, respectively. Expression of NLRP3 associated proteins was assessed in liver biopsies of a well-characterized group of patients with the full spectrum of NAFLD. Nlrp3(-/-) mice were protected from long-term feeding CDAA-induced hepatomegaly, liver injury, and infiltration of activated macrophages. More importantly, Nlrp3(-/-) mice showed marked protection from CDAA-induced liver fibrosis. After 4 weeks on CDAA diet, wild-type (WT) animals showed isolated hepatic steatosis while Nlrp3 knock-in mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis. In the liver samples of patients with NAFLD, inflammasome components were significantly increased in those patients with nonalcoholic steatohepatitis (NASH) when compared to those with non-NASH NAFLD with mRNA levels of pro-IL1 beta correlated to levels of COL1A1. Our study uncovers a crucial role for the NLRP3 inflammasome in the development of NAFLD. These findings may lead to novel therapeutic strategies aimed at halting the progression of hepatic steatosis to the more severe forms of this disease. Key message: Mice with NLRP3 inflammasome loss of function are protected from diet-induced steatohepatitis. NLRP3 inflammasome gain of function leads to early and severe onset of diet-induced steatohepatitis in mice. Patients with severe NAFLD exhibit increased levels of NLRP3 inflammasome components and levels of pro-IL1β mRNA correlate with the expression of COL1A1.

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl4) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP(+) cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl4 liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles.

Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior reports that used a reporter gene fate-mapping strategy are limited in their ability to investigate the functional significance of epithelial cell-derived mesenchymal proteins during fibrogenesis. We found that lung epithelial cell-derived collagen I activates fibroblast collagen receptor discoidin domain receptor-2, contributes significantly to fibrogenesis, and promotes resolution of lung inflammation. Alveolar epithelial cells undergoing transforming growth factor-β-mediated mesenchymal transition express several other secreted profibrotic factors and are capable of activating lung fibroblasts. These studies provide direct evidence that activated epithelial cells produce mesenchymal proteins that initiate a cycle of fibrogenic effector cell activation, leading to progressive fibrosis. Therapy targeted at epithelial cell production of type I collagen offers a novel pathway for abrogating this progressive cycle and for limiting tissue fibrosis but may lead to sustained lung injury/inflammation.

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

This study tests the hypothesis that silicon and calcium ions combinatorially target gene expression during osteoblast differentiation. MC3T3-E1 subclone 4 osteoblast progenitors (transformed mouse calvarial osteoblasts) were exposed to Si(4+) (from Na(2)SiO(3)) and Ca(2+) (from CaCl(2):H(2)O) ion treatments both individually (0.4 mM each + control treatment) and combinatorially (0.4 mM Si(4+) + 0.4 mM Ca(2+) + control treatment) and compared to control treated (α-minimum essential medium, 10% fetal bovine serum, and 1% penicillin-streptomycin) cells. Cell proliferation studies showed no significant increase in cell density between treatments over 5 days of culture. Cellular differentiation studies involved addition of ascorbic acid (50 mg/L) for all treatments. Relative gene expression was determined for collagen type 1 (Col(I)α1/Col(I)α2), core-binding factor a (cbfa1/Runx2), and osteocalcin (OCN), which indicated osteoblast progenitor differentiation into a mineralizing phenotype. Increased Si(4+) or Ca(2+) ion treatments enhanced Col(I)α1, Col(I)α2, Runx2, and OCN expression, while increased Si(4+) + Ca(2+) ion treatments enhanced OCN expression. Moreover, it was found that a Si(4+)/Ca(2+) ratio of unity was optimal for maximal expression of OCN. Collagen fiber bundles were dense, elongated, and thick within extracellular matrices (ECM) exposed to Si(4+) and Si(4+) + Ca(2+) treatments, while collagen fiber bundles were sparse, short, and thin within Ca(2+) and control treated ECM. These results indicated that individual ions enhance multiple osteogenic gene expression, while combined ion treatments enhance individual gene expression. In addition, these results indicated that Si(4+) enhanced osteoblast gene expression and ECM formation at higher levels than Ca(2+). These results support the larger concept that ions (possibly released from bioactive glasses) could control bone formation by targeting osteoblast marker expression.

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF(165/121) antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair.

Osteogenesis imperfecta (OI), also known as brittle bone disease, is a clinically and genetically heterogeneous disorder primarily characterized by susceptibility to fracture. Although OI generally results from mutations in the type I collagen genes, COL1A1 and COL1A2, the relationship between genotype and phenotype is not yet well understood. To provide additional data for genotype-phenotype analyses and to determine the proportion of mutations in the type I collagen genes among subjects with lethal forms of OI, we sequenced the coding and exon-flanking regions of COL1A1 and COL1A2 in a cohort of 63 subjects with OI type II, the perinatal lethal form of the disease. We identified 61 distinct heterozygous mutations in type I collagen, including five non-synonymous rare variants of unknown significance, of which 43 had not been seen previously. In addition, we found 60 SNPs in COL1A1, of which 17 were not reported previously, and 82 in COL1A2, of which 18 are novel. In three samples without collagen mutations, we found inactivating mutations in CRTAP and LEPRE1, suggesting a frequency of these recessive mutations of approximately 5% in OI type II. A computational model that predicts the outcome of substitutions for glycine within the triple helical domain of collagen alpha1(I) chains predicted lethality with approximately 90% accuracy. The results contribute to the understanding of the etiology of OI by providing data to evaluate and refine current models relating genotype to phenotype and by providing an unbiased indication of the relative frequency of mutations in OI-associated genes.

The Mov-13 strain of mice was created by the insertion of the murine Moloney leukemia virus into the first intron of the alpha 1 (I) collagen gene. Consequently, Mov-13 embryos do not transcribe alpha 1 (I) collagen mRNA and lack type I collagen protein in the extracellular matrix (ECM). Homozygotes die within 12-14 days of embryonic development, in part from the rupture of large blood vessels, and also exhibit deficiencies in hematopoesis and assembly of the ECM (Lohler et al. [1984] Cell 38:597-607). Several matricellular proteins, proteoglycans, and growth factors bind to type I collagen, e.g., fibronectin, secreted protein acidic and rich in cysteine (SPARC), decorin, and transforming growth factor-beta. Here we investigate the expression and function of SPARC in the absence of type I collagen. We show that fibroblasts isolated from Mov-13 homozygous, heterozygous, and wild-type embryos transcribed and translated SPARC mRNA in vitro. However, accumulation of extracellular SPARC was severely affected in the tissues of Mov-13 homozygotes, whereas extracellular deposition of the secreted glycoproteins fibronectin and type III collagen was not altered. Since SPARC has been shown to be a regulator of cell shape, the functional consequences of the absence of extracellular SPARC were evaluated in collagen gel contraction assays. Fibroblasts isolated from homozygous Mov-13 mice did not contract native type I collagen gels as efficiently as fibroblasts from heterozygous littermates; however, addition of exogenous SPARC enhanced the contraction of collagen by homozygous Mov-13 fibroblasts. The stimulatory effect of SPARC was blocked by antibodies specific for the amino terminus of the protein. These results provide evidence that type I collagen is one of the major extracellular proteins that binds SPARC in vivo. Furthermore, the capacity of fibroblasts to contract ECM in vitro is enhanced by extracellular SPARC. We therefore propose that the remodeling of ECM by cells in vivo is regulated in part by a specific interaction between SPARC and type I collagen.