Irisin deficiency disturbs bone metabolism.

Balancing the process of bone formation and resorption is important in the maintenance of healthy bone. Therefore, the discovery of novel factors that can regulate bone metabolism remains needed. Irisin is a newly identified hormone-like peptide. Recent studies have reported the involvement of irisin in many physiological and pathological conditions with bone mineral density changes, including osteopenia and osteoporotic fractures. In this study, we generated the first line of Osx-Cre:FNDC5/irisin KO mice, in which FNDC5/irisin was specifically deleted in the osteoblast lineage. Gene and protein expressions of irisin were remarkably decreased in bones but no significant differences in other tissues were observed in knockout mice. FNDC5/irisin deficient mice showed a lower bone density and significantly delayed bone development and mineralization from early-stage to adulthood. Our phenotypical analysis exhibited decreased osteoblast-related gene expression and increased osteoclast-related gene expression in bone tissues, and reduced adipose tissue browning due to bone-born irisin deletion. By harvesting and culturing MSCs from the knockout mice, we found that osteoblastogenesis was inhibited and osteoclastogenesis was increased. By using irisin stimulated wildtype primary cells as a gain-of-function model, we further revealed the effects and mechanisms of irisin on promoting osteogenesis and inhibiting osteoclastogenesis in vitro. In addition, positive effects of exercise, including bone strength enhancement and body weight loss were remarkably weakened due to irisin deficiency. Interestingly, these changes can be rescued by supplemental administration of recombinant irisin during exercise. Our study indicates that irisin plays an important role in bone metabolism and the crosstalk between bone and adipose tissue. Irisin represents a potential molecule for the prevention and treatment of bone metabolic diseases.

Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow.

OBJECTIVE: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. CONCLUSIONS: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

Differential Roles of Endothelial Cell-Derived and Smooth Muscle Cell-Derived Fibronectin Containing Extra Domain A in Early and Late Atherosclerosis.

OBJECTIVE: The extracellular matrix of atherosclerotic arteries contains abundant deposits of cellular Fn-EDA (fibronectin containing extra domain A), suggesting a functional role in the pathophysiology of atherosclerosis. Fn-EDA is synthesized by several cell types, including endothelial cells (ECs) and smooth muscle cells (SMCs), which are known to contribute to different stages of atherosclerosis. Although previous studies using global Fn-EDA-deficient mice have demonstrated that Fn-EDA is proatherogenic, the cell-specific role of EC versus SMC-derived-Fn-EDA in atherosclerosis has not been investigated yet. Approach and Results: To determine the relative contribution of different pools of Fn-EDA in atherosclerosis, we generated mutant strains lacking Fn-EDA in the ECs (Fn-EDAEC-KO) or smooth muscle cells (Fn-EDASMC-KO) on apolipoprotein E-deficient (Apoe-/-) background. The extent of atherosclerotic lesion progression was evaluated in whole aortae, and cross-sections of the aortic sinus in male and female mice fed a high-fat Western diet for either 4 weeks (early atherosclerosis) or 14 weeks (late atherosclerosis). Irrespective of sex, Fn-EDAEC-KO, but not Fn-EDASMC-KO mice, exhibited significantly reduced early atherogenesis concomitant with decrease in inflammatory cells (neutrophil and macrophage) and VCAM-1 (vascular cell adhesion molecule-1) expression levels within the plaques. In late atherosclerosis model, irrespective of sex, Fn-EDASMC-KO mice exhibited significantly reduced atherogenesis, but not Fn-EDAEC-KO mice, that was concomitant with decreased macrophage content within plaques. Lesional SMCs, collagen content, and plasma inflammatory cytokines (TNF-α [tumor necrosis factor-α] and IL-1ß [interleukin-1ß]), total cholesterol, and triglyceride levels were comparable among groups. CONCLUSIONS: EC-derived Fn-EDA contributes to early atherosclerosis, whereas SMC-derived Fn-EDA contributes to late atherosclerosis.

Phosphorylated fibronectin enhances cell attachment and upregulates mechanical cell functions.

A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosphorylation of fibronectin by a casein kinase II (CKII) significantly upregulated cell traction forces and total strain energy generated by fibroblasts on nanopillar arrays, and consequently other elementary cell functions including cell spreading and metabolic activity. Mass spectrometry of plasma fibronectin from healthy human donors then identified a constitutively phosphorylated site in the C-terminus, and numerous other residues that became phosphorylated by the CKII kinase in vitro. Our findings open up novel strategies for translational applications including targeting diseased ECM, or to develop assays that probe the phosphorylation state of the ECM or blood as potential cancer markers.

Endothelial FN (Fibronectin) Deposition by α5ß1 Integrins Drives Atherogenic Inflammation.

Objective- Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to an FN (fibronectin)-rich matrix preceding lesion development. Endothelial cell interactions with FN prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic FN accumulation remain unknown. We previously demonstrated that oxLDL (oxidized low-density lipoprotein) promotes endothelial proinflammatory gene expression by activating the integrin α5ß1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting α5ß1 (blocking antibodies, α5 knockout cells) completely inhibits oxLDL-induced FN deposition. Consistent with this, inducible endothelial-specific α5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGFß (transforming growth factor ß)-induced FN deposition, oxLDL does not induce FN expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived FN differentially affect endothelial function, with only cell-derived FN capable of supporting oxLDL-induced VCAM-1 (vascular cell adhesion molecule 1) expression, despite plasma FN deposition by oxLDL. The inclusion of alternative exon EIIIA (EDA) of FN (EIIIA) and alternative exon EIIIB (EDB) of FN (EIIIB) domains in cell-derived FN mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggest that EIIIA/EIIIB-positive cellular FN is required for maximal α5ß1 recruitment to focal adhesions and FN fibrillogenesis. Conclusions- Taken together, our data demonstrate that endothelial α5 integrins drive oxLDL-induced FN deposition and early atherogenic inflammation. Additionally, we show that α5ß1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.

Deletion of Extra Domain A of Fibronectin Reduces Acute Myocardial Ischaemia/Reperfusion Injury in Hyperlipidaemic Mice by Limiting Thrombo-Inflammation.

BACKGROUND: Fibronectin splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for Toll-like receptor 4 (TLR4), is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with co-morbid conditions including diabetes and hyperlipidaemia, which are risk factors for myocardial infarction (MI). Very little is known about the role of Fn-EDA in the pathophysiology of acute MI under these co-morbid conditions. MATERIALS AND METHODS: We determined the role of Fn-EDA in myocardial ischaemia/reperfusion (I/R) injury in the hyperlipidaemic apolipoprotein E-deficient (ApoE-/-) mice. Infarct size, plasma cardiac troponin I (cTnI) levels, intravascular thrombosis (CD41-positive), neutrophil infiltration (Ly6 B.2-positive), neutrophil extracellular traps (citrullinated H3-positive) and myocyte apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive) were assessed in myocardial I/R injury model (1-hour ischaemia/23 hours of reperfusion). RESULTS: Irrespective of gender, Fn-EDA-/-ApoE-/- mice exhibited smaller infarct size and decreased cTnI levels concomitant with reduced post-ischaemic intra-vascular thrombi, neutrophils influx, neutrophil extracellular traps and myocyte apoptosis (p < 0.05 vs. ApoE-/- mice). Genetic deletion of TLR4 attenuated myocardial I/R injury in ApoE-/- mice (p < 0.05 vs. ApoE-/- mice), but did not further reduce in Fn-EDA-/- ApoE-/- mice suggesting that Fn-EDA requires TLR4 to mediate myocardial I/R injury. Bone marrow transplantation experiments revealed that Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the haematopoietic cells. Infusion of a specific inhibitor of Fn-EDA, 15 minutes post-reperfusion, into ApoE-/- mice attenuated myocardial I/R injury. CONCLUSION: Fn-EDA exacerbates TLR4-dependent myocardial I/R injury by promoting post-ischaemic thrombo-inflammatory response. Targeting Fn-EDA may reduce cardiac damage following coronary artery re-canalization after acute MI.

Fibronectin EDA forms the chronic fibrotic scar after contusive spinal cord injury.

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is an impediment to axonal regeneration. Fibrotic scarring is characterized by the accumulation of fibronectin, collagen, and fibroblasts at the lesion site. The mechanisms regulating fibrotic scarring after SCI and its effects on axonal elongation and functional recovery are not well understood. In this study, we examined the effects of eliminating an isoform of fibronectin containing the Extra Domain A domain (FnEDA) on both fibrosis and on functional recovery after contusion SCI using male and female FnEDA-null mice. Eliminating FnEDA did not reduce the acute fibrotic response but markedly diminished chronic fibrotic scarring after SCI. Glial scarring was unchanged after SCI in FnEDA-null mice. We found that FnEDA was important for the long-term stability of the assembled fibronectin matrix during both the subacute and chronic phases of SCI. Motor functional recovery was significantly improved, and there were increased numbers of axons in the lesion site compared to wildtype mice, suggesting that the chronic fibrotic response is detrimental to recovery. Our data provide insight into the mechanisms of fibrosis after SCI and suggest that disruption of fibronectin matrix stability by targeting FnEDA represents a potential therapeutic strategy for promoting recovery after SCI.

Fibronectin Containing Extra Domain A Induces Plaque Destabilization in the Innominate Artery of Aged Apolipoprotein E-Deficient Mice.

OBJECTIVE: Fibronectin containing extra domain A (Fn-EDA) is an endogenous ligand of TLR4 (toll-like receptor 4) and is abundant in the extracellular matrix of advanced atherosclerotic lesions in human and mice. Irrespective of sex, deletion of Fn-EDA reduces early atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. However, the contribution of Fn-EDA in advanced atherosclerosis remains poorly characterized. We determined the contribution of Fn-EDA in advanced atherosclerotic lesions of aged (1-year-old) Apoe-/- mice. APPROACH AND RESULTS: Plaque composition was determined in the innominate artery, a plaque instability site that is known to mimic several histological features of vulnerable human plaques. Female Apoe-/-, Fn-EDA-/-Apoe-/-, TLR4-/-Apoe-/-, and Fn-EDA-/-TLR4-/-Apoe-/- mice were fed a high-fat Western diet for 44 weeks. Fn-EDA-/-Apoe-/- mice exhibited reduced plaque size characterized by smaller necrotic cores, thick fibrous caps containing abundant vascular smooth muscle cells and collagen, reduced CD68/MMP9 (matrix metalloproteinase 9)-positive content, less accumulation of MMP-cleaved extracellular matrix aggrecan, and decreased vascular smooth muscle cell and macrophage apoptosis (P<0.05 versus Apoe-/- mice). Together these findings suggest that Fn-EDA induces plaque destabilization. Deletion of TLR4 reduced histological features of plaque instability in Apoe-/- mice but did not further reduce features of plaque destabilization in Fn-EDA-/-Apoe-/- mice, suggesting that TLR4 may contribute to Fn-EDA-induced plaque destabilization. Fn-EDA potentiated TLR4-dependent MMP9 expression in bone marrow-derived macrophages, suggesting that macrophage TLR4 may contribute to Fn-EDA-mediated plaque instability. CONCLUSIONS: Fn-EDA induces histological features of plaque instability in established lesions of aged Apoe-/- mice. The abundance of Fn-EDA in advanced atherosclerotic lesions may increase the risk of plaque destabilization.

Circulating fibronectin contributes to mesangial expansion in a murine model of type 1 diabetes.

Fibronectin is ubiquitously expressed in the extracellular matrix, and its accumulation in the glomerular mesangium in diabetic nephropathy is associated with deterioration of renal function in these patients. However, the exact role of fibronectin in the pathogenesis of diabetic nephropathy remains unknown. To clarify this, we administered fluorescent-labeled plasma fibronectin to wild-type mice and found it to accumulate in the mesangium. Using liver-specific conditional-knockout mice to decrease circulating fibronectin, we reduced circulating fibronectin by more than 90%. In streptozotocin-induced diabetes of these knockout mice, the pronounced fall in circulating fibronectin resulted in a decrease in mesangial expansion by 25% and a decline in albuminuria by 30% compared to diabetic control mice. Indeed, the amount of fibronectin in the kidney was reduced, as was the total amount of collagen. In vitro experiments confirmed that matrix accumulation of fibronectin was enhanced by increasing fibronectin only, glucose only, or the combination of both. Thus, circulating fibronectin contributes to mesangial expansion and exacerbation of albuminuria in a murine model of type 1 diabetes.

NS5ABP37 inhibits liver cancer by impeding lipogenesis and cholesterogenesis.

The molecular mechanism underlying non-alcoholic fatty liver disease progression to hepatocellular carcinoma (HCC) remains unknown. In this study, immunohistochemistry staining results showed that NS5ABP37 protein, which is in a state of lower expression in tumor tissues, decreased with increasing degree of HCC malignancy. Two cell models, HepG2 and L02, were used to analyze the mechanism between NS5ABP37 and HCC. In agreement, NS5ABP37 protein overexpression significantly suppressed cell proliferation, caused G1 /S cell cycle arrest, and induced apoptosis by increasing caspase-3/7 activity and cleaved caspase-3 levels. In addition, NS5ABP37 overexpression resulted in decreased intracellular triglyceride and total cholesterol contents, with level reduction in sterol regulatory element-binding proteins (SREBPs) and downstream effectors. Furthermore, NS5ABP37 overexpression decreased SREBP1c and SREBP2 levels by reducing their respective promoters. Finally, reactive oxygen species levels and endoplasmic reticulum stress were both induced by NS5ABP37 overexpression. These findings together indicate that NS5ABP37 inhibits cancer cell proliferation and promotes apoptosis, by altering SREBP-dependent lipogenesis and cholesterogenesis in HepG2 and L02 cells and inducing oxidative stress and endoplasmic reticulum stress.

FNDC5 Alleviates Hepatosteatosis by Restoring AMPK/mTOR-Mediated Autophagy, Fatty Acid Oxidation, and Lipogenesis in Mice.

Fibronectin type III domain-containing 5 (FNDC5) protein induces browning of subcutaneous fat and mediates the beneficial effects of exercise on metabolism. However, whether FNDC5 is associated with hepatic steatosis, autophagy, fatty acid oxidation (FAO), and lipogenesis remains unknown. Herein, we show the roles and mechanisms of FNDC5 in hepatic steatosis, autophagy, and lipid metabolism. Fasted FNDC5-/- mice exhibited severe steatosis, reduced autophagy, and FAO, and enhanced lipogenesis in the liver compared with wild-type mice. Energy deprivation-induced autophagy, FAO, and AMPK activity were attenuated in FNDC5-/- hepatocytes, which were restored by activating AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Inhibition of mammalian target of rapamycin (mTOR) complex 1 with rapamycin enhanced autophagy and FAO and attenuated lipogenesis and steatosis in FNDC5-/- livers. FNDC5 deficiency exacerbated hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. Exogenous FNDC5 stimulated autophagy and FAO gene expression in hepatocytes and repaired the attenuated autophagy and palmitate-induced steatosis in FNDC5-/- hepatocytes. FNDC5 overexpression prevented hyperlipemia, hepatic FAO and autophagy impairment, hepatic lipogenesis, and lipid accumulation in obese mice. These results indicate that FNDC5 deficiency impairs autophagy and FAO and enhances lipogenesis via the AMPK/mTOR pathway. FNDC5 deficiency aggravates whereas FNDC5 overexpression prevents the HFD-induced hyperlipemia, hepatic lipid accumulation, and impaired FAO and autophagy in the liver.

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis.

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-ß activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-ß1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-ß1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-ß bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.

Genetic Ablation of Extra Domain A of Fibronectin in Hypercholesterolemic Mice Improves Stroke Outcome by Reducing Thrombo-Inflammation.

BACKGROUND: The fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe(-/-)) mouse. METHODS AND RESULTS: In a transient cerebral ischemia/reperfusion injury model, Apoe(-/-) mice expressing fibronectin deficient in EDA (Fn-EDA(-/-)Apoe(-/-) mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 versus Apoe(-/-) mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho-nuclear factor-κB p65, phospho-IκB kinase α/ß, interleukin 1ß, and tumor necrosis factor-α) within lesions of Fn-EDA(-/-)Apoe(-/-) mice were markedly decreased (P<0.05 versus Apoe(-/-) mice). In an FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA(-/-)Apoe(-/-) mice (P<0.05 versus Apoe(-/-) mice). Genetic ablation of TLR4 improved stroke outcome in Apoe(-/-) mice (P<0.05) but had no effect on stroke outcome in Fn-EDA(-/-)Apoe(-/-) mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell-derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe(-/-) mouse 15 minutes after reperfusion significantly improved stroke outcome. CONCLUSIONS: Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

Fibronectin Splicing Variants Containing Extra Domain A Promote Atherosclerosis in Mice Through Toll-Like Receptor 4.

OBJECTIVE: Cellular fibronectin containing extra domain A (EDA(+)-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA(+)-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA(+)-FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E-deficient (Apoe(-/-)) mouse. APPROACH AND RESULTS: The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA(-/-)Apoe(-/-) mice (which lack EDA(+)-FN), EDA(fl/fl)Apoe(-/-) mice (which constitutively express EDA(+)-FN), and control Apoe(-/-) mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA(fl/fl)Apoe(-/-) mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA(-/-)Apoe(-/-) mice exhibited reduced atherosclerotic lesions (P<0.05 versus Apoe(-/-), n=10-12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA(fl/fl)Apoe(-/-) mice (P<0.05) but had no effect on atherosclerotic lesions in EDA(-/-)Apoe(-/-) mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1ß) in bone marrow-derived macrophages from EDA(-/-)Apoe(-/-) mice but not from EDA(-/-)TLR4(-/-)Apoe(-/-) mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA(+)-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques. CONCLUSIONS: Our findings reveal that TLR4 signaling contributes to EDA(+)-FN-mediated exacerbation of atherosclerosis. We suggest that EDA(+)-FN could be a therapeutic target in atherosclerosis.

Fibronectin extra domain A stabilises atherosclerotic plaques in apolipoprotein E and in LDL-receptor-deficient mice.

The primary transcript of fibronectin undergoes alternative splicing in the cassette-type EDA and EDB exons and in the IIICs segment to generate different protein isoforms. Human carotid atherosclerotic plaques with a more stable phenotype are enriched with EDA containing fibronectin (FN-EDA). The aim of this study was to investigate the role of EDA containing fibronectin during atherogenesis. Mice constitutively expressing or lacking the EDA domain of fibronectin (EDA+/+ or EDA-/-)were crossed with ApoE-/- or LDL-R-/- mice and fed with a western type diet for 12 weeks. Lack of FN-EDA resulted in reduced atherosclerosis and in a plaque phenotype characterised by decreased calponin positive VSMC's (-15 %) and increased macrophages (+20 %). This was paralleled by increased MMP2, MMP9, and reduced TIMP2, collagen 1A1, 1A2 and 3A1 gene expression compared to that of wild-type and EDA+/+ mice. In vitro, VSMCs and macrophages isolated from EDA-/- miceshowed increased MMPs expression and activity compared to wild-type or EDA+/+ mice. Albumin-Cre recombinase/EDA+/+/ApoE-/- mice, which produceEDA containing FN only in peripheral tissues, presented an extension, a composition and a gene expression pattern in the atherosclerotic lesions similar to that of controls. The inclusion of EDA in FN results in larger atherosclerotic plaques compared to mice lacking EDA but with a more favourable phenotype in two animals models of atherosclerosis. This effect depends on the EDA-containing fibronectin produced by cells in the vasculature but not in the liver. These observations set the stage for investigating the properties of circulating EDA containing FN in improving plaque stability.

Alternative splicing of endothelial fibronectin is induced by disturbed hemodynamics and protects against hemorrhage of the vessel wall.

OBJECTIVE: Abnormally low-flow conditions, sensed by the arterial endothelium, promote aneurysm rupture. Fibronectin (FN) is among the most abundant extracellular matrix proteins and is strongly upregulated in human aneurysms, suggesting a possible role in disease progression. Altered FN splicing can result in the inclusion of EIIIA and EIIIB exons, generally not expressed in adult tissues. We sought to explore the regulation of FN and its splicing and their possible roles in the vascular response to disturbed flow. APPROACH AND RESULTS: We induced low and reversing flow in mice by partial carotid ligation and assayed FN splicing in an endothelium-enriched intimal preparation. Inclusion of EIIIA and EIIIB was increased as early as 48 hours, with negligible increases in total FN expression. To test the function of EIIIA and EIIIB inclusion, we induced disturbed flow in EIIIAB(-/-) mice unable to include these exons and found that they developed focal lesions with hemorrhage and hypertrophy of the vessel wall. Acute deletion of floxed FN caused similar defects in response to disturbed flow, consistent with a requirement for the upregulation of the spliced isoforms, rather than a developmental defect. Recruited macrophages promote FN splicing because their depletion by clodronate liposomes blocked the increase in endothelial EIIIA and EIIIB inclusion in the carotid model. CONCLUSIONS: These results uncover a protective mechanism in the inflamed intima that develops under disturbed flow, by showing that splicing of FN mRNA in the endothelium, induced by macrophages, inhibits hemorrhage of the vessel wall.

FibronectinEDA promotes chronic cutaneous fibrosis through Toll-like receptor signaling.

Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (Fn(EDA)) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of Fn(EDA) was preferentially stimulated by transforming growth factor-ß in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous Fn(EDA) was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic Fn(EDA) responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of Fn(EDA) or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the Fn(EDA)-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant Fn(EDA) accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

Mesenchymal stromal cells: inhibiting PDGF receptors or depleting fibronectin induces mesodermal progenitors with endothelial potential.

Realizing the full therapeutic potential of mesenchymal stromal/stem cells (MSCs) awaits improved understanding of mechanisms controlling their fate. Using MSCs cultured as spheroids to recapitulate a three-dimensional cellular environment, we show that perturbing the mesenchymal regulators, platelet-derived growth factor (PDGF) receptors or fibronectin, reverts MSCs toward mesodermal progenitors with endothelial potential that can potently induce neovascularization in vivo. MSCs within untreated spheroids retain their mesenchymal spindle shape with abundant smooth muscle α-actin filaments and fibronectin-rich matrix. Inhibiting PDGF receptors or depleting fibronectin induces rounding and depletes smooth muscle α-actin expression; these cells have characteristics of mesenchymoangioblasts, with enhanced expression of mesendoderm and endoderm transcription factors, prominent upregulation of E-cadherin, and Janus kinase signaling-dependent expression of Oct4A and Nanog. PDGF receptor-inhibited spheroids also upregulate endothelial markers platelet endothelial cell adhesion molecule 1 and vascular endothelial-cadherin and secrete many angiogenic factors, and in vivo they potently stimulate neovascularization, and their MSCs integrate within functional blood vessels that are perfused by the circulation. Thus, MSC potency and vascular induction are regulated by perturbing mesenchymal fate.

Fibronectin extra domain-A promotes hepatic stellate cell motility but not differentiation into myofibroblasts.

BACKGROUND & AIMS: Myofibroblasts are the primary cell type involved in physiologic wound healing and its pathologic counterpart, fibrosis. Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA) is up-regulated during these processes and is believed to promote myofibroblast differentiation. We sought to determine the requirement for EIIIA in fibrosis and differentiation of myofibroblasts in rodent livers. METHODS: We used a mechanically tunable hydrogel cell culture system to study differentiation of primary hepatic stellate cells and portal fibroblasts from rats into myofibroblasts. Liver fibrosis was induced in mice by bile duct ligation or administration of thioacetamide. RESULTS: EIIIA was not required for differentiation of rat hepatic stellate cells or portal fibroblasts into fibrogenic myofibroblasts. Instead, hepatic stellate cells cultured on EIIIA-containing cellular fibronectin formed increased numbers of lamellipodia; their random motility and chemotaxis also increased. These increases required the receptor for EIIIA, the integrin α(9)ß(1). In contrast, the motility of portal fibroblasts did not increase on EIIIA, and these cells expressed little α(9)ß(1). Male EIIIA(-/-) mice were modestly protected from thioacetamide-induced fibrosis, which requires motile hepatic stellate cells, but not from bile duct ligation-induced fibrosis, in which portal fibroblasts are more important. Notably, myofibroblasts developed during induction of fibrosis with either thioacetamide or bile duct ligation in EIIIA(-/-) mice. CONCLUSIONS: EIIIA is dispensable for differentiation of hepatic stellate cells and portal fibroblasts to myofibroblasts, both in culture and in mouse models of fibrosis. Our findings, however, indicate a role for EIIIA in promoting stellate cell motility and parenchymal liver fibrosis.

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5ß1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.