Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells

Introduction

Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface.

Methods

AECA activity in sera from patients with collagen diseases was measured with flow cytometry by using human umbilical vein endothelial cells (HUVECs). A cDNA library of HUVECs was retrovirally transfected into a rat myeloma cell line, from which AECA-positive clones were sorted with flow cytometry. cDNA of the cells was analyzed to identify an autoantigen, and then the clinical characteristics and the functional significance of the autoantibody were evaluated.

Results

Two distinct AECA-positive clones were isolated by using serum immunoglobulin G (IgG) from a patient with systemic lupus erythematosus (SLE). Both clones were identical to cDNA of fibronectin leucine-rich transmembrane protein 2 (FLRT2). HUVECs expressed FLRT2 and the prototype AECA IgG bound specifically to FLRT2-transfected cells. Anti-FLRT2 antibody activity accounted for 21.4% of AECAs in SLE. Furthermore, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2-expressing cells.

Conclusions

We identified the membrane protein FLRT2 as a novel autoantigen of AECAs in SLE patients by using the retroviral vector system. Anti-FLRT2 antibody has the potential to induce direct endothelial cell cytotoxicity in about 10% of SLE patients and could be a novel molecular target for intervention. Identification of such a cell-surface target for AECAs may reveal a comprehensive mechanism of vascular injury in collagen diseases.     

A Protocadherin-Cadherin-FLRT3 Complex Controls Cell Adhesion and Morphogenesis

Background

Paraxial protocadherin (PAPC) and fibronectin leucine-rich domain transmembrane protein-3 (FLRT3) are induced by TGFβ signaling in Xenopus embryos and both regulate morphogenesis by inhibiting C-cadherin mediated cell adhesion.

Principal Findings

We have investigated the functional and physical relationships between PAPC, FLRT3, and C-cadherin. Although neither PAPC nor FLRT3 are required for each other to regulate C-cadherin adhesion, they do interact functionally and physically, and they form a complex with cadherins. By itself PAPC reduces cell adhesion physiologically to induce cell sorting, while FLRT3 disrupts adhesion excessively to cause cell dissociation. However, when expressed together PAPC limits the cell dissociating and tissue disrupting activity of FLRT3 to make it effective in physiological cell sorting. PAPC counteracts FLRT3 function by inhibiting the recruitment of the GTPase RND1 to the FLRT3 cytoplasmic domain.

Conclusions/Significance

PAPC and FLRT3 form a functional complex with cadherins and PAPC functions as a molecular “governor” to maintain FLRT3 activity at the optimal level for physiological regulation of C-cadherin adhesion, cell sorting, and morphogenesis.     

Active systemic lupus erythematosus is associated with a reduced cytokine production by B cells in response to TLR9 stimulation

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break in self-tolerance reflected by a production of antinuclear autoantibodies. Since autoantibody production can be activated via nucleic acid Toll-like receptor 9 (TLR9), the respective pathway has been implicated in the development of SLE and pathogenic B cell responses. However, the response of B cells from SLE patients to TLR9 stimulation remains incompletely characterized.

Methods

In the current study, the response of B cells from SLE patients and healthy donors upon TLR9 stimulation was analyzed in terms of proliferation and cytokine production and correlated with the lupus disease activity and anti-dsDNA titers.

Results

B cells from SLE patients showed a reduced response to TLR9 agonist compared to B cells from healthy donors in terms of proliferation and activation. B cells from SLE patients with higher disease activity produced less interleukin (IL)-6, IL-10, vascular endothelial growth factor, and IL-1ra than B cells from healthy donors. Further analyses revealed an inverse correlation of cytokines produced by TLR9-stimulated B cells with lupus disease activity and anti-dsDNA titer, respectively.

Conclusion

The capacity of B cells from lupus patients to produce cytokines upon TLR9 engagement becomes less efficient with increasing disease activity, suggesting that they either enter an exhausted state or become tolerant to TLR stimulation for cytokine production when disease worsens.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0477-1) contains supplementary material, which is available to authorized users.     

Critical Role of FLRT1 Phosphorylation in the Interdependent Regulation of FLRT1 Function and FGF Receptor Signalling

Background

Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation.

Principal Findings

Here we show that FLRT1 is a target for tyrosine phosphorylation mediated by FGFR1 and implicate a non-receptor Src family kinase (SFK). We identify the target tyrosine residues in the cytoplasmic domain of FLRT1 and show that these are not direct substrates for Src kinase suggesting that the SFK may exert effects via potentiation of FGFR1 kinase activity. We show that whilst FLRT1 expression results in a ligand-dependent elevation of MAP kinase activity, a mutant version of FLRT1, defective as an FGFR1 kinase substrate (Y3F-FLRT1), has the property of eliciting ligand-independent chronic activation of the MAP kinase pathway which is suppressed by pharmacological inhibition of either FGFR1 or Src kinase. Functional investigation of FGFR1 and FLRT1 signalling in SH-SY5Y neuroblastoma cells reveals that FLRT1 alone acts to induce a multi-polar phenotype whereas the combination of FLRT1 and FGFR activation, or expression of Y3F-FLRT1, acts to induce neurite outgrowth via MAPK activation. Similar results were obtained in a dendrite outgrowth assay in primary hippocampal neurons. We also show that FGFR1, FLRT1 and activated Src are co-localized and this complex is trafficked toward the soma of the cell. The presence of Y3F-FLRT1 rather than FLRT1 resulted in prolonged localization of this complex within the neuritic arbour.

Conclusions

This study shows that the phosphorylation state of FLRT1, which is itself FGFR1 dependent, may play a critical role in the potentiation of FGFR1 signalling and may also depend on a SFK-dependent phosphorylation mechanism acting via the FGFR. This is consistent with an ‘in vivo’ role for FLRT1 regulation of FGF signalling via SFKs. Furthermore, the phosphorylation-dependent futile cycle mechanism controlling FGFR1 signalling is concurrently crucial for regulation of FLRT1-mediated neurite outgrowth.     

Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells

Background

Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells.

Methodology/Principal Findings

Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected.

Conclusion/Significance

These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury.     

Ginsenoside Rb1 Prevents H2O2-Induced HUVEC Senescence by Stimulating Sirtuin-1 Pathway

Purposes

We have previously reported that Ginsenoside Rb1 may effectively prevent HUVECs from senescence, however, the detailed mechanism has not demonstrated up to now. Recent studies have shown that sirtuin-1 (Sirt1) plays an important role in the development of endothelial senescence. The purpose of this study was to explore whether Sirt1 is involved in the action of Ginsenoside Rb1 regarding protection against H₂O₂-induced HUVEC Senescence.

Methods and Results

Senescence induced by hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs) was examined by analyzing plasminogen activator inhibitor-1 (PAI-1) expression, cell morphology, and senescence-associated beta-galactosidase (SA-β-gal) activity. The results revealed that 42% of control-treated HUVECs were SA-β-gal positive after treatment by 60 µmol/L H₂O₂, however, this particular effect of H₂O₂ was decreased more than 2-fold (19%) in the HUVECs when pretreated with Rb1 (20 µmol/L) for 30 min. Additionally, Rb1 decreased eNOS acetylation, as well as promoted more NO production that was accompanied by an increase in Sirt1 expression. Furthermore, upon knocking down Sirt1, the effect of Rb1 on HUVEC senescence was blunted.

Conclusions

The present study indicated that Ginsenoside Rb1 acts through stimulating Sirt1 in order to protect against endothelial senescence and dysfunction. As such, Sirt1 appears to be of particular importance in maintaining endothelial functions and delaying vascular aging.     

Autoantibodies to endothelial cell surface ATP synthase, the endogenous receptor for hsp60, might play a pathogenic role in vasculatides

Background

Heat shock protein (hsp) 60 that provides “danger signal” binds to the surface of resting endothelial cells (EC) but its receptor has not yet been characterized. In mitochondria, hsp60 specifically associates with adenosine triphosphate (ATP) synthase. We therefore examined the possible interaction between hsp60 and ATP synthase on EC surface.

Methodology/Principal Findings

Using Far Western blot approach, co-immunoprecipitation studies and surface plasmon resonance analyses, we demonstrated that hsp60 binds to the β-subunit of ATP synthase. As a cell surface-expressed molecule, ATP synthase is potentially targeted by anti-EC-antibodies (AECAs) found in the sera of patients suffering vasculitides. Based on enzyme-linked immunosorbent assay and Western blotting techniques with F1-ATP synthase as substrate, we established the presence of anti-ATP synthase antibodies at higher frequency in patients with primary vasculitides (group I) compared with secondary vasculitides (group II). Anti-ATP synthase reactivity from group I patients was restricted to the β-subunit of ATP synthase, whereas those from group II was directed to the α-, β- and γ-subunits. Cell surface ATP synthase regulates intracellular pH (pHi). In low extracellular pH medium, we detected abnormal decreased of EC pHi in the presence of anti-ATP synthase antibodies, irrespective of their fine reactivities. Interestingly, soluble hsp60 abrogated the anti-ATP synthase-induced pHi down-regulation.

Conclusions/Significance

Our results indicate that ATP synthase is targeted by AECAs on the surface of EC that induce intracellular acidification. Such pathogenic effect in vasculitides can be modulated by hsp60 binding on ATP synthase which preserves ATP synthase activity.     

Predictors of the first cardiovascular event in patients with systemic lupus erythematosus - a prospective cohort study

Introduction

Cardiovascular disease (CVD) is a major cause of premature mortality among Systemic lupus erythematosus (SLE) patients. Many studies have measured and evaluated risk factors for premature subclinical atherosclerosis, but few studies are prospective and few have evaluated risk factors for hard endpoints, i.e. clinically important cardiovascular events (CVE). We investigated the impact of traditional and lupus associated risk factors for the first ever CVE in a longitudinal cohort of SLE patients.

Methods

A total of 182 SLE patients (mean age 43.9 years) selected to be free of CVE were included. Cardiovascular and autoimmune biomarkers were measured on samples collected after overnight fasting at baseline. Clinical information was collected at baseline and at follow up. End point was the first ever CVE (ischemic heart, cerebrovascular or peripheral vascular disease or death due to CVD). Impact of baseline characteristics/biomarkers on the risk of having a first CVE was evaluated with Cox regression.

Results

Follow up was 99.5% after a mean time of 8.3 years. Twenty-four patients (13%) had a first CVE. In age-adjusted Cox regression, any positive antiphospholipid antibody (aPL), elevated markers of endothelial activation (von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1)) and fibrinogen predicted CVEs. Of SLE manifestations, arthritis, pleuritis and previous venous occlusion were positively associated with future CVEs while thrombocytopenia was negatively associated. Among traditional risk factors only age and smoking were significant predictors. In a multivariable Cox regression model age, any positive aPL, vWf and absence of thrombocytopenia were all predictors of the first CVE.

Conclusions

In addition to age, positive aPL, biomarkers indicating increased endothelial cell activity/damage, and absence of thrombocytopenia were independent predictors of CVEs in this prospective study. Our results indicate that activation of the endothelium and the coagulation system are important features in SLE related CVD. Furthermore, we observed that the risk of CVEs seems to differ between subgroups of SLE patients.     

Inflammatory expression profiles in monocyte-to-macrophage differentiation in patients with systemic lupus erythematosus and relationship with atherosclerosis

Introduction

Our objectives were to examine mononuclear cell gene expression profiles in patients with systemic lupus erythematosus (SLE) and healthy controls and to compare subsets with and without atherosclerosis to determine which genes’ expression is related to atherosclerosis in SLE.

Methods

Monocytes were obtained from 20 patients with SLE and 16 healthy controls and were in vitro-differentiated into macrophages. Subjects also underwent laboratory and imaging studies to evaluate for subclinical atherosclerosis. Whole-genome RNA expression microarray was performed, and gene expression was examined.

Results

Gene expression profiling was used to identify gene signatures that differentiated patients from controls and individuals with and without atherosclerosis. In monocytes, 9 out of 20 patients with SLE had an interferon-inducible signature compared with 2 out of 16 controls. By looking at gene expression during monocyte-to-macrophage differentiation, we identified pathways which were differentially regulated between SLE and controls and identified signatures based on relevant intracellular signaling molecules which could differentiate SLE patients with atherosclerosis from controls. Among patients with SLE, we used a previously defined 344-gene atherosclerosis signature in monocyte-to-macrophage differentiation to identify patient subgroups with and without atherosclerosis. Interestingly, this signature further classified patients on the basis of the presence of SLE disease activity and cardiovascular risk factors.

Conclusions

Many genes were differentially regulated during monocyte-to-macrophage differentiation in SLE patients compared with controls. The expression of these genes in mononuclear cells is important in the pathogenesis of SLE, and molecular profiling using gene expression can help stratify SLE patients who may be at risk for development of atherosclerosis.     

Detection of Circulating B Cells Producing Anti-GPIb Autoantibodies in Patients with Immune Thrombocytopenia

Background

We previously reported that an enzyme-linked immunospot (ELISPOT) assay for detecting anti-GPIIb/IIIa antibody-secreting B cells is a sensitive method for identifying patients with immune thrombocytopenia (ITP). Here we assessed the clinical significance of measuring circulating B cells producing antibodies to GPIb, another major platelet autoantigen.

Methods

Anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells were simultaneously measured using ELISPOT assays in 32 healthy controls and 226 consecutive thrombocytopenic patients, including 114 with primary ITP, 25 with systemic lupus erythematosus (SLE), 30 with liver cirrhosis, 39 with post-hematopoietic stem cell transplantation (post-HSCT), and 18 non-ITP controls (aplastic anemia and myelodysplastic syndrome).

Results

There were significantly more circulating anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells in primary ITP, SLE, liver cirrhosis, and post-HSCT patients than in healthy controls (P<0.05 for all comparisons). For diagnosing primary ITP, the anti-GPIb ELISPOT assay had 43% sensitivity and 89% specificity, whereas the anti-GPIIb/IIIa ELISPOT assay had 86% sensitivity and 83% specificity. When two tests were combined, the sensitivity was slightly improved to 90% without a reduction in specificity. In primary ITP patients, the anti-GPIb antibody response was associated with a low platelet count, lack of Helicobacter pylori infection, positive anti-nuclear antibody, and poor therapeutic response to intravenous immunoglobulin.

Conclusion

The ELISPOT assay for detecting anti-GPIb antibody-secreting B cells is useful for identifying patients with ITP, but its utility for diagnosing ITP is inferior to the anti-GPIIb/IIIa ELISPOT assay. Nevertheless, detection of the anti-GPIb antibody response is useful for subtyping patients with primary ITP.     

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

Semaphorin 3A is a marker for disease activity and a potential immunoregulator in systemic lupus erythematosus

Introduction

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen induced arthritis. This study was undertaken to evaluate the role of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients.

Methods

Thirty two SLE and 24 rheumatoid arthritis (RA) patients were assessed and compared with 40 normal individuals. Sema3A serum levels were measured and correlated with SLE disease activity. The in vitro effect of sema3A in reducing Toll-like receptor 9 (TLR-9) expression in B cells of SLE patients was evaluated.

Results

Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (55.04 ± 16.30 ng/ml versus 65.54 ± 14.82 ng/ml, P = 0.018) and lower yet than in normal individuals (55.04 ± 16.30 ng/ml versus 74.41 ± 17.60 ng/ml, P < 0.0001). Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, when sema3A was co-cultured with cytosine-phosphodiester-guanine oligodeoxynucleotides (CpG-ODN)-stimulated B cells of SLE patients, their TLR-9 expression was significantly reduced, by almost 50% (P = 0.001).

Conclusions

This is the first study in which a reduced serum level of sema3A was found in association with SLE disease activity. It also raises the possibility that sema3A may have a regulatory function in SLE.     

Altered expression of protein tyrosine phosphatase,non-receptor type 22 isoforms in systemic lupus erythematosus

Introduction

A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human protein tyrosine phosphatase, non-receptor type 22 (PTPN22) complementary DNA (cDNA) is associated with an increased risk of systemic lupus erythematosus (SLE). How the overall activity of PTPN22 is regulated and how the expression of PTPN22 differs between healthy individuals and patients with lupus are poorly understood. Our objectives were to identify novel alternatively spliced forms of PTPN22 and to examine the expression of PTPN22 isoforms in healthy donors and patients with lupus.

Methods

Various human PTPN22 isoforms were identified from the GenBank database or amplified directly from human T cells. The expression of these isoforms in primary T cells and macrophages was examined with real-time polymerase chain reaction. The function of the isoforms was determined with luciferase assays. Blood samples were collected from 49 subjects with SLE and 15 healthy controls. Correlation between the level of PTPN22 isoforms in peripheral blood and clinical features of SLE was examined with statistical analyses.

Results

Human PTPN22 was expressed in several isoforms, which differed in their level of expression and subcellular localization. All isoforms except one were functionally interchangeable in regulating NFAT activity. SLE patients expressed higher levels of PTPN22 than healthy individuals and the levels of PTPN22 were negatively correlated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC-DI).

Conclusions

The overall activity of PTPN22 is determined by the functional balance among all isoforms. The levels of PTPN22 isoforms in peripheral blood could represent a useful biomarker of SLE.     

Desflurane Preconditioning Induces Oscillation of NF-κB in Human Umbilical Vein Endothelial Cells

Background

Nuclear factor kappa B (NF-κB) has been implicated in anesthetic preconditioning (APC) induced protection against anoxia and reoxygenation (A/R) injury. The authors hypothesized that desflurane preconditioning would induce NF-κB oscillation and prevent endothelial cells apoptosis.

Methods

A human umbilical vein endothelial cells (HUVECs) A/R injury model was used. A 30 minute desflurane treatment was initiated before anoxia. NF-κB inhibitor BAY11-7082 was administered in some experiments before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V–fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT) assay. The cellular superoxide dismutases (SOD) activitiy were tested by water-soluble tetrazolium salt (WST-1) assay. NF-κB p65 subunit nuclear translocation was detected by immunofluorescence staining. Expression of inhibitor of NF-κB-α (IκBα), NF-κB p65 and cellular inhibitor of apoptosis 1 (c-IAP1), B-cell leukemia/lymphoma 2 (Bcl-2), cysteine containing aspartate specific protease 3 (caspases-3) and second mitochondrial-derived activator of caspase (SMAC/DIABLO) were determined by western blot.

Results

Desflurane preconditioning caused phosphorylation and nuclear translocation of NF-κB before anoxia, on the contrary, induced the synthesis of IκBα and inhibition of NF-κB after reoxygenation. Desflurane preconditioning up-regulated the expression of c-IAP1 and Bcl-2, blocked the cleavage of caspase-3 and reduced SMAC release, and decreased the cell death of HUVECs after A/R. The protective effect was abolished by BAY11-7082 administered before desflurane.

Conclusions

The results demonstrated that desflurane activated NF-κB during the preconditioning period and inhibited excessive activation of NF-κB in reperfusion. And the oscillation of NF-κB induced by desflurane preconditioning finally up-regulated antiapoptotic proteins expression and protected endothelial cells against A/R.