Phosphatidylinositol-4-phosphate 5-kinase and GEP100/Brag2 protein mediate antiangiogenic signaling by semaphorin 3E-plexin-D1 through Arf6 protein

The semaphorins are a family of secreted or membrane-bound proteins that are known to guide axons in the developing nervous system. Genetic evidence revealed that a class III semaphorin, semaphorin 3E (Sema3E), and its receptor Plexin-D1 also control the vascular patterning during development. At the molecular level, we have recently shown that Sema3E acts on Plexin-D1 expressed in endothelial cells, thus initiating a novel antiangiogenic signaling pathway that results in the retraction of filopodia in endothelial tip cells. Sema3E induces the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix. This process requires the activation of small GTPase Arf6 (ADP-ribosylation factor 6), which regulates intracellular trafficking of β1 integrin. However, the molecular mechanisms by which Sema3E-Plexin-D1 activates Arf6 remained to be identified. Here we show that GEP100 (guanine nucleotide exchange protein 100)/Brag2, a guanine nucleotide exchange factor for Arf6, mediates Sema3E-induced Arf6 activation in endothelial cells. We provide evidence that upon activation by Sema3E, Plexin-D1 recruits phosphatidylinositol-4-phosphate 5-kinase, and its enzymatic lipid product, phosphatidylinositol 4,5-bisphosphate, binds to the pleckstrin homology domain of GEP100. Phosphatidylinositol 4,5-bisphosphate binding to GEP100 enhances its guanine nucleotide exchange factor activity toward Arf6, thus resulting in the disassembly of integrin-mediated focal adhesions and endothelial cell collapse. Our present study reveals a novel phospholipid-regulated antiangiogenic signaling pathway whereby Sema3E activates Arf6 through Plexin-D1 and consequently controls integrin-mediated endothelial cell attachment to the extracellular matrix and migration.     

Sema3E/plexin-D1 mediated epithelial-to-mesenchymal transition in ovarian endometrioid cancer

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration.     

The role and mechanism-of-action of Sema3E and Plexin-D1 in vascular and neural development

Class 3 secreted semaphorins (Sema3A–3G) participate in many aspects of axon guidance through holoreceptor complexes that include Neuropilin-1 (Npn-1) or Neuropilin-2 and one of the four class A plexin proteins. However, unlike other Sema3 family proteins, Sema3E directly binds to Plexin-D1 without neuropilins. Its biological function was first explored in intersomitic vessel formation and since its initial discovery, Sema3E–Plexin-D1 signaling has been found to participate in the many biological systems in addition to vascular development, via seemingly different mode of actions. For example, temporal and spatial control of ligand vs. receptor results in two different mechanisms governing vascular patterning. Interactions with other transmembrane proteins such as neuropilin and VEGFR2 result in different axonal behaviors. Ligand receptor localization on pre- vs. post-synaptic neurons is used to control different types of synapse formation. Perhaps different downstream effectors will also result in different functional outcomes. Given the limited number of ligands and receptors in the genome and their multifunctional nature, we expect that more modes of action will be discovered in the future. In this review, we highlight current advances on the mechanisms of how Sema3E–Plexin-D1 interaction shapes the networks of multiple biological systems, in particular the vascular and nervous systems.     

Semaphorin-4A, an activator for T-cell-mediated immunity, suppresses angiogenesis via Plexin-D1

Originally identified as axon guidance molecules, semaphorins are now known to be widely expressed mediators that play significant roles in immune responses and organ morphogenesis. However, not much is known about the signaling pathways via which they exert their organ-specific effects. Here we demonstrate that Sema4A, previously identified as an activator of T-cell-mediated immunity, is expressed in endothelial cells, where it suppresses vascular endothelial growth factor (VEGF)-mediated endothelial cell migration and proliferation in vitro and angiogenesis in vivo. Mice lacking Sema4A exhibit enhanced angiogenesis in response to VEGF or inflammatory stimuli. In addition, binding and functional experiments revealed Plexin-D1 to be a receptor for Sema4A on endothelial cells, indicating that Sema4A exerts organ-specific activities via different receptor-mediated signaling pathways: via Plexin-D1 in the endothelial cells and via T-cell immunoglobulin and mucin domain-2 in T cells. The effects of Sema4A on endothelial cells are dependent on its ability to suppress VEGF-mediated Rac activation and integrin-dependent cell adhesion. It thus appears that Sema4A-Plexin-D1 signaling negatively regulates angiogenesis.     

Systemic Sclerosis Sera Impair Angiogenic Performance of Dermal Microvascular Endothelial Cells: Therapeutic Implications of Cyclophosphamide

In systemic sclerosis (SSc), dermal capillaries are progressively lost with consequent chronic tissue hypoxia insufficiently compensated by angiogenesis. Clinical studies reported that intravenous cyclophosphamide (CYC) may improve SSc-related peripheral microvascular damage. Recently, we showed that CYC treatment may normalize SSc sera-induced abnormalities in endothelial cell-matrix interactions. Our objective was to evaluate in vitro the effects of sera from treatment-naïve or CYC-treated SSc patients on dermal blood microvascular endothelial cell (dMVEC) angiogenesis, migration, proliferation and apoptosis. dMVECs were challenged with sera from 21 SSc patients, treatment-naïve (n = 8) or under CYC treatment (n = 13), and 8 healthy controls. Capillary morphogenesis on Geltrex matrix was significantly reduced upon challenge with sera from naïve SSc patients compared with healthy controls. When dMVECs were challenged with sera from CYC-treated SSc patients, their angiogenic capacity was comparable to that of cells treated with healthy sera. Wound healing capacity and chemotaxis in Boyden chamber were both significantly decreased in the presence either of naïve or CYC-treated SSc sera compared with healthy sera. WST-1 assay revealed that cell proliferation was significantly decreased in dMVECs challenged with sera from naïve SSc patients compared with healthy sera. Conversely, dMVEC proliferation was not impaired in the presence of sera from CYC-treated SSc patients. Accordingly, the percentage of TUNEL-positive apoptotic dMVECs was significantly higher in the presence of sera from naïve SSc patients than healthy controls, while CYC-treated SSc sera did not induce dMVEC apoptosis. Levels of the angiostatic mediators endostatin, pentraxin 3, angiostatin and matrix metalloproteinase-12 were all significantly elevated in sera from naïve SSc patients compared with sera from both healthy controls and CYC-treated SSc patients. In SSc, CYC treatment might boost angiogenesis and consequently improve peripheral microangiopathy through the normalization of the endothelial cell-matrix interactions, reduction of endothelial cell apoptosis and rebalance of dysregulated angiostatic factors.     

Decreased expression of the endothelial cell-derived factor EGFL7 in systemic sclerosis: potential contribution to impaired angiogenesis and vasculogenesis

Introduction

Microvascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc.

Methods

Serum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting.

Results

Serum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively).

Conclusions

Our findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.     

Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells

The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC “routes”). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC “target”). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.     

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

Associations between circulating endostatin levels and vascular organ damage in systemic sclerosis and mixed connective tissue disease: an observational study

IntroductionSystemic sclerosis (SSc) and mixed connective tissue disease (MCTD) are chronic immune-mediated disorders complicated by vascular organ damage. The aim of this study was to examine the serum levels of the markers of neoangiogenesis: endostatin and vascular endothelial growth factor (VEGF), in our unselected cohorts of SSc and MCTD.MethodsSera of SSc patients (N = 298) and MCTD patients (N = 162) from two longitudinal Norwegian cohorts were included. Blood donors were included as controls (N = 100). Circulating VEGF and endostatin were analyzed by enzyme immunoassay.ResultsMean endostatin levels were increased in SSc patients 93.7 (37) ng/ml (P < .001) and MCTD patients 83.2 (25) ng/ml (P < .001) compared to controls 65.1 (12) ng/ml. Median VEGF levels were elevated in SSc patients 209.0 (202) pg/ml compared to MCTD patients 181.3 (175) pg/ml (P = .017) and controls 150.0 (145) pg/ml (P < .001). Multivariable analysis of SSc subsets showed that pulmonary arterial hypertension (coefficient 15.7, 95 % CI: 2.2–29.2, P = .023) and scleroderma renal crisis (coefficient 77.6, 95 % CI: 59.3–100.0, P < .001) were associated with elevated endostatin levels. Multivariable analyses of MCTD subsets showed that digital ulcers were associated with elevated endostatin levels (coefficient 10.5, 95 % CI: 3.2–17.8, P = .005). The risk of death increased by 1.6 per SD endostatin increase (95 % CI: 1.2–2.1, P = .001) in the SSc cohort and by 1.6 per SD endostatin increase (95 % CI: 1.0–2.4, P = .041) in the MCTD cohort after adjustments to known risk factors.ConclusionsEndostatin levels were elevated in patients with SSc and MCTD, particularly SSc patients with pulmonary arterial hypertension and scleroderma renal crisis, and MCTD patients with digital ulcers. Elevated endostatin levels were also associated with increased all-cause mortality during follow-up in both groups of patients. We propose that endostatin might indicate the degree of vascular injury in SSc and MCTD patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0756-5) contains supplementary material, which is available to authorized users.     

Epigenetic down-regulation of microRNA-126 in scleroderma endothelial cells is associated with impaired responses to VEGF and defective angiogenesis

Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA-126 (miR-126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty-related protein-1 (SPRED1), and phosphoinositide-3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR-126 expression was measured by qPCR and in situ hybridization. Matrigel-based tube assembly was used to test angiogenesis. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by hsa-miR-126 Mimic. Epigenetic regulation of miR-126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR-126 promoter region. MiR-126 expression, as well as EGFL7 (miR-126 host gene), in SSc-MVECs and skin, was significantly down-regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR-126 in NL-MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR-126 in SSc-MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR-126 and EGFL7 expression levels in SSc-MVECs. Heavy methylation in miR-126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down-regulation of miR-126 results in impaired VEGF responses.     

Estradiol promotes the development of a fibrotic phenotype and is increased in the serum of patients with systemic sclerosis

Introduction

Systemic sclerosis (SSc) is more prevalent in women. Our goal is to determine the effects of 17β-estradiol (E2) on the development of fibrosis and to compare circulating levels of estrogens in SSc patients and healthy controls.

Methods

Using primary human dermal fibroblasts, we evaluated the effect of E2 on fibronectin (FN) expression with and without the estrogen receptor (ER) antagonist ICI 182,780, inhibitors of signaling, propyl-pyrazole-triol, an ERα specific ligand, and genistein, an ERβ selective ligand, to identify the signaling pathways mediating E2''s effect. We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model. Lastly, we measured levels of E2 and estrone in serum samples from SSc patients with diffuse cutaneous involvement and healthy controls using mass spectrometry.

Results

E2 increased expression of FN in dermal fibroblasts. ICI 182,780, inositol-1,4,5-triphosphate inhibitor, and p38 mitogen-activated protein kinase inhibitor blocked the effects of E2 on FN. Propyl-pyrazole-triol, but not genistein, significantly increased FN expression. Ex vivo, E2 induced fibrosis of human skin. The effects of E2 were abrogated by ICI 182,780. Circulating levels of E2 and estrone were significantly increased in sera of patients with diffuse cutaneous SSc.

Conclusion

Our findings implicate estrogens in the fibrotic process and may explain the preponderance of SSc in women. ICI 182,780 or other ER signaling antagonists may be effective agents for the treatment of fibrosis.     

Semaphorin 3E Initiates Antiangiogenic Signaling through Plexin D1 by Regulating Arf6 and R-Ras

Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.Pathological angiogenesis characterizes numerous human diseases, ranging from chronic inflammation, atherosclerosis, diabetic retinopathy, and age-related macular degeneration to cancer (5, 11, 30). Thus, elucidating the mechanisms underlying normal and aberrant blood vessel growth may provide new therapeutic options for many highly prevalent disease conditions. Ultimately, normal angiogenesis results from a precise balance between pro- and antiangiogenic mediators. Among the former, the family of vascular endothelial growth factors (VEGFs), basic fibroblastic growth factor (bFGF), sphingosine-1-phosphate (S1P), and the chemokines interleukin-8/CXCL8 and SDF-1/CXCL12 and their receptors are some of the most widely investigated (reviewed in references 3, 5, 8, and 17). The best-known angiogenesis inhibitors are proteolytic cleavage products of extracellular matrix (ECM) or serum components, such as endostatin, angiostatin, arresten, and tumstatin (reviewed in references 11 and 20). Antiangiogenic cytokines have also been described, including interferons and certain interleukins, which appear to act indirectly by limiting the expression of proangiogenic mediators or inducing antiangiogenic molecules (reviewed in references 11 and 20). In contrast, there are few known developmentally regulated, naturally occurring antiangiogenic molecules, which include platelet factor 4 (18), thrombospondin 1 (14), and pigment epithelium-derived factor (PEDF) (9). Their precise mechanism of action is not fully understood, thus limiting the ability to design new molecularly based antiangiogenic strategies.Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of the netrin, slit, eph, and semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological blood vessel development (6). For example, multiple secreted class III semaphorins, which regulate developmental axonal growth (23, 27), are now known to act through their receptors, the A family plexins (plexins A1, A2, and A3), and their coreceptors, neuropilin 1 and neuropilin 2, to initiate pro- and antiangiogenic responses (reviewed in references 6 and 19). However, neuropilins also act as coreceptors for multiple angiogenic factors, such as VEGF, thus limiting our ability to distinguish the downstream signaling events initiated by semaphorins from those resulting from their interplay with endothelial growth and motility factors (19). In this regard, recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, independently of neuropilins to control endothelial cell (EC) positioning and patterning of the developing vasculature (13, 15). These findings prompted us to explore whether Sema3E acts as a natural antiangiogenic molecule and, if so, to investigate the underlying molecular mechanism. Indeed, we show here that Sema3E is a potent inhibitor of adult and tumor-induced angiogenesis. Sema3E causes filopodial retraction in endothelial tip cells and inhibits endothelial cell adhesion by disrupting integrin-mediated adhesive structures. At the molecular level, this process involves the stimulation of plexin D1 by Sema3E, which in turn interferes with R-Ras function and leads to the rapid activation of Arf6, thus revealing a novel physiological antiangiogenic signaling route.     

Serum neurotrophin profile in systemic sclerosis

Background

Neurotrophins (NTs) are able to activate lymphocytes and fibroblasts; they can modulate angiogenesis and sympathic vascular function. Thus, they can be implicated in the three pathogenic processes of systemic sclerosis (SSc). The aims of this study are to determine blood levels of Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT-3) in SSc and to correlate them with clinical and biological data.

Methods

Serum samples were obtained from 55 SSc patients and 32 control subjects to measure NTs levels by ELISA and to determine their relationships with SSc profiles.

Findings

Serum NGF levels were higher in SSc patients (288.26±170.34 pg/mL) than in control subjects (170.34±50.8 pg/mL, p<0.001) and correlated with gammaglobulins levels and the presence of both anti-cardiolipin and anti-Scl-70 antibodies (p<0.05). In contrast, BDNF levels were lower in SSc patients than in controls (1121.9±158.1 vs 1372.9±190.9 pg/mL, p<0.0001), especially in pulmonary arterial hypertension and diffuse SSc as compared to limited forms (all p<0.05). NT-3 levels were similar in SSc and in the control group (2657.2±2296 vs 2959.3±2555 pg/mL, NS). BDNF levels correlated negatively with increased NGF levels in the SSc group (and not in controls).

Conclusion

Low BDNF serum levels were not previously documented in SSc, particularly in the diffuse SSc subset and in patients with pulmonary hypertension or anti-Scl-70 antibodies. The negative correlation between NGF and BDNF levels observed in SSc and not in healthy controls could be implicated in sympathic vascular dysfunction in SSc.     

Serum IL-15 in patients with early systemic sclerosis: a potential novel marker of lung disease

The pathogenesis of systemic sclerosis (SSc) is characterized by autoimmunity, vasculopathy and fibrosis. IL-15 is a pleiotropic cytokine that has impact on immune, vascular and connective tissue cells. We therefore investigated IL-15 in the circulation of patients with early SSc and explored possible associations of serum IL-15 with vasculopathy and fibrosis. Serum levels of IL-15 were analysed in 63 consecutive patients with SSc of disease duration less than 4 years and without disease-modifying treatment. Thirty-three age-matched healthy control individuals were enrolled. Serum IL-15 levels were increased in the sera of SSc patients compared with that of healthy control individuals (P < 0.01). Serum IL-15 levels correlated with impaired lung function, assessed both by the vital capacity (P < 0.05) and by the carbon monoxide diffusion capacity (P < 0.05). The association between IL-15 and the vital capacity remained after multiple linear regression analysis. Patients with intermediate serum IL-15 levels had a higher prevalence of increased systolic pulmonary pressure compared with patients with either low or high serum IL-15 levels (P < 0.05). Moreover, increased serum IL-15 levels were associated with a reduced nailfold capillary density in multivariable logistic regression analysis (P < 0.01). Serum IL-15 levels also correlated inversely with the systolic blood pressure (P < 0.01). We conclude that IL-15 is associated with fibrotic as well as vascular lung disease and vasculopathy in early SSc. IL-15 may contribute to the pathogenesis of SSc. IL-15 could also be a candidate biomarker for pulmonary involvement and a target for therapy in SSc.     

Endothelial Expression of Endothelin Receptor A in the Systemic Capillary Leak Syndrome

Idiopathic systemic capillary leak syndrome (SCLS) is a rare and potentially fatal vascular disorder characterized by reversible bouts of hypotension and edema resulting from fluid and solute escape into soft tissues. Although spikes in permeability-inducing factors have been linked to acute SCLS flares, whether or not they act on an inherently dysfunctional endothelium is unknown. To assess the contribution of endothelial-intrinsic mechanisms in SCLS, we derived blood-outgrowth endothelial cells (BOEC) from patients and healthy controls and examined gene expression patterns. Ednra, encoding Endothelin receptor A (ETA)—the target of Endothelin 1 (ET-1)—was significantly increased in SCLS BOEC compared to healthy controls. Although vasoconstriction mediated by ET-1 through ETA activation on vascular smooth muscle cells has been well characterized, the expression and function of ETA receptors in endothelial cells (ECs) has not been described. To determine the role of ETA and its ligand ET-1 in SCLS, if any, we examined ET-1 levels in SCLS sera and functional effects of endothelial ETA expression. ETA overexpression in EAhy926 endothelioma cells led to ET-1-induced hyper-permeability through canonical mechanisms. Serum ET-1 levels were elevated in acute SCLS sera compared to remission and healthy control sera, suggesting a possible role for ET-1 and ETA in SCLS pathogenesis. However, although ET-1 alone did not induce hyper-permeability of patient-derived BOEC, an SCLS-related mediator (CXCL10) increased Edrna quantities in BOEC, suggesting a link between SCLS and endothelial ETA expression. These results demonstrate that ET-1 triggers classical mechanisms of vascular barrier dysfunction in ECs through ETA. Further studies of the ET-1-ETA axis in SCLS and in more common plasma leakage syndromes including sepsis and filovirus infection would advance our understanding of vascular integrity mechanisms and potentially uncover new treatment strategies.     

Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

IntroductionAbnormal oxidative stress has been described in systemic sclerosis (SSc) and previous works from our laboratory demonstrated an increased generation of reactive oxygen species (ROS) by SSc fibroblasts and monocytes. This study investigated the ability of SSc T lymphocytes to produce ROS, the molecular pathway involved, and the biological effects of ROS on SSc phenotype.MethodsPeripheral blood T lymphocytes were isolated from serum of healthy controls or SSc patients by negative selection with magnetic beads and activated either with PMA or with magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Intracellular ROS generation was measured using a DCFH-DA assay in a plate reader fluorimeter or by FACS analysis. CD69 expression and cytokine production were analyzed by FACS analysis. Protein expression was studied using immunoblotting techniques and mRNA levels were quantified by real-time PCR. Cell proliferation was carried out using a BrdU incorporation assay.ResultsPeripheral blood T lymphocytes from SSc patients showed an increased ROS production compared to T cells from healthy subjects. Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox. Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.ConclusionsSSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production. These data confirm lymphocytes as key cellular players in the pathogenesis of systemic sclerosis and suggest a crucial link between ROS and T cell activation.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0591-8) contains supplementary material, which is available to authorized users.     

Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3a ^loxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.