Claudia Ceccarelli

Overexpression of VEGF165b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

Rationale: Systemic sclerosis (SSc) is characterized by widespread microangiopathy, fibrosis, and autoimmunity. Despite the lack of angiogenesis, the expression of vascular endothelial growth factor A (VEGF) was shown to be upregulated in SSc skin and circulation; however, previous studies did not distinguish between proangiogenic VEGF165 and antiangiogenic VEGF165b isoforms, which are generated by alternative splicing in the terminal exon of VEGF pre-RNA. Objective: We investigated whether VEGF isoform expression could be altered in skin and circulation of patients with SSc. Methods and Results: Here, we show that the endogenous antiangiogenic VEGF165b splice variant is selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF165b expression correlated with increased expression of profibrotic transforming growth factor-&bgr;1 and serine/arginine protein 55 splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. Circulating levels of VEGF165b were significantly higher in patients with SSc than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF165b than healthy MVECs. Transforming growth factor-&bgr;1 upregulated the expression of VEGF165b and serine/arginine protein 55 in both SSc and healthy MVECs. In SSc MVECs, VEGF receptor-2 was overexpressed, but its phosphorylation was impaired. Recombinant VEGF165b and SSc-MVEC–conditioned medium inhibited VEGF165-mediated VEGF receptor-2 phosphorylation and capillary morphogenesis in healthy MVECs. The addition of anti-VEGF165b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC–conditioned medium. Capillary morphogenesis was severely impaired in SSc MVECs and could be ameliorated by treatment with recombinant VEGF165 and anti-VEGF165b blocking antibodies. Conclusions: In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia.

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro

Objective To characterise bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic sclerosis (SSc) for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis and fibrosis. The study also analysed whether the production/release of bioactive mediators by MSCs were affected by stimulation with cytokines found upregulated in SSc serum and tissues, and whether MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis. Methods MSCs obtained from five patients with early severe diffuse SSc (SSc-MSCs) and five healthy donors (H-MSCs) were stimulated with vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ) or stromal cell-derived factor-1 (SDF-1). Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFβ1 and receptors TβRI and TβRII were evaluated by quantitative real-time PCR, western blotting and confocal microscopy. VEGF, SDF-1 and TGFβ1 secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. Results In SSc-MSCs the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared with H-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation and CXCR4 redistributed from the cytoplasm to the cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFβ and SDF-1 stimulation upregulated TGFβ1, TβRI and TβRII in SSc-MSCs. TβRII redistributed from the cytoplasm to focal adhesion contacts. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than H-MSCs. Experiments with blocking antibodies showed that MSC-derived cytokines were responsible for this potent proangiogenic effect. Conclusion SSc-MSCs constitutively overexpress and release bioactive mediators/proangiogenic factors and potentiate dermal MVEC angiogenesis.

Differential expression of junctional adhesion molecules in different stages of systemic sclerosis.

OBJECTIVE Systemic sclerosis (SSc) is characterized by early perivascular inflammation, microvascular endothelial cell (MVEC) activation/damage, and defective angiogenesis. Junctional adhesion molecules (JAMs) regulate leukocyte recruitment to sites of inflammation and ischemia-reperfusion injury, vascular permeability, and angiogenesis. This study was undertaken to investigate the possible role of JAMs in SSc pathogenesis. METHODS JAM-A and JAM-C expression levels in skin biopsy samples from 25 SSc patients and 15 healthy subjects were investigated by immunohistochemistry and Western blotting. Subcellular localization of JAMs in cultured healthy dermal MVECs and SSc MVECs was assessed by confocal microscopy. Serum levels of soluble JAM-A (sJAM-A) and sJAM-C in 64 SSc patients and 32 healthy subjects were examined by enzyme-linked immunosorbent assay. RESULTS In control skin, constitutive JAM-A expression was observed in MVECs and fibroblasts. In early-stage SSc skin, JAM-A expression was strongly increased in MVECs, fibroblasts, and perivascular inflammatory cells. In late-stage SSc, JAM-A expression was decreased compared with controls. JAM-C was weakly expressed in control and late-stage SSc skin, while it was strongly expressed in MVECs, fibroblasts, and inflammatory cells in early-stage SSc. Surface expression of JAM-A was higher in early-stage SSc MVECs and increased in healthy MVECs stimulated with early-stage SSc sera. JAM-C was cytoplasmic in resting healthy MVECs, while it was recruited to the cell surface upon challenge with early-stage SSc sera. Early-stage SSc MVECs exhibited constitutive surface JAM-C expression. In SSc, increased levels of sJAM-A and sJAM-C correlated with early disease and measures of vascular damage. CONCLUSION Our findings indicate that JAMs may participate in MVEC activation, inflammatory processes, and impaired angiogenesis in different stages of SSc.

Increased circulating levels of interleukin 33 in systemic sclerosis correlate with early disease stage and microvascular involvement

Early activation/damage of endothelial cells and multiple profibrotic T helper type 2 (Th2)-associated cytokines play an important role in the pathogenesis of systemic sclerosis (SSc).1 Interleukin 33 (IL-33) was recently identified as a member of the IL-1 family and a ligand for the orphan receptor ST2L, which mediates the action of IL-33 on polarised Th2 lymphocytes and other leucocyte subsets, and tissue-resident cells, such as vascular endothelial cells and fibroblasts/myofibroblasts.2 IL-33 has been shown to induce IL-13-dependent cutaneous fibrosis and stimulate angiogenesis and vascular permeability.3 4 In healthy human tissues IL-33 protein is constitutively highly expressed in the nuclei of resting endothelial cells, but rapidly lost upon angiogenic or proinflammatory activation.2 Indeed, IL-33 is thought to function as an endogenous ‘alarmin’ that is rapidly released after endothelial cell damage to alert the cells of the innate and adaptive immune system.2 Recently, we have shown that microvascular endothelial cells in inflamed skin from patients with early-stage SSc lacked or had decreased nuclear IL-33 protein expression.5 Instead, IL-33 transcript levels were even upregulated suggesting that, …

RANK-RANKL-OPG in hemophilic arthropathy: from clinical and imaging diagnosis to histopathology.

Objective. Hemarthrosis triggers hemophilic arthropathy, involving the target joints. The histopathogenesis of blood-induced joint damage remains unclear. The triad of receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG; RANK-RANKL-OPG) controls bone turnover. Our aim was to evaluate RANK-RANKL-OPG expression in the synovium of hemophilic patients with severe arthropathy. Methods. Synovial biopsies were obtained from 18 patients with hemophilic arthropathy and 16 with osteoarthritis (OA) who were undergoing total knee replacement and synovectomy. The severity of hemophilic arthropathy was evaluated according to ultrasonography score, the World Federation of Hemophilia (WFH) orthopedic joint scale, and the radiographic Pettersson score. RANK-RANKL-OPG expression was examined by immunohistochemistry and Western blotting. Serum levels of soluble RANKL (sRANKL) and OPG from an extended group of 67 patients with hemophilic arthropathy and 30 healthy controls were measured by ELISA. Results. The mean ultrasonography, WFH orthopedic joint scale, and Pettersson scores in patients with hemophilic arthropathy indicated severe arthropathy. A decreased expression of OPG was found in hemophilic arthropathy synovium compared with patients with OA. RANK and RANKL immunopositivity was strong in the lining and sublining layers in hemophilic arthropathy synovial tissue. Western blotting confirmed the immunohistological findings. Serum levels of sRANKL and OPG in patients with hemophilia were lower than in healthy controls. Conclusion. In hemophilic arthropathy, the synovium highly expressed RANK and RANKL, whereas OPG immunopositivity decreased, suggesting an osteoclastic activation. Low tissue expression of OPG paralleled the serum levels of this protein and the severity of hemophilic arthropathy assessed by ultrasonography, Pettersson, and WFH orthopedic joint scale scores.

Overexpression of VEGF 165 b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by widespread microangiopathy, fibrosis, and autoimmunity that affects the skin and internal organs. Although in SSc there is a lack of sufficient angiogenic response to chronic tissue ischemia culminating in the loss of capillary vessels, the expression of vascular endothelial growth factor-A (VEGF) has paradoxically been shown to be upregulated in SSc skin and circulation. However, previous studies in the field did not distinguish between the proangiogenic VEGF 165 and antiangiogenic VEGF 165 b isoforms that are generated by alternative splicing in the terminal exon of VEGF pre-RNA. In the present study, we investigated whether VEGF isoform expression could be altered in skin and circulation of SSc patients. Using RT-PCR, quantitative real-time PCR, Western blotting, immunohistochemistry and confocal microscopy, we could show that the VEGF 165 b splice variant was selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF 165 b expression correlated with increased expression of profibrotic transforming growth factor-β1 (TGF-β1) and serine/arginine protein 55 (SRp55) splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. ELISA on plasma samples revealed that circulating levels of VEGF 165 b were significantly higher in SSc patients than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF 165 b than healthy MVECs (H-MVECs). TGF-β1 upregulated the expression of VEGF 165 b and SRp55 in both SSc- and H-MVECs. In SSc-MVECs, VEGF receptor-2 (VEGFR-2) was overexpressed, but its phosphorylation and ERK1/2 downstream signaling were impaired. Recombinant human VEGF 165 b and SSc-MVEC–conditioned medium inhibited VEGF 165 -mediated VEGFR-2 phosphorylation, ERK1/2 activation and capillary morphogenesis on Matrigel in H-MVECs. The addition of anti-VEGF 165 b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC–conditioned medium. Capillary morphogenesis was severely impaired in SSc-MVECs and could be ameliorated by treatment with recombinant VEGF 165 and anti-VEGF 165 b blocking antibodies. In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia. The combination of proangiogenic VEGF 165 administration and VEGF 165 b neutralization might represent a potential therapeutic strategy to promote effective angiogenesis and capillary regeneration in SSc.

Differential expression of junctional adhesion molecules in systemic sclerosis (SSc) skin

Objective Junctional adhesion molecule (JAM)-A and JAM-C regulate leucocyte-endothelial cell (EC) interactions. JAMs also interact at inter-EC junctions regulating vascular permeability and are implicated in pathophysiological processes involving leucocyte transmigration, tight junction assembly and angiogenesis. SSc is characterised by early perivascular inflammatory infiltrates, EC damage and defective angiogenesis. We investigated the expression of JAMs in SSc skin and dermal microvascular ECs (MVECs) challenged with SSc sera. Methods Skin biopsies were obtained from the involved skin of 16 SSc patients (10 early, 6 late phase) and from 10 controls. Skin sections were stained with primary antibodies (Abs) anti-human JAM-A or JAM-C followed by fluorochrome-conjugated secondary Abs. The lymphatic EC specific marker podoplanin (D2-40) was used to differentiate blood (D2-40–) and lymphatic (D2-40+) vessels. Human dermal MVECs were stimulated with VEGF or early SSc (n=5) and control (n=4) sera for 1, 6, 24 h. MVECs were double-immunolabeled for JAM-C and the human tight junction protein zonula occludens-1 (ZO-1), or for JAM-A and the pro-angiogenic αVβ3 integrin. Immunostained tissue sections and cells were examined by confocal laser scanning microscopy. Densitometric analysis of staining intensity was performed using ImageJ software. Results In control skin, constitutive expression of JAM-A was observed in blood and lymphatic ECs, fibroblasts and keratinocytes. In early SSc skin, JAM-A expression was increased in blood and lymphatic vessels and fibroblasts. Moreover, perivascular inflammatory cells showed strong JAM-A positivity. In late SSc, JAM-A expression was weaker than in controls. JAM-C was weakly expressed in controls. In early SSc, JAM-C expression was markedly observed in blood and lymphatic ECs, fibroblasts, and inflammatory cells. Instead, in late SSc JAM-C expression was similar to controls. Stimulation of MVECs with early SSc sera increased the cell surface expression of JAM-A and αVβ3 integrin. JAM-C expression was found in MVEC cytoplasm at basal conditions and under stimulation with control sera. Upon early SSc sera challenging, JAM-C was rapidly recruited to tight junctions where it colocalised with ZO-1, with the maximun effect after 1 h. Similar effects were observed after 1 h VEGF stimulation. Conclusions JAMs are differentially expressed in early and late SSc skin. Early SSc sera affect the expression and subcellular localisation of JAMs in dermal MVECs. In SSc, JAMs may participate in early inflammatory process, EC activation and impaired angiogenesis.

Involvement of junctional adhesion molecules in lymphatic vascular remodelling in rheumatoid arthritis

Background and objective Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, in which neovascularisation of the synovium is regarded as a crucial step in its development and progression. Lymphatic neovascularisation has been observed in RA joints. Junctional adhesion molecule A (JAM-A) and JAM-C regulate leucocyte-endothelial cell (EC) interactions. JAMs also interact at inter-EC junctions regulating vascular permeability and are implicated in pathophysiological processes involving leukocyte transmigration, tight junction assembly and angiogenesis. The aim of this study was to investigate the role of JAM-A and JAM-C in mediating the lymphoangiogenic process in RA synovium. Materials and methods Synovial biopsies from RA and osteoarthritic (OA) patients, used as control, has been collected during arthroplasty or synovectomy. Synovial sections were stained with primary antibodies (Abs) antihuman JAM-A or JAM-C followed by fluorochrome-conjugated secondary Abs. The lymphatic EC specific marker podoplanin (D2-40) was used to differentiate blood (D2-40−) and lymphatic (D2-40+) vessels. Results Immunohistochemical analysis showed, in RA synovium, the presence of abundant D2-40+ structures, identifiable as lymphatic vessels. Many D2-40+ cells were also found in the intimal lyning layer, specially in the hyperplasic areas. Lymphatic vessels were distributed in the sublining, specially around blood vessels. Clusters of D2-40+ cells were also found in the inflammatory infiltrate, in structures resembling new lymphatic vessels. In all sections from RA, JAM-A and JAM-C were observed on almost all vasculature including the lymphatic (D2-40+) and blood vessels (D2-40−). Initial lymphatic vessels reacted with anti-JAM-A and anti-JAM-C. In the synovial lining, JAM-A and JAM-C were expressed on D2-40+ macrophage-like synoviocites. D2-40+ cells in the inflammatory infiltrate showed positivity for both JAM-A and JAM-C. The immunostaining for both JAMs was weaker in OA than in RA synovium. Conclusion This results confirm the presence of an increased number of lymphatic vessels in RA synovial tissue. Moreover, the authors observed D2-40+ structures, mostly around pre-existing blood vessels, indicating the development of a new lymphatic network in the synovial sublining. The presence of D2-40+ cells in the inflammatory infiltrate and in the macrophage-like synoviocites, lead us to hypothesise that in RA, macrophages are activated and may function as lymphatic precursors. In RA synovium, both JAM-A and JAM-C are expressed in all D2-40+ cells, indicating their participation in the inflammatory process and intercellular junction assembly. Their expression in new lymphatic vessels and in D2-40+ cells stimulated to form new vessels, confirm their involvement in the lymphoangiogenic process occurring in RA.