Evidence of telomere attrition and a potential role for DNA damage in systemic sclerosis.

BACKGROUND: To investigate the role of cell senescence in systemic sclerosis (SSc), we analyzed telomere shortening (TS) in SSc patients and the effect of targeting DNA damage in the bleomycin model of skin fibrosis. RESULTS: Telomere length (TL) in blood leukocytes of 174 SSc patients and 68 healthy controls was measured by Southern blot, and we found shorter age-standardized TL in SSc patients compared to healthy controls. TL was shorter in SSc patients with ILD compared to those without ILD and in anti-topoisomerase I positive compared to anti-centromere positive patients. To analyze the potential role of DNA damage in skin fibrosis, we evaluated the effects of the DNA protective GSE4 peptide in the bleomycin mouse model of scleroderma and the fibrotic response of cultured human dermal fibroblasts. Administration of GSE4-nanoparticles attenuated bleomycin-induced skin fibrosis as measured by Masson's staining of collagen and reduced Acta2 and Ctgf mRNA expression, whereas transduction of dermal fibroblasts with a lentiviral GSE4 expression vector reduced COL1A1, ACTA2 and CTGF gene expression after stimulation with bleomycin or TGF-ß, in parallel to a reduction of the phospho-histone H2A.X marker of DNA damage. CONCLUSIONS: SSc is associated with TS, particularly in patients with lung disease or anti-topoisomerase I antibodies. Administration of GSE4 peptide attenuated experimental skin fibrosis and reduced fibroblast expression of profibrotic factors, supporting a role for oxidative DNA damage in scleroderma.

TFAM-deficient mouse skin fibroblasts - an ex vivo model of mitochondrial dysfunction.

Mitochondrial dysfunction associates with several pathological processes and contributes to chronic inflammatory and ageing-related diseases. Mitochondrial transcription factor A (TFAM) plays a critical role in maintaining mtDNA integrity and function. Taking advantage of Tfamfl/fl UBC-Cre/ERT2+/+ mice to investigate mitochondrial dysfunction in the stromal cell component, we describe an inducible in vitro model of mitochondrial dysfunction by stable depletion of TFAM in primary mouse skin fibroblasts (SK-FBs) after 4-hydroxytamoxifen (4-OHT) administration. Tfam gene deletion caused a sustained reduction in Tfam and mtDNA-encoded mRNA in Cre(+) SK-FBs cultured for low (LP) and high (HP) passages that translated into a loss of TFAM protein. TFAM depletion led to a substantial reduction in mitochondrial respiratory chain complexes that was exacerbated in HP SK-FB cultures. The assembly pattern showed that the respiratory complexes fail to reach the respirasome in 4-OHT-treated Cre(+) SK-FBs. Functionally, mito-stress and glycolysis-stress tests showed that mitochondrial dysfunction developed after long-term 4-OHT treatment in HP Cre(+) SK-FBs and was compensated by an increase in the glycolytic capacity. Finally, expression analysis revealed that 4-OHT-treated HP Cre(+) SK-FBs showed a senescent and pro-inflammatory phenotype.

IL6/sIL6R regulates TNFα-inflammatory response in synovial fibroblasts through modulation of transcriptional and post-transcriptional mechanisms.

INTRODUCTION: The clinical efficacy of specific interleukin-6 inhibitors has confirmed the central role of IL6 in rheumatoid arthritis (RA). However the local role of IL6, in particular in synovial fibroblasts (SF) as a direct cellular target to IL6/sIL6R signal is not well characterized. The purpose of the study was to characterize the crosstalk between TNFα and IL6/sIL6R signaling to the effector pro-inflammatory response of SF. METHODS: SF lines were stimulated with either TNFα, IL6/sIL6R, or both together, for the time and dose indicated for each experiment, and where indicated, cells were treated with inhibitors actinomycin D, adalimumab, ruxolitinib and cycloheximide. mRNA expression of cytokines, chemokines and matrix metalloproteases (MMPs) were analyzed by quantitative RT-PCR. Level of IL8/CXCL8 and CCL8 in culture supernatants was measured by ELISA. Mononuclear and polymorphonuclear cells migration assays were assessed by transwell using conditioned medium from SF cultures. Statistical analyses were performed as indicated in the corresponding figure legends and a p-value < 0.05 was considered statistically significant. RESULTS: The stimulation of SF with IL6/sIL6R and TNFα, cooperatively promotes the expression of mono- and lymphocytic chemokines such as IL6, CCL8 and CCL2, as well as matrix degrading enzymes such as MMP1, while inhibiting the induction of central neutrophil chemokines such as IL8/CXCL8. These changes in the pattern of chemokines expression resulted in reduced polymorphonuclear (PMN) and increased mononuclear cells (MNC) chemoattraction by SF. Mechanistic analyses of the temporal expression of genes demonstrated that the cooperative regulation mediated by these two factors is mostly induced through de novo transcriptional mechanisms activated by IL6/sIL6R. Furthermore, we also demonstrate that TNFα and IL6/sIL6R cooperation is partially mediated by the expression of secondary factors signaling through JAK/STAT pathways. CONCLUSIONS: These results point out to a highly orchestrated response to IL6 in TNFα-induced SF and provide additional insights into the role of IL6/sIL6R in the context of RA, highlighting the contribution of IL6/sIL6R to the interplay of SF with other inflammatory cells.

Senescent synovial fibroblasts accumulate prematurely in rheumatoid arthritis tissues and display an enhanced inflammatory phenotype.

BACKGROUND: Accumulation of senescent cells has been associated with pro-inflammatory effects with deleterious consequences in different human diseases. The purpose of this study was to analyze cell senescence in human synovial tissues (ST), and its impact on the pro-inflammatory function of synovial fibroblasts (SF). RESULTS: The expression of the senescence marker p16INK4a (p16) was analyzed by immunohistochemistry in rheumatoid arthritis (RA), osteoarthritis (OA), and normal ST from variably aged donors. The proportion of p16(+) senescent cells in normal ST from older donors was higher than from younger ones. Although older RA and OA ST showed proportions of senescent cells similar to older normal ST, senescence was increased in younger RA ST compared to age-matched normal ST. The percentage of senescent SA-ß-gal(+) SF after 14 days in culture positively correlated with donor's age. Initial exposure to H2O2 or TNFα enhanced SF senescence and increased mRNA expression of IL6, CXCL8, CCL2 and MMP3 and proteins secretion. Senescent SF show a heightened IL6, CXCL8 and MMP3 mRNA and IL-6 and IL-8 protein expression response upon further challenge with TNFα. Treatment of senescent SF with the senolytic drug fenofibrate normalized IL6, CXCL8 and CCL2 mRNA expression. CONCLUSIONS: Accumulation of senescent cells in ST increases in normal aging and prematurely in RA patients. Senescence of cultured SF is accelerated upon exposure to TNFα or oxidative stress and may contribute to the pathogenesis of synovitis by increasing the production of pro-inflammatory mediators.

Hif-1α Knockdown Reduces Glycolytic Metabolism and Induces Cell Death of Human Synovial Fibroblasts Under Normoxic Conditions.

Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O2 environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1α target genes in hypoxia demonstrated the presence of functional HIF-1α in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1α target even in normoxia. HIF-1α silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1α targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1α plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1α provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases.

CD271(+) stromal cells expand in arthritic synovium and exhibit a proinflammatory phenotype.

BACKGROUND: CD271(+) stromal cells (SCs) with multipotent stem cell capacity have been identified in synovial tissues, but their functional significance is unknown. We analyzed the distribution of CD271(+) cells in inflammatory synovial tissues as well as their ex vivo immunomodulatory and inflammatory phenotypes. METHODS: CD271 expression was analyzed by immunohistochemistry in synovial tissues and by flow cytometry in primary adherent synovial cell cultures from rheumatoid arthritis (RA), osteoarthritis (OA), and non-inflammatory control tissues. Isolation of CD271(+) synovial SCs was carried out by magnetic cell sorting. Allogeneic T-cell/SC cocultures were performed to analyze the regulatory capacity of these cells on T-cell proliferation and cytokine production. The production of inflammatory mediators was analyzed in cultures of sorted CD271(+)/(-) SCs. The capacity of CD271(+)/(-) SCs to induce inflammatory cell recruitment in vivo was evaluated in subcutaneous implants in immunodeficient mice. RESULTS: CD271(+) SC were detected in non-inflammatory as well as in arthritic synovial tissues with a specific perivascular distribution. CD271(+) SC density was increased in RA and OA compared with normal synovial tissues. T-cell proliferation and cytokine synthesis were similarly modified by CD271(+) and CD271(-) SCs. Sorted CD271(+) SCs from OA synovial tissues released significantly more interleukin (IL)-6, matrix metalloproteinase (MMP)-1, and MMP-3 than CD271(-) SCs. In immunodeficient mice, implants of CD271(+) SCs induced significantly higher myeloid cell infiltration than CD271(-) SCs. CONCLUSIONS: Our results demonstrate that CD271(+) perivascular SCs expand in RA and OA synovial tissues. CD271(+) cells showed enhanced proinflammatory properties ex vivo and in vivo, whereas immunoregulatory properties were equivalent in CD271(+) and CD271(-) SC.

Clinicopathological correlations of podoplanin (gp38) expression in rheumatoid synovium and its potential contribution to fibroblast platelet crosstalk.

INTRODUCTION: Synovial fibroblasts (SF) undergo phenotypic changes in rheumatoid arthritis (RA) that contribute to inflammatory joint destruction. This study was undertaken to evaluate the clinical and functional significance of ectopic podoplanin (gp38) expression by RA SF. METHODS: Expression of gp38 and its CLEC2 receptor was analyzed by immunohistochemistry in synovial arthroscopic biopsies from RA patients and normal and osteoarthritic controls. Correlation between gp38 expression and RA clinicopathological variables was analyzed. In patients rebiopsied after anti-TNF-α therapy, changes in gp38 expression were determined. Platelet-SF coculture and gp38 silencing in SF were used to analyze the functional contribution of gp38 to SF migratory and invasive properties, and to SF platelet crosstalk. RESULTS: gp38 was abundantly but variably expressed in RA, and it was undetectable in normal synovial tissues. Among clinicopathologigal RA variables, significantly increased gp38 expression was only found in patients with lymphoid neogenesis (LN), and RF or ACPA autoantibodies. Cultured synovial but not dermal fibroblasts showed strong constitutive gp38 expression that was further induced by TNF-α. In RA patients, anti-TNF-α therapy significantly reduced synovial gp38 expression. In RA synovium, CLEC2 receptor expression was only observed in platelets. gp38 silencing in cultured SF did not modify their migratory and invasive properties but reduced the expression of IL-6 and IL-8 genes induced by SF-platelet interaction. CONCLUSIONS: In RA, synovial expression of gp38 is strongly associated to LN and it is reduced after anti-TNF-α therapy. Interaction between gp38 and CLEC2 platelet receptor is feasible in RA synovium in vivo and can specifically contribute to gene expression by SF.

Topical vitamin D analogue calcipotriol reduces skin fibrosis in experimental scleroderma.

Vitamin D analogues can reduce TGF-ß pro-fibrotic signaling in dermal fibroblasts, but they may also induce a potentially pro-fibrotic thymic stromal lymphopoietin (TSLP)-dependent Th2 cytokine local response. We have analyzed the net effect of topical vitamin D analogue calcipotriol (CPT) on the cytokine profile and the development of fibrosis in experimental model of bleomycin-induced fibrosis. Mice were simultaneously treated with topical CPT or vehicle cream and skin fibrosis was measured by collagen deposition, Masson's trichrome staining and hydroxyproline content. Cytokine and TSLP gene expression was evaluated by quantitative RT-PCR. We showed that in bleomycin injected skin, CPT administration significantly enhanced TSLP and IL-13 gene expression, but did not modify the expression of other cytokines. Skin fibrosis and hydroxyproline content were significantly reduced in CPT compared to vehicle-treated mice. In normal skin, topical administration of CPT lacked a direct pro-fibrotic effect. Our results demonstrate that topical CPT superinduces the expression of the TSLP/IL-13 Th2 axis in fibrotic skin, but it has a net anti-fibrotic effect. These data support the therapeutic use of topical vitamin D analogues for skin fibrosis.

A profibrotic role for thymic stromal lymphopoietin in systemic sclerosis.

OBJECTIVE: [corrected] Systemic sclerosis (SSc) is an autoimmune disease characterised by progressive fibrosis. Although SSc shares pathogenetic features with other autoimmune diseases, the participation of profibrotic Th2 cytokines is unique to SSc, but the mechanisms of Th2 skewing are unknown. We have analysed the expression and function of thymic stromal lymphopoietin (TSLP), a central regulator of Th2-mediated allergic inflammation, in human SSc, primary lung fibrosis and in a mouse model of scleroderma. METHODS: TSLP expression was analysed by immunohistochemistry in human SSc skin, primary lung fibrosis and mouse bleomycin-induced skin fibrosis, and by quantitative RT-PCR in mouse skin and cultured fibroblasts. The regulation of TSLP expression by specific toll-like receptors (TLR)-2, -3 and -4 agonists was analysed in human dermal fibroblast cultures. The role of TSLP in skin fibrosis and local cytokine expression was analysed in TSLP receptor (TSLPR)-deficient mice. RESULTS: TSLP was overexpressed by epithelial cells, mast cells and fibroblasts in human SSc skin and lung fibrosis, and in the bleomycin model of scleroderma. In cultured human and mouse skin fibroblasts, TSLP expression was inducible by activation of TLR, particularly TLR3. In TSLPR-deficient mice, bleomycin-induced fibrosis was significantly reduced in parallel with significantly reduced local expression of IL-13. CONCLUSIONS: These data provide the first evidence of TSLP overexpression in SSc and other non-allergic fibrotic conditions, and demonstrate a profibrotic role that is potentially meditated by specific changes in the local cytokine milieu. Thus, modulating TSLP may have antifibrotic therapeutic implications.

CXCL12γ isoform is expressed on endothelial and dendritic cells in rheumatoid arthritis synovium and regulates T cell activation.

OBJECTIVE: CXCL12γ is an alternative splicing isoform of CXCL12 with enhanced affinity for heparan sulfate (HS) proteoglycans. This study was undertaken to investigate the distribution and potential function of CXCL12γ in rheumatoid arthritis (RA) synovium and normal lymphoid tissue, where its immobilization to HS may be relevant in pathologic or homeostatic immune cell migration and activation. METHODS: Expression of CXCL12 or CXCL12γ was immunodetected in RA and normal synovium, lymphoid tissue, and cultured cells with anti-pan-CXCL12 or anti-CXCL12γ-specific monoclonal antibodies. CXCL12α and CXCL12γ messenger RNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Binding of wild-type CXCL12 isoforms or their HS binding-defective mutants to monocyte-derived dendritic cells (DCs) was analyzed by flow cytometry. The effect of DC-bound CXCL12α and CXCL12γ on T cell activation was analyzed in DC/T cell allogeneic cultures. RESULTS: CXCL12γ expression was increased in RA compared to normal synovium and preferentially located in endothelia and DC-SIGN-positive cells. This distribution was also observed in lymphoid organs. Surface-bound CXCL12γ was detected in a fraction of freshly isolated DCs. Monocyte-derived DCs, but not monocytes, showed a high capacity to bind CXCL12γ in an HS-dependent manner. Surface-bound CXCL12α and CXCL12γ on monocyte-derived DCs were potent inhibitors of allogeneic T cell activation, in contrast to the T cell-stimulatory effects of soluble CXCL12 proteins. CONCLUSION: CXCL12γ shows a specific and similar distribution in RA synovium and lymphoid tissue, consistent with its higher HS binding affinity. Presentation of CXCL12 to T cells on membrane HS in DCs can play a distinct regulatory role in T cell activation.

Transcriptome analysis reveals specific changes in osteoarthritis synovial fibroblasts.

OBJECTIVE: Changes in rheumatoid arthritis synovial fibroblast (RASF) gene expression are usually defined by a comparison to osteoarthritis synovial fibroblasts (OASFs). This study was undertaken to analyse the transcriptome of OASFs as compared to RASFs and healthy synovial fibroblasts (HSFs). METHODS: The authors used microarray messenger RNA expression profiling of synovial fibroblasts cultured from osteoarthritis (OA), rheumatoid arthritis and normal synovial tissues. Quantitative real-time PCR of selected genes was performed to validate microarray data. Analysis of variance, Student t test and the Benjamini-Hochberg multiple testing correction method for multiple testing correction were used to determine the statistical significance of the changes between the three groups. RESULTS: Larger numbers of transcripts showed a differential expression in OASFs versus the other groups, rather than in RASFs versus HSFs. Cluster analysis confirmed that the differences between the three groups were mostly due to the differences between OA and the other groups. Functional classification identified a significant number of genes related to growth factor activities, cell adhesion, neurotransmission and Ras signalling that are differentially expressed in OASFs. Classical proinflammatory factors or proteases involved in cartilage degradation were not found to be overexpressed in OASFs. CONCLUSION: Cultured OASFs display a more homogeneous transcriptomic profile than RASFs when compared to HSFs. This supports the participation of synovial fibroblasts in the pathogenesis of OA and may reflect global defects in the mesenchyma-derived lineages of the different tissues in OA joints. These data support individual heterogeneity among RASFs and advise against the use of OASFs as controls.

Fibroblast abnormalities in the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a chronic systemic disease characterized by autoimmunity, vascular lesions and progressive fibrosis. The fibrotic component is dominant in SSc compared with other vascular or autoimmune diseases and determines its prognosis and therapeutic refractoriness. Fibroblasts are responsible for abnormal extracellular matrix accumulation. Studies in cultured SSc skin fibroblasts have facilitated the identification of potential pathways involved in their profibrotic phenotype. Profibrotic fibroblasts characterized by abnormal growth and extracellular matrix synthesis may differentiate or expand from normal resident fibroblasts. Recruitment of bone marrow-derived progenitors and transdifferentiation of different cell lineages might also be involved. Multiple factors and signaling pathways appear to be involved in the development or persistence of the SSc fibroblast phenotype. Although their relative relevance and interplay are unclear, aberrant TGF-ß signaling seems pivotal and constitutes the best characterized therapeutic target.

Synovial fibroblast hyperplasia in rheumatoid arthritis: clinicopathologic correlations and partial reversal by anti-tumor necrosis factor therapy.

OBJECTIVE: Synovial fibroblast (SF) hyperplasia contributes to the pathogenesis of rheumatoid arthritis (RA), but quantitative information on this process is scarce. This study was undertaken to evaluate the fibroblast-specific marker Hsp47 as a quantitative marker for SFs and to analyze its clinicopathologic correlates and evolution after anti-tumor necrosis factor α (anti-TNFα) therapy. METHODS: Synovial biopsy samples were obtained from 48 patients with RA and 20 controls who were healthy or had osteoarthritis (OA). Twenty-five RA patients who had active disease at the time of biopsy underwent a second biopsy after anti-TNFα therapy. Immunolabeling for Hsp47, inflammatory cells, and vascular cell markers was performed. Hsp47-positive lining and sublining fractional areas were quantified, and their correlation with clinicopathologic variables was analyzed. RESULTS: In normal and diseased synovial tissue, Hsp47 was specifically and uniformly expressed by lining, sublining, and perivascular fibroblasts. Lining SF area was significantly increased in both RA and late OA tissue compared to normal tissue. Sublining SF area was increased in RA tissue but not in late OA tissue compared to normal tissue. Lining SF area was positively correlated with macrophage density, Disease Activity Score in 28 joints, and RA disease duration. In contrast, sublining SF area was negatively correlated with RA disease duration and activity. A significant reduction in lining SF area but not sublining SF area was observed after anti-TNFα therapy. CONCLUSION: Our findings indicate that Hsp47 is a reliable marker for quantifying SFs in human synovial tissue. Our data suggest that lining and sublining SFs undergo different dynamics during the course of the disease. Lining SF expansion parallels the activity and temporal progression of RA and can be partially reversed by anti-TNFα therapy.

CXCL12 gene expression is upregulated by hypoxia and growth arrest but not by inflammatory cytokines in rheumatoid synovial fibroblasts.

OBJECTIVES: CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions. METHODS: Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5'CXCL12 -1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts. RESULTS: CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the -1.4 kb promoter. CONCLUSIONS: Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.

The transcriptional response of normal and rheumatoid arthritis synovial fibroblasts to hypoxia.

OBJECTIVE: Hypoxia is a prominent feature in rheumatoid arthritis (RA) synovium. However, its contribution to the pathogenesis of RA remains unclear. We undertook this study to systematically characterize the changes in gene expression induced by hypoxia in synovial fibroblasts. METHODS: We used microarray expression profiling in paired normoxic and hypoxic cultures of healthy synovial fibroblasts (HSFs) and RA synovial fibroblasts (RASFs). We used Student's paired t-test with Benjamini and Hochberg multiple testing correction to determine statistical significance. Validation of microarray data was performed by quantitative real-time reverse transcription-polymerase chain reaction analysis of selected genes. Biologic pathways differentially modulated by hypoxia in RASFs or HSFs were identified using unsupervised Ingenuity Pathways Analysis. RESULTS: Hypoxia induced significant changes in the expression of a large group of genes in both HSFs and RASFs. In RASFs, we observed a lower number of hypoxia-regulated genes and partial differences in their functional categories. The number of differentially expressed genes in RASFs compared with HSFs was significantly increased by hypoxia. Multiple gene sets involved in energy metabolism, intracellular signal transduction, angiogenesis, and immune and inflammatory pathways were significantly modified, the last in both proinflammatory and antiinflammatory directions. CONCLUSION: These data demonstrate that hypoxia induces significant changes in gene expression in HSFs and RASFs and identify differences between RASF and HSF profiles. The hypoxia-induced gene expression program in synovial fibroblasts identifies new factors and pathways relevant to understanding their contribution to the pathogenesis of chronic arthritis.

Immature blood vessels in rheumatoid synovium are selectively depleted in response to anti-TNF therapy.

BACKGROUND: Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists. METHODOLOGY/PRINCIPAL FINDINGS: Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy. CONCLUSION/SIGNIFICANCE: RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the development of anti-angiogenic interventions in RA.

Human inflammatory synovial fibroblasts induce enhanced myeloid cell recruitment and angiogenesis through a hypoxia-inducible transcription factor 1alpha/vascular endothelial growth factor-mediated pathway in immunodeficient mice.

OBJECTIVE: Hyperplasia and phenotypic changes in fibroblasts are often observed in chronic inflammatory lesions, and yet the autonomous pathogenic contribution of these changes is uncertain. The purpose of this study was to analyze the intrinsic ability of fibroblasts from chronically inflamed synovial tissue to drive cell recruitment and angiogenesis. METHODS: Fibroblasts from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), as well as fibroblasts from healthy synovial tissue and healthy skin, were cultured and subcutaneously engrafted into immunodeficient mice. Cell infiltration and angiogenesis were analyzed in the grafts by immunohistochemical studies. The role of vascular endothelial growth factor (VEGF), CXCL12, and hypoxia-inducible transcription factor 1alpha (HIF-1alpha) in these processes was investigated using specific antagonists or small interfering RNA (siRNA)-mediated down-regulation of HIF-1alpha in fibroblasts. RESULTS: Inflammatory (OA and RA) synovial fibroblasts, compared with healthy dermal or synovial tissue fibroblasts, induced a significant enhancement in myeloid cell infiltration and angiogenesis in immunodeficient mice. These activities were associated with increased constitutive and hypoxia-induced expression of VEGF, but not CXCL12, in inflammatory fibroblasts compared with healthy fibroblasts. VEGF and CXCL12 antagonists significantly reduced myeloid cell infiltration and angiogenesis. Furthermore, targeting of HIF-1alpha expression by siRNA or of HIF-1alpha transcriptional activity by the small molecule chetomin in RA fibroblasts significantly reduced both responses. CONCLUSION: These results demonstrate that chronic synovial inflammation is associated with stable fibroblast changes that, under hypoxic conditions, are sufficient to induce inflammatory cell recruitment and angiogenesis, both of which are processes relevant to the perpetuation of chronic inflammation.

Autoimmune lymphoproliferative syndrome (ALPS) in a patient with a new germline Fas gene mutation.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphoproliferation, autoimmune manifestations and expansion of TCRalphabeta+CD4-CD8- lymphocytes. The main pathogenic factor is a defective Fas-mediated apoptosis generally caused by mutations in the Fas gene. This report describes a new heterozygous Fas gene mutation in a boy with clinical and immunological features of ALPS. In vitro, T-cell blasts from the patient are completely resistant to the effects on the anti-Fas cytotoxic mAb CH-11, they also have a higher proliferation rate than T cells from healthy donors, while PHA-induced AICD is normal. The location of the mutation (I246S) found in the intracytoplasmic death domain, and the conservation of that residue in four different species from human suggest that I246 is an essential amino acid for Fas function. The patient has inherited the mutation from his father who also shows defective Fas-mediated apoptosis but the clinical and immunological manifestations are much less severe. These results provide evidence that the penetrance of genetic defects in Fas is variable and that other factors may influence the phenotype of the disease.

A modified cord blood collection method achieves sufficient cell levels for transplantation in most adult patients.

Umbilical cord blood transplantation (UCBT) has been used increasingly in both pediatric and adult patients. The total nucleated cell (NC) dose infused is the most critical factor in determining speed of engraftment and survival. Using standard collection techniques, the mean NC content of UCB units is about 10 x 10(8) and only 25% of these units reach the target cell dose of 2 x 10(7)/kg in UCBT patients weighing 50-70 kg. We have designed a modified placental/umbilical two-step collection method in which a standard blood fraction obtained by umbilical venipuncture is combined with a second fraction harvested after placental perfusion with 50 ml heparinized 0.9% saline. This second fraction contributed 32% volume and 15% NCs to the whole UCB unit (123.7 +/- 50.1 ml and 1.26 +/- 0.52 x 10(9) NC). The proportion of progenitor cells in both fractions was not significantly different, indicating that the hematopoietic potential of these larger units is 20% (range, 2%-100%) higher than UCB units collected by standard methods. In addition, the bacterial contamination rate associated with this novel collection method (2.78%) compares favorably. Since 1998 we have further enriched our units by processing only UCB units over 0.8 x 10(9) NCs, resulting in a 36% cell increment (1.46 +/- 0.52 x 10(9) NCs). Thus, 84% and 54% of the Madrid UCB Bank inventory would fulfill the target cell dose of 2 x 10(7)/kg in patients weighing 50 and 65 kg, respectively. This significant UCB banking improvement gives larger pediatric and adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT.

HLA haplotypes associated with hemochromatosis mutations in the Spanish population.

BACKGROUND: The present study is an analysis of the frequencies of HLA-A and -B antigens and HLA haplotypes in two groups of individuals homozygous for the two main HFE mutations (C282Y and H63D) and a group heterozygous for the S65C mutation. METHODS: The study population includes: 1123 healthy individuals, 100 homozygous for the C282Y mutation, 138 homozygous for the H63D mutation and 17 heterozygous for the S65C mutation. HFE and HLA alleles were detected using DNA-based and microlymphocytotoxicity techniques respectively. RESULTS: An expected significant association between C282Y and the HLA-A3/B7 haplotype was found, but other HLA haplotypes carrying the -A3 antigen were found: HLA-A3/B62 and HLA-A3/B44. Also, a significant association between H63D mutation and HLA-A29/B44 haplotype was found, and again other HLA haplotypes carrying the HLA-A29 antigen were also found: HLA-A29/B14 and HLA-A29/B62. In addition, the S65C mutation seems to be associated with a HLA haplotype carrying the HLA-A26 antigen. CONCLUSION: These findings clearly suggest that HLA-A3/B7 and HLA-A29/B44 are the ancestral haplotypes from which the C282Y and H63D mutations originated, respectively. The frequencies of these mutations in different populations, their geographical distribution, and the degree of the statistical association to the ancestral haplotypes, suggest that the H63D mutation must have occurred earlier than the C282Y mutation.