Biomarkers of haemodynamic severity of systemic sclerosis-associated pulmonary arterial hypertension by serum proteome analysis.

OBJECTIVES: To mine the serum proteome of patients with systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH) and to detect biomarkers that may assist in earlier and more effective diagnosis and treatment. METHODS: Patients with limited cutaneous SSc, no extensive interstitial lung disease and no PAH-specific therapy were included. They were classified as cases if they had PAH confirmed by right heart catheterisation (RHC) and serum collected on the same day as RHC; and as controls if they had no clinical evidence of PAH. RESULTS: Patients were mostly middle-aged females with anticentromere-associated SSc. Among 1129 proteins assessed by a high-throughput proteomic assay (SOMAscan), only 2 were differentially expressed and correlated significantly with pulmonary vascular resistance (PVR) in SSc-PAH patients (n=15): chemerin (ρ=0.62, p=0.01) and SET (ρ=0.62, p=0.01). To validate these results, serum levels of chemerin were measured by ELISA in an independent cohort. Chemerin levels were confirmed to be significantly higher (p=0.01) and correlate with PVR (ρ=0.42, p=0.04) in SSc-PAH patients (n=24). Chemerin mRNA expression was detected in fibroblasts, pulmonary artery smooth muscle cells (PA-SMCs)/pericytes and mesothelial cells in SSc-PAH lungs by single-cell RNA-sequencing. Confocal immunofluorescence revealed increased expression of a chemerin receptor, CMKLR1, on SSc-PAH PA-SMCs. SSc-PAH serum seemed to induce higher PA-SMC proliferation than serum from SSc patients without PAH. This difference appeared neutralised when adding the CMKLR1 inhibitor α-NETA. CONCLUSION: Chemerin seems an interesting surrogate biomarker for PVR in SSc-PAH. Increased chemerin serum levels and CMKLR1 expression by PA-SMCs may contribute to SSc-PAH pathogenesis by inducing PA-SMC proliferation.

Hemodynamic Response to Oral Vasodilator Therapy in Systemic Sclerosis-Related Pulmonary Hypertension.

PURPOSE: Although classified as group 1 pulmonary arterial hypertension (PAH), patients with systemic sclerosis-related pulmonary hypertension (SSc-PH) experience poorer clinical response to PAH therapy and increased mortality compared to those with idiopathic PAH. Due to heterogeneity in phenotypes, identifying patients likely to respond to therapy is challenging. The goal of this study was to determine clinical factors associated with hemodynamic response, defined by a > 20% reduction in pulmonary vascular resistance on repeat right heart catheterization. METHODS: We applied a time-to-event model using a retrospective cohort of 39 patients with precapillary SSc-PH, defined by a mean pulmonary artery pressure of ≥ 25 mmHg and pulmonary arterial wedge pressure (PAWP) ≤ 15 mmHg on right heart catheterization. RESULTS: Patients with PAWP ≤ 8 mmHg were nearly fourfold more likely to achieve a hemodynamic response compared to those with PAWP > 8 mmHg (HR 3.88; 95% CI: 1.20, 12.57); each 1 mmHg increase in PAWP was associated with a decreased hazard for hemodynamic response (HR 0.84; 95% CI: 0.70, 1.00). CONCLUSION: In patients with precapillary SSc-PH, PAWP was associated with time to hemodynamic response, suggesting the importance of subclinical cardiac disease in determining hemodynamic response to oral vasodilator therapy.

A Clinical Decision Tool for Risk Stratifying Patients with Systemic Sclerosis-Related Pulmonary Hypertension.

We devised a scoring system to identify patients with systemic sclerosis (SSc) at risk for pulmonary hypertension (PH) and predict all-cause mortality. Using 7 variables obtained via pulmonary function testing, echocardiography, and computed tomographic chest imaging, we applied the score to a retrospective cohort of 117 patients with SSc. There were 60 (51.3%) who were diagnosed with PH by right heart catheterization. Using a scoring threshold ≥ 0, our decision tool predicted PH with a sensitivity, specificity, and accuracy of 0.87 (95% CI 0.75, 0.94), 0.74 (95% CI 0.60, 0.84), and 0.80 (95% CI 0.72, 0.87), respectively. When adjusted for age at PH diagnosis, sex, and receipt of pulmonary arterial vasodilators, each one-point score increase was associated with an adjusted HR of 1.19 (95% CI 1.05, 1.34) for all-cause mortality. With further validation in external cohorts, our simplified clinical decision tool may better streamline earlier detection of PH in SSc.

Thymic stromal lymphopoietin is up-regulated in the skin of patients with systemic sclerosis and induces profibrotic genes and intracellular signaling that overlap with those induced by interleukin-13 and transforming growth factor ß.

OBJECTIVE: To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin-13 (IL-13) and transforming growth factor ß (TGFß). METHODS: Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild-type, IL-4Rα1-, and TSLP-deficient mice were treated with TGFß, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFß, IL-13, poly(I-C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho-Smad2 and macrophage marker CD163 were tested. RESULTS: TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP-treated mice showed up-regulated clusters of gene expression that overlapped strongly with those in IL-13- and TGFß-treated mice. TSLP up-regulated specific genes, including CXCL9, proteasome, and interferon (IFN)-regulated genes. TSLP treatment in IL-4Rα1-deficient mice promoted similar cutaneous inflammation as in wild-type mice, though TSLP-induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up-regulated tumor necrosis factor α, Mx-1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP-deficient mice treated with TGFß showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I-C)-treated mice showed high levels of cutaneous TSLP. CONCLUSION: TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFß and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.

Role of Left Ventricular Dysfunction in Systemic Sclerosis-Related Pulmonary Hypertension.

BACKGROUND: In systemic sclerosis (SSc), pulmonary hypertension remains a significant cause of morbidity and mortality. Although conventionally classified as group 1 pulmonary arterial hypertension, systemic sclerosis-related pulmonary hypertension (SSc-PH) is a heterogeneous disease. The contribution of left-sided cardiac disease in SSc-PH remains poorly understood. RESEARCH QUESTION: How often does left ventricular (LV) dysfunction occur in SSc among patients undergoing right heart catheterization and how does coexistent LV dysfunction with SSc-PH affect all-cause mortality in this patient population? STUDY DESIGN AND METHODS: We conducted a retrospective, observational study of 165 patients with SSc who underwent both echocardiography and right heart catheterization. LV dysfunction was identified using LV global longitudinal strain (GLS) on speckle-tracking echocardiography based on a defined threshold of > -18%. SSc-PH was defined by a mean pulmonary artery pressure > 20 mmHg. RESULTS: Among patients with SSc who have undergone right heart catheterization, LV dysfunction occurred in 74.2% with SSc-PH and 51.2% without SSc-PH. The median survival of patients with SSc-PH and LV dysfunction was 67.9 (95% CI, 38.3-102.0) months, with a hazard ratio of 12.64 (95% CI, 1.73-92.60) for all-cause mortality when adjusted for age, sex, SSc disease duration, and FVC compared with patients with SSc without pulmonary hypertension with normal LV function. INTERPRETATION: LV dysfunction is common in SSc-PH. Patients with SSc-PH and LV dysfunction by LV GLS have increased all-cause mortality. This suggests that LV GLS may be helpful in identifying underlying LV dysfunction and in risk assessment of patients with SSc-PH.

Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis.

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Extensive and Persistent Extravascular Dermal Fibrin Deposition Characterizes Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive multiorgan fibrosis. While the cause of SSc remains unknown, a perturbed vasculature is considered a critical early step in the pathogenesis. Using fibrinogen as a marker of vascular leakage, we found extensive extravascular fibrinogen deposition in the dermis of both limited and diffuse systemic sclerosis disease, and it was present in both early and late-stage patients. Based on a timed series of excision wounds, retention on the fibrin deposit of the splice variant domain, fibrinogen αEC, indicated a recent event, while fibrin networks lacking the αEC domain were older. Application of this timing tool to SSc revealed considerable heterogeneity in αEC domain distribution providing unique insight into disease activity. Intriguingly, the fibrinogen-αEC domain also accumulated in macrophages. These observations indicate that systemic sclerosis is characterized by ongoing vascular leakage resulting in extensive interstitial fibrin deposition that is either continually replenished and/or there is impaired fibrin clearance. Unresolved fibrin deposition might then incite chronic tissue remodeling.

A Multimodal Prediction Model for Diagnosing Pulmonary Hypertension in Systemic Sclerosis.

OBJECTIVE: Diagnosis of pulmonary hypertension (PH) in systemic sclerosis (SSc) requires an invasive right heart catheterization (RHC), often based on an elevated estimated pulmonary artery systolic pressure on screening echocardiography. However, because of the poor specificity of echocardiography, a greater number of patients undergo RHC than necessary, exposing patients to potentially avoidable complication risks. The development of improved prediction models for PH in SSc may inform decision-making for RHC in these patients. METHODS: We conducted a retrospective study of 130 patients with SSc; 66 (50.8%) were diagnosed with PH by RHC. We used data from pulmonary function testing, electrocardiography, echocardiography, and computed tomography to identify and compare the performance characteristics of 3 models predicting the presence of PH: 1) random forest, 2) classification and regression tree, and 3) logistic regression. For each model, we generated receiver operating curves and calculated sensitivity and specificity. We internally validated models using a train-test split of the data. RESULTS: The random forest model performed best with an area under the curve of 0.92 (95% confidence interval [95% CI] 0.83-1.00), sensitivity of 0.95 (95% CI 0.75-1.00), and specificity of 0.80 (95% CI 0.56-0.94). The 2 most important variables in our random forest model were pulmonary artery diameter on chest computed tomography and diffusing capacity for carbon monoxide on pulmonary function testing. CONCLUSIONS: In patients with SSc, a random forest model can aid in the detection of PH with high sensitivity and specificity and may allow for better patient selection for RHC, thereby minimizing patient risk.

Single Cell Transcriptomics of Fibrotic Lungs Unveils Aging-associated Alterations in Endothelial and Epithelial Cell Regeneration.

Lung regeneration deteriorates with aging leading to increased susceptibility to pathologic conditions, including fibrosis. Here, we investigated bleomycin-induced lung injury responses in young and aged mice at single-cell resolution to gain insights into the cellular and molecular contributions of aging to fibrosis. Analysis of 52,542 cells in young (8 weeks) and aged (72 weeks) mice identified 15 cellular clusters, many of which exhibited distinct injury responses that associated with age. We identified Pdgfra + alveolar fibroblasts as a major source of collagen expression following bleomycin challenge, with those from aged lungs exhibiting a more persistent activation compared to young ones. We also observed age-associated transcriptional abnormalities affecting lung progenitor cells, including ATII pneumocytes and general capillary (gCap) endothelial cells (ECs). Transcriptional analysis combined with lineage tracing identified a sub-population of gCap ECs marked by the expression of Tropomyosin Receptor Kinase B (TrkB) that appeared in bleomycin-injured lungs and accumulated with aging. This newly emerged TrkB + EC population expressed common gCap EC markers but also exhibited a distinct gene expression signature associated with aberrant YAP/TAZ signaling, mitochondrial dysfunction, and hypoxia. Finally, we defined ACKR1 + venous ECs that exclusively emerged in injured lungs of aged animals and were closely associated with areas of collagen deposition and inflammation. Immunostaining and FACS analysis of human IPF lungs demonstrated that ACKR1 + venous ECs were dominant cells within the fibrotic regions and accumulated in areas of myofibroblast aggregation. Together, these data provide high-resolution insights into the impact of aging on lung cell adaptability to injury responses.

Metabolomic Profiles Differentiate Scleroderma-PAH From Idiopathic PAH and Correspond With Worsened Functional Capacity.

BACKGROUND: The prognosis and therapeutic responses are worse for pulmonary arterial hypertension associated with systemic sclerosis (SSc-PAH) compared with idiopathic pulmonary arterial hypertension (IPAH). This discrepancy could be driven by divergence in underlying metabolic determinants of disease. RESEARCH QUESTION: Are circulating bioactive metabolites differentially altered in SSc-PAH vs IPAH, and can this alteration explain clinical disparity between these PAH subgroups? STUDY DESIGN AND METHODS: Plasma biosamples from 400 patients with SSc-PAH and 1,082 patients with IPAH were included in the study. Another cohort of 100 patients with scleroderma with no PH and 44 patients with scleroderma with PH was included for external validation. More than 700 bioactive lipid metabolites, representing a range of vasoactive and immune-inflammatory pathways, were assayed in plasma samples from independent discovery and validation cohorts using liquid chromatography/high-resolution mass spectrometry-based approaches. Regression analyses were used to identify metabolites that exhibited differential levels between SSc-PAH and IPAH and associated with disease severity. RESULTS: From hundreds of circulating bioactive lipid molecules, five metabolites were found to distinguish between SSc-PAH and IPAH, as well as associate with markers of disease severity. Relative to IPAH, patients with SSc-PAH carried increased levels of fatty acid metabolites, including lignoceric acid and nervonic acid, as well as eicosanoids/oxylipins and sex hormone metabolites. INTERPRETATION: Patients with SSc-PAH are characterized by an unfavorable bioactive metabolic profile that may explain the poor and limited response to therapy. These data provide important metabolic insights into the molecular heterogeneity underlying differences between subgroups of PAH.

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation.

Previous studies have shown that the transforming growth factor (TGF)ß/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFß. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFß requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFß-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFß/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.

The c-Abl tyrosine kinase controls protein kinase Cδ-induced Fli-1 phosphorylation in human dermal fibroblasts.

OBJECTIVE: We have previously demonstrated that in response to transforming growth factor ß (TGFß), Fli-1 activity is repressed through a series of sequential posttranslational modifications, consisting of protein kinase Cδ (PKCδ)-induced Thr312 phosphorylation, acetylation by p300/CREB binding protein-associated factor, and detachment from the collagen promoter. The purpose of this study was to further investigate the upstream events that lead to Fli-1 phosphorylation in response to TGFß. METHODS: Dermal fibroblasts were isolated from systemic sclerosis (SSc) patients and healthy control subjects matched for age, sex, and ethnicity. Western blotting was used to analyze protein levels and real-time quantitative reverse transcription-polymerase chain reaction analysis was used to measure messenger RNA expression. Cells were transduced with constitutively active PKCδ adenovirus or were transiently transfected with a Bcr-Abl-overexpressing plasmid. Subcellular localization of PKCδ was examined by immunocytochemistry. RESULTS: Western blot analysis of cell lysates demonstrated that the levels of phospho-Fli-1 (Thr312) were up-regulated in SSc fibroblasts, correlating with increased levels of type I collagen and c-Abl protein. Experiments using a constitutively activated form of c-Abl, small interfering RNA against c-Abl and the specific tyrosine kinase inhibitor imatinib, demonstrated the requirement of c-Abl for the TGFß-induced phosphorylation of Fli-1. Additionally, we showed that c-Abl kinase activity was required for nuclear localization of PKCδ. CONCLUSION: Our results demonstrate that in SSc fibroblasts, c-Abl is an upstream regulator of the profibrotic PKCδ/phospho-Fli-1 pathway, via induction of PKCδ nuclear localization. Additionally, the finding that Fli-1 is phosphorylated at higher levels in SSc fibroblasts supports the notion that the c-Abl/PKCδ/phospho-Fli-1 pathway is constitutively activated in these cells. Thus, blocking the TGFß/c-Abl/PKCδ/phospho-Fli-1 pathway could be an attractive alternative approach to therapy for scleroderma.

Clinical characteristics and outcomes in pulmonary manifestations of systemic sclerosis: Contribution from pulmonary hypertension and interstitial lung disease severity.

Patients with systemic sclerosis complicated by both pulmonary hypertension (SSc-PH) and interstitial lung disease (SSc-PH-ILD) have poor prognosis compared to those with SSc-PH or SSc-ILD alone. Little is known of how ILD severity affects outcomes in those with SSc-PH, or how PH severity affects outcomes in those with SSc-ILD. Herein, we aimed to delineate clinical features of patients with SSc-PH and SSc-ILD and determine to what degree PH and ILD severity contribute to mortality in patients with SSc. We conducted parallel retrospective studies in cohorts of patients with SSc-PH and SSc-ILD. We categorized ILD severity by pulmonary function testing and PH severity by cardiopulmonary hemodynamics. Our primary outcome was all-cause mortality from time of PH or ILD diagnosis for the SSc-PH and SSc-ILD cohorts, respectively. We calculated adjusted risks of time to all-cause mortality using Cox proportional hazards models. In patients with SSc-PH, severe ILD (HR: 3.54; 95% CI: 1.05, 11.99) was associated with increased hazards for all-cause mortality. By contrast, mild and moderate ILD were not associated with increased mortality risk. In patients with SSc-ILD, both moderate (HR: 2.65; 95% CI: 1.12, 6.31) and severe PH (HR: 6.60; 95% CI: 2.98, 14.61) were associated with increased hazards for all-cause mortality, while mild PH was not. Through our parallel study design, the risk of all-cause mortality increases as severity of concomitant ILD or PH worsens. Therapies that target slowing disease progression earlier in the disease course may be beneficial.

Periostin overexpression in scleroderma cardiac tissue and its utility as a marker for disease complications.

OBJECTIVE: To evaluate the levels of periostin in patients with systemic sclerosis (SSc) and their association with features of systemic sclerosis. METHODS: The levels of periostin were assessed in the serum of 106 SSc patients and 22 healthy controls and by immunofluorescence staining in cardiac tissue from 4 SSc patients and 4 controls. Serum periostin was measured via enzyme-linked immunosorbent assay. The results were analyzed using Mann-Whitney test or Kruskal-Wallis test followed by Dunn's multiple comparisons tests and Spearman's test for correlations. Cardiac tissue from SSc patients and controls was stained for periostin and co-stained for periostin and collagen type I using immunofluorescence. RESULTS: Periostin levels were higher in patients with SSc compared to controls and directly correlated to modified Rodnan skin score and echocardiography parameters of left ventricular measurements. Immunofluorescence staining in SSc cardiac tissue showed patchy periostin expression in all SSc patients, but not in controls. Furthermore, there was extensive periostin expression even in areas without collagen deposition, while all established fibrotic areas showed colocalization of collagen and periostin. There was no association between periostin levels and interstitial lung disease, pulmonary hypertension or other vascular complications. CONCLUSION: Periostin is elevated in SSc cardiac tissue in vivo and circulating levels of periostin are increased in SSc, correlating with the extent of disease duration, degree of skin fibrosis, and left ventricular structural assessments. Periostin may be a potential biomarker that can provide further pathogenic insight into cardiac fibrosis in SSc.

Prognostic Value of Cardiac Axis Deviation in Systemic Sclerosis-Related Pulmonary Hypertension.

OBJECTIVE: Systemic sclerosis-related pulmonary hypertension (SSc-PH) is a common complication of SSc associated with accelerated mortality. The present study was undertaken to investigate whether cardiac axis deviation indicates abnormalities in cardiac function allowing for prognostication of disease severity and mortality. METHODS: This was a retrospective study in which electrocardiograms (ECGs) were reviewed for cardiac axis deviation and their association with echocardiography and cardiopulmonary hemodynamics on right-sided heart catheterization. The primary outcome observed was all-cause mortality from the time of PH diagnosis. RESULTS: ECG results were reviewed from 169 patients with SSc-PH. Right axis deviation (RAD) and left axis deviation (LAD) occurred in 28.4% and 30.8% of patients with SSc-PH, respectively. Compared to those without RAD, patients with RAD exhibited predominantly right-sided cardiac disease on echocardiography and increased PH severity by cardiopulmonary hemodynamics including a greater mean ± SD pulmonary artery pressure (42.0 ± 12.5 mm Hg versus 29.8 ± 7.0 mm Hg) and mean ± SD pulmonary vascular resistance (645.6 ± 443.2 dynes · seconds/cm5 versus 286.3 ± 167.7 dynes · seconds/cm5 ). LAD was associated with predominantly left-sided cardiac disease on echocardiography but was not associated with PH severity on cardiopulmonary hemodynamics. Both RAD (hazard ratio 10.36 [95% confidence interval 4.90-21.93], P < 0.001) and LAD (hazard ratio 2.94 [95% confidence interval 1.53-5.68], P = 0.001) were associated with an increased hazard for all-cause mortality. CONCLUSION: RAD and LAD reflect structural cardiac abnormalities and are associated with poor prognosis in patients with SSc-PH. These findings support the importance of electrocardiography, an inexpensive, widely available noninvasive test, in risk stratification.

Akt inhibition up-regulates MMP1 through a CCN2-dependent pathway in human dermal fibroblasts.

Akt is a key signalling molecule that was found to be down-regulated in chronic wounds. Akt blockade has dual antifibrotic effects in human dermal fibroblasts, by up-regulating matrix metalloproteinase 1 (MMP1) and down-regulating collagen gene expression (J Invest Dermatol 2008: 128: 1906). The aim of this study was to gain additional insights into the mechanism of MMP1 up-regulation following Akt blockade. As previous studies showed that CCN2 can be a positive regulator of MMP1, we examined the effects of Akt inhibition on CCN2 expression. Akt blockade using a specific pharmacological inhibitor and Akt siRNA resulted in a significant up-regulation of CCN2, which correlated with the increase in MMP1. The MMP1 up-regulation following Akt blockade was partially suppressed by CCN2 siRNA, suggesting that CCN2 is contributing to this effect. Additional experiments showed that CCN2 induces phosphorylation of ERK1/2, Ets1 and c-Jun. Consistent with the stimulatory role of ERK1/2/Ets1 in the expression of MMP1, the ERK1/2 inhibitor UO126 prevented the phosphorylation of ERK1/2 and Ets1 and completely abrogated the induction of MMP1 after CCN2 overexpression, while having no effect on c-Jun activation. Taken together these results establish CCN2 as a key regulator of MMP1 induction via activation of the ERK1/2/Ets1 pathway. Down-regulation of Akt signalling leads to inappropriate activation of the CCN2/MMP1 pathway that may contribute to the pathogenesis of chronic wounds. Coordinate expression of CCN2, Akt and MMP1 could be important for normal wound healing to occur. Thus, targeting these specific proteins may represent a promising approach to the therapy of dysregulated wound healing.

Fli1 Downregulation in Scleroderma Myeloid Cells Has Profibrotic and Proinflammatory Effects.

Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation, vasculopathy, and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc, playing key roles in disease pathogenesis. However, the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes, B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC), showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1 flox/flox LysMCre+/+ mice, and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I, independent of TGFß upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation, to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc, the top most upregulated pathways were hallmark IFN-γ and IFN-α response. Additionally, several genes previously linked to SSc pathogenesis and fibrosis in general were also induced, including CCL2, CCL7, MMP12, and CXCL10. ANKRD1, a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients, with higher expression of profibrotic markers and activation of interferon responsive genes, thus suggesting that dysregulation of Fli1 in myeloid cells may contribute to SSc pathogenesis.

Basic Calcium Phosphate Crystal Periarthritis Involving the Distal Interphalangeal Joints in a Patient with Systemic Lupus Erythematosus.

BACKGROUND Increased serum levels of basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) are found in patients on dialysis, following trauma, and are associated with connective tissue diseases (CTDs), including dermatomyositis, scleroderma, and systemic lupus erythematosus (SLE). The shoulder is the joint most commonly associated with BCP crystal periarthritis. A report is presented of a case of BCP crystal periarthritis involving the distal interphalangeal (DIP) joints in a patient with SLE. CASE REPORT A 34-year-old woman with SLE presented with destructive arthritis of the DIP joints that developed during a two-year period, despite immunosuppressive therapy. Aspiration of synovial fluid from a DIP joint showed a lack of inflammatory cells, but the fluid was positive for the presence of crystals on alizarin red S histochemical staining. CONCLUSIONS A case of BCP crystal periarthritis is reported in a patient with SLE with chronic joint symptoms that were unresponsive to immunosuppressive therapy. This case has shown that chronic joint symptoms that are unresponsive to immunosuppressive therapy may be due to causes other than connective tissue disease (CTD) and that imaging studies and diagnostic workup that includes synovial fluid examination may support the diagnosis of BCP crystal periarthritis.

Comparison of oral versus parenteral methotrexate in the treatment of rheumatoid arthritis: A meta-analysis.

OBJECTIVE: Studies suggest that parenteral MTX may be more efficacious than the oral form at equivalent doses for the treatment of rheumatoid arthritis. We carried out a meta-analysis to compare the efficacy of oral versus parenteral MTX in RA. METHODS: PubMed, Web of Science and Embase were systematically searched from inception to June 8th 2017 and reviewed following PRISMA 2009 guidelines, by two independent reviewers. To be included, trials had to study adults with RA randomized to the same dose of either oral or parenteral MTX. The primary endpoint was ACR20 at 6 months. Intention-to-treat analysis results were used when possible. Data from direct comparisons between oral and parenteral methotrexate quantitatively analyzed using maximum likelihood random effects meta-analysis. Relative treatment effects were generated as an odds ratio [OR] (OR>1 indicated a benefit for parenteral therapy). RESULTS: The search yielded 357 papers or abstracts. After review of titles or abstracts and full text papers, we found 4 that met inclusion criteria with 703 patients randomized. Dose of MTX started at 15mg/week and increased up to 25mg/week. The summary OR for achieving ACR20 using parenteral vs. oral MTX was 3.02 (95% CI 1.41, 6.46), with no significant difference in the risk for all adverse events. CONCLUSION: Parenteral MTX therapy had significantly higher odds than oral MTX of achieving reduction in disease activity. We propose that parenteral MTX is more effective than weekly oral MTX; its widespread use may lead to better control of disease and a decrease in demand for biologic agents.