Masataka Kuwana

Derivation of multipotent progenitors from human circulating CD14+ monocytes

Circulating CD14(+) monocytes are originated from hematopoietic stem cells in the bone marrow and believed to be committed precursors for phagocytes, such as macrophages. Recently, we have reported a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which has a fibroblast-like morphology in culture and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from circulating CD14(+) monocytes, but circulating precursors for MOMCs still remain undetermined. Comparative analysis of gene expression profiles of MOMCs and other monocyte-derived cells has revealed that embryonic stem cell markers, Nanog and Oct-4, are specifically expressed by MOMCs. In vitro generation of MOMCs requires binding to fibronectin and exposure to soluble factors derived from activated platelets. MOMCs contain progenitors with capacity to differentiate into a variety of nonphagocytes, including bone, cartilage, fat, skeletal and cardiac muscle, neuron, and endothelium, indicating that circulating monocytes are more multipotent than previously thought. In addition, MOMCs are capable of promoting ex vivo expansion of human hematopoietic progenitor cells through direct cell-to-cell contact and secretion of a variety of hematopoietic growth factors. These findings obtained from the research on MOMCs indicate that CD14(+) monocytes in circulation are involved in a variety of physiologic functions other than innate and acquired immune responses, such as repair and regeneration of the damaged tissue.

Anti-Melanoma Differentiation-Associated Gene 5 Is Associated With Rapidly Progressive Lung Disease and Poor Survival in US Patients With Amyopathic and Myopathic Dermatomyositis.

Clinically amyopathic dermatomyositis (CADM) is a subset of dermatomyositis (DM) presenting with the characteristic rash of DM without objective muscle weakness. Asian studies report that anti–melanoma differentiation–associated gene 5 (anti–MDA‐5) autoantibody in CADM is associated with interstitial lung disease (ILD), particularly rapidly progressive ILD (RPILD). These associations have not been established in US myositis patients. The goal of our study was to determine the association of anti–MDA‐5 autoantibody with ILD, RPILD, and survival in US patients with CADM and classic DM.

Interleukin-6 as a Potential Therapeutic Target for Pulmonary Arterial Hypertension

Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of biologic activities in immune regulation, hematopoiesis, inflammation, and oncogenesis. Recent accumulating evidence indicates a pathologic role for IL-6 in promoting proliferation of both smooth muscle and endothelial cells in the pulmonary arterioles, resulting in development of pulmonary arterial hypertension (PAH). Here, we describe a patient with mixed connective tissue disease and severe, refractory PAH. Her functional activity and hemodynamic parameters dramatically responded to tocilizumab, a humanized monoclonal antibody to human IL-6 receptor, which was aimed at treating multicentric Castlemans disease. It appears that IL-6 blockade may hold promise as an adjunct drug in treatment of PAH in idiopathic form as well as in association with connective tissue disease.

Anti‐MDA5 is associated with rapidly progressive lung disease and poor survival in U.S. patients with amyopathic and myopathic dermatomyositis

Clinically amyopathic dermatomyositis (CADM) is a subset of dermatomyositis (DM) presenting with the characteristic rash of DM without objective muscle weakness. Asian studies report that anti–melanoma differentiation–associated gene 5 (anti–MDA‐5) autoantibody in CADM is associated with interstitial lung disease (ILD), particularly rapidly progressive ILD (RPILD). These associations have not been established in US myositis patients. The goal of our study was to determine the association of anti–MDA‐5 autoantibody with ILD, RPILD, and survival in US patients with CADM and classic DM.

Initial combination therapy with ambrisentan and tadalafil in connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH): subgroup analysis from the AMBITION trial.

Background Patients with connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH), in particular systemic sclerosis (SSc), had an attenuated response compared with idiopathic PAH in most trials. Thus, there is uncertainty regarding the benefit of PAH-targeted therapy in some forms of CTD-PAH. Objective To explore the safety and efficacy of initial combination therapy with ambrisentan and tadalafil versus ambrisentan or tadalafil monotherapy in patients with CTD-PAH and SSc-PAH enrolled in the AMBITION trial. Methods This was a post hoc analysis of patients with CTD-PAH and SSc-PAH from AMBITION, an event-driven, double-blind trial in patients with WHO functional class II/III PAH. Treatment-naive patients were randomised 2:1:1 to once-daily initial combination therapy with ambrisentan plus tadalafil or monotherapy with ambrisentan or tadalafil, respectively. The primary endpoint was time to the first clinical failure event (first occurrence of death, hospitalisation for worsening PAH, disease progression or unsatisfactory long-term clinical response). Results In the primary analysis set (N=500), 187 patients had CTD-PAH, of whom 118 had SSc-PAH. Initial combination therapy reduced the risk of clinical failure versus pooled monotherapy in each subgroup: CTD-PAH (HR 0.43 (95% CI 0.24 to 0.77)) and SSc-PAH (0.44 (0.22 to 0.89)). The most common AE was peripheral oedema, which was reported more frequently with initial combination therapy than monotherapy in the two PAH subgroups. The relative frequency of adverse events between those on combination therapy versus monotherapy was similar across subgroups. Conclusions This post hoc subgroup analysis provides evidence that CTD-PAH and SSc-PAH patients benefit from initial ambrisentan and tadalafil combination therapy. Trial registration number NCT01178073, post results.

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies.

Objective Autoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies. Methods Here we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated. Results In patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006). Conclusion Our newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.

Association of hepatocyte growth factor promoter polymorphism with severity of interstitial lung disease in Japanese patients with systemic sclerosis

OBJECTIVE To examine associations of single-nucleotide polymorphisms (SNPs) within genes for hepatocyte growth factor (HGF) and its receptor c-met with disease susceptibility and organ involvement in Japanese patients with systemic sclerosis (SSc). METHODS Four SNPs (HGF -1652 C/T, +44222 C/T, and +63555 G/T, and c-met -980 T/A) were analyzed in 159 SSc patients and 103 healthy control subjects with the use of a polymerase chain reaction-based assay. The influence of the HGF -1652 SNP on transcription activity was evaluated with a luciferase reporter assay and an electrophoretic mobility shift assay (EMSA). RESULTS There was no difference in the distribution of HGF/c-met SNPs between SSc patients and controls. HGF -1652 TT was found much more frequently in SSc patients with end-stage lung disease (ESLD) than in those without (41% versus 8%; P = 0.0004). This association was confirmed by a replication study involving a separate cohort of 155 SSc patients. Kaplan-Meyer analysis revealed that HGF -1652 TT carriers had a higher probability of developing ESLD than did CT or CC carriers. The HGF promoter carrying the HGF -1652 T allele had lower transcription activity than did the promoter carrying the C allele. EMSA showed the presence of a potential negative transcription regulator that binds specifically to the HGF promoter carrying a T allele at position -1652. Finally, TT carriers had a relative inability to increase circulating HGF levels even in the presence of advanced interstitial lung disease. CONCLUSION A SNP in the HGF promoter region may modulate the severity of interstitial lung disease by controlling the transcriptional efficiency of the HGF gene.

Elevated Levels of Pentraxin 3 in Systemic Sclerosis: Associations With Vascular Manifestations and Defective Vasculogenesis

To clarify the role of pentraxin 3 (PTX3), a multifunctional pattern recognition protein that can suppress fibroblast growth factor 2 (FGF‐2), in systemic sclerosis (SSc)–related vasculopathy.

Quantification of circulating endothelial progenitor cells in systemic sclerosis: a direct comparison of protocols

Background It has been proposed that dysfunctional endothelial progenitor cells (EPCs) play a role in pathogenic vasculopathy in systemic sclerosis (SSc). However, there is some debate as to whether the EPC count is reduced in SSc. The European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group recently proposed recommendations for evaluating EPCs. Objective To validate the proposed EUSTAR recommendations by a side-by-side comparison of methods for quantifying EPCs. Methods Peripheral blood samples were obtained from 11 patients with SSc and 11 age-matched healthy controls. EPCs were simultaneously quantified by two methods: flow cytometry combined with immunomagnetic CD34+ cell enrichment or rosette-based lineage-negative (Lin−) cell enrichment. EPCs, defined as CD34+CD133+VEGFR2+ cells, were counted with and without fluorosphere calibration. Results EPC counts measured with fluorosphere calibration correlated well with each other, regardless of the enrichment procedure used. In contrast, EPC counts from protocols that did not use fluorospheres correlated poorly with results from other protocols. Conclusions The EUSTAR recommendations are valid when they are combined with fluorosphere calibration.

Enhanced angiogenic potency of monocytic endothelial progenitor cells in patients with systemic sclerosis

IntroductionMicrovasculopathy is one of the characteristic features in patients with systemic sclerosis (SSc), but underlying mechanisms still remain uncertain. In this study, we evaluated the potential involvement of monocytic endothelial progenitor cells (EPCs) in pathogenic processes of SSc vasculopathy, by determining their number and contribution to blood vessel formation through angiogenesis and vasculogenesis.MethodsMonocytic EPCs were enriched and enumerated using a culture of peripheral blood mononuclear cells and platelets on fibronectin in 23 patients with SSc, 22 patients with rheumatoid arthritis (RA), and 21 healthy controls. To assess the capacity of monocytic EPCs to promote vascular formation and the contribution of vasculogenesis to this process, we used an in vitro co-culture system with human umbilical vein endothelial cells (HUVECs) on Matrigel® and an in vivo murine tumor neovascularization model.ResultsMonocytic EPCs were significantly increased in SSc patients than in RA patients or healthy controls (P = 0.01 for both comparisons). Monocytic EPCs derived from SSc patients promoted tubular formation in Matrigel® cultures more than those from healthy controls (P = 0.007). Transplantation of monocytic EPCs into immunodeficient mice resulted in promotion of tumor growth and blood vessel formation, and these properties were more prominent in SSc than healthy monocytic EPCs (P = 0.03 for both comparisons). In contrast, incorporation of SSc monocytic EPCs into the tubular structure was less efficient in vitro and in vivo, compared with healthy monocytic EPCs.ConclusionsSSc patients have high numbers of aberrant circulating monocytic EPCs that exert enhanced angiogenesis but are impaired in vasculogenesis. However, these cells apparently cannot overcome the anti-angiogenic environment that characterizes SSc-affected tissues.