Miyuki Niisato

Heterogeneous Characteristics of Lymphatic Microvasculatures Associated with Pulmonary Sarcoid Granulomas

RATIONALE Pulmonary sarcoidosis is a disorder characterized by noncaseating epithelioid granulomas that are anatomically distributed along lymphogenous routes. Currently, limited information is available about lymphangiogenesis in pulmonary sarcoidosis. OBJECTIVE To clarify the characteristics of lymphangiogenesis in pulmonary sarcoidosis. METHODS The concentrations of vascular endothelial growth factor (VEGF), VEGF-C, and VEGF-D in serum and bronchoalveolar lavage fluid from 65 patients with pulmonary sarcoidosis, 10 with idiopathic pulmonary fibrosis, and 29 healthy volunteers were measured by ELISA. Paraffin-embedded lung tissues obtained from 19 patients were used for immunohistochemical analyses, using primary antibodies against VEGF, VEGF-C, VEGF-D, podoplanin, VEGF receptor (VEGFR)-2, VEGFR-3, and CD73. RESULTS The serum and bronchoalveolar lavage fluid concentrations of VEGF and VEGF-C were significantly increased in patients with pulmonary sarcoidosis. Immunohistochemical analysis showed that VEGF and VEGF-C were expressed in sarcoid granulomas. Immunostaining with anti-podoplanin antibody for the detection of lymphatic vasculatures showed the presence of usual lymphatics and atypical tubular structures around sarcoid granulomas. Atypical tubular structures were characterized by a thin membrane, with weak expression of podoplanin and a membrane deficit in a part of the borderline. The structures were observed in around 58.6% of the total of 193 granulomas, whereas usual lymphatics were limited in 15.6%. Atypical tubular structures were coexpressed with VEGFR-2, but not VEGFR-3, whereas VEGFR-3 was expressed in usual lymphatics. Part of the tubular structures was connected to CD73(+) afferent lymphatics. CONCLUSION These results indicate the presence and the importance of heterogeneous lymphatic microvasculature around sarcoid granulomas in pulmonary sarcoidosis.

Lymphangiogenic factors are associated with the severity of hypersensitivity pneumonitis

Background Antigen presenting cells play a pivotal role in the adaptive immune response in hypersensitivity pneumonitis (HP). It was hypothesised that lymphangiogenesis is involved in the pathophysiology of HP via cell transport. Objective To determine the clinical significance of lymphangiogenic factors in HP. Methods Levels of vascular endothelial growth factors (VEGF)-A, VEGF-C, VEGF-D and CCL21 in the serum and bronchoalveolar lavage fluid (BALF) were measured in 29 healthy volunteers, 14 patients with idiopathic pulmonary fibrosis (IPF) and 26 patients with HP by ELISA. Additionally, immunohistochemical analyses were performed using lung specimens of patients with HP (n=8) and IPF (n=10). Results BALF VEGF-D levels were significantly elevated in patients with HP compared to the other groups. BALF VEGF–D levels in patients with HP correlated significantly with the BALF total cell and lymphocyte counts (r=0.485, p=0.014 and r=0.717, p<0.0001, respectively). BALF VEGF-C and CCL21 levels were increased in patients with HP compared to healthy volunteers, but not patients with IPF. BALF CCL21 levels were negatively correlated with the forced expiratory volume in 1 s percentage and diffuse capacity of the lung for carbon monoxide (r=−0.662, p=0.007 and r=−0.671, p=0.024, respectively). According to the immunohistochemical analyses, CCL21 was expressed in the lymphatic endothelium in both conditions and CCR7+ cells were aggregated around lymphatics in patients with HP, but not in patients with IPF. Conclusions Lymphangiogenic factors might be associated with the inflammatory and functional severity of HP. The increased BALF VEGF-D levels were associated with lymphatic alveolitis intensity, and CCL21 with lung function impairment.

Analysis of pulmonary allergic vasculitis with eosinophil infiltration in asthma model of mice

ABSTRACT Here the authors report pulmonary allergic vasculitis with eosinophil infiltration in an asthma model of mice and investigated its pathogenesis. C57BL/6 and BALB/c mice were sensitized with ovalbumin (OVA). After the inhalation of OVA, the authors measured the cell number and cytokine concentration in the blood and bronchoalveolar lavage fluid (BALF). The authors also examined the histological changes of the pulmonary. The number of eosinophils increased in the blood and BALF in both strains; however, the number in C57BL/6 in BALF was significantly higher than that in BALB/c. Histological analysis demonstrated severe vasculitis of the pulmonary arteries with derangement of the muscle layer and smooth muscle cell hyperplasia in C57BL/6. Semiquantitative analysis of the severity of vasculitis in the pulmonary arteries revealed that the internal vascular space was highly reduced by smooth muscle hyperplasia in C57BL/6 compared to BALB/c mice. The concentrations of interleukin (IL)-4, IL-5, and interferon (IFN)-γ in BALF of C57BL/6 were significantly high compared to those of BALB/c. C57BL/6 mice exhibited severe allergic vasculitis in the pulmonary arteries compared to BALB/c mice. The high concentrations of IL-4, IL-5, and IFN-γ in the lung may play a critical role in the pathogenesis of allergic vasculitis in C57BL/6 mice.

Chaperone protein L-isoaspartate (D-aspartyl) O-methyltransferase as a novel predictor of poor prognosis in lung adenocarcinoma.

Endoplasmic reticulum stress and chaperone dysfunction have recently been associated with poor prognoses in various cancers. The newly discovered chaperone protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) regulates the viability of cancer cells in various cancers, although no clinical information regarding the relationship between lung cancer and PIMT expression has been reported. In this study, we aimed to elucidate the relationship between PIMT expression and the prognosis of lung adenocarcinoma. Paraffin-embedded lung tissues obtained from 208 patients with surgically resected lung adenocarcinoma were subjected to immunohistochemical analyses using primary antibodies against PIMT. Kaplan-Meier curves, log-rank tests, and the Cox proportional hazards model were used to analyze the association between PIMT expression and patient survival. Strong PIMT expression was detected in 106 (50.9%) patients, being particularly observed in patients with advanced stages of lung adenocarcinoma. Strong PIMT expression was associated with that of 78-kDa glucose-regulated protein, a marker of endoplasmic reticulum stress. Patients with strong PIMT expression had a shorter survival time (Kaplan-Meier analysis, P<.001). Multivariate Cox hazard regression analysis demonstrated that strong PIMT expression was an independent predictor of poor prognosis of lung adenocarcinoma, including those with stage I disease (hazard ratios, 6.45 and 6.81, respectively; 95% confidence intervals, 2.46-16.9 and 1.79-25.8, respectively; P<.001 and P=.005, respectively). Collectively, strong PIMT expression was a predictive marker of poor prognosis for surgically resected lung adenocarcinoma, and this finding might help clinicians determine the need for postoperative adjuvant chemotherapy in patients with stage I lung adenocarcinoma.

A phosphatidylinositol 3-kinase inhibitor strongly suppressed pulmonary vascular remodeling of allergic vasculitis in a murine model

ABSTRACT Objectives: We investigated the effects of pan-class I PI3K inhibitor, ZSTK474 on vascular remodeling using a murine model of allergic vasculitis with eosinophil infiltration. Methods: C57BL/6 mice were sensitized with OVA. The positive controls were exposed to aerosolized OVA daily for 7 days. The other group of mice were administered ZSTK474 (30 mg/kg, p.o. daily) in parallel with daily exposure to aerosolized OVA for 7 days. On the 3rd and 7th day, bronchoalveolar lavage (BAL) was performed and the lungs were excised for pathological analysis. Cell differentials were determined and the concentrations of IL-4, IL-5, IL-13 and TGF-βin BAL fluid were measured. Results: The total cell numbers and eosinophil numbers in BALF were greatly reduced in the ZSTK474-treated group on the 3rd and 7th day after exposure to OVA. The numbers of total white blood cells and eosinophils in the peripheral blood were significantly reduced in the ZSTK474-treated group on the 3rd and 7th day after exposure to OVA. The concentrations of IL-4, IL-5, and IL-13 in BAL fluids were also reduced significantly on the 3rd day in the ZSTK474-treated group. The concentrations of TGF-β in BAL fluids were also reduced significantly on the 3rd and 7th day in the ZSTK474-treated group. The pathological scores reduced significantly in the ZSTK474-treated group compared to the control group. Conclusion: The PI3K inhibitor, ZSTK474 suppressed pulmonary vascular remodeling in the murine model of allergic vasculitis with eosinophil infiltration. PI3K signal transduction may have a critical role in the immunological process that induces allergic vasculitis.

Deficiency of protein-L-isoaspartate (D-aspartate) O -methyl-transferase expression under endoplasmic reticulum stress promotes epithelial mesenchymal transition in lung adenocarcinoma

A prognostic association between the novel chaperone protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) and lung adenocarcinoma has recently been reported. Here, we evaluated the functional roles of PIMT in the progression of lung adenocarcinoma. PIMT expression was detectable in 6 lung adenocarcinoma cell lines: A549, H441, H460, H1650, Calu 1, and Calu 6 cell lines. In A549 and H441 cells, knockdown by PIMT using silencing RNA of PIMT (si-PIMT) and/or small hairpin-RNA (sh-PIMT) induced a decrease in the expression of E-cadherin with an increase in vimentin expression, indicating that the epithelial to mesenchymal transition (EMT) was induced. Cell mobility, including migration and invasion capability, was increased in sh-PIMT A549 stable and si-PIMT H441 cells compared to in control cells. Endoplasmic reticulum (ER) stress, such as Thapsigargin (Tg) stress and hypoxia, induced EMT in A549 cells but not in other cell types, with an increase in GRP78 expression, whereas overexpression of PIMT reduced the EMT and cell invasion under stress conditions. The expression of hypoxia inducible factor-1 alpha (HIF1α) and Twist increased in sh-PIMT A549 and si-PIMT H441 cells, and Tg stress increased HIF1α expression levels in A549 cells in a dose-dependent manner. Moreover, LW6, an HIF1α inhibitor, reduced EMT, cancer invasion, and the levels of Twist in sh-PIMT A549 cells. Our results indicate that deficiency of supplemental PIMT expression under ER stress facilitates EMT and cell invasion in some cell types of lung adenocarcinoma.