Kota Ishizawa

Bone Marrow-Derived Progenitor Cells Are Important for Lung Repair after Lipopolysaccharide-Induced Lung Injury

Tissue repair often occurs in organs damaged by an inflammatory response. Inflammatory stimuli induce a rapid and massive release of inflammatory cells including neutrophils from the bone marrow. Recently, many studies suggested that bone marrow cells have the potential to differentiate into a variety of cell types. However, whether inflammatory stimuli induce release of bone marrow-derived progenitor cells (BMPCs), or how much impact the suppression of BMPCs has on the injured organ is not clear. Here we show that LPS, a component of Gram-negative bacterial cell walls, in the lung airways, induces a rapid mobilization of BMPCs into the circulation in mice. BMPCs accumulate within the inflammatory site and differentiate to become endothelial and epithelial cells. Moreover, the suppression of BMPCs by sublethal irradiation before intrapulmonary LPS leads to disruption of tissue structure and emphysema-like changes. Reconstitution of the bone marrow prevents these changes. These data suggest that BMPCs are important and required for lung repair after LPS-induced lung injury.

Bone-marrow-derived cells contribute to lung regeneration after elastase-induced pulmonary emphysema

All‐trans retinoic acid (ATRA) is known to reverse the anatomic and physiologic signs of pulmonary emphysema. However, the origin of the progenitor cells involved in this lung regeneration remains unclear. Recently, it was shown that bone marrow could be the source of progenitor cells for several cell types. Mice with elastase‐induced emphysema were treated with ATRA, granulocyte colony‐stimulating factor (G‐CSF), or a combination of both. ATRA or G‐CSF promoted lung regeneration and increased bone marrow‐derived cell (BMC) numbers in alveoli. Combined treatment of both had an additive effect, which indicated that BMC mobilization might be important in lung regeneration.

Increased circulating endothelial progenitor cells in patients with bacterial pneumonia: evidence that bone marrow derived cells contribute to lung repair

Background: Tissue repair often occurs in organs damaged by various inflammatory diseases including pneumonia. Inflammatory stimuli induce a rapid and massive release of inflammatory cells from the bone marrow. Recent studies have suggested that bone marrow cells have the potential to differentiate into a variety of cell types. It has been shown that administration of lipopolysaccharide (LPS) to murine lungs induces a rapid release of endothelial progenitor cells (EPCs) into the circulation, and that bone marrow derived progenitor cells including EPCs contribute to lung repair after lung injury in mice. This study was undertaken to investigate the mobilisation of EPCs in humans following acute pneumonia. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood taken from 23 patients with pneumonia during both the acute and convalescent phase. 1×106 PBMCs were plated on fibronectin coated culture slides and cultured in culture medium for endothelium. The numbers of EPCs were counted 8 days after plating. Results: The number of circulating EPCs significantly increased in patients with pneumonia (p<0.0001). Patients with low EPC counts tended to have persistent fibrotic changes in their lungs even after their recovery from pneumonia. Conclusions: Inflammatory stimuli induce a rapid release of EPCs into the circulation in humans. A sufficient number of EPCs is necessary for proper lung repair following bacterial pneumonia.

Randomized Phase II Trial Comparing Nitroglycerin Plus Vinorelbine and Cisplatin With Vinorelbine and Cisplatin Alone in Previously Untreated Stage IIIB/IV Non–Small-Cell Lung Cancer

PURPOSE To investigate the efficacy and safety of nitroglycerin plus vinorelbine and cisplatin in patients with previously untreated stage IIIB/IV non-small-cell lung cancer (NSCLC) as the experimental arm for the next phase III trial. PATIENTS AND METHODS One hundred twenty patients with stage IIIB/IV NSCLC were randomly assigned to vinorelbine 25 mg/m2 on days 1 and 8 and cisplatin 80 mg/m2 on day 1, with transdermally applied nitroglycerin (25 mg/patient daily for 5 days; arm A) or with placebo patch (arm B) every 3 weeks for a maximum of four cycles in a double-blind and controlled trial. Primary efficacy end points were the best confirmed response rate and time to disease progression (TTP). RESULTS The response rate in arm A (72%; 43 of 60 patients) was significantly higher than that for patients in arm B (42%; 25 of 60 patients; P < .001). Median TTP in arm A was longer than that in arm B (327 v 185 days). No severe adverse effect was recognized for either arm. The rate of grade 1 to 2 headache in arm A (30%; 18 of 60 patients) was significantly higher than that in arm B (2%; one of 60 patients; P < .001, chi(2) test). CONCLUSION Use of nitroglycerin combined with vinorelbine and cisplatin may improve overall response and TTP in patients with stage IIIB/IV NSCLC. The arm A regimen is being evaluated in a large phase III trial.

The increase in surface CXCR4 expression on lung extravascular neutrophils and its effects on neutrophils during endotoxin-induced lung injury.

Inflammatory stimuli, such as a microbes or lipopolysaccharides, induce a rapid release of neutrophils from the bone marrow and promote neutrophil migration into inflamed sites to promote host defense. However, an excess accumulation and retention of neutrophils in inflamed tissue can cause severe tissue injuries in the later stages of inflammation. Recent studies have reported that both CXCL12 levels in injured lungs and its receptor, CXCR4, on accumulated neutrophils in injured lungs, increased; furthermore, these studies showed that the CXCL12/CXCR4 signaling pathway participated in neutrophil accumulation in the later stages of lipopolysaccharide (LPS)-induced lung injury. However, the mechanisms underlying this increase in surface CXCR4 expression in neutrophils remain unclear. In this study, we found that surface CXCR4 expression increased in extravascular, but not intravascular, neutrophils in the lungs of LPS-induced lung injury model mice. Furthermore, ex vivo studies revealed that CXCL12 acted not only as a chemoattractant, but also as a suppressor of cell death for the lung neutrophils expressing CXCR4. Sulfatide, one of the native ligands for L-selectin, induced the increase of surface CXCR4 expression on isolated circulating neutrophils, suggesting that the activation of L-selectin may be involved in the increase in surface CXCR4. Our findings show that surface CXCR4 levels on neutrophils increase after extravasation into injured lungs, possibly through the activation of L-selectin. The CXCL12/CXCR4 signaling pathway plays an important role in the modulation of neutrophil activity during acute lung injury, not only by promoting chemotaxis but also by suppressing cell death.

Clinical Utility of Patient-Derived Xenografts to Determine Biomarkers of Prognosis and Map Resistance Pathways in EGFR-Mutant Lung Adenocarcinoma

PURPOSE Although epidermal growth factor receptor (EGFR) -mutated adenocarcinomas initially have high response rates to EGFR tyrosine kinase inhibitors (TKIs), most patients eventually develop resistance. Patient-derived xenografts (PDXs) are considered preferred preclinical models to study the biology of patient tumors. EGFR-mutant PDX models may be valuable tools to study the biology of these tumors and to elucidate mechanisms of resistance to EGFR-targeted therapies. METHODS Surgically resected early-stage non-small-cell lung carcinoma (NSCLC) tumors were implanted into nonobese diabetic severe combined immune deficient (NOD-SCID) mice. EGFR TKI treatment was initiated at tumor volumes of 150 μL. Gene expression analysis was performed using a microarray platform. RESULTS Of 33 lung adenocarcinomas with EGFR activating mutations, only 6 (18%) engrafted and could be propagated beyond passage one. Engraftment was associated with upregulation of genes involved in mitotic checkpoint and cell proliferation. A differentially expressed gene set between engrafting and nonengrafting patients could identify patients harboring EGFR-mutant tumor with significantly different prognoses in The Cancer Genome Atlas Lung Adenocarcinoma datasets. The PDXs included models with variable sensitivity to first- and second-generation EGFR TKIs and the monoclonal antibody cetuximab. All EGFR-mutant NSCLC PDXs studied closely recapitulated their corresponding patient tumor phenotype and clinical course, including response pattern to EGFR TKIs. CONCLUSION PDX models closely recapitulate primary tumor biology and clinical outcome. They may serve as important laboratory models to investigate mechanisms of resistance to targeted therapies, and for preclinical testing of novel treatment strategies.

The effects of vinblastine on the expression of human immunodeficiency virus type 1 long terminal repeat

Previous work by our group has demonstrated induction of the HIV-LTR following exposure of cells to various DNA-damaging agents such as ultraviolet (UV) light, cisplatin, and doxorubicin. The current experiments were designed to determine the relative effects of the anti-mitotic drug vinblastine on expression of the HIV-LTR. Using human cervical carcinoma (HeLa) cells stably transfected with the chloramphenicol acetyl transferase (CAT) reporter transcriptionally driven by the HIV-LTR promoter, we demonstrated a 9-10-fold induction at 48-72 h following vinblastine treatment. Previous experiments had demonstrated repression of cisplatin or doxorubicin-mediated HIV induction by treatment with salicylic acid. The vinblastine induction also was repressed by salicylic acid treatment, but not by treatment with indomethacin, suggesting a role for the NF kappa B pathway in the inductive response. When UV exposure was coupled to the vinblastine treatment, there was no additive or synergistic effect evident, suggesting similar paths of induction between the two agents. Northern blots demonstrated that these agents were operating at the level of transcription and that salicylic acid inhibited vinblastine-mediated induction of HIV-LTR-CAT mRNA only if administered at the same time as vinblastine; addition of salicylic acid 2 h later had no effect on transcript accumulation. All combinations of treatments with vinblastine and/or salicylic acid markedly reduced cell survival.

Receptor for advanced glycation end products expressed on alveolar epithelial cells is the main target for hyperoxia-induced lung injury

BACKGROUND Receptor for advanced glycation end products (RAGE) is abundantly expressed on alveolar epithelial cells (AECs) and participates in innate immune responses such as apoptosis and inflammation. However, it is unclear whether RAGE-mediated apoptosis of AECs is associated with hyperoxia-induced lung injury. METHODS We used wild-type and RAGE-knockout C57BL6/J mice in this study. In addition, we developed bone marrow chimeric mouse models expressing RAGE on hematopoietic or non-hematopoietic cells, including lung parenchymal cells, and compared survival ratios and changes in the permeability of the alveolar-capillary barrier after hyperoxia exposure. Further, we prepared single cell suspensions of lung cells and evaluated the apoptosis of AECs or microvascular endothelial cells (MVECs) by using a combination of antibodies and JC-1 dye. We also examined whether RAGE inhibition decreased hyperoxia-induced apoptosis of human lung epithelial cells in vitro. RESULTS After hyperoxia exposure, mice expressing RAGE on lung cells showed lower survival rate and increased alveolar-capillary permeability than mice expressing RAGE on hematopoietic cells. RAGE-expressing AECs showed significantly higher apoptosis than RAGE-knockout AECs after in vivo hyperoxia exposure. The level of hyperoxia-induced apoptosis was not different in MVECs. However, RAGE-null lung epithelial cells showed lower apoptosis than RAGE-expressing cells in vitro. CONCLUSION These results indicated that RAGE on AECs mainly contributed to hyperoxia-induced lung injury and alveolar-capillary barrier disruption.

Comment on “Cutting Edge: TLR4 Deficiency Confers Susceptibility to Lethal Oxidant Lung Injury”

We read with interest the article by Zhang et al. published in the October 15, 2005 issue ([1][1]). They reported that the absence of TLR4 increases susceptibility to hyperoxia-induced lung injury. They clearly demonstrated that antiapoptotic responses were attenuated in TLR4-deficient mice. However

A Novel SDHB IVS2-2A>C Mutation Is Responsible for Hereditary Pheochromocytoma/Paraganglioma Syndrome

Pheochromocytomas and paragangliomas are neuroendocrine tumors which arise from adrenal medulla, and sympathetic or parasympathetic nerves, respectively. Hereditary cases afflicted by both or either pheochromocytomas and paragangliomas have been reported: these are called hereditary pheochromocytoma/paraganglioma syndromes (HPPS). Many cases of HPPS are caused by mutations of one of the succinate dehydrogenase (SDH) genes; mainly SDHB and SDHD that encode subunits for the mitochondrial respiratory chain complex II. In this study, we investigated mutations of SDH genes in six HPPS patients from four Japanese pedigrees using peripheral blood lymphocytes (from one patient with pheochromocytoma and five patients with neck paraganglioma) and tumor tissues (from two patients with paraganglioma). Results showed that all of these pedigrees harbor germline mutations in one of the SDH genes. In two pedigrees, a novel IVS2-2A>C mutation in SDHB, at the acceptor-site in intron 2, was found, and the tumor RNA of the patient clearly showed frameshift caused by exon skipping. Each of the remaining two pedigrees harbors a reported missense mutation, R242H in SDHB or G106D in SDHD. Importantly, all these mutations are heterozygous in constitutional DNAs, and two-hit mutations were evident in tumor DNAs. We thus conclude that the newly identified IVS2-2A>C mutation in SDHB is responsible for HPPS. The novel mutation revealed by our study may contribute to improvement of clinical management for patients with HPPS.