Takeshi Oya

Regulatory Mechanisms for Adipose Tissue M1 and M2 Macrophages in Diet-induced Obese Mice

OBJECTIVE To characterize the phenotypic changes of adipose tissue macrophages (ATMs) under different conditions of insulin sensitivity. RESEARCH DESIGN AND METHODS The number and the expressions of marker genes for M1 and M2 macrophages from mouse epididymal fat tissue were analyzed using flow cytometry after the mice had been subjected to a high-fat diet (HFD) and pioglitazone treatment. RESULTS Most of the CD11c-positive M1 macrophages and the CD206-positive M2 macrophages in the epididymal fat tissue were clearly separated using flow cytometry. The M1 and M2 macrophages exhibited completely different gene expression patterns. Not only the numbers of M1 ATMs and the expression of M1 marker genes, such as tumor necrosis factor-α and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by subsequent pioglitazone treatment, suggesting the correlation with whole-body insulin sensitivity. We also found that the increased number of M2 ATMs after an HFD was associated with the upregulated expression of interleukin (IL)-10, an anti-inflammatory Th2 cytokine, in the adipocyte fraction as well as in adipose tissue. The systemic overexpression of IL-10 by an adenovirus vector increased the expression of M2 markers in adipose tissue. CONCLUSIONS M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.

A functional SNP in CILP , encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T → C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-β1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-β1–mediated induction of cartilage matrix genes through direct interaction with TGF-β1 and inhibition of TGF-β1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-β1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-β signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-β.

PDGF Receptor β Is a Potent Regulator of Mesenchymal Stromal Cell Function

Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet‐derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)β gene by Cre‐loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF‐A, ‐B, ‐C, and ‐D as well as PDGFRα and β was detected. Depletion of PDGFRβ in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT‐PCR. PDGF‐BB, but not PDGF‐AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF‐BB were eliminated by depletion of PDGFRβ in MSCs except that PDGF‐BB still suppressed osterix expression in PDGFRβ‐depleted MSCs. Depletion of PDGFRβ significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFRβ gene deletion, we showed that PDGFRβ signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFRα on the osteogenic differentiation were very subtle. PDGFRβ could represent an important target for guided tissue regeneration or tissue engineering of bone.

A Functional Polymorphism in COL11A1, Which Encodes the α1 Chain of Type XI Collagen, Is Associated with Susceptibility to Lumbar Disc Herniation

Lumbar disc herniation (LDH), degeneration and herniation of the nucleus pulposus of the intervertebral disc (IVD) of the lumbar spine, is one of the most common musculoskeletal diseases. Its etiology and pathogenesis, however, remain unclear. Type XI collagen is important for cartilage collagen formation and for organization of the extracellular matrix. We identified an association between one of the type XI collagen genes, COL11A1, and LDH in Japanese populations. COL11A1, which encodes the alpha 1 chain of type XI collagen, was highly expressed in IVD, but its expression was decreased in the IVD of patients with LDH. The expression level was inversely correlated with the severity of disc degeneration. A single-nucleotide polymorphism (c.4603C-->T [rs1676486]) had the most significant association with LDH (P=3.3 x 10(-6)), and the transcript containing the disease-associated allele was decreased because of its decreased stability. These observations indicate that type XI collagen is critical for IVD metabolism and that its decrease is related to LDH.

Telmisartan attenuates progression of steatohepatitis in mice: role of hepatic macrophage infiltration and effects on adipose tissue.

Background/Aims: Non‐alcoholic fatty liver disease and non‐alcoholic steatohepatitis (NASH) are the hepatic manifestation of metabolic syndrome. However, its therapeutic strategy has not been established. Recently, an angiotensin II type 1 receptor blocker, telmisartan (Tel), has received a great deal of attention as a therapeutic tool for metabolic syndrome. The aim of this study was to investigate the efficacy and mechanisms of Tel on a murine NASH model.

Mouse brains deficient in neuronal PDGF receptor-beta develop normally but are vulnerable to injury.

Platelet‐derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null‐knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the β subunit of PDGFR (PDGFR‐β) was genetically deleted in CNS neurons to elucidate the role of PDGFR‐β, particularly in the post‐natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR‐β detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild‐type mice was depleted, but PDGFR‐β detected in blood vessels remained unaffected. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT‐mediated dUTP‐biotin nick end labeling (TUNEL)‐positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR‐β expressed in neurons protects them from cryogenic injury and NMDA‐induced excitotoxicity.

Platelet-derived growth factor-b expression induced after rat peripheral nerve injuries

Schwann cells are crucially important for peripheral nerve regeneration. These cells synthesize several factors that are supposed to enhance axonal regeneration when injured. Platelet‐derived growth factor (PDGF) B‐chain and its β‐receptor are expressed in Schwann cells in both normal peripheral nerves and culture. To elucidate the role of PDGF‐B in peripheral nerve regeneration, we investigated its expression in cut or crush‐injured rat sciatic nerves for up to 28 days. Northern blotting identified substantial increase of PDGF B‐chain transcripts in injured nerves. Immunohistochemistry demonstrated that protein products of the transcripts were augmented at the distal tip of swollen axons in proximal nerve segments and in regenerating axons. Soon after both types of injury, considerable amounts of PDGF‐B accumulated in numerous Schwann cells in distal segments of both models. With restoration of the axon–Schwann cell relationship in the crush model, levels of PDGF‐B tended to decrease, eventually returning to normal. In the cut model in which the relationship cannot be restored, the PDGF‐B was depleted to a very low level. The spatiotemporal correlation between PDGF‐B and cell proliferation was very close throughout the study. These results differed strikingly from those of our previous study of rat optic nerve transection, in which PDGF‐B was expressed only in a few recruited macrophages and glial cells. Augmented PDGF‐B expression after sciatic nerve injury might contribute to peripheral nerve regeneration because PDGF‐B is a mitogen and survival factor for Schwann cells and because it has trophic activity on neurons. GLIA 38:303–312, 2002.

Decreased neural damage after spinal cord injury in tPA-deficient mice

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin. It plays an important role in the nervous system, including the processes of neuronal migration, neurite outgrowth, and neuronal plasticity. tPA has also been suggested to have a role in several neuropathological conditions, such as cerebral ischemia, seizures, and demyelinating diseases. To investigate the role of tPA in spinal cord injury, wild-type mice and mice with homozygous tPA deficiency (tPA(-/-) mice) were subjected to spinal cord contusion and the differences of hindlimb function, electrophysiological changes, and histopathological changes were assessed for 6 weeks. Functional recovery was greater in tPA(-/-) mice than in wild-type mice throughout the observation period. The time course of myoelectric motor-evoked potentials supported the hindlimb functional findings. Histological examination showed that injured areas were smaller in tPA(-/-) mice than wild-type mice on Luxol fast blue staining or myelin basic protein and neurofilament protein immunostaining at 6 weeks after contusion. Electron microscopy showed that the white matter was better preserved in tPA(-/-) mice than in wild-type mice. The expression of tPA protein was widespread on the first day after contusion and this expression was detected for at least a week. Activation of microglia/macrophages and apoptotic cell death were significantly reduced in tPA(-/-) mice after contusion. This study shows that neural damage is decreased in tPA(-/-) mice after spinal cord injury. Suppression of tPA production may help to decrease secondary injury after spinal cord contusion.

Involvement of aquaporin-5 in differentiation of human gastric cancer cells

Litttle is known about the function of aquaporin (AQP) water channels in human gastric cancer. In the upper or middle part of human stomach, we found that expression level of AQP5 protein in intestinal type of adenocarcinoma was significantly higher than that in accompanying normal mucosa. AQP5 was localized in the apical membrane of the cancer cells. On the other hand, both AQP3 and AQP4 were not up-regulated in the adenocarcinoma. To elucidate the role of AQP5 in cancer cells, AQP5 was exogenously expressed in a cell line of poorly differentiated human gastric adenocarcinoma (MKN45). The AQP5 expression significantly increased the proportion of differentiated cells with a spindle shape, the activity of alkaline phosphatase, a marker for the intestinal epithelial cell type of cancer cells, and the expression level of laminin, an epithelial cell marker. Treatment of the MKN45 cells stably expressing AQP5 with HgCl2, an inhibitor of aquaporins, significantly decreased the proportion of differentiated cells and the activity of alkaline phosphatase. Our results suggest that up-regulation of AQP5 may be involved in differentiation of human gastric cancer cells.

Activation of MAP kinases, Akt and PDGF receptors in injured peripheral nerves

A number of receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol‐3‐kinase (PI3K)/Akt and mitogen‐activated protein (MAP) kinase signaling pathways have been critically involved in peripheral nerve regeneration. Here, we examined the activation of PI3K/Akt and MAP kinase pathways, and platelet‐derived growth factor receptors (PDGFRs) in the distal segments of crushed rat sciatic nerve from 3 to 28 days after injury. In Western blot analyses, the phosphorylated forms of extracellular signal‐regulated protein kinase (ERK) and c‐Jun NH2‐terminal kinases (JNKs) were highly augmented on days 3 and 7 and on days 7 and 14 after injury, respectively. Phosphorylated Akt and p38 consistently increased from 3 to 28 days after injury. Phosphorylated PDGFR‐α and ‐β were also increased from 3 to 14 days. In the immunohistological analyses, phosphorylated ERK and PDGFR‐α were co‐localized in many activated Schwann cells and regrowing axons 3 days after injury, while PDGFR‐β was localized in a few spindle‐shaped cells. The detected temporal profile of RTK signaling appears to be crucial for the regulation of Schwann cell proliferation and following redifferentiation. Furthermore, the immunohistological studies suggested a role of ERK and PDGFR‐α in axon regeneration as well.