Yasuhiro Setoguchi

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

AbstractPulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM.

Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents ischemic brain injury after transient middle cerebral artery occlusion in rats

To examine a possible protective effect of exogenous glial cell line-derived neurotrophic factor (GDNF) gene expression against ischemic brain injury, a replication-defective adenoviral vector containing GDNF gene (Ad-GDNF) was directly injected into the cerebral cortex at 1 day before 90 minutes of transient middle cerebral artery occlusion (MCAO) in rats. 2,3,5-Triphenyltetrazolium chloride staining showed that infarct volume of the Ad-GDNF-injected group at 24 hours after the transient MCAO was significantly smaller than that of vehicle- or Ad-LacZ-treated group. Enzyme-linked immunosorbent assay (ELISA) for immunoreactive GDNF demonstrated that GDNF gene products in the Ad-GDNF-injected group were higher than those of vehicle-treated group at 24 hours after transient MCAO. Immunoreactive GDNF staining was obviously detected in the cortex around the needle track just before or 24 hours after MCAO in the Ad-GDNF group, whereas no or slight GDNF staining was detected in the vehicle group. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons induced in the ipsilateral cerebral cortex at 24 hours after transient MCAO were markedly reduced by the Ad-GDNF group. These results suggest that the successful exogenous GDNF gene transfer ameliorates ischemic brain injury after transient MCAO in association with the reduction of apoptotic signals.

Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis

Effects of adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) were studied in transgenic (Tg) mice model for amyotrophic lateral sclerosis (ALS). Adenoviral vector containing GDNF gene (Ad-GDNF), E. coli lacZ (Ad-LacZ), or vehicle was injected once a week from 35 weeks of age into the right gastrocnemius muscle of Tg mice carrying mutant human Cu/Zn superoxide dismutase (SOD1) gene, and histological analysis was performed at 46 W. Clinical data showed a tendency of improvement, but was not significantly different among the three animal groups. In contrast, total number of and phospho-Akt (p-Akt) positive large motor neurons in the treated side was significantly preserved in Ad-GDNF-treated group than in vehicle- and Ad-LacZ-treated groups (*p < 0.05). Immunoreactivity of phospho-ERK (p-ERK) and active caspases-3 and -9 showed no difference. These results indicate that the Ad-GDNF treatment prevented motor neuron loss with preserving survival p-Akt signal and without affecting caspase activations, suggesting a future possibility for the therapy of the disease.

In vivo adenovirus-mediated gene transfer and the expression in ischemic and reperfused rat brain

In an attempt to study whether ischemic brain could express a foreign gene in vivo, a replication-defective adenoviral vector containing the Escherichia coli lacZ gene was directly injected into the ischemic or reperfused cerebral cortex of rat, and temporal and spatial profiles of the exogenous gene expression were compared with that of the control brain. Right middle cerebral artery (MCA) of rat was continuously occluded by an insertion of nylon thread for 2 days, or only transiently occluded for 90 min and then the blood flow was restored for 21 days. The adenoviral vector was administered just after the MCA occlusion or reperfusion in the case of continuous ischemia and reperfusion, respectively. Adenoviral vector was transferred into the continuous ischemic brain, and the lacZ gene was expressed until 2 days of the occlusion in the cerebral cortex of the occluded MCA territory with the number of expressing cells smaller and the staining just weaker than that of the control brain. In contrast, expression of the lacZ gene was not or only minimally observed in the reperfused brain until 2 days. However, the expression dramatically exploded at 7 days of reperfusion at a level similar to that of the control, and the expression diminished by 21 days. A few neurons in the ipsilateral thalamus, hypothalamus, and basal ganglia, and in the contralateral cerebral cortex expressed the lacZ gene at 7 days after reperfusion, a phenomenon similar to the case of the control. The majority of brain cells that expressed the lacZ gene were neurons, and a part (5-10%) were astroglial cells. Traumatic injury and immunological response in the brain were minimal both in the cases of control and ischemia/reperfusion. The present study shows an effective gene transfer and the expression in neural cells of ischemic and reperfused brains in vivo, and suggests a great potential of the gene therapy for ischemic stroke patients in the future.

Impaired retrograde axonal transport of adenovirus-mediated E. coli LacZ gene in the mice carrying mutant SOD1 gene

A replication-defective recombinant adenoviral vector containing E. coli lacZ gene was injected into the gastrocnemius muscles of transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 40 weeks of age. After 60 and 90 h of the injection, lacZ staining was observed at the distal ends of the sciatic nerves in both mice groups, with the number and the distances greatly reduced in the transgenic mice. Mean velocities of retrograde transport for lacZ was estimated to be 2.1 and 0.05 mm/24 h in non-transgenic and transgenic mice, respectively. These results indicate that the retrograde axonal transport of foreign gene product is impaired in the mice model for familial amyotrophic lateral sclerosis.

TGF-β/Smad signaling inhibits IFN-γ and TNF-α-induced TARC (CCL17) production in HaCaT cells

Abstract Background: A Th2 chemokine, thymus and activation regulated chemokine (TARC/CCL17), produced by keratinocytes, is implicated in the development of atopic dermatitis by recruiting CLA+CCR4+ lymphocytes into lesional skin and its expression was induced by proinflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). However, it remains unknown how TARC expression is negatively regulated in keratinocytes. Objective: We sought to determine whether transforming growth factor-β1 (TGF-β1) regulated TARC expression in keratinocytes. Methods: The effect of TGF-β1 on mRNA and protein expression of IFN-γ and TNF-α-induced TARC in a human keratinocyte cell line, HaCaT cells, was evaluated by using RT-PCR and ELISA. Adenovector-mediated gene transfer was used to determine the effect of Smad proteins on TARC expression in HaCaT cells. Results: TGF-β1 inhibited mRNA and protein expression of IFN-γ and TNF-α-induced TARC in HaCaT cells. The inhibitory effect of TGF-β1 on the TARC expression was suppressed by overexpression of Smad7, a major inhibitory regulator of Smad pathway for transforming growth factor-β (TGF-β) signaling, but not by PD98059, an inhibitor for ERK/mitogen-activated protein kinase (MAPK) pathway. In addition, overexpression of Smad2 or Smad3, major signal transducing Smads, was sufficient to inhibite the IFN-γ and TNF-α-induced TARC production in HaCaT cells. Conclusion: TGF-β1 inhibited IFN-γ and TNF-α-induced TARC production in HaCaT cells via Smad2/3, suggesting that modulation of TGF-β/Smad signaling pathway may be beneficial for the treatment of atopic dermatitis.

Smads regulate collagen gel contraction by human dermal fibroblasts

Backgroundu2003 Transforming growth factor (TGF)‐β induces fibroblast contraction that is implicated in efficient wound healing. The Smad family of proteins mediates signal transduction of the TGF‐β superfamily. However, its role in fibroblast contraction remains unclear.

Smad7 Suppresses the Inhibitory Effect of TGF-β2 on Corneal Endothelial Cell Proliferation and Accelerates Corneal Endothelial Wound Closure In Vitro

Purpose. The inhibitory activity of transforming growth factor-&bgr;2 (TGF-&bgr;2) on corneal endothelial cell proliferation is thought to be a cause of the limited regenerative capacity of corneal endothelial cells that may be related to impaired corneal transparency when many corneal endothelial cells are lost due to various stresses. We determined whether Smad7, an intracellular antagonist of TGF-&bgr; signaling, regulated the inhibitory activity of TGF-&bgr;2 or aqueous humor on corneal endothelial cell proliferation. Methods. The effect of Smad7 on TGF-&bgr;2– or aqueous humor–mediated inhibition of corneal endothelial cell proliferation was evaluated using thymidine uptake assay with cultured rabbit corneal endothelial cells infected with adenovirus carrying Smad7. Expression of Smad or cell cycle–related proteins was detected by immunoblotting. In addition, a small scrape wound was made across a monolayer of Smad7-expressing cultured rabbit corneal endothelial cells to examine the effect of Smad7 on the wound-healing process in vitro. Results. Overexpression of Smad7 abolished the inhibitory effect of TGF-&bgr;2 or aqueous humor on the proliferation of cultured rabbit corneal endothelial cells associated with the inhibition of phosphorylation of Smad2 and downregulation of p27kip1. Smad7-overexpressing cultured rabbit corneal endothelial cells exhibited shorter wound closure time in the presence of aqueous humor than LacZ-expressing cells. Conclusion. Overexpression of Smad7 suppressed the inhibitory effect of TGF-&bgr;2 or aqueous humor on corneal endothelial cell proliferation and accelerated corneal endothelial wound closure in vitro. Modification of Smad7 expression in corneal endothelial cells may thus have applicability in the treatment of wounded corneal endothelium.

A patient with TSC1 germline mutation whose clinical phenotype was limited to lymphangioleiomyomatosis

Background:u2002 Lymphangioleiomyomatosis (LAM) can occur as in isolated form (sporadic LAM) or as a pulmonary manifestation of tuberous sclerosis complex (TSC) (TSC‐associated LAM). Recent studies, however, revealed that both forms of LAM are genetically related but that sporadic LAM is a distinct clinical entity caused by somatic mutations of TSC2 (not TSC1) rather than a forme fruste of TSC carrying either of the TSC1 or TSC2 germline mutations.

Protection Against Fas-Mediated and Tumor Necrosis Factor Receptor 1-Mediated Liver Injury by Blockade of FADD Without Loss of Nuclear Factor-κB Activation

ObjectiveTo investigate the role of FADD (Fas-associated protein with death domain) in Fas and tumor necrosis factor receptor 1 (TNFR1)-mediated hepatic injury and inflammatory response in vivo. Summary Background DataFas and TNFR1 are cell surface molecules that trigger apoptosis or inflammation on engagement by a specific ligand or antibody. FADD is recruited to the cytoplasmic domain of these receptors on their activation and works as a common mediator to induce apoptosis. It is known that a blockade of FADD can inhibit apoptosis mediated by Fas or TNFR1 in vitro. However, it is not known whether the blockade can prevent organ injury and whether the inflammatory cascade is affected in vivo. MethodsA FADD deletion mutant lacking the death effector domain was introduced into mice by transduction with an adenovirus vector, and the effect of this FADD dominant negative mutant was examined in several liver injury models. ResultsHepatic injury induced by anti-Fas monoclonal antibody or tumor necrosis factor (TNF)-&agr; plus D-galactosamine was markedly ameliorated by the FADD dominant negative transduction, which abrogated the death rate. Further, the FADD dominant negative transduction efficiently blocked T cell- mediated concanavalin A-induced hepatitis while not affecting TNF-&agr; production or TNF-&agr;-induced nuclear factor-&kgr;B activation in the liver. ConclusionsThese results provide the basis for a novel therapeutic modality in which an unfavorable apoptotic process can be inhibited without affecting a favorable response for liver regeneration; this would be relevant to the clinical treatment of acute and chronic liver diseases as well as to some inflammatory disorders with hypercytokinemia, such as sepsis.