Takeya Sato

H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis

Our study reports a preliminary investigation into the role of human H2 relaxin in prostate tumor growth. A luciferase‐expressing human prostate cancer cell line, PC‐3, was generated and termed PC3‐Luc. PC3‐Luc cells were transduced with lentiviral vectors engineering the expression of either enhanced green fluorescent protein (eGFP) or both H2 relaxin and eGFP in a bicistronic format. These transduced cells were termed PC3‐Luc‐eGFP and PC3‐Luc‐H2/eGFP, respectively. To gauge effects, PC3‐Luc‐H2/eGFP and PC3‐Luc‐eGFP cells were injected into NOD/SCID mice and monitored over 6 weeks. PC‐3 tumor xenografts overexpressing H2 relaxin exhibited greater tumor volumes compared to control tumors. Circulating H2 relaxin levels in sera increased with the relative size of the tumor, with moderately elevated H2 relaxin levels in mice bearing PC3‐Luc‐H2/eGFP tumors compared to PC3‐Luc‐eGFP tumors. Zymographic analysis demonstrated that proMMP‐9 enzyme activity was significantly downregulated in H2 relaxin‐overexpressing tumors. An advanced angiogenic phenotype was observed in H2 relaxin‐overexpressing tumors indicated by greater intratumoral vascularization by immunohistochemical staining of endothelial cells with anti‐mouse CD31. Moreover, PC3‐Luc‐H2/eGFP tumors exhibited increased VEGF transcript by reverse‐transcription PCR, compared to basal levels in control animals. Taken together, our study provides the first account of a potential role of H2 relaxin in prostate tumor development.

Storage of Lipid Droplets in and Production of Extracellular Matrix by Hepatic Stellate Cells (Vitamin A- Storing Cells) in Long-Evans Cinnamon-Like Colored (LEC) Rats

LEC rats spontaneously develop hepatocellular carcinoma with cholangiofibrosis after chronic hepatitis, but the mechanism of development of the hepatic injury is not clear. To investigate the role of hepatic stellate cells in induction or suppression of hepatic fibrosis, we morphologically examined the liver of LEC rats. Accumulation of copper was analyzed by the Danscher‐Timms sulfide‐silver method. Histopathological changes were evaluated by hematoxylin and eosin staining, and by Massons trichrome method. Activated stellate cells were identified by immunostaining method for α‐smooth muscle actin. Cytological alterations of the stellate cells were investigated by transmission electron microscopy. To evaluate the lipid content in the stellate cells, we analyzed the area of lipid droplets of the cells by morphometric analysis. Also for evaluation of the changes in the number of stellate cells, the numbers of nucleated stellate cells and parenchymal cells were counted and statistically analyzed. Hepatic parenchymal cells showed excessive accumulation of copper at 5 weeks of age. Submassive necrosis was observed at 19 weeks of age. The liver of LEC rats 1.5 years of age showed cholangiofibrosis and subcellular injury of hepatic parenchymal cells. However, no diffuse hepatic fibrosis was observed in the liver, and hepatic stellate cells around the regions of cholangiofibrosis were negative for α‐smooth muscle actin. The area of lipid droplets of a stellate cell in the liver of LEC rats was 1.6 to 1.8 times as large as that of normal Wistar rats. The hepatic stellate cells did not participate in the accumulation of collagen fibers around themselves when the cells contained a large amount of vitamin A‐lipid droplets, even though the development of hepatic lesions was in progress. Our present data are consistent with our previous hypothesis that there is an antagonistic relationship between the storage of vitamin A and the production of collagen in stellate cells. Anat Rec 258:338–348, 2000.

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Glomerular endothelium exhibits enhanced expression of costimulatory adhesion molecules, CD80 and CD86, by warm ischemia/reperfusion injury in rats.

Recent studies suggested that the vascular endothelial cells function as a resident antigen-presenting cell (APC) in certain situations such as organ transplantation, and the ischemia/reperfusion injury, an inevitable event in organ transplantation, leads to an enhanced biosynthesis of cell adhesion molecules. We have demonstrated that the hepatic sinusoidal endothelial cells have potential ability as APCs by expressing the costimulatory adhesion molecule proteins, CD80 (B7–1) and CD86 (B7–2), of which expression was enhanced by warm ischemia/reperfusion of the rat liver. In this study, we assessed the localization of CD80, CD86, and intercellular adhesion molecule 1 in the rat kidneys and the influence of warm ischemia/reperfusion with or without a hypercreatinemic condition on the expression of these adhesion molecules in the renal tissues. Wistar male rats weighing 150 to 230 g were divided into group A, receiving a sham-operation (control), group B, receiving 1-hour clamping of the left renal pedicle (temporary ischemia), and group C, receiving right nephrectomy and 1-hour clamping of the left renal pedicle (temporary ischemia with hypercreatinemia). The left kidneys were submitted to immunohistochemical and molecular analyses sequentially for the period of 14 days. We found that CD80, CD86, and intercellular adhesion molecule 1 proteins localized on the glomerular and peritubular endothelium and were up-regulated after ischemia/reperfusion. The up-regulation of these three proteins was enhanced by the hypercreatinemic condition. The relative mRNA levels analyzed by real-time reverse transcription polymerase chain reaction showed that CD80 and CD86 expressions were constitutively observed and significantly increased for 14 days after the warm ischemia reperfusion with a peak level at Day 3 (6.7- and 20.8-fold increase for CD80 and CD86, respectively). Our results suggested that the glomerular endothelial cells will play a pivotal role as a APC by expressing CD80 and CD86 in the induction of renal tissue injury associated with the ischemia/reperfusion at renal transplantation surgery, as well as the peritubular endothelium.

The engineered thymidylate kinase (TMPK)/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

We previously described a novel suicide (or ‘cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

Cell Fate Control Gene Therapy Based on Engineered Variants of Human Deoxycytidine Kinase

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

Potentiation of potassium currents by beta-adrenoceptor agonists in human urinary bladder smooth muscle cells: a possible electrical mechanism of relaxation.

We examined the effects of β-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca2+-activated K+ (KCa) channels in relaxation by β-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 µmol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 µmol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 µmol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 µmol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of β2-adrenoceptors in the cells. These results suggest that Ca2+-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance KCa channels could be increased by stimulation of β2-adrenoceptors.

CLIC4 interacts with histamine H3 receptor and enhances the receptor cell surface expression

Histamine H3 receptor (H3R), one of G protein-coupled receptors (GPCRs), has been known to regulate neurotransmitter release negatively in central and peripheral nervous systems. Recently, a variety of intracellular proteins have been identified to interact with carboxy (C)-termini of GPCRs, and control their intracellular trafficking and signal transduction efficiencies. Screening for such proteins that interact with the C-terminus of H3R resulted in identification of one of the chloride intracellular channel (CLIC) proteins, CLIC4. The association of CLIC4 with H3R was confirmed in in vitro pull-down assays, coimmunoprecipitation from rat brain lysate, and immunofluorescence microscopy of rat cerebellar neurons. The data from flowcytometric analysis, radioligand receptor binding assay, and cell-based ELISA indicated that CLIC4 enhanced cell surface expression of wild-type H3R, but not a mutant form of the receptor that failed to interact with CLIC4. These results indicate that, by binding to the C-terminus of H3R, CLIC4 plays a critical role in regulation of the receptor cell surface expression.

Mitochonic Acid 5 (MA-5), a Derivative of the Plant Hormone Indole-3-Acetic Acid, Improves Survival of Fibroblasts from Patients with Mitochondrial Diseases

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Lebers hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.

Direct injection of kit ligand-2 lentivirus improves cardiac repair and rescues mice post-myocardial infarction.

Myocardial infarction (MI) and subsequent adverse remodeling cause heart failure. Previously we demonstrated a role for Kit ligand (KL) in improving cardiac function post-MI. KL has two major isoforms; KL-1 is secreted whereas KL-2 is predominantly membrane bound. We demonstrate here first that KL-2-deficient mice have worse survival and an increased heart/bodyweight ratio post-MI compared to mice with reduced c-Kit receptor expression. Next we synthesized recombinant lentiviral vectors (LVs) that engineered functional expression of murine KL-1 and KL-2. For in vivo analyses, we directly injected these LVs into the left ventricle of membrane-bound KL-deficient Sl/Sl(d) or wild-type (WT) mice undergoing MI. Control LV/enGFP injection led to measurable reporter gene expression in hearts. Injection of LV/KL-2 attenuated adverse left ventricular remodeling and dramatically improved survival post-MI in both Sl/Sl(d) and WT mice (from 12 to 71% and 35 to 73%, respectively, versus controls). With regard toward beginning to understand the possible salutary mechanisms involved in this effect, differential staining patterns of Sca-1 and Ly49 on peripheral blood (PB) cells from therapeutically treated animals was found. Our data show that LV/KL-2 gene therapy is a promising treatment for MI.