Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation.

Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.

CDK8/19 inhibition plays an important role in pancreatic ß-cell induction from human iPSCs.

BACKGROUND: Transplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs. METHODS: We evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose-response assays, single-cell RNA-sequencing and in vivo efficacy study. RESULTS: We observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used ß-cell inducer but no other tested ALK5 inhibitors induced ß-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced ß-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII. CONCLUSION: Our findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the ß-cell differentiation mechanism.

Insulin-Deficient Diabetic Condition Upregulates the Insulin-Secreting Capacity of Human Induced Pluripotent Stem Cell-Derived Pancreatic Endocrine Progenitor Cells After Implantation in Mice.

The host environment is a crucial factor for considering the transplant of stem cell-derived immature pancreatic cells in patients with type 1 diabetes. Here, we investigated the effect of insulin (INS)-deficient diabetes on the fate of immature pancreatic endocrine cell grafts and the underlying mechanisms. Human induced pluripotent stem cell-derived pancreatic endocrine progenitor cells (EPCs), which contained a high proportion of chromogranin A+ NK6 homeobox 1+ cells and very few INS+ cells, were used. When the EPCs were implanted under the kidney capsule in immunodeficient mice, INS-deficient diabetes accelerated increase in plasma human C-peptide, a marker of graft-derived INS secretion. The acceleration was suppressed by INS infusion but not affected by partial attenuation of hyperglycemia by dapagliflozin, an INS-independent glucose-lowering agent. Immunohistochemical analyses indicated that the grafts from diabetic mice contained more endocrine cells including proliferative INS-producing cells compared with that from nondiabetic mice, despite no difference in whole graft mass between the two groups. These data suggest that INS-deficient diabetes upregulates the INS-secreting capacity of EPC grafts by increasing the number of endocrine cells including INS-producing cells without changing the graft mass. These findings provide useful insights into postoperative diabetic care for cell therapy using stem cell-derived pancreatic cells.

GPR40 full agonism exerts feeding suppression and weight loss through afferent vagal nerve.

GPR40/FFAR1 is a Gq protein-coupled receptor expressed in pancreatic ß cells and enteroendocrine cells, and mediates insulin and incretin secretion to regulate feeding behavior. Several GPR40 full agonists have been reported to reduce food intake in rodents by regulating gut hormone secretion in addition to their potent glucose-lowering effects; however, detailed mechanisms of feeding suppression are still unknown. In the present study, we characterized T-3601386, a novel compound with potent full agonistic activity for GPR40, by using in vitro Ca2+ mobilization assay in Chinese hamster ovary (CHO) cells expressing FFAR1 and in vivo hormone secretion assay. We also evaluated feeding suppression and weight loss after the administration of T-3601386 and investigated the involvement of the vagal nerve in these effects. T-3601386, but not a partial agonist fasiglifam, increased intracellular Ca2+ levels in CHO cells with low FFAR1 expression, and single dosing of T-3601386 in diet-induced obese (DIO) rats elevated plasma incretin levels, suggesting full agonistic properties of T-3601386 against GPR40. Multiple doses of T-3601386, but not fasiglifam, in DIO rats showed dose-dependent weight loss accompanied by feeding suppression and durable glucagon-like peptide-1 elevation, all of which were completely abolished in Ffar1-/- mice. Immunohistochemical analysis in the nuclei of the solitary tract demonstrated that T-3601386 increased the number of c-Fos positive cells, which also disappeared in Ffar1-/- mice. Surgical vagotomy and drug-induced deafferentation counteracted the feeding suppression and weight loss induced by the administration of T-3601386. These results suggest that T-3601386 exerts incretin release and weight loss in a GPR40-dependent manner, and that afferent vagal nerves are important for the feeding suppression induced by GPR40 full agonism. Our novel findings raise the possibility that GPR40 full agonist can induce periphery-derived weight reduction, which may provide benefits such as less adverse effects in central nervous system compared to centrally-acting anti-obesity drugs.

SCO-267, a GPR40 Full Agonist, Improves Glycemic and Body Weight Control in Rat Models of Diabetes and Obesity.

The GPR40/FFA1 receptor is a G-protein-coupled receptor expressed in the pancreatic islets and enteroendocrine cells. Here, we report the pharmacological profiles of (3S)-3-cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-methylpyridin-2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)methoxy]pyridin-4-yl}propanoic acid (SCO-267), a novel full agonist of GPR40. Ca2+ signaling and insulin and glucagon-like peptide-1 (GLP-1) secretion were evaluated in GPR40-expressing CHO, MIN6, and GLUTag cells. Hormone secretions and effects on fasting glucose were tested in rats. Single or repeated dosing effects were evaluated in neonatally streptozotocin-induced diabetic rats (N-STZ-1.5 rats), diet-induced obese (DIO) rats, and GPR40-knockout (Ffar1-/- ) mice. Treatment with SCO-267 activated Gq signaling in both high- and low-FFAR1-expressing CHO cells, stimulated insulin secretion in MIN6 cells, and induced GLP-1 release in GLUTag cells. When administered to normal rats, SCO-267 increased insulin, glucagon, GLP-1, glucose-dependent insulinotropic peptide, and peptide YY (PYY) secretions under nonfasting conditions. These results show the full agonistic property of SCO-267 against GPR40. Hypoglycemia was not induced in SCO-267-treated rats during the fasting condition. In diabetic N-STZ-1.5 rats, SCO-267 was highly effective in improving glucose tolerance in single and 2-week dosing studies. DIO rats treated with SCO-267 for 2 weeks showed elevated plasma GLP-1 and PYY levels, reduced food intake, and decreased body weight. In wild-type mice, SCO-267 induced GLP-1 secretion, food intake inhibition, and body weight reduction; however, these effects were abolished in Ffar1-/- mice, indicating a GPR40-dependent mechanism. In conclusion, SCO-267 stimulated islet and gut hormone secretion, improved glycemic control in diabetic rats, and decreased body weight in obese rats. These data suggest the therapeutic potential of SCO-267 for the treatment of diabetes and obesity.

Trelagliptin (SYR-472, Zafatek), Novel Once-Weekly Treatment for Type 2 Diabetes, Inhibits Dipeptidyl Peptidase-4 (DPP-4) via a Non-Covalent Mechanism.

Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation ≈ 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin.

Reduction of fibroproliferative changes in irradiated rat lung with soluble transforming growth factor-ß receptor.

The present study investigated whether established fibroproliferative changes in the irradiated rat lung are histopathologically reduced by an adenovirus­mediated soluble transforming growth factor (TGF)­ß type II receptor. Replication­defective adenoviral vectors expressing a type II human TGF­ß receptor (AdTß­ExR) were prepared. Male Fisher­344 rats were divided into the C, R and R + T groups. The rats in the C group did not receive irradiation or treatment. The rats in the R and R + T group each received 30 Gy irradiation to the right lung. Eight weeks following irradiation, the rats in the R and R + T group were treated with saline or AdTß­ExR, respectively. To analyze the TGF­ß expression, myofibroblast proliferation and macrophage/monocyte infiltration, sections of the lung were immunohistochemically stained at 16 weeks following irradiation. Silver staining was performed for semi­quantitative evaluation of the fibroproliferative changes. Definitive TGF­ß expression, myofibroblast proliferation and macrophage/monocyte infiltration were observed in the lungs of the R group, but were significantly lower in the lungs of the R + T group. With respect to the fibroproliferative changes, the proportion of red­stained areas in the R + T group was markedly lower than that in the R group. These data indicate that fibroproliferative changes induced by radiation are reversible and that TGF­ß has a critical role in fibroproliferative changes in the irradiated lung. The present results suggest that gene therapy with an adenoviral vector expressing a soluble TGF­ß receptor may be effective in reducing the established pulmonary fibrosis caused by radiation.

Hypoxia induces CD133 expression in human lung cancer cells by up-regulation of OCT3/4 and SOX2.

CD133 has been recognized as a specific cell surface marker for cancer stem cells in various tumors, although its biological functions and transcriptional regulation remain unclear. We found that the CD133 expression level was up-regulated in the lung cancer cell lines N417, H358, and A549, when these cell lines were cultured under hypoxic conditions. Among the five promoters (P1-P5) of human CD133 gene loci, P1 promoter was most strongly associated with hypoxia-induced promoter activity of CD133 gene expression. The P1 promoter possesses several cis-regulatory elements, including RUNT, GATA, ETS, OCT, SRY, and CREB-binding sites. A series of deletion and base substitution mutants of the P1 promoter revealed that OCT- and SRY-binding sites are important for hypoxia-induced promoter activity. The chromatin immunoprecipitation assay further confirmed the direct binding of Octamer biding trans-cription factor 3/4 (OCT4) and/or SRY-box containing gene 2 (SOX2) to the P1 promoter region of CD133 gene loci. In addition, the enhancement of both OCT4 and SOX2 expression by the α subunit of hypoxia-inducible factors (HIF1α and HIF2α) was required for hypoxia-induced CD133 expression. Knockdown of OCT4 or SOX2 expression in N417 cells with stabilized HIF1α and/or HIF2α abolished CD133P1 activity, while ectopic OCT4 or SOX2 expression triggers CD133P1 activity in the absence of HIF1α or HIF2α. Thus, in the hypoxic conditions, OCT4 and SOX2, both of which are induced by HIF1α/HIF2α. promote CD133 expression in the lung cancer cells via their direct interaction with the P1 promoter.

Risk stratification based on metabolic syndrome as well as non- metabolic risk factors in the assessment of carotid atherosclerosis.

AIM: We aimed to develop a new approach to risk stratification using metabolic syndrome as well as traditional non-metabolic risk factors, and to examine its validity in carotid atherosclerosis. METHODS: A total of 1,189 men and women aged 21-93 years old were stratified according to the absence or presence of metabolic syndrome defined by Japanese criteria, non-metabolic risk factors, and a past history of coronary heart disease. The risk stratification was as follows: (S-1) persons without a past history, non-metabolic risk factors and metabolic syndrome, (S-2a) those with metabolic syndrome only, (S-2b) those with non-metabolic risk factors only, (S-3) those with non-metabolic risk factors and metabolic syndrome but no past history, and (S-4) those with a past history. Carotid atherosclerosis was defined as maximum intima-media thickness ≥1.1 mm of the far wall of the common carotid artery. RESULTS: Compared with individuals without these three risk components (S-1), the odds ratio was 7.2 (2.8-18.6) for a past history (S-4), 4.3 (1.7-10.9) for non-metabolic risk factors plus metabolic syndrome but no past history (S-3), 2.6 (1.1-6.4) for non-metabolic risk factors only (S-2b) and 0.5 (0.0-5.7) for metabolic syndrome only (S-2a). Net reclassification improvement from metabolic syndrome only (presence versus absence) to our risk stratification (≥S-3 versus < S-3) was 16.4% (p< 0.0001), suggesting that our risk stratification improved the classification of atherosclerosis in comparison to metabolic syndrome only. CONCLUSION: Risk stratification based on traditional non-metabolic risk factors plus metabolic syndrome rather than metabolic syndrome only appears to be more useful for the clinical assessment of atherosclerosis, and probably in the prevention and control of atherosclerotic disease.

Rabbit plaque models closely resembling lesions in human coronary artery disease.

BACKGROUND: A suitable animal model is required to investigate plaque biology. Here, we examined 6 rabbit models of plaque generated by balloon injury and sequential combinations of normal and high-cholesterol diets. METHODS AND RESULTS: Fifty-eight male Japanese White rabbits were used. Lipid-rich macrophages accumulated in the center of the intima, and smooth muscle cells were located on the luminal side of the intima (similar to stable plaques in human coronary arteries) of a model in which balloon injury was followed by a normal diet for 4 weeks and then by a high-cholesterol diet for 4 weeks. Extending the high-cholesterol diet for a further 4 weeks increased accumulation of lipid-rich macrophages, diminished the amounts of elastic fibers and smooth muscle cells in the intima and caused the expression of matrix metalloproteinase-9 and tissue factor. All of these features are characteristic of unstable plaques. Moreover, quantitative analysis revealed that matrix metalloproteinase-9 expression and elastic-fiber content inversely correlated with statistical significance (R(2) = 0.52, p = 0.0003). CONCLUSION: A high-cholesterol diet for 0 to 8 weeks after a normal diet for the first 4 weeks following balloon injury induced various arterial lesions resembling the diffuse intimal thickening, as well as stable and unstable plaques that accumulate in human coronary arteries. The present models might be useful for plaque studies.

Effect of endothelial cell proliferation on atherogenesis: a role of p21(Sdi/Cip/Waf1) in monocyte adhesion to endothelial cells.

OBJECTIVE: Uniform laminar shear stress (LS) and disturbed turbulent shear stress (DS) are thought to play opposite roles in preventing or inducing atherosclerosis. Endothelial cell (EC) growth and monocyte adhesion to ECs, an early event in atherosclerosis, are also oppositely regulated by LS and DS. However, how atherogenesis is affected by the regulation of growth by blood flow is unknown. Here we examined the role of p21(Sdi/Cip/Waf1) (p21), a growth inhibitor induced by LS, in monocyte adhesion to ECs. METHODS: p21 was overexpressed by transfecting a p21-expressing adenoviral vector into ECs. Factors linking EC growth, monocyte adhesion, and p21 were examined by microarray analysis, PCR and Western blotting. RESULTS: Compared with DS, in the presence or absence of TNFalpha, LS significantly inhibited EC growth and monocyte adhesion to ECs. Both EC proliferation and monocyte adhesion induced by DS were inhibited by p21-overexpression. LS suppressed the expression of thioredoxin-interacting protein (TXNIP). Thioredoxin (TRX) activity, which is suppressed by TXNIP, was therefore higher under LS than DS, as reported previously. p21-overexpression significantly suppressed the DS-induced TXNIP expression, and inhibited the expression of vascular cell adhesion molecule 1 and chemokine (C-C motif) ligand 5 (CCL5/RANTES), which stimulates leukocyte recruitment and is downregulated by ROS scavenging. CONCLUSION: p21 may function to prevent atherogenesis by regulating the redox balance, which leads to the inhibition of adhesion molecule and chemokine expression in ECs under LS.

Experimental study of antiangiogenic gene therapy targeting VEGF in oral cancer.

It is well known that tumor angiogenesis plays an important role in local growth and metastasis of oral cancer; therefore, inhibiting angiogenesis is considered to be effective for treating oral cancer. This study aimed to investigate the effectiveness of systemically available antiangiogenic gene therapy targeting vascular endothelial growth factor (VEGF), which is one of the most important angiogenesis accelerators. We administered a soluble form of VEGF receptor-expressing gene incorporated into adenovirus (AdVEGF-ExR) intraperitoneally to nude mice to which oral cancer cell lines (SAS, HSC-3, and Ca9-22) had been transplanted subcutaneously in vivo to inhibit angiogenesis and tumor proliferation. Then, we measured tumor volumes over time, and tumors were enucleated and examined histopathologically and immunohistologically at 28 days after AdVEGF-ExR administration. Compared to the controls to which we administered AdLacZ or saline, significant antiproliferative effects were observed (P < 0.05) in the AdVEGF-ExR administration group, and extensive tumor necrosis was found histopathologically. Immunohistochemical analysis with CD34 (NU-4A1) revealed tumor angiogenesis was suppressed significantly (P < 0.05), and that with ssDNA revealed apoptosis induction was significantly high (P < 0.05) in the AdVEGF-ExR group. However, analysis with Ki-67 (MIB-1) revealed tumor proliferative capacity was not significantly different between the groups. Consequently, we consider that AdVEGF-ExR administration achieved tumor growth suppression by inhibiting angiogenesis and inducing apoptosis, but not by inhibiting the proliferative capacity of tumor cells. Neither topical administration of a soluble form of VEGF receptor (sVEGFR) to the tumor nor a megadose was needed to achieve this inhibition effect. These results suggest gene therapy via sVEGFR would be an effective oral cancer therapy and benefit future clinical applications.

Blockade of TGF-ß by catheter-based local intravascular gene delivery does not alter the in-stent neointimal response, but enhances inflammation in pig coronary arteries.

BACKGROUND: Extracellular matrix (ECM) accumulation significantly contributes to in-stent restenosis. In this regard, transforming growth factor (TGF)-ß, a positive regulator of ECM deposition, may be implicated in in-stent restenosis. The goal of this study was to assess the effect of blockade of TGF-ß on stent-induced restenosis in porcine coronary arteries. METHODS: An adenovirus expressing the ectodomain of the TGF-ß type II receptor (AdTß-ExR) was applied onto a coronary arterial segment of a pig (n=10) using an Infiltrator, followed by stent deployment. Controls consisted of adenoviruses expressing ß-galactosidase (AdLacZ) or phosphate-buffered saline (PBS) applied onto the other segment (n=10) of the same pig. RESULTS: Computer-based pathological morphometric analysis of stented coronary arteries, performed 4 weeks after stenting, demonstrated no significant difference in morphometric parameters such as in-stent neointimal area and % area stenosis between the AdTß-ExR group and control (n=7 for each). However the AdTß-ExR group had increased neointimal cell density, infiltration of inflammatory cells mostly consisting of CD3+ T cell, accumulation of hyaluronan, cell proliferation rate, and adventitial matrix metalloproteinase-1 (MMP-1) expression compared with control. The expression of connective tissue growth factor mRNA, measured by reverse transcription PCR, in cultured rat arterial smooth muscle cells was inhibited by AdTß-ExR at moi 60. CONCLUSIONS: Blockade of TGF-ß by catheter-based local intravascular gene delivery does not reduce stent-induced neointima formation 4 weeks after stenting in spite of modest inhibition of ECM accumulation, but it induces vascular inflammation and associated pathological changes that may potentially aggravate lesion progression.

Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling.

Painful neuropathy is one of the most common complications of diabetes, one hallmark of which is tactile allodynia (pain hypersensitivity to innocuous stimulation). The underlying mechanisms of tactile allodynia are, however, poorly understood. Emerging evidence indicates that, following nerve injury, activated microglia in the spinal cord play a crucial role in tactile allodynia. However, it remains unknown whether spinal microglia are activated under diabetic conditions and whether they contribute to diabetes-induced tactile allodynia. In the present study, using streptozotocin (STZ)-induced diabetic rats that displayed tactile allodynia, we found several morphological changes of activated microglia in the dorsal horn. These included increases in Iba1 and OX-42 labeling (markers of microglia), hypertrophic morphology, the thickness and the retraction of processes, and in the number of activated microglia cells. Furthermore, in the dorsal horn of STZ diabetic rats, extracellular signal-regulated protein kinase (ERK) and an upstream kinase, Src-family kinase (SFK), both of which are implicated in microglial functions, were activated exclusively in microglia. Moreover, inhibition of ERK phosphorylation in the dorsal horn by intrathecal administration of U0126, an inhibitor of ERK activation, produced a striking alleviation of existing, long-term tactile allodynia of diabetic rats. We also found that a single administration of U0126 reduced the expression of allodynia. Together, these results suggest that activated dorsal horn microglia may be a crucial component of diabetes-induced tactile allodynia, mediated, in part, by the ERK signaling pathway. Thus, inhibiting microglia activation in the dorsal horn may represent a therapeutic strategy for treating diabetic tactile allodynia.

Stage-specific role of endogenous Smad2 activation in cardiomyogenesis of embryonic stem cells.

The role of Smads and their specific ligands during cardiomyogenesis in ES cells was examined. Smad2 was activated bimodally in the early and late phases of cardiac differentiation, whereas Smad1 was activated after the middle phase. Nodal and Cripto were expressed in the early stage and then downregulated, whereas transforming growth factor-beta and activin were expressed only in the late phase. Suppression of early Smad2 activation by SB-431542 produced complete inhibition of endodermal and mesodermal induction but augmented neuroectodermal differentiation, followed by poor cardiomyogenesis, whereas inhibition during the late phase alone promoted cardiomyogenesis. Inhibitory effect of Smad2 on cardiomyogenesis in the late phase was mainly mediated by transforming growth factor-beta, and inhibition of transforming growth factor-beta-mediated Smad2 activation resulted in a greater replicative potential in differentiated cardiac myocytes and enhanced differentiation of nonmyocytes into cardiac myocytes. Thus, endogenous Smad2 activation is indispensable for endodermal and mesodermal induction in the early phase. In the late phase, endogenous transforming growth factor-beta negatively regulates cardiomyogenesis through Smad2 activation by modulating proliferation and differentiation of cardiac myocytes.

Properties of acetylcholine-induced hyperpolarization in smooth muscle cells of the mouse mesenteric artery.

The properties of smooth muscle cell hyperpolarization produced by acetylcholine (ACh) were investigated in mesenteric arteries isolated from mice. The resting membrane potential of the smooth muscle cells was about -60 mV. When ACh (10 microM) was applied for 1 min, the membrane hyperpolarized with a peak amplitude of about 5 mV which was reached in about 1 min, following which the potential slowly reverted to the resting level over about 7 min following withdrawal of ACh from the superfusate (recovery component). Exposure of the artery to 0.5 mM Ba(2+), an inhibitor of inward rectifier K-channels, depolarized the membrane by about 13 mV, increased the amplitude of the ACh-induced hyperpolarization to about 10 mV, and facilitated the visualization of the recovery component. Indomethacine (10 microM), an inhibitor of cyclooxygenase, inhibited the recovery component and as a consequence reduced the duration of the hyperpolarization. The ACh-induced response was not markedly altered by either N(omega)-nitro-L-arginine (10 microM), an inhibitor of nitric oxide (NO) production, or catalase (130 U/ml), a super oxide scavenger. Exogenously applied hydrogen peroxide (H(2)O(2), 300 microM) hyperpolarized the membrane by about 5 mV, which was abolished by catalase. These results suggest that in the mouse mesenteric artery, the ACh-induced hyperpolarization has two components, both an indomethacin-sensitive and an indomethacin-insensitive component. The former component may be produced by prostanoids, while the latter may be produced by factors other than NO or H(2)O(2). The results also suggested that the inward rectifier K-channels may be important for producing the resting membrane potential, but they may not be the main contributor to the ACh-induced hyperpolarization of smooth muscle cell membranes in the mouse mesenteric artery.

Combination gene therapy of HGF and truncated type II TGF-beta receptor for rat liver cirrhosis after partial hepatectomy.

BACKGROUND: In a cirrhotic liver, the regenerative ability and specific functions are impaired; a hepatic resection increases the possibility of postoperative liver failure. Hepatocyte growth factor (HGF) stimulates liver regeneration, accelerates restoration of hepatic function, and improves fibrosis. A truncated type II transforming growth factor-beta receptor (TbetaTR), which specifically inhibits TGF-beta signaling as a dominant-negative receptor, appears to prevent the progression of liver fibrosis. We demonstrated the therapeutic efficacy of adenovirus-mediated HGF and TbetaTR gene transduction after partial hepatectomy for liver cirrhosis. METHODS: Rats were treated with dimethylnitrosamine for 3 weeks, and they all had severe cirrhosis. After partial hepatectomy (10%), we injected adenovirus expressing bacterial beta-galactosidase (AdLacZ), adenovirus expressing a truncated type II TGF-beta receptor (AdTbetaTR), adenovirus expressing hepatocyte growth factor (AdHGF), or AdTbetaTR + AdHGF into the portal vein, which was followed by an additional 2-week dimethylnitrosamine treatment. RESULTS: On histologic examination, fibrotic tissue had decreased in the livers of the AdTbetaTR + AdHGF-treated rats compared with rats that were treated by AdLacZ, AdTbetaTR alone, and AdHGF alone. Liver function, which included serum levels of alanine aminotransferase, improved significantly in AdTbetaTR + AdHGF-treated rats compared with all other groups. The number of hepatocytes that were positive for proliferating-cell nuclear antigen was greater (P < .05) in AdHGF alone and AdTbetaTR + AdHGF-treated rat livers than in AdLacZ- and AdTbetaTR-treated rats. All AdTbetaTR + AdHGF-treated rats survived >60 days, and AdTbetaTR + AdHGF treatment markedly improved the survival rate after a partial hepatectomy. CONCLUSION: Our results suggest that the combination of HGF and TbetaTR gene therapy may increase the possibility of hepatectomy in a cirrhotic liver by improving fibrosis, hepatic function, and hepatocyte regeneration.

In vivo growth of transitional and renal cell carcinoma cell lines can be suppressed by the adenovirus-mediated expression of a soluble form of vascular endothelial growth factor receptor.

Antiangiogenic therapy is a promising strategy for the treatment of cancer since tumor development and metastases require angiogenesis. Vascular endothelial growth factor (VEGF) is one of the most important factors in tumor angiogenesis. In the present study, we investigated the antitumor effect of an adenovirus (AdVEGF-ExR) expressing the extracellular domain of the human VEGF receptor (flt-1) using two different urological tumor/mouse systems. RENCA, a renal cell carcinoma of BALB/c origin, and MBT-2, a poorly differentiated transitional carcinoma of C3H/He origin, were used. Both types of tumor were in vitro infected with AdVEGF-ExR and inoculated subcutaneously into the abdomens of syngenenic mice, and tumor growth was measured twice weekly. In some experiments, BALB/c mice with established RENCA tumors were injected intramuscularly with AdVEGF-ExR as a therapeutic model. The cytotoxicity of spleen cells from the tumor-rejected mice was assessed by 51Cr-release assay. Although the in vitro cell growth of either MBT-2 or RENCA was not affected by infection with AdVEGF-ExR, the in vivo growth of both AdVEGF-ExR-infected tumors was significantly suppressed in the syngeneic mice. In addition, although 2 of 5 mice rejected the AdVEGF-ExR-infected RENCA, tumor-specific cytotoxic T lymphocytes were not generated from their spleen cells, thus suggesting no cellular immune response. In a therapeutic model, intramuscular injections of AdVEGF-ExR at a remote site also significantly suppressed the growth of the subcutaneously established RENCA. These results indicate that the adenovirus-mediated expression of a soluble VEGF receptor can be an effective therapy for urological cancer treatment; however, such VEGF-targeted gene therapy is not necessarily accompanied by subsequent antitumor T cell immunity.

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis.

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.

Angiotensin II-induced activation of p21-activated kinase 1 requires Ca2+ and protein kinase C{delta} in vascular smooth muscle cells.

ANG II promotes remodeling of vascular smooth muscle cells (VSMCs) in cardiovascular diseases. It has been shown to activate p21-activated kinase (PAK)1, a critical component of signaling pathways implicated in growth and migration. However, the detailed signaling mechanism by which ANG II induces PAK1 activation in VSMCs remains unclear. Therefore, we have examined the mechanism required for activation of PAK1 by ANG II in VSMCs. ANG II, through activation of the ANG II type 1 receptor, rapidly promotes phosphorylation of PAK1 in VSMCs via a pathway independent of transactivation of the epidermal growth factor receptor. Using selective agonists and inhibitors, we demonstrated that mobilization of intracellular Ca(2+) and PKCdelta activation are required for ANG II-induced PAK1 phosphorylation. Rottlerin, a PKCdelta inhibitor, significantly blocked ANG II-induced PAK1 phosphorylation. Further support for this notion was provided through infection of VSMCs with adenovirus encoding a dominant-negative (dn)PKCdelta, which also markedly reduced phosphorylation of PAK1 by ANG II. In this pathway, Ca(2+) acts upstream of PKCdelta because a Ca(2+) ionophore rapidly induced PKCdelta phosphorylation at Tyr311 and Ca(2+)-dependent PAK1 phosphorylation was blocked by rottlerin. In addition, dnPYK-2, dnRac, and antioxidants inhibited ANG II-induced PAK1 phosphorylation, suggesting that PYK-2, Rac, and reactive oxygen species are involved in the upstream signaling. Finally, dnPAK1 markedly inhibited ANG II-induced protein synthesis in VSMCs. These data provide a novel signaling pathway by which ANG II may contribute to vascular remodeling.