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It is widely accepted that angiogenesis is required for tumor progression. Vascular endothelial growth factor (VEGF) is a key molecule for tumor angiogenesis; however, its expressional regulation is not well understood during all stages of tumorigenesis. Using cell lines and surgical specimens of human non–small cell lung cancers (NSCLCs), we here show that platelet-derived growth factor-AA (PDGF-AA) is an essential autocrine regulator for VEGF expression. To directly assess the expression of PDGF-AA–dependent VEGF and its roles in tumorigenesis, we stably transfected established cell lines with their antisense genes. In addition, the levels of PDGF-AA and VEGF expression in surgical sections were measured and compared with clinicopathologic findings such as tumor size and patient prognosis. PDGF-AA tightly regulated VEGF expression and had a greater effect on tumor size and patient prognosis than did VEGF in both cell lines and surgical sections. PDGF-AA expression was not seen in the atypical adenomatous hyperplasia at all, whereas VEGF was occasionally seen. Furthermore, the frequency of VEGF expression was higher in advanced NSCLCs than in precancerous lesions, which was tightly correspondent to the results for PDGF-AA. These results indicate that PDGF-AA is an important regulator of the frequency and level of VEGF expression during the transition from a precancerous lesion to advanced cancer. The PDGF-AA/VEGF axis, therefore, may be a ubiquitous autocrine system for enhancing angiogenic signals, and PDGF-AA, and its related pathways could be a more efficient target of antiangiogenic therapy for cancers than VEGF and its pathways.

Supplementary Figure S1 from Platelet-Derived Growth Factor-AA Is an Essential and Autocrine Regulator of Vascular Endothelial Growth Factor Expression in Non–Small Cell Lung Carcinomas

Spindle cell carcinoma (SpCC) of the breast is quite a rare modality classified to the metaplastic carcinoma of the breast. Regarding its biological behavior and the prognosis of the patients with this rare tumor, it has been remaining controversial. We herein report an 88 year-old woman who had a huge bleeding tumor on the right breast. She was a high-aged woman with low activities of daily life, even with some suspicion of distant organ metastasis. While the tumor proved to drastically bleed due to the tumor disintegration, a right simple mastectomy was performed. According to the histopathologic examinations, sarcomatoid spindle cells with severe atypia were observed. By an immunohistochemical examination, the tumor had proved to express neither estrogen receptor, progesterone receptor nor HER2 receptor. Moreover an immunohistochemical expression of AE1/3 and CAM5.2, defining an epithelial neoplasm were observed in addition to an expression of vimentin. From these findings, this bleeding tumor was diagnosed as spindle cell carcinoma of the breast. J. Med. Invest. 67 : 365-367, August, 2020.

Genomewide association studies have shown that a nonsynonymous single nucleotide polymorphism in PRKCH is associated with cerebral infarction and atherosclerosis-related complications. We examined the role of PKCη in lipid metabolism and atherosclerosis using apolipoprotein E-deficient (Apoe−/−) mice. PKCη expression was augmented in the aortas of mice with atherosclerosis and exclusively detected in MOMA2-positive macrophages within atherosclerotic lesions. Prkch+/+Apoe−/− and Prkch−/−Apoe−/− mice were fed a high-fat diet (HFD), and the dyslipidemia observed in Prkch+/+Apoe−/− mice was improved in Prkch−/−Apoe−/− mice, with a particular reduction in serum LDL cholesterol and phospholipids. Liver steatosis, which developed in Prkch+/+Apoe−/− mice, was improved in Prkch−/−Apoe−/− mice, but glucose tolerance, adipose tissue and body weight, and blood pressure were unchanged. Consistent with improvements in LDL cholesterol, atherosclerotic lesions were decreased in HFD-fed Prkch−/−Apoe−/− mice. Immunoreactivity against 3-nitrotyrosine in atherosclerotic lesions was dramatically decreased in Prkch−/−Apoe−/− mice, accompanied by decreased necrosis and apoptosis in the lesions. ARG2 mRNA and protein levels were significantly increased in Prkch−/−Apoe−/− macrophages. These data show that PKCη deficiency improves dyslipidemia and reduces susceptibility to atherosclerosis in Apoe−/− mice, showing that PKCη plays a role in atherosclerosis development.

An 85-year old woman who had a large tumor in the left breast came to our out-patient clinic. Computed tomography showed multiple lung tumors in addition to a huge tumor in the left breast. A needle biopsy brought about a histological diagnosis of ductal carcinoma. A simple mastectomy was performed and a histological examination using the resected specimen demonstrated a coexistence of an adenoid structure and a false ductal structure according the histologic characteristics of adenoid cystic carcinoma, which is quite rare among breast tumors. J. Med. Invest. 65:289-291, August, 2018.

A 69-year-old man, who had undergone distal gastrectomy for duodenal ulcer, was diagnosed with remnant gastric cancer and jejunal mesenteric lymph node metastasis. To improve curability, we planned 2 courses of S-1 and cisplatin therapy. After chemotherapy, primary lesion and lymph node metastases reduced in size drastically. Completion gastrectomy and lymph node dissection were performed with curative intent. The tumor was found to have a pathological complete response(pCR) to chemotherapy on histological examination.

Previously we have generated transgenic (Tg) mice developing severe diabetes early in life with a profound depletion of β-cells with β-cell-directed expression of inducible cAMP early repressor-Iγ. Only male mice continue to demonstrate hyperglycemia throughout life. To investigate this sexual dimorphism, we treated severely diabetic male Tg mice with orchiectomy (ORX) or 17β-estradiol (E2) pellet implantation alone or in combination with ORX and E2-implantation to change the circulating levels and patterns of the ratio of estradiol to androgens. In the Tg-ORX group, the blood-glucose levels decreased to a certain level within several weeks but never reached the female Tg-control level. In contrast, the Tg-ORX+E2 or Tg-E2 group showed a more rapid drop in blood glucose to the basal level with a substantial increase in β-cells, thus preventing the occurrence of severe diabetes in the male mice. The β-cells, not only within islet but also in and adjacent to ducts and scattered β-cell clusters, were strongly induced by 1 week after treatment, and the islet morphology dramatically changed. Enhanced β-cell induction in the ducts occurred concomitantly with markedly increased levels of pancreatic duodenal homeobox-1 and related transcription factors. The glucose-lowering and β-cell-increasing effects were independent of the age at which the treatment is started. These data provide evidence that the circulating level of E2 and the ratio of E2 to T greatly affect the blood glucose levels, the β-cell induction, and the islet morphology in diabetic male Tg mice. This novel mechanism offers great potential for developing strategies to increase the number of β-cells in vivo.

There is little information regarding whether patients with chronic kidney disease (CKD) have a high incidence of vulnerable plaques in their coronary arteries. To gain additional evidence on this, we conducted a population-based study by randomly selecting 126 subjects from 844 consecutive autopsies of elderly residents of Hisayama, Japan. We then determined the relationships of CKD with neovascularization and intraplaque hemorrhage in coronary atherosclerosis with the subjects classified into four categories based on their estimated glomerular filtration rate (eGFR). Areas of oxidized low-density lipoprotein (oxLDL) and vascular endothelial growth factor (VEGF) expression, assessed by immunohistochemistry in a total of 375 coronary arteries, increased significantly with decreasing eGFR. A lower eGFR was also associated with increased numbers of newly formed blood vessels. These relationships remained substantially unchanged after adjustment for confounding factors. The multivariate-adjusted odds ratio of the presence of intraplaque hemorrhages was 6.2 (95% confidence interval, 1.1-35.0) in patients with an eGFR30 ml/min/1.73 m(2) compared with those with an eGFR of ≥ 60 ml/min/1.73 m(2). Thus, elderly patients with CKD have intimal neoangiogenesis and an increased risk of intraplaque hemorrhage in coronary arteries, possibly favored by local accumulation of oxLDL and VEGF.

Pleomorphic carcinoma is an uncommon neoplasm that is known to arise in diverse sites, including the lung, breast, pancreas, gallbladder and prostate. We encountered a rare case of pleomorphic carcinoma of undetected origin associated with production of granulocyte-colony stimulating factor (G-CSF). The patient was a 62-year-old man who presented with cough and general fatigue. Chest computed tomography (CT) demonstrated a mass lesion in the mediastinum, so mediastinoscopic tumor biopsy was performed. However, making the diagnosis was difficult despite the suspected malignant potential. CT-supported positron emission tomography showed multiple uptake lesions in the small intestine, duodenum and right adrenal gland, in addition to the mediastinum. The patient subsequently presented with right-side weakness and sensory impairment. Brain CT showed multiple intracerebral lesions, indicating metastatic tumors. For the definitive diagnosis and to clarify options for chemotherapeutic management, the patient underwent surgical craniotomy. The resected specimen was considered to represent sarcomatoid carcinoma with positive immunoreactive staining for cytokeratin as an epithelial marker, indicating pleomorphic carcinoma. Furthermore, laboratory data showed leukocytosis without any infections and elevated serum G-CSF levels during hospitalization. The patient developed complications of occlusive intussusception in the small intestine caused by probably metastatic lesions and died from disease progression 82 days after initial admission. Although the presence of G-CSF-producing pleomorphic carcinoma was confirmed, the primary origin of this pleomorphic carcinoma could not be indentified clearly even at autopsy.

We herein report two cases of combined pulmonary fibrosis and emphysema (CPFE), whose histological patterns of lung pathology could not be categorized into any subset of idiopathic interstitial pneumonias(IIPs). Case 1 was a 62-year-old man, who presented with dyspnea on exertion and cough. Case 2 was a 51-year-old man with a dry cough. The CT findings of both cases fit the definition of CPFE. Surgical lung biopsies of both patients revealed alveolar septal widening due to collagen deposition, with emphysema and respiratory bronchiolitis mainly in the subpleural parenchyma. These cases suggest that the fibrosis of CPFE includes smoking-related interstitial fibrosis other than the known histological patterns of IIPs.

A 60s male was admitted to our hospital because of appetite loss and nausea. After examination, he was diagnosed with type 3 advanced gastric cancer in the antrum. Abdominal computed tomography showed gastric cancer invasion to the left liver lobe. We initiated neoadjuvant chemotherapy using S-1 plus CDDP after laparoscopic gastrojejunostomy. S-1 was orally administered for 3 weeks followed by a 2-week drug-free period. CDDP was administered intravenously on day 8 of each course. After 5 courses of chemotherapy, the gastric cancer was reduced in size. We therefore performed total gastrectomy with D2-affiliated left liver resection. S-1 plus CDDP is expected to improve outcomes in unresectable or locally advanced gastric cancer.

Objective— The goal of this study was to investigate the effects of stimulants for a nucleotide-binding domain, leucine-rich repeat-containing (NLR) protein family on human artery endothelial cells and murine arteries. Methods and Results— Human coronary artery endothelial cells were challenged in vitro with microbial components that stimulate NLRs or Toll-like receptors. We found stimulatory effects of NLR and Toll-like receptor ligands on the adhesion molecule expression and cytokine secretion by human coronary artery endothelial cells. On the basis of these results, we examined the in vivo effects of these ligands in mice. Among them, FK565, 1 of the nucleotide-binding oligomerization domain (Nod)-1 ligands induced strong site-specific inflammation in the aortic root. Furthermore, coronary arteritis/valvulitis developed after direct oral administration or ad libitum drinking of FK565. The degree of the respective vascular inflammation was associated with persistent high expression of proinflammatory chemokine/cytokine and matrix metallopeptidase ( Mmp ) genes in each tissue in vivo by microarray analysis. Conclusion— This is the first coronary arteritis animal model induced by oral administration of a pure synthetic Nod1 ligand. The present study has demonstrated an unexpected role of Nod1 in the development of site-specific vascular inflammation, especially coronary arteritis. These findings might lead to the clarification of the pathogenesis and pathophysiology of coronary artery disease in humans.

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors, but the functional capacity of DC must be assessed in detail, especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant, providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol, resulting in full maturation of monocyte-derived DC(s). Both RIG-I–dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1–specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors, or TLRs, do not show these functional features. Therefore, SeV-infected DC have the potential for DC-directed immunotherapy.

Podoplanin is expressed in a variety of malignant cells, and is generally regarded as a factor promoting tumor progression in conventional studies. Conversely, a recent clinicopathological study has revealed that low podoplanin in cancer cells was correlated with poor prognosis of patients with stage IB lung squamous cell carcinoma (LSCC). We here evaluated the clinicopathological relationship between cancer-cell podoplanin expression and clinicopathological parameters in 40 cases of LSCC (stage I–III). Immunohistochemical podoplanin expression significantly correlated with N classification and pathological stage, but not with other clinicopathological parameters. Notably, all 16 cases with high podoplanin expression unexceptionally exhibited pathological N0 status. Cases without nodal metastasis showed a significantly higher podoplanin-positive score. Furthermore, patients with high podoplanin expression exhibited a significantly longer survival time and disease-free time. These findings suggest that immunohistochemical analysis for podoplanin may serve as a marker of risk of nodal metastasis and prognosis in patients with LSCC.

25歳，男性。消防士で，腹部に摩擦の加わる訓練をしていた。3週間前に臍から臍右側に移動する痛みを自覚し，臍右側の皮下結節に気づいた。臍周囲から下腹部にかけて多毛を認め，臍右側に約3×2cmの硬い皮下結節を認めた。エコー所見では，内部エコーが不均一な iso-hypo echoic mass を認め，単純CTでは，境界不明瞭な淡い濃度上昇部を認めた。手術所見では，皮下脂肪織内に黄白色調の硬い結節を認め，割面に体毛を確認した。病理組織学的には，皮下の毛幹の周囲に好中球，組織球の密な浸潤と異物型巨細胞を含む異物反応を認め，膿瘍を伴う異物肉芽腫の像を呈していた。以上の所見より，臍右側に皮下結節を呈した pilonidal disease と診断した。多毛傾向のある若い男性の臍周囲に生じる皮下結節のひとつとして本疾患を挙げる必要があると考えた。

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8+ T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC.

Here we examined whether new β-cell formation occurs when β cells face being severely destroyed and hyperglycemia is restored. Animals were made diabetic by a single i.p. injection of a high dose of streptozotocin, and blood glucose levels were kept in the normal range with twice-daily Detemir (long-acting human insulin analog) injection or islet transplantation for 10 weeks. Although Detemir injection could effectively reverse hyperglycemia and glycemic control was successful, there was no β-cell increase, new formation, or recovery of islet morphology in Detemir-treated mice. In contrast, β-cell regeneration was restored when hyperglycemia was reversed by islet transplantation. The number of β cells and islets was increased, and islet structure was greatly recovered. We further evaluated whether replication or new formation contributes to the recovery. Newly born β cells, as observed as scattered singlets-doublets of insulin-positive cells or clusters of less than 6 β cells across, were frequently seen in transplanted mice, suggesting that neogenesis of β cells was enhanced in transplanted mice. Ki67-positive islets were increased in transplanted mice, suggesting that β-cell proliferation is enhanced. Thus, this recovery involved both increased new formation and replication. Our results suggest that the effects of Detemir on pancreatic β cells were very different from those of islet transplantation and that islet transplantation could be a trigger for the induction of new formation and replication.

Objective— The angiopoietin (Ang)–Tie-2 system plays a critical role during fetal and adult angiogenesis. Herein, we explored the Tie-2 shedding–related molecular mechanisms and the pathophysiological significance. Methods and Results— By using a mouse hindlimb ischemia model, we observed dissociated expression between the full-length Tie-2 (fTie-2) protein and Tie-2 mRNA in thigh muscles 1 day after an ischemic operation, suggesting that fTie-2 expression was modified through the posttranscriptional regulation in vivo. A soluble form of Tie-2 produced in human umbilical vein endothelial cells was dramatically suppressed by treatment with siRNA–matrix metalloproteinase (MMP) 14 or tissue inhibitor of metalloproteinase 3, resulting in an increase in cellular fTie-2 and thereby enhancing Ang-1–dependent Akt phosphorylation and Akt-dependent endothelial functions, such as Ang-2 downregulation or an increase of endothelial viability. Phorbol-12-myristate-13 acetate (PMA) upregulates MMP-14 mRNA via protein kinase C–extracellular signal–regulated kinase pathways, and enhanced soluble Tie-2 production in an MMP-14–dependent manner, resulting in a reduction of cellular fTie-2. In addition, the PMA-induced soluble Tie-2 was mediated by the protein kinase C–extracellular signal–regulated kinase signaling pathways. Finally, downregulation of tissue inhibitor of metalloproteinase 3 and upregulation of MMP-14 mRNA were confirmed in ischemic thigh muscles 1 day after the operation. Conclusion— An autocrine linkage between the endothelial protein kinase C–MMP-14 axis and Tie-2 shedding was shown to be a novel regulatory mechanism for the Ang–Tie-2 system and may play a role in modulating endothelial function during angiogenesis.

Background Chronic kidney disease (CKD) is associated with increased risk of coronary heart disease. However, information regarding the histopathologic characteristics of coronary atherosclerosis in individuals with CKD is scarce. This study investigated the relationship between CKD and severity of coronary atherosclerosis in population-based autopsy samples. Study Design Cross-sectional study. Setting & Participants 126 individuals randomly selected from 844 consecutive population-based autopsy samples. Predictor Estimated glomerular filtration rate (eGFR) calculated using the 6-variable Modification of Diet in Renal Disease (MDRD) Study equation. Outcomes Severity of atherosclerosis in 3 main coronary arteries, including atherosclerotic lesion types defined using the American Heart Association classification; stenosis rates; and coronary calcified lesions. Measurements The relationship between CKD and severity of coronary atherosclerosis was evaluated using generalized estimating equation methods. Results Frequencies of advanced atherosclerotic lesions increased gradually as eGFR decreased (33.6%, 41.7%, 52.3%, and 52.8% for eGFRs ≥60, 45-59, 30-44, and 2 , respectively; P for trend=0.006). This relationship was substantially unchanged even after adjustment for potential confounding factors (ORs, 1.40 [95% CI, 0.76-2.55], 2.02 [95% CI, 0.99-4.15], and 3.02 [95% CI, 1.22-7.49] for eGFRs of 45-59, 30-44, and 2 , respectively). Frequencies of calcified lesions of coronary arteries also increased gradually with lower eGFRs ( P for trend=0.02). Hypertension and diabetes were associated with increased risk of advanced coronary atherosclerosis and calcification of coronary arteries in individuals with decreased eGFR. Limitations Cross-sectional study, absence of data for proteinuria, and extremely high proportion of aged people. Conclusions The autopsy findings presented here suggest that CKD is associated significantly with severity of coronary atherosclerosis. Patients with CKD should be considered a high-risk population for advanced coronary atherosclerosis.

Emerging evidence indicates that the tight communication between vascular endothelial cells and mural cells using platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process. However, little is known about the related regulator that determines PDGF-BB expression. Using murine models of therapeutic neovascularization, we here show that a typical lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action. The blockade of VEGF type 3 receptor (VEGFR3) using neutralizing antibody AFL-4 abrogated FGF-2-mediated limb salvage and blood flow recovery in severely ischemic hindlimb. Interestingly, inhibition of VEGFR3 activity not only diminished lymphangiogenesis, but induced marked dilatation of capillary vessels, showing mural cell dissociation. In these mice, VEGF-C and PDGF-B were upregulated in the later phase after induced ischemia, on day 7, when exogenous FGF-2 expression had already declined, and blockade of VEGFR3 or PDGF-BB activities diminished PDGF-B or VEGF-C expression, respectively. These results clearly indicate that VEGF-C is a critical mediator, not only for lymphangiogenesis, but also for capillary stabilization, the essential molecular mechanism of communication between endothelial cells and mural cells during neovascularization.

To investigate risk factors for coronary atherosclerosis in men and women in the recent general Japanese population, we examined coronary arteries obtained from subjects autopsied in the Hisayama cohort study (autopsy rate: 78.7%). The subjects were over 40 years of age and consisted of 125 men and 108 women. They underwent an antemortem medical examination in 1988 and were subject to autopsy at death during an 8-year follow-up period. Atherosclerosis was globally assessed by examining 14 specimens taken from wide areas of epicardial coronary arteries and classified into 6 grades. The frequency of more severe grades of coronary atherosclerosis increased with age in both genders and was greater in men than in women of the same age. Multiple regression analysis revealed that age, systolic blood pressure, serum total cholesterol, and hemoglobin A(1C) were significant risk factors for men. Age, systolic blood pressure, and waist to hip ratio were risk factors for women. Smoking was not significantly correlated with the grade of coronary atherosclerosis in either gender. Thus, aging, hypertension, hypercholesterolemia, obesity, and glucose intolerance are risk factors for coronary atherosclerosis in recent Japanese populations, and the significance of the metabolic risk factors is different between men and women.

Numerous past studies have suggested a critical role of the paracrine effect between tumor vascular endothelial growth factor (VEGF)-C and lymphatic FLT-4 in solid tumor-associated lymphangiogenesis. In contrast, the pathophysiological role of tumor cell-associated FLT-4 in tumor progression remains to be elucidated. Here, we investigated this role using a tumor implantation model. SAS cells, an oral squamous carcinoma cell line expressing both VEGF-C and FLT-4 but neither FLK-1/KDR nor VEGF-D were adopted for experiments. Stable transformants of dominant-negative (dn) SAS cells were established in which the cytoplasmic domain-deleted FLT-4 was exogenously overexpressed, which can lead to inactivation of endogenous FLT-4 through competitive antagonism and is associated with down-activation of endogenous FLT-4-related intracellular signals. In vitro and in vivo proliferation assays showed lower proliferative activity of dn-SAS cells. An immunohistochemical study revealed that the tumor lymphangiogenesis was significantly suppressed, and the level of human VEGF-C mRNA was significantly lower in dn-SAS cell-derived tumor tissues. Moreover, in vitro studies demonstrated that the significant suppression of VEGF-C and VEGF-A expression was evident in dn-SAS cells or wild-type SAS cells treated with either the FLT-4 kinase inhibitor MAZ51 or the inhibitor of FLT-4-related signals. These findings together suggested that the VEGF-C/FLT-4 autocrine loop in tumor cells was a potential enhancer system to promote cancer progression, and FLT-4 in tumor tissue might become an effective target for cancer therapy.

Pulmonary arterial hypertension (PAH) is an intractable disease of the small pulmonary artery that involves multiple inflammatory factors. We hypothesized that a redox-sensitive transcription factor, nuclear factor kappaB (NF-kappaB), which regulates important inflammatory cytokines, plays a pivotal role in PAH. We investigated the activity of NF-kappaB in explanted lungs from patients with PAH and in a rat model of PAH. We also examined a nanotechnology-based therapeutic intervention in the rat model. Immunohistochemistry results indicated that the activity of NF-kappaB increased in small pulmonary arterial lesions and alveolar macrophages in lungs from patients with PAH compared with lungs from control patients. In a rat model of monocrotaline-induced PAH, single intratracheal instillation of polymeric nanoparticles (NPs) resulted in delivery of NPs into lungs for

Objective— We have demonstrated that vascular endothelial growth factor (VEGF) expression is upregulated in injured vascular wall, and blockade of VEGF inhibited monocyte infiltration and neointima formation in several animal models. In the present study, we aimed to clarify relative role of two VEGF receptors, flt-1 versus flk-1/KDR, in neointima formation after injury using flt-1 tyrosine kinase-deficient (Flt-1 TK −/− ) mice and soluble Flt-1(sFlt-1) gene transfer. Methods and Results— Neointima formation was comparable between wild-type and Flt-1 TK −/− mice 28 days after intraluminal wire injury in femoral arteries. By contrast, neointima formation was significantly suppressed by sFlt-1 gene transfer into Flt-1 TK −/− mice that blocks VEGF action on flk-1 (intima/media ratio: 2.8±0.4 versus 1.4±0.4, P −/− mice. Conclusions— These results suggest that endogenous VEGF accelerates neointima formation through flk-1 by regulating MCP-1 expression in VSMCs and macrophage-mediated inflammation in injured vascular wall in murine model of wire injury.

Objectives The objective of this study was to formulate a nanoparticle (NP)-eluting drug delivery stent system by a cationic electrodeposition coating technology. Background Nanoparticle-mediated drug delivery systems (DDS) are poised to transform the development of innovative therapeutic devices. Therefore, we hypothesized that a bioabsorbable polymeric NP-eluting stent provides an efficient DDS that shows better and more prolonged delivery compared with dip-coating stent. Methods We prepared cationic NP encapsulated with a fluorescence marker (FITC) by emulsion solvent diffusion method, succeeded to formulate an NP-eluting stent with a novel cation electrodeposition coating technology, and compared the in vitro and in vivo characteristics of the FITC-loaded NP-eluting stent with dip-coated FITC-eluting stent and bare metal stent. Results The NP was taken up stably and efficiently by cultured vascular smooth muscle cells in vitro. In a porcine coronary artery model in vivo, substantial FITC fluorescence was observed in neointimal and medial layers of the stented segments that had received the FITC-NP-eluting stent until 4 weeks. In contrast, no substantial FITC fluorescence was observed in the segments from the polymer-based FITC-eluting stent or from bare metal stent. The magnitudes of stent-induced injury, inflammation, endothelial recovery, and neointima formation were comparable between bare metal stent and NP-eluting stent groups. Conclusions Therefore, this NP-eluting stent is an efficient NP-mediated DDS that holds as an innovative platform for the delivery of less invasive nano-devices targeting cardiovascular disease.

Few studies regarding the topographical expression of heme oxygenase-1 (HO-1) and its pathophysiological role in human coronary atherosclerotic lesions, particularly in relation to type 2 diabetes mellitus (DM) and intimal angiogenesis, have been reported. HO-1 expression was immunohistochemically examined in 312 tissue blocks of coronary arteries obtained from 53 Japanese autopsy cases in Hisayama cohort study that included 19 diabetic subjects and 34 age- and sex-matched non-diabetic subjects (56-93 years old, mean+/-S.D.: 73+/-10). The HO-1 was ubiquitously distributed in atherosclerotic intima, and was mainly expressed by macrophages and endothelial cells, and partly by smooth muscle cells. The prevalence of HO-1 expression increased as the lesion type (as classified by the American Heart Association (AHA) Committee) and stenotic grade progressed (p0.0001), and was significantly higher in diabetic than in non-diabetic subjects (p0.01). This HO-1 overexpression was associated with greater CD-68-positive macrophage infiltration (p=0.005). Interestingly, the distribution of HO-1-positive cells was accentuated in coronary atherosclerotic lesions with intimal microvessels in diabetic subjects (p0.05), particularly those with hypercholesterolemia (p0.05), and was preferentially distributed in the shoulder region of atherosclerotic lesion type IV in the AHA classification (p0.01). In conclusion, HO-1 expression was distributed in overall human coronary atherosclerotic lesions, particularly in diabetic subjects, indicating that HO-1 expression is intimately associated with atherogenesis and may play an important role as an adaptive molecule in the inflammatory-repair process. The association of HO-1 overexpression with a greater extent of intraplaque angiogenesis suggests a multi-faceted role for HO-1 in modulating the progression of atherosclerosis.

Twin-twin transfusion syndrome (TTTS) carries a significant risk of perinatal morbidity and mortality. Serial amnioreduction and fetoscopic laser photocoagulation have decreased perinatal mortality, thereby shifting attention towards short-term and long-term morbidity. Most morbidity occurs within the central nervous system. Ischemic lesions, however, such as congenital skin loss and limb necrosis may occur anywhere in the vasculature of recipient twins. Ischemia may also occur during serial amnioreduction and fetoscopic laser photocoagulation. We report a case of ileal perforation in the donor twin of untreated TTTS. Histopathology showed a focal area of erosion, possibly from ischemia. We also discuss the clinical features of ischemic intestinal complications of TTTS and review the literature.

We describe biochemical assessment of maternal circulation in a case of massive fetomaternal hemorrhage at term associated with intraplacental choriocarcinoma. Markedly elevated maternal serum hCG level at 37 weeks of gestation suggested choriocarcinoma as a cause of fetomaternal hemorrhage in this case. Measurement of maternal hCG may be a useful parameter when intraplacental choriocarcinoma is in the differential diagnosis. In addition, the placenta should be examined in all cases of fetomaternal hemorrhage.

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Loss of tumor suppressors and activation of oncogenes lead to carcinogenesis. Abnormal expression of CHFR, a novel checkpoint gene, or of Aurora kinases, key regulators of mitosis, has been detected in a variety of solid tumors. Recently, CHFR has been revealed to ensure chromosomal stability by controlling the expression level of Aurora-A in vitro. However, the frequency of aberrant expression of these proteins and the association with clinicopathologic parameters remain poorly defined in nonsmall-cell lung cancer (NSCLC). In this study, we investigated the immunohistochemical protein expression of CHFR and Aurora-A in 157 NSCLC cases and evaluated the association between clinicopathologic parameters statistically. The relationship between CHFR protein and mRNA levels and the association between this relationship and promoter methylation of the CHFR gene were also examined in 20 frozen sections of NSCLC. Overexpression of Aurora-A and reduced expression of CHFR were found in 94 cases (59.8%) and 62 cases (39%) of NSCLC, respectively, and those were significantly correlated with tumor differentiation and size. Moreover, diminished CHFR expression was significantly associated with smoking-related squamous cell carcinoma cases and poor prognosis. Multivariate analysis revealed that CHFR expression was an independent prognostic factor. A statistical correlation was evident between CHFR protein and mRNA expression. In conclusion, our results suggest the aberrant expression of Aurora-A and/or of CHFR contributed to the increase in the malignant potential of NSCLC. We also revealed that CHFR expression was predominantly impaired in smoking-related squamous cell carcinoma and might be a useful prognostic marker in NSCLC. © 2008 Wiley-Liss, Inc.

3-Hydroxy-3-methyl-glutaryl CoA reductase inhibitors, or statins, have pleiotropic effects and can protect the vasculature in a manner independent of their lipid-lowering effect. The effectiveness of statins in reducing the risk of coronary events has been shown even in patients with diabetes, and their effects on diabetic complications have been reported. Using a model of severe hindlimb ischemia in streptozotocin-induced diabetic mice (STZ-DM), we investigated the effects and mechanisms of statin therapy in diabetic angiopathy in ischemic hindlimbs. As a result, STZ-DM mice frequently lost their hindlimbs after induced ischemia, whereas non-DM mice did not. Supplementation with statins significantly prevented autoamputation. We previously showed that diabetic vascular complications are caused by impaired expression of PDGF-BB, but statin therapy did not enhance PDGF-BB expression. Statins helped enhance endogenous endothelial nitric oxide (NO) synthase (eNOS) expression. Furthermore, the inhibition of NO synthesis by the administration of Nω-nitro-l-arginine methyl ester impaired the ability of statins to prevent STZ-DM mouse limb autoamputation, indicating that the therapeutic effect of statins in hindlimb ischemia in STZ-DM mice occurs via the eNOS/NO pathway. A combination therapy of statins and PDGF-BB gene supplementation was more effective for diabetic angiopathy than either therapy alone. In conclusion, these findings indicate that statin therapy might be useful for preventing intractable diabetic foot disease in patients with diabetic angiopathy.

Background Recombinant Sendai virus vectors (rSeV) constitute a new class of cytoplasmic RNA vectors that have shown efficient gene transfer in various organs, including retinal tissue; however, the related immune responses remain to be overcome in view of clinical applications. We recently developed a novel rSeV from which all envelope-related genes were deleted (rSeV/dFdMdHN) and, in the present study, assess host immune responses following retinal gene transfer. Methods rSeV/dFdMdHN or conventional F-gene deleted rSeV (rSeV/dF) was injected into subretinal space of adult Wistar rats or C57BL/6 mice. The transgene expression and histopathological findings were assessed at various time points. Immunological assessments, including the expression of proinflammatory cytokines, natural killer (NK)-cell activity, as well as SeV-specific cytotoxic T lymphocytes (CTLs) and antibodies, were performed following vector injection. Results rSeV/dFdMdHN showed high gene transfer efficiency into the retinal pigment epithelium at an equivalent level to that seen with rSeV/dF. In the early phase, the upregulation of proinflammatory cytokines, local inflammatory cell infiltration and tissue damage that were all prominently seen in rSeV/dF injection were dramatically diminished using rSeV/dFdMdHN. NK cell activity was also decreased, indicating a reduction of the innate immune response. In the later phase, on the other hand, CTL activity and anti-SeV antibodies were similarly induced, even using rSeV/dFdMdHN, and resulted in transient transgene expression in both vector types. Conclusions Deletion of envelope-related genes of rSeV dramatically reduces the vector-induced retinal damage and may extend the utility for ocular gene transfer; however, further studies regulating the acquired immune response are required to achieve long-term transgene expression of rSeV. Copyright © 2007 John Wiley & Sons, Ltd.

The anticholinergic propiverine (1-methyl-4-piperidyl diphenylpropoxyacetate), which is used for the treatment of overactive bladder syndrome, has functionally active metabolites [M-1 (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide) and M-2 (1-methyl-4-piperidyl benzilate N-oxide)], but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1 and M-2). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca(2+) currents (I(Ca)) using patch-clamp techniques and fluorescent Ca(2+) imaging [after electrical field stimulation (EFS) and acetylcholine (ACh)] in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of I(Ca) in a concentration-dependent manner at a holding potential of -60 mV (propiverine, K(i) = 10 microM; M-1, K(i) = 118 microM). M-1 shifted the steady-state inactivation curve of I(Ca) to the left at -90 mV by 7 mV. Carbachol (CCh) reversibly inhibited I(Ca). This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, because nanomolar concentrations of 4-diphenylacetoxy-N-methyl-piperidine greatly reduced this inhibition, whereas pirenzepine or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at concentrations up to 1 microM was almost ineffective. In the presence of M-2, the CCh-induced inhibition of I(Ca) was blocked. In fluorescent Ca(2+) imaging, M-2 inhibited EFS-induced and ACh-induced Ca(2+) transients. These results suggest that M-1 acts, at least in part, as a Ca(2+) channel antagonist (as it inhibited I(Ca)), whereas M-2 has more direct antimuscarinic actions.

A patient presenting with Pancoast syndrome was definitely diagnosed to have pulmonary leiomyosarcoma. The patient underwent a right upper lobectomy combined with resection of the chest wall, including the 1st to 3rd ribs. The disease recurred rapidly at the local and distant sites. No previous reports about pulmonary sarcoma presenting as a Pancoast tumor were found in the literature. The possibility of primary pulmonary leiomyosarcoma should be considered, and early detection and surgical resection are necessary when a round or oval tumor with necrosis is observed in the apex of the lung.

Recent clinical studies revealed 'proof of concept' of gene therapy targeting hematopoietic stem cells (HSCs) to treat hematopoietic disorders. However, vector integration-related adverse events of retroviral vectors have slowed progress in this field. As an initial step to overcoming this hurdle, we examined the potential of an improved cytoplasmic RNA vector, temperature-sensitive mutant non-transmissible recombinant Sendai virus (ts-rSeV/dF), for gene transfer to murine HSCs and progenitors. Both conventional vector and ts-rSeV/dF-GFP showed efficient gene transfer to T-lymphocyte-depleted syngeneic bone marrow cells (BMCs) (>85%), but only BMCs treated with ts-rSeV/dF-GFP but not with conventional vector efficiently repopulated in the recipient mice, associated with multilineage differentiation in vitro and in vivo. To our knowledge, this is the first demonstration of the in vivo reconstruction of hematopoietic series by cytoplasmically gene transferred BMCs, that warrants further investigation to realize this strategy in clinical settings.

Objective— The present study was designed to clarify the morphological features of early human atherosclerosis and to determine whether specific extracellular matrix proteoglycans play a role in early atherogenesis. Methods and Results— Step and serial sections were obtained from right coronary arteries with no or early atherosclerosis. Atherosclerosis was classified into 4 grades according to the amount of lipid deposition. Coronary arteries with Grade 0 showed diffuse intimal thickening (DIT) with no lipid deposits. The extracellular matrix proteoglycans, biglycan and decorin, were localized in the outer layer of DIT. Most cases of Grade 1 and Grade 2 exhibited fatty streaks with extracellular lipids colocalizing with biglycan and decorin in the outer layer of the intima. As lipid grades increased, macrophages increased in number and were present in the deeper layers. Most cases of Grade 3 exhibited pathologic intimal thickening (PIT) with extracellular lipids underneath a layer of foam cell macrophages. Conclusions— In early human coronary atherosclerosis, fatty streaks develop via extracellular deposition of lipids associated with specific types of proteoglycans in the outer layer of preexisting DIT. As the amount of the lipid increases in fatty streaks, macrophages infiltrate toward the deposited lipid to form PIT with foam cells.

We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.

Cerebral infarction is the most common type of stroke and often causes long-term disability. To investigate the genetic contribution to cerebral infarction, we conducted a case-control study using 52,608 gene-based tag SNPs selected from the JSNP database. Here we report that a nonsynonymous SNP in a member of protein kinase C (PKC) family, PRKCH, was significantly associated with lacunar infarction in two independent Japanese samples (P = 5.1 x 10(-7), crude odds ratio of 1.40). This SNP is likely to affect PKC activity. Furthermore, a 14-year follow-up cohort study in Hisayama (Fukuoka, Japan) supported involvement of this SNP in the development of cerebral infarction (P = 0.03, age- and sex-adjusted hazard ratio of 2.83). We also found that PKCeta was expressed mainly in vascular endothelial cells and foamy macrophages in human atherosclerotic lesions, and its expression increased as the lesion type progressed. Our results support a role for PRKCH in the pathogenesis of cerebral infarction.

Objective—Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine that is known as an inflammatory/arteriogenic factor. Angiogenesis contributes to the inflammatory process; however, the molecular and cellular mechanisms of the links among the inflammatory pathway, arteriogenesis, and angiogenesis have not been well elucidated.Methods and Results—Using murine models of fibroblast growth factor-2 (FGF-2)–mediated therapeutic neovascularization, we here show that FGF-2 targets nonendothelial mesenchymal cells (NEMCs) enhancing both angiogenic (vascular endothelial growth factor [VEGF]) and arteriogenic (MCP-1) signals via independent signal transduction pathways. Severe hindlimb ischemia stimulated MCP-1 expression that was strongly enhanced by FGF-2 gene transfer, and a blockade of MCP-1 activity via a dominant negative mutant as well as a deficiency of its functional receptor CCR2 resulted in the diminished recovery of blood flow attributable to adaptive and therapeutic neovascularization. Tumor necrosis factor (TNF)-α stimulated MCP-1 expression in all cell types tested, whereas FGF-2–mediated upregulation of MCP-1 was found only in NEMCs but not in others, a finding that was not affected by VEGF in vitro and in vivo.Conclusions—These results indicate that FGF-2 targets NEMCs independently, enhancing both angiogenic (VEGF) as well as inflammatory/arteriogenic (MCP-1) pathways. Therefore, MCP-1/CCR2 plays a critical role in adaptive and FGF-2–mediated therapeutic neovascularization.

Background and purpose: ATP-sensitive K+ channels (KATP channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. Experimental approach: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. Key results: ZD0947 (≥0.1 μM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil>ZD0947>diazoxide. In conventional whole-cell configuration, ZD0947 (≥1 μM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (≤1 μM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki=4.0 μM) and the diazoxide-induced currents (high-affinity site, Ki1=42.4 nM; low-affinity site, Ki2=84.5 μM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. Conclusions and Implications: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder. British Journal of Pharmacology (2006) 149, 542–550. doi:10.1038/sj.bjp.0706893

Background. AUosensitization is a major risk factor for graft failure in clinical bone marrow transplantation, even with an human leukocyte antigen (HLA)-matched combination under radiation-based conditioning regimens. The critical components of immunological memory in donor bone marrow graft rejection in allosensitized hosts remain unclear at present. Methods. C57BL/6-recipient mice, which had been intraperitoneally injected with splenocytes from donor C3H mice on day -35 (sensitized recipients), had been lethally irradiated with 10-Gy whole-body irradiation and were intravenously injected with T-cell-depleted bone marrow cells (TCD-BMC) from C3H mice or third-party SJL mice. Results. Lethally irradiated recipient mice, which had been sensitized by donor splenocytes 5 weeks before the transplantation of TCD-BMC, completely rejected the donor-BMC in a donor-specific manner, whereas none of the nonsensitized recipient mice, all of which showed full allogeneic chimerism, rejected the donor TCD-BMC. Antibody-mediated T cell and/or Natural Killer (NK) cell depletion did not improve the ability of the sensitized recipients to overcome the rejection even when a megadose of TCD-BMC was administered to the sensitized recipients. Furthermore, BMC rejection occurred in sensitized B cell-deficient mice. In adoptive transfer experiments, naive mice, which received a transfer of purified T cells from sensitized mice, rejected the donor BMC, but not those from nonsensitized mice. Moreover, naive mice, which received a transfer of serum containing antidonor immunoglobulin, rejected the donor BMC. Conclusions. These findings suggest that alloreactive memory T cells and antidonor immunoglobulin independently function to reject donor BMC in sensitized recipients.

Background: Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantations, is attributed to the release of inflammatory cytokines, especially the tumor necrosis factor- (TNF) a, from activated Kupffer cells (KC). Therefore, the specific inhibition of TNF-a could improve the viability of the hepatic graft upon reperfusion. Methods: We assessed the efficacy of TNF-a antisense (TNF-AS) oligodeoxynucleotides (ODNs) delivery to KC in a rodent liver transplantation model. Results: Seventy-one percent of the animals that received 6 hours preserved grafts in baths of lactated Ringer's solution (4 1C) and were treated with TNF-AS survived for over 14 days. Eighty percent of the animals treated with vehicle, sense ODNs, or balanced salt saline (BSS) died. Four hours after reperfusion of the liver, a significant reduction was noted in livers treated with TNF-AS in the release of cytosolic enzymes from the hepatocytes and the serum TNF-a (Po0.05). The expressions of TNF-a on KC and of intercellular adhesion molecule-1 on sinusoidal endothelial cells were completely suppressed in TNF-AS-treated livers. Conclusions: TNF-AS delivery improves the viability of the hepatic graft, and this technique may solve hepatic graft nonfunction in a clinical setting.

MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28alpha/beta, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.

We encountered one case of duodenal atresia complicated by massive intrauterine hemorrhage due to the perforation of an umbilical cord ulceration (UCU). UCU is an extremely rare complication in the perinatal period. Although the prenatal diagnosis of upper intestinal atresia has been established, little is known about the association between UCU and upper intestinal atresia. In this article, we report our case, review past articles, and discuss the underlying pathophysiological mechanisms of the cause of an UCU. Given the characteristic sites of upper intestinal atresia, we speculate that regurgitation of gastric or intestinal juice into the amniotic fluid could be responsible for the development of UCU. We also believe that close observation is required for patients who have upper intestinal atresia.

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.