Regulation of vascular permeability in cancer metastasis.

Enhancement of vascular permeability is indispensable for cancer metastasis. Weakened endothelial barrier function enhances vascular permeability. Circulating tumor cells moving in the microvasculature tend to invade into stromal tissue at the location where vascular permeability is enhanced. Many basic studies have identified permeability factors by using gene-modified animals and cells. These factors directly/indirectly interact with endothelial cells. Here, we review vascular permeability factors and their molecular mechanisms. Interactions between tumor cells and endothelial cells are also discussed in the process of extravasation, one of the most critical steps in tumor metastasis. In some cases, primary tumors can manipulate permeability in a remote organ by secreting permeability factors. In addition, the importance of glycocalyx, which covers the endothelial cell surface, in controlling vascular permeability and tumor metastasis is also described. Furthermore, analysis of the hyperpermeable region found in a mouse model study is introduced. It clearly showed that tumor-bearing mouse lungs had a hyperpermeable region due to the influence of a remote primary tumor, and fibrinogen deposition was observed in that region. Given that fibrinogen was reported to be a permeability factor and a key regulator of inflammation, eliminating fibrinogen deposition may prevent future metastasis.

Cloning of Helicobacter suis cholesterol α-glucosyltransferase and production of an antibody capable of detecting it in formalin-fixed, paraffin-embedded gastric tissue sections.

Helicobacter suis (H. suis), formerly called Helicobacter heilmannii type 1 (H. heilmannii), is a gram-negative bacterium of the Helicobacter species. This pathogen infects the stomach of humans and animals such as dogs, cats, pigs, and rodents, the latter giving rise to zoonotic infection. Here, we generated a H. suis-specific antibody useful for immunohistochemistry with formalin-fixed, paraffin-embedded tissue sections. To do so, we began by cloning the gene encoding H. suis cholesterol α-glucosyltransferase (αCgT). αCgT is the key enzyme responsible for biosynthesis of cholesteryl α-D-glucopyranoside (CGL), a major cell wall component of Helicobacter species including H. suis. The deduced amino acid sequence of H. suis αCgT had 56% identity with the corresponding Helicobacter pylori (H. pylori). We then developed a polyclonal antibody (anti-Hh-I205R) by immunizing rabbits with a 205 amino acid H. suis αCgT fragment. Immunohistochemistry with the anti-Hh-I205R antibody could differentiate H. suis from H. pylori in gastric mucosa sections derived from mice infected with either pathogen. We then probed formalin-fixed, paraffin-embedded sections of human gastric mucosa positive for H. suis infection with the anti-Hh-I205R antibody and detected positive staining. These results indicate that anti-Hh-I205R antibody is specific for H. suis αCgT and useful to detect H. suis in gastric specimens routinely analyzed in pathological examinations.

Superoxide-Generating Nox5α Is Functionally Required for the Human T-Cell Leukemia Virus Type 1-Induced Cell Transformation Phenotype.

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and transforms T cells in vitro. To our knowledge, the functional role of reactive oxygen species (ROS)-generating NADPH oxidase 5 (Nox5) in HTLV-1 transformation remains undefined. Here, we found that Nox5α expression was upregulated in 88% of 17 ATL patient samples but not in normal peripheral blood T cells. Upregulation of the Nox5α variant was transcriptionally sustained by the constitutive Janus family tyrosine kinase (Jak)-STAT5 signaling pathway in interleukin-2 (IL-2)-independent HTLV-1-transformed cell lines, including MT1 and MT2, whereas it was transiently induced by the IL-2-triggered Jak-STAT5 axis in uninfected T cells. A Nox inhibitor, diphenylene iodonium, and antioxidants such as N-acetyl cysteine blocked proliferation of MT1 and MT2 cells. Ablation of Nox5α by small interfering RNAs abrogated ROS production, inhibited cellular activities, including proliferation, migration, and survival, and suppressed tumorigenicity in immunodeficient NOG mice. The findings suggest that Nox5α is a key molecule for redox-signal-mediated maintenance of the HTLV-1 transformation phenotype and could be a potential molecular target for therapeutic intervention in cancer development. IMPORTANCE: HTLV-1 is the first human oncogenic retrovirus shown to be associated with ATL. Despite the extensive study over the years, the mechanism underlying HTLV-1-induced cell transformation is not fully understood. In this study, we addressed the expression and function of ROS-generating Nox family genes in HTLV-1-transformed cells. Our report provides the first evidence that the upregulated expression of Nox5α is associated with the pathological state of ATL peripheral blood mononuclear cells and that Nox5α is an integral component of the Jak-STAT5 signaling pathway in HTLV-1-transformed T cells. Nox5α-derived ROS are critically involved in the regulation of cellular activities, including proliferation, migration, survival, and tumorigenicity, in HTLV-1-transformed cells. These results indicate that Nox5α-derived ROS are functionally required for maintenance of the HTLV-1 transformation phenotype. The finding provides new insight into the redox-dependent mechanism of HTLV-1 transformation and raises an intriguing possibility that Nox5α serves as a potential molecular target to treat HTLV-1-related leukemia.

ROS-generating oxidases Nox1 and Nox4 contribute to oncogenic Ras-induced premature senescence.

Activated oncogenes induce premature cellular senescence, a permanent state of proliferative arrest in primary rodent and human fibroblasts. Recent studies suggest that generation of reactive oxygen species (ROS) is involved in oncogenic Ras-induced premature senescence. However, the signaling mechanism controlling this oxidant-mediated irreversible growth arrest is not fully understood. Here, we show that through the Ras/MEK pathway, Ras oncogene up-regulated the expression of superoxide-generating oxidases, Nox1 in rat REF52 cells and Nox4 in primary human lung TIG-3 cells, leading to an increase in intracellular level of ROS. Ablation of Nox1 and Nox4 by small interfering RNAs (siRNAs) blocked the RasV12 senescent phenotype including ß-galactosidase activity, growth arrest and accumulation of tumor suppressors such as p53 and p16Ink4a. This suggests that Nox-generated ROS transduce senescence signals by activating the p53 and p16Ink4a pathway. Furthermore, Nox1 and Nox4 siRNAs inhibited both Ras-induced DNA damage response and p38MAPK activation, whereas overexpression of Nox1 and Nox4 alone was able to induce senescence. The involvement of Nox1 in Ras-induced senescence was also confirmed with embryonic fibroblasts derived from Nox1 knockout mice. Together, these findings suggest that Nox1- and Nox4-generated ROS play an important role in Ras-induced premature senescence, which may involve DNA damage response and p38MAPK signaling pathways.

Impairment of flow-mediated dilation correlates with aortic dilation in patients with Marfan syndrome.

Marfan syndrome is an inherited disorder characterized by genetic abnormality of microfibrillar connective tissue proteins. Endothelial dysfunction is thought to cause aortic dilation in subjects with a bicuspid aortic valve; however, the role of endothelial dysfunction and endothelial damaging factors has not been elucidated in Marfan syndrome. Flow-mediated dilation, a noninvasive measurement of endothelial function, was evaluated in 39 patients with Marfan syndrome. Aortic diameter was measured at the aortic annulus, aortic root at the sinus of Valsalva, sinotubular junction and ascending aorta by echocardiography, and adjusted for body surface area (BSA). The mean value of flow-mediated dilation was 6.5 ± 2.4 %. Flow-mediated dilation had a negative correlation with the diameter of the ascending thoracic aorta (AscAd)/BSA (R = -0.39, p = 0.020) and multivariate analysis revealed that flow-mediated dilation was an independent factor predicting AscAd/BSA, whereas other segments of the aorta had no association. Furthermore, Brinkman index had a somewhat greater influence on flow-mediated dilation (R = -0.42, p = 0.008). Although subjects who smoked tended to have a larger AscAd compared with non-smokers (AscA/BSA: 17.3 ± 1.8 versus 15.2 ± 3.0 mm/m(2), p = 0.013), there was no significant change in flow-mediated dilation, suggesting that smoking might affect aortic dilation via an independent pathway. Common atherogenic risks, such as impairment of flow-mediated dilation and smoking status, affected aortic dilation in subjects with Marfan syndrome.

A crucial role for Nox 1 in redox-dependent regulation of Wnt-ß-catenin signaling.

Canonical Wnt signaling critically regulates cell fate and proliferation in developmental stages and adult tissues. Redox regulation through nucleoredoxin (NRX) has recently been identified in canonical Wnt signaling. However, the source of reactive oxygen species (ROS) affecting the redox state of NRX remains elusive. Our principal aim in this study was to investigate whether superoxide-generating NADPH oxidase1 (Nox1) is involved in NRX-regulated Wnt signaling in intestinal and colon epithelial cells. Here, we demonstrate that Wnt treatment of mouse intestinal cells induces production of ROS through Nox1. This Nox1 action is regulated by Rac1 GTPase through Wnt-induced activation of the Rac1 guanine nucleotide exchange factor Vav2 by Src-mediated tyrosine phosphorylation. Nox1-generated ROS oxidize and inactivate NRX, thereby releasing the NRX-dependent suppression of Wnt-ß-catenin signaling through dissociation of NRX from Dvl. Nox1 small-interference RNA inhibits cell response to Wnt, including stabilization of ß-catenin, expression of cyclin D1 and c-Myc via the TCF transcription factor, and accelerated cell proliferation. Nox1 mediates Wnt-induced cell growth in colon cancer cells with the normal Wnt pathway, but not in APC-deficient colon cancer cells, which are constitutively active in Wnt signaling. Together, these results suggest the mediating role of Nox1 in redox-dependent regulation of canonical Wnt-ß-catenin signaling and provide further insight into the regulatory mechanism of the Wnt pathway.

Circulating transforming growth factor ß-1 level in Japanese patients with Marfan syndrome.

Marfan syndrome (MFS) is an inherited connective tissue disorder mainly caused by the fibrillin-1 mutation. Deficient fibrillin-1 is thought to result in the failed sequestration of transforming growth factor ß (TGFß) and subsequent activation of the TGFß signaling pathway, suggesting that the circulating TGFß level may be elevated in MFS, although its accurate measurement is complex due to ex vivo release from platelet stores upon platelet activation. We measured the plasma TGFß1 levels of 32 Japanese MFS patients (22 medically untreated, 10 treated, 20 males, 30.1 ± 9.6 years old) and 30 healthy volunteers (19 males, 29.5 ± 5.8 years old) by ruthenium-based electrochemiluminescence platform (ECL). PF4 was also measured by enzyme immunoassay (EIA) as a platelet degranulation marker. There was no significant difference in the mean plasma TGFß1 level between the MFS group (1.31 ± 0.40 ng/mL) and controls (1.17 ± 0.33 ng/mL) (P = 0.16, NS). Also, there was no significant difference between the untreated (1.24 ± 0.37 ng/mL) and treated (1.46 ± 0.45 ng/mL) MFS patients (P = 0.15, NS). We also measured PF4, which showed wide deviations but no significant difference between the two groups (P = 0.50). A difference in circulating TGFß1 levels between MFS patients and controls was not detected in this Japanese population. Circulating TGFß1 is not a diagnostic and therapeutic marker for Japanese MFS patients, although our findings do not eliminate the possible association of TGFß with the pathogenesis of MFS.

Suppression of CEBPδ recovers exhaustion in anti-metastatic immune cells.

The pre-metastatic microenvironment consists of pro-metastatic and anti-metastatic immune cells in the early stages of cancer, when the primary tumor begins to proliferate. Redundantly, pro-inflammatory immune cells predominated during tumor growth. Although it is well known that pre-metastatic innate immune cells and immune cells fighting primary tumor cells become exhausted, the mechanism by which this occurs is unknown. We discovered that anti-metastatic NK cells were mobilized from the liver to the lung during primary tumor progression and that the transcription factor CEBPδ, which was upregulated in a tumor-stimulated liver environment, inhibited NK cell attachment to the fibrinogen-rich bed in pulmonary vessels and sensitization to the environmental mRNA activator. CEBPδ-siRNA treated anti-metastatic NK cells regenerated the binding proteins that support sitting in fibrinogen-rich soil, such as vitronectin and thrombospondin, increasing fibrinogen attachment. Furthermore, CEBPδ knockdown restored an RNA-binding protein, ZC3H12D, which captured extracellular mRNA to increase tumoricidal activity. Refreshed NK cells using CEBPδ-siRNA with anti-metastatic abilities would work at metastatic risk areas in the pre-metastatic phase, resulting in a reduction in lung metastasis. Furthermore, tissue-specific siRNA-based therapy in lymphocyte exhaustion may be beneficial in the treatment of early metastases.

Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis.

Primary tumor cells metastasize to a distant preferred organ. However, the most decisive host factors that determine the precise locations of metastases in cancer patients remain unknown. We have demonstrated that post-translational citrullination of fibrinogen creates a metastatic niche in the vulnerable spots. Pulmonary endothelial cells mediate the citrullination of fibrinogen, changing its conformation, surface charge, and binding properties with serum amyloid A proteins (SAAs), to make it a host tissue-derived metastatic pathogen. The human-specific SAAs-citrullinated fibrinogen (CitFbg) complex recruits cancer cells to form a protein-metastatic cell aggregation in humanized SAA cluster mice. Furthermore, a CitFbg peptide works as a competitive inhibitor to block the homing of metastatic cells into the SAAs-CitFbg sites. The potential metastatic sites in the lungs of patients are clearly visualized by our specific antibody for CitFbg. Thus, CitFbg deposition displays metastatic risks for cancer patients, and the citrullinated peptide is a new type of metastasis inhibitor.

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia.

Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of PtdIns(3,4,5)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.

Extracellular mRNA transported to the nucleus exerts translation-independent function.

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether "naked nonvesicular extracellular mRNA" (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1ß (IL1ß)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3'-untranslated region of nex-IL1ß-mRNA and transports it to the nucleus. The nex-IL1ß-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.

Impact of Pathogenic FBN1 Variant Types on the Progression of Aortic Disease in Patients With Marfan Syndrome.

BACKGROUND: Marfan syndrome can cause life-threatening aortic complications. We investigated the relationship between FBN1 genotype and severe aortopathy (aortic root replacement, type A dissections, and related death). METHODS: We evaluated 248 patients with pathogenic or likely pathogenic FBN1 variants. The variants were classified as haploinsufficient type (HI, n=93) or dominant-negative type (DN, n=155) based on their location and predicted amino acid alterations, and we examined the effects of the FBN1 genotype on severe aortic events (aortic root replacement, type A dissections, and related death). RESULTS: The cumulative event-free probability was significantly lower in the HI group than in the DN group (adjusted hazard ratio, 2.1; 95% confidence interval, 1.4 -3.2; P<0.001). CONCLUSIONS: DN-CD+HI patients should be monitored more carefully than DN-nonCD patients for rapid development of aortic root aneurysms.

Perioperative Factors Associated With Chronic Central Pain After the Resection of Intramedullary Spinal Cord Tumor.

OBJECTIVE: Some patients experience severe chronic pain after intramedullary spinal cord tumor (IMSCT) resection, but the underlying mechanisms have yet to be fully elucidated. We aimed to investigate perioperative factors associated with chronic pain after IMSCT resection. MATERIALS AND METHODS: We analyzed data from a postal survey and the medical records of patients who had undergone IMSCT resection in our institution between 2000 and 2008. Chronic pain was assessed using the Neuropathic Pain Symptom Inventory score, and its associations with factors related to tumor pathology, patient demographics, neurological findings, surgery, anesthesia, and perioperative management were determined. RESULTS: Seventy-eight consecutive patients (55 men and 23 women; age 17 to 79 y) were included in the statistical analysis of the present study. In univariate analyses, sex, body mass index, preoperative tumor-related pain, preoperative nonsteroidal anti-inflammatory drugs, intraoperative hypotension, postoperative corticosteroids, and decrease in Japanese Orthopaedic Association (JOA) scores were found to be associated with postsurgical chronic central pain. Logistic regression analysis identified 3 significant factors: a decline in JOA scores compared with preoperative values (odds ratio [OR], 3.33; 95% confidence interval [CI], 1.18-9.42; P=0.023), intraoperative hypotension (OR, 3.01; 95% CI, 1.02-8.97; P=0.047), and postoperative corticosteroids (OR, 3.21; 95% CI, 1.02-10.09; P=0.046). DISCUSSION: Decline in JOA score, intraoperative hypotension, and postoperative corticosteroids are independently associated with postsurgical chronic central pain. Intraoperative hypotension and the use of postoperative corticosteroids can be avoided or modified during perioperative management. As results from animal studies have indicated that the administration of corticosteroids may intensify chronic pain, further studies in larger cohorts are required to definitively determine the effect of corticosteroids on postsurgical central pain.

The ROS-generating oxidase Nox1 is required for epithelial restitution following colitis.

Accumulating evidence suggests that reactive oxygen species (ROS) generated by endogenous metabolic enzymes are involved in a variety of intracellular mechanisms. In particular, superoxide-generating NADPH oxidase (Nox) 1 is highly expressed in the colon and has been implicated in physiological and pathophysiological states of colon tissues. However, its role in tissue repair following colitis has not been fully elucidated. Our study using experimental colitis in mice showed that repair of the mucosal layer did not occur in Nox1-deficient mice following dextran sulfate sodium-induced colitis. This was accompanied by inhibition of proliferation, cell survival, migration, and terminal differentiation (generation of goblet cells) of crypt progenitor cells, as determined by histochemical analyses. Furthermore, Nox1 expression as well as ROS production in the colon crypt was increased during the repair process, and Nox1 deficiency suppressed these events. The results suggest that Nox1 promotes colon mucosal wound repair by sustaining the bioactivity of crypt progenitor cells and plays a crucial role in the epithelial restitution in the case of damage associated with colitis.

Amlodipine increases nitric oxide production in exhaled air during exercise in patients with essential hypertension.

BACKGROUND: Endothelial production of nitric oxide (NO) is attenuated in patients with essential hypertension. We investigated whether treatment with amlodipine increased exhaled NO output (VNO) at rest and during exercise in patients with essential hypertension. METHODS: We studied the effect of amlodipine in seven untreated hypertensive patients. Cardiopulmonary exercise testing and NO measurement of exhaled air were performed on these patients before and after 2 months of amlodipine treatment. RESULTS: Amlodipine decreased blood pressure (BP) both at rest and during exercise (at rest: 147.1 +/- 6.4 [SEM]/89.9 +/- 4.4 v 133.6 +/- 5.4/82.7 +/- 3.9 mm Hg, P <.05; at peak exercise: 224.9 +/- 8.0/113.1 +/- 5.3 v 207.0 +/- 6.0/100.7 +/- 5.0 mm Hg, P <.05) without affecting heart rate (at rest: 67.6 +/- 3.9 v 70.4 +/- 4.5 beats/min, P =.33; peak exercise: 146.4 +/- 7.4 v 144.0 +/- 7.2 beats/min, P =.49). Amlodipine did not affect minute ventilation (VE) at rest or during exercise. It did not alter anaerobic threshold, peak oxygen uptake (peak VO(2)), or peak workload. However, after amlodipine treatment, VNO was significantly greater both at rest (130.8 +/- 19.4 v 180.4 +/- 24.8 nL/min, P <.05) and at peak exercise (380.0 +/- 47.5 v 582.6 +/- 74.3 nL/min, P <.05). CONCLUSIONS: Amlodipine increased NO production, at least in the pulmonary circulation, in patients with essential hypertension. In addition to its antihypertensive effect, the enhancement of NO production by amlodipine in the vasculature of other organs may contribute to its beneficial effects on the cardiovascular system.

Nox4-derived ROS signaling contributes to TGF-ß-induced epithelial-mesenchymal transition in pancreatic cancer cells.

UNLABELLED: Transforming growth factor (TGF)-ß induces epithelial-mesenchymal transition (EMT) in pancreatic adenocarcinoma. In this study, we investigated how NADPH oxidase (Nox) 4-generated reactive oxygen species (ROS) regulate TGF-ß-induced EMT in pancreatic cancer cells. MATERIALS AND METHODS: Pancreatic cancer cells were transfected with Nox4 siRNAs or PTP1B mutants and subjected to TGF-ß-induced EMT assay. Expression of Nox4, TGF-ß, and N-cadherin was immunohistochemically-examined with patient tumor samples. RESULTS: Treatment of pancreatic cancer cells with TGF-ß induced Nox4 expression, indicating that Nox4 represents a major source for ROS production. The Nox4 inhibitor diphenylene iodonium and Nox4 siRNAs blocked TGF-ß-induced EMT phenotype including morphological changes, augmented migration, and altered expression of E-cadherin and Snail. Furthermore, PTP1B as a redox-sensor for Nox4-derived ROS participated in TGF-ß-promoted EMT. Nox4, TGF-ß, and N-cadherin were up-regulated in tumors from pancreatic cancer patients. CONCLUSIONS: These findings suggest that Nox4-derived ROS, at least in part, transmit TGF-ß-triggered EMT signals through PTP1B in pancreatic cancer.

Cardiac rehabilitation decreases plasma pentraxin 3 in patients with cardiovascular diseases.

BACKGROUND: Inflammatory markers such as serum C-reactive protein (CRP), serum amyloid A (SAA), and plasma pentraxin 3 (PTX3), which belong to the pentraxin superfamily, increase due to various inflammatory diseases. Some studies demonstrated that serum CRP and SAA are predictors of cardiovascular diseases, and cardiac rehabilitation (CR) induces anti-inflammatory effects. In the present study, we investigated the effects of CR on pentraxins (serum CRP, SAA, and plasma PTX3) in patients with cardiovascular diseases. METHODS: Fifty patients with cardiovascular diseases [61 ± 13 (mean ± SD) years old, male/female 44/6] participated. Each subject performed CR using aerobic bicycle exercise two or three times per week for 3-6 months. We measured resting serum high-sensitivity CRP (hsCRP), SAA, and plasma PTX3 before and 3 and 6 months after CR, and compared them with VO(2peak) determined using a standard increment cycle ergometer protocol, B-type natriuretic peptide (BNP), and other biochemical data such as HbA1c. RESULTS: There was a significant positive correlation between hsCRP and SAA (r = 0.92, p < 0.001), but no relations between these parameters and PTX3. Plasma PTX3 significantly decreased time dependently during CR (at baseline 3.2 ± 2.0 ng/ml, at 3 months 2.3 ± 0.8 ng/ml, at 6 months 2.1 ± 0.7 ng/ml; all p < 0.05). Serum hsCRP tended to decrease, but not statistically significantly. At baseline, plasma PTX3 was negatively correlated with the percentage of the predicted values of VO(2peak) and positively correlated with BNP. CR significantly increased the percentage of the predicted values of VO(2peak) and decreased BNP. CONCLUSIONS: Plasma PTX3, an inflammatory marker, which was quite different from CRP and SAA, decreased during cardiac rehabilitation with an improvement of exercise capacity in patients with cardiovascular diseases.

Evaluating Japanese patients with the Marfan syndrome using high-throughput microarray-based mutational analysis of fibrillin-1 gene.

Marfan syndrome (MS) is an inherited connective tissue disorder, and detailed evaluations of multiple organ systems are required for its diagnosis. Genetic testing of the disease-causing fibrillin-1 gene (FBN1) is also important in this diagnostic scheme. The aim of this study was to define the clinical characteristics of Japanese patients with MS and enable the efficient and accurate diagnosis of MS with mutational analysis using a high-throughput microarray-based resequencing system. Fifty-three Japanese probands were recruited, and their clinical characteristics were evaluated using the Ghent criteria. For mutational analysis, an oligonucleotide microarray was designed to interrogate FBN1, and the entire exon and exon-intron boundaries of FBN1 were sequenced. Clinical evaluation revealed more pulmonary phenotypes and fewer skeletal phenotypes in Japanese patients with MS compared to Caucasians. The microarray-based resequencing system detected 35 kinds of mutations, including 23 new mutations. The mutation detection rate for patients who fulfilled the Ghent criteria reached 71%. Of note, splicing mutations accounted for 19% of all mutations, which is more than previously reported. In conclusion, this comprehensive approach successfully detected clinical phenotypes of Japanese patients with MS and demonstrated the usefulness and feasibility of this microarray-based high-throughput resequencing system for mutational analysis of MS.

NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.

Generation of reactive oxygen species (ROS) has been implicated in carcinogenic development of melanoma, but the underlying molecular mechanism has not been fully elucidated. We studied the expression and function of the superoxide-generating NADPH oxidase (Nox)4 in human melanoma cells. Nox4 was up-regulated in 13 of 20 melanoma cell lines tested. Silencing of Nox4 expression in melanoma MM-BP cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenecity in nude mice. Consistently, a general Nox inhibitor, diphenylene iodonium, and antioxidants vitamine E and pyrrolidine dithiocarbamate blocked cell proliferation of MM-BP cells. Flow cytometric analysis indicated that Nox4 small interfering RNAs and diphenylene iodonium induced G(2)-M cell cycle arrest, which was also observed with another melanoma cell line, 928mel. This was accompanied by induction of the Tyr-15 phosphorylated, inactive form of cyclin-dependent kinase 1 (a hallmark of G(2)-M checkpoint) and hyperphosphorylation of cdc25c leading to its increased binding to 14-3-3 proteins. Ectopic expression of catalase, a scavenger of ROS, also caused accumulation of cells in G(2)-M phase. Immunohistochemistry revealed that expression of Nox4 was detected in 31.0% of 13 melanoma patients samples, suggesting the association of Nox4 expression with some steps of melanoma development. The findings suggest that Nox4-generated ROS are required for transformation phenotype of melanoma cells and contribute to melanoma growth through regulation of G(2)-M cell cycle progression.