Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12

OBJECTIVE: Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells. METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques. RESULTS: Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis. CONCLUSIONS: Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.     

Three-dimensional magnetic resonance imaging for stringent diagnosis of advanced fibrosis associated with nonalcoholic steatohepatitis

Background

The definitive diagnosis of nonalcoholic steatohepatitis (NASH) is currently based on histopathological assessment. This study aimed to elucidate the utility of a novel noninvasive method, three-dimensional magnetic resonance imaging (3D-MRI), for diagnosing advanced fibrosis in patients with NASH, using histopathological diagnosis as the reference standard.

Methods

This retrospective study included 30 consecutive patients who had been diagnosed with NASH by histopathology and had undergone 3D-MRI before biopsy. 3D-MRI provided a three-dimensional reconstruction of the liver from contrast-enhanced hepatobiliary phase MR images. In the present study, histopathological advanced fibrosis was defined as stage 3 and 4 NASH. Advanced fibrosis, diagnosed by 3D-MRI, was considered to be diffuse irregularity of the entire surface of the liver. The diagnostic features of 3D-MRI and the noninvasive evaluation systems (APRI, FIB-4 index, and BARD score) for identifying advanced and nonadvanced fibrosis of NASH were determined and compared.

Results

Nine (30 %) of the 30 study patients were diagnosed histopathologically with advanced fibrosis, and 11 (37 %) of 30 patients were diagnosed with advanced fibrosis using 3D-MRI. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 3D-MRI for diagnosing advanced fibrosis were 100, 90, 82, and 100 %, respectively. The sensitivities of APRI, the FIB-4 index, and BARD score ranged from 78 to 89 %, the specificities from 71 to 90 %, the PPVs from 54 to 78 %, and the NPVs from 88 to 94 %.

Conclusion

Compared with the common noninvasive methods for diagnosing advanced fibrosis associated with NASH, 3D-MRI was more accurate.     

Expression of metalloproteinase-2 (gelatinase A) in labial salivary glands of patients with Sj?gren's syndrome with HTLV-I infection.

OBJECTIVE: To determine whether gelatinase A (MMP-2) plays a significant role in the pathogenesis of Sj?gren's syndrome (SS) with or without HTLV-I infection. METHODS: We examined 24 patients with SS (14 HTLV-I-seropositive and 8 HTLV-I-seronegative). Labial salivary gland tissue samples were analysed immunohistochemically using anti-MMP-2 monoclonal antibodies. RESULTS: In normal salivary glands, MMP-2 expression was not detected. All biopsy samples of 8 SS patients with HTLV-I-associated myelopathy (HAM) and 3 of 6 HTLV-I-seropositive SS patients without manifestation of HAM stained positively for MMP-2. However, the other samples were negative for MMP-2. CONCLUSION: Our study showed the MMP-2 expression in labial salivary glands of HTLV-I seropositive SS patients, especially in all SS patients with HAM. The presence of MMP-2 in the salivary glands of these patients suggests that it may play a role in cellular infiltration and destruction in salivary glands of SS.     

In vivo biomechanical stability of osseointegrating mesoporous TiO2 implants

Mesoporous materials are of high interest as implant coatings to receive an enhanced osseointegration. In this study, titanium implants coated with mesoporous TiO₂ thin films have been evaluated both in vitro and in vivo. Material characterization showed that, with partly crystalline TiO₂ (anatase), long-range-ordered hydrophilic mesoporous thin films with a pore size of 6 nm were obtained. Evaluation of the mechanical resistance showed that the films were robust enough to withstand the standard implantation procedure. In vitro apatite formation was studied using simulated body fluids, showing that the pores are accessible for ions and that formation of apatite was increased due to the presence of the mesopores. An in vivo study using a rabbit model was executed in which the removal torque and histomorphometry were evaluated. The results show that the biomechanical stability of the TiO₂ coating was unaffected by the presence of mesopores and that osseointegration was achieved without any signs of inflammation.     

Urinary iodine kinetics after oral loading of potassium iodine

We studied the kinetics of iodine in various forms, in order to establish appropriate guidelines of iodine prophylaxis for thyroid blockade at nuclear emergency in "iodine rich areas", such as Japan. First, the effect of equivalent dose of potassium iodide (KI) (solution vs. tablet) was evaluated with excretions of urinary iodine (UI) at Nagasaki, Japan, and it was revealed that there was no difference of iodine kinetics between solution and tablet. We also performed the same study at Gomel, Belarus, which was known to be an iodine deficient area, and obtained the same results. Second, the kinetics of iodine included in "iodine rich" food was also evaluated with excretions of UI. Interestingly, the ratio of UI excretion with iodine rich food was significantly lower than that with KI tablets until 6 h after the intake (ANOVA, p = 0.02). These results proved that 1) KI solution as well as its tablet is useful for prophylaxis and 2) prophylaxis by iodine rich food is not effective for rapid blockade of thyroid gland at nuclear emergency. Finally, we emphasized that the approach from social medicine is definitely important to establish an effective iodine prophylaxis.     

Ecology of hantaviruses in Mexico: Genetic identification of rodent host species and spillover infection

In our recent epidemiological survey conducted in Mexico for hantavirus infection, we identified three distinct viruses circulating in Mexican wild rodents, namely Montano virus (MTNV), Huitzilac virus (HUIV), and Carrizal virus (CARV). To gain a detailed understanding of hantavirus epidemiology and its associated hosts, 410 rodents were captured at eight collecting points in Morelos and Guerrero, Mexico, and examined for hantavirus seroprevalence, the presence of viral RNA, and rodent host species identification using cytochrome b gene sequences. Of the 32 species captured, seven species were positive for hantavirus: Peromyscus beatae (31/127; 24.4%), Reithrodontomys sumichrasti (6/15; 40%), Reithrodontomys megalotis (2/25; 8%), Peromyscus aztecus evides (1/1; 100%), Peromyscus megalops (1/41; 2.4%), Megadontomys thomasi (1/9; 11.1%), and Neotoma picta (1/6; 16.7%), with an overall prevalence of 10.5%; virus genome persisted in the majority of seropositive rodents. Nucleotide sequence and phylogenetic analysis showed that the viruses belonged mainly to the three lineages previously identified. The data showed that MTNV and CARV were primarily carried by P. beatae and R. sumichrasti, respectively. In addition, the data revealed an apparent complex interaction between hantaviruses and their hosts, suggesting active transmission and/or spillover infections within sympatric rodent species.     

Ets-1 activates overexpression of JunB and CD30 in Hodgkin's lymphoma and anaplastic large-cell lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.     

Clinical characteristics of young-onset and medical treatment-requiring hypertension identified by targeted screening in university health check-up

Based on targeted screening for hypertension at a university health check-up, we previously reported a high incidence of white-coat hypertension and estimated prevalence of hypertension requiring medical treatments (HT) as around 0.1% in young population aged less than 30. In spite of such low prevalence, continuous screening for seven consecutive years (2003-2009) increased the number of HT students to 20 (19 males and 1 female). We presently assessed the clinical characteristics of these HTs. Renovascular hypertension was found in the only female HT and aortic valve regurgitation in two HTs. Resting 17 HTs were diagnosed as having essential hypertension (EH). A father and/or a mother had EH in 16 out of 17 EHs, and blood pressure (BP) at home was slightly elevated (135-145 mm Hg in systolic) except three obese EHs (body mass index more than 30) who demonstrated more than 160 mm Hg in systolic. Plasma aldosterone-renin ratio (ARR) of EHs did not differ from that of normal controls, and Pearson correlation coefficient (R) between ARR and systolic BP (SBP) was -0.2. Its partial correlation coefficient, however, was statistically significant (R = -0.55, P = .026) after correcting for body mass index, which was significantly correlated with both SBP (P = .006, after correcting for ARR) and ARR (P = .004, after correcting for SBP). In conclusion, most of young-onset HTs are male EHs, and aortic valve regurgitation should be carefully checked. Excess plasma renin activity would be one of additional characteristics of young-onset EH to male gender, genetic background, and increased body mass.     

Relationship between reduced serum IGF-I levels and accumulation of visceral fat in Japanese men

OBJECTIVE: To investigate whether the changes in IGF-I concentrations after weight reduction in Japanese overweight men are associated with changes in visceral and subcutaneous fat. DESIGN: Cross-sectional and longitudinal clinical intervention study with exercise education. SUBJECTS: One-hundred and twelve Japanese overweight men aged 30-59 y (body mass index (BMI) 28.4+/-2.5 kg/m(2)) and 33 normal-weight men aged 30-39 y (BMI 22.1+/-1.5 kg/m(2)) at baseline. From the participants, 56 randomly selected overweight men (BMI 28.8+/-2.8) were further enrolled into a 1 y exercise program. MEASUREMENTS: Fat distribution was evaluated by visceral fat (V) and subcutaneous fat (S) areas measured with computed tomography scanning at umbilical levels, metabolic parameters and hormones including insulin, leptin and IGF-I at baseline and after 1 y. RESULTS: In 112 overweight subjects at baseline, insulin (10.5+/-5.0 microU/ml) and leptin (6.4+/-3.7 ng/ml) significantly correlated with both V (r=0.260, P=0.0073; r=0.410, P<0.0001) and S areas (r=0.377, P<0.0001; r=0.613, P<0.0001), respectively. IGF-I (156.8+/-48.7 microU/ml) significantly and negatively correlated with V area (r=-0.242, P=0.0125) and age (r=-0.192, P=0.0480). In normal-weight men aged 30-39 y (n=33) and age-matched subjects (n=30) selected from the 112 overweight men, the serum IGF-I further tightly correlated with V area (r=-0.467, P<0.0001). Visceral fat area and age were independently related to serum IGF-I levels by multiple regression analysis. By intervention with exercise education, 56 overweight subjects showed an increase in daily steps (6224+/-2781 to 7898+/-4141 steps/day) and reduction of BMI (28.8+/-2.8 to 27.7+/-2.9). deltaIGF-I significantly correlated with deltaV area (r=-0.432, P=0.0009) but not with DeltaS area or deltaBMI. CONCLUSION: The present study indicated a negative correlation between IGF-I levels and visceral fat at baseline as well as an association between the reduction in visceral fat and increase in IGF-I levels after an exercise intervention.