Teiji Oda

Hyperbilirubinemia-related behavioral and neuropathological changes in rats: A possible schizophrenia animal model

BACKGROUND Patients with schizophrenia show a significantly higher frequency of hyperbilirubinemia than patients suffering from other psychiatric disorders and the general healthy population. We examined the hyperbilirubinemia on behavioral and neuropathological changes in rats as a possible animal model of schizophrenia. METHODS Gunn rats with severe hyperbilirubinemia (j/j), Gunn rats without severe hyperbilirubinemia (+/j), and Wistar rats were examined by open-field, social interaction, and prepulse inhibition tests. TUNEL, AgNOR and Ki-67 were also assayed on paraffin-embedded brain sections of these rats. RESULTS Compared to Wistar rats, both Gunn j/j and +/j rats showed hyperlocomotion, high sniffing scores, and low defecation scores. They showed significantly more aggressive behaviors and impaired prepulse inhibition. The numbers of Ki-67-labeled cells and AgNOR were lower and the number of TUNEL-positive cells was higher than that of Wistar rats. CONCLUSIONS These results might support the neurodevelopmental hypothesis of schizophrenia. Both Gunn j/j and +/j rats may be a useful animal model and provide clues to the role of hyperbilirubinemia in schizophrenia.

Proteomic analysis of calcified abdominal and thoracic aortic aneurysms

Aortic aneurysm is a complex multifactorial disease with genetic and environmental risk factors. It is often accompanied by aortic calcification. Here, to uncover proteins that are significantly changed in calcified abdominal aortic aneurysms (CAAs) and calcified thoracic aortic aneurysms (CTAs) compared with those in adjacent normal aorta tissues, comprehensive analysis of differentially expressed proteins in their tissues was performed by a quantitative proteomic approach with iTRAQ labeling in combination with nanoLC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis. The proteomic analysis revealed 138 and 134 proteins differentially expressed in CAAs and CTAs in contrast to neighboring normal aorta tissues with high confidence, respectively. Significantly increased expression (≥1.3-fold) was found in 41 and 28 proteins, whereas decreased expression (<0.77-fold) was found in 4 and 60 proteins in CAAs and CTAs, respectively. Among them, we identified already known proteins involved in aneurysm formation and vascular calcification, such as type I and III collagen, matrix Gla protein, and α-2-HS-glycoprotein in CAAs and fibrinogen α, β and γ chains and α-2-HS-glycoprotein in CTAs with increased expression and mimecan in CAAs and fibulin-5 in CTAs with decreased expression. Based on the Panther pathway and Genesis clustering analyses, some of the proteins could be linked to corresponding biochemical pathways, such as the integrin signaling pathway with increased expression in CAAs, the blood coagulation pathway with increased expression in CTAs, and the inflammation mediated by chemokine and cytokine signaling pathway and the glycolysis pathway with decreased expression in CTAs. Interestingly, it was found by clustering analysis that samples from CAAs of patients with both CAAs and CTAs were clustered outside the samples of patients with CAAs and were clustered with samples of patients with CTAs. Our results provide a comprehensive patient-based proteomic analysis for the identification of potential biomarkers for CAAs and CTAs.

Noticeable Decreased Expression of Tenascin-X in Calcific Aortic Valves

Calcification of aortic valves results in valvular aortic stenosis and is becoming a common valvular condition in elderly populations. An understanding of the molecular mechanisms of this valve lesion is important for revealing potential biomarkers associated with the development and progression of this disease. In order to identify proteins that are differentially expressed in calcific aortic valves (CAVs) compared with those in adjacent normal valvular tissues, comprehensive analysis of differentially expressed proteins in the tissues was done by a quantitative proteomic approach with isobaric tag for absolute and relative quantitation labeling followed by nanoliquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The proteomic analysis revealed 105 proteins differentially expressed in CAVs in contrast to adjacent normal valvular tissues with high confidence. Significantly increased expression (>_1.3-fold) was found in 34 proteins, whereas decreased expression (<0.77-fold) was found in 39 proteins in CAVs. Among them, α-2-HS-glycoprotein showed the greatest increase in expression (6.54-fold) and tenascin-X showed the greatest decrease in expression (0.37-fold). Numerous extracellular matrix proteins such as collagens were identified as proteins with significantly decreased expression. Panther pathway analysis showed that some of the identified proteins were linked to blood coagulation and integrin signaling pathways. Cluster analysis of the 105 proteins differentially expressed in CAVs based on the expression pattern revealed that tenascin-X was clustered with proteins controlling collagen structure and function, especially collagen fibrillogenesis, such as decorin and fibromodulin. We confirmed decreased levels of these proteins in CAVs by Western blot analyses. These results indicated that massive destruction of the extracellular matrix occurs in CAVs.

Proteomic profiling for the identification of serum diagnostic biomarkers for abdominal and thoracic aortic aneurysms.

BackgroundAortic aneurysm is an increasingly common vascular disorder with fatal implication. However, there is no established diagnosis other than that based on aneurysmal size. For this purpose, serum protein biomarkers for aortic aneurysms are valuable. Although most of the studies on serum biomarker discovery have been based on comparison of serum proteins from the patient group with those from the healthy group, we considered that comparison of serial protein profiles such as those in presurgical and postsurgical sera within one patient would facilitate identification of biomarkers since the variability of serial protein profiles within one patient is smaller than that between groups. In this study, we examined serum proteins with differential levels in postsurgery compared with those in presurgery after the removal of aneurysmal tissues in abdominal aortic aneurysm (AAA) and thoracic aortic aneurysm (TAA) patients in order to identify potential serum biomarkers for AAAs and TAAs.ResultsA proteomic approach with an isobaric tag for relative and absolute quantitation (iTRAQ) labeling followed by nano liquid chromatography (nanoLC)-matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF/TOF)-tandem mass spectrometry (MS/MS) was used. In the sera of patients with AAAs and TAAs, a total of 63 and 71 proteins with differential levels were further narrowed down to 6 and 8 increased proteins (≧1.3 fold, postsurgical vs. presurgical) (p < 0.05, patient vs. control) and 12 and 17 decreased proteins (< 0.77 fold, postsurgical vs. presurgical) (p < 0.05, patient vs. control) in postsurgical sera compared with those in presurgical sera, respectively. All of the increased proteins in postsurgical sera of both AAA and TAA patients included several known acute-phase proteins. On the other hand, in the decreased proteins, we found intriguing molecules such as α-2-macroglobulin, gelsolin, kallistatin, and so on. Among them, we confirmed that kallistatin in both AAA and TAA patients and α-2-macroglobulin in TAA patients showed decrease levels in postsurgical sera similar to those in control sera by Western blot analysis with other sera from AAA and TAA patients.ConclusionsTaken together, our findings suggest that Kallistatin and α-2-macroglobulin are potential serum biomarkers for both AAA and TAA and TAA, respectively.

Hypothermia produces rat liver proteomic changes as in hibernating mammals but decreases endoplasmic reticulum chaperones.

Hypothermia is used in the clinic for protection of organs such as the brain against ischemic injury during aortic/complex congenital cardiac surgery or post-resuscitation encephalopathy. The principal mechanism of hypothermic protection is suppression of metabolism, however, the pleiotropic effects of cooling are incompletely understood. Here, we used a rat model system to evaluate metabolic changes induced by deep hypothermia. The hypothermia-induced changes were identified using fluorescence-based two-dimensional (2-D) difference gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry. Rats were randomly assigned to a normothermic control group (37°C, n=6) or hypothermia group (23°C, n=6) that received surface cooling for 3h. Liver tissue was excised for assessment. Functional profiling of differently expressed proteins was performed as an enrichment analysis of Gene Ontology (GO) terms and pathways. We found that the livers of anesthetized rats with deep hypothermia showed significant downregulation of proteins in the endoplasmic reticulum and mitochondria, and of those involved in ATP binding, amino acid metabolism and urea cycle, response to oxidative stress, anti-apoptosis, negative regulation of apoptosis. The changes in the proteome of the hypothermic rats showed similarities, except with regard to endoplasmic reticulum chaperones, to those identified elsewhere in mammals undergoing hibernation.

Proteomic comparison between abdominal and thoracic aortic aneurysms

The pathogenesis of abdominal aortic aneurysms (AAAs) and that of thoracic aortic aneurysms (TAAs) is distinct. In this study, to reveal the differences in their biochemical properties, we performed quantitative proteomic analysis of AAAs and TAAs compared with adjacent normal aorta (NA) tissues. The proteomic analysis revealed 176 non-redundant differentially expressed proteins in the AAAs and 189 proteins in the TAAs which were common in at least 5 samples within 7 samples of each. Among the identified proteins, 55 and 68 proteins were unique to the AAAs and TAAs, respectively, whereas 121 proteins were identified in both the AAAs and TAAs. Panther overrepresentation analysis of the unique proteins in the AAAs and TAAs revealed a significant downregulation of the blood coagulation pathway in the AAAs and that of the integrin signaling pathway in the TAAs. On the other hand, Genesis analysis revealed distinct expression patterns of 58 proteins among the 121 proteins. Panther overrepresentation analysis of these 58 proteins revealed that the expression of these proteins in the blood coagulation and the plasminogen activating cascade was decreased in the AAAs, whereas it was increased in the TAAs compared with the NA tissues. On the other hand, the protein expression in the integrin signaling pathway was increased in the AAAs, whereas it was decreased in the TAAs compared with the NA tissues. Thus, the data presented in this study indicate that the proteins that show differential expression patterns in AAAs and TAAs may be involved in the distinct pathogenesis of AAAs and TAAs.

Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries

Deep hypothermic circulatory arrest (DHCA) is a protective method against brain ischemia in aortic surgery. However, the possible effects of DHCA on the plasma proteins remain to be determined. In the present study, we used novel high-throughput technology to compare the plasma proteomes during DHCA (22°C) with selective cerebral perfusion (SCP, n=7) to those during normothermic cardiopulmonary bypass (CPB, n=7). Three plasma samples per patient were obtained during CPB: T1, prior to cooling; T2, during hypothermia; T3, after rewarming for the DHCA group and three corresponding points for the normothermic group. A proteomic analysis was performed using isobaric tag for relative and absolute quantification (iTRAQ) labeling tandem mass spectrometry to assess quantitative protein changes. In total, the analysis identified 262 proteins. The bioinformatics analysis revealed a significant upregulation of complement activation at T2 in normothermic CPB, which was suppressed in DHCA. These findings were confirmed by the changes of the terminal complement complex (SC5b-9) levels. At T3, however, the level of SC5b-9 showed a greater increase in DHCA compared to normothermic CPB, while 48 proteins were significantly downregulated in DHCA. The results demonstrated that DHCA and rewarming potentially exert a significant effect on the plasma proteome in patients undergoing aortic surgery.

Impairment of platelet retention rate in patients with severe aortic valve stenosis.

BACKGROUND Recent reports revealed the presence of acquired von Willebrand syndrome type 2A in patients with aortic valve stenosis (AS). von Willebrand factor (vWF) has been shown to play a vital role in platelet adhesion. Therefore, we measured the platelet retention rates, which reflect platelet adhesion, in patients with severe AS. METHODS In addition to echocardiography, routine blood screening tests were performed and the platelet retention rates were measured using collagen-coated bead columns in 21 patients with severe AS and in 21 control subjects. RESULTS Patients with severe AS showed the maximum aortic valve pressure gradients of 110.9±22.7 mmHg, and effective orifice areas of 0.59±0.20 cm2. The results of routine blood tests in patients with severe AS were comparable to those of control subjects; however, the platelet retention rates in the AS patients (7.3±5.0%) were significantly lower than those in control subjects (30.5±11.8%, p<0.001). A significant negative correlation was observed between maximum aortic valve pressure gradients and platelet retention rates (r = -0.81, p<0.001). In 8 patients with severe AS, the platelet retention rates increased from 5.8±3.6% to 16.0±2.4% after aortic valve replacement (p<0.001). CONCLUSION These findings suggest that impairment of platelet retention rate is seen in almost all patients with severe AS. Clinicians should be aware of the possibilities of vWF-mediated platelet dysfunction and bleeding tendency in patients with severe AS.

Does the Rewarmed Heart Restore the Myocardial Proteome to That of the Pre-Cooled State? : A Proteomic Analysis of Surgical Samples

BACKGROUND Hypothermia is utilized in cardiac and aortic surgery to protect organs from ischemic reperfusion injury. Although the cooled body is invariably rewarmed after the procedure, it is still unknown whether the rewarmed body regains its former biological state. This study determined the modulatory effects of hypothermia on the human myocardial proteome and whether subsequent rewarming restores the proteome to the state prior to cooling. METHODSANDRESULTS A quantitative proteomic analysis was performed using isobaric tags for relative and absolute quantification labeling tandem mass spectrometry. Right atrial samples were taken 3 times (pre, during and post cooling) during deep hypothermic cardiopulmonary bypass (CPB) from 8 patients with aortic arch aneurysms and 3 corresponding time points during normothermic CPB from 8 patients with ascending aortic or valsalva aneurysms. In total, 697 proteins were identified, with 222 proteins having high protein confidence. Bioinformatic analyses revealed significant downregulation of 19 proteins associated with energy production at hypothermic cardioplegic arrest. On rewarmed beating, 10 proteins remained downregulated, including those regulating cardiac contraction and adaptor proteins, although levels of the aforementioned 19 downregulated proteins returned to their initial values. Additional echocardiographic evaluation demonstrated that hypothermia preserved the variables of diastolic function to a greater extent than normothermic surgery. CONCLUSIONS Rewarming restores the human myocardial proteome to the pre-cooled state, except for proteins regulating cardiac contraction and adaptor proteins.

Fatal septicemia and endotoxic shock due to Aeromonas hydrophila

Summary Background: Although rare, bloodstream infections caused by Aeromonas tend to be very severe and progress rapidly. Case Report: We report a case of an 81-year-old man with fetal septicemia and endotoxin shock caused by Aeromonas hydrophila. The patient had dilated cardiomyopathy, paroxysmal atrial fibrillation, interstitial pneumonitis and renal dysfunction was admitted to our hospital with chest pain and dyspnea. Transthoracic echocardiography demonstrated impaired left ventricular wall motion and severe mitral regurgitation due to tethering. Cardiac catheterization revealed severe stenotic lesions in the left anterior descending artery and the right coronary artery. Surgery for coronary artery bypass grafts and mitral annuloplasty were performed. However, 2 days after surgery, he suddenly developed a high-grade fever and his hemodynamics deteriorated rapidly. His blood cultures revealed gram-negative Bacillus and the endotoxin concentration in the blood was elevated. Despite intensive support efforts, the patient died 1 day after the sudden change. His blood culture revealed A. hydrophila. Conclusions: Whenever Aeromonas is found in a patient’s bloodstream, clinicians should start appropriate and intensive treatment immediately.