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Background— Acute aortic dissection (AAD) is a life-threatening vascular disease without effective pharmaceutical therapy. Matrix metalloproteinases (MMPs) are implicated in the development of chronic vascular diseases including aneurysm, but the key effectors and mechanism of action remain unknown. To define further the role of MMPs in AAD, we screened circulating MMPs in AAD patients, and then generated a novel mouse model for AAD to characterize the mechanism of action. Methods and Results— MMP9 and angiotensin II were elevated significantly in blood samples from AAD patients than in those from the patients with nonruptured chronic aortic aneurysm or healthy volunteers. Based on the findings, we established a novel AAD model by infusing angiotensin II to immature mice that had been received a lysyl oxidase inhibitor, β-aminopropionitrile monofumarate. AAD was developed successfully in the thoracic aorta by angiotensin II administration to β-aminopropionitrile monofumarate-treated wild-type mice, with an incidence of 20%, 80%, and 100% after 6, 12, and 24 hours, respectively. Neutrophil infiltrations were observed in the intima of the thoracic aorta, and the overexpression of MMP9 in the aorta was demonstrated by reverse transcription polymerase chain reaction, gelatin zymography, and immunohistochemistry. The incidence of AAD was reduced significantly by 40% following the administration of an MMP inhibitor and was almost blocked completely in MMP −/− mice without any influence on neutrophil infiltration. Neutrophil depletion by injection of anti-granulocyte-differentiation antigen-1 (anti-Gr-1) antibody also significantly decreased the incidence of AAD. Conclusions— These data suggest that AAD is initiated by neutrophils that have infiltrated the aortic intima and released MMP9 in response to angiotensin II.

To compare the diagnostic performance of computed tomographic (CT) attenuation and CT attenuation ratio at different tube voltages for ex vivo plaque characterization.Human coronary arteries were obtained at the time of autopsy in 15 subjects. The coronary arteries were serially cut into 5-mm-long segments and scanned ex vivo using 4 sets of tube voltages and tube currents (80 kV, 660 mA; 100 kV, 500 mA; 120 kV, 400 mA; and 140 kV, 340 mA). The CT attenuation value at the center of each plaque was obtained, and the ratio of the CT attenuation value at the 80-kV setting divided by that at the 140-kV setting (Hounsfield ratio [HR], 80:140) was calculated. Separate receiver operating characteristic (ROC) analyses were used to assess the usefulness of the CT attenuation value and the 80:140 HR for the differential diagnosis of lipid-rich plaques from other types of plaques.A total of 93 coronary plaques were detected macroscopically. Histological examination revealed 39 lipid-rich, 24 calcified, and 30 fibrotic plaques. At all the tube voltages, the CT attenuation values of the lipid-rich plaques were lower than those of the calcified plaques, whereas the CT attenuation values of the lipid-rich and fibrotic plaques overlapped. An ROC analysis showed that the area under the curve (AUC) for the differential diagnosis of lipid-rich plaques from fibrotic plaques was 0.813 at 80 kV, 0.772 at 100 kV, 0.682 at 120 kV, or 0.651 at 140 kV. Regarding the 80:140 HR, the AUC was 0.952 (0.029). The AUC was significantly larger at 80 and 100 kV and 80:140 HR compared with the AUC at 120 kV.The diagnostic performance of CT analysis for ex vivo plaque characterization was superior at lower energy settings and using the dual-energy method.

Background— Rupture of the chordae tendineae cordis (CTC) is a well-known cause of mitral regurgitation. Despite its importance, the mechanisms by which the CTC is protected and the cause of its rupture remain unknown. CTC is an avascular tissue. We investigated the molecular mechanisms underlying the avascularity of CTC and the correlation between avascularity and CTC rupture. Methods and Results— We found that tenomodulin, which is a recently isolated antiangiogenic factor, was expressed abundantly in the elastin-rich subendothelial outer layer of normal rodent, porcine, canine, and human CTC. Conditioned medium from cultured CTC interstitial cells strongly inhibited tube formation and mobilization of endothelial cells; these effects were partially inhibited by small-interfering RNA against tenomodulin. The immunohistochemical analysis was performed on 12 normal and 16 ruptured CTC obtained from the autopsy or surgical specimen. Interestingly, tenomodulin was locally absent in the ruptured areas of CTC, where abnormal vessel formation, strong expression of vascular endothelial growth factor-A and matrix metalloproteinases, and infiltration of inflammatory cells were observed, but not in the normal or nonruptured area. In anesthetized open-chest dogs, the tenomodulin layer of tricuspid CTC was surgically filed, and immunohistological analysis was performed after several months. This intervention gradually caused angiogenesis and expression of vascular endothelial growth factor-A and matrix metalloproteinases in the core collagen layer in a time-dependent manner. Conclusions— These findings provide evidence that tenomodulin is expressed universally in normal CTC in a concentric pattern and that local absence of tenomodulin, angiogenesis, and matrix metalloproteinase activation are associated with CTC rupture.

STUDY DESIGN Experimental study on age-related changes in expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) associated with transition from notochordal nucleus pulposus (NP) to fibrocartilaginous NP in rabbit intervertebral disc (IVD). OBJECTIVES To identify roles of notochordal NP in extracellular matrix (ECM) metabolism of IVD. SUMMARY OF BACKGROUND DATA One of most interesting properties of TIMP-3 is to inhibit aggrecanases in addition to matrix metalloproteinases. Balance of aggrecanase/TIMP-3 is critical to maintain homeostasis of ECM metabolism. METHODS Four-week-old and 160-week-old male Japanese white rabbits were used. Age-related changes in IVDs were evaluated histologically using previously established grading system. Immunohistochemistry of TIMP-3 and semiquantitative reverse transcriptase-polymerase reaction (RT-PCR) of TIMP-3, a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS) 4, 5, and transforming growth factor-beta1 (TGF-beta1), were conducted. RESULTS Semiquantitative assessment of histologic changes indicated that 4-week-old rabbit was equivalent to fetus to 2-year-old human and 160-week-old rabbit was equivalent to 11- to 30-year-old human, particularly 11- to 16-year-old, which corresponds to transition period from notochordal to fibrocartilaginous NP. Immunohistochemistry revealed that TIMP-3 was positive in 4-week-old rabbit only. Semiquantitative RT-PCR revealed that levels of expressions of TGF-beta1 and TIMP-3 mRNAs in 4-week-old were significantly higher than those in 160-week-old rabbits. There was no significant difference in expression of ADAMTS4 mRNA. ADAMTS5 mRNA was not detected or extremely low in both groups. Expression of TIMP-3 mRNA in NP was upregulated by TGF-beta1 but was not affected by IL-1beta. On the contrary, expression of ADAMTS4 mRNA was not upregulated by TGF-beta1 but was upregulated by IL-1beta. CONCLUSIONS Levels of expression of TIMP-3 in notochordal NP were significantly lower in 160-week-old rabbits than those in 4-week-old rabbits. Decrease in expression of TIMP-3, possibly mediated in part by TGF-beta1, may cause imbalance of ADAMTS4/TIMP-3 ratio at transition period from notochordal to fibrocartilaginous NP.

Jay D. Raman*, Carla Caruso, Zhaohai Yang, David DeGraff, Hershey, PA; Shahrokh Shariat, Vienna, Austria; Jose Karam, Christopher Wood, Houston, TX; Alon Weizer, Ann Arbor, MI; Mesut Remzi, Andrea Haitel, Vienna, Austria; Karim Bensalah, Nathalie Rioux-Leclerq, Rennes, France; Christian Bolenz, Mannheim, Germany; Marco Roscigno, Bergamo, Italy; Laura-Maria Krabbe, Payal Kapur, Yair Lotan, Vitaly Margulis, Dallas, TX

Study design Examination of ADAMTS-4 expression, and cellular lineages, distribution, and numbers of ADAMTS-4 (aggrecanase-1) expressing cells in herniated lumbar intervertebral discs. Objective To determine the expression of ADAMTS-4, a metalloproteinase capable of digesting aggrecan, and its role in herniated lumbar intervertebral disc degradation. Summary of background data Matrix metalloproteinases degrade extracellular matrix of herniated discs, but the mechanism of aggrecan degradation, the major component of intervertebral discs, is poorly understood. Methods Surgically resected herniated lumbar intervertebral discs from 22 patients were subclassified into protrusion, subligamentous extrusion, transligamentous extrusion, and sequestration types. Reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry were used to evaluate ADAMTS-4 messenger ribonucleic acid and protein expression. Results Expression of ADAMTS-4 messenger ribonucleic acid and protein was shown in the samples of herniated lumbar intervertebral discs. Immunohistochemical staining showed that ADAMTS-4 was mainly localized in CD68-positive mononuclear cells in granulation and adjacent disc tissues. ADAMTS-4 positive cell counts were significantly higher in transligamentous extrusion and sequestration than protrusion and subligamentous extrusion types. Alcian blue staining showed a decrease of proteoglycan in transligamentous extrusion and sequestration cases. Conclusions Macrophages infiltrating granulation and adjacent disc tissues express ADAMTS-4, suggesting its involvement in herniated disc regression.

association between gain of 8q and age (p1⁄40.23), sex (p1⁄40.42), and CCI (p1⁄40.59) was seen. Gain of chromosome 8q was associated with a 6.56 (95% CI, 3.06-14.05, p

INTRODUCTION AND OBJECTIVES: Many transcriptional programs governing somatic cell reprogramming and cancer progression have similarities. Cancer cells show heterogeneity in terms of drug resistance and cancer stemness is predicted to show enhanced drug resistance. We aimed to apply the knowledge of the similarity of transcriptional program between cancer progression and somatic cell reprogramming to identify a new clinically available drug to overcome the docetaxel-resistant prostate cancer (PCa), focusing on gene network of cancer stemness. METHODS: We retrospectively analyzed the clinical records and evaluated the OCT4 expression by immunohistochemistry in 164 patients with PCa who underwent radical prostatectomy. We enriched DU145 and LNCaP cells by using a vector system with endogenous OCT4 promoter activity, used in human induced pluripotent stem cells purification. The vector contains GFP and the puromycin resistance gene driven by the OCT4 enhancer. To find a new drug, we focused transciptome signature through Connectivity Map. We screened the drugs which converted the signature genes expression. RESULTS: Strong nuclear OCT4 staining in high stage tumors was observed. OCT4 expression was significantly associated with higher pathological T stage (p

Three different membrane-type matrix metalloproteinases (MT1, 2, 3-MMP) which can activate proMMP-2 (progelatinase A) are thought to have an important role in various human carcinoma invasions and metastases. We examined the mRNA expression of MT-MMPs and the tissue immunolocalization of MT1-MMP in human urothelial carcinomas.mRNA was extracted from 27 clinical urothelial carcinomas and 10 normal urothelial mucosa tissues remote from the tumor. RT-PCR using specific primers was performed, and PCR products were hybridized to 32P-labeled internal probes and analyzed by a bioimage analyzer. Immunolocalization was studied using a monoclonal antibody against MT1-MMP (114-6G6).MT1-MMP and MT2-MMP mRNA expressions in urothelial carcinomas were significantly higher than those in the normal mucosa. In contrast, MT3-MMP mRNA was little expressed in both tissues, and the amount of MT3-MMP mRNA appeared to be much lower than MT1-MMP and MT2-MMP in the tissue samples. In terms of the tumor multiplicity, MT1-MMP and MT2-MMP mRNA expressions in the group of multiple tumors were significantly higher than those in the solitary tumor group. The carcinoma cells were immunostained for MT1-MMP predominantly in invasive and superficial carcinoma cells. The immunoreactivity was more intense in the invasive type than in the superficial type.It is suggested that MT1-MMP and MT2-MMP play an important role in the development of human urothelial carcinomas and reflect some aspects of the pathogenesis of multifocal occurrence. In spite of the possible contribution to the invasive and metastatic phenotype, MT1-MMP mRNA and its product are thought to be expressed already in the clinical superficial stage in some cases of this tumor type.

elevated pre-CRP level and not receiving adjuvant chemotherapy, post-CRP normalization was observed in 62 (64.6%) after RNU. The 5-year cancer-specific survival rates were 73.4% in patients in the normalized post-CRP group and 47.7% in their counterparts (p 0.011). Multivariate analysis demonstrated that post-CRP normalization was an independent predictor for cancer-specific mortality (HR 2.20), in addition to pT stage. CONCLUSIONS: Serum CRP could provide useful information on cancer-specific mortality in UTUC patients treated with RNU. In the elevated pre-CRP group, post-CRP normalization was an independent predictor for better outcome. We propose that serum CRP may become a useful biomarker in UTUC patients, due to its low cost and easy accessibility.

[Background] Congestive heart failure (CHF) is characterized by activation of the sympathetic nervous system (SNS) with depletion of norepinephrine (NE) stores, which was initially considered to be the result of excess NE secretion and the loss of noradrenergic nerve terminals. Recent studies however have revealed that it is caused by down regulation of NE synthesis and re-uptake, although the molecular mechanism of down regulation of the sympathetic neuronal function remains unknown. We recently found in an animal model of CHF that the cardiac SNS switches the neurotransmitter property from catecholaminergic to cholinergic, mediated by cytokines LIF and CT-1 secreted from failing myocardium. This study was designed to investigate whether or not this cholinergic transdifferentiation of cardiac SNS occurs in patients with CHF. [Methods & Results] (1) We analyzed 8 samples from patients who died of non-cardiac causes obtained at autopsy (control group), and 8 samples from patients with CHF (CHF group). Five of them died of CHF, and 3 were obtained from native hearts of transplant recipients. (2) The heart weight was significantly higher in the CHF group. (3) The gross morphology of the cardiac SNS did not differ between the two groups. HE and Masson trichrome staining showed disorganized cardiomyocytes and interstitial fibrosis in CHF. (4) Immunostaining for tyrosine hydroxylase (TH, sympathetic nerve marker) revealed that the epicardial nerve bundles and stellate ganglia of the control group had a predominance of TH + nerves, whereas those of CHF group were significantly decreased. (5) Immunostaining for choline transporter (CHT, cholinergic neuron marker) revealed that CHT + neurons were markedly increased in the epicardial nerve bundles of CHF hearts compared with the control group. Some nerves co-expressed both TH and CHT markers. (6) Immunostaining for choline acetyl transferase (ChAT, a cholinergic neuron marker) revealed that stellate ganglia had a lot of ChAT + neurons compared with the control. (7) Nissl staining showed that there was no difference between the two groups in neuron number in the stellate ganglia. [Conclusions] These results indicated that in patients with CHF the cardiac sympathetic nerves also had cholinergic nerve properties.

247 RANKL EXPRESSION IN CLEAR CELL RENAL CELL CARCINOMAS AND ITS ASSOCIATION WITH BONE METASTASIS AND CANCER CELL MIGRATION Shuji Mikami*, Ken-ichi Katsube, Mototsugu Oya, Satsuki Mochizuki, Masayuki Shimoda, Ryuichi Mizuno, Masaru Ishida, Tohru Ikeda, Makio Mukai, Yasunori Okada. Tokyo, Japan, and Nagasaki, Japan. INTRODUCTION AND OBJECTIVE: Bone metastasis occurs in ~30% of patients with renal cell carcinoma (RCC) and represents the most serious clinical complication. However, it has been unclear

199 NUCLEAR EXPRESSION OF ARYL HYDROCARBON RECEPTOR PREDICTS DISEASE SPECIFIC SURVIVAL OF UROTHELIAL CARCINOMA OF THE UPPER URINARY TRACT Masaru Ishida*, Eiji Kikuchi, Shuji Mikami, Takeo Kosaka, Akira Miyajima, Makio Mukai, Yasunori Okada, Mototsugu Oya. Tokyo, Japan. INTRODUCTION AND OBJECTIVE: Cigarette smoke contributes to the development of urothelial cancer (UC). The aryl hydrocarbon receptor (AhR), which is the receptor for xenobiotics such as cigarette smoke, induces the CYP1 family, which becomes involved in tumor initiation and progression. AhR overexpression is observed in a few cancers, such as lung and pancreatic cancer, but expression of AhR in UCs, including bladder cancer, has not been investigated. We detected