Assessment of left ventricular ejection fraction using low radiation dose computed tomography.

BACKGROUND: Cardiac CT is a non-invasive modality with the ability to estimate LVEF. However, given its limited temporal resolution and radiation, there has been initial resistance to use CT to measure LVEF. Developing an accurate, fast, low radiation dose protocol is desirable. OBJECTIVE: The objective of this study is to demonstrate that a 'low radiation dose' 64 slice cardiac computed tomography (CT) protocol is feasible and can accurately measure left ventricular ejection fraction (LVEF) while delivering a radiation dose lower than radionuclide angiography (RNA). METHODS: Patients undergoing RNA were prospectively screened and enrolled to undergo a 'low-dose' 64 slice CT LVEF protocol. LVEF measures, duration of each study and radiation dose between CT and RNA were compared. RESULTS: A total of 77 patients (mean age = 61.8 ± 12.2 years and 58 men) were analyzed. The mean LVEF measured by CT and RNA were 41.9 ± 15.2% and 39.4 ± 13.9%, respectively, (P = 0.154) with a good correlation (r = 0.863). Bland-Altman plot revealed a good agreement between the CT and RNA LVEF (mean difference of -2.4). There was good agreement between CT LVEF and RNA for identifying patients with LVEF ≤30% (kappa = 0.693) and LVEF ≥50% (kappa = 0.749). The mean dose estimated effective dose for CT and RNA were 4.7 ± 1.6 and 9.5 ± 1.0 mSv, respectively. The mean CT LVEF imaging duration (4:32 ± 3:05 minutes) was significantly shorter than the RNA image acquisition time (9:05 ± 2:36 minutes; p < 0.001). CONCLUSION: The results of our study suggest that low-dose CT LVEF protocol is feasible, accurate, and fast while delivering a lower radiation dose than traditional RNA.

Discordance between Framingham Risk Score and atherosclerotic plaque burden.

AIM: Clinical predictors are routinely used to identify individuals who may benefit from aggressive risk factor modification. However, clinical predictors cannot account for all genetic and environmental variables. The objective of this study is to investigate the association of Framingham Risk Score (FRS) with computed tomography angiography (CTA) measures of coronary atherosclerosis. METHODS AND RESULTS: Consecutive patients who underwent CTA were prospectively enrolled and categorized according to clinical predictors such as FRS and pre-test probability for obstructive coronary artery disease (CAD). Atherosclerotic calcific and non-calcific plaques were assessed. Of the 1507 patients without a history of diabetes mellitus, myocardial infarction, and not on statin therapy, coronary atherosclerosis was present in 63.5% of the patients. Of the 1173 patients with low and intermediate FRS, atherosclerotic plaque was visually present in 47.6 and 72.7% of the patients, respectively. A higher proportion of low FRS patients had isolated non-calcific plaque (14.8%) compared with patients in the intermediate (10.1%) or high (7.2%) FRS groups, and 11.7% of high FRS patients had no visual evidence of plaque. The correlation between FRS and plaque was fair (r = 0.48; P < 0.001). CONCLUSION: Although clinical variables are predictive of CAD events, CTA identified coronary atherosclerosis in a significant proportion of patients with low to intermediate FRS, and a small minority of patients with high FRS had no evidence of atherosclerosis. Prospective studies are required to determine the potential value of identifying coronary atherosclerosis using CTA and to assess whether modifying therapies based on these results are warranted.

Identification of vascular breast tumor markers by laser capture microdissection and label-free LC-MS.

Blood vessels in tumors frequently show abnormal characteristics, such as tortuous morphology or leakiness, but very little is known about protein expression in tumor vessels. In this study, we have used laser capture microdissection (LCM) to isolate microvessels from clinical samples of invasive ductal carcinoma (IDC), the most common form of malignant breast cancer, and from patient-matched adjacent nonmalignant tissue. This approach eliminates many of the problems associated with the heterogeneity of clinical tumor tissues by controlling for differences in protein expression between both individual patients and different cell types. Proteins from the microvessels were trypsinized and the resulting peptides were quantified by a label-free nanoLC-MS method. A total of 86 proteins were identified that are overexpressed in tumor vessels relative to vessels isolated from the adjacent nonmalignant tissue. These proteins include well-known breast tumor markers such as Periostin and Tenascin C but also proteins with lesser-known or emerging roles in breast cancer and tumor angiogenesis (i.e., Serpin H1, Clic-1, and Transgelin 2). We also identified 40 proteins that were relatively under-expressed in IDC tumor vessels, including several components of the basement membrane whose lower expression could be responsible for weakening tumor vessels. Lastly, we show that a subset of 29 proteins, derived from our list of differentially expressed proteins, is able to predict survival in three publicly available clinical breast cancer microarray data sets, which suggests that this subset of proteins likely plays a functional role in cancer progression and outcome.

Obesity and coronary artery calcification: Can it explain the obesity-paradox?

The inverse relationship between obesity and adverse cardiovascular outcomes has been coined the 'obesity-paradox'. We sought to determine the relationship between measures of obesity [body mass index (BMI), body surface area (BSA) and body fat percentage (BF%)] and coronary artery calcification (CAC). We retrospectively analyzed patients who underwent CAC using the Agatston score. Baseline demographics were collected and BMI, BSA and BF% were calculated. A two-stage regression modeling approach was used to evaluate the association between BMI, BSA, BF% and Agatston score. Of the 6661 patients [mean age = 57.1 ± 10.8 years, men = 54.3%, median Agatston score = 14 (0, 163)], 0.1% were underweight, 21.3% had normal BMI, 39.1% were overweight and 39.4% were obese. The mean BMI, BSA and BF% were 29.6 ± 6.1 kg/m(2), 1.97 ± 0.25 m(2) and 37 ± 10 %, respectively. There was an independent association between the presence of CAC and BMI (5 kg/m(2) increments) (OR 1.05, CI 1.00-1.11, P = 0.038) and BF% (OR 2.38, CI 1.05-5.41, P = 0.038). Neither BMI categories nor large BSA independently predicted the presence of CAC. BF% predicted the extent of CAC in men but not in women, and higher BF% was associated with higher category of CAC severity in men only. BMI and BF% were independent predictors of the presence of CAC. BF% was associated with the extent of CAC and higher BF% was associated with higher category of CAC severity in men only. These results suggest that further study is needed to better understand the obesity-paradox.

Site-directed mutagenesis of the type II TGF-beta receptor indicates a ligand-binding site distinct from that of the type II activin receptor.

Site-directed mutagenesis was used to map the ligand-binding surface of the type II transforming growth factor-beta receptor extracellular domain (TbetaRII-ECD). Two putative ligand-binding sites were probed, the first being a predicted hydrophobic patch, the second being the finger 1 surface loop. Nine residues were mutated in the context of full-length TbetaRII and the effect of these mutations on ligand-binding and receptor signaling was analyzed. Complementary information was obtained by examining 'natural' evolutionary TbetaRII mutations. Together, the results indicate that residues within the finger 1 region, but not the hydrophobic patch, of the TbetaRII-ECD are required for productive ligand-binding. We conclude that, surprisingly, the ECDs of TbetaRII and type II activin receptor utilize distinct interacting surfaces for binding their respective ligands.

Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses.

Laser-capture microdissection (LCM) is a technique that enables selective extraction of desired cells from heterogeneous tissues compatible with subsequent molecular analyses. The specific visualization of desired cell types prior to LCM is essential for achieving selective capture. We have developed rapid and selective staining protocols for LCM extraction of microvessels from human and rat brain. Vessels in human and rat brain sections were visualized by a 2 min exposure to fluorescein-labeled lectins Ulex Europeaus Agglutinin I (UEA I) and Ricinus Communis Agglutinin I (RCA I), respectively. Immunohistochemical staining for the endothelial-specific marker, Factor VIII-related antigen (FVIII-rAg), co-localized with that for either UEA I or RCA I, confirming the selective staining of vascular structures with these lectins. Both brain vessels and perivascular parenchyma were captured using LCM, followed by RNA isolation. RT-PCR analyses demonstrated the enrichment of LCM-captured vessels and parenchyma in FVIII-rAg and GFAP mRNA, respectively. LCM-captured human vessels also expressed the tight junction-specific gene, zonula occludens 1 (ZO-1). LCM extraction of vessels from brain sections can be used to perform molecular fingerprinting of neurovascular unit in various brain pathologies.

Prognostic value of Rb-82 positron emission tomography myocardial perfusion imaging in coronary artery bypass patients.

AIMS: We sought to determine the prognostic value of positron emission tomography (PET) myocardial perfusion imaging (MPI) in patients with prior coronary artery bypass graft (CABG) surgery. PET MPI has recently been shown to provide incremental risk stratification for patients with suspected coronary artery disease (CAD), but the prognostic utility of PET MPI in CABG patients has not been well studied. METHODS AND RESULTS: A multi-centre PET registry of 7061 patients who underwent Rb-82 PET MPI from four participating centres was screened. Nine hundred and fifty-three CABG patients were identified and their images were analysed. Outcomes of all-cause mortality and cardiac death were collected. With a mean follow-up of 2.4 ± 1.4 years, 128 (13.4%) all-cause deaths and 44 (4.6%) cardiac deaths were observed. Multivariable analyses, adjusted for clinical variables, demonstrated that the summed stress score (SSS) was a significant independent predictor of both all-cause mortality [HR: 1.60 (per 1 category increase in SSS); 95% CI: 1.34-1.92; P < 0.001] and cardiac death (HR: 1.80; 95% CI: 1.33, 2.44; P < 0.001). The receiver-operator characteristic (ROC) curves showed that the addition of SSS increased the area under the curve (AUC) from 0.645 to 0.693 (P = 0.014) for all-cause mortality, and from 0.612 to 0.704 (P = 0.027) for cardiac death. SSS also improved the net reclassification improvement (NRI) for all-cause mortality (category-free NRI = 0.422; 95% CI: 0.240-0.603; P < 0.001) and cardiac death (category-free NRI = 0.552; 95% CI: 0.268-0.836; P < 0.001). CONCLUSIONS: PET MPI provides independent and incremental prognostic value to clinical variables in predicting all-cause mortality and cardiac death in CABG patients.

Insulin-like growth factor binding protein 7 exhibits tumor suppressive and vessel stabilization properties in U87MG and T98G glioblastoma cell lines.

Insulin-like growth factor binding protein 7 (IGFBP7) is downregulated in several solid cancers. IGFBP7 has been proposed to act as a tumor suppressor gene through mechanisms involving senescence and apoptotic pathways. The tumor suppressor effect of IGFBP7 in glioblastoma multiforme (GBM) was examined in this study using two human GBM cell lines, U87MG and T98G. Exogenously applied IGFBP7 (20 and 100 nM) significantly reduced U87MG (~70 and ~75%, respectively) and T98G (~37 and ~50%, respectively) cell growth in soft agar. IGFBP7 stimulated senescence-associated ß-galactosidase in both U87MG and T98G cells without stimulating apoptosis (annexin V and propidium iodide staining, expression of SMARCB1 or BNIP3L and caspase cleavage) or affecting phosphorylation of p44/42 MAPK. The inhibitory effect of IGFBP7 on U87MG cell growth was further assessed in vivo using U87MG cells grafted on the chick chorioallantoic membrane. In this model, U87MG cells formed solid and highly vascularized tumors that were reduced in size (~40%) when treated with 500 nM IGFBP7 compared with control tumors. Vessels in IGFBP7-treated tumors were clustered, unevenly distributed and associated with higher number of α-SMA positive cells compared with those in untreated tumors. IGFBP7 induced both aortic smooth muscle cell (AoSMC) chemoattraction and endothelial cell (EC) transdifferentiation into a SM-like cell phenotype. U87MG conditioned media-induced IGFBP7 expression in ECs was significantly inhibited by the cross-talk/interaction with SMCs. This study indicates that IGFBP7 suppresses U87MG tumor cell growth, induces cell senescence and participates in tumor vessel stabilization by promoting SMC/pericyte recruitment and differentiation.

Molecular markers of extracellular matrix remodeling in glioblastoma vessels: microarray study of laser-captured glioblastoma vessels.

Glioblastoma multiforme (GBM) are the most malignant and vascularized brain tumors. The aberrant vascular phenotype of GBM could be exploited for diagnosis or therapeutic targeting. This study identified new molecular markers of GBM vessels, using a combination of laser capture microdissection (LCM) microscopy, RNA amplification, and microarray analyses to compare vessels from nonmalignant human brain and GBM tumors. Forty-two genes were differentially expressed in GBM vessels compared to nonmalignant brain vessels. Validation of differentially expressed genes was performed by literature mining, Q-PCR, and immunohistochemistry. Among the differentially expressed genes, only 64% were previously associated with vessels, angiogenesis, gliomas, and/or cancer. The upregulation of genes encoding secreted extracellular proteins IGFBP7 and SPARC was confirmed by Q-PCR in LCM-captured vessels. Whereas SPARC and IGFBP7 protein were absent in nonmalignant brain vessels, a distinct immunoreactivity patterns were observed in GBM sections whereby SPARC was strongly expressed in perivascular cells adjacent to GBM vessels while GBM endothelial cells were immunostained for IGFBP7. IGFBP7 immunoreactivity was also detected on the abluminal side of GBM vessels deposited between strands of vascular basal lamina. The study discerns unique molecular characteristics of GBM vessels compared with nonmalignant brain vessels that could potentially be used for diagnostic or therapeutic purposes.

Purification and characterization of recombinant microsomal prostaglandin E synthase-1.

Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation.