Differential expression of Oct3/4 in human breast cancer and normal tissues.

Oct3/4, a transcription factor specifically expressed in mammalian totipotent embryonic stem and germ cells, has a critical role in the regulation and maintenance of pluripotency and self-renewal. However, reactivation of Oct3/4 expression is observed in several human breast cancer cell lines, but not in non­malignant cells. To examine Oct3/4 expression in human primary breast carcinomas and normal breast tissues, we obtained breast tumor tissues from 28 patients and normal breast tissues from 9 women. According to quantitative polymerase chain reaction, all of the tumor tissues, irrespective of tumor type or clinicopathological status, expressed Oct3/4 mRNA at 10- to 100- fold higher levels than that in the normal breast tissues. Expression of the Oct3/4 protein in tumors was confirmed by western blot analysis and immunofluorescent staining. Additionally, rapid amplification of cDNA ends and DNA sequencing revealed expression of multiple Oct4 gene transcripts from chromosome 6 (POU5F1) in normal breast tissues and the non­malignant breast epithelial cell line MCF­10A; by contrast, the breast tumors and malignant breast cancer cell line MCF­7 predominantly expressed transcripts of an Oct4-like gene (POU5F1B) from chromosome 8, which was termed Oct3 in the current study. The deduced amino acid sequences of full-length Oct3 and Oct4 are 96% identical. The findings of the current study indicated that Oct3, rather than Oct4, may serve as a novel clinical marker and a potential target for gene-specific therapy of breast cancer.

A novel combined imaging/morphometrical method for the analysis of human sural nerve biopsies for clinical diagnosis.

Nerve Morphometry is one tool employed in the clinical assessment of peripheral sural nerve pathological abnormalities. A new method is presented in this chapter incorporating an unbiased approach to quantitative sural nerve evaluation. Using conventional epoxy embedded nerves processed for electron microscopy, confocal microscopy, and interactive digital assessment, this method produces a rigorous, accurate reproducible record for use in clinical diagnosis.

Cell adhesion molecule 1 (CADM1) is ubiquitously present in the endothelium and smooth muscle cells of the human macro- and micro-vasculature.

Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin cell adhesion molecule family. Recently, we identified CADM1 to be a novel risk factor for venous thrombosis in a large, protein C deficient, thrombophilic family and showed, for the first time, the expression of CADM1 in endothelial cells (Hasstedt et al. in Blood 114:3084-3091, 2009). To further investigate its role in venous thrombosis, as well as other vasculopathies, we undertook a systematic confocal microscopic investigation for the presence of CADM1 in the vasculature of 28 different human tissues. Paraffin embedded tissue sections were dual immunostained with an antibody against CADM1, together with an antibody against either von Willebrand factor (to identify endothelial cells), or α-smooth muscle actin (to identify smooth muscle cells). The results showed that CADM1 was ubiquitously present in endothelial cells and smooth muscle cells in the vasculature from all 28 tissues, though its representation in the various classes of vessels was tissue dependent.

Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm.

Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores - ranging in emission from 435 to 665 nm - applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent.

Valves of the deep venous system: an overlooked risk factor.

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.

Imaging aspects of cardiovascular disease at the cell and molecular level.

Cell and molecular imaging has a long and distinguished history. Erythrocytes were visualized microscopically by van Leeuwenhoek in 1674, and microscope technology has evolved mightily since the first single-lens instruments, and now incorporates many types that do not use photons of light for image formation. The combination of these instruments with preparations stained with histochemical and immunohistochemical markers has revolutionized imaging by allowing the biochemical identification of components at subcellular resolution. The field of cardiovascular disease has benefited greatly from these advances for the characterization of disease etiologies. In this review, we will highlight and summarize the use of microscopy imaging systems, including light microscopy, electron microscopy, confocal scanning laser microscopy, laser scanning cytometry, laser microdissection, and atomic force microscopy in conjunction with a variety of histochemical techniques in studies aimed at understanding mechanisms underlying cardiovascular diseases at the cell and molecular level.

A novel dual staining method for identification of apoptotic cells reveals a modest apoptotic response in infarcted mouse myocardium.

Confocal scanning laser microscopy was used to investigate the myocardium of control C57BL/6 and plasminogen activator inhibitor 1 knockout (PAI-1KO) mice 3 days following persistent ligation of the left descending coronary artery. Paraffin sections taken from infarcted areas of the left ventricle were stained with antibodies recognizing cardiomyocytes, neutrophils, macrophages and apoptotic cells. In both animal groups, a strong neutrophil response was noted in the infarcted myocardium, with a large proportion of these cells also displaying staining for anti-alpha-sarcomeric actin in the PAI-1KO animals. Abundant macrophages were also identified in the infarcted regions of both animal groups, forming demonstrable streams at the border region in the C57BL/6 control animals. Surprisingly, only sparse cells from both animal groups were labeled with the apoptotic markers anti-cleaved caspase 3 antibody and anti-single stranded DNA antibody (following formamide treatment). A dual immunostaining protocol was developed to localize both of these apoptotic markers in the same cell. Again, only scattered cells were found displaying both markers in the zones of infarction, suggesting that 3 days of persistent ischemia results in a robust necrotic response, but only a very minor apoptotic response in this mouse model.

Group v secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice.

OBJECTIVE: Group V secretory phospholipase A2 (GV sPLA2) has been detected in both human and mouse atherosclerotic lesions. This enzyme has potent hydrolytic activity towards phosphatidylcholine-containing substrates, including lipoprotein particles. Numerous studies in vitro indicate that hydrolysis of high density lipoproteins (HDL) and low density lipoproteins (LDL) by GV sPLA2 leads to the formation of atherogenic particles and potentially proinflammatory lipid mediators. However, there is no direct evidence that this enzyme promotes atherogenic processes in vivo. METHODS AND RESULTS: We performed gain-of-function and loss-of-function studies to investigate the role of GV sPLA2 in atherogenesis in LDL receptor-deficient mice. Compared with control mice, animals overexpressing GV sPLA2 by retrovirus-mediated gene transfer had a 2.7 fold increase in lesion area in the ascending region of the aortic root. Increased atherosclerosis was associated with an increase in lesional collagen deposition in the same region. Mice deficient in bone marrow-derived GV sPLA2 had a 36% reduction in atherosclerosis in the aortic arch/thoracic aorta. CONCLUSIONS: Our data in mouse models provide the first in vivo evidence that GV sPLA2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease.

Quantitative analysis of atherosclerotic lesion composition in mice.

Comparative quantitation has become an increasingly desirable tool in determining compositional differences of aortic plaque lesion in transgenically altered mice. To this end, methodology has been developed to identify lipid, cellularity, collagen, and elastin components using traditional bright-field microscopy, fluorescence, and polarized light microscopy, employing both confocal and wide-field imaging systems. Subsequent imaging processing and analysis on the digitally captured images reveals differences in compositional components as influenced by diet, age and gender. This method can be expanded to employ a rich variety of histochemical and immunohistochemical staining protocols.

Attenuation of accumulation of neointimal lipid by pioglitazone in mice genetically deficient in insulin receptor substrate-2 and apolipoprotein E.

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE(-/-)) and in ApoE(-/-) mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2(+/-) ApoE(-/-) mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2(+/-) ApoE(-/-) mice. The extent of the aortic intima occupied by lesion was increased in the IRS2(+/-) ApoE(-/-) compared with ApoE(-/-) mice (79 +/- 3% compared with 68 +/- 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 +/- 6% to 30 +/- 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 +/- 1% to 19 +/- 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

Delineation of the evolution of compositional changes in atheroma.

In a previous manuscript, we described a method combining immunohistochemistry, confocal scanning laser microscopy, and computer-assisted image analysis for the determination of the composition of atherosclerotic plaques [Taatjes et al. (2000) Histochemistry and Cell Biology 113:161-173). We now present an enhanced technique, and its use for age- and gender-related comparative analysis of lesion composition in ApoE knockout mice. Cryosections from aorta were stained with oil red O to detect lipid, SYTOX Green to detect cellularity, and Picrosirius red to delineate collagen fibers. The stained sections were imaged by brightfield light microscopy, epifluorescence microscopy, and polarized light microscopy. Digital images were collected, processed to isolate the lesions, and subjected to computer-assisted image analysis. The average percentage of the vessel wall occupied by lesion increased 1.5-fold in animals from 10 to 20 weeks. Although the amount of lipid in the lesions increased in animals from 10 to 20 weeks, the percentage composition in the lesion remained constant because of the increase in lesion size. Average cellularity showed a modest decrease over the same interval. However, the percentage composition of plaque attributable to collagen increased 2.5-fold in 20-week-old female animals compared with that in males or females of 10 weeks of age and males of 20 weeks of age.