Identification of a 'snapshot' of co-expressed angiogenic markers in laser-dissected vessels from unstable carotid plaques with targeted arrays.

BACKGROUND/AIMS: Angiogenesis is a feature of the atherogenic process, with intimal neovascularisation arising from vessels in the adventitia, adjacent to a plaque. Immature, leaky blood vessels from unstable plaques proliferate abnormally and, being poorly invested with smooth muscle cells, may contribute to instability of the plaque by facilitation of inflammatory cell infiltration and haemorrhagic complications. METHODS: We used laser-capture microdissection to isolate angiogenic areas of the extracellular matrix (containing CD105/flt-1-positive, fragile thin-walled vessels) and non-angiogenic vascular areas (CD105-negative, with smooth muscle cell covering) of complicated endarterectomy plaques, and specifically designed angiogenesis-TaqMan real-time PCR microarrays to identify gene expression. RESULTS: Important pro-angiogenic components, including Notch-3, delta-like-4 (DLL4), Tie-2, angiopoietin-1 (Angio-1) and receptor for advanced glycation end products (RAGE), and one anti-angiogenic factor, endostatin, were up-regulated in these regions. Immunohistochemistry demonstrated localisation within intimal, active (CD105-positive) microvessels and co-localisation of Notch-3 and DLL4/Tie-2 and Angio-1 in the same vessels indicating multiple/synergistic signalling mechanisms associated with vessel development. CONCLUSION: These data, although providing only a snapshot of information, demonstrate that plaque vascularisation occurs in the presence of multiple angiogenically active factors. Knowledge of their combined effects could help in the formulation of novel therapeutics designed to stabilise or prevent their formation in the treatment of atherosclerosis.

B-cell translocation gene 2 is over-expressed in peri-infarct neurons after ischaemic stroke.

OBJECTIVES: Recovery from stroke is dependent on the survival of neurons in the dynamic peri-infarcted region. Although several markers of neuronal injury and apoptotic cell death have been described, administration of neuroprotective drugs directed at specific molecules has had limited success. A complete understanding of deregulated genes associated with neuronal death would be beneficial. Our previous microarray studies identified increased expression of a novel protein, the B-cell translocation gene 2 (BTG2), in infarcted regions. METHODS: We have used immunohistochemistry and Western blotting to examine the expression and localization of BTG2 in stroked brain tissue and immunofluorescent staining of human fetal brain neurons to determine if oxygen-glucose deprivation affected its expression. RESULTS: We show that BTG2 is strongly expressed in peri-infarcted and infarcted regions of brain tissue, localizing in neuronal nuclei and cytoplasm, whilst being absent or very weakly expressed in normal looking contralateral tissue. Exposure of human fetal brain neurons to oxygen-glucose deprivation also induced BTG2 expression in the cytoplasm and perinuclear regions of cells staining positive for propidium iodide (a marker of nuclear damage). CONCLUSIONS: BTG2 may be a modulator of cell survival and differentiation and could help to protect against cell death by inhibition of necrosis and/or apoptotic signalling pathways.

Receptor-mediated gene transfer vectors: progress towards genetic pharmaceuticals.

Although specific delivery to tissues and unique cell types in vivo has been demonstrated for many non-viral vectors, current methods are still inadequate for human applications, mainly because of limitations on their efficiencies. All the steps required for an efficient receptor-mediated gene transfer process may in principle be exploited to enhance targeted gene delivery. These steps are: DNA/vector binding, internalization, subcellular trafficking, vesicular escape, nuclear import, and unpacking either for transcription or other functions (i.e., antisense, RNA interference, etc.). The large variety of vector designs that are currently available, usually aimed at improving the efficiency of these steps, has complicated the evaluation of data obtained from specific derivatives of such vectors. The importance of the structure of the final vector and the consequences of design decisions at specific steps on the overall efficiency of the vector will be discussed in detail. We emphasize in this review that stability in serum and thus, proper bioavailability of vectors to their specific receptors may be the single greatest limiting factor on the overall gene transfer efficiency in vivo. We discuss current approaches to overcome the intrinsic instability of synthetic vectors in the blood. In this regard, a summary of the structural features of the vectors obtained from current protocols will be presented and their functional characteristics evaluated. Dissecting information on molecular conjugates obtained by such methodologies, when carefully evaluated, should provide important guidelines for the creation of effective, targeted and safe DNA therapeutics.

[Clinicopathological and morphometrical analysis of 5 cases from 4 families of fibronectin glomerulopathy].

Fibronectin glomerulopathy (FNG) is an inherited disease, characterized by massive fibronectin (FN) deposits in the glomeruli. We semiquantitatively analyzed glomerular lesions and their progression in 5 cases with FNG from 4 different families: a 4 year-old male, a 19 year-old female, a 27 year-old male, a 58 year-old male (the father of the former case) and a 75 year-old male. All subjects showed a 201 times higher value of mean glomerular-tuft area (GA) and a 2.0 times higher mean number of mesangial cells (No. of MC) relative to control (p < 0.001). Strong positive correlations were observed between GA and the No. of MC (r = 0.86, p < 0.001). The younger cases showed markedly higher value of GA and No. of MC than the older cases. Mean individual capillary luminal area was decreased in all but one case and the mean total capillary luminal area, which is roughly estimated as the glomerular filtration area, was less changed compared with the control. The number of capillary loops tended to increase, indicating elongation of the capillary loops. The fractional area of FN (%FN), collagen IV (%Coll. IV) and laminin (%Lam) were high in all cases except for the first Bx in the 27 year-old case. The %FN strongly correlated with %Coll. IV and %Lam (r = 0.86, r = 0.69, p < 0.001, respectively). Serial biopsy (Bx) with a 10 year-interval was examined in the 27 year-old case and his father: GA and No. of MC were increased 1.4 and 1.9 times in the son, compared with his first Bx (p < 0.001, respectively), while no change was observed in the father. The %FN, %Coll. IV and %Lam were significantly increased in their second Bx (p < 0.001). These results suggest that 1) enlargement of the glomeruli in FNG is caused by intraglomerular accumulation of FN, Coll. IV and Lam and proliferated mesangial cells, 2) there is a strong influence from the aging factor, and 3) compensatory elongation of the loops (increase in the capillary luminal area) may maintain the glomerular filtration.

[The antero-posterior diameter of the cervical spinal canal in the ossification of the posterior longitudinal ligament (author's transl)].

1. The antero-posterior diameter (APD-Fig. 1A) of the cervical spinal canals in 38 cases of symptomatic ossification of the posterior longitudinal ligament (OPLL) and 29 cases of asymptomatic OPLL was measured for each vertebra, and the thecoperiosteal diameter (TPD-Fig. 1B) of the cervical spinal canals in the same cases was measured. 2. The APD in the cases of symptomatic OPLL was found to be significantly smaller than those of asymptomatic OPLL. 3. The TPD in symptomatic OPLL was also found to be more significantly smaller than those of asymptomatic OPLL. Measurements of 9 mm or less were considered liable to be associated with cord compression. 4. The growing rate of the ossification was calculated in 13 cases of symptomatic OPLL which we have followed up radiologically for more than 2 years. The growing rate varied considerably from case to case. The average growing rate per one year was 4.07 mm in axial length and 0.67 mm in thickness on antero- posterior direction. 5. The incidence of OPLL was examined.