The onset of angiogenesis in cancer often involves down-regulation of endogenous angiogenesis inhibitors, of which thrombospondin-1 (TSP-1) is a paradigm. As this effect is thought to occur under the influence of transforming genetic lesions (e.g., expression of the mutant ras oncogene), its nature is regarded as intrinsic to cancer cells themselves. Here, we show that ras-transformed cancer cells can also induce TSP-1 down-regulation in their adjacent nontransformed stromal fibroblasts, but not in endothelial cells, in a paracrine and distance-dependent manner. Indeed, several H-ras-expressing fibrosarcoma (528ras1, B6ras, and NIH3T3Ras) and carcinoma (DLD-1 and IEC18Ras3) cells were found to release soluble factors capable of suppressing TSP-1 protein, mRNA, and promoter activity in nontumorigenic, immortalized dermal fibroblastic cell lines in culture (e.g., in fibroblasts expressing enhanced green fluorescent protein/TSP-1 reporter). This effect was abrogated in Id1−/− fibroblasts. At least two low molecular weight (<3 kDa), heat-labile, and trypsin-resistant mediators of TSP-1 suppression were found to be released from 528ras1 cells. Their effects on normal fibroblasts were inhibited (albeit to different extents) by pertussis toxin and, in one case, by dimethylsphingosine, none of which affected TSP-1 expression by 528ras1 cells. Collectively, our study suggests that the effect of mutant ras on tumor neovascularization is not limited to changes in angiogenic properties of cancer cells themselves. Rather, mutant ras, through a different signaling mechanism, may modulate the properties of the adjacent normal stroma, thus eliciting a proangiogenic field effect.

Percutaneous coronary intervention has become the most common and widely implemented method of heart revascularization. However, the development of restenosis remains the major limitation of this method. Photodynamic therapy (PDT) recently emerged as a new and promising method for the prevention of arterial restenosis. Here the efficacy of chlorin e6 in PDT was investigated in vitro using human vascular smooth muscle cells (TG/HA-VSMCs) as one of the cell types crucial in the development of restenosis. PDT-induced cell death was studied on many levels, including annexin V staining, measurement of the generation reactive oxygen species (ROS) and caspase-3 activity, and assessment of changes in mitochondrial membrane potential and fragmentation of DNA. Photosensitization of TG/HA-VSMCs with a 170 μM of chlorin e6 and subsequent illumination with the light of a 672-nm diode laser (2 J/cm²) resulted in the generation of ROS, a decrease in cell membrane polarization, caspase-3 activation, as well as DNA fragmentation. Interestingly, the latter two apoptotic events could not be observed in photosensitized and illuminated NIH3T3 fibroblasts, suggesting different outcomes of the model of PDT in various types of cells. The results obtained with human VSMCs show that chlorin e6 may be useful in the PDT of aerial restenosis, but its efficacy still needs to be established in an animal model.

Gadolinium-doped nanoparticles (NPs) are regarded as promising luminescent probes. In this report, we studied details of toxicity mechanism of low doses of NaGdF₄-based fluorescent nanoparticles in activated RAW264.7, J774A.1 macrophages. These cell lines were specifically sensitive to the treatment with nanoparticles. Using nanoparticles of three different sizes, but with a uniform zeta potential (about −11 mV), we observed rapid uptake of NPs by the cells, resulting in the increased lysosomal compartment and subsequent superoxide induction along with a decrease in mitochondrial potential, indicating the impairment of mitochondrial homeostasis. At the molecular level, this led to upregulation of proapoptotic Bax and downregulation of anti-apoptotic Bcl-2, which triggered the apoptosis with phosphatidylserine externalization, caspase-3 activation and DNA fragmentation. We provide a time frame of the toxicity process by presenting data from different time points. These effects were present regardless of the size of nanoparticles. Moreover, despite the stability of NaGdF₄ nanoparticles at low pH, we identified cell acidification as an essential prerequisite of cytotoxic reaction using acidification inhibitors (NH₄Cl or Bafilomycin A1). Therefore, approaching the evaluation of the biocompatibility of such materials, one should keep in mind that toxicity could be revealed only in specific cells. On the other hand, designing gadolinium-doped NPs with increased resistance to harsh conditions of activated macrophage phagolysosomes should prevent NP decomposition, concurrent gadolinium release, and thus the elimination of its toxicity.