Tetiana Zaichuk

Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium–derived factor

Natural inhibitors of angiogenesis are able to block pathological neovascularization without harming the preexisting vasculature. Here we show that two such inhibitors, thrombospondin-1 and pigment epithelium–derived factor, derive specificity for remodeling vessels from their dependence on Fas/Fas ligand (FasL)-mediated apoptosis to block angiogenesis. Both inhibitors upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas/CD95 receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-generated FasL. The anti-angiogenic activity of thrombospondin-1 and pigment epithelium–derived factor both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis. This example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis provides one explanation for the ability of inhibitors to select remodeling capillaries for destruction.

Id1 regulates angiogenesis through transcriptional repression of thrombospondin-1

Id proteins are helix-loop-helix transcription factors that regulate tumor angiogenesis. In order to identify downstream effectors of Id1 involved in the regulation of angiogenesis, we performed PCR-select subtractive hybridization on wild-type and Id1 knockout mouse embryo fibroblasts (MEFs). Here we demonstrate that thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis, is a target of transcriptional repression by Id1. We also show that Id1-null MEFs secrete an inhibitor of endothelial cell migration, which is completely inactivated by depletion of TSP-1. Furthermore, in vivo studies revealed decreased neovascularization in matrigel assays in Id1-null mice compared to their wild-type littermates. This decrease was completely reversed by a TSP-1 neutralizing antibody. We conclude that TSP-1 is a major target for Id1 effects on angiogenesis.

Bacteria-induced uroplakin signaling mediates bladder response to infection.

Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli.

Nuclear Factor of Activated T Cells Balances Angiogenesis Activation and Inhibition

It has been demonstrated that vascular endothelial cell growth factor (VEGF) induction of angiogenesis requires activation of the nuclear factor of activated T cells (NFAT). We show that NFATc2 is also activated by basic fibroblast growth factor and blocked by the inhibitor of angiogenesis pigment epithelial–derived factor (PEDF). This suggests a pivotal role for this transcription factor as a convergence point between stimulatory and inhibitory signals in the regulation of angiogenesis. We identified c-Jun NH2-terminal kinases (JNKs) as essential upstream regulators of NFAT activity in angiogenesis. We distinguished JNK-2 as responsible for NFATc2 cytoplasmic retention by PEDF and JNK-1 and JNK-2 as mediators of PEDF-driven NFAT nuclear export. We identified a novel NFAT target, caspase-8 inhibitor cellular Fas-associated death domain–like interleukin 1β–converting enzyme inhibitory protein (c-FLIP), whose expression was coregulated by VEGF and PEDF. Chromatin immunoprecipitation showed VEGF-dependent increase of NFATc2 binding to the c-FLIP promoter in vivo, which was attenuated by PEDF. We propose that one possible mechanism of concerted angiogenesis regulation by activators and inhibitors may be modulation of the endothelial cell apoptosis via c-FLIP controlled by NFAT and its upstream regulator JNK.

NF-κB balances vascular regression and angiogenesis via chromatin remodeling and NFAT displacement

Extracellular factors control the angiogenic switch in endothelial cells (ECs) via competing survival and apoptotic pathways. Previously, we showed that proangiogenic and antiangiogenic factors target the same signaling molecules, which thereby become pivots of angiogenic balance. Here we show that in remodeling endothelium (ECs and EC precursors) natural angiogenic inhibitors enhance nuclear factor-kappaB (NF-kappaB) DNA binding, which is critical for antiangiogenesis, and that blocking the NF-kappaB pathway abolishes multiple antiangiogenic events in vitro and in vivo. NF-kappaB induction by antiangiogenic molecules has a dual effect on transcription. NF-kappaB acts as an activator of proapoptotic FasL and as a repressor of prosurvival cFLIP. On the FasL promoter, NF-kappaB increases the recruitment of HAT p300 and acetylated histones H3 and H4. Conversely, on cFLIP promoter, NF-kappaB increases histone deacetylase 1 (HDAC1), decreases p300 and histone acetylation, and reduces the recruitment of NFAT, a transcription factor critical for cFLIP expression. Finally, we found a biphasic effect, when HDAC inhibitors (HDACi) were used to test the dependence of pigment epithelial-derived factor activity on histone acetylation. The cooperative effect seen at low doses switches to antagonistic as the concentrations increase. Our study defines an interactive transcriptional network underlying angiogenic balance and points to HDACi as tools to manipulate the angiogenic switch.

In vivo upregulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metronomic doxorubicin treatment.

Antiangiogenic thrombospondin-1 (TSP1) induces endothelial cell death via a CD95-mediated cascade. We used this signaling pathway, where CD95/Fas is a rate-limiting intermediate, as a target to optimize the efficacy of TSP1 active peptide, DI-TSP. Like TSP1, DI-TSP upregulated endothelial CD95L in vivo. To modulate CD95 levels, we chose chemotherapy agent doxorubicin (DXR). DXR caused sustained upregulation of CD95 in the activated endothelium at 1/100 of the maximal tolerated dose. DI-TSP and DXR synergistically induced endothelial apoptosis in vitro, and in vivo, in developing murine vessels. Fas decoy, TSP1 receptor antibody and Pifithrin, a p53 inhibitor, severely decreased apoptosis and restored angiogenesis by DXR–DI-TSP combination, evidencing critical roles of CD95 and TSP1. Combined therapy synergistically blocked neovascularization and progression of the bladder and prostate carcinoma. Such informed design of a complex antiangiogenic therapy based on the rate-limiting molecular targets is a novel concept, which may yield new approaches to cancer treatment.

Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning

Significance This paper presents a high-resolution map of nucleosome positions of Schizosaccharomyces pombe. Comparison with the high-resolution map of Saccharomyces cerevisiae has provided important insights into nucleosome–DNA interaction and mechanistic variation in nucleosome positioning. The map shows a preponderance of linker lengths centered on 4/5 bp, placing adjacent nucleosomes on opposite faces of the DNA. The dinucleotide signature for nucleosome positioning is equally strong in exons as in introns. Unexpectedly, S. pombe nucleosomes have a preference for A/T residues surrounding the nucleosome dyad, and nucleosome occupancy is very mildly affected by poly (dA-dT) tracts. The preference for A/T residues around the dyad and its role in nucleosome phasing suggest a coevolution of genomes with the DNA binding preferences of nucleosomes across species. Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the −1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.

Internalization and retention of nanodiamonds by MCF-7 cells in vitro: a potential drug delivery platform.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #2134 Background: Selective uptake and prolonged retention of drugs will facilitate pharmacotherapy to the breast. This may be accomplished by complexing of drugs to carbon-based nanodiamond (ND) particles, 2-8nm size. ND particles are attractive for this purpose because they are inert and non-toxic.We tested the uptake and retention of ND particles by the estrogen receptor positive breast cancer cell line MCF-7. Methods: Polylysine coated ND were labeled with fluorescein isothiocyanate (FITC-ND), and examined by UV-visible spectra and found to be stable. MCF-7 cells were cultured on Lab-Tek II chamber slides using DMEM with 10% FCS, insulin, sodium pyruvate, L-glutamine, and Pen/Strep. To assess the efficiency of ND internalization, we added 10ug/ml ND-FITC in complete media to the chamber slides and incubated for 30 min, 1 hr, 3 hr, 4 hr, 17 hr, and 24 hr. FITC-ND was withdrawn from the medium after an initial exposure of 17 hr by washing of cells, and retention of fluorescence was measured at intervals, up to 3 days after withdrawal. To assess the ability of MCF-7 cells to take up NDs under low metabolic conditions MCF-7 cells were incubated in serum-free medium. As a control soluble FITC alone was incubated at 10 ug/ml with MCF-7 cells. Cells were imaged on the laser confocal microscope and FITC detection at 488nm was analyzed by three dimensional Z stack imaging. Results: Fluorescence was initially associated with the membrane surface, and later was visualized in the cytoplasm and in the perinuclear zone. Cellular uptake of FITC and ND-FITC occurred at 30 minutes and reached a maximum at 17 hr of incubation. After washing of cells, the retention studies showed little decrease in the intensity of the ND-FITC signal within the cells up to 96 hours. The FITC molecule was readily released from the cells by passive diffusion within 24 hours while NDs appeared to be compartmentalized in cytoplasmic vesicles. ![][1] Conclusions: Internalization of ND particles within the cell cytoplasm occurs within a relatively short time, and the ND particles are retained for at least 96 hours following removal from the culture medium. This prolonged retention is likely to be an advantage when considering novel routes of drug delivery to the breast, such as transdermal or intraductal approaches. Funded by Avon. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2134. [1]: /embed/graphic-1.gif