Inhibition of human immunodeficiency virus type 1 infection in macrophages by an alpha-v integrin blocking antibody.

Macrophages are key cells for HIV infection and HIV spreading inside the organism. Macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (M-CSF). In the monocyte to macrophage differentiation process with M-CSF, alphav-integrins are upregulated concomitantly with the capacity of HIV to generate a productive virus infection. In the present study we show that an anti-alphav antibody, 17E6, inhibited HIV-1 infection of primary macrophages. The effect of 17E6 on HIV-1 BaL replication in acutely infected macrophages was dose-dependent, with a 50% effective concentration (EC50) of 17+/-2 microg/ml in the absence of cytotoxicity. Similarly, a monoclonal antibody targeting the alphavbeta6 integrin (14D9.F8) also inhibited HIV-1 BaL infection in this cell type. 17E6 reduced the detection of HIV-1 BaL proviral DNA in acutely infected macrophages, but was completely ineffective against HIV-1 BaL production in chronically infected macrophages, suggesting that 17E6 inhibited HIV infection at an early stage of the virus cycle. Finally, a small molecular weight antagonist of the alphavbeta6 integrin, EMD 409849, reduced HIV replication at subtoxic concentrations. Therefore, our results suggest that alphav-containing integrins could play a role in HIV replication in macrophages and suggest that small-molecular-weight compounds might interfere with HIV replication in macrophages through the interaction with alphav integrins.

Use of siRNAs and antisense oligonucleotides against survivin RNA to inhibit steps leading to tumor angiogenesis.

The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.

Synthesis of oligonucleotide-peptide conjugates carrying the c-myc peptide epitope as recognition system.

Oligonucleotide-peptide conjugates 1-3 were prepared by sequential addition of the appropriate Boc-protected amino acids, followed by nucleoside phosphoramidites in the same support. These molecules are designed to be used for triplex formation and for the directed assembly of nanomaterials. The structures of the desired oligonucleotide-peptide conjugates were confirmed by mass spectrometry on small oligonucleotide-peptide conjugates, by gel electrophoresis, and by hybridization with complementary oligonucleotides. Oligonucleotides carrying the c-myc peptide were specifically recognized by the anti-c-myc monoclonal antibody.

Synthesis and biological evaluation of novel indolocarbazoles with anti-angiogenic activity.

A novel series of indolocarbazoles were synthesized and their antiproliferative activity against HUVEC, LoVo, DLD-1 and ST-486 cell lines, was investigated. Those staurosporine analogs in which a substituted dimethylaminoalkoxy chain was attached to the indolic nitrogen showed interesting activity and selectivity with respect to HUVEC proliferation. The effect on capillary tube formation in 3-dimensional matrigel matrix was studied using the most active compounds. Evaluation of their in vivo anti-angiogenic activity in a murine Lewis lung cancer model was also analyzed.

Anti-migratory and anti-angiogenic effect of p16: a novel localization at membrane ruffles and lamellipodia in endothelial cells.

Recent evidence has established different functions for the tumor suppressor protein, p16(INK4A) aside from controlling the cell cycle. Here we report that cdk4/6 inhibition blocked both human umbilical vein endothelial cells (HUVEC) spreading on a vitronectin matrix and HUVEC migration on vitronectin. p16 can also act as an anti-angiogenic molecule in vitro since HUVEC and HMEC cells transfected with Ad-p16 or treated with Antennapedia p16 peptides are unable to differentiate on a Matrigel matrix. Both, p16, cyclin D1, cdk4 and cdk6 were immuno-colocalized with Ezrin, Rac, Vinculin, alphav-integrin, and FAK proteins in the ruffles and lamellipodia of migratory cells. Our results indicate that p16 is a key component of a new cytoplasmic pathway controlling angiogenesis of endothelial cells via the alphavbeta3-integrin-mediated migration.