E-selectin-targeted copolymer reduces atherosclerotic lesions, adverse cardiac remodeling, and dysfunction.

Endothelial activation with up-regulation of E-selectin adhesion molecules mediates leukocyte rolling along the vascular wall and controls inflammation in many diseases including atherosclerosis and heart failure. Therefore, we aimed to test the hypothesis that inhibition of E-selectin-mediated interactions by a new E-selectin-targeted copolymer could inhibit the progression of atherosclerosis. To target E-selectin on activated endothelium, we developed a new N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer with or without dexamethasone (Dex) (designated P-(Esbp)-Dex and P-Esbp, respectively). To determine the effect of P-(Esbp)-Dex and P-Esbp on atherosclerosis, we allocated ApoE (-/-) mice on a high fat diet, to weekly intra-peritoneal injections of either 1) P-Esbp; 2) P-(Esbp)-Dex; 3) free Dex (1 mg/kg) or 4) saline, for four weeks. Aortic atherosclerosis and left ventricular (LV) remodeling and function were assessed by serial ultrasound studies and histology. Monocytes and macrophages were characterized by flow cytometry. After four weeks of treatment, P-Esbp effectively targeted aortic atherosclerotic lesions. Both P-Esbp and P-(Esbp)-Dex reduced wall thickening of the ascending aortas. However, only the drug-free copolymer (P-Esbp) significantly decreased the areas of necrotic core in the plaques and switched spleen macrophages toward an anti-inflammatory (M2) phenotype. Furthermore, P-Esbp attenuated adverse LV remodeling and dysfunction in ApoE (-/-) mice. In summary, P-Esbp copolymer targets activated endothelial cells, regresses and stabilizes atherosclerotic plaques, and prevents adverse LV remodeling and dysfunction in ApoE (-/-) mice. Our results suggest a new, drug-free macromolecular therapy to treat vascular inflammation.

Assessing the therapeutic efficacy of VEGFR-1-targeted polymer drug conjugates in mouse tumor models.

Polymer-drug conjugates that can actively target the tumor vasculature have emerged as an attractive technology for improving the therapeutic efficacy of cytotoxic drugs. We have recently provided, for the first time, in vivo evidence showing the significant advantage of the E-selectin-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin conjugate, P-(Esbp)-DOX, in inhibiting primary tumor growth and preventing the formation and development of cancer metastases. Here, we describe the design of a vascular endothelial growth factor receptor (VEGFR)-1-targeted HPMA copolymer-DOX conjugate (P-(F56)-DOX) that can actively and simultaneously target different cell types in the tumor microenvironment, such as endothelial cells (ECs), bone marrow-derived cells and many human cancer cells of diverse tumor origin. The VEGFR-1-targeted copolymer was tested for its binding, internalization and in vitro cytotoxicity in ECs (bEnd.3 and cEND cells) and cancer cells (B16-F10, 3LL and HT29). The in vivo anti-cancer activity of P-(F56)-DOX was then tested in two tumor-bearing mice (TBM) models (i.e., primary Lewis lung carcinoma (3LL) tumors and B16-F10 melanoma pulmonary metastases), relative to that of the E-selectin-targeted system (P-(Esbp)-DOX) that solely targets ECs. Our results indicate that the binding and internalization profiles of the VEGFR-1-targeted copolymer were superior towards ECs as compared to cancer cells and correlated well to the level of VEGFR-1 expression in cells. Accordingly, the VEGFR-1-targeted copolymer (P-(F56)-DOX) was more toxic towards bEnd.3 cells than to cancer cells, and exhibited significantly higher cytotoxicity than did the non-targeted control copolymer. P-(F56)-DOX inhibited 3LL tumor growth and significantly prolonged the survival of mice with B16-F10 pulmonary metastases. When compared to a system that actively targets only tumor vascular ECs, P-(F56)-DOX and P-(Esbp)-DOX exhibited comparable efficacy in slowing the growth of primary 3LL tumors and prolonging the survival of these mice. Still, P-(Esbp)-DOX had more pronounced anti-tumor activity in mice bearing B16-F10 lung metastases after a single intravenous injection, at an equivalent DOX dose. Overall, our results indicate that the VEGFR-1- and E-selectin-targeted drug delivery systems evaluated here show enhanced anti-cancer activity, and prolonged the survival of mice after a single intravenous injection. This is thus the first study comparing the anti-tumor activity of VEGFR-1- and E-selectin-targeted polymer drug conjugates in the same TBM models at an equivalent drug dose.

Oligomerization of the mitochondrial protein voltage-dependent anion channel is coupled to the induction of apoptosis.

Accumulating evidence implicates that the voltage-dependent anion channel (VDAC) functions in mitochondrion-mediated apoptosis and as a critical player in the release of apoptogenic proteins, such as cytochrome c, triggering caspase activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c-conducting channel remain unknown. Here the relationship between VDAC oligomerization and the induction of apoptosis was examined. We demonstrated that apoptosis induction by various stimuli was accompanied by highly increased VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using bioluminescence resonance energy transfer technology. VDAC oligomerization was induced in all cell types and with all apoptosis inducers used, including staurosporine, curcumin, As(2)O(3), etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-α), H(2)O(2), and UV irradiation, all acting through different mechanisms yet all involving mitochondria. Moreover, correlation between the levels of VDAC oligomerization and apoptosis was observed. Furthermore, the apoptosis inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited VDAC oligomerization. Finally, a caspase inhibitor had no effect on VDAC oligomerization and cytochrome c release. We propose that VDAC oligomerization is involved in mitochondrion-mediated apoptosis and may represent a general mechanism common to numerous apoptogens acting via different initiating cascades. Thus, targeting the oligomeric status of VDAC, and hence apoptosis, offers a therapeutic strategy for combating cancers and neurodegenerative diseases.

Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c, AIF and Smac/Diablo.

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2 or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.