Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell-stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the up-regulation of a previous un-described downstream effector- αB-crystallin (CRYAB). Short interfering RNA-mediated knockdown of two major UPR proteins-IRE1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell-stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here we report that the molecular chaperone-CRYAB) was significantly increased and co-localized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells co-cultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling.

Preclinical studies have established tumor angiogenesis as a potential therapeutic target for breast cancer. However, there is an urgent need to either improve existing anti-angiogenic agents or devise new anti-angiogenic therapy for a subset of breast cancer patients with resistance to current anti-angiogenic regimes. The purpose of this study is to develop an anti-angiogenic therapeutic model for breast cancer by a combination of 1) siRNA-based therapy intratumorally delivered by self-complementary adeno-associated virus serotype 2 (scAAV2) vector to target tumor vasculature, and 2) non-invasive monitoring for tumor response to anti-angiogenic agents by serial photoacoustic imaging. We have reported previously that under the stress conditions caused by tumor microenvironment or/and anti-VEGF therapies, endothelial cells adopt the up-regulation of IRE1α/XBP-1 and ATF6. This in turn maintains VEGF intracrine signaling for endothelial cell survival. Here we identified that scAAV2 septuplet-Y-F mutant vector was able to transfect mice microvascular endothelial cells with high efficiency. scAAV2 septuplet-tyrosine mutant vectors encoding the siRNAs against IRE1α or XBP-1 or ATF6 significantly inhibited breast cancer-induced angiogenesis in vitro by, in part, inhibiting endothelial cell survival. Acoustic-resolution photoacoustic microscopy (ARPAM) can provide non-invasive, label-free, high resolution vascular imaging. Utilizing ARAM, we showed that intratumoral delivering the siRNAs against IRE1α or XBP-1 or ATF6 by scAAV2 septuplet-tyrosine mutant vector resulted in a significant decrease in tumor growth and tumor angiogenesis in breast cancer xenograft models. These data have generated a proof-to-concept model with important implications for the development of novel anti-angiogenic targeted therapies for patients with breast cancer.

Pericytes are distinctive regulators of vascular morphologenesis and function during vascular development and homeostasis. Pericytes have recently come into focus as implications of aberrant interactions between pericytes and endothelial cells in number of pathological angiogenesis conditions, including diabetic retinopathy and tumor angiogenesis. Pericyte dropout is a hallmark of early diabetic retinopathy. Abnormal angiopoietin (Ang)-Tie-2 signaling is one principal system participating in pericyte/endothelial cell dissociation during early stages of diabetic retinopathy. Angiopoietin 2 (Ang-2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularization and cause pericyte loss. Furthermore, high levels of VEGF synergize Ang-Tie-2 signaling during the development of diabetic retinopathy. An accelerated rate of clinical development Ang-Tie-2-manipulating drugs requests a better mechanistic understanding the connection between pericytes and Ang-Tie-2 systems both under normal and disease conditions. We summarize recent advances in pericyte study in conjunction with Ang-Tie-2 signaling and also discuss possible therapeutic strategies for diabetic retinopathy by targeting pericytes through manipulating Ang-Tie-2 signaling.

Supplementary Data from αB-Crystallin, an Effector of Unfolded Protein Response, Confers Anti-VEGF Resistance to Breast Cancer via Maintenance of Intracrine VEGF in Endothelial Cells