Background Pleiotrophin (PTN) is a heparin-binding growth factor with significant role(s) in tumour growth and angiogenesis. Although implication of endogenous PTN has been studied in several in vivomodels of tumour angiogenesis, its role in physiological angiogenesis has not been addressed. In the present work, we studied expression and functional significance of endogenous PTN during angiogenesis in the chicken embryo chorioallantoic membrane (CAM). Methods Using molecular, cellular and biochemical assays, we studied the expression pattern of PTN in CAM and human endothelial cells and its possible interaction with nucleolin (NCL). CAM cells were transfected with a pCDNA3.1 vector, empty (PC) or containing full length cDNA for PTN in antisense orientation (AS-PTN). Angiogenesis was estimated by measuring total vessel length. In vitro, human endothelial cells migration was studied by using a transwell assay, and down-regulation of NCL was performed by using a proper siRNA. Results Endogenous PTN mRNA and protein levels, as well as protein levels of its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) were maximal at early stages, when CAM angiogenesis is active. Application of AS-PTN onto CAM at days of active angiogenesis was not toxic to the tissue and led to dose-dependent decreased expression of endogenous PTN, ERK1/2 activity and angiogenesis. Interestingly, endogenous PTN was also immunolocalized at the endothelial cell nucleus, possibly through interaction with NCL, a protein that has a significant role in the nuclear translocation of many proteins. Down-regulation of NCL by siRNA in human endothelial cells significantly decreased nuclear PTN, verifying this hypothesis. Moreover, it led to abolishment of PTN-induced endothelial cell migration, suggesting, for the first time, that PTN-NCL interaction has a functional significance. Conclusions Expression of endogenous PTN correlates with and seems to be involved in angiogenesis of the chicken embryo CAM. Our data suggest that NCL may have a role, increasing the number of growth factors whose angiogenic/tumorigenic activities are mediated by NCL.

GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (K_i = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion induction, a steroid hormone that contributes to the pathology of postmyocardial infarction (MI) heart failure (HF), is mediated by both G_q/11proteins andβ‐arrestins, both of which couple to the AngII type 1 receptors (AT₁Rs) of adrenocortical zona glomerulosa (AZG) cells. Over the past several years, AngII analogs with increased selectivity (“bias”) towardβ‐arrestin‐dependent signaling at the AT₁R have been designed and described, starting with SII, the gold‐standardβ‐arrestin‐”biased” AngII analog. In this study, we examined the relative potencies of an extensive series of AngII peptide analogs at relative activation of G proteins versusβ‐arrestins by the AT₁R. The major structural difference of these peptides from SII was their varied substitutions at position 5, rather than position 4 of native AngII. Three of them were found biased forβ‐arrestin activation and extremely potent at stimulating aldosterone secretion in AZG cells in vitro, much more potent than SII in that regard. Finally, the most potent of these three ([Sar¹, Cys(Et)⁵, Leu⁸]‐AngII, CORET) was further examined in post‐MI rats progressing to HF and overexpressing adrenalβ‐arrestin1 in vivo. Consistent with the in vitro studies, CORET was found to exacerbate the post‐MI hyperaldosteronism, and, consequently, cardiac function of the post‐MI animals in vivo. Finally, our data suggest that increasing the size of position 5 of the AngII peptide sequence results in directly proportional increases in AT₁R‐dependentβ‐arrestin activation. These findings provide important insights for AT₁R pharmacology and future AngII‐targeted drug development.

We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.