We hypothesized that PDGF-B/PDGFR-␤-signaling is important in the cardiac contribution of epicardiumderived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf-b؊/؊ and Pdgfr-␤؊/؊ mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for ␣SMA, periostin, ephrinB2, EphB4, VEGFR-2, Dll1, and NCAM was performed on wild-type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest-related innervation deficits. This implies a role for PDGF-B/PDGFR-␤-signaling specifically in the contribution of these cell lineages to cardiac development. Developmental Dynamics 237:494 -503, 2008.

Enhanced epidermal growth factor receptor (EGFR) activity has been strongly linked to breast cancer progression and mediators of EGFR endocytosis may well be involved. We developed a semi-automated high-content fluorescence microscopy-based EGFR endocytosis screen to identify proteins that mediate EGFR endocytosis in human HBL100 breast cancer cells. Knockdown of 172 individual endocytosis and actin-regulatory genes with small interfering RNAs led to the identification of 14 genes of which the contribution to EGFR endocytosis in breast cancer is until now poorly defined, including DNAJC6, GDI2, FGD6, HAX1, NECAP2 and AnxA2. We show that depletion of the actin and endocytosis regulatory protein annexin A2 (AnxA2) in a panel of four triple negative breast cancer (TNBC) cell lines affected EGFR endocytosis. Depletion of AnxA2 in the aggressive and highly metastatic MDA-MB-231 TNBC cell line resulted in the inhibition of EGFR transport beyond the early endosomes. This inhibition coincided with enhanced epidermal growth factor (EGF)-induced cell migration and downstream signaling via c-Jun N-terminal kinase (JNK) and Akt. Moreover, AnxA2 knockdown increased lung metastasis formation in mice. The effect of AnxA2 knockdown on EGFR endocytosis in MDA-MB-231 was related to dephosphorylation/activation of the actin-severing protein cofilin, as re-expression of an inactive S3E-cofilin mutant, but not an active S3A-cofilin mutant, re-established EGFR endocytosis to control levels. Together, our data provide evidence for AnxA2 as a mediator of EGFR endocytosis and signaling in breast cancer via regulation of cofilin activation.

No abstract

The need for noninvasive imaging to distinguish stable from vulnerable atherosclerotic plaques is evident. Activated macrophages play a role in atherosclerosis and express folate receptor folate receptor β (FR-β). The feasibility of folate targeting to detect atherosclerosis was demonstrated in human and mouse plaques, and it was suggested that molecular imaging of FR-β through folate conjugates might be a specific marker for plaque vulnerability. However, these studies did not allow differentiation between stable and vulnerable atherosclerotic plaques. We investigated the feasibility of a folate-based radiopharmaceutical (111)In-EC0800) with high-resolution animal single-photon emission computed tomography/computed tomography (SPECT/CT) to differentiate between stable and vulnerable atherosclerotic plaques in apolipoprotein E(−/−) mice in which we can induce plaques with the characteristics of stable and vulnerable plaques by placing a flow-modifying cast around the common carotid artery. Both plaques showed (111)In-EC0800 uptake, with higher uptake in the vulnerable plaque. However, the vulnerable plaque was larger than the stable plaque. Therefore, we determined tracer uptake per plaque volume and demonstrated higher accumulation of (111)In-EC0800 in the stable plaque normalized to plaque volume. Our data show that (111)In-EC0800 is not a clear-cut marker for the detection of vulnerable plaques but detects both stable and vulnerable atherosclerotic plaques in a mouse model of atherosclerosis.