Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the ␤-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments. INTRODUCTIONThe actin cytoskeleton plays an essential role in the structural changes that initially establish neuronal shape and subsequently contribute to the morphological differentiation of neurons. Actin filaments have been implicated in the initial sprouting of neurites, whereas microtubules strengthen and support the new extensions (Smith, 1988(Smith, , 1994.Tropomyosin (Tm) isoforms, integral components of actin microfilaments, form coiled-coil head-to-tail dimers that bind along the major groove of actin polymers (Phillips et al., 1979). Tms are derived from four highly conserved genes known as the ␣Tm fast , ␤Tm, ␥Tm (Tm5NM), and ␦Tm genes that, via alternative splicing, give rise to Ͼ40 isoforms (LeesMiller and Helfman, 1991;Dufour et al., 1998;Cooley and Bergtrom, 2001). Despite the well understood function of Tms in muscle where, together with the troponin complex, they regulate contraction in a calcium-dependent manner, little is known about their role in nonmuscle cells. In vitro studies have implicated Tms in the stabilization of the actin cytoskeleton by protecting actin filaments from the severing action of gelsolin (Ishikawa et al., 1989) and the depolymerizing action of ADF/cofilin (Bernstein and Bamburg, 1982). Gene transfection studies have demonstrated that tropomyosin isoforms can regulate the organization of actin filaments in transformed cells (Prasad et al., 1993;Boyd et al., 1995;Gimona et al., 1996) and the neuroepithelial cell line B35 (Bryce et al., 2003).In neurons, a strict repertoire of Tm isoforms is known to be expressed. These include TmBr3, Tm5a and Tm5b from the ␣Tm fast gene; multiple products from the ␥Tm gene; and Tm4 from the ␦Tm gene. Most interestingly, Tm isoforms have been previously shown to be spatially and temporally regulated, identifying distinct subcellular compartments. The Tm5a and Tm5b isoforms are enric...

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Neovascularization in cancer or retinopathy is driven by pathological changes that foster abnormal sprouting of endothelial cells. Mouse genetic studies indicate that the stress-induced small GTPase RhoB is dispensable for normal physiology but required for pathogenic angiogenesis. In diabetic retinopathy, retinopathy of prematurity (ROP) or age-related wet macular degeneration (AMD), progressive pathologic anatomic changes and ischemia foster neovascularization are characterized by abnormal sprouting of endothelial cells. This process is driven by the angiogenic growth factor VEGF, which induces and supports the formation of new blood vessels. While injectable biologics targeting VEGF have been used to treat these pathological conditions, many patients respond poorly, prompting interest in other types of mechanism-based therapy.Here we report the preclinical efficacy of a monoclonal antibody that specifically targets RhoB, a signaling molecule that is genetically dispensable for normal physiology but required for pathogenic retinal angiogenesis. In murine models of proliferative retinal angiogenesis or oxygen-induced retinopathy, administering a monoclonal RhoB antibody (7F7) was sufficient to block neoangiogenesis or avascular pathology, respectively. Our findings offer preclinical proof of concept for antibody targeting of RhoB to limit diabetic retinopathy, ROP or wet AMD and perhaps other diseases of neovasculogenesis such as hemangioma or hemangiosarcoma nonresponsive to existing therapies. K E Y W O R D S animal model, diabetic retinopathy, macular degeneration, monoclonal antibody, oxygen-induced retinopathy (OIR), retinopathy of prematurity (ROP), Rho GTPase

Angiopoeitin-2 (Ang2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. Combination of an anti-Ang2 antibody and aflibercept, a VEGF trap, has been shown to inhibit tumor growth and decrease tumor vascularity in mouse xenograft tumor models (Daly et al., Cancer Res (2013) 73(1):108). Multiple investigational anti-Ang2 antibody therapies are currently in clinical trials. LY3127804 is a humanized and engineered IgG4 isotype antibody that selectively binds to Ang2 with high affinity and neutralizes Ang2 induced phospho-Tie2. LY3127804 inhibits sprouting angiogenesis and increases pericyte coverage in a mouse developmental retinal angiogenesis model and in mice bearing PC3 xenograft tumors. Combination of LY3127804 and DC101, a potent anti-VEGFR2 antibody, exhibits enhanced efficacy when compared to monotherapy in multiple patient derived xenograft models including NSCLC and ovarian cancers. Anti-Ang2 antibody monotherapy alone resulted in marginal reduction of tumor growth and improved overall survival, while DC101monotherapy had greater reduction in tumor volume with no survival benefit in MDA-MB-231 breast orthotopic model. Combination of anti-Ang2 antibody with anti-VEGFR2 antibody shows reduction in tumor volume and improved overall survival. This robust pre-clinical evidence supports testing the combination of anti-Ang2 and anti-VEGFR2 antibodies in the clinic. LY3127804 is currently in Phase 1 clinical trials (NCT02597036) Citation Format: Sudhakar R. Chintharlapalli, Johnny E. Croy, Donmienne Leung, Damien Gerald, Jirong Lu, Philip W. Iversen, Linda N. Lee, Lysiane Huber, Jonathan Tetreault, Rowena Almonte-Baldonado, Jianghuai Xu, Bharathi Ramamurthy, Jennifer A. Pereira, Chi-Kin Chow, Axel-Rainer Hanauske, Volker Wacheck, Laura Benjamin, Ling Liu. LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3259.

<p>Figure S3: S1P1 is largely restricted to tumor vessels. Six different tumor models were assessed for S1P1 expression by immunofluorescence. In five of the models S1P1 expression was restricted to tumor vessels. The SK-Hep 1 tumor model was the only model in which some S1P1 expression was also seen on tumor cells in addition to on tumor vessels.</p>