Search

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis., American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, National Science Foundation (U.S.), Massachusetts Institute of Technology. Presidential Fellowship

Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2 K270E -activated Gα relative to Gpa2 + Gα. Deletion of sck1 + or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2 + or Gpa2 K270E , suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2 K270E GDP-GTP exchange rate mutant, GTPase-defective Gpa2 R176H weakly binds Sck1 in the two-hybrid screen and a deletion of sck1 + in a Gpa2 R176H strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting sck1 + in a gpa2 Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow S. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.

Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.

While the counterselectable Schizosaccharomyces pombe ura4 + gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4 + confers 5FOA-resistant (5FOAR) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOAR mutants. Relative to the same approach using the homologous URA3 + gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOAR colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOAR strains carry mutations in either of two genes; ura4 + and ura5 +. We then cloned the S. pombe ura5 + orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5 + together with the S. pombe lys7 + gene. Using this doubly marked cassette to disrupt the sck1 + kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4 − and ura5 − mutants by screening 5FOAR colonies for the loss of the lys7 + marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOAR colonies in our studies arose from background ura4 − and ura5 − mutations.

Background: Epoxyeicosatrienoic acids (EETs) are lipid autacoids biosynthesized by cytochrome P450 epoxygenases and metabolized by soluble epoxide hydrolase (sEH) to less active dihydroxyeicosatrienoic acids (DHETs). EETs are autocrine and paracrine mediators of arachidonic acid-induced vasorelaxation in the cardiovascular and renal systems. Thus, inhibitors of sEH, which raise endogenous EET levels, are being considered for long-term use in hypertension, diabetes, stroke and other medical conditions. 20-hydroxeicosatetraenoic acid (20-HETE) is a small molecule biosynthesized by cytochrome P450 ω-hydroxylases. We recently demonstrated that EETs stimulate multiorgan and lymph node metastasis, a major cause of disease dissemination and death. However, the mechanism of EETs in tumor lymphangiogenesis is unknown. Thus, we hypothesize that EETs stimulate lymph node metastasis via tumor lymphangiogenesis. Methods and Results: Using two murine tumor lines transfected with VEGFC, B16F10-VEGFC melanoma and T241-VEGFC fibrosarcoma tumors, we demonstrate that EETs stimulate primary tumor growth via tumor lymphangiogenesis. Systemic administration of 14,15-EET and/or 20-HETE by osmotic minipump accelerated primary B16F10-VEGFC melanoma and T241-VEGFC fibrosarcoma tumor growth. Inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and also promoted tumor growth: sEH inhibitors (TUPS and AUCB) stimulated primary B16F10-VEGFC tumor growth. Conversely, systemic administration of a 20-HETE antagonist (HET0016) and/or EET antagonist (14,15-EEZE) regressed established primary B16F10-VEGFC tumors. 14,15-EET and 11,12-EET stimulated lymphatic endothelial cell proliferation, viability and migration. To determine whether tumor lymphangiogenesis contributes to the increase in B16F10-VEGFC tumor growth in 14,15-EET treated mice, we analyzed tumors for the specific lymphangiogenesis markers podoplanin and LYVE-1. Immunohistochemistry studies revealed an increase in podoplanin-positive and LYVE-1-positive vessels in B16F10-VEGFC tumors treated with 14,15-EET compared to vehicle treatment. 14,15-EET, 11,12-EET, and 20-HETE increased the production of VEGFC by B16F10-VEGFC and T241 fibrosarcoma tumor cells. Using a well-established model in which resection of a primary tumor (Lewis lung carcinoma) reproducibly stimulates the development of distant metastases 14-17 days after resection, we investigated whether EET antagonists inhibit spontaneous metastatic growth. The EET antagonist 14,15-EEZE and/or siRNA to VEGFR3 inhibited lung metastasis and the combination showed additive inhibition. Conclusion: EETs and 20-HETE stimulate tumor lymphangogenesis, offering a mechanistic rationale for using EET and 20-HETE antagonists as novel anti-cancer therapeutics to inhibit lymph node metastasis and tumor lymphangiogenesis. Citation Format: Dayna K. Mudge, Karolina Serhan, Diane R. Bielenberg, Sung Hee Hwang, Bruce D. Hammock, Mark W. Kieran, Dipak Panigrahy. Control of tumor lymphangiogenesis by epoxyeicosanoids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4961. doi:10.1158/1538-7445.AM2013-4961