Signalling and functions of angiopoietin-1 in vascular protection

Angiopoietin-1 (Ang1) has powerful vascular protective effects: suppressing plasma leakage, inhibiting vascular inflammation, and preventing endothelial death. Preclinical studies indicate that Ang1 may be therapeutically useful in a number of situations, including treatment of edema, endotoxemia, and transplant arteriosclerosis. However, the ligand has also been implicated in vessel remodeling, induction of angiogenesis and pulmonary hypertension, indicating that strategies to minimize any deleterious effects while optimizing vessel protection are likely to be needed. This review surveys the published data on vascular protective effects of Ang1 and highlights the therapeutic potential of this ligand, as well as possible limitations to its use. We also consider the data on Ang1 receptors and speculate on how to maximize therapeutic benefit by targeting the Tie receptors

Simultaneous targeting of VEGF-receptors 2 and 3 with immunoliposomes enhances therapeutic efficacy

<p><i>Background</i>: Tumor progression depends on angiogenesis. Vascular endothelial growth factor (VEGF) receptors (VEGFRs) are the main signal transducers that stimulate endothelial cell migration and vessel sprouting. At present, only VEGFR2 is targeted in the clinical practice.</p> <p><i>Purpose</i>: To develop new, anti-angiogenic nanoparticles (immunoliposomes, ILs), that redirect cytotoxic compounds to tumor-associated vascular cells.</p> <p><i>Methods</i>: Pegylated liposomal doxorubicin (PLD) was targeted against VEGFR2- and VEGFR3-expressing cells by inserting anti-VEGFR2 and/or anti-VEGFR3 antibody fragments into the lipid bilayer membrane of PLD. These constructs were tested <i>in vitro</i>, and <i>in vivo</i> in the Rip1Tag2 mouse model of human cancer.</p> <p><i>Results</i>: The combination treatment with anti-VEGFR2-ILs-dox and anti-VEGFR3-ILs-dox was superior to targeting only VEGFR2 cells and provides a highly efficient approach of depleting tumor-associated vasculature. This leads to tumor starvation and pronounced reduction of tumor burden.</p> <p><i>Conclusion</i>: Nanoparticles against VEGFR2 and −3 expressing tumor-associated endothelial cells represent a promising and novel anti-cancer strategy.</p

Induction of definitive endoderm (DE).

<p>A. Protocol for definitive endoderm differentiation. ActA = Activin A; NaB = Sodium butyrate. B. Immunocytochemistry for FOXA2, OCT4, SOX17 and VIM at day 5 (Scale bars 100 μm). C. Gene expression of ES-cell markers <i>OCT4</i>, <i>SOX2</i>; mesendodermal marker <i>BRA</i>; and mesenchymal <i>VIM</i> (mean ± SEM; n = 4). D. Upregulation of <i>FOXA2</i>, <i>SOX17</i>, <i>GSC</i> and <i>GATA4</i> during differentiation (mean ± SEM; n = 4). E. Cytometry analysis of DE-specific marker SOX17 (n = 5) and endodermal cell surface marker CXCR4 (n = 11) for day 5 cells (mean ± SD).</p

HPSC-derived organoids express intestinal marker genes at levels comparable to human intestinal epithelium.

<p>Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. A. Gene expression of intestinal differentiation markers <i>CDX2</i>, <i>KRT20</i>, <i>KLF5</i>, <i>IFABP2</i> and <i>HOXA13</i>. B. Expression of crypt area/intestinal stem cell markers <i>LGR5</i>, <i>ASCL2</i> and <i>SOX9</i>. Data represent the mean ± SEM of 3–9 samples. (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4; 2D, initial monolayer culture; HI, human intestinal epithelium samples.)</p

HPSC-derived organoids express intestinal marker genes at levels comparable to human intestinal epithelium.

<p>Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. A. Gene expression of intestinal differentiation markers <i>CDX2</i>, <i>KRT20</i>, <i>KLF5</i>, <i>IFABP2</i> and <i>HOXA13</i>. B. Expression of crypt area/intestinal stem cell markers <i>LGR5</i>, <i>ASCL2</i> and <i>SOX9</i>. Data represent the mean ± SEM of 3–9 samples. (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4; 2D, initial monolayer culture; HI, human intestinal epithelium samples.)</p

Inhibition of Fibroblast Growth Factor activity by both a genetic and pharmacological approach blocks mouse mammary 66c14 carcinoma tumor cell proliferation.

<p>(A) Expression pattern of FGF ligands and receptors mRNA in 66c14 carcinoma tumor cells was determined by standard RT-PCR. (B) Expression of the FGFR-2DN mRNA, receptor truncated for its intracellular tyrosine kinase domain, is only detected in mouse mammary 66c14 carcinoma cells stably transfected with the FGFR-2DN construct (clones C4 and C22) but not with the empty plasmid (Control). (C) Expression of FGFR-1 (black) and FGFR-2 mRNA (white) was determined by quantitative RT-PCR in 66c14 carcinoma cells transfected with control, FGFR-1, FGFR-2 or both FGFR-1 and FGFR-2 (FGFR-1/R-2) siRNA. (D) <i>In vitro</i> cell proliferation is inhibited in FGFR-2DN-expressing 66c14 cells compared to mock-transfected cells (Control). (E) 66c14 (Control) carcinoma cells proliferation <i>in vitro</i> is decreased when treated with increasing doses of the FGFR inhibitor, PD-173074. (F) Cell proliferation is decreased in FGFR-1, FGFR-2 and both FGFR-1/R-2 siRNA-transfected 66c14 carcinoma cells as compared to control siRNA condition. (*p<0.05 versus respective control group).</p

HPSC-derived hindgut cells form intestinal organoids in the absence of R-spondin1.

<p>Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. For CHIR+F cells see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134551#pone.0134551.s004" target="_blank">S4 Fig</a>. A. Timeline representing the differentiation process B. Light microscopic images showing budding (left) vs. bubble-like (right) morphologies (scale bars 500 μm). C. Number of organoids in tested conditions (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4). Error bars represent SD. D. Percentages of budding and bubble-like structures in the tested conditions. E. Hematoxylin-eosin (HE) stainings for organoid sections showing both tightly packed well-polarized structures (left panel) versus more loosely organized epithelium (righ panel). F. Immunohistochemistry for E-CADHERIN (E-CAD), VIMENTIN (VIM), CHROMOGRANIN A (CHRA), CYTOKERATIN 20 (KRT20), KI67 and MUCIN2 (MUC2) in organoids (scale bars 50 μm). G. Whole mount confocal immunocytochemistry for E-CAD and LYZ (magnification 40 x).</p

FGF4 suppresses unwanted hepatic differentiation.

<p>A. Expression of hepatic markers <i>AFP</i> (n = 4–15) and <i>ALB</i> (n = 4–14) during hindgut differentiation induced by different concentrations (ng/ml) of WNT3A (W) and FGF4 (F) and 3 μM CHIR99021 (mean ± SEM). B. Double immunocytochemistry for CDX2 and AFP at day 9. C. Expression levels of <i>AFP</i> and <i>ALB</i> during differentiation with varying concentrations of CHIR, (mean ± SEM; n = 3). D. Immunocytochemistry for VIM at day 9. Scale bars 100 μm.</p

FGFR signaling stimulates expression of lymphangiogenic genes in lymphatic endothelial cells.

<p>(A) VEGFR-2 and VEGFR-3 mRNA expression is increased in FGF-2-treated (white) human dermal microvascular lymphatic endothelial cells (HMVEC-dLys) as compared to control (untreated cells, black). (B) Netrin-1 (left panel), Prox1 (middle panel) and integrin α9 (right panel) mRNA expression is stimulated by FGF-2 (white) in HMVEC-dLys as compared to control (untreated cells, black). (*p<0.05 versus respective control group).</p

Schematic representation of FGFs-mediated tumor growth, metastasis and lymphangiogenesis.

<p>Tumor-secreted FGFs (red) play a central role in the induction of tumor metastasis, both directly by stimulating cancer cell proliferation and indirectly by upregulating VEGF-C expression in tumor cells (black). Tumor secreted FGFs might also induce directly lymphatic tube formation as previously demonstrated <i>in vitro</i> (dashed black line). Thus, tumor VEGF-C activates its VEGFR-2 and VEGFR-3 receptors on lymphatic endothelial cells, leading to lymphatic vessel formation. Tumor FGFs promote also pro-lymphatic gene expression (such as VEGFR-2, VEGFR-3, netrin-1, prox1 and integrin α9) in lymphatic endothelial cells (blue). Both tumor growth and lymphangiogenesis lead to tumor metastasis.</p