Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin

AbstractOsteopontin (OPN) is a secreted adhesive glycoprotein with a gly-arg-gly-asp-ser (GRGDS) cell binding domain. Several independent studies have suggested that OPN functions in tumor growth and metastasis, and one likely possibility is that OPN facilitates tumor invasion by promoting tumor cell migration. Consistent with this hypothesis, immobilized OPN promoted concentration-dependent tumor cell migration (i.e., haptotaxis) in modified Boyden chambers. In particular, cleavage of OPN by thrombin, which likely occurs in the tumor microenvironment, resulted in enhancement of OPNs haptotactic activity; and assays performed with purified preparations of the two individual OPN thrombin-cleavage fragments demonstrated that all detectable activity was associated with the GRGDS-containing fragment. In contrast to the activity of both OPN and its GRGDS-containing fragment in promoting haptotaxis, neither of these proteins in solution promoted chemotaxis, indicating that each must be immobilized to promote cell migration. In haptotaxis assays, antibody LM609 to integrin αvβ3 blocked > 80% cell migration towards the GRGDS-containing OPN fragment, implicating αvβ3 as its principal functional receptor. In comparison with equimolar quantities of other adhesive proteins, the GRGDS-containing OPN thrombin-cleavage fragment was not only > 2-fold more effective than intact OPN at promoting haptotaxis, but also > 8-fold and > 6-fold more effective than fibrinogen and vitronectin, respectively, indicating that this OPN fragment is highly active relative to other αvβ3 ligands

Akt promotes Endocardial-Mesenchyme Transition

Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1–DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury

Correction: Overexpression of MyrAkt1 in Endothelial Cells Leads to Erythropoietin- and BMP4-Independent Splenic Erythropoiesis in Mice.

[This corrects the article on p. e55095 in vol. 8.]

MyrAkt1 overexpression in endothelial cells in vivo leads to a transient increase in IgM^hiIgD^loCD24^intCD43^−/lo cells in the spleen.

<p>At 4 weeks off tetracycline, endothelial myrAkt1 mice displayed an increase in spleen weight (<b>A</b>) p<0.04. The total number of nucleated cells in the spleen increased transiently at 1–2 weeks but returned to normal at 3–4 weeks (<b>B</b>) p<0.03. The absolute numbers of CD45+ (<b>C</b>) and CD45+B220+ (<b>D</b>) cells displayed a transient increase at one week p<0.02. The relative percentage of IgM^loIgD^hi and IgM^hiIgD^lo cells was altered at one week (<b>E</b>) p<0.02. Absolute numbers of B220⁺IgM^hiIgD^loCD24^intCD43^−/lo cells were increased at one week (<b>F</b>) p<0.01. Representative plots of B220⁺ fractionation by IgM and IgD (<b>G</b>), and B220⁺IgM^hiIgD^lo fractionation by CD24 and CD43 (<b>H</b>).</p

Increased spleen weight in myrAkt1 overexpressing animals is due to increased erythropoiesis.

<p>An increased percentage of reticulocytes was found in the blood at 3–4 weeks (<b>A</b>) p<0.02. Staining against TER119 revealed an increase in red cell area in the spleens of endothelial myrAkt1 compared with control mice (<b>B</b>) (5x). The total number of TER119^hi cells increased by 2 weeks and remained elevated in the spleen (<b>C</b>) p<0.04. Further characterization using CD71 and TER119 showed no differences in erythroid differentiation in the spleen (<b>D</b>). The total number of TER119^hi cells in the bone marrow remained constant (<b>E</b>), but myrAkt1 animals displayed an increase in stages I and II with a complementary decrease in III (<b>F</b>) p<0.007.</p

Initial splenic erythropoiesis is not mediated by erythropoietin or BMP4.

<p>Addition of BMP4 to methylcellulose cultures did not increase the number of colonies compared with erythropoietin alone (<b>A</b>). Levels of BMP4 mRNA (<b>B</b>) and protein (<b>C</b>) were unchanged in the spleen. Splenic BMP4+ macrophages recruited in response to chemotherapeutically-induced anemia (<b>D</b>) were not found in spleens of control (<b>E</b>) or myrAkt1 (<b>F</b>) animals (40x). Plasma erythropoietin protein was not significantly elevated until 4 weeks off tetracycline (<b>E</b>) p<0.0005, and levels of erythropoietin mRNA were not elevated until 3 weeks off tetracycline in the kidney (<b>F</b>) or liver (<b>G</b>) p<0.05. Endothelial cells isolated from myrAkt1 kidneys displayed downregulation of phosphorylated and total Akt1 in the presence of tetracycline (<b>J</b>). TER119⁺ erythroblasts showed no significant decrease in Annexin V positivity in spleen or bone marrow (<b>K</b>), but TUNEL staining showed a transient decrease in apoptotic nuclei in myrAkt1 spleens at one week p<0.006 (<b>L)</b>.</p