VEGF overexpression cannot rescue angiogenic deficit.

<p>A) Extensor digitorum longus muscle VEGF expression in wildtype and MUC1-VEGF mice. MUC1-VEGF human VEGF (•) plus mouse VEGF (○) resulted in a 45% increase in total content (▪) <i>vs.</i> wildtype. B) MUC1-VEGF mice had higher initial C∶F than wildtype. Capillary sprouting with or without platelets was no different from wildtype. *<i>P</i><0.05 <i>vs.</i> C57 BL/6 controls, +<i>P</i><0.05 <i>vs.</i> contralateral.</p

<i>In Vivo</i> Evidence for Platelet-Induced Physiological Angiogenesis by a COX Driven Mechanism

<div><p>We sought to determine a role for platelets in <i>in vivo</i> angiogenesis, quantified by changes in the capillary to fibre ratio (C∶F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C∶F (1.42±0.02 <i>vs.</i> contralateral 1.29±0.02, <i>P</i><0.001) that was abolished by platelet depletion, while the significant C∶F increase caused by splitting (1.40±0.03 <i>vs.</i> control 1.28±0.03, <i>P</i><0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation <i>via</i> collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis <i>in vivo</i>, likely <i>via</i> COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.</p></div

Granulocytes/monocytes do not mediate endothelial sprouting.

<p>A) Representative whole blood smears showing lymphocytes (black arrows) and granulocytes/monocytes (white arrow) were visualised in control animals. B) Following i.p. administration of anti-Ly6G/6C antibody only lymphocytes were readily visualised at 4, 24, and 48 h. C, Granulocyte/monocyte depletion did not alter the endothelial sprouting response normally observed (<i>P</i><0.01).</p

Angiogenesis is independent of collagen-induced platelet activation.

<p>A) Anti-GPVI resulted in ∼70% GPVI shedding. Shaded grey peak, GPVI shed; clear black peak, control; clear grey peak, GPVI shed IgG expression. B) GPVI shedding did not alter capillary sprouting, with significantly increased C∶F. *<i>P</i><0.05 <i>vs.</i> contralateral.</p

Platelet depletion differentially affects skeletal muscle angiogenesis.

<p>A) Capillary sprouting produced a significant capillary to fibre ratio (C∶F) increase, abolished by platelet depletion following i.p. injection of anti-GPIbα(denoted by ‘-’). Depletion did not affect C∶F of untreated or contralateral limbs. B) Longitudinal splitting caused a significant increase in C∶F which was unaffected by platelet depletion. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs.</i> untreated. C) Detail of representative images of lectin-stained mouse muscle cross-section showing fibres (asterisks) and capillaries (arrows) for extirpation with (i) or without (ii) platelets, and prazosin with (iii) or without (iv) platelets.</p

Platelets differentially affect angiogenesis <i>via</i> COX signalling.

<p>A) Dual clopidogrel/ASA regimen inhibited sprouting angiogenesis. Single regimens identified ASA as the active agent, with clopidogrel unable to alter the angiogenic response. B) Longitudinal splitting was unaffected by ASA or lower dose (LD)-ASA. C) PGF1α dropped significantly with overload + LD-ASA. D) Induction of longitudinal splitting did not alter PGF1α levels. *<i>P</i><0.05, **<i>P</i><0.01 between columns (A), or <i>vs.</i> untreated controls (B).</p

Platelet depletion did not affect proliferation of capillary-associated cells or myocytes.

<p>A) The total number of proliferating cells increased slightly in tissue after induction of capillary sprouting, but was not affected by platelet depletion. B) Capillary-associated cell proliferation was unaffected by platelet depletion. C) The increase in proliferating cell number with sprouting results from increased interstitial cell proliferation. D) The absence of platelets did not impair the proliferation of myocytes following muscle overload. E) BrdU labelling was performed in conjunction with rhodamine <i>Griffonia simplicifolia</i> lectin-1 staining (bottom left panel) for visualisation of vasculature, and DAPI (top right panel) to ensure BrdU staining observed was localised to nuclei. A merge (bottom right panel) of the three other panels with three BrdU-labelled interstitial cells (white arrows) is shown. Imaging was performed with fluorescent light microscopy as described in the methods at ×40 magnification. *<i>P</i><0.05 <i>vs.</i> contralateral.</p

Different forms of angiogenesis and experimental protocols.

<p>A) Mechanisms of capillary sprouting and longitudinal splitting. B) Extirpation induced endothelial sprouting, with anti-GPIbα, anti-Ly6G/6C or anti-GPVI administration 24 h after surgery. C) Prazosin induced longitudinal splitting with anti-GPIbα. Sampling always occurred on the eighth day.</p

(A) Effect of PP2 on PKCδ tyrosine phosphorylation.

<p>Human washed platelets were treated with 0.1 or 1 U/ml thrombin for 1 min in the presence or absence of PP2 (10 µM, 5 min). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (B) Effect of PP2 on pleckstrin phosphorylation in thrombin-stimulated platelets. Washed human platelets were labelled with radioactive ³²P-orthophosphate and stimulated with 1 U/ml thrombin for 1 min. PP2 (10 µM, 5 min) or Ro31-8220 (10 µM, 1 min) were added where indicated. After separation by SDS-PAGE, the resulting gel was analysed using a phosphoimager to obtain radioactivity levels. Histogram shows the level of pleckstrin phosphorylation compared to basal levels. Data from one experiment, representative of two. (C) Tyrosine phosphorylation of PKCδ in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin in the absence or presence of Ro31-8220 (10 µM, 1 min) (left), or with 1 U/ml thrombin for the times shown (right) in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. Histogram shows mean density of bands, normalised according to the basal value. (D) Tyrosine phosphorylation of PKCε in mouse platelets. Washed mouse platelets were stimulated with 3 µg/ml CRP or 1 U/ml thrombin for 1 min. Immunoprecipitations and reprobes were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003793#s2" target="_blank">methods</a>. All studies described in this figure were performed in the presence of apyrase (2.5 U/ml) and indomethacin (10 µM). Histograms show mean density of bands, normalised according to the basal value: *signifies values significant from basal, ^#signifies significant from equivalent time point without inhibitor. Data is from 3 separate experiments.</p

Pf4-Cre genetically marks the adult stem cell and early progenitor cell compartment.

<p>Bone marrow from Rosa26-tdRFP⁺;Pf4-Cre⁺ and litter matched control mice was isolated and (<b>A</b>) the Sca-1⁻c-kit⁺ cells identified in the Lin⁻ cell compartment. LK compartment was subfractionated using side scatter (SSC) and RFP expression to identify the total number of RFP⁻ (black) and RFP⁺ (red) cells. (<b>B</b>) The LK population was subfractionated using CD34 and CD16/32 to identify RFP⁺ (red) cells in the megakaryocyte-erythrocyte progenitor (MEP) (Compartment I), common myeloid progenitor (CMP) (Compartment II) and granulocyte-macrophage progenitor (GMP) (Compartment III) cell populations. (<b>C</b>) RFP expression in HSCs and primitive progenitors in the LSK compartment. The LSK cell compartment was subfractionated using CD48 and CD150 to identify the frequencies of RFP⁺ (red) cells in LSK CD150⁺CD48⁻ HSC (Compartment I), LSK CD150⁻CD48⁻ (Compartment II), LSK CD150⁺CD48⁺ (Compartment III) and LSK CD150⁻CD48⁺ (Compartment IV) compartments. Dot plots and histograms are representative figures of three independent experiments with the mean±SEM from three independent experiments.</p