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

Substrate metabolism in the isolated perfused control and MH heart.

<p>Hearts were perfused with ¹⁴C-labelled glucose and ³H-palmitic acid and metabolism estimated from recovered ¹⁴CO₂ and ³H₂O, respectively. (A) Glucose oxidation was estimated in the absence and presence of insulin (100mU/L). (B) Palmitate oxidation was estimated in the absence and presence of insulin (100mU/L). Data represents mean ± SD (n = 6). Statistical significance is indicated: effects of maternal hypoxia †† <i>P</i><0.01; effects of insulin * <i>P</i><0.05, ** <i>P</i><0.01.</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

Calculated myocardial tissue oxygen partial pressure (PO₂) and metabolic rate (MO₂) for control and MH hearts.

<p>Probability (A) and cumulative probability (B) for tissue PO₂ was calculated with reference to histological images to calculate capillary domain area, and citrate synthase activity to estimate the relative changes in tissue oxygen tension for control (CON) and hearts following chronic maternal hypoxia (CHU). Metabolic rate for individual myocytes (C) and cumulative probability (D) for metabolic rates were calculated from domain area and estimates of citrate synthase activity (for further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127424#sec002" target="_blank">Methods</a>).</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

Changes to cardiac protein expression caused by MH in rats.

<p>(A) An example of reducing SDS polyacrylamide gel electrophoresis for control and MH heart tissue. (B) Proteomic analysis of the excised band corresponding to 78kd proteins isolated from gel, eluted from acrylamide and subjected to tryptic digestion before separation by HPLC and analysis by mass spectrometry (for further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127424#sec002" target="_blank">Methods</a>). Data represents mean ± SD (n = 4). Statistical significance is indicated: effects of maternal hypoxia † <i>P</i><0.05.</p

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

Maternal hypoxia and effects on cardiac pyruvate dehydrogenase activities.

<p>Effects of maternal hypoxia on absolute rates of pyruvate dehydrogenase activity (A) and the proportion of pyruvate dehydrogenase that formed the active component (B). Pyruvate dehydrogenase enzyme was isolated from heart homogenates from control and MH hearts (for further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127424#sec002" target="_blank">Methods</a>). Data represents mean ± SD (n = 6). Statistical significance is indicated: effects of maternal hypoxia † <i>P</i><0.05, †† <i>P</i><0.01; effects of insulin *** <i>P</i><0.001.</p

The effect of maternal hypoxia on adult offspring at post-mortem and cardiac parameters for perfused hearts.

<p>Control and maternal hypoxia (MH) rats (age = 14weeks) were anaesthetised and hearts excised and perfused as detailed in the methods section. Data represents Mean ± SD (n = 6). Statistical significance represented as: effects of maternal hypoxia * P<0.05, ** P<0.01, *** P<0.001: effects of insulin †† P<0.01, ††† P<0.001.</p