HOX expression validation by qPCR.

<p>a-e) HOXD1, D3, D4, D8 and D9 mRNA expression in BOECs, pulmonary artery ECs (PAECs), Aortic ECs (AECs), umbilical vein ECs (HUVECs), microvascular lymphatic ECs (HMECs), lung microvascular ECs (HMLECs). Expressed in relative arbitrary units. f) MicroRNA10 expression in BOECs and PAECs. Data was analyzed by ANOVA *p<0.05 **p<0.01.</p

The BOEC is functionally a mature endothelial cell.

<p>a) Phase microscopy of BOECs in culture demonstrates typical endothelial “cobblestone” morphology. Confocal microscopy of BOECs with nuclear counterstaining. b) CD31 (red), c) CD146 (green), d) fibronectin (green). e) Network formation in matrix gel, f) vacoulised BOECs in fibronectin/collagen matrix. g) Electron microscopy (EM) of whole BOEC showing extensive vesicles (Ves), h) EM close up of W-P bodies, i) EM of W-P bodies with immunogold staining for vWF. j-l) Neutrophil rolling adhesion and transmigration after stimulation with TNF in a flow-based assay.</p

BOECs have a near identical mRNA microarray profile to PAECs but differ significantly in HOX expression.

<p>a) Microarray expression of BOECs vs PAECs (n = 4) demonstrates high concordance of gene expression b) Heat map of HOX genes in BOECs vs PAECs. c) Venn diagram showing convergence of gene expression, d) Heat maps of differentially expressed HOXs. Additional lineage associated transcripts showing endothelial expression pattern, e) PCA analysis of BOEC vs 131 tissue arrays. Principal components analysis; Graph 1-blood derived cells (red), bowel (blue), brain (green); Graph 2 PAECs (blue), BOECs (red) demonstrating similarity of BOECs and PAECs when compared to other mature cell types.</p

Heat map and hierarchical clustering of publically available microarray data of 61 cell types using HOXD expression only.

<p>There remains a similar endothelial hierarchy, with the exception of uterine microvascular cells.</p

Heat map and hierarchical clustering of publically available microarray data from 53 endothelial cell types.

<p>Hierarchical analysis demonstrates that microvascular cells are ordered in hierarchies (within the red box). Adult and foetal cell lines segregate in separated hierarchies, as do cardiovascular-derived endothelial cells.</p

Gene ontology analysis - differentially regulated genes between BOECs and PAECs using 2 fold cut-off.

<p>Gene ontology analysis - differentially regulated genes between BOECs and PAECs using 2 fold cut-off.</p

Microarray data from mouse ES cells differentiated towards endothelial phenotype in vitro also segregates according to HOX expression.

<p>Heat map and hierarchical clustering of publically available microarray data from mouse ES cells differentiated towards an endothelial lineage (Flk +ve) at 84 hrs, 95 hrs and 8 days.</p

Schemata of HOXD expression patterns according to cellular distance from the cardiogenic area and vessel size.

<p>Schemata of HOXD expression patterns according to cellular distance from the cardiogenic area and vessel size.</p

Heat map and hierarchical clustering of publically available microarray data of 61 cell types using HOX expression.

<p>Endothelial cells all cluster within the same hierarchy (within red box) with the exception of cardiac microvascular cells.</p

Adult human tissue immunostaining of HOXD9 confirms endothelial expression.

<p>Tissue expression of HOXD9. a) Aorta, low power (x200) and b) high power (x400), c) staining of small vessels surrounding the aorta (x630) d) Main pulmonary artery low power (x200) and e) high power (x400), f) small arteries in small bowel low power (x200) and g) high power (x630), h) small pulmonary arteries (x200), i) bladder with small arteries and epithelium visible (x630), j) stomach with small vessels and mucosal glands (x400), k) skin with microvasculature and epithelium at low power (x200) and l) skin microvasculature at high power (x400), m) small arteries within fat surrounding muscle (x400), n) placental villi with central small arteries (x200), o) negative control in pulmonary vessel (x200). Scale bars 100 μm.</p