Transcript analysis reveals a specific HOX signature associated with positional identity of human endothelial cells

Abstract

The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole "HOX transcriptome" of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity.

Figure 1. The BOEC is functionally a mature endothelial cell.

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.

Figure 2. BOECs have a near identical mRNA microarray profile to PAECs but differ significantly in HOX expression.

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.

Figure 3. HOX expression validation by qPCR.

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.

Figure 4. Heat map and hierarchical clustering of publically available microarray data from 53 endothelial cell types.

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.

Figure 5. Adult human tissue immunostaining of HOXD9 confirms endothelial expression.

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.

Figure 6. Heat map and hierarchical clustering of publically available microarray data of 61 cell types using HOX expression.

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

Figure 7. Heat map and hierarchical clustering of publically available microarray data of 61 cell types using HOXD expression only.

There remains a similar endothelial hierarchy, with the exception of uterine microvascular cells.

Figure 8. Microarray data from mouse ES cells differentiated towards endothelial phenotype in vitro also segregates according to HOX expression.

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.

Figure 9. Tissue expression of HOXD8 and HOXD9 in the developing fetus demonstrates cardiovascular expression.

(magnificationx100). a–c) HOXD8 d–f) HOXD9. Low power magnification (x40) of g) HOXD8 and h) HOXD9. i) Negative control.

Figure 10. Fetal lung sections from distinct timepoints during fetal development demonstrate endothelial expression of HOXD8 and HOXD9.

Expression of HOXD8 (a–e) and HOXD9 (f–j) during embryo and fetal development at 47 days (vasculogenesis), days 121 and 144 (angiogenesis), term and in adult lung. Whole lung day 47 (magnification ×40); developing bronchus/airway with surrounding islands of vasculogenesis day 47 (x100); vessel during angiogenesis (V) with adjacent airway (A), day 121 (x400); at day 144 the vessel has lumenised and is perfused (x200); term (x400) and adult lung (x200). B =  bronchus, V = vessel. Scale bars 100 μm.

Figure 11. Schemata of HOXD expression patterns according to cellular distance from the cardiogenic area and vessel size.