Enhanced platelet adhesion induces angiogenesis in intestinal inflammation and inflammatory bowel disease microvasculature

Abstract

Although angiogenesis is viewed as a fundamental component of inflammatory bowel disease (IBD) pathogenesis, we presently lack a thorough knowledge of the cell type(s) involved in its induction and maintenance in the inflamed intestinal mucosa. This study aimed to determine whether platelet (PLT) adhesion to inflamed intestinal endothelial cells of human origin may favour angiogenesis. Unstimulated or thrombin-activated human PLT were overlaid on resting or tumour necrosis factor (TNF)-α-treated human intestinal microvascular endothelial cells (HIMEC), in the presence or absence of blocking antibodies to either vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, integrin α(v)β(3) , tissue factor (TF) or fractalkine (FKN). PLT adhesion to HIMEC was evaluated by fluorescence microscopy, and release of angiogenic factors (VEGF and soluble CD40L) was measured by ELISA. A matrigel tubule formation assay was used to estimate PLT capacity to induce angiogenesis after co-culturing with HIMEC. TNF-α up-regulated ICAM-1, α(v)β(3) and FKN expression on HIMEC. When thrombin-activated PLT were co-cultured with unstimulated HIMEC, PLT adhesion increased significantly, and this response was further enhanced by HIMEC activation with TNF-α. PLT adhesion to HIMEC was VCAM-1 and TF independent but ICAM-1, FKN and integrin α(v)β(3) dependent. VEGF and sCD40L were undetectable in HIMEC cultures either before or after TNF-α stimulation. By contrast, VEGF and sCD40L release significantly increased when resting or activated PLT were co-cultured with TNF-α-pre-treated HIMEC. These effects were much more pronounced when PLT were derived from IBD patients. Importantly, thrombin-activated PLT promoted tubule formation in HIMEC, a functional estimate of their angiogenic potential. In conclusion, PLT adhesion to TNF-α-pre-treated HIMEC is mediated by ICAM-1, FKN and α(v)β(3) , and is associated with VEGF and sCD40L release. These findings suggest that inflamed HIMEC may recruit PLT which, upon release of pro-angiogenic factors, actively contribute to inflammation-induced angiogenesis.

Fig 1

Expression of adhesion molecules after exposure to TNF-α. HIMEC were treated with TNF-α, and then labelled with monoclonal antibodies directed against VCAM-I, ICAM-I, integrin α_Vβ₃, TF and FKN prior to flow cytometry analysis. A representative experiment out of six with similar results is shown in (A). Black histograms depict isotypic controls. The mean fluorescence intensity ratios of histogram distributions recorded in these experiments have been pooled and are shown as mean and S.D. in (B). *P < 0.05 and **P < 0.01 compared with HIMEC that were not challenged with TNF-α.

Fig 2

Assays of PLT adhesion to HIMEC. Prior to the adhesion assay, PLT and HIMEC were either left untouched or activated with thrombin and TNF-α, respectively, as detailed in ‘Materials and methods’. The bar graph (mean ± S.D.) summarizes the results obtained in six independent experiments. *P < 0.05 compared with PLT adhesion to resting HIMEC; **P < 0.01 compared with adhesion of resting PLT to HIMEC; §P < 0.01 compared with adhesion of resting PLT to TNF-α-activated HIMEC.

Fig 3

PLT adhesion to HIMEC after antibody-mediated neutralization of adhesion molecules. Neutralising antibodies to VCAM-I, TF, ICAM-1, integrin αVβ3 and FKN were provided to the HIMEC-PLT co-cultures. The number of adhering PLT was counted and plotted in the y-axis. The adhesion of thrombin-activated PLT to TNF-α-activated HIMEC was measured as positive control. The addition of neutralising antibodies to ICAM-I, integrin α_Vβ₃ and FKN translated into a statistically significant reduction of the number of adhering PLT. The bar graph (mean ± S.D.) summarizes the results obtained in six independent experiments. *P < 0.01 compared with adhesion of activated PLT to TNF-α-activated HIMEC.

Fig 4

Release of VEGF upon co-culture of HIMEC with PLT from healthy controls and patients with active IBD. PLT from either healthy controls (n= 8; empty columns) or patients with active IBD (8 UC and 9 CD; shaded columns) were co-cultured with HIMEC that were either left untouched or activated with TNF-α. VEGF release was measured with conventional ELISA in culture supernatants. The bar graph (mean ± S.D.) summarizes the results obtained in six independent experiments.

Fig 5

Release of sCD40L upon co-culture of HIMEC with PLT from healthy controls and patients with active IBD. PLT from either healthy controls (n= 8; empty columns) or patients with active IBD (8 UC and 9 CD; shaded columns) were co-cultured with HIMEC that were either left untouched or activated with TNF-α. The release of sCD40L was measured with conventional ELISA in culture supernatants. The bar graph (mean ± S.D.) summarizes the results obtained in six independent experiments.

Fig 6

Tubule formation assay and visualization of PLT-HIMEC interaction. (A) PLT from healthy controls were either left untouched or activated with thrombin. Tubule formation was evaluated as detailed in ‘Materials and methods’. One representative experiment out of six with similar results is shown. (B) PLT from healthy controls were either left untouched or activated with thrombin. The interaction of HIMEC (green) and PLT (red) was visualized by confocal microscopy. One representative experiment out of four with similar results is shown.