Low molecular weight heparin and aspirin exacerbate human endometrial endothelial cell responses to antiphospholipid antibodies

Abstract

Problem: Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications despite treatment with low molecular weight heparin (LMWH) or aspirin (ASA). aPL recognizing beta₂ glycoprotein I can target the uterine endothelium, however, little is known about its response to aPL. This study characterized the effect of aPL on human endometrial endothelial cells (HEECs), and the influence of LMWH and ASA.

Method of study: HEECs were exposed to aPL or control IgG, with or without low-dose LMWH and ASA, alone or in combination. Chemokine and angiogenic factor secretion were measured by ELISA. A tube formation assay was used to measure angiogenesis.

Results: aPL increased HEEC secretion of pro-angiogenic VEGF and PlGF; increased anti-angiogenic sFlt-1; inhibited basal secretion of the chemokines MCP-1, G-CSF, and GRO-α; and impaired angiogenesis. LMWH and ASA, alone and in combination, exacerbated the aPL-induced changes in the HEEC angiogenic factor and chemokine profile. There was no reversal of the aPL inhibition of HEEC angiogenesis by either single or combination therapy.

Conclusion: By aPL inhibiting HEEC chemokine secretion and promoting sFlt-1 release, the uterine endothelium may contribute to impaired placentation and vascular transformation. LMWH and ASA may further contribute to endothelium dysfunction in women with obstetric APS.

Keywords: angiogenic; antiphospholipid antibody; chemokine; endothelium; pregnancy.

Figure 1. Effect of aPL on HEEC angiogenic factor and chemokine secretion

HEECs were treated with no treatment (NT), aPL, or control IgG. Data are pooled from 9 independent experiments. Box plots show secreted levels of VEGF; PlGF; sFlt-1; sEndoglin; MCP-1; G-CSF; GRO-α; IL-8; and GM-CSF as determined by ELISA and multiplex analysis. *p<0.05 relative to the NT control.

Figure 2. Effect of aPL on HUVEC angiogenic factor and chemokine secretion

HEECs were treated with no treatment (NT), aPL, or control IgG. Data are pooled from 3 independent experiments. Box plots show secreted levels of VEGF; PlGF; sFlt-1; sEndoglin; MCP-1; G-CSF; GRO-α; IL-8; and GM-CSF as determined by ELISA. *p<0.05 relative to the NT control.

Figure 3. Effect of TLR4 inhibition on aPL-induced HEEC angiogenic factor and chemokine secretion

HEECs were treated with no treatment (NT) or aPL in the presence of media or LPS-RS. Supernatants were measured by ELISA for: VEGF; PlGF; sFlt-1; MCP-1; G-CSF; and GRO-α. Data are pooled from 3 independent experiments. *p<0.05 relative to the matching NT control for each condition (media or LPS-RS) unless otherwise indicated.

Figure 4. Effect of aPL on HEEC tube formation

HEECs were seeded on Matrigel-coated plates and treated with no treatment (NT), aPL, or control IgG. After 8hrs, tube-like vessels were imaged and quantitative analysis performed. Images are from one representative field of one representative experiment (magnification 10X). Barchart shows the number of tubes counted per field and pooled from 5 independent experiments. *p<0.05 relative to the NT control.

Figure 5. Effect of ASA and LMWH on HEEC aPL-induced HEEC angiogenic factor and chemokine secretion

HEECs were treated no treatment (NT), ASA, LMWH or both (LMWH + ASA) in the presence of media or aPL. Supernatants were measured by ELISA for: VEGF; PlGF; sFlt-1; MCP-1; G-CSF; and GRO-α. Data are pooled from 3 independent experiments. *p<0.05 relative to the NT/media control unless otherwise indicated.

Figure 6. Effect of ASA and LMWH on HEEC tube formation in the presence and absence of aPL

HEECs were seeded on Matrigel-coated plates and treated with no treatment (NT), ASA, LMWH or both (LMWH + ASA) in the presence of media or aPL. After 8hrs, tube-like vessels were imaged and quantified. Images are from one representative field of one representative experiment (magnification 10X). Barchart shows the number of tubes counted per field and pooled from 4 independent experiments. *p<0.05 relative to the NT/media control unless otherwise indicated.