Immune checkpoint inhibitors promote endothelial inflammatory responses
D N Krueger, M Tangos, G R Y De Meyer, N Hamdani, P J Guns, C FranssenAbstract
Background
Immune checkpoint inhibitors (ICI) have revolutionised cancer therapy, yet their efficacy can be compromised by immune-related adverse events (irAEs). Although initially, research focused on myocarditis, more recent data suggest an increased incidence of vascular events, such as myocardial infarction, after initiation of ICI-treatment. With the expanding indications (and duration) of ICI treatment, cardiovascular safety gains importance even more.
Purpose
This study explored a possible relationship between ICI treatment and acute endothelial inflammation and activation, using a combination of in vivo mouse models and in vitro human and murine cellular assays.
Methods
ICI were evaluated in 3 different models: 1. C57BL/6J mice; 2. Hyperlipidaemic ApoE-/- mice; 3. C57BL/6J pretreated with doxorubicin (DOX, for 3 weeks, 4 mg/kg/week) to induce an inflammatory phenotype. Mice received three doses of anti-CTLA-4 and anti-PD-1 (7 mg/kg, each, i.p.) as combination over one week or Vehicle injections. Afterwards, vascular reactivity of the thoracic aorta was analysed ex vivo and molecular analyses were conducted. Additionally, primary murine cardiac microvascular endothelial cells (EC) were treated with either one or both ICI, and with or without DOX. Finally, functional in vitro experiments were conducted with primary human umbilical vein EC (HUVEC) treated with anti-CTLA-4.
Results
ICI did not alter phenylephrine-induced contraction, nor affected acetylcholine-induced relaxation. However, administration of ICI in vivo led to increased mRNA expression of adhesion molecules (VCAM-1, ICAM-1) and elevated TNF-α in the aorta, suggestive of endothelial inflammation and activation. Notably, mice pretreated with DOX showed an overall increased inflammatory response, which was enhanced after ICI treatment. Interestingly, Serpina3n expression was higher than ICAM-1, VCAM-1, and TNF-α after ICI treatment in mice. Primary murine EC exhibited a 4-fold increase in Vcam-1 and Icam-1 expression in response to DOX alone and DOX combined with ICI. In HUVECs, anti-CTLA-4 treatment increased cell migration and angiogenesis, indicative of an activated phenotype (Figure 1), with upregulation of TNF-α, IL-6 and VCAM-1 (Figure 2).
Conclusion
Our results indicate a direct inflammatory activation of EC by ICI. Moreover, the effect of ICI was more pronounced after pretreatment with DOX. Given the increasing use of ICI, particularly in combination with other cancer therapies, further research is essential to ensure cardiovascular safety.Figure 1. Figure 2.