Anti‐Cancer Therapy Provokes Human Microvascular Endothelial Dysfunction via Circulating Mitochondrial DNA and TLR9 Activation

Abstract

Rationale The detrimental impact of anti-cancer therapy (CTx) on endothelial function has emerged as a novel putative contributor to cardiovascular toxicity in cancer patients and survivors. Ongoing work in our laboratory has established impaired microvascular endothelial function in breast cancer patients following CTx, yet the causative mechanisms have not been identified. Mitochondrial DNA (mtDNA) is susceptible to damage from CTx, which stimulates release of cell-free mtDNA (cf-mtDNA) fragments that are recognized by toll-like receptor 9 (TLR9) on endothelial cells. We sought to determine whether elevated plasma cf-mtDNA and TLR9 activation following CTx are sufficient to induce endothelial dysfunction in healthy arterioles. Methods cf­-mtDNA levels were assessed (via PCR) in plasma obtained from breast cancer patients ~1 mo following CTx (doxorubicin and/or anti-HER2 therapy) and in plasma from CTx-naïve breast cancer patients (n = 7-9). HEK-Blue™-hTLR9 reporter cells (Invivogen) were treated with plasma to assess TLR9 activation (n = 4-5). We then exposed healthy human adipose arterioles to plasma from either CTx patients or healthy controls (CTRL). Freshly isolated resistance vessels were cannulated and filled with 10% plasma. Following overnight incubation, endothelium-dependent dilation was assessed as the percent change in internal diameter (videomicroscopy) in response to flow (FMD; n = 3-4) or acetylcholine (ACh; n = 2). Smooth muscle vasodilatory function was assessed in response to papaverine (n = 3-4). Vessels transfected with siRNA targeted to TLR9 were used to determine specificity of the response. Data are shown as mean ± SE. Dilation is expressed relative to maximal diameter. Results cf-mtDNA levels were 50 ± 26% higher in plasma from CTx patients relative to CTx-naïve patients (Fig. 1A) and elicited 70 ± 31% greater TLR9 activation (P < 0.05). Endothelium-dependent dilation was diminished in arterioles treated with plasma from CTx patients compared to healthy controls (Fig. 1B, FMD: 64 ± 5% vs. 86 ± 3%; ACh: 34 ± 3% vs. 90 ± 1%, P < 0.05). Maximal smooth muscle-dependent dilation to papaverine was not affected (CTx plasma: 92 ± 7% vs. CTRL plasma: 96 ± 2%; P = 0.62). In an initial observation, targeted knockdown of TLR9 prevented the detrimental effect of CTx plasma on endothelial function (FMD: 91%, n = 1). Conclusions These results demonstrate that plasma obtained from breast cancer patients 1 mo following CTx induces endothelial dysfunction in healthy human arterioles. Our preliminary findings suggest that circulating cf-mtDNA and activation of TLR9 may contribute to microvascular endothelial dysfunction in cancer patients following treatment.