Can nucleotide scavenging prevent anti-cancer therapy-induced microvascular endothelial dysfunction?

Introduction: Cancer survivors have an increased risk of cardiovascular (CV) events compared to the general population. To date, most research has focused on how anti-cancer therapy (CTx) impacts the heart, but increasing evidence suggests altered microvascular function following CTx. Since impairments in microvascular function can precede and contribute to the onset of CV events, understanding how CTx impacts the microcirculation is of critical importance. Our preliminary studies suggest a role for toll-like receptor 9 (TLR9) mediated inflammation in CTx-induced microvascular dysfunction. TLR9 is activated by hypomethylated nucleotides (e.g., mitochondrial DNA) which may be released into the plasma following CTx-induced damage of healthy and/or cancer cells. Polyamidoamine dendrimers (PAMAM) scavenge circulating nucleic acids and prevent TLR9 activation and tumor metastasis in preclinical cancer models. As such, the objective of this study was to assess whether PAMAM could prevent TLR9 activation, and thus the manifestation of microvascular dysfunction caused by plasma from CTx-treated patients. We hypothesized that treating plasma from CTx-treated patients with PAMAM would prevent TLR9 activation in HEK293 reporter cells and the onset of microvascular dysfunction in human arterioles compared to untreated CTx plasma samples. Methods: Plasma from patients diagnosed with hepatocellular carcinoma (HCC) was obtained one month following CTx treatment. Samples were diluted (1:9 in PBS or OptiMEM) and incubated with or without 50 ug/mL PAMAM (HCC+PAMAM) for 1-hr. TLR9 activation was assessed by treating HEK293 reporter cells and their null line with HCC (n=7) or HCC+PAMAM (n=7) plasma for 24-hr. Data are presented as %change in mean absorbance±SD from the null and compared using 2-way ANOVA. Microvascular function was assessed in human adipose arterioles obtained from surgical discard tissue from healthy donors. Arterioles were incubated overnight with plasma from healthy donors (healthy n=5), HCC (n=8), or HCC+PAMAM (n=6). Pressure myography was used to assess endothelium-dependent flow-mediated dilation (FMD) and -independent (100µM Papaverine) dilation. Pressure gradients between 5 and 100 cmH2O were used to induce flow, with data presented as %max diameter±SD. Data were compared using 2-way RM-ANOVA. Significance for all tests was set to p<0.05. Results: In TLR9-expressing HEK293 cells, absorbance values were significantly elevated compared to null cells following exposure to HCC plasma (15±11%, p<0.001). This elevation was significantly reduced when HCC plasma was incubated with PAMAM (-1±9%, p<0.001). In null cells, there was no difference in absorbance between HCC and HCC+PAMAM (data not shown, p=0.10). There was a significant pressure gradient X group interaction (p=0.017) for FMD in microvessels treated with healthy, HCC, and HCC+PAMAM. Vessels treated with HCC alone had significant reductions in FMD at all pressure gradients compared to vessels treated with healthy plasma (all p<0.05). FMD for HCC+PAMAM was significantly greater than HCC at pressure gradients 20 cmH2O above (all p<0.05) yet remained significantly lower than FMD for healthy plasma (all p<0.05). No group differences to papaverine were detected (all p>0.05). Conclusions: These preliminary data suggest plasma from CTx-patients may contain cellular debris that activates TLR9 and causes microvascular dysfunction. This is prevented with the addition of a nucleotide scavenger. Future studies will seek to determine the specificity of TLR9 in CTx-induced microvascular dysfunction, determine the source/contents of the debris, and assess whether TLR9 activation increases from Pre- to Post-CTx. Supported by NIH T35HL072483 (JMV), NIH T32 HL134643 and the MCW CVC’s Pilot Award and A.O. Smith Fellowship Scholars Program (STH), and NIH R01HL173549 (AMB). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.