Abstract Body: Introduction:
Atherosclerosis is driven in part by dysfunctional macrophages exhibiting altered lysosomal acid lipase (LAL) activity. Previous studies from our group demonstrated that oxidized low-density lipoprotein (oxLDL) induces mitochondrial reactive oxygen species (mtROS) through binding to its receptor CD36, contributing to atherogenesis. However, the regulatory mechanisms underlying LAL activity in response to atherogenic stimuli, particularly the role of mtROS, remain poorly understood.

Hypothesis:
We hypothesized that chronic oxLDL exposure modulates LAL activity through the generation of mtROS in macrophages.

Methods:
Wild-type murine peritoneal macrophages were chronically treated with oxLDL (20 µg/mL) for up to 14 days to simulate prolonged exposure. LAL activity in live cells was measured using an LAL-specific hydrolysis substrate and flow cytometry. MtROS levels were assessed using the fluorescent probe MitoNeoD and modulated with the mitochondrial-targeted antioxidant MitoTempo to determine the dependency of LAL activity on mtROS. Experiments were also conducted in transgenic mice macrophages overexpressing mitochondrial catalase, which suppresses mtROS.

Results:
In wild-type macrophages exposed to chronic oxLDL, LAL activity increased significantly in comparison to controls. An initial phase peaking between 48 - 72 hours (244.7% ± 32.5%, p = 0.0002, n = 8). After a transient reduction, LAL activity rose again to match the initial peak and remained elevated up to 7 days (257.6% ± 16.5%, p < 0.0001, n = 8) and even up to 14 days (309.9% ± 22.4%, p < 0.0001, n = 3). Notably, mtROS levels closely paralleled the changes in LAL activity (n = 3). However, the oxLDL-induced increase in LAL activity was absent in macrophages overexpressing mitochondrial catalase (n = 4). Moreover, treatment with the ROS quencher MitoTempo lowered mtROS levels and prevented the acute surge in LAL activity in response to oxLDL (n = 5). These findings suggest a complex relationship between LAL activity, mtROS, and prolonged oxLDL exposure.

Conclusions:
In conclusion, chronic exposure to oxLDL leads to a sustained upregulation of LAL activity in macrophages, mediated by mtROS. These findings reveal a dynamic interplay between oxLDL, mtROS and LAL activity, highlighting a potential mitochondria-lysosome crosstalk mechanism in atherogenic macrophages.