Background: The circle of Willis (CoW) is the most common location for aneurysms to form in humans. Although the major cell types of the intracranial vasculature are well known, the heterogeneity and relative contributions of the different cells in healthy and aneurysmal vessels have not been well characterized. Here, we present the first comprehensive analysis of the lineage heterogeneity and altered transcriptomic profiles of vascular cells from healthy and aneurysmal mouse CoW using single-cell RNA sequencing. Methods: Cerebral aneurysms (CAs) were induced in adult male mice using an elastase model. Single-cell RNA sequencing was then performed on CoW samples obtained from animals that either had aneurysms form or rupture 14 days post-induction. Sham-operated animals served as controls. Results: Unbiased clustering analysis of the transcriptional profiles from >3900 CoW cells identified 19 clusters representing ten cell lineages: vascular smooth muscle cells, endothelial cells fibroblasts, pericytes and immune cells (macrophages, T and B lymphocytes, dendritic cells, mast cells, and neutrophils). The 5 vascular smooth muscle cell subpopulations had distinct transcriptional profiles and were classified as proliferative, stress-induced senescent, quiescent, inflammatory-like, or hyperproliferative. The transcriptional signature of the metabolic pathways of ATP generation was found to be downregulated in 2 major vascular smooth muscle cell clusters when CA was induced. Aneurysm induction led to significant expansion of the total macrophage population, and this expansion was further increased with rupture. Both inflammatory and resolution-phase macrophages were identified, and a massive spike of neutrophils was seen with CA rupture. Additionally, the neutrophil-to-lymphocyte ratio (NLR), which originated from CA induction mirrored what happens in humans. Conclusions: Our data identify CA disease-relevant transcriptional signatures of vascular cells in the CoW and is searchable via a web-based R/shiny interface.

Traumatic brain injury (TBI) rapidly triggers proinflammatory activation of microglia, contributing to secondary brain damage post-TBI. Although the governing role of energy metabolism in shaping the inflammatory phenotype and function of immune cells has been increasingly recognized, the specific alterations in microglial bioenergetics post-TBI remain poorly understood. Itaconate, a metabolite produced by the enzyme aconitate decarboxylase 1 [IRG1; encoded by immune responsive gene 1 ( Irg1 )], is a pivotal metabolic regulator in immune cells, particularly in macrophages. Because microglia are macrophages of the brain parenchyma, the IRG1/itaconate pathway likely modulates microglial inflammatory responses. In this study, we explored the role of the IRG1/itaconate pathway in regulating microglial bioenergetics and inflammatory activation post-TBI using a mouse controlled cortical impact (CCI) model. We isolated microglia before and 4 and 12 hours after TBI and observed a swift but transient increase in glycolysis coupled with a prolonged disruption of mitochondrial metabolism after injury. Despite an up-regulation of Irg1 expression, itaconate in microglia declined after TBI. Microglia-specific Irg1 gene knockout ( Irg1 -Mi-KO) exacerbated metabolic changes, intensified proinflammatory activation and neurodegeneration, and worsened certain long-term neurological deficits. Supplementation with 4-octyl itaconate (OI) reinstated the use and oxidative metabolism of glucose, glutamine, and fatty acid, thereby enhancing microglial bioenergetics post-TBI. OI supplementation also attenuated proinflammatory activation and neurodegeneration and improved long-term neurological outcomes. These results suggest that therapeutically targeting the itaconate pathway could improve microglial energy metabolism and neurological outcomes after TBI.

Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.