Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre (Adipoq-Cre) strain to activate a ligand-independent Notch1 intracellular domain transgene (N1ICD) to drive constitutive Notch signaling in the adipose tissues (N1ICD;Adipoq-Cre). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus N1ICD;Adipoq-Cre mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the N1ICD;Adipoq-Cre mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology.

Over a hundred years ago, mutants were detected in Drosophila melanogaster that led to a NOTCH in the wing tip. This original phenotype was reflected in the nomenclature of the gene family that was later cloned and characterized in the 1980s and found to be conserved across metazoans. NOTCH signaling relies on transmembrane ligands and receptors that require cellular contact for receptor activation, reflecting its role in multicellular organisms as an intercellular signaling strategy. In humans, mutations in genes encoding NOTCH and their ligands have been shown to promote human disease; these aspects have been extensively reviewed. Notch signaling plays important roles in vascular development (vasculogenesis and angiogenesis) and homeostasis. NOTCH signaling is also active in adipose tissue and contributes to adipocyte differentiation. In addition, NOTCH activity regulates functions of other metabolic organs. This review focuses on NOTCH activity in perivascular adipose tissue within the vascular microenvironment as defined by mouse studies and summarizes expression and potential signaling of the NOTCH signaling network in human perivascular adipose tissue. Due to the strong activity of NOTCH in regulation of metabolic function, activation of the NOTCH network in specific cell types in perivascular adipose tissue has implications for signaling to the underlying blood vessel and control of vascular health and disease.