Vascular smooth muscle cells (VSMCs), known for their remarkable lifelong phenotypic plasticity, play a pivotal role in vascular pathologies through their ability to transition between different phenotypes. Our group discovered that the deficiency of the mitochondrial protein Poldip2 induces VSMC differentiation both in vivo and in vitro. Further comprehensive biochemical investigations revealed Poldip2's specific interaction with the mitochondrial ATPase caseinolytic protease chaperone subunit X (CLPX), which is the regulatory subunit for the caseinolytic protease proteolytic subunit (ClpP) that forms part of the ClpXP complex - a proteasome-like protease evolutionarily conserved from bacteria to humans. This interaction limits the protease's activity, and reduced Poldip2 levels lead to ClpXP complex activation. This finding prompted the hypothesis that ClpXP complex activity within the mitochondria may regulate the VSMC phenotype. Employing gain-of-function and loss-of-function strategies, we demonstrated that ClpXP activity significantly influences the VSMC phenotype. Notably, both genetic and pharmacological activation of ClpXP inhibits VSMC plasticity and fosters a quiescent, differentiated, and anti-inflammatory VSMC phenotype. The pharmacological activation of ClpP using TIC10, currently in phase III clinical trials for cancer, successfully replicates this phenotype both in vitro and in vivo and markedly reduces aneurysm development in a mouse model of elastase-induced aortic aneurysms. Our mechanistic exploration indicates that ClpP activation regulates the VSMC phenotype by modifying the cellular NAD+/NADH ratio and activating Sirtuin 1. Our findings reveal the crucial role of mitochondrial proteostasis in the regulation of the VSMC phenotype and propose the ClpP protease as a novel, actionable target for manipulating the VSMC phenotype.
Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.

The mitochondrial Poldip2 (protein polymerase interacting protein 2) is required for the activity of the tricarboxylic acid cycle. As a consequence, Poldip2 deficiency induces metabolic reprograming with repressed mitochondrial respiration and increased glycolytic activity. Though homozygous deletion of Poldip2 is lethal, heterozygous mice are viable and show protection against aneurysm and injury-induced neointimal hyperplasia, diseases linked to loss of vascular smooth muscle differentiation. Thus, we hypothesize that the metabolic reprograming induced by Poldip2 deficiency controls VSMC differentiation.
To determine the role of Poldip2-mediated metabolic reprograming in phenotypic modulation of VSMC.
We show that Poldip2 deficiency in vascular smooth muscle in vitro and in vivo induces the expression of the SRF (serum response factor), myocardin, and MRTFA (myocardin-related transcription factor A) and dramatically represses KLF4 (Krüppel-like factor 4). Consequently, Poldip2-deficient VSMC and mouse aorta express high levels of contractile proteins and, more significantly, these cells do not dedifferentiate nor acquire macrophage-like characteristics when exposed to cholesterol or PDGF (platelet-derived growth factor). Regarding the mechanism, we found that Poldip2 deficiency upregulates the hexosamine biosynthetic pathway and OGT (O-linked N-acetylglucosamine transferase)-mediated protein O-GlcNAcylation. Increased protein glycosylation causes the inhibition of a nuclear ubiquitin proteasome system responsible for SRF stabilization and KLF4 repression and is required for the establishment of the differentiated phenotype in Poldip2-deficient cells.
Our data show that Poldip2 deficiency induces a highly differentiated phenotype in VSMCs through a mechanism that involves regulation of metabolism and proteostasis. Additionally, our study positions mitochondria-initiated signaling as key element of the VSMC differentiation programs that can be targeted to modulate VSMC phenotype during vascular diseases.

Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown.
Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4(-/-), Mcpt5(-/-)), Mcpt4(-/-) but not Mcpt5(-/-) had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4(-/-) mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4(-/-) mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although Kit(W-sh/W-sh) mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4(-/-) mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis.
High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4(-/-) mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.

Mouse models of human cancers may provide a valuable resource for the discovery of cancer biomarkers. We have developed a practical strategy for profiling specific proteins in mouse plasma using low-volume sandwich-immunoassays. We used this method to profile the levels of 14 different cytokines, acute-phase reactants, and other cancer markers in plasma from a mouse models of intestinal tumors and their wild-type littermates, using as little as 1.5 microliters of diluted plasma per assay. Many of the proteins were significantly and consistently up-regulated in the mutant mice. The mutant mice could be distinguished nearly perfectly from the wild-type mice based on the combined levels of as few as three markers. Many of the proteins were up-regulated even in the mutant mice with few or no tumors, suggesting the presence of a systemic host response at an early stage of cancer development. These results have implications for the study of host responses in mouse models of cancers and demonstrate the value of a new low-volume, high-throughput sandwich-immunoassay method for sensitively profiling protein levels in cancer.