The mitochondrial protease ClpP is a druggable target that controls VSMC phenotype by a SIRT1-dependent mechanism

Abstract

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.

Keywords: Aneurysms; ClpXP complex; Clpp; Metabolism; Mitochondria; NAD(+); Sirtuin 1; TIC10; VSMC; Vascular smooth mucle cell; Vasculature.

Fig. 1

ClpXP loss-of-function reduces differentiation and increases inflammatory cytokines expression in VSMCs. Representative western blots of protein expressed in HASMCs transfected with siRNA control or against CLPX or siClpP (left). Box-and-whisker plots illustrate the distribution of protein expression levels across multiple experiments (right). The box represents the interquartile range, encompassing the 25th to 75th percentiles of the dataset, with the median protein expression level indicated by a line within the box. Whiskers extend to the 5th and 95th percentiles, denoting the range of data of 4–5 independent experiments. A threshold of P < 0.05 was considered significant and is indicated by * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.

Fig. 2

ClpXP gain-of-function induces differentiation and increases inflammatory cytokines expression in VSMCs. Representative western blots showing protein expression in human aortic smooth muscle cells (HASMCs) transduced with either an adenovirus expressing CLPX or a control adenovirus containing no insert (left). Cells were treated with either platelet-derived growth factor (PDGF) at a concentration of 10 ng/mL or a vehicle control for 48 h. Box-and-whisker plots illustrate the distribution of protein expression levels across multiple experiments (right). The box represents the interquartile range, encompassing the 25th to 75th percentiles of the dataset, with the median protein expression level indicated by a line within the box. Whiskers extend to the 5th and 95th percentiles, denoting the range of data of 5–6 independent experiments. A threshold of P < 0.05 was considered significant and is indicated by * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.

Fig. 3

Metabolic changes induced by ClpXP gain-of-function in VSMCs. (A) Profile of mitochondrial respiration over time in HASMCs expressing Ad_Control or Ad_CLPX. Basal OCR measurements were made, and 1 μg/mL oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 10 μM antimycin-A and 1 μM rotenone were then sequentially injected. Data are presented as mean ± SE (B) Box-and-whisker plots from the data obtained from the analysis of OCR profiles (basal corresponds to antimycin-A–inhibitable and ATP-linked corresponds to oligomycin-inhibitable). (C) Energy map showing the metabolic reprogramming (D) Box-and-whisker plots depicting total cellular NAD⁺ and NADH levels and (E) Box-and-whisker plots depicting NAD⁺/NADH ratio induced by ClpXP gain-of-function in VSMCs. Box-and-whisker plots illustrate the distribution of protein expression levels across multiple experiments (right). The box represents the interquartile range, encompassing the 25th to 75th percentiles of the dataset, with the median protein expression level indicated by a line within the box. Whiskers extend to the 5th and 95th percentiles, denoting the range of data of 4–5 independent experiments. A threshold of P < 0.05 was considered significant and is indicated by * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.

Fig. 4

ClpXP-induced VSMC phenotypic modulation is mediated by SIRT1 activation in VSMCs. (A) Human aortic smooth muscle cells (HASMCs) were transduced with either an adenovirus expressing CLPX or a control adenovirus containing no insert and SIRT1 activity was measured as described in the methods section. (B) Representative western blots of protein expression in HASMCs transduced with either an adenovirus expressing CLPX or a control adenovirus containing no insert in cells transfected with control siRNA or SIRT1 directed siRNA. (C) Box-and-whisker plots depicting NAD⁺/NADH ratio induced by ClpXP gain-of-function in VSMCs. Box-and-whisker plots illustrate the distribution of protein expression levels across multiple experiments. The box represents the interquartile range, encompassing the 25th to 75th percentiles of the dataset, with the median protein expression level indicated by a line within the box. Whiskers extend to the 5th and 95th percentiles, denoting the range of data of 4–5 independent experiments. A threshold of P < 0.05 was considered significant and is indicated by * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.

Fig. 5

Pharmacological activation of ClpP induces a differentiated and anti-inflammatory phenotype in VSMCs. Representative western blots of protein expression in human aortic smooth muscle cells treated with TIC10 (1 μM) or ADEP1 (1 μM) for 48 h (right). Box-and-whisker plots illustrate the distribution of protein expression levels across multiple experiments (right). The box represents the interquartile range, encompassing the 25th to 75th percentiles of the dataset, with the median protein expression level indicated by a line within the box. Whiskers extend to the 5th and 95th percentiles, denoting the range of data of 5–6 independent experiments. A threshold of P < 0.05 was considered significant and is indicated by * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.

Fig. 6

TIC10 treatment results in attenuated aneurysm formation. (A) Model illustrating the timeline of the experiment, TIC10 (50 mg/kg) injections, and differences in aortic diameters between baseline and at 14 days, quantified by ECHO. The recording and subsequent analysis were blinded to the treatment regimen assignment. (B) (Left) Representative images from the experimental animals in each group at day 14. (Right) Scatter dot plots illustrating the aortic diameter value for each experimental animal, with the median value indicated by a line. (C) (Left) Differences in aortic diameters between baseline and at 14 days were quantified by ECHO for the same experimental animal. Representative ECHO images are shown. The recording and subsequent analysis were blinded to the assignment of the treatment regimen. (Right) Scatter dot plots illustrate the value for the % of maximal aortic dilation at day 14 for each experimental animal, with the median value indicated by a line. Each experimental group was composed of 10 animals.