Aaron T. Place

PAI-1–regulated extracellular proteolysis governs senescence and survival in Klotho mice

Significance Plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of cellular senescence in vitro and is one of the biochemical fingerprints of senescence in vivo. Klotho-deficient (kl/kl) mice display a complex phenotype reminiscent of human aging and exhibit age-dependent increases in PAI-1 in tissues and in plasma. Thus, we hypothesized that PAI-1 contributes to the aging-like phenotype of kl/kl mice. We observed that either genetic deficiency or pharmacological inhibition of PAI-1 in kl/kl mice was associated with reduced evidence of senescence, preserved organ structure and function, and a fourfold increase in median lifespan. These findings indicate that PAI-1 is a critical mediator of senescence in vivo and defines a novel target for the prevention and treatment of age-related disorders in man. Cellular senescence restricts the proliferative capacity of cells and is accompanied by the production of several proteins, collectively termed the “senescence-messaging secretome” (SMS). As senescent cells accumulate in tissue, local effects of the SMS have been hypothesized to disrupt tissue regenerative capacity. Klotho functions as an aging-suppressor gene, and Klotho-deficient (kl/kl) mice exhibit an accelerated aging-like phenotype that includes a truncated lifespan, arteriosclerosis, and emphysema. Because plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor (SERPIN), is elevated in kl/kl mice and is a critical determinant of replicative senescence in vitro, we hypothesized that a reduction in extracellular proteolytic activity contributes to the accelerated aging-like phenotype of kl/kl mice. Here we show that PAI-1 deficiency retards the development of senescence and protects organ structure and function while prolonging the lifespan of kl/kl mice. These findings indicate that a SERPIN-regulated cell-nonautonomous proteolytic cascade is a critical determinant of senescence in vivo.

MiR-125b is Critical for Fibroblast-to-Myofibroblast Transition and Cardiac Fibrosis

Background— Cardiac fibrosis is the pathological consequence of stress-induced fibroblast proliferation and fibroblast-to-myofibroblast transition. MicroRNAs have been shown to play a central role in the pathogenesis of cardiac fibrosis. We identified a novel miRNA-driven mechanism that promotes cardiac fibrosis via regulation of multiple fibrogenic pathways. Methods and Results— Using a combination of in vitro and in vivo studies, we identified that miR-125b is a novel regulator of cardiac fibrosis, proliferation, and activation of cardiac fibroblasts. We demonstrate that miR-125b is induced in both fibrotic human heart and murine models of cardiac fibrosis. In addition, our results indicate that miR-125b is necessary and sufficient for the induction of fibroblast-to-myofibroblast transition by functionally targeting apelin, a critical repressor of fibrogenesis. Furthermore, we observed that miR-125b inhibits p53 to induce fibroblast proliferation. Most importantly, in vivo silencing of miR-125b by systemic delivery of locked nucleic acid rescued angiotensin II–induced perivascular and interstitial fibrosis. Finally, the RNA-sequencing analysis established that miR-125b altered the gene expression profiles of the key fibrosis-related genes and is a core component of fibrogenesis in the heart. Conclusions— In conclusion, miR-125b is critical for induction of cardiac fibrosis and acts as a potent repressor of multiple anti-fibrotic mechanisms. Inhibition of miR-125b may represent a novel therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases.

Response to Letter Regarding Article, "MiR-125b Is Critical for Fibroblast-to-Myofibroblast Transition and Cardiac Fibrosis".

We appreciate Li and colleagues for their interest in our recent publication on miR-125b and cardiac fibrogenesis.1 In their letter, the authors commented that “it is not known whether miR-125b is cell specific.” In fact, miR-125b is a highly conserved microRNA throughout diverse species from nematodes to humans and is expressed in different types of organs. Notably, we have previously reported upregulation of miR-125b in cardiac endothelial-to-mesenchymal transition, demonstrating that miR-125b is indeed not fibroblast specific.2 The authors were also concerned about potential side effects of inhibition of miR-125b in cardiomyocytes. We reported that miR-125b was upregulated during cardiac fibrosis, and the primary focus of our study was to normalize the levels of …

A null mutation in SERPINE1 protects against biological aging in humans

Humans with a rare gene mutation in SERPINE1 live longer and show evidence of protection from aging-related morbidity. Plasminogen activator inhibitor–1 (PAI-1) has been shown to be a key component of the senescence-related secretome and a direct mediator of cellular senescence. In murine models of accelerated aging, genetic deficiency and targeted inhibition of PAI-1 protect against aging-like pathology and prolong life span. However, the role of PAI-1 in human longevity remains unclear. We hypothesized that a rare loss-of-function mutation in SERPINE1 (c.699_700dupTA), which encodes PAI-1, could play a role in longevity and metabolism in humans. We studied 177 members of the Berne Amish community, which included 43 carriers of the null SERPINE1 mutation. Heterozygosity was associated with significantly longer leukocyte telomere length, lower fasting insulin levels, and lower prevalence of diabetes mellitus. In the extended Amish kindred, carriers of the null SERPINE1 allele had a longer life span. Our study indicates a causal effect of PAI-1 on human longevity, which may be mediated by alterations in metabolism. Our findings demonstrate the utility of studying loss-of-function mutations in populations with geographic and genetic isolation and shed light on a novel therapeutic target for aging.

PAI-1 is a critical regulator of FGF23 homeostasis

Pharmacological inhibition of PAI-1 augments proteolytic clearance of FGF23. Elevated levels of fibroblast growth factor 23 (FGF23), a bone-derived phosphaturic hormone, are associated with a number of pathologic conditions including chronic kidney disease, cardiac hypertrophy, and congestive heart failure. Currently, there are no specific treatments available to lower plasma FGF23 levels. We have recently reported that genetic plasminogen activator inhibitor–1 (PAI-1) deficiency provided a significant reduction in circulating FGF23 levels while simultaneously prolonging the life span of Klotho-deficient mice. We extend our investigations into the effect of PAI-1 on FGF23 homeostasis. Transgenic overexpression of PAI-1 resulted in threefold increase in FGF23 levels compared to wild-type littermates. Moreover, pharmacological modulation of PAI-1 activity with the small-molecule PAI-1 antagonist TM5441 significantly reduced FGF23 levels in PAI-1 transgenic and Klotho-deficient mice. In addition, TM5441 treatment or PAI-1 deficiency significantly accelerated the clearance of endogenous FGF23 and recombinant human FGF23 from circulation in mice with acute kidney injury. On the basis of these observations, we studied the effects of plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), on FGF23. We demonstrate that both PAs directly cleave FGF23; however, it is not known whether the PA-generated FGF23 peptides retain or acquire functions that affect binding and/or signaling properties of intact FGF23. PAI-1 inhibits the PA-dependent cleavage of FGF23, and TM5441 inhibition of PAI-1 restores the proteolysis of FGF23. Furthermore, top-down proteomic analysis indicates that tPA cleaves FGF23 at multiple arginines including the proconvertase sensitive site R176. In summary, our results indicate that PAI-1 prevents the PA-driven proteolysis of FGF23 and PAI-1 inhibition provides a novel therapeutic approach to prevent the pathologic consequences of increased FGF23.