Osteopontin promotes age-related adipose tissue remodeling through senescence-associated macrophage dysfunction.

Adipose tissue macrophages (ATMs) play an important role in obesity and inflammation, and they accumulate in adipose tissue (AT) with aging. Furthermore, increased ATM senescence has been shown in obesity-related AT remodeling and dysfunction. However, ATM senescence and its role are unclear in age-related AT dysfunction. Here, we show that ATMs (a) acquire a senescence-like phenotype during chronological aging; (b) display a global decline of basic macrophage functions such as efferocytosis, an essential process to preserve AT homeostasis by clearing dysfunctional or apoptotic cells; and (c) promote AT remodeling and dysfunction. Importantly, we uncover a major role for the age-associated accumulation of osteopontin (OPN) in these processes in visceral AT. Consistently, loss or pharmacologic inhibition of OPN and bone marrow transplantation of OPN-/- mice attenuate the ATM senescence-like phenotype, preserve efferocytosis, and finally restore healthy AT homeostasis in the context of aging. Collectively, our findings implicate pharmacologic OPN inhibition as a viable treatment modality to counter ATM senescence-mediated AT remodeling and dysfunction during aging.

Diabetic cardiomyopathy: the need for adjusting experimental models to meet clinical reality.

Diabetic cardiomyopathy (CM), occurring in the absence of hypertension, coronary artery disease, and valvular or congenital heart disease, is now recognized as a distinct, multifactorial disease leading to ventricular hypertrophy and abnormal myocardial contractility that correlates with an array of complex molecular and cellular changes. Animal models provide the unique opportunity to investigate mechanistic aspects of diabetic CM, but important caveats exist when extrapolating findings obtained from preclinical models of diabetes to humans. Indeed, animal models do not recapitulate the complexity of environmental factors, most notably the duration of the exposure to insulin resistance that may play a crucial role in the development of diabetic CM. Moreover, most preclinical studies are performed in animals with uncontrolled or poorly controlled diabetes, whereas patients tend to undergo therapeutic intervention. Finally, whilst type 2 diabetes mellitus prevalence trajectory mainly increases at 40- < 75 years (with a currently alarming increase at younger ages, however), it is a legitimate concern how closely rodent models employing young animals recapitulate the disease developing in old people. The aim of this review is to identify the current limitations of rodent models and to discuss how future mechanistic and preclinical studies should integrate key confounding factors to better mimic the diabetic CM phenotype.

Adipose tissue senescence is mediated by increased ATP content after a short-term high-fat diet exposure.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

Dysregulated Phenylalanine Catabolism Plays a Key Role in the Trajectory of Cardiac Aging.

BACKGROUND: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging. The present study aimed to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine and how it may promote age-related cardiac dysfunction. METHODS: We studied cardiac structure and function, together with phenylalanine catabolism in wild-type (WT) and p21-/- mice (male; 2-24 months), with the latter known to be protected from cellular senescence. To explore phenylalanine's effects on cellular senescence and ectopic phenylalanine catabolism, we treated cardiomyocytes (primary adult rat or human AC-16) with phenylalanine. To establish a role for phenylalanine in driving cardiac aging, WT male mice were treated twice a day with phenylalanine (200 mg/kg) for a month. We also treated aged WT mice with tetrahydrobiopterin (10 mg/kg), the essential cofactor for the phenylalanine-degrading enzyme PAH (phenylalanine hydroxylase), or restricted dietary phenylalanine intake. The impact of senescence on hepatic phenylalanine catabolism was explored in vitro in AML12 hepatocytes treated with Nutlin3a (a p53 activator), with or without p21-targeting small interfering RNA or tetrahydrobiopterin, with quantification of PAH and tyrosine levels. RESULTS: Natural aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, whereas p21 deletion delayed these changes. Phenylalanine treatment induced premature cardiac deterioration in young WT mice, strikingly akin to that occurring with aging, while triggering cellular senescence, redox, and epigenetic changes. Pharmacological restoration of phenylalanine catabolism with tetrahydrobiopterin administration or dietary phenylalanine restriction abrogated the rise in plasma phenylalanine and reversed cardiac senescent alterations in aged WT mice. Observations from aged mice and human samples implicated age-related decline in hepatic phenylalanine catabolism as a key driver of elevated plasma phenylalanine levels and showed increased myocardial PAH-mediated phenylalanine catabolism, a novel signature of cardiac aging. CONCLUSIONS: Our findings establish a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. They highlight phenylalanine/PAH modulation as a potential therapeutic strategy for age-associated cardiac impairment.

The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice.

Oxidative stress is a major contributor to chronic lung diseases. Antioxidants such as N-acetylcysteine (NAC) are broadly viewed as protective molecules that prevent the mutagenic effects of reactive oxygen species. Antioxidants may, however, increase the risk of some forms of cancer and accelerate lung cancer progression in murine models. Here, we investigated chronic NAC treatment in aging mice displaying lung oxidative stress and cell senescence due to inactivation of the transcription factor JunD, which is downregulated in diseased human lungs. NAC treatment decreased lung oxidative damage and cell senescence and protected from lung emphysema but concomitantly induced the development of lung adenocarcinoma in 50% of JunD-deficient mice and 10% of aged control mice. This finding constitutes the first evidence to our knowledge of a carcinogenic effect of antioxidant therapy in the lungs of aged mice with chronic lung oxidative stress and warrants the utmost caution when considering the therapeutic use of antioxidants.

Cardiac adenylyl cyclase overexpression precipitates and aggravates age-related myocardial dysfunction.

AIMS: Increase of cardiac cAMP bioavailability and PKA activity through adenylyl-cyclase 8 (AC8) overexpression enhances contractile function in young transgenic mice (AC8TG). Ageing is associated with decline of cardiac contraction partly by the desensitization of ß-adrenergic/cAMP signalling. Our objective was to evaluate cardiac cAMP signalling as age increases between 2 months and 12 months and to explore whether increasing the bioavailability of cAMP by overexpression of AC8 could prevent cardiac dysfunction related to age. METHODS AND RESULTS: Cardiac cAMP pathway and contractile function were evaluated in AC8TG and their non-transgenic littermates (NTG) at 2- and 12 months old. AC8TG demonstrated increased AC8, PDE1, 3B and 4D expression at both ages, resulting in increased phosphodiesterase and PKA activity, and increased phosphorylation of several PKA targets including sarco(endo)plasmic-reticulum-calcium-ATPase (SERCA2a) cofactor phospholamban (PLN) and GSK3α/ß a main regulator of hypertrophic growth and ageing. Confocal immunofluorescence revealed that the major phospho-PKA substrates were co-localized with Z-line in 2-month-old NTG but with Z-line interspace in AC8TG, confirming the increase of PKA activity in the compartment of PLN/SERCA2a. In both 12-month-old NTG and AC8TG, PLN and GSK3α/ß phosphorylation was increased together with main localization of phospho-PKA substrates in Z-line interspaces. Haemodynamics demonstrated an increased contractile function in 2- and 12-month-old AC8TG, but not in NTG. In contrast, echocardiography and tissue Doppler imaging (TDI) performed in conscious mice unmasked myocardial dysfunction with a decrease of systolic strain rate in both old AC8TG and NTG. In AC8TG TDI showed a reduced strain rate even in 2-month-old animals. Development of age-related cardiac dysfunction was accelerated in AC8TG, leading to heart failure (HF) and premature death. Histological analysis confirmed early cardiomyocyte hypertrophy and interstitial fibrosis in AC8TG when compared with NTG. CONCLUSION: Our data demonstrated an early and accelerated cardiac remodelling in AC8TG mice, leading to the development of HF and reduced lifespan. Age-related reorganization of cAMP/PKA signalling can accelerate cardiac ageing, partly through GSK3α/ß phosphorylation.

Carbon monoxide-induced metabolic switch in adipocytes improves insulin resistance in obese mice.

Obesity is characterized by accumulation of adipose tissue and is one the most important risk factors in the development of insulin resistance. Carbon monoxide-releasing (CO-releasing) molecules (CO-RMs) have been reported to improve the metabolic profile of obese mice, but the underlying mechanism remains poorly defined. Here, we show that oral administration of CORM-401 to obese mice fed a high-fat diet (HFD) resulted in a significant reduction in body weight gain, accompanied by a marked improvement in glucose homeostasis. We further unmasked an action we believe to be novel, by which CO accumulates in visceral adipose tissue and uncouples mitochondrial respiration in adipocytes, ultimately leading to a concomitant switch toward glycolysis. This was accompanied by enhanced systemic and adipose tissue insulin sensitivity, as indicated by a lower blood glucose and increased Akt phosphorylation. Our findings indicate that the transient uncoupling activity of CO elicited by repetitive administration of CORM-401 is associated with lower weight gain and increased insulin sensitivity during HFD. Thus, prototypic compounds that release CO could be investigated for developing promising insulin-sensitizing agents.

Visceral Adipose Tissue Drives Cardiac Aging Through Modulation of Fibroblast Senescence by Osteopontin Production.

BACKGROUND: Aging induces cardiac structural and functional changes linked to the increased deposition of extracellular matrix proteins, including OPN (osteopontin), conducing to progressive interstitial fibrosis. Although OPN is involved in various pathological conditions, its role in myocardial aging remains unknown. METHODS: OPN deficient mice (OPN-/-) with their wild-type (WT) littermates were evaluated at 2 and 14 months of age in terms of cardiac structure, function, histology and key molecular markers. OPN expression was determined by reverse-transcription polymerase chain reaction, immunoblot and immunofluorescence. Luminex assays were performed to screen plasma samples for various cytokines/adipokines in addition to OPN. Similar explorations were conducted in aged WT mice after surgical removal of visceral adipose tissue (VAT) or treatment with a small-molecule OPN inhibitor agelastatin A. Primary WT fibroblasts were incubated with plasma from aged WT and OPN-/- mice, and evaluated for senescence (senescence-associated ß-galactosidase and p16), as well as fibroblast activation markers (Acta2 and Fn1). RESULTS: Plasma OPN levels increased in WT mice during aging, with VAT showing the strongest OPN induction contrasting with myocardium that did not express OPN. VAT removal in aged WT mice restored cardiac function and decreased myocardial fibrosis in addition to a substantial reduction of circulating OPN and transforming growth factor ß levels. OPN deficiency provided a comparable protection against age-related cardiac fibrosis and dysfunction. Intriguingly, a strong induction of senescence in cardiac fibroblasts was observed in both VAT removal and OPN-/- mice. The addition of plasma from aged OPN-/- mice to cultures of primary cardiac fibroblasts induced senescence and reduced their activation (compared to aged WT plasma). Finally, Agelastatin A treatment of aged WT mice fully reversed age-related myocardial fibrosis and dysfunction. CONCLUSIONS: During aging, VAT represents the main source of OPN and alters heart structure and function via its profibrotic secretome. As a proof-of-concept, interventions targeting OPN, such as VAT removal and OPN deficiency, rescued the heart and induced a selective modulation of fibroblast senescence. Our work uncovers OPN's role in the context of myocardial aging and proposes OPN as a potential new therapeutic target for a healthy cardiac aging.

Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

Clinical Implications of Echocardiographic Phenotypes of Patients With Diabetes Mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) may alter cardiac structure and function, but obesity, hypertension (HTN), or aging can induce similar abnormalities. OBJECTIVES: This study sought to link cardiac phenotypes in T2DM patients with clinical profiles and outcomes using cluster analysis. METHODS: Baseline echocardiography and a composite endpoint (cardiovascular mortality and hospitalization) were evaluated in 842 T2DM patients from 2 prospective cohorts. A cluster analysis was performed on echocardiographic variables, and the association between clusters and clinical profiles and outcomes was assessed. RESULTS: Three clusters were identified. Cluster 1 patients had the lowest left ventricular (LV) mass index and ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e') ratio, had the highest left ventricular ejection fraction (LVEF), and were predominantly male with the lowest rate of obesity or HTN. Cluster 2 patients had the highest strain and highest E/e' ratio, were the oldest, were predominantly female, and had the lowest rate of isolated T2DM (without HTN or obesity). Cluster 3 patients had the highest LV mass index and volumes and the lowest LVEF and strain, were predominantly male, and shared similar age and rate of obesity and HTN as cluster 1 patients. After follow-up of 67 months (interquartile range: 40 to 87), the composite endpoint occurred in 56 of 521 patients (10.8%). Clusters 2 (hazard ratio: 2.37; 95% confidence interval: 1.15 to 4.88) and 3 (hazard ratio: 2.19; 95% confidence interval: 1.00 to 4.82) had a similar outcome, which was worse than cluster 1. CONCLUSIONS: Cluster analysis of echocardiographic variables identified 3 different echocardiographic phenotypes of T2DM patients that were associated with distinct clinical profiles and highlighted the prognostic value of LV remodeling and subclinical dysfunction.

Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

Short-term high-fat diet compromises myocardial function: a radial strain rate imaging study.

AIM: Long-term high-fat diet (HFD) induces both cardiac remodelling and myocardial dysfunction in murine models. The aim was to assess the time course and mechanisms of metabolic and cardiac modifications induced by short-term HFD in wild-type (WT) mice. METHODS AND RESULTS: Thirty-three WT mice were subjected to HFD (60% fat, n = 16) and chow diet (CD, 13% fat, n = 17). Metabolic and echocardiographic data were collected at baseline and every 5 weeks for 20 weeks. Invasive haemodynamic data and myocardial samples were collected at 5 and 20 weeks. Echocardiographic data included left ventricular (LV) diameters and thickness, and systolic function using radial strain rate (SR). Histological assessment of cardiomyocyte and adipocyte sizes, interstitial fibrosis, and apoptosis index were performed. During follow-up, body weight, and glycaemia levels were higher in HFD than in CD mice, in association with an early adipose tissue remodelling. Despite no difference between both groups in blood pressure and LV mass at 5 weeks, an early LV dysfunction was observed in HFD mice as assessed by radial SR (21 ± 0.8 vs. 27 ± 0.8 unit/s, P < 0.001) and haemodynamic assessment. During follow-up, both groups demonstrated a progressive systolic and diastolic LV dysfunction and remodelling including dilatation and hypertrophy, which were more severe in HFD mice. Compared with CD mice, the early LV impairment in HFD mice was coupled with a higher cardiomyocyte apoptosis level (0.95 vs. 0.02%, P < 0.05) associated with an interstitial fibrosis process (2.3 vs. 0.2%, P < 0.05), which worsen during follow-up. CONCLUSION: The HFD promoted early metabolic and cardiac dysfunctions, and adipose and myocardial tissues remodelling.

Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity.

Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Extracellular Calpain/Calpastatin Balance Is Involved in the Progression of Pulmonary Hypertension.

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Selective Tuberous Sclerosis Complex 1 Gene Deletion in Smooth Muscle Activates Mammalian Target of Rapamycin Signaling and Induces Pulmonary Hypertension.

Constitutive activation of the mammalian target of rapamycin (mTOR) complexes mTORC1 and mTORC2 is associated with pulmonary hypertension (PH) and sustained growth of pulmonary artery (PA) smooth muscle cells (SMCs). We investigated whether selective mTORC1 activation in SMCs induced by deleting the negative mTORC1 regulator tuberous sclerosis complex 1 gene (TSC1) was sufficient to produce PH in mice. Mice expressing Cre recombinase under SM22 promoter control were crossed with TSC1(LoxP/LoxP) mice to generate SM22-TSC1(-/-) mice. At 8 weeks of age, SM22-TSC1(-/-) mice exhibited PH with marked increases in distal PA muscularization and Ki67-positive PASMC counts, without systemic hypertension or cardiac dysfunction. Marked activation of the mTORC1 substrates S6 kinase and 4E-BP and the mTORC2 substrates p-Akt(Ser473) and glycogen synthase kinase 3 was found in the lungs and pulmonary vessels of SM22-TSC1(-/-) mice when compared with control mice. Treatment with 5 mg/kg rapamycin for 3 weeks to inhibit mTORC1 and mTORC2 fully reversed PH in SM22-TSC1(-/-) mice. In chronically hypoxic mice and SM22-5HTT(+) mice exhibiting PH associated with mTORC1 and mTORC2 activation, PH was maximally attenuated by low-dose rapamycin associated with selective mTORC1 inhibition. Cultured PASMCs from SM22-TSC1(-/-), SM22-5HTT(+), and chronically hypoxic mice exhibited similar sustained growth-rate enhancement and constitutive mTORC1 and mTORC2 activation; both effects were abolished by rapamycin. Deletion of the downstream mTORC1 effectors S6 kinase 1/2 in mice also activated mTOR signaling and induced PH. We concluded that activation of mTORC1 signaling leads to increased PASMC proliferation and subsequent PH development.