Dietary intake and glutamine-serine metabolism control pathologic vascular stiffness.

Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.

Allele-specific control of rodent and human lncRNA KMT2E-AS1 promotes hypoxic endothelial pathology in pulmonary hypertension.

Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N-methyltransferase 2E-antisense 1 (KMT2E-AS1) and histone lysine N-methyltransferase 2E (KMT2E). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1/KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

Frataxin deficiency disrupts mitochondrial respiration and pulmonary endothelial cell function.

Deficiency of iron­sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency.

Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4-Human Leukocyte Antigen-DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension.

Pulmonary hypertension (PH) is a vascular disease characterized by elevated pulmonary arterial pressure, leading to right ventricular failure and death. Pathogenic features of PH include endothelial apoptosis and vascular inflammation, which drive vascular remodeling and increased pulmonary arterial pressure. Re-analysis of the whole transcriptome sequencing comparing human pulmonary arterial endothelial cells (PAECs) isolated from PH and control patients identified AREG, which encodes Amphiregulin, as a key endothelial survival factor. PAECs from PH patients and mice exhibited down-regulation of AREG and its receptor epidermal growth factor receptor (EGFR). Moreover, the deficiency of AREG and EGFR in ECs in vivo and in vitro heightened inflammatory leukocyte recruitment, cytokine production, and endothelial apoptosis, as well as diminished angiogenesis. Correspondingly, hypoxic mice lacking Egfr in ECs (cdh5 cre/+ Egfr fl/fl) displayed elevated RVSP and pulmonary remodeling. Computational analysis identified NCOA6, PHB2, and RRP1B as putative genes regulating AREG in endothelial cells. The master transcription factor of hypoxia HIF-1⍺ binds to the promoter regions of these genes and up-regulates their expression in hypoxia. Silencing of these genes in cultured PAECs decreased inflammation and apoptosis, and increased angiogenesis in hypoxic conditions. Our pathway analysis and gene silencing experiments revealed that BCL2-associated agonist of cell death (BAD) is a downstream mediator of AREG BAD silencing in ECs lacking AREG mitigated inflammation and apoptosis, and suppressed tube formation. In conclusion, loss of Amphiregulin and its receptor EGFR in PH is a crucial step in the pathogenesis of PH, promoting pulmonary endothelial cell death, influx of inflammatory myeloid cells, and vascular remodeling.

Computational repurposing of therapeutic small molecules from cancer to pulmonary hypertension.

Cancer therapies are being considered for treating rare noncancerous diseases like pulmonary hypertension (PH), but effective computational screening is lacking. Via transcriptomic differential dependency analyses leveraging parallels between cancer and PH, we mapped a landscape of cancer drug functions dependent upon rewiring of PH gene clusters. Bromodomain and extra-terminal motif (BET) protein inhibitors were predicted to rely upon several gene clusters inclusive of galectin-8 (LGALS8). Correspondingly, LGALS8 was found to mediate the BET inhibitor­dependent control of endothelial apoptosis, an essential role for PH in vivo. Separately, a piperlongumine analog's actions were predicted to depend upon the iron-sulfur biogenesis gene ISCU. Correspondingly, the analog was found to inhibit ISCU glutathionylation, rescuing oxidative metabolism, decreasing endothelial apoptosis, and improving PH. Thus, we identified crucial drug-gene axes central to endothelial dysfunction and therapeutic priorities for PH. These results establish a wide-ranging, network dependency platform to redefine cancer drugs for use in noncancerous conditions.

Simultaneous Pharmacologic Inhibition of Yes-Associated Protein 1 and Glutaminase 1 via Inhaled Poly(Lactic-co-Glycolic) Acid-Encapsulated Microparticles Improves Pulmonary Hypertension.

Background Pulmonary hypertension (PH) is a deadly disease characterized by vascular stiffness and altered cellular metabolism. Current treatments focus on vasodilation and not other root causes of pathogenesis. Previously, it was demonstrated that glutamine metabolism, as catalyzed by GLS1 (glutaminase 1) activity, is mechanoactivated by matrix stiffening and the transcriptional coactivators YAP1 (yes-associated protein 1) and transcriptional coactivator with PDZ-binding motif (TAZ), resulting in pulmonary vascular proliferation and PH. Pharmacologic inhibition of YAP1 (by verteporfin) or glutaminase (by CB-839) improved PH in vivo. However, systemic delivery of these agents, particularly YAP1 inhibitors, may have adverse chronic effects. Furthermore, simultaneous use of pharmacologic blockers may offer additive or synergistic benefits. Therefore, a strategy that delivers these drugs in combination to local lung tissue, thus avoiding systemic toxicity and driving more robust improvement, was investigated. Methods and Results We used poly(lactic-co-glycolic) acid polymer-based microparticles for delivery of verteporfin and CB-839 simultaneously to the lungs of rats suffering from monocrotaline-induced PH. Microparticles released these drugs in a sustained fashion and delivered their payload in the lungs for 7 days. When given orotracheally to the rats weekly for 3 weeks, microparticles carrying this drug combination improved hemodynamic (right ventricular systolic pressure and right ventricle/left ventricle+septum mass ratio), histologic (vascular remodeling), and molecular markers (vascular proliferation and stiffening) of PH. Importantly, only the combination of drug delivery, but neither verteporfin nor CB-839 alone, displayed significant improvement across all indexes of PH. Conclusions Simultaneous, lung-specific, and controlled release of drugs targeting YAP1 and GLS1 improved PH in rats, addressing unmet needs for the treatment of this deadly disease.

Frataxin deficiency promotes endothelial senescence in pulmonary hypertension.

The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S-containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell-derived endothelial cells from Friedreich's ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency-dependent senescence drove vessel inflammation, remodeling, and PH, whereas pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes.

Interleukin-6 mediates neutrophil mobilization from bone marrow in pulmonary hypertension.

Myeloid cells, such as neutrophils, are produced in the bone marrow in high quantities and are important in the pathogenesis of vascular diseases such as pulmonary hypertension (PH). Although neutrophil recruitment into sites of inflammation has been well studied, the mechanisms of neutrophil egress from the bone marrow are not well understood. Using computational flow cytometry, we observed increased neutrophils in the lungs of patients and mice with PH. Moreover, we found elevated levels of IL-6 in the blood and lungs of patients and mice with PH. We observed that transgenic mice overexpressing Il-6 in the lungs displayed elevated neutrophil egress from the bone marrow and exaggerated neutrophil recruitment to the lungs, resulting in exacerbated pulmonary vascular remodeling, and dysfunctional hemodynamics. Mechanistically, we found that IL-6-induced neutrophil egress from the bone marrow was dependent on interferon regulatory factor 4 (IRF-4)-mediated CX3CR1 expression in neutrophils. Consequently, Cx3cr1 genetic deficiency in hematopoietic cells in Il-6-transgenic mice significantly reduced neutrophil egress from bone marrow and decreased neutrophil counts in the lungs, thus ameliorating pulmonary remodeling and hemodynamics. In summary, these findings define a novel mechanism of IL-6-induced neutrophil egress from the bone marrow and reveal a new therapeutic target to curtail neutrophil-mediated inflammation in pulmonary vascular disease.

SCUBE1 Controls BMPR2-Relevant Pulmonary Endothelial Function: Implications for Diagnostic Marker Development in Pulmonary Arterial Hypertension.

Utilizing publicly available ribonucleic acid sequencing data, we identified SCUBE1 as a BMPR2-related gene differentially expressed between induced pluripotent stem cell-endothelial cells derived from pulmonary arterial hypertension (PAH) patients carrying pathogenic BMPR2 mutations and control patients without mutations. Endothelial SCUBE1 expression was decreased by known triggers of PAH, and its down-regulation recapitulated known BMPR2-associated endothelial pathophenotypes in vitro. Meanwhile, SCUBE1 concentrations were reduced in plasma obtained from PAH rodent models and patients with PAH, whereas plasma concentrations were tightly correlated with hemodynamic markers of disease severity. Taken together, these data implicate SCUBE1 as a novel contributor to PAH pathogenesis with potential therapeutic, diagnostic, and prognostic applications.

Long Range Endocrine Delivery of Circulating miR-210 to Endothelium Promotes Pulmonary Hypertension.

RATIONALE: Unproven theories abound regarding the long-range uptake and endocrine activity of extracellular blood-borne microRNAs into tissue. In pulmonary hypertension (PH), microRNA-210 (miR-210) in pulmonary endothelial cells promotes disease, but its activity as an extracellular molecule is incompletely defined. OBJECTIVE: We investigated whether chronic and endogenous endocrine delivery of extracellular miR-210 to pulmonary vascular endothelial cells promotes PH. METHODS AND RESULTS: Using miR-210 replete (wild-type [WT]) and knockout mice, we tracked blood-borne miR-210 using bone marrow transplantation and parabiosis (conjoining of circulatory systems). With bone marrow transplantation, circulating miR-210 was derived predominantly from bone marrow. Via parabiosis during chronic hypoxia to induce miR-210 production and PH, miR-210 was undetectable in knockout-knockout mice pairs. However, in plasma and lung endothelium, but not smooth muscle or adventitia, miR-210 was observed in knockout mice of WT-knockout pairs. This was accompanied by downregulation of miR-210 targets ISCU (iron-sulfur assembly proteins)1/2 and COX10 (cytochrome c oxidase assembly protein-10), indicating endothelial import of functional miR-210. Via hemodynamic and histological indices, knockout-knockout pairs were protected from PH, whereas knockout mice in WT-knockout pairs developed PH. In particular, pulmonary vascular engraftment of miR-210-positive interstitial lung macrophages was observed in knockout mice of WT-knockout pairs. To address whether engrafted miR-210-positive myeloid or lymphoid cells contribute to paracrine miR-210 delivery, we studied miR-210 knockout mice parabiosed with miR-210 WT; Cx3cr1 knockout mice (deficient in myeloid recruitment) or miR-210 WT; Rag1 knockout mice (deficient in lymphocytes). In both pairs, miR-210 knockout mice still displayed miR-210 delivery and PH, thus demonstrating a pathogenic endocrine delivery of extracellular miR-210. CONCLUSIONS: Endogenous blood-borne transport of miR-210 into pulmonary vascular endothelial cells promotes PH, offering fundamental insight into the systemic physiology of microRNA activity. These results also describe a platform for RNA-mediated crosstalk in PH, providing an impetus for developing blood-based miR-210 technologies for diagnosis and therapy in this disease.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

Inflammatory Macrophage Expansion in Pulmonary Hypertension Depends upon Mobilization of Blood-Borne Monocytes.

Pulmonary inflammation, which is characterized by the presence of perivascular macrophages, has been proposed as a key pathogenic driver of pulmonary hypertension (PH), a vascular disease with increasing global significance. However, the mechanisms of expansion of lung macrophages and the role of blood-borne monocytes in PH are poorly understood. Using multicolor flow cytometric analysis of blood in mouse and rat models of PH and patients with PH, an increase in blood monocytes was observed. In parallel, lung tissue displayed increased chemokine transcript expression, including those responsible for monocyte recruitment, such as Ccl2 and Cx3cl1, accompanied by an expansion of interstitial lung macrophages. These data indicate that blood monocytes are recruited to lung perivascular spaces and differentiate into inflammatory macrophages. Correspondingly, parabiosis between congenically different hypoxic mice demonstrated that most interstitial macrophages originated from blood monocytes. To define the actions of these cells in PH in vivo, we reduced blood monocyte numbers via genetic deficiency of cx3cr1 or ccr2 in chronically hypoxic male mice and by pharmacologic inhibition of Cx3cl1 in monocrotaline-exposed rats. Both models exhibited decreased inflammatory blood monocytes, as well as interstitial macrophages, leading to a substantial decrease in arteriolar remodeling but with a less robust hemodynamic effect. This study defines a direct mechanism by which interstitial macrophages expand in PH. It also demonstrates a pathway for pulmonary vascular remodeling in PH that depends upon interstitial macrophage-dependent inflammation yet is dissociated, at least in part, from hemodynamic consequences, thus offering guidance on future anti-inflammatory therapeutic strategies in this disease.

Specific circulating microRNAs display dose-dependent responses to variable intensity and duration of endurance exercise.

Circulating microRNAs (c-miRNAs), plasma-based noncoding RNAs that control posttranscriptional gene expression, mediate processes that underlie phenotypical plasticity to exercise. The relationship and biological relevance between c-miRNA expression and variable dose exercise exposure remains uncertain. We hypothesized that certain c-miRNAs respond to changes in exercise intensity and/or duration in a dose-dependent fashion. Muscle release of such c-miRNAs may then deplete intracellular stores, thus facilitating gene reprogramming and exercise adaptation. To address these hypotheses, healthy men participated in variable intensity ( n = 12, 30 × 1 min at 6, 7, and 8 miles/h, order randomized) and variable duration ( n = 14, 7 × 1 mile/h for 30, 60, and 90 min, order randomized) treadmill-running protocols. Muscle-enriched c-miRNAs (i.e., miRNA-1 and miRNA-133a) and others with known relevance to exercise were measured before and after exercise. c-miRNA responses followed three profiles: 1) nonresponsive (miRNA-21 and miRNA-210), 2) responsive to exercise at some threshold but without dose dependence (miRNA-24 and miRNA-146a), and 3) responsive to exercise with dose dependence to increasing intensity (miRNA-1) or duration (miRNA-133a and miRNA-222). We also studied aerobic exercise-trained mice, comparing control, low-intensity (0.5 km/h), or high-intensity (1 km/h) treadmill-running protocols over 4 wk. In high- but not low-intensity-trained mice, we found increased plasma c-miR-133a along with decreased intracellular miRNA-133a and increased serum response factor, a known miR-133a target gene, in muscle. Characterization of c-miRNAs that are dose responsive to exercise in humans and mice supports the notion that they directly mediate physiological adaptation to exercise, potentially through depletion of intracellular stores of muscle-specific miRNAs. NEW & NOTEWORTHY In this study of humans and mice, we define circulating microRNAs in plasma that are dose responsive to exercise. Our data support the notion that these microRNAs mediate physiological adaptation to exercise potentially through depletion of intracellular stores of muscle-specific microRNAs and releasing their inhibitory effects on target gene expression.