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MicroRNAs (miRNAs) have recently received great attention for their regulatory roles in diverse cellular processes and for their contribution to several human pathologies. Modulation ...of miRNAs in vivo provides beneficial therapeutic strategies for the treatment of many diseases, as evidenced by various preclinical studies. However, specific issues regarding the in vivo use of miRNA inhibitors (antimiRs) such as organ-specific delivery, optimal dosing and formulation of the best chemistry to obtain efficient miRNA inhibition remain to be addressed. Here, we aimed at comparing the in vivo efficacy of different chemistry-based antimiR oligonucleotides to inhibit cardiac expression of miR-199b, a highly promising therapeutic target for the treatment of pressure overload-induced cardiac dysfunction. For this purpose, four different designs of oligonucleotides to inhibit miR-199b were initially developed. Systemic administration to wildtype mice on three consecutive days was followed by organ harvesting, seven days after the first injection, in order to quantify the dose-dependent changes in miR-199b expression levels. When comparing the efficiency of each inhibitor at the highest applied dose we observed that the antagomir was the only inhibitor inducing complete inhibition of miR-199b in the heart. LNA reduced expression in the heart by 50 percent while the Zen-AMO and F/MOE chemistries failed to repress miR-199b expression in the heart at any given dose, in vivo. Further optimization was achieved by subjecting the antagomir and LNA nucleotides to additional chemical modifications. Interestingly, antagomir modification by replacing the cholesterol moiety from the 3′ to the 5′ end of the molecule significantly improved the inhibitory capacity, as reflected by a 75 percent downregulation of miR-199b expression already at a concentration of 5 mg/kg/day. Similar results could be obtained with a LNA-RNA molecule but upon administration of 80 mg/kg/day. These findings show that, from all the chemistries tested by us, an antagomir carrying the cholesterol group at the 5′ end was the most efficient inhibitor of miR-199b in the heart, in vivo. Moreover, our data also emphasize the importance of chemistry optimization and best dose range finding to achieve the greatest efficacy in miRNA inhibition in vivo.
As mediators of intercellular communication, extracellular vesicles containing molecular cargo, such as microRNAs, are secreted by cells and taken up by recipient cells to influence their cellular ...phenotype and function. Here we report that cardiac stress-induced differential microRNA content, with miR-200c-3p being one of the most enriched, in cardiomyocyte-derived extracellular vesicles mediates functional cross-talk with endothelial cells. Silencing of miR-200c-3p in mice subjected to chronic increased cardiac pressure overload resulted in attenuated hypertrophy, smaller fibrotic areas, higher capillary density, and preserved cardiac ejection fraction. We were able to maximally rescue microvascular and cardiac function with very low doses of antagomir, which specifically silences miR-200c-3p expression in non-myocyte cells. Our results reveal vesicle transfer of miR-200c-3p from cardiomyocytes to cardiac endothelial cells, underlining the importance of cardiac intercellular communication in the pathophysiology of heart failure.
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Ottaviani et al. report a new mechanism used by cardiac muscle cells under stress to influence neighboring cell function by releasing small vesicles containing miR-200c-3p. When taken up by endothelial cells, this miRNA impairs angiogenic function, worsening cardiac function. Inhibition of this process is a potential therapeutic target for heart failure.
While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated ...tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.
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Da Costa Martins and colleagues identified miR-216a as a pro-angiogenic microRNA, enriched in cardiac microvascular endothelial cells and downregulated under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization due to unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.
Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure ...overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling.
These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling.
Raso et al. show that miR-148a is elevated in concentric hypertrophy and decreased in dilated cardiomyopathy. Adeno-associated viral delivery of miR-148a protects the mouse heart from cardiac dilation. Mechanistically, miR-148a regulates the sensitivity of the heart to extracellular cytokines.
Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that ...Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determine the role of PNUTS in endothelial cell aging. We confirm that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. Findings are validate in vivo using endothelial-specific inducible PNUTS-deficient mice (Cdh5-CreERT2;PNUTS
), termed PNUTS
. Two weeks after PNUTS deletion, PNUTS
mice present severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTS
mice reveal that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restores barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.
Aging increases the risk of age-related diseases, imposing substantial healthcare and personal costs. Targeting fundamental aging mechanisms pharmacologically can promote healthy aging and reduce ...this disease susceptibility. In this work, we employed transcriptome-based drug screening to identify compounds emulating transcriptional signatures of long-lived genetic interventions. We discovered compound 60 (Cmpd60), a selective histone deacetylase 1 and 2 (HDAC1/2) inhibitor, mimicking diverse longevity interventions. In extensive molecular, phenotypic, and bioinformatic assessments using various cell and aged mouse models, we found Cmpd60 treatment to improve age-related phenotypes in multiple organs. Cmpd60 reduces renal epithelial-mesenchymal transition and fibrosis in kidney, diminishes dementia-related gene expression in brain, and enhances cardiac contractility and relaxation for the heart. In sum, our two-week HDAC1/2 inhibitor treatment in aged mice establishes a multi-tissue, healthy aging intervention in mammals, holding promise for therapeutic translation to promote healthy aging in humans.
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•Transcriptome-based drug screening finds Cmpd60 as longevity intervention•Renal aging: Cmpd60 curtails epithelial-mesenchymal transition and fibrosis•Brain aging: Cmpd60 diminishes dementia-related gene expression•Cardiac aging: Cmpd60 enhances ventricular contractility
Drugs; Molecular biology; Epigenetics; Omics; Transcriptomics
Indoxyl sulfate (IS) is an accumulative protein-bound uremic toxin found in patients with kidney disease. It is reported that IS impairs the vascular endothelium, but a comprehensive overview of all ...mechanisms active in IS-injury currently remains lacking. Here we performed RNA sequencing in human umbilical vein endothelial cells (HUVECs) after IS or control medium treatment and identified 1293 genes that were affected in a IS-induced response. Gene enrichment analysis highlighted pathways involved in altered vascular formation and cell metabolism. We confirmed these transcriptome profiles at the functional level by demonstrating decreased viability and increased cell senescence in response to IS treatment. In line with the additional pathways highlighted by the transcriptome analysis, we further could demonstrate that IS exposure of HUVECs promoted tubule formation as shown by the increase in total tubule length in a 3D HUVECs/pericytes co-culture assay. Notably, the pro-angiogenic response of IS and increased ROS production were abolished when
, one of the main target genes that was highly upregulated by IS, was silenced. This observation indicates IS-induced ROS in endothelial cells is
-dependent. Taken together, our findings demonstrate that IS promotes angiogenesis and
is an important factor in IS-activated angiogenic response.
Abstract Nitric oxide (NO) produced by endothelial NO synthase (eNOS) exerts beneficial effects in a variety of cardiovascular disease states. Studies on the benefit of eNOS activity in ...pressure-overload cardiac hypertrophy and dysfunction produced by aortic stenosis are equivocal, which may be due to different expression levels of eNOS or different severities of pressure-overload. Consequently, we investigated the effects of eNOS-expression level on cardiac hypertrophy and dysfunction produced by mild or severe pressure-overload. To unravel the impact of eNOS on pressure-overload cardiac dysfunction we subjected eNOS deficient, wildtype and eNOS overexpressing transgenic (eNOS-Tg) mice to 8 weeks of mild or severe transverse aortic constriction (TAC) and studied cardiac geometry and function at the whole organ and tissue level. In both mild and severe TAC, lack of eNOS ameliorated, whereas eNOS overexpression aggravated, TAC-induced cardiac remodeling and dysfunction. Moreover, the detrimental effects of eNOS in severe TAC were associated with aggravation of TAC-induced NOS-dependent oxidative stress and by further elevation of eNOS monomer levels, consistent with enhanced eNOS uncoupling. In the presence of TAC, scavenging of reactive oxygen species with N-acetylcysteine reduced eNOS S-glutathionylation, eNOS monomer and NOS-dependent superoxide levels in eNOS-Tg mice to wildtype levels. Accordingly, N-acetylcysteine improved cardiac function in eNOS-Tg but not in wildtype mice with TAC. In conclusion, independent of the severity of TAC, eNOS aggravates cardiac remodeling and dysfunction, which appears due to TAC-induced eNOS uncoupling and superoxide production.
Heart failure is a complex syndrome involving various pathophysiological processes. An increasing body of evidence shows that the myocardial microvasculature is essential for the homeostasis state ...and that a decompensated heart is associated with microvascular dysfunction as a result of impaired endothelial angiogenic capacity. The intercellular communication between endothelial cells and cardiomyocytes through various signaling molecules, such as vascular endothelial growth factor, nitric oxide, and non-coding RNAs is an important determinant of cardiac microvascular function. Non-coding RNAs are transported from endothelial cells to cardiomyocytes, and vice versa, regulating microvascular properties and angiogenic processes in the heart. Small-exocytosed vesicles, called exosomes, which are secreted by both cell types, can mediate this intercellular communication. The purpose of this review is to highlight the contribution of the microvasculature to proper heart function maintenance by focusing on the interaction between cardiac endothelial cells and myocytes with a specific emphasis on non-coding RNAs (ncRNAs) in this form of cell-to-cell communication. Finally, the potential of ncRNAs as targets for angiogenesis therapy will also be discussed.
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