Charles D. Searles

MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity.

Mechanical forces associated with blood flow play an important role in regulating vascular signaling and gene expression in endothelial cells (ECs). MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. miRNAs are known to have an important role in modulating EC biology, but their expression and functions in cells subjected to shear stress conditions are unknown. We sought to determine the miRNA expression profile in human ECs subjected to unidirectional shear stress and define the role of miR-21 in shear stress-induced changes in EC function. TLDA array and qRT-PCR analysis performed on HUVECs exposed to prolonged unidirectional shear stress (USS, 24h, 15 dynes/cm(2)) identified 13 miRNAs whose expression was significantly upregulated (p<0.05). The miRNA with the greatest change was miR-21; it was increased 5.2-fold (p=0.002) in USS-treated versus control cells. Western analysis demonstrated that PTEN, a known target of miR-21, was downregulated in HUVECs exposed to USS or transfected with pre-miR-21. Importantly, HUVECs overexpressing miR-21 had decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO(*)) production. These data demonstrate that shear stress forces regulate the expression of miRNAs in ECs, and that miR-21 influences endothelial biology by decreasing apoptosis and activating the NO(*) pathway. These studies advance our understanding of the mechanisms by which shear stress forces modulate vascular homeostasis.

Identification of Therapeutic Covariant MicroRNA Clusters in Hypoxia-Treated Cardiac Progenitor Cell Exosomes Using Systems Biology

Rationale: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects. Objective: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions. Methods and Results: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-&bgr;–stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue–signal–response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia–reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis. Conclusions: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart.

In vitro quantification of specific microRNA using molecular beacons

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)–DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.

Actin Cytoskeleton Organization and Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase During Cell Growth

Posttranscriptional regulation of endothelial nitric oxide synthase (eNOS) expression is an important mechanism by which endothelial cells respond to various physiological and pathophysiological stimuli. Previously, we showed that eNOS expression was dramatically altered by the state of cell growth and that the mechanism responsible for this regulation was entirely posttranscriptional, occurring via changes in eNOS mRNA stability. The present study identifies a role for actin cytoskeleton organization in the posttranscriptional regulation of eNOS during cell growth and examines the relationship between the state of actin polymerization and eNOS expression. We identified monomeric actin (globular [G]-actin) as the major component of a 51-kDa ribonucleoprotein that binds to the eNOS mRNA 3′ untranslated region in UV-crosslinking analysis. Binding activity of the ribonucleoprotein complex correlated with the relative concentration of G-actin versus filamentous actin (F-actin). ENOS transcripts colocalized with cytoplasmic G-actin in cells subjected to fluorescence in situ hybridization and G-actin fluorescence staining. In subcellular fractionation studies, eNOS transcripts were enriched in the free polysomal fraction of nonproliferating cells and enriched in the cell matrix-associated polysomal fraction of proliferating cells. Furthermore, an inverse relationship between the concentration of G-actin and eNOS expression was observed in endothelial cells subjected to pharmacological alteration of their cytoskeleton; lower G/F-actin ratios correlated with increased eNOS expression. Our findings provide some insight into how endothelial cells may use the dynamic organization of the actin cytoskeleton to regulate expression of an enzyme that is crucial to vascular homeostasis.

MicroRNA Expression Profile in CAD Patients and the Impact of ACEI/ARB.

Coronary artery disease (CAD) is the largest killer of males and females in the United States. There is a need to develop innovative diagnostic markers for this disease. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in important cellular processes, and we hypothesized that the miRNA expression profile would be altered in whole blood samples of patients with CAD. We performed a microarray analysis on RNA from the blood of 5 male subjects with CAD and 5 healthy subjects (mean age 53 years). Subsequently, we performed qRT-PCR analysis of miRNA expression in whole blood of another 10 patients with CAD and 15 healthy subjects. We identified 11 miRNAs that were significantly downregulated in CAD subjects (P < .05). Furthermore, we found an association between ACEI/ARB use and downregulation of several miRNAs that was independent of the presence of significant CAD. In conclusion, we have identified a distinct miRNA signature in whole blood that discriminates CAD patients from healthy subjects. Importantly, medication use may significantly alter miRNA expression. These findings may have significant implications for identifying and managing individuals that either have CAD or are at risk of developing the disease.

Statin Treatment and 3' Polyadenylation of eNOS mRNA

Objective—Statins have been shown to increase endothelial nitric oxide synthase expression via enhanced mRNA stability. Because the poly(A) tail is an important determinant of transcript stability, we sought to characterize the effect of statins on eNOS mRNA 3′ polyadenylation. Methods and Results—Endothelial cells treated with statins had a time- and dose-dependent increase in eNOS transcripts with long poly(A) tails (75 to 160 adenosines). This effect was dependent on 3-hydroxy-3-methylglutaryl (HMG)-coenxyme A (CoA) reductase inhibition and was observed with both lipophilic (simvastatin) and hydrophilic (rosuvastatin) statins. In mRNA stability assays, polyadenylated eNOS transcripts from statin-treated cells were 2- to 3-fold more stable than transcripts from untreated cells. The effect of statins on eNOS polyadenylation was related to cytoskeleton organization; there was increased eNOS mRNA polyadenylation after Rho inhibition and cytochalasin D treatment. Further, we found increased phosphorylation of RNA polymerase II in statin-treated cells, suggesting that statin-induced polyadenylation involved modulation of RNA polymerase II activity. Conclusions—Our data provide insight into a mechanism by which statins enhance eNOS mRNA stability and increase eNOS protein: statins increase eNOS mRNA polyadenylation through Rho-mediated changes in the actin cytoskeleton.

miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export

Skeletal muscle atrophy is prevalent in chronic diseases, and microRNAs (miRs) may play a key role in the wasting process. miR-23a was previously shown to inhibit the expression of atrogin-1 and muscle RING-finger protein-1 (MuRF1) in muscle. It also was reported to be regulated by cytoplasmic nuclear factor of activated T cells 3 (NFATc3) in cardiomyocytes. The objective of this study was to determine if miR-23a is regulated during muscle atrophy and to evaluate the relationship between calcineurin (Cn)/NFAT signaling and miR-23a expression in skeletal muscle cells during atrophy. miR-23a was decreased in the gastrocnemius of rats with acute streptozotocin-induced diabetes, a condition known to increase atrogin-1 and MuRF1 expression and cause atrophy. Treatment of C2C12 myotubes with dexamethasone (Dex) for 48 h also reduced miR-23a as well as RCAN1.4 mRNA, which is transcriptionally regulated by NFAT. NFATc3 nuclear localization and the amount of miR-23a decreased rapidly within 1 h of Dex administration, suggesting a link between Cn signaling and miR-23a. The level of miR-23a was lower in primary myotubes from mice lacking the α- or β-isoform of the CnA catalytic subunit than wild-type mice. Dex did not further suppress miR-23a in myotubes from Cn-deficient mice. Overexpression of CnAβ in C2C12 myotubes prevented Dex-induced suppression of miR-23a. Finally, miR-23a was present in exosomes isolated from the media of C2C12 myotubes, and Dex increased its exosomal abundance. Dex did not alter the number of exosomes released into the media. We conclude that atrophy-inducing conditions downregulate miR-23a in muscle by mechanisms involving attenuated Cn/NFAT signaling and selective packaging into exosomes.

Laminar Shear Stress and 3′ Polyadenylation of eNOS mRNA

The 3′ poly(A) tail is important in messenger RNA stability and translational efficiency. In somatic tissues, 3′ polyadenylation of mRNAs has been thought to largely be a constitutively active process. We have reported that laminar shear stress causes a brief increase in endothelial nitric oxide synthase (eNOS) transcription, followed by a prolonged increase in eNOS mRNA stability. We sought to determine whether shear stress and other stimuli affected eNOS 3′ polyadenylation in endothelial cells. Under basal (static) conditions, eNOS mRNA possessed short 3′ poly(A) tails of <25 nt. In contrast, laminar shear stress increased expression of eNOS transcripts with long poly(A) tails. ENOS transcripts with longer poly(A) tails had prolonged half-lives (6 hours in static cells versus 18 hours in sheared cells). Polysome analysis revealed that eNOS mRNA from sheared cells was shifted into more translationally active polysome fractions compared with eNOS mRNA from static cells. Shear-induced lengthening of the eNOS 3′ poly(A) tail was the result of increased nuclear polyadenylation. Furthermore, hydrogen peroxide and HMG Co-A reductase inhibitors, other stimuli known to modulate eNOS expression posttranscriptionally, also induced eNOS 3′ poly(A) tail lengthening. These results support the concept that shear stress modulates eNOS mRNA stability and translation via increased 3′ polyadenylation. We suggest that mRNA 3′ polyadenylation is a posttranscriptional mechanism used by endothelial cells to regulate gene expression.

The interaction of nitric oxide, bradykinin, and the angiotensin II type 2 receptor: lessons learned from transgenic mice.

Despite intensive study over the past few years, several aspects of the renin-angiotensin system remain poorly understood. Such is the case for the angiotensin II type 2 receptor (AT2), one of the two receptor subtypes that mediate the actions of angiotensin II. In fetal tissues, this receptor is expressed at high levels and appears to have a role in growth, differentiation, and maturation of cells in various organs, including the vasculature (1–4). As development progresses, the level of expression of the AT2 is downregulated. In adult animals, in contrast to the wide distribution of angiotensin II type 1 receptor (AT1), AT2 is expressed at low levels and is restricted to the adrenal gland, brain, ovary, uterus, kidney, and heart. Importantly, there appears to be upregulation of AT2 in pathological states such as salt depletion, heart failure, experimental cardiac hypertrophy, myocardial infarction, and vascular injury (3–8). Because of the low level of AT2 expression in normal tissues, there is substantial debate as to its role under normal circumstances. A growing body of research suggests that there is crosstalk between AT1 and AT2 in mediating the physiologic effects of angiotensin II. Based largely on pharmacological studies, stimulation of the AT2 seems to antagonize several of the effects caused by AT1 stimulation. Thus, AT1 stimulation activates the mitogen-activated protein kinases ERK1 and ERK2, whereas AT2 stimulation suppresses this pathway, perhaps by activating ERK phosphatase 1 (MKP-1) (4, 9, 10). AT1 stimulation promotes cellular growth and hypertrophy, while the AT2 antagonizes them (3–6). AT1 stimulation facilitates angiogenesis, while the AT2 inhibits this process (11). AT1 activation induces vasoconstriction, while AT2 activation causes vasodilation (12). These cellular and organ-level effects appear to act in intact animal models as well. In cardiomyopathic hamsters, AT2 expression is upregulated in cardiac fibroblasts of the failing heart and appears to antagonize AT1–mediated progression of interstitial fibrosis and cardiac remodeling (6). Overexpression of AT2 in balloon-injured vascular smooth muscle cells attenuates neointimal formation (6). In a rat model of ischemic cardiomyopathy, the beneficial effects of AT1 blockade on cardiac remodeling and hemodynamics are inhibited by AT2 blockade (5). Recent work has suggested that some of the beneficial effects of AT2 stimulation may be meditated through the bradykinin/nitric oxide (NO) cascade (5, 13–15). Endothelial cells contain bradykinin type 2 receptors (B2), which, when activated, potently stimulate production of NO. Although the effects of angiotensin II and NO seem to vary with the concentration of NO and the cell type involved, these two factors appear to play opposing roles in the cardiovascular system: angiotensin II is a potent stimulus for vasoconstriction and vascular smooth muscle hypertrophy, whereas NO has a vasodepressor effect and has been shown to be an antiproliferative agent. Thus, in spontaneously hypertensive rats, AT2 activation has been shown to increase vascular cyclic guanosine 3′,5′-monophosphate (cGMP) levels, an effect that could be inhibited by B2 blockade or by inhibition of NO synthase (15). Likewise in conscious rats, salt depletion, which activates the renin-angiotensin system, increases cGMP levels in the renal interstitial fluid (14), an effect that can be prevented by blockade of either NO synthase or the AT2. Following myocardial infarction in rats, either angiotensin I–converting enzyme inhibitors or AT1 antagonists can prevent remodeling of the left ventricle, as assessed by collagen deposition, myocyte size, and left ventricular diameter (5), and in either case the effect could be blocked by B2 inhibition. Importantly, these studies not only underscore the antagonistic interactions between AT1 and AT2 but also introduce the notion that the bradykinin/NO system mediates this interaction. In the previous issue of the JCI, Tsutsumi et al. (16) provided additional compelling evidence linking AT2 to the bradykinin/NO cascade. These authors targeted overexpression of AT2 to the vascular smooth muscle in transgenic mice, achieving a 5-fold increase in expression of this receptor. These animals exhibited an attenuated pressor response to angiotensin II infusion. Pretreatment of these transgenic mice with an AT2 antagonist, a B2-receptor antagonist, or an NO synthase inhibitor restored the pressor response to angiotensin II. Angiotensin II produced a paradoxical decrease in blood pressure after AT1 blockade in these animals, suggesting that selective AT2 stimulation had a vasodepressor effect. Furthermore, the authors showed that the AT2–mediated vasodepressor effect was associated with an endothelium-dependent increase in aortic production of cGMP and activation of the kinin-kallikrein system. In the authors’ interpretation of the results, angiotensin II stimulates AT2 in vascular smooth muscle, which leads to activation of the kinin-kallikrein system and bradykinin release. Bradykinin then binds to its receptor on adjacent endothelial cells, causing the release of NO and stimulation of cGMP. As with other work investigating the physiologic role of AT2, this study relied heavily on pharmacologic manipulation, and the question arises as to what extent the results could be attributed to the specificity of the various agents used. Despite this concern, the transgene clearly altered the effects of exogenously administered angiotensin II, and therefore this work provides important information regarding interactions between angiotensin II and NO in the vasculature. This study is the first to directly demonstrate AT2 stimulation of vascular smooth muscle kininogenase activity, which, in turn, explains increased vascular production of bradykinin and NO in response to angiotensin II. Certainly, more work has do be done to verify and elucidate the physiologic importance of this angiotensin II/bradykinin/NO cascade and its relevance to heart failure, myocardial infarction, and vascular injury. One intriguing notion that has evolved from this type of work is that failure of normal crosstalk between AT1 and AT2 may worsen development of cardiovascular disease. Thus, absence or decreased activity of AT2 may allow the deleterious effects of AT1 stimulation to go unchecked. Interestingly, NO has been reported to downregulate AT1 expression (17). Therefore, enhanced NO production in response to AT2 stimulation may diminish AT1 responsiveness directly. Conversely, selective pharmacotherapeutic stimulation of the AT2 may have beneficial effects in treatment of cardiovascular diseases. Treatment with AT1 antagonists, which leave AT2 unblocked, may in part achieve this end. As the role of the AT2 rises from physiologic obscurity, a more thorough understanding of its interactions with the AT1 and the NO system is emerging. Understanding these interactions may allow greater insight into the full impact of angiotensin II in disease.

Hypoxia mediates mutual repression between microRNA-27a and PPARγ in the pulmonary vasculature.

Pulmonary hypertension (PH) is a serious disorder that causes significant morbidity and mortality. The pathogenesis of PH involves complex derangements in multiple pathways including reductions in peroxisome proliferator-activated receptor gamma (PPARγ). Hypoxia, a common PH stimulus, reduces PPARγ in experimental models. In contrast, activating PPARγ attenuates hypoxia-induced PH and endothelin 1 (ET-1) expression. To further explore mechanisms of hypoxia-induced PH and reductions in PPARγ, we examined the effects of hypoxia on selected microRNA (miRNA or miR) levels that might reduce PPARγ expression leading to increased ET-1 expression and PH. Our results demonstrate that exposure to hypoxia (10% O2) for 3-weeks increased levels of miR-27a and ET-1 in the lungs of C57BL/6 mice and reduced PPARγ levels. Hypoxia-induced increases in miR-27a were attenuated in mice treated with the PPARγ ligand, rosiglitazone (RSG, 10 mg/kg/d) by gavage for the final 10 d of exposure. In parallel studies, human pulmonary artery endothelial cells (HPAECs) were exposed to control (21% O2) or hypoxic (1% O2) conditions for 72 h. Hypoxia increased HPAEC proliferation, miR-27a and ET-1 expression, and reduced PPARγ expression. These alterations were attenuated by treatment with RSG (10 µM) during the last 24 h of hypoxia exposure. Overexpression of miR-27a or PPARγ knockdown increased HPAEC proliferation and ET-1 expression and decreased PPARγ levels, whereas these effects were reversed by miR-27a inhibition. Further, compared to lungs from littermate control mice, miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. Knockdown of either SP1 or EGR1 was sufficient to significantly attenuate miR-27a expression in HPAECs. Collectively, these studies provide novel evidence that miR-27a and PPARγ mediate mutually repressive actions in hypoxic pulmonary vasculature and that targeting PPARγ may represent a novel therapeutic approach in PH to attenuate proliferative mediators that stimulate proliferation of pulmonary vascular cells.