Endothelial specific prolyl hydroxylase domain-containing protein 2 deficiency attenuates aging-related obesity and exercise intolerance.

Obesity and exercise intolerance greatly reduce the life quality of older people. Prolyl hydroxylase domain-containing protein 2 (PHD2) is an important enzyme in modulating hypoxia-inducible factor-alpha (HIF) protein. Using vascular endothelial cell-specific PHD2 gene knockout (PHD2 ECKO) mice, we investigated the role of endothelial PHD2 in aging-related obesity and exercise capacity. Briefly, PHD2 ECKO mice were obtained by crossing PHD2-floxed mice with VE-Cadherin (Cdh5)-Cre transgenic mice. The effect of PHD2 ECKO on obesity and exercise capacity in PHD2 ECKO mice and control PHD2f/f mice were determined in young mice (6 to 7 months) and aged mice (16-18 months). We found that aged PHD2 ECKO mice, but not young mice, exhibited a lean phenotype, characterized by lower fat mass, and its ratio to lean weight, body weight, or tibial length, while their food uptake was not reduced compared with controls. Moreover, as compared with aged control mice, aged PHD2 ECKO mice exhibited increased oxygen consumption at rest and during exercise, and the maximum rate of oxygen consumption (VO2 max) during exercise. Furthermore, as compared with corresponding control mice, both young and aged PHD2 ECKO mice demonstrated improved glucose tolerance and lower insulin resistance. Together, these data demonstrate that inhibition of vascular endothelial PHD2 signaling significantly attenuates aging-related obesity, exercise intolerance, and glucose intolerance.

Inhibition of NK1.1 signaling attenuates pressure overload-induced heart failure, and consequent pulmonary inflammation and remodeling.

Background: Inflammation contributes to heart failure (HF) development, the progression from left ventricular failure to pulmonary remodeling, and the consequent right ventricular hypertrophy and failure. NK1.1 plays a critical role in Natural killer (NK) and NK T (NKT) cells, but the role of NK1.1 in HF development and progression is unknown. Methods: We studied the effects of NK1.1 inhibition on transverse aortic constriction (TAC)-induced cardiopulmonary inflammation, HF development, and HF progression in immunocompetent male mice of C57BL/6J background. Results: We found that NK1.1+ cell-derived interferon gamma+ (IFN-γ+) was significantly increased in pulmonary tissues after HF. In addition, anti-NK1.1 antibodies simultaneously abolished both NK1.1+ cells, including the NK1.1+NK and NK1.1+NKT cells in peripheral blood, spleen, and lung tissues, but had no effect on cardiopulmonary structure and function under control conditions. However, systemic inhibition of NK1.1 signaling by anti-NK1.1 antibodies significantly rescued mice from TAC-induced left ventricular inflammation, fibrosis, and failure. Inhibition of NK1.1 signaling also significantly attenuated TAC-induced pulmonary leukocyte infiltration, fibrosis, vessel remodeling, and consequent right ventricular hypertrophy. Moreover, inhibition of NK1.1 signaling significantly reduced TAC-induced pulmonary macrophage and dendritic cell infiltration and activation. Conclusions: Our data suggest that inhibition of NK1.1 signaling is effective in attenuating systolic overload-induced cardiac fibrosis, dysfunction, and consequent pulmonary remodeling in immunocompetent mice through modulating the cardiopulmonary inflammatory response.

IL-12α deficiency attenuates pressure overload-induced cardiac inflammation, hypertrophy, dysfunction, and heart failure progression.

IL-12α plays an important role in modulating inflammatory response, fibroblast proliferation and angiogenesis through modulating macrophage polarization or T cell function, but its effect on cardiorespiratory fitness is not clear. Here, we studied the effect of IL-12α on cardiac inflammation, hypertrophy, dysfunction, and lung remodeling in IL-12α gene knockout (KO) mice in response to chronic systolic pressure overload produced by transverse aortic constriction (TAC). Our results showed that IL-12α KO significantly ameliorated TAC-induced left ventricular (LV) failure, as evidenced by a smaller decrease of LV ejection fraction. IL-12α KO also exhibited significantly attenuated TAC-induced increase of LV weight, left atrial weight, lung weight, right ventricular weight, and the ratios of them in comparison to body weight or tibial length. In addition, IL-12α KO showed significantly attenuated TAC-induced LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and lung inflammation and remodeling (such as lung fibrosis and vessel muscularization). Moreover, IL-12α KO displayed significantly attenuated TAC-induced activation of CD4+ T cells and CD8+ T cells in the lung. Furthermore, IL-12α KO showed significantly suppressed accumulation and activation of pulmonary macrophages and dendritic cells. Taken together, these findings indicate that inhibition of IL-12α is effective in attenuating systolic overload-induced cardiac inflammation, heart failure development, promoting transition from LV failure to lung remodeling and right ventricular hypertrophy.

Sustained Elevated Circulating Activin A Impairs Global Longitudinal Strain in Pregnant Rats: A Potential Mechanism for Preeclampsia-Related Cardiac Dysfunction.

Mediators of cardiac injury in preeclampsia are not well understood. Preeclamptic women have decreased cardiac global longitudinal strain (GLS), a sensitive measure of systolic function that indicates fibrosis and tissue injury. GLS is worse in preeclampsia compared to gestational hypertension, despite comparable blood pressure, suggesting that placental factors may be involved. We previously showed that Activin A, a pro-fibrotic factor produced in excess by the placenta in preeclampsia, predicts impaired GLS postpartum. Here, we hypothesized that chronic excess levels of Activin A during pregnancy induces cardiac dysfunction. Rats were assigned to sham or activin A infusion (1.25-6 µg/day) on a gestational day (GD) 14 (n = 6-10/group). All animals underwent blood pressure measurement and comprehensive echocardiography followed by euthanasia and the collection of tissue samples on GD 19. Increased circulating activin A (sham: 0.59 ± 0.05 ng/mL, 6 µg/day: 2.8 ± 0.41 ng/mL, p < 0.01) was associated with impaired GLS (Sham: -22.1 ± 0.8%, 6 µg/day: -14.7 ± 1.14%, p < 0.01). Activin A infusion (6 µg/day) increased beta-myosin heavy chain expression in heart tissue, indicating cardiac injury. In summary, our findings indicate that increasing levels of activin A during pregnancy induces cardiac dysfunction and supports the concept that activin A may serve as a possible mediator of PE-induced cardiac dysfunction.

Integrin affinity modulation critically regulates atherogenic endothelial activation in vitro and in vivo.

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5ß1 integrins in endothelial cells in vitro, and α5ß1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5ß1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5ß1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5ß1 integrins in talin1 L325R endothelial cells suggest that NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.

Nck1, But Not Nck2, Mediates Disturbed Flow-Induced p21-Activated Kinase Activation and Endothelial Permeability.

Background Alteration in hemodynamic shear stress at atheroprone sites promotes endothelial paracellular pore formation and permeability. The molecular mechanism remains unknown. Methods and Results We show that Nck (noncatalytic region of tyrosine kinase) deletion significantly ameliorates disturbed flow-induced permeability, and selective isoform depletion suggests distinct signaling mechanisms. Only Nck1 deletion significantly reduces disturbed flow-induced paracellular pore formation and permeability, whereas Nck2 depletion has no significant effects. Additionally, Nck1 re-expression, but not Nck2, restores disturbed flow-induced permeability in Nck1/2 knockout cells, confirming the noncompensating roles. In vivo, using the partial carotid ligation model of disturbed flow, Nck1 knockout prevented the increase in vascular permeability, as assessed by Evans blue and fluorescein isothiocyanate dextran extravasations and leakage of plasma fibrinogen into the vessel wall. Domain swap experiments mixing SH2 (phosphotyrosine binding) and SH3 (proline-rich binding) domains between Nck1 and Nck2 showed a dispensable role for SH2 domains but a critical role for the Nck1 SH3 domains in rescuing disturbed flow-induced endothelial permeability. Consistent with this, both Nck1 and Nck2 bind to platelet endothelial adhesion molecule-1 (SH2 dependent) in response to shear stress, but only Nck1 ablation interferes with shear stress-induced PAK2 (p21-activated kinase) membrane translocation and activation. A single point mutation into individual Nck1 SH3 domains suggests a role for the first domain of Nck1 in PAK recruitment to platelet endothelial cell adhesion molecule-1 and activation in response to shear stress. Conclusions This work provides the first evidence that Nck1 but not the highly similar Nck2 plays a distinct role in disturbed flow-induced vascular permeability by selective p21-activated kinase activation.

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor.

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

Cyclic Peptidomimetics as Inhibitor for miR-155 Biogenesis.

miR-155 plays key promoting roles in several cancers and emerges as an important anticancer therapeutic target. However, the discovery of small molecules that target RNAs is challenging. Peptidomimetics have been shown to be a rich source for discovering novel ligands to regulate cellular proteins. However, the potential of using peptidomimetics for RNA targeting is relatively unexplored. To this end, we designed and synthesized members of a novel 320 000 compound macrocyclic peptidomimetic library. An affinity-based screening protocol led to the identification of a pre-miR-155 binder that inhibits oncogenic miR-155 maturation in vitro and in cell and induces cancer cell apoptosis. The results of this investigation demonstrate that macrocyclic peptidomimetics could serve as a new scaffold for RNA targeting.

Design, synthesis and activity of light deactivatable microRNA inhibitor.

miRNAs are key cellular regulators and their dysregulation is associated with many human diseases. They are usually produced locally in a spatiotemporally controlled manner to target mRNAs and regulate gene expression. Thus, developing chemical tools for manipulating miRNA with spatiotemporal precise is critical for studying miRNA. Herein, we designed a strategy to control miRNA biogenesis with light controllable inhibitor targeting the pre-miRNA processing by Dicer. By conjugating two non-inhibiting units, a low affinity Dicer inhibitor and a pre-miRNA binder, through a photocleavable linker, the bifunctional molecule obtained could inhibit miRNA production. Taking advantage of the photocleavable property of the linker, the bifunctional inhibitor can be fragmented into separate non-inhibiting units and therefore be deactivated by light. We expect that this strategy could be applied to generate chemical biological tools that allow light-mediated spatiotemporal control of miRNA maturation and contribute to the study of miRNA function.

Regulating miRNA-21 Biogenesis By Bifunctional Small Molecules.

We report a new strategy to regulate microRNAs (miRNAs) biogenesis by using bifunctional small molecules that consist of a pre-miRNA binding unit connected by a linker to a Dicer inhibiting unit. In this effort, fluorescence polarization-based screening was used to identify neomycin as a pre-miR-21 binding ligand. Although neomycin cannot inhibit miR-21 maturation, linking it to the RNase inhibitor 1 forms the bifunctional conjugate 7A, which inhibits the production of miR-21. We expect that this strategy will be applicable to design other molecules for miRNA regulation.