Chronic anemia is associated with systemic endothelial dysfunction.

Background: In acute myocardial infarction and heart failure, anemia is associated with adverse clinical outcomes. Endothelial dysfunction (ED) is characterized by attenuated nitric oxide (NO)-mediated relaxation responses which is poorly studied in chronic anemia (CA). We hypothesized that CA is associated with ED due to increased oxidative stress in the endothelium. Methods: CA was induced by repeated blood withdrawal in male C57BL/6J mice. Flow-Mediated Dilation (FMD) responses were assessed in CA mice using ultrasound-guided femoral transient ischemia model. Tissue organ bath was used to assess vascular responsiveness of aortic rings from CA mice, and in aortic rings incubated with red blood cells (RBCs) from anemic patients. In the aortic rings from anemic mice, the role of arginases was assessed using either an arginase inhibitor (Nor-NOHA) or genetic ablation of arginase 1 in the endothelium. Inflammatory changes in plasma of CA mice were examined by ELISA. Expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS), myeloperoxidase (MPO), 3-Nitrotyrosine levels, and 4-Hydroxynonenal (4-HNE) were assessed either by Western blotting or immunohistochemistry. The role of reactive oxygen species (ROS) in ED was assessed in the anemic mice either supplemented with N-Acetyl cysteine (NAC) or by in vitro pharmacological inhibition of MPO. Results: The FMD responses were diminished with a correlation to the duration of anemia. Aortic rings from CA mice showed reduced NO-dependent relaxation compared to non-anemic mice. RBCs from anemic patients attenuated NO-dependent relaxation responses in murine aortic rings compared to non-anemic controls. CA results in increased plasma VCAM-1, ICAM-1 levels, and an increased iNOS expression in aortic vascular smooth muscle cells. Arginases inhibition or arginase1 deletion did not improve ED in anemic mice. Increased expression of MPO and 4-HNE observed in endothelial cells of aortic sections from CA mice. NAC supplementation or inhibition of MPO improved relaxation responses in CA mice. Conclusion: Chronic anemia is associated with progressive endothelial dysfunction evidenced by activation of the endothelium mediated by systemic inflammation, increased iNOS activity, and ROS production in the arterial wall. ROS scavenger (NAC) supplementation or MPO inhibition are potential therapeutic options to reverse the devastating endothelial dysfunction in chronic anemia.

Circulating MicroRNA-122-5p Is Associated With a Lack of Improvement in Left Ventricular Function After Transcatheter Aortic Valve Replacement and Regulates Viability of Cardiomyocytes Through Extracellular Vesicles.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is a well-established treatment option for high- and intermediate-risk patients with severe symptomatic aortic valve stenosis. A majority of patients exhibit improvements in left ventricular ejection fraction (LVEF) after TAVR in response to TAVR-associated afterload reduction. However, a specific role for circulating microRNAs (miRNAs) in the improvement of cardiac function for patients after TAVR has not yet been investigated. Here, we profiled the differential expression of miRNAs in circulating extracellular vesicles (EVs) in patients after TAVR and, in particular, the novel role of circulating miR-122-5p in cardiomyocytes. METHODS: Circulating EV-associated miRNAs were investigated by use of an unbiased Taqman-based human miRNA array. Several EV miRNAs (miR-122-5p, miR-26a, miR-192, miR-483-5p, miR-720, miR-885-5p, and miR-1274) were significantly deregulated in patients with aortic valve stenosis at day 7 after TAVR compared with the preprocedural levels in patients without LVEF improvement. The higher levels of miR-122-5p were negatively correlated with LVEF improvement at both day 7 (r=-0.264 and P=0.015) and 6 months (r=-0.328 and P=0.0018) after TAVR. RESULTS: Using of patient-derived samples and a murine aortic valve stenosis model, we observed that the expression of miR-122-5p correlates negatively with cardiac function, which is associated with LVEF. Mice with graded wire injury-induced aortic valve stenosis demonstrated a higher level of miR-122-5p, which was related to cardiomyocyte dysfunction. Murine ex vivo experiments revealed that miR-122-5p is highly enriched in endothelial cells compared with cardiomyocytes. Coculture experiments, copy-number analysis, and fluorescence microscopy with Cy3-labeled miR-122-5p demonstrated that miR-122-5p can be shuttled through large EVs from endothelial cells into cardiomyocytes. Gain- and loss-of-function experiments suggested that EV-mediated shuttling of miR-122-5p increases the level of miR-122-5p in recipient cardiomyocytes. Mechanistically, mass spectrometry, miRNA pulldown, electrophoretic mobility shift assay, and RNA immunoprecipitation experiments confirmed that miR-122-5p interacts with the RNA-binding protein hnRNPU (heterogeneous nuclear ribonucleoprotein U) in a sequence-specific manner to encapsulate miR-122-5p into large EVs. On shuttling, miR-122-5p reduces the expression of the antiapoptotic gene BCL2 by binding to its 3' untranslated region to inhibit its translation, thereby decreasing the viability of target cardiomyocytes. CONCLUSIONS: Increased levels of circulating proapoptotic EV-incorporated miR-122-5p are associated with reduced LVEF after TAVR. EV shuttling of miR-122-5p regulates the viability and apoptosis of cardiomyocytes in a BCL2-dependent manner.

Safety and efficacy of iron supplementation after myocardial infarction in mice with moderate blood loss anaemia.

AIMS: Iron deficiency is frequently observed in patients with acute coronary syndrome and associates with poor prognosis after acute myocardial infarction (AMI). Anaemia is linked to dysregulation of iron metabolism, red blood cell dysfunction, and increased reactive oxygen species generation. Iron supplementation in chronic heart failure is safe and improves cardiac exercise capacity. Increases in iron during ischaemia or immediately after reperfusion are associated with detrimental effects on left ventricular (LV) function. The safety and applicability of iron during or immediately after reperfusion of AMI in anaemia are not known. We aimed to study the safety and efficacy of iron supplementation within 1 h or deferred to 24 h after reperfusion of AMI by analysing LV function and infarct size. METHODS AND RESULTS: In a mouse model of moderate blood loss anaemia (n = 6-8 mice/group), the effects of iron supplementation (20 mg iron as ferric carboxymaltose per kg body weight) within 1 h and deferred to 24 h after ischaemia/reperfusion were assessed. Cardiac function was analysed in vivo by echocardiography at baseline (Day 3) with and without anaemia, after AMI (24 h), and after administration of intravenous iron. Anaemia was characterized by iron deficiency and a trend towards increased haemolysis, which was supported by increased plasma free-haemoglobin [sham vs. anaemia (n = 8/group): P < 0.05]. Anaemia increased heart rate, LV end-diastolic volume, stroke volume, and cardiac output, while LV end-systolic volume remained unchanged at baseline. Superimposition of AMI deteriorated global LV function, whereas infarct sizes remained unaffected [sham vs. anaemia (n = 6/group): P = 0.9]. Deferred iron supplementation 24 h after ischaemia/reperfusion resulted in reversal of end-systolic volume increase and reduced infarct size [% of area at risk: sham vs. anaemia + iron after 24 h; (n = 6/group); 48 ± 7 vs. 38 ± 7; P < 0.05], whereas administration within 1 h after reperfusion was neutral [sham vs. anaemia + iron; (n = 6/group); 48 ± 7 vs. 42 ± 8; P = 0.56]. Moreover, iron application after reperfused AMI showed unaltered mortality compared with sham. CONCLUSIONS: Iron supplementation 24 h after reperfusion of AMI is safe and reversed enlargement of end-systolic volume after AMI resulting in increased stroke volume and cardiac output. This highlights its potential as adjunctive treatment in anaemia with ID after reperfused AMI. Time point of iron application after reperfusion appears critical.

Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation.

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

YAP/TAZ Are Required to Suppress Osteogenic Differentiation of Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) represent the prevailing cell type of arterial vessels and are essential for blood vessel structure and homeostasis. They have substantial potential for phenotypic plasticity when exposed to various stimuli in their local microenvironment. How VSMCs maintain their differentiated contractile phenotype is still poorly understood. Here we demonstrate that the Hippo pathway effectors YAP and TAZ play a critical role in maintaining the differentiated contractile phenotype of VSMCs. In the absence of YAP/TAZ, VSMCs lose their differentiated phenotype and undergo osteogenic differentiation, which results in vascular calcification. Osteogenic transdifferentiation was accompanied by the upregulation of Wnt target genes. The absence of YAP/TAZ in VSMCs led to Disheveled 3 (DVL3) nuclear translocation and upregulation of osteogenesis-associated genes independent of canonical Wnt/ß-catenin signaling activation. Our data indicate that cytoplasmic YAP/TAZ interact with DVL3 to avoid its nuclear translocation and osteogenic differentiation, thereby maintaining the differentiated phenotype of VSMCs.

The Lipopeptide MALP-2 Promotes Collateral Growth.

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA (MALP-2) to promote blood flow recovery in mice. Hypercholesterolemic apolipoprotein E (Apoe)-deficient mice were subjected to microsurgical ligation of the femoral artery. MALP-2 significantly improved blood flow recovery at early time points (three and seven days), as assessed by repeated laser speckle imaging, and increased the growth of pre-existing collateral arteries in the upper hind limb, along with intimal endothelial cell proliferation in the collateral wall and pericollateral macrophage accumulation. In addition, MALP-2 increased capillary density in the lower hind limb. MALP-2 enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) release from endothelial cells and improved the experimental vasorelaxation of mesenteric arteries ex vivo. In vitro, MALP-2 led to the up-regulated expression of major endothelial adhesion molecules as well as their leukocyte integrin receptors and consequently enhanced the endothelial adhesion of leukocytes. Using the experimental approach of femoral artery ligation (FAL), we achieved promising results with MALP-2 to promote peripheral blood flow recovery by collateral artery growth.

Orphan G Protein-Coupled Receptor GPRC5B Controls Smooth Muscle Contractility and Differentiation by Inhibiting Prostacyclin Receptor Signaling.

BACKGROUND: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear. METHODS: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM-Gprc5b-KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo. RESULTS: Mesenteric arteries from SMC-specific Gprc5b-KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM-Gprc5b-KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap. CONCLUSIONS: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.

Myogenic vasoconstriction requires G12/G13 and LARG to maintain local and systemic vascular resistance.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Deletion of endothelial arginase 1 does not improve vasomotor function in diabetic mice.

Endothelial arginase 1 was ablated to assess whether this prevents hyperglycemia-induced endothelial dysfunction by improving arginine availability for nitric oxide production. Endothelial Arg1-deficient mice (Arg1-KOTie2 ) were generated by crossing Arg1fl/fl (controls) with Tie2Cretg/- mice and analyzed by immunohistochemistry, measurements of hemodynamics, and wire myography. Ablation was confirmed by immunohistochemistry. Mean arterial blood pressure was similar in conscious male control and Arg1-KOTie2 mice. Depletion of circulating arginine by intravenous infusion of arginase 1 or inhibition of nitric oxide synthase activity with L-NG -nitro-arginine methyl ester increased mean arterial pressure similarly in control (9 ± 2 and 34 ± 2 mmHg, respectively) and Arg1-KOTie2 mice (11 ± 3 and 38 ± 4 mmHg, respectively). Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Arg1-KOTie2 and control animals by wire myography. Diabetes was induced in 10-week-old control and Arg1-KOTie2 mice with streptozotocin, and vasomotor responses were studied 10 weeks later. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in normoglycemic control and Arg1-KOTie2 mice. The relaxing response to acetylcholine was dependent on the availability of extracellular l-arginine. In the diabetic mice, arterial relaxation responses to endothelium-dependent hyperpolarization and to exogenous nitric oxide were impaired. The data show that endothelial ablation of arginase 1 in mice does not markedly modify smooth muscle and endothelial functions of a resistance artery under normo- and hyperglycemic conditions.

Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release.

Arterial blood pressure is controlled by vasodilatory factors such as nitric oxide (NO) that are released from the endothelium under the influence of fluid shear stress exerted by flowing blood. Flow-induced endothelial release of ATP and subsequent activation of Gq/G11-coupled purinergic P2Y2 receptors have been shown to mediate fluid shear stress-induced stimulation of NO formation. However, the mechanism by which fluid shear stress initiates these processes is unclear. Here, we have shown that the endothelial mechanosensitive cation channel PIEZO1 is required for flow-induced ATP release and subsequent P2Y2/Gq/G11-mediated activation of downstream signaling that results in phosphorylation and activation of AKT and endothelial NOS. We also demonstrated that PIEZO1-dependent ATP release is mediated in part by pannexin channels. The PIEZO1 activator Yoda1 mimicked the effect of fluid shear stress on endothelial cells and induced vasorelaxation in a PIEZO1-dependent manner. Furthermore, mice with induced endothelium-specific PIEZO1 deficiency lost the ability to induce NO formation and vasodilation in response to flow and consequently developed hypertension. Together, our data demonstrate that PIEZO1 is required for the regulation of NO formation, vascular tone, and blood pressure.

Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.

Endothelial arginine resynthesis contributes to the maintenance of vasomotor function in male diabetic mice.

AIM: Argininosuccinate synthetase (ASS) is essential for recycling L-citrulline, the by-product of NO synthase (NOS), to the NOS substrate L-arginine. Here, we assessed whether disturbed arginine resynthesis modulates endothelium-dependent vasodilatation in normal and diabetic male mice. METHODS AND RESULTS: Endothelium-selective Ass-deficient mice (Assfl/fl/Tie2Cretg/- = Ass-KOTie2) were generated by crossing Assfl/fl mice ( = control) with Tie2Cre mice. Gene ablation in endothelial cells was confirmed by immunohistochemistry. Blood pressure (MAP) was recorded in 34-week-old male mice. Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Ass-KOTie2 and control animals. At the age of 10 weeks, diabetes was induced in control and Ass-KOTie2 mice by streptozotocin injections. Vasomotor responses of diabetic animals were studied 10 weeks later. MAP was similar in control and Ass-KOTie2 mice. Depletion of circulating L-arginine by arginase 1 infusion or inhibition of NOS activity with L-NAME resulted in an increased MAP (10 and 30 mmHg, respectively) in control and Ass-KOTie2 mice. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in healthy control and Ass-KOTie2 mice. However, in diabetic Ass-KOTie2 mice, relaxation responses to acetylcholine and endothelium-derived NO (EDNO) were significantly reduced when compared to diabetic control mice. CONCLUSIONS: Absence of endothelial citrulline recycling to arginine did not affect blood pressure and systemic arterial vasomotor responses in healthy mice. EDNO-mediated vasodilatation was significantly more impaired in diabetic Ass-KOTie2 than in control mice demonstrating that endothelial arginine recycling becomes a limiting endothelial function in diabetes.

Endothelium-dependent hyperpolarization-related relaxations diminish with age in murine saphenous arteries of both sexes.

BACKGROUND AND PURPOSE: We investigated the effects of aging on the contributions of NO and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation in saphenous arteries of male and female C57BL/6J mice aged 12, 34 and 64 weeks. EXPERIMENTAL APPROACH: Vasomotor responses of saphenous arteries were analysed by wire myography in the absence and presence of stimuli of the endothelium, inhibitors of NOS, and inhibitors and stimulants of small (KCa 2.3) and intermediate (KCa 3.1) conductance calcium-activated potassium channels. KEY RESULTS: Arterial relaxing responses to sodium nitroprusside and to ACh in the absence of pharmacological inhibitors (indomethacin and L-NAME), were similar in all age groups and sexes, but those mediated by endothelium-derived NO were slightly but significantly increased in 64-week-old male mice. In the presence of inhibitors, 12-week-old animals showed pronounced ACh-induced relaxation, which was significantly reduced in 34- and 64-week-old mice of both sexes. The EDH-related component of ACh-induced relaxations was abolished by TRAM-34 (KCa 3.1 blocker) or UCL 1684 (KCa 2.3 blocker). Although the maximal relaxation induced by NS309 (KCa activator) was not affected by aging, the sensitivity for NS309 significantly decreased with aging. The presence of SKA-31 (KCa modulator) potentiated relaxations induced by ACh in arteries of 12-week-old but not older mice. CONCLUSION AND IMPLICATIONS: In a small muscular artery of mice of either sex, total endothelium-dependent relaxation is not affected by age. However, possibly due to changes in KCa channel function, the contribution of EDH to endothelium-dependent relaxations decreased with age. The contribution of endothelium-derived NO increases in old male mice.