Tissue ischemia, such as transient myocardial ischemia, leads to release of cellular RNA including microRNA(miRNA) into the circulation and extracellular (ex-) space, but the biological function of the ex-RNA is poorly understood. We recently reported that cardiac RNA of both human and rodent origins induced cytokine production and immune cell activation. However, the identity of the ex-RNA responsible for the proinflammatory effect remains unclear. In the current study, using an miRNA array, we profiled the plasma miRNAs 4 h after transient myocardial ischemia (45 min) or sham procedure. Among 38 plasma miRNAs that were elevated following ischemia, eight were tested for their ability to induce cytokine response in macrophages and cardiomyocytes. We found that six miRNA mimics (miR-34a, -122, -133a, -142, -146a, and -208a) induced cytokine production in a dose-dependent manner. The effects of miRNAs (miR-133a, -146a, and -208a) were diminished by uridine→adenosine mutation and by RNase pretreatment. The miRNA-induced cytokine (MIP-2, TNF-α, and IL-6) production was abolished in cells deficient of TLR7 or MyD88, or by a TLR7 antagonist, but remained the same in TLR3- or Trif-deficient cells. In vivo, mice i.p. injected with miR-133a or miR-146a had marked peritoneal neutrophil and monocyte migration, which was significantly attenuated in TLR7 mice. Moreover, locked nucleic acid anti-miRNA inhibitors of these six miRNAs markedly reduced cardiac RNA-induced cytokine production. Taken together, these data demonstrate that ex-miRNA mimics (miR-34a, -122, -133a, -142, -146a, and -208a) are potent innate immune activators and that the miRNAs most likely induce cytokine production and leukocyte migration through TLR7 signaling.

Background: Necrotic extracellular RNA induces inflammation and may contribute to ischemic myocardial injury, but the underlying mechanism is unclear. Results: Isolated cardiac RNA induced cytokine production in cardiomyocytes/immune cells as well as acute peritonitis. Genetic deletion of TLR7 or MyD88 markedly attenuates RNA-mediated cytokine production. Conclusion: Cellular RNA induces cytokine production via TLR7-MyD88 signaling. Significance: Cardiac RNA is a pro-inflammatory mediator sensed by TLR7.

Complement factor B (cfB) is an essential component of alternative pathway (AP) and plays an important role in the pathogenesis of polymicrobial sepsis. However, the mechanism leading to cfB production and AP activation during sepsis remains poorly understood. In this study, we found that the plasma cell-free RNA was significantly increased following cecal ligation and puncture (CLP), an animal model of polymicrobial sepsis, and was closely associated with sepsis severity. qRT-PCR and miRNA array analysis revealed an increase in bacterial RNA and multiple host microRNAs (miR-145, miR-146a, miR-122, miR-210) in the blood following CLP. Treatment with tissue RNA or synthetic miRNA mimics (miR-145, miR-146a, miR-122, miR-34a) induced a marked increase in cfB production in cardiomyocytes or macrophages. The newly synthesized cfB released into medium was biologically active as it participated in the AP activation initiated by cobra venom factor. Genetic deletion of TLR7 or MyD88, but not TLR3, and inhibition of the MAP kinases (JNK and p38) or NF-κB abolished miR-146a-induced cfB production. In vivo, CLP led to a significant increase in splenic cfB expression, which was correlated with the plasma RNA or miRNA levels. Peritoneal injection of RNA or miR-146a led to a rise in cfB expression in the peritoneal space, which was attenuated in MyD88-KO or TLR7-KO mice, respectively. These findings demonstrate that host cellular RNA and specific miRNAs are released into the circulation during polymicrobial sepsis and may function as extracellular mediators capable of promoting cfB production and AP activation through the specific TLR7 and MyD88 signaling.

ObjectivesThe velocity vector imaging (VVI) technique is useful to assess regional myocardial mechanics. The aim of this study was to evaluate the usefulness of this technology in assessing regional right ventricular longitudinal functions in the fetus and to establish a nomogram of the right ventricle (RV). MethodsWe studied 170 healthy fetuses that were divided into five groups based on gestational age. Dynamic digital images of four chambers were collected and analyzed off-line. The longitudinal VVI parameters were calculated in the right free wall and ventricular septum, respectively.Results A total of 151 out of 170 fetuses (89%) were successfully analyzed using VVI, with good interand intra-observer agreements. Normal values for velocity, strain, and strain rate were established. The tissue velocity gradually decreased from basal to apical segment (P < 0.05), whereas strain and strain rate remained stable. The tissue velocity increased with gestational age (P < 0.05), whereas strain and strain rate were stable (P > 0.05). ConclusionFetal myocardial velocity, strain, and strain rate measurements are easy to obtain and are reproducible. From mid-to-late gestation, the longitudinal tissue velocity of the RV increases with gestational age, whereas strain and strain rate remain stable. These results indicate that myocardial contractility is established in mid-gestation and remains constant throughout gestation.

Objectives To use three-dimensional (3D) power Doppler ultrasound to investigate cerebral blood flow perfusion in fetuses with congenital heart disease (CHD). Methods The vascularization index (VI), flow index (FI) and vascularization flow index (VFI) in the total MDI (r = 0.243 and 0.203, P = 0.126 and 0.204, respectively

No abstract

Objectives Toll-like receptors (TLRs) and complement are two components of the innate immunity. Factor B (cfB) is essential for the alternative pathway (AP) of complement activation. We have recently reported that cfB is significantly up-regulated in the kidney and may contribute to acute tubular injury in an animal model of sepsis. The current study investigates the mechanisms responsible for the cfB up-regulation and its role in sodium transporter expression in tubular cells during sepsis. Design Animal study. Setting Laboratory investigation. Subjects C57BL/6 J wide-type (WT), cfB−/−, and Nfkb1tm1Bal p50−/− mice. Interventions Human proximal tubular cells (HK-2) and mouse tubular epithelial cells (MTECs) were stimulated with TLR agonists. Bay 11-7082 was used to block NF-κB pathway. AP activation was detected by C3 zymosan deposition. Polymicrobial sepsis was created by cecal ligation and puncture (CLP). Sodium transporter gene expression was determined by qRT-PCR. Measurements and Main Results The agonists for TLR4 (LPS) or TLR3 (poly I:C) induced a marked increase in cfB expression in HK-2 and MTECs both at gene and protein levels. The TLR1/2 agonist, Pam3cys, induced cfB production only in HK-2 cells, not MTEC. The TLR9 ligand, CpG, failed to induce cfB production either in HK-2 cells or MTEC. LPS/poly I:C-induced cfB up-regulation was blocked by Bay 11-7082, a potent inhibitor of NF-κB signaling, and in MTECs deficient in p50 subunit of NF-κB. Media from the LPS-treated MTEC cultures contained de novo synthesized cfB and led to functional AP activation. In a CLP model, WT septic mice had down-regulated expression of sodium transporters in the kidney compared with the sham. In comparison, cfB−/− mice or mice treated with anti-cfB displayed preserved levels of Na+/K+ ATPase-α1 following sepsis. Conclusions 1) TLR3/4 activation is sufficient to induce cfB production via NF-κB pathway and to enhance AP activation in the kidney tubular epithelial cells; 2) cfB may contribute to the down-regulation of certain sodium transporter expression during sepsis.

Objectives To establish Z‐scores for the diameter and blood flow volume of the umbilical vein (UV) in normal fetuses. Methods This was a prospective study involving 907 normal fetuses. We measured the diameter (Duv) of two different segments of the UV (FUV: the free loop of the UV; FIUV: the fetal intra‐abdominal UV). Next, we calculated the blood flow volume (Quv). Z‐scores were created for both Duv and Quv using gestational age, femur length, and biparietal diameter as independent variables. Results We successfully acquired 858 (94.6%) normal fetal measurements. Between 20 and 39 weeks, the Duv of the FUV and FIUV increased from 0.38 to 0.80 cm and from 0.33 to 0.70 cm, respectively. The Quv of the FUV and FIUV increased from 32.66 to 381.88 ml/min and from 31.50 to 360.15 ml/min, respectively. Linear or quadratic regression models were best fitted between the parameters of UV and the independent variables. Z‐scores were successfully determined for both the Duv and Quv. Conclusions The calculation of Z‐scores for the Duv and Quv is simple by applying standard statistical methods. These Z‐scores may be useful to evaluate placental circulation and provide a rationale for monitoring and evaluating the prognosis of fetuses.