[Effect of the ischemic post-conditioning on the prevention of the cardio-renal damage in patients with acute ST-segment elevation myocardial infarction after primary percutaneous coronary intervention].

Objective: To evaluate the effect of the ischemic post-conditioning (IPC) on the prevention of the cardio-renal damage in patients with acute ST-segment elevation myocardial infarction (STEMI) after primary percutaneous coronary intervention (PPCI). Methods: A total of 251 consecutive STEMI patients underwent PPCI in the heart center of Tianjin Third Central Hospital from January 2012 to June 2014 were enrolled in this prospective, randomized, control, single-blinded, clinical registry study. Patients were randomly divided into IPC group (123 cases) and control group (128 cases) with random number table. Patients in IPC group underwent three times of inflation/deflation with low inflation pressure using a balloon catheter within one minute after culprit vessel blood recovery, and then treated by PPCI. Patients in control group received PPCI procedure directly. The basic clinical characteristics, incidence of reperfusion arrhythmia during the procedure, the rate of electrocardiogram ST-segment decline, peak value of myocardial necrosis markers, incidence of contrast induced acute kidney injury(CI-AKI), and one-year major adverse cardiovascular events(MACE) which including myocardial infarction again, malignant arrhythmia, rehospitalization for heart failure, repeat revascularization, stroke, and death after the procedure were analyzed between the two groups. Results: The age of IPC group and control group were comparable((61.2±12.6) vs. (64.2±12.1) years old, P=0.768). The incidence of reperfusion arrhythmia during the procedure was significantly lower in the IPC group than in the control group(42.28% (52/123) vs. 57.03% (73/128), P=0.023). The rate of electrocardiogram ST-segment decline immediately after the procedure was significantly higher in the IPC group than in the control group (77.24% (95/123) vs. 64.84% (83/128), P=0.037). The peak value of myocardial necrosis markers after the procedure were significantly lower in the IPC group than in the control group(creatine kinase: 1 257 (682, 2 202) U/L vs. 1 737(794, 2 816)U/L, P=0.029; creatine kinase-MB: 123(75, 218)U/L vs.165(95, 288)U/L, P=0.010). The rate of CI-AKI after the procedure was significantly lower in the IPC group than in the control group(5.69%(7/123) vs. 14.06%(18/128), P=0.034). The rate of the one-year MACE was significantly lower in the IPC group than in the control group(7.32%(9/123) vs. 15.63% (20/128), P=0.040). Conclusion: The IPC strategy performed eight before PPCI can reduce myocardial ischemia- reperfusion injury, decline the rates of CI-AKI and one-year MACE significantly in STEMI patients, thus has a significant protective effect on heart and kidney in STEMI patients. Clinical Trial Registration Chinese Clinical Trials Registry, ChiCTR-ICR-15006590.

Analysis of antithrombotic therapy in elderly patients with atrial fibrillation.

This study aimed to analyze the impact factors and outcome of antithrombotic therapy in elderly patients over 65 years old that suffered from atrial fibrillation (AF). A total of 256 elderly patients with AF over 65 years old were divided into 3 groups: 65-74 years old (N = 86), 75-84 years old (N = 122), and over 85 years old (N = 48). The clinical characteristics, antithrombotic therapy, and its related impact factors were retrospectively analyzed. Of all patients, 187 received antithrombotic therapy. In the 65-74 year-old group, 78 patients received antiplatelet treatment (90.7%) and 5 patients received anticoagulation treatment (5.8%). In the 75-84 year-old group, 76 patients received antiplatelet treatment (62.3%) and 14 patients received anticoagulation treatment (11.5%). In the group of over 85 year-olds, 33 patients received antiplatelet therapy (68.8%) and 4 patients received anticoagulation treatment (8.3%). Eleven patients had deep vein thrombosis and atrial thrombosis during antiplatelet therapy (5.9%), 5 patients had gastrointestinal hemorrhage after antiplatelet therapy (2.7%), 2 patients had gastrointestinal bleeding, and 3 patients had brain hemorrhage after anticoagulation treatment (21.7%). Suboptimal antithrombotic therapy was observed in the elderly patients with AF, partly owing to the risks of both thromboembolism and bleeding.

Circ_CHFR expedites cell growth, migration and inflammation in ox-LDL-treated human vascular smooth muscle cells via the miR-214-3p/Wnt3/ß-catenin pathway.

OBJECTIVE: Atherosclerosis (AS) is a representative inflammatory vascular disease. This study explored the molecular pathogenesis of AS based on circular RNA (circRNA), the checkpoint with forkhead-associated and ring-finger domains (circ_CHFR). PATIENTS AND METHODS: The cell model of AS in vitro was established by stimulating human vascular smooth muscle cells (VSMCs) with oxidized low-density lipoprotein (ox-LDL). The RNA expression was measured by quantitative Real-time polymerase chain reaction (qRT-PCR). Cell viability and colony formation ability were separately evaluated using 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) and colony formation assay. Cell migration was assessed via the transwell assay. The inflammation injury was analyzed by enzyme-linked immunosorbent assay (ELISA). Associated proteins were determined through Western blot. The combination of hypothetic targets was ascertained using Dual-Luciferase reporter assay. RESULTS: Circ_CHFR was up-regulated in AS serums and ox-LDL-stimulated VSMCs. Circ_CHFR depletion weakened the ox-LDL-induced promotion of cell growth, migration and inflammation in VSMCs. Circ_CHFR positively regulated Wnt3 expression and the downregulation of Wnt3 abrogated the ox-LDL-triggered injuries in VSMCs. Circ_CHFR functioned as the sponge of microRNA-214-3p (miR-214-3p) and miR-214-3p targeted Wnt3. Circ_CHFR regulated cell growth, migration and inflammation via regulating the expression of Wnt3 as a competitive endogenous RNA (ceRNA) of miR-214 in ox-LDL-treated VSMCs. Circ_CHFR/miR-214-3p axis mediated the Wnt3/ß-catenin signal pathway. CONCLUSIONS: Circ_CHFR contributed to the progression of AS through the miR-214-3p/Wnt3/ß-catenin signals, which illuminated the molecular mechanism of AS and suggested circ_CHFR might be an index for AS treatment.

Neuronal differentiation rescued by implantation of Weaver granule cell precursors into wild-type cerebellar cortex.

The migration of postmitotic neurons away from compact, germinal zones is a critical step in neuronal differentiation in the developing brain. To study the molecular signals necessary for cerebellar granule cell migration in situ, precursor cells from the neurological mutant mouse weaver, an animal with phenotypic defects in migration, were implanted into the external germinal layer (EGL) of wild-type cerebellar cortex. In this region, labeled weaver precursor cells of the EGL progressed through all stages of granule neuron differentiation, including the extension of parallel fibers, migration through the molecular and Purkinje cell layers, positioning in the internal granule cell layer, and extension of dendrites. Thus, the weaver gene acts nonautonomously in vivo, and local cell interactions may induce early steps in neuronal differentiation that are required for granule cell migration.

Two-amino acid molecular switch in an epithelial morphogen that regulates binding to two distinct receptors.

Ectodysplasin, a member of the tumor necrosis factor family, is encoded by the anhidrotic ectodermal dysplasia (EDA) gene. Mutations in EDA give rise to a clinical syndrome characterized by loss of hair, sweat glands, and teeth. EDA-A1 and EDA-A2 are two isoforms of ectodysplasin that differ only by an insertion of two amino acids. This insertion functions to determine receptor binding specificity, such that EDA-A1 binds only the receptor EDAR, whereas EDA-A2 binds only the related, but distinct, X-linked ectodysplasin-A2 receptor (XEDAR). In situ binding and organ culture studies indicate that EDA-A1 and EDA-A2 are differentially expressed and play a role in epidermal morphogenesis.

Axon extension and retraction by leech neurons: severing early projections to peripheral targets prevents normal retraction of other projections.

During leech embryogenesis, interactions between homologous neurons in neighboring segments lead to the selective retraction of longitudinal axonal projections by midbody AP and AE neurons, which maintain lateral axonal projections to the periphery. Results of experiments reported here show that disconnecting the lateral projections from the periphery rescues the projections normally fated to retract. We propose that these neurons normally progress through two states during early development, one in which they are insensitive to interactions with their homologs (state A) and a second in which they are sensitive (state B). Establishment of lateral connections with their targets triggers the switch from state A to state B; cutting these projections puts neurons back to state A.

Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears.

For mammalian cochlear hair cells, fate determination is normally completed by birth. We report here that overexpression of Math1, a mouse homolog of the Drosophila gene atonal, in postnatal rat cochlear explant cultures resulted in extra hair cells. Surprisingly, we found that the source of the ectopic hair cells was columnar epithelial cells located outside the sensory epithelium in the greater epithelial ridge, which normally give rise to inner sulcus cells. Moreover, Math1 expression also facilitated conversion of postnatal utricular supporting cells into hair cells. Thus Math1 was sufficient for the production of hair cells in the ear, and immature postnatal mammalian inner ears retained the competence to generate new hair cells.

miR-302/367/LATS2/YAP pathway is essential for prostate tumor-propagating cells and promotes the development of castration resistance.

Clinical intervention for patients with advanced prostate cancer (PCa) remains challenging due to the inevitable recurrence of castration-resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT). Cancer stem cells (CSCs) with serial tumor-propagating capacity are considered to be the driving force for PCa progression and recurrence. In this study, we report that the miR-302/367 cluster, a previously identified potent pluripotency regulator, is upregulated in prostate tumors. Specifically, the forced expression of the miR-302/367 cluster accelerates the in vitro and in vivo growth of PCa cells and their resistance to androgen ablation, whereas the knockdown of the miR-302/367 cluster using anti-sense RNA suppresses the incidence of formation, growth rate and endpoint weight of PCa cell tumors. Mechanistically, we find that LATS2, a key component of the tumor-suppressive Hippo signaling pathway, acts as a direct target of the miR-302/367 cluster in PCa cells. The downregulation of LATS2 by the miR-302/367 cluster reduces the phosphorylation and enhances the nuclear translocation of the YAP oncoprotein. Conversely, the restoration of LATS2 expression abrogates the tumor-promoting effects of forced miR-302/367 cluster expression. Collectively, the potent pluripotency regulator-triggered miR-302/367/LATS2/YAP pathway is essential for prostate tumor-propagating cells and promotes castration resistance. Thus, targeting this signaling axis may represent a promising therapeutic strategy for CRPC.

Dynamics of notch expression during murine prostate development and tumorigenesis.

Notch signaling has been widely demonstrated to be responsible for cell fate determination during normal development and implicated in human T-cell leukemia and mouse mammary carcinomas. Here we show that Notch signaling may be involved in prostatic development and cancer cell growth. In situ hybridization and reverse transcription-PCR analyses revealed that Notch1 was expressed in prostate epithelial cells during normal development and in prostate cancer cells. Characterization of Notch1-green fluorescent protein transgenic mice, in which the expression of reporter green fluorescent protein is under the control of the Notch1 promoter, indicated that Notch1-expressing cells were associated with the basal epithelial cell population in the prostate. Examination of the transgenic adenocarcinoma of the mouse prostate showed that expression of Notch1 was elevated in malignant prostatic epithelial cells of primary and metastatic tumors. Expression of Notch ligands, however, was low or undetectable in cultured prostate cancer cells or in malignant prostatic epithelial cells in transgenic adenocarcinoma of the mouse prostate. Furthermore, overexpression of a constitutively active form of Notch1 inhibited the proliferation of various prostate cancer cells, including DU145, LNCaP, and PC3 cells. Taken together, our data indicate for the first time that Notch signaling may play a role in murine prostatic development and tumorigenesis.

Shp2 promotes metastasis of prostate cancer by attenuating the PAR3/PAR6/aPKC polarity protein complex and enhancing epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT), marked by the dissolution of cell-cell junctions, loss of cell polarity and increased cell motility, is one of the essential steps for prostate cancer metastasis. However, the underlying mechanism has not been fully explored. We report in this study that Shp2 is upregulated in prostate cancers and is associated with a poor disease outcome, namely tumor metastasis and shortened patient survival. Overexpression of wild-type Shp2 or an oncogenic Shp2 mutant leads to increased prostate cancer cell proliferation, colony and sphere formation, and in vivo tumor formation. Opposite effects are seen in Shp2-knockdown cells. Moreover, Shp2 promotes in vitro migration and in vivo metastasis of prostatic tumor cells. Mechanistically, Shp2 interacts with PAR3 (partitioning-defective 3) via its Src homology-2 domain. Ectopic expression of Shp2 attenuates the phosphorylation of PAR3 and the formation of the PAR3/PAR6/atypical protein kinase C polarity protein complex, resulting in disrupted cell polarity, dysregulated cell-cell junctions and increased EMT. These findings provide a novel mechanism by which oncogenic signal-transduction molecules regulate cell polarity and induction of EMT.

Distinct expression patterns of notch family receptors and ligands during development of the mammalian inner ear.

The cochlea and vestibular structures of the inner ear labyrinth develop from the otic capsule via step-wise regional and cell fate specification. Each inner ear structure contains a sensory epithelium, composed of hair cells, the mechanosensory transducers, and supporting cells. We examined the spatio-temporal expression of genes in the Notch signaling pathway, Notch receptors (Notch1-4) and two ligands, Jagged1 and Delta1, in the developing mammalian inner ear. Our results show that Notch1 and Jagged1 are first expressed in the otic vesicle, likely involved in differentiation of the VIIIth nerve ganglion neurons, and subsequently within the inner ear sensory epithelia, temporally coincident with initial hair cell differentiation. Notch1 expression is specific to hair cells and Jagged1 to supporting cells. Their expression persists into adult. Notch2, Notch3, Notch4, and Delta1 are excluded from the inner ear epithelia. These data support the hypothesis that Notch signaling is involved in hair cell differentiation during inner ear morphogenesis.

Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.

The utility of lentivirus as a gene delivery vector in the cochlea was evaluated in vitro and in vivo. Lentivirus transduction was assessed through expression analysis of a reporter gene, green fluorescent protein (GFP), integrated within the viral genome. In vitro characterization of lentivirus-GFP was assessed by infection of explants from cochleas of neonatal rat. The lentiviral vector transduced both spiral ganglion neurons (SGNs) and glial cells. In vivo characterization of lentivirus-GFP was assessed by directly infusing the vector into the guinea pig cochlea via an osmotic minipump. Sections of lentivirus-infused cochlea revealed a highly restricted fluorescence pattern limited to the periphery of the perilymphatic space. Transduction of SGNs and glial cells by lentivirus in vitro but not in vivo suggests limited dissemination of the viral vector from the perilymphatic space. The cellular and tissue architecture of the lentivirus-infused cochlea was intact and free of inflammation. Restricted transduction of cell types confined to the periphery of the perilymphatic space by the lentivirus is ideal for stable production of gene products secreted into the perilymph.

Therapeutic potential of neurotrophins for treatment of hearing loss.

Degeneration of spiral ganglion neurons (SGNs) and hair cells in the cochlea induced by aging, injury, ototoxic drugs, acoustic trauma, and various diseases is the major cause of hearing loss. Discovery of growth factors that can either prevent SGN and hair-cell death or stimulate hair-cell regeneration would be of great interest. Studies over the past several years have provided evidence that specific neurotrophins are potent survival factors for SGNs and protect these neurons from ototoxic drugs in vitro and in vivo. Current research focuses more on understanding the mechanism of hair-cell regeneration/differentiation and identification of growth factors that can stimulate hair-cell regeneration. SGNs are required to relay the signal to the central nervous system even when a cochlear implant is used to replace hair-cell function or in the case that cochlear sensory epithelium can be stimulated to regenerate new hair cells successfully. Therefore, neurotrophins may have their therapeutic value in prevention and treatment of hearing impairment.

Segmental differentiation in the leech central nervous system: proposed segmental homologs of the heart accessory neurons.

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.

Role of neurotrophins and lectins in prevention of ototoxicity.

Degeneration of hair cells (HC) and/or spiral ganglion neurons (SGN) is a major cause of hearing loss. Postnatal rat cochlear explant cultures are used to study the toxic actions of different classes of ototoxins and to identify molecules that can protect SGN and HC from ototoxic damage. Various ototoxins induce differential damage to HC and/or SGN. While gentamicin preferentially causes HC death, sodium salicylate selectively induces degeneration of SGN. In contrast, cisplatin results in destruction of both SGN and HC. Specific neurotrophins, including NT-4/5, BDNF, and NT-3, greatly protect SGN from all three types of ototoxins. In contrast, NGF and other growth factors have no effect. Of the 51 compounds examined, only concanavalin A (Con A), a lectin molecule, significantly protects HC from gentamicin. A dose-dependent study of Con A shows that maximal protection occurred at 100 nM. Further experiments indicates that preincubation of Con A with gentamicin does not form a complex, and coaddition of Con A and gentamicin to bacterial cultures, such as E. Coli cultures, does not interfere with the antibiotic activity of gentamicin. When the other 21 lectins are examined, Erythrina cristagalli lectin and Detura stramonium lectin also show activity similar to Con A. These findings may help elucidate the mechanisms of ototoxins and suggest that specific neurotrophins and lectins may be of therapeutic value in the prevention of ototoxin-induced hearing loss.

Establishment of conditionally immortalized rat utricular epithelial cell lines using a retrovirus-mediated gene transfer technique.

Supporting cells in the inner ear sensory epithelium are most likely hair cell progenitors. In an effort to establish an in vitro model system of hair cell differentiation, we developed immortalized epithelial cell lines by transferring the tsA58 allele of the SV40 large T antigen oncogene into neonatal rat utricular supporting cells using a retrovirus. The established cell lines have been stably maintained continuously for more than 25 passages and display many features similar to primary supporting cells. They grow in patches and assume a polygonal morphology. Immunocytochemical characterization of the established cell lines reveals that these cells can be labeled by epithelial cell markers, but not by fibroblast, glial or neuronal markers. The immortalized cells grow rapidly in serum medium at permissive temperature, but the majority cease proliferation when cultured in serum free medium at non-permissive temperature. These cells respond to mitogenic growth factors including bFGF, EGF and TGF-alpha and express growth factor receptors in a manner similar to the primary supporting cells. Furthermore, we find that the cells undergo a morphological differentiation when cultured in serum free medium at non-permissive temperature in the presence of bFGF. Under these conditions, the cells shrink in size, become elongated, and express early hair cell markers such as calretinin and calmodulin. The utricular epithelial cell line we have established may potentially provide an invaluable system for studying hair cell differentiation and regeneration.

Roles of microRNAs during prostatic tumorigenesis and tumor progression.

Prostate cancer (PCa) is considered to be a frequently diagnosed cancer in males with high mortality worldwide, but the molecular mechanism responsible for prostate tumorigenesis and progression remains unclear. Increasing evidence has shown that microRNAs (miRNAs) play an important role in PCa. In this review, we focus on the current advances about the role of miRNAs in regulating tumorigenesis and progression of PCa, mainly in suppressing or promoting PCa growth and metastasis, and maintaining the pluripotency of PCa stem cells (PCSC). More studies on miRNAs will provide a better understanding of their regulatory mechanisms in PCa.

Cyclin B1 turnover and the mechanism causing insensitivity of fully grown mouse oocytes to cycloheximide inhibition of meiotic resumption.

Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.