Research Progress on the Pathogenesis of Aortic Dissection.

Aortic dissection is a critical cardiovascular disease due to the separation of media and adventitia caused by the rupture of vascular wall intima. The disease has a high mortality rate of about 1%-3% for each additional hour, since the adventitia of the aorta can rupture and bleed to death at any time. Although great progress has been made in clinical treatment of aortic dissection, and the mortality rate has been significantly reduced, the pathogenesis is still not very clear. At present, related studies have confirmed that inflammation of aortic wall promotes the occurrence and development of Aortic dissection. Although the mechanism of aortic dissection is more complicated, some studies have shown that the infiltration of monocytes/macrophages into the aortic wall is the main pathogenic mechanism of the disease. This review introduces the latest research results on the mechanism of macrophage infiltration and plasticity in aortic dissection.

Novel Fluorescent Probe toward Fe3+ Based on Rhodamine 6G Derivatives and Its Bioimaging in Adult Mice, Caenorhabditis elegans, and Plant Tissues.

A new fluorescent probe LXY based on the rhodamine 6G platforms has been designed, synthesized, and characterized, which could recognize Fe3+ effectively in HEPES buffer (10 mM, pH = 7.4)/CH3CN (2:3, v/v). And the distinct color change and the rapid emergence of fluorescence emission at 550 nm achieved "naked eye" detection of Fe3+. The interaction mode between them was achieved by Job's plot, MS, SEM, and X-ray single-crystal diffraction. Importantly, the crystal structures proved that Fe3+ could induce the rhodamine moiety transform the closed-cycle form to the open-cycle form. But it is interesting that Fe3+ did not appear in the crystal structures. Meanwhile, the limit of detection (LOD) of LXY to Fe3+ was calculated to be 3.47 × 10-9. In addition, the RGB experiment, test papers, and silica gel plates all indicated that the probe LXY could be used to distinguish Fe3+ quantitatively and qualitatively on-site. Moreover, the probe LXY has also been successfully applied to Fe3+ image in Caenorhabditis elegans, adult mice, and plant tissues. Thus, LXY was considered to have some potential for application in bioimaging.

Exploration of the glutamate-mediated retinal excitotoxic damage: a rat model of retinal neurodegeneration.

AIM: To explore the more suitable concentration of glutamate or N-methyl-D-aspartic acid (NMDA) for intravitreal injection to establish a rat model of retinal neurodegeneration. METHODS: We injected different doses of glutamate (20 or 50 nmol) or NMDA (40 nmol) into the vitreous chambers of rats, then measured the concentration of glutamate and retinal thickness, quantified apoptotic cells and determined the degree of tau hyperphosphorylation at different time points. T-test was used for comparison of two groups. One-way ANOVA and Turkey's multiple comparisons test were used for comparisons of different groups, and P values below 0.05 were considered statistically significant. RESULTS: The glutamate level in the rats treated with 50 nmol of glutamate was twice that of the control group and persisted two weeks. Seven days after intravitreal injection of 50 nmol of glutamate, three parameters [inner retinal thickness (IRT), retinal thickness (RT) and ganglion cell layer (GCL) cell number] were reduced significantly. Furthermore, numerous TUNEL-positive cells were observed in the GCL one day after intravitreal injection of 50 nmol of glutamate, the expression of the apoptosis-related factor cleaved casepase-3 was markedly increased compared with the expression levels in the other treatment groups, and the expression levels of tau s396 and tau s404 were significantly increased compared with those in the control group. CONCLUSION: This study demonstrates that the intravitreal injection of 50 nmol of glutamate can establish the more effective retinal neurodegeneration animal model relative to other treatment groups.

Notch Signaling Modulates Macrophage Polarization and Phagocytosis Through Direct Suppression of Signal Regulatory Protein α Expression.

The Notch pathway plays critical roles in the development and functional modulation of myeloid cells. Previous studies have demonstrated that Notch activation promotes M1 polarization and phagocytosis of macrophages; however, the downstream molecular mechanisms mediating Notch signal remain elusive. In an attempt to identify Notch downstream targets in bone marrow-derived macrophages (BMDMs) using mass spectrometry, the signal regulatory protein α (SIRPα) appeared to respond to knockout of recombination signal-binding protein Jk (RBP-J), the critical transcription factor of Notch pathway, in macrophages. In this study, we validated that Notch activation could repress SIRPα expression likely via the Hes family co-repressors. SIRPα promoted macrophage M2 polarization, which was dependent on the interaction with CD47 and mediated by intracellular signaling through SHP-1. We provided evidence that Notch signal regulated macrophage polarization at least partially through SIRPα. Interestingly, Notch signal regulated macrophage phagocytosis of tumor cells through SIRPα but in a SHP-1-independent way. To access the translational value of our findings, we expressed the extracellular domains of the mouse SIRPα (mSIRPαext) to block the interaction between CD47 and SIRPα. We demonstrated that the soluble mSIRPαext polypeptides could promote M1 polarization and increase phagocytosis of tumor cells by macrophages. Taken together, our results provided new insights into the molecular mechanisms of notch-mediated macrophage polarization and further validated SIRPα as a target for tumor therapy through modulating macrophage polarization and phagocytosis.

Exosomes derived from human umbilical vein endothelial cells promote neural stem cell expansion while maintain their stemness in culture.

The neural stem cell (NSC) niche in subventricular zone (SVZ) of adult mammalian brain contains dense vascular plexus, where endothelial cells (ECs) regulate NSCs by releasing plenty of angiocrine factors. However, the role of ECs-derived exosomes, a novel type of mediators of intercellular communications, in the regulation of NSCs remains unclear. In the current study, primary NSCs isolated from embryonic mouse brains form more neurospheres when cultured in the presence of human umbilical vein endothelial cells (HUVECs). The supportive role of ECs in the coculture was significantly attenuated when GW4869, a blocker of exosome formation, was included, suggesting that HUVECs-derived exosomes played a significant role in supporting NSCs. In order to investigate the role of ECs-derived exosomes on NSCs, we collected exosomes from HUVECs. We found that HUVECs-derived exosomes could significantly promote the formation of neurospheres by primary murine NSCs. EdU incorporation and TUNEL assays indicated that the proliferation of NSCs increased while apoptosis decreased when cultured in the presence of HUVECs-derived exosomes. NSCs incubated with the HUVECs-derived exosomes maintained their potential of multi-lineage differentiation potentials. The expression of stemness-related genes was up-regulated. These data suggested that ECs-derived exosomes could play an importantly role in NSC niche, and they might be used as a reagent for ex vivo NSC amplification for medical application.

Cytotherapy with M1-polarized macrophages ameliorates liver fibrosis by modulating immune microenvironment in mice.

BACKGROUND & AIMS: Macrophages play vital roles in chronic liver injury, and have been tested as a tool for cytotherapy in liver fibrosis. However, macrophages possess ontogenic and functional heterogeneities. Some subsets are pro-fibrotic, whereas others are anti-fibrotic. This study aimed to clarify which macrophage subset is efficient for cytotherapy in liver fibrosis and to elucidate the underlying mechanisms. METHODS: Liver fibrosis was induced in mice by carbon tetrachloride injection or bile duct ligation. Bone-marrow-derived macrophages (BMDMs) were polarized into M0, M1, or M2 macrophages, respectively. BMDMs were infused into mice through the tail vein at different stages of fibrogenesis. Fibrosis progression, hepatic cell populations, and related molecular changes were evaluated. RESULTS: Both M0 and M1 BMDMs significantly ameliorated liver fibrosis, but M1 exhibited stronger therapeutic effects than M0. M2 macrophages were not effective on liver fibrosis. M1 macrophages reduced the number and activation of hepatic stellate cells (HSCs), which could be attributed at least partly to increased HSC apoptosis. M1 macrophages enhanced the recruitment of endogenous macrophages into fibrotic liver, which displayed the phenotype of Ly6Clo restorative macrophages and produced matrix metalloproteinases (MMPs) and hepatic growth factor (HGF) to enhance collagen degradation and hepatocyte proliferation, respectively. M1 macrophages also increased the number of total and activated natural killer (NK) cells in the fibrotic liver, which released TNF-related apoptosis-inducing ligand (TRAIL), inducing HSC apoptosis. CONCLUSIONS: M1 macrophages, which modulate the immune microenvironment to recruit and modify the activation of endogenous macrophages and NK cells, are effective for cytotherapy in experimental liver fibrosis. Lay summary: M1 Bone marrow-derived macrophages (BMDMs) exhibit a stronger therapeutic effect by modulating the hepatic microenvironment to recruit and modify the activation of endogenous macrophages and natural killer (NK) cells, which likely lead to hepatic stellate cells (HSCs) apoptosis and hampered fibrogenesis.

The Notch ligand delta-like 3 promotes tumor growth and inhibits Notch signaling in lung cancer cells in mice.

Although it has been suggested that Dll3, one of the Notch ligands, promotes the proliferation and inhibits the apoptosis of cancer cells, the role of Dll3 in cancers remains unclear. In this study, we found that in the murine Lewis lung carcinoma (LLC) cells, the level of Dll3 mRNA changed upon tumor microenvironment (TME) stimulation, namely, decreased under hypoxia or stimulated with tumor necrosis factor (TNF)-α. Dll3 was also expressed at higher level in human lung carcinoma tissues than in the para-carcinoma tissues. Overexpression of Dll3 in LLC cells promoted cell proliferation and reduced apoptosis in vitro, and enhanced tumor growth when inoculated in vivo in mice. The Dll3-mediated proliferation could be due to increased Akt phosphorylation in LLC cells, because an Akt inhibitor counteracted Dll3-induced proliferation. Moreover, Dll3 overexpression promoted PI3K/Akt signaling through inhibiting Notch signaling.

miR-342-5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor ß Signaling.

BACKGROUND: Endothelial cells (ECs) form blood vessels through angiogenesis that is regulated by coordination of vascular endothelial growth factor (VEGF), Notch, transforming growth factor ß, and other signals, but the detailed molecular mechanisms remain unclear. METHODS AND RESULTS: Small RNA sequencing initially identified miR-342-5p as a novel downstream molecule of Notch signaling in ECs. Reporter assay, quantitative reverse transcription polymerase chain reaction and Western blot analysis indicated that miR-342-5p targeted endoglin and modulated transforming growth factor ß signaling by repressing SMAD1/5 phosphorylation in ECs. Transfection of miR-342-5p inhibited EC proliferation and lumen formation and reduced angiogenesis in vitro and in vivo, as assayed by using a fibrin beads-based sprouting assay, mouse aortic ring culture, and intravitreal injection of miR-342-5p agomir in P3 pups. Moreover, miR-342-5p promoted the migration of ECs, accompanied by reduced endothelial markers and increased mesenchymal markers, indicative of increased endothelial-mesenchymal transition. Transfection of endoglin at least partially reversed endothelial-mesenchymal transition induced by miR-342-5p. The expression of miR-342-5p was upregulated by transforming growth factor ß, and inhibition of miR-342-5p attenuated the inhibitory effects of transforming growth factor ß on lumen formation and sprouting by ECs. In addition, VEGF repressed miR-342-5p expression, and transfection of miR-342-5p repressed VEGFR2 and VEGFR3 expression and VEGF-triggered Akt phosphorylation in ECs. miR-342-5p repressed angiogenesis in a laser-induced choroidal neovascularization model in mice, highlighting its clinical potential. CONCLUSIONS: miR-342-5p acts as a multifunctional angiogenic repressor mediating the effects and interaction among angiogenic pathways.

Deletion of RBP-J in adult mice leads to the onset of aortic valve degenerative diseases.

Transcription factor RBP-J-mediated Notch signaling has been implicated in several inherited cardiovascular diseases including aortic valve diseases (AVD). But whether Notch signal plays a role in AVD in adults has been unclear. This study aims to test whether the deletion of RBP-J in adult mice would lead to AVD and to investigate the underlying mechanisms. Cre-LoxP-mediated gene deletion was employed to disrupt Notch signal in adult mice. Immunofluorescence and electron microscope observations showed that deletion of RBP-J in adult mice led to early morphological changes of AVD. The size of aortic valve was enlarged. The endothelial homeostasis was perturbed, probably due to the up-regulation of VEGFR2. The endothelial cells exhibited increased proliferation and loose endothelial junctions. The valvular mesenchyme displayed significant fibrosis, consistent with the up-regulation of TGF-ß1 and activation of endothelial-mesenchymal transition. We observed melanin-producing cells in aortic valves. The number of melanin-producing cells increased significantly, and their location changed from the mesenchyme to subendothelial layer of valve cusps in RBP-J deficient mice. These results suggest that RBP-J-mediated Notch signaling in aortic valves may be critically involved in valve homeostasis and valve diseases as well. These findings will be helpful for the understanding of the molecular mechanisms of AVD in adults.

[Influence of Pyk2 on hepatic metastasis and ultrastructure of human colonic cancer cells].

OBJECTIVE: To study the influence of proline-rich tyrosine kinase 2 (Pyk2) on hepatic metastasis and microstructure of colon cancer cells in nude mice. METHODS: Hepatic metastases of colonic cancer were established in Babl/c nu/nu nude mice by splenic injection of colonic cancer cells SW480 which were transfected with sense-Pyk2 plasmid and antisense-Pyk2 plasmid. After six weeks, we counted the number of liver metastases in each group and observed the ultrastructure of tumor cells in each group by electron microscopy; furthermore, we tried to find out the correlationship between Pyk2 expression and the number of colonic cancer liver metastases. RESULTS: Difference was found between the nude mice injected with sense-Pyk2 plasmid and antisense-Pyk2 plasmid, and there was correlation between Pyk2 expression and the number of liver metastases, Pyk2 could inhabit the liver metastasis of colonic adenocarcinoma. In contrast, the ultrastructure of tumor cell had less heteromorphism in nude mice transfected with sense-Pyk2 plamisd. CONCLUSION: Pyk2 can reduce the number of liver metastasis of colonic adenocarcinoma, and also the heteromorphism of tumor cells which inhabit the progress of colonic adenocarcinoma liver metastasis.

Modulation of intracellular calcium transient in response to beta-adrenoceptor stimulation in the hearts of 4-wk-old rats during simulated weightlessness.

Modulation of intracellular calcium ([Ca(2+)](i)) transient in response to beta-adrenoceptor stimulation in the hearts of hindlimb unweighted (HLU) rats during simulated weightlessness has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness. Effects of simulated microgravity on beta-adrenoceptor responsiveness were then studied. Mean arterial blood pressure, left ventricular pressure (LVP), systolic function [maximum positive change in pressure over time (+dP/dt(max))], and diastolic function [maximum negative change in pressure over time (-dP/dt(max))] were monitored during the in vivo experiment. beta-Adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. +/-dP/dt(max), myocyte contraction, intracellular [Ca(2+)](i) transient, and L-type calcium current in response to beta-adrenoceptor stimulation with isoproterenol were measured. Compared with the control group, no significant changes were found in heart weight, body weight, and mean arterial blood pressure, whereas LVP and +/-dP/dt(max) were significantly reduced. LVP and +/-dP/dt(max) were significantly attenuated in the HLU group in response to isoproterenol administration. In the in vitro study, the beta-adrenoceptor density was unchanged. Effects of isoproterenol on electrically induced single-cell contraction and [Ca(2+)](i) transient in myocytes of ventricles in HLU rats were significantly attenuated. The enhanced L-type Ca(2+) current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The above results indicate that impaired function of L-type Ca(2+) current and decreased [Ca(2+)](i) transient cause the depressed responsiveness of the beta-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.

Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells.

Since Notch signaling plays a critical role in stem cells and oncogenesis, we hypothesized that Notch signaling might play roles in cancer stem cells and cancer cells with a stem cell phenotype. In this study, we accessed potential functions of the Notch pathway in the formation of cancer stem cells using human glioma. Using RT-PCR, we found that most human astrogliomas of different grades expressed moderate to high level of Notch receptors and ligands. mRNA of Hes5 but not Hes1, both of which are major downstream molecules of the Notch pathway, was also detected. In human glioma cell lines BT325, U251, SHG-44, and U87, mRNA encoding different types of Notch receptors were detected, but active form of Notch1 (NIC) was only detected in SHG-44 and U87 by Western blot. Interestingly, proliferation of these two glioma cell lines appeared faster than that of the other two lines in which NIC was not detected. We have over-expressed NIC of Notch1 in SHG-44 cells by constitutive transfection to evaluate the effects of Notch signaling on glioma cells. Our results showed that over-expression of NIC in SHG-44 cells promoted the growth and the colony-forming activity of SHG-44 cells. Interestingly, over-expression of NIC increased the formation neurosphere-like colonies in the presence of growth factors. These colonies expressed nestin, and could be induced to cells expressing neuron-, astrocyte-, or oligodendrocyte-specific markers, consistent with phenotypes of neural stem cells. These data suggest that Notch signaling promote the formation of cancer stem cell-like cells in human glioma.

[Reoperation about recurrent heart valve disease in 221 cases].

OBJECTIVE: To retrospectively review the experience of reoperation after closed mitral commissurotomy, valvuloplasty, perivalvular leakage and dysfunction of bioprosthetic valve in 221 cases. METHODS: Two hundred and twenty-one patients underwent heart valve reoperation from January 1998 to August 2005. Among them, 8 cases was emergency operation. The reasons of reoperation included 105 cases suffered from mitral valve restenosis after closed mitral commisurotomy, 37 cases suffered from valve lesion after mitral or aortic valvuloplasty, 29 cases suffered from perivalvular leakage after valve replacement. Eighteen cases suffered from bioprosthetic valve decline, 9 cases suffered from dysfunction of machine valve, 7 cases suffered from tricuspid insufficiency of Ebstein, 5 cases suffered from prosthetic valve endocarditis and 11 cases suffered from other valve disease. The re-operations were mitral valve replacement, mitral and aortic valve replacement, aortic valve replacement and tricuspid valve replacement. The interval from first operation to next operation was 1 - 21 years. RESULTS: The early-stage postoperative mortality was 8.6% (19/221). And the reasons were low cardiac output syndrome, arrhythmia, multiple organ dysfunction failure (MODF) and renal failure. Among these the emergency operative mortality was 3/8. And the mortality was 14.5% (9/62) in class IV of cardiac function (NYHA). CONCLUSIONS: The risk factors of reoperation about heart valve disease include emergency operation, low preoperative cardiac function, MODF, long time of cardiopulmonary bypass and aortic blocking. Therefore it is emphasized that mastering and treating the risk factors promptly, which could decrease the mortality and incidence of complication.

[Fontan operation with extracardiac conduit on beating hearts].

OBJECTIVE: To evaluate the results of Fontan operation with extracardiac conduit on beating hearts. METHODS: Forty-two patients (31 males and 11 females) age ranged from 3 to 19 years old included in this study. There were 19 double inlet-ventricle, 10 tricuspid atresia, and 3 patients with mitral atresia, 10 patients with other complex congenital cardiac malformations. Fontan operations with extracardiac conduit were performed in all patients with the help of cardiopulmonary bypass without hypothermia in this study. Atrial septal fenestration was performed in 8 patients. In one patient, bi-directional cardiopulmonary procedure was performed 2 years before Fontan operation. RESULTS: There was one early death caused by acute hepatic function failure and one late death caused by repeated lung infections. The follow-up of 1 to 4.5 years showed that all patients' cardiac functions were grade I to II, and arterial oxygen saturation was 92% - 96%. CONCLUSIONS: The early and mid-term outcome of Fontan operation with extracardiac conduit on beating hearts is good and the method can be used in the single ventricle repair.