MiR-92a regulates endothelial progenitor cells (EPCs) by targeting GDF11 via activate SMAD2/3/FAK/Akt/eNOS pathway.

BACKGROUND: The effects of miR-92a on EPCs are still poorly elucidated. This study aimed to investigate the effects of miR-92a on EPCs (Endothelial progenitor cells) in a model of hypoxia (HO) or high glucose (HG)-induced EPCs injury by targeting GDF11 (Differentiation growth factor 11). METHODS: The effects of miR-92a on EPCs subjected to HO or HG were investigated firstly. Subsequently, the action mechanism of miR-92a on EPCs by targeting GDF11 was elucidated. Proliferation, apoptosis, migration, angiogenesis was measured with MTT, flow cytometry, transwell, tube formation respectively. After 24 h, levels of reactive oxygen species (ROS) were measured by fluorescence intensity. LDH and NO (nitric oxide) levels were determined by ELISA. The expression of FLK-1 (fetal liver kinase 1) and vWF (von Willebrand factor) was detected by immunofluorescence. mRNA and protein expression levels were examined using PCR and western blotting respectively. The interaction between miR-92a and GDF11 was evaluated by dual-luciferase reporter assay. RESULTS: Our results showed that HO or HG increased apoptosis, production of LDH and generation of ROS, but decreased the ability of migration and tube formation and generation of NO in EPCs; inhibiting of miR-92a decreased HO or HG-induced injury of EPCs, whereas miR-92a over-expression had the opposite effect; the protective effects induced by inhibiting of miR-92a on EPCs could be reversed by GDF11 siRNA and the harmful effects induced by over-expression of miR-92a could be rescued by over-expression of GDF11, which showed that the harmful effects of miR-92a be related to its inhibition of GDF11 and subsequent inactivation of the SMAD2/3/FAK/Akt/eNOS signaling pathway. CONCLUSIONS: Inhibiting miR-92a can protect EPCs from HO or HG-induced injury. The effect of miR-92a on EPCs are mediated by regulating of GDF11 and downstream SMAD2/3/FAK/Akt/eNOS signaling pathway.

High plasma D-dimer level is associated with decreased survival in patients with lung cancer: a meta-analysis.

An elevated plasma D-dimer level indicates the activation of coagulation and fibrinolysis. Several studies suggested that high level of plasma D-dimer was associated with the prognosis of lung cancer. In the present study, we performed a meta-analysis to evaluate the relationship between plasma D-dimer level and the prognosis of lung cancer based on larger sample size. We retrieved the literature, assessed and selected the data, and performed the statistical analysis according to the RevMan 5.0 guidelines. Literature-based searching was guided to gather data, and fixed-effects model was used to pool the hazard ratio according to the test of heterogeneity. A total of seven eligible studies including 1,377 lung cancer patients were analyzed. Survival time was significantly better in patients in the low D-dimer group than those in the high D-dimer group (hazard ratio for high D-dimer group = 1.12; 95% confidence interval 1.02 to 1.23). Patients with high levels of D-dimer have a poorer overall survival compared with those patients with low levels of D-dimer.

[Effect of the valved stent on closure of patent ductus arteriosus in a miniswine model].

OBJECTIVE: To evaluate the feasibility of a valved stent on closure patent ductus arteriosus in a Chinese miniswine model. METHODS: Self-expandable nitinol stents were made of Ni-Ti shape memory alloy (9 mm in diameter). Bovine pericardium was shaped and sutured onto the stents. Fluid passing test, pre-releasing test and static test of pressure in tube were performed in all devices before use. In eight Chinese miniswine, vascular grafts (PTFE vascular prosthesis) were surgically inserted between the descending thoracic aorta and pulmonary artery for establishment of patent ductus arteriosus model. Valved stents were deployed to occlude the patent ductus arteriosus. Echocardiography was performed two hours post operation. Aortic angiography was made 30 days post operation in survived animals. Animals were then sacrificed for autopsy and electron microscopy examinations. RESULTS: In vitro testing showed that the closure of the valved stent leaflets was satisfactory and fluid flows were not restricted in the opposite direction. The valved stents could be released through catheter, expanded completely, rapidly fixed in the tube. Closure of patent ductus arteriosus was succeeded in 6 out of 8 animals. One animal died of respiratory failure 2 hours post operation, another one died of pulmonary embolism due to valved stent displacement. Resident shunt was not evidenced by echocardiography, aortic angiography and dissection examinations in the remaining 6 animals. The new endothelial tissue fully covered the pulmonary and aortic sides of patent ductus arteriosus in 4 and 3 animals respectively. The electron microscopic observation revealed endothelial coverage of dives. CONCLUSION: The valved stent could effectively close artificial patent ductus arteriosus in vivo with satisfactory new intima covering on both sides of patent ductus arteriosus.

[Coronary flow obstruction in percutaneous aortic valve replacement using self-expandable valved stent: an in vitro study].

OBJECTIVE: To study the effects of self-expandable and orthotopically implanted percutaneous aortic valved stent on coronary artery flow in vitro. METHODS: Self-expandable valved stent was developed with nitinol stent and bovine pericardium. The ascending aorta of Chinese mini swine hearts was cut proximal to the brachiocephalic trunk. The right and left main coronary arteries were dissected. In vitro coronary flow tests were performed. Firstly, baseline coronary flow with the native aortic valve was measured (n = 12). Secondly, the valved stent was deployed orthotopically. The commissures of prosthesis were positioned randomly. Through an endoscope, the effects of valved stent and native valve on coronary ostium were obtained and coronary flow measurements repeated (valve preservation group, n = 12). Then the distance from coronary ostium to native leaflet free edge was measured. Native leaflets were removed before similar valved stent deployment. Coronary flow measurements and endoscopic inspections were repeated post-implantation (valve removal group, n = 12). RESULTS: In valve preservation group, valved stent implantation resulted in a significant decrease in left coronary flow (29.46%, P < 0.05). The obstruction was due to native leaflets sandwiched between the stent and aortic wall. The left ostia were obstructed totally in 3 and partially in 4. The flow of right coronary decreased 7.34% (P > 0.05). The right ostia were obstructed partially in 3. In valve removal group, 6.82% and 5.37% decrement in left and right coronary flow were observed after valved stent placement (P > 0.05). The distances from right coronary ostia to annulus were farther than from left coronary ostia. In two groups, the commissures of prosthesis obstructed partially left coronary ostia in 4 and right coronary ostia in 1. CONCLUSION: Orthotopic implantation of a percutaneous self-expandable aortic valved stent would obstruct the left coronary ostium with the native valve. Coronary ostium may be obstructed partly by the commissures of prosthesis.

Results of radiofrequency ablation for permanent atrial fibrillation in patients undergoing mitral valve surgery.

OBJECTIVES: The study aim was to evaluate the safety and feasibility of radiofrequency ablation for the surgical treatment of permanent atrial fibrillation in patients with degenerative mitral valve disease. DESIGN: From August 2000 to August 2003, 40 consecutive patients (mean age 69.0 +/- 9.3 years) with permanent atrial fibrillation and degenerative mitral valve disease underwent surgical radiofrequency ablation in conjunction with 22 mitral valve repairs and 18 mitral valve replacements. The mean duration of chronic AF was 5.1 +/- 3.4 years. The completeness of follow-up was 100%. The mean follow-up time was 4.6 +/- 2.0 years (range 0 to 7.8 years). RESULTS: Thirty-day mortality was 2.5% (1 patient), the cause of death was cardiac failure. Cardiac failure and temporary A-V block were the most common postoperative complications. Both occurred in 10% (4 patients). No complication was related to the ablation procedure. At discharge, 65% (26/40) of the patients were in sinus rhythm. Overall incidence of sinus rhythm at the end of the follow-up was 56.4% (22/39).The 1-, 3- and 5-year survival was 97.5%, 91.8% and 85.9%, respectively. CONCLUSION: Mitral valve surgery combined with radiofrequency ablation is a safe and effective procedure in patients with permanent atrial fibrillation and degenerative mitral valve disease. The result is encouraging in restoring sinus rhythm, and an excellent postoperative survival rate can be achieved.

[Effect of atorvastatin on postoperative atrial fibrillation in patients undergoing coronary artery bypass grafting].

OBJECTIVE: To evaluate the effect of atorvastatin on postoperative atrial fibrillation (AF) in patients undergoing coronary artery bypass grafting (CABG). METHODS: A cohort of 140 consecutive patients without a history of documented AF or previous statin use, who were scheduled to undergo selective CABG, were enrolled. Included patients were randomly assigned to atorvastatin group (n = 71) who were administered atorvastatin 20 mg/d or to control group (n = 69). After CABG, subjects were monitored continuously by electrocardiographic monitors at least 7 days. During the initial postoperative 7 d, the incidence and duration of AF were recorded. And the levels of high-sensitivity C-reactive protein (hs-CRP) were measured before and 24 hours, 72 hours, 7 days after operation, respectively. The statistical software package SPSS (version 13.0) were used to analyze the data. The differences between groups were evaluated by chi(2)-test for discrete variables and student t-test for continuous variables. Multivariate logistic regression analysis was performed to determine the independent predictors of early postoperative AF. RESULTS: During initial postoperative 7 d, AF occurred at least once in 10 cases in atorvastatin group, with a prevalence of roughly 14%, and in 23 cases in control group, with a prevalence of approximately 34% (P = 0.009). The mean duration of single AF was 3.6 +/- 0.4 hours in atorvastatin group and 5.7 +/- 0.5 hours in control group (P < 0.01), respectively. The multivariate logistic analysis showed that perioperative atorvastatin administration was an independently risk factor for early postoperative AF (OR = 0.219, 0.076-0.633, P = 0.005). There was also statistical difference in hs-CRP after CABG between the two groups. CONCLUSIONS: Perioperative atorvastatin administration may inhibit inflammatory reaction, reduce the incidence and duration of postoperative AF, hence may prevent and treat postoperative AF.

[Combined use of transmyocardial stents with gene therapy in the treatment of acute myocardial infarction].

OBJECTIVES: To determine the efficacy of combined use of transmyocardial stent with gene therapy to treat acute myocardial infarction in porcine model. METHODS: 24 Chinese mini swines have been divided into 4 groups randomly: group myocardial infarction (group MI n(1) = 6), group transmyocardial stent (group ST n(2) = 6), group vascular endothelial growth factor (group VEGF n(3) = 6), group transmyocardial stent and VEGF (group ST + VEGF n(4) = 6). In group MI, acute myocardial infarction animal model has been established by the ligation of the left descending coronary artery. In group ST, after the establishment of the model, 3 transmyocardial stents were implanted. In group VEGF, an expression plasmid containing the gene-encoding VEGF(165) (300 microg) was administered directly in the myocardium at 6 sites. In group ST + VEGF, both transmyocardial stents and expression plasmid containing the gene-encoding VEGF(165) are applied. 4 weeks later, the animals are sacrificed and echocardiography and pathological analysis have been done. RESULTS: The density of blood vessel in group ST, VEGF and ST + VEGF are significantly higher than group MI. And capillary density in group ST + VEGF is the highest in these groups statistically. Expression of VEGF was detected in group ST, VEGF and ST + VEGF, but in group VEGF and ST + VEGF the level of expression are higher. CONCLUSION: Combined use of transmyocardial stent with gene therapy has synthetic effect for the treatment of acute myocardial ischemia in porcine model and can significantly increase the vascular density.

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library.

A single-chain fragment containing antibody V domains (scFv) isolated from a lupus antibody library displayed the ability to bind gp120 and the conserved gp120 determinant composed of residues 421-436. The scFv neutralized R5 and X4-dependent HIV-1 strains from clades B, C, and D. The lupus repertoire may be useful as a source of neutralizing antibodies to HIV.

DC-SIGN: binding receptor for HCV?

DC-SIGN, a dendritic Cell-specific adhesion receptor and a type II transmembrane mannose-binding C-type lectin, is very important in the function of DC, both in mediating naive T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by viral and bacterial pathogens including Human Immunodeficiency Virus (HIV), HCV, Ebola Virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropriate entry receptors. Recent work showed that DC-SIGN are high-affinity binding receptors for HCV. Besides playing a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental for the interaction of DC with T cells during antigen presentation. The clinical strategies that target DC-SIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.

VIPase autoantibodies in Fas-defective mice and patients with autoimmune disease.

The immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) was cleaved by purified IgG from Fas-defective C3H/gld mice, lupus patients, and autoimmune thyroiditis patients. No VIPase activity was detected in IgG from control mice and humans. Kinetic analyses of VIPase IgG preparations suggested low-affinity recognition of VIP. Yet the VIPase activity was VIP selective, judged by lack of correlation with other protease activities expressed by the IgG and by noninterference of unrelated peptides in the activity. Recombinant Fv constructs selected from a human lupus phage show library displayed VIPase activity, confirming that the active site is located in the V domains. Inhibition of the VIPase activity by di-isopropylfluorophosphate suggested a serine protease-like mechanism of catalysis. Irreversible binding of a biotinyated phosphonate diester by the IgG and Fv preparations was observed, consistent with the presence of activated nucleophiles similar to those in enzymes capable of covalent catalysis. These observations show that VIP is a target for specific catalytic autoantibodies in autoimmune disease.

Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120.

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.

Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity.

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421- 431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421- 436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421- 436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.

Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice.

Autoantibodies to the recombinant extracellular domain of epidermal growth factor receptor (exEGFR) were detected by ELISA in the serum of Fas-defective old MRL/MpJ/lpr and C3H/HeJ/gld mice, but not young mice from these strains, or nonautoimmune young and old BALB/c, MRL/MpJ/++, and C3H/HeJ/MMTV mice. Compared with control human subjects without autoimmune disease, the frequency of exEGFR-binding autoantibodies was increased in scleroderma (systemic sclerosis) patients and to a lesser extent in lupus patients. Phage autoantibodies (Fv fragments) isolated from a lupus library by selection on a linear epitope of EGFR (residues 294-310) displayed the ability to bind exEGFR. Treatment of EGFR-expressing A431 cells with autoantibodies purified by affinity chromatography on immobilized exEGFR resulted in specific staining of the cells. Short-lived but strong inhibition of cellular DNA synthesis was observed in the presence of the autoantibodies. We concluded that autoantibody responses to EGFR hold the potential of fulfilling a pathogenic role in autoimmune disease.

A mechanism-based probe for gp120-Hydrolyzing antibodies.

An antigenic peptide analogue consisting of HIV gp120 residues 421-431 (an antigen recognition site probe) with diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies.

Prospects for immunotherapeutic proteolytic antibodies.

Monoclonal antibodies are suitable for therapeutic applications by virtue of their excellent target binding characteristics (specificity, affinity) and long half-life in vivo. Catalytic antibodies (CAbs) potentially represent a new generation of therapeutics with enhanced antigen inactivation capability. Here, we describe prospects for development of therapeutic CAbs to the envelope protein gp120 of HIV. The strategy consists of exploiting the natural tendency of the immune system to synthesize germline-encoded, serine protease-like CAbs. Lupus patients were found to develop antibodies to a conserved component of the CD4 binding site of gp120, potentially offering a means to obtain human antibodies expressing broad reactivity with various HIV strains. Covalently reactive antigen analogs (CRAs) capable of selective recognition of nucleophilic Abs were synthesized and applied to isolate Fv and L chain catalysts from lupus phage repertoires. CRA binding by the recombinant Ab fragments was statistically correlated with catalytic cleavage of model peptide substrates. A peptidyl CRA composed of residues 421-431 with a phosphonate diester moiety at its C terminus was validated as a reagent that combines noncovalent and covalent binding interactions in recognition of a gp120ase L chain. A general challenge in the field is the apparent instability of the catalytic conformation of the Abs. In reference to therapy of HIV infection, assurance is required that the Abs recognize the native conformation of gp120 expressed as a trimer on the virus surface.

Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs.

Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies.