A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency

Background

RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied.

Results

To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system.

Conclusions

We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.

     

Visfatin Destabilizes Atherosclerotic Plaques in Apolipoprotein E–Deficient Mice

Objectives

Although there is evidence that visfatin is associated with atherogenesis, the effect of visfatin on plaque stability has not yet been explored.

Methods

In vivo, vulnerable plaques were established by carotid collar placement in apolipoprotein E–deficient (ApoE^−/−) mice, and lentivirus expressing visfatin (lenti-visfatin) was locally infused in the carotid artery. The lipid, macrophage, smooth muscle cell (SMC) and collagen levels were evaluated, and the vulnerability index was calculated. In vitro, RAW264.7 cells were stimulated with visfatin, and the MMPs expressions were assessed by western blot and immunofluorescence. And the mechanism that involved in visfatin-induced MMP-8 production was investigated.

Results

Transfection with lenti-visfatin significantly promoted the expression of visfatin which mainly expressed in macrophages in the plaque. Lenti-visfatin transfection significantly promoted the accumulation of lipids and macrophages, modulated the phenotypes of smooth muscle cells and decreased the collagen levels in the plaques, which significantly decreased the plaque stability. Simultaneously, transfection with lenti-visfatin significantly up-regulated the expression of MMP-8 in vivo, as well as MMP-1, MMP-2 and MMP-9. Recombinant visfatin dose- and time-dependently up-regulated the in vitro expression of MMP-8 in macrophages. Visfatin promoted the translocation of NF-κB, and inhibition of NF-κB significantly reduced visfatin-induced MMP-8 production.

Conclusions

Visfatin increased MMP-8 expression, promoted collagen degradation and increased the plaques vulnerability index.     

β-COP as a Component of Transport Vesicles for HDL Apolipoprotein-Mediated Cholesterol Exocytosis

Objective

HDL and its apolipoproteins protect against atherosclerotic disease partly by removing excess cholesterol from macrophage foam cells. But the underlying mechanisms of cholesterol clearance are still not well defined. We investigated roles of vesicle trafficking of coatomer β-COP in delivering cholesterol to the cell surface during apoA-1 and apoE-mediated lipid efflux from fibroblasts and THP-1 macrophages.

Methods

shRNA knockout, confocal and electron microscopy and biochemical analysis were used to investigate the roles of β-COP in apolipoprotein-mediated cholesterol efflux in fibroblasts and THP-1 macrophages.

Results

We showed that β-COP knockdown by lentiviral shRNA resulted in reduced apoA-1-mediated cholesterol efflux, while increased cholesterol accumulation and formation of larger vesicles were observed in THP-1 macrophages by laser scanning confocal microscopy. Immunogold electron microscopy showed that β-COP appeared on the membrane protrusion complexes and colocalized with apoA-1 or apoE during cholesterol efflux. This was associated with releasing heterogeneous sizes of small particles into the culture media of THP-1 macrophage. Western blotting also showed that apoA-1 promotes β-COP translocation to the cell membrane and secretion into culture media, in which a total of 17 proteins were identified by proteomics. Moreover, β-COP exclusively associated with human plasma HDL fractions.

Conclusion

ApoA-1 and apoE promoted transport vesicles consisting of β-COP and other candidate proteins to exocytose cholesterol, forming the protrusion complexes on cell surface, which were then released from the cell membrane as small particles to media.     

Myeloid-Specific Deletion of Diacylglycerol Lipase α Inhibits Atherogenesis in ApoE-Deficient Mice

Background

The endocannabinoid 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Despite its high concentration in vascular tissue, the role of 2-AG in atherogenesis has not yet been examined.

Methods

ApoE-deficient mice were sublethally irradiated and reconstituted with bone marrow from mice with a myeloid-specific knockout of the 2-AG synthesising enzyme diacylglycerol lipase α (Dagla) or control bone marrow with an intact 2-AG biosynthesis. After a cholesterol-rich diet for 8 weeks, plaque size and plaque morphology were examined in chimeric mice. Circulating inflammatory cells were assessed by flow cytometry. Aortic tissue and plasma levels of endocannabinoids were measured using liquid chromatography-multiple reaction monitoring.

Results

Mice with Dagla-deficient bone marrow and circulating myeloid cells showed a significantly reduced plaque burden compared to controls. The reduction in plaque size was accompanied by a significantly diminished accumulation of both neutrophil granulocytes and macrophages in atherosclerotic lesions of Dagla-deficient mice. Moreover, CB2 expression and the amount of oxidised LDL within atherosclerotic lesions was significantly reduced. FACS analyses revealed that levels of circulating inflammatory cells were unaltered in Dagla-deficient mice.

Conclusions

Myeloid synthesis of the endocannabinoid 2-AG appears to promote vascular inflammation and atherogenesis. Thus, myeloid-specific disruption of 2-AG synthesis may represent a potential novel therapeutic strategy against atherosclerosis.     

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity

Background

Osteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.

Aims

To investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.

Methods

We compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.

Results

OPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.

Conclusions

OPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.     

Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells

Background

Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.

Principal Finding

Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID₅₀ of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.

Conclusion

RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.     

Mesenchymal Stem Cells Stabilize Atherosclerotic Vulnerable Plaque by Anti-Inflammatory Properties

Background and objectives

Formation and progression of atherosclerotic vulnerable plaque (VP) is the primary cause of many cardio-cerebrovascular diseases such as acute coronary syndrome and stroke. It has been reported that bone marrow mesenchymal stem cells (MSC) exhibit protective effects against many kinds of diseases including myocardial infarction. Here, we examined the effects of intravenous MSC infusion on a VP model and provide novel evidence of its influence as a therapy in this animal disease model.

Subjects and methods

Thirty healthy male New Zealand white rabbits were randomly divided into a MSC, VP or stable plaque (SP) group (n = 10/group) and received high fat diet and cold-induced common carotid artery intimal injury with liquid nitrogen to form atherosclerotic plaques. Serum hs-CRP, TNF-α, IL-6 and IL-10 levels were measured by ELISA at 1, 2, 3, 7, 14, 21 and 28 days after MSC transplantation. The animals were sacrificed at 4 weeks after MSC transplantation. Lesions in the right common carotid were observed using H&E and Masson staining, and the fibrous cap/lipid core ratio of atherosclerotic plaques were calculated. The expression of nuclear factor κB (NF-κB) and matrix metalloproteinase 1, 2, 9 (MMP-1,2,9) in the plaque were detected using immunohistochemistry, and apoptotic cells in the plaques were detected by TUNEL. In addition, the level of TNF-α stimulated gene/protein 6 (TSG-6) mRNA and protein were measured by quantitative Real-Time PCR and Western blotting, respectively.

Results

Two rabbits in the VP group died of lung infection and cerebral infarction respectively at 1 week after plaque injury by liquid nitrogen. Both H&E and Masson staining revealed that the plaques from the SP and MSC groups had more stable morphological structure and a larger fibrous cap/lipid core ratio than the VP group. Serum hs-CRP, TNF-α and IL-6 were significantly down-regulated, whereas IL-10 was significantly up-regulated in the MSC group compared with the VP group. .Immunohistochemistry analysis revealed that NF-κB and MMP expression was reduced in the MSC and SP groups compared to the VP group. Cell apoptosis decreased significantly in both the MSC and SP groups in comparison to the VP group. TSG-6 mRNA and protein expression were higher in the plaques of the MSC group compared to the VP and SP groups.

Conclusions

Our study results suggest that MSC transplantation can effectively stabilize vulnerable plaques in atherosclerotic rabbits. This may potentially offer a new clinical application of MSC in atherosclerosis.     

An Investigation of Two-Dimensional Ultrasound Carotid Plaque Presence and Intima Media Thickness in Middle-Aged South Asian and European Men Living in the United Kingdom

Objectives

Ultrasound studies of carotid intima media thickness (cIMT) and plaques are limited in South Asians, a group at elevated cardiovascular disease (CVD) risk. We determined whether South Asians have a difference in these ultrasound markers compared to Europeans living in the United Kingdom and whether measured risk factor(s) could account for any such differences.

Methods

One hundred South Asian men, aged 40 to 70 years and 100 European men of similar age and BMI, without diagnosed CVD or diabetes, underwent carotid ultrasound for measurement of cIMT and carotid plaque presence. Physical activity, cardiorespiratory fitness, anthropometry and blood pressure were assessed, fasted blood taken for measurement of cardiometabolic risk factors and demographic and lifestyle factors recorded.

Results

Age-adjusted mean (SD) cIMT was similar in South Asians and Europeans (0.64 (0.16) mm v 0.65 (0.12) mm, p = 0.64). Plaque was present in 48 South Asians and 37 Europeans and overall, there was no age-adjusted difference between South Asian and Europeans for plaque score(odds ratio 1.49, 95% CI, 0.86-2.80, p = 0.16), however, South Asians appeared to have more plaques at a younger age than Europeans; at age 40-50 years the odds of South Asians having plaques was 2.63 (95% CI, 1.16-5.93) times that for Europeans.

Conclusions

cIMT is similar between healthy South Asian and European men. Whilst there was no overall difference in plaque presence in South Asians, there is an indication of greater plaque prevalence at younger ages - an observation requiring further investigation. Prospective studies linking plaques to CVD outcomes in South Asians are needed to investigate whether these measures help improve CVD risk prediction.     

Dystroglycan Depletion Impairs Actin-Dependent Functions of Differentiated Kasumi-1 Cells

Background

Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils.

Purpose

In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells.

Methods

We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells.

Results

Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1β and expression of markers of differentiation are not altered.

Conclusion

Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.     

Pharmacological Treatment with Annexin A1 Reduces Atherosclerotic Plaque Burden in LDLR-/- Mice on Western Type Diet

Objective

To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR^-/- mice.

Methods

Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of ^99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR^-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.

Results

Hr-anxA1 bound PS in Ca²⁺-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating ^99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P).

Conclusion

Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.     

Circulating Autoantibodies against the Apolipoprotein B-100 Peptides p45 and p210 in Relation to the Occurrence of Carotid Plaques in 64-Year-Old Women

Objectives

Immune responses against oxidized low density lipoprotein (LDL) play a key role in atherosclerosis. Previous studies have indicated inverse associations between autoantibodies to epitopes in oxidized LDL and cardiovascular disease. In this study we investigated the associations between autoantibodies against the apolipoprotein B-100 (apoB-100) peptides p45 and p210 and occurrence of carotid plaques.

Design

The study cohort consisted of a population-based sample of 64-year-old women with varying degrees of glucose tolerance (n=594). To identify and record the occurrence of carotid atherosclerotic plaques ultrasonography was used. Measurements of plasma IgM and IgG autoantibodies against the native and malondialdehyde (MDA)-modified apoB-100 peptides p45 and p210 were performed by ELISA.

Results

Women with carotid plaques were found to have lower levels of IgM MDA-p210 autoantibodies compared to plaque-free women. The number of carotid plaques in each subject and the total carotid plaque area correlated inversely with IgM MDA-p210 levels (r=-0.11, P=0.009 and r=-0.11, P=0.013, respectively). Furthermore, levels of IgM MDA-p210 above the lowest tertile were associated with an odds ratio of 0.55 (95% CI 0.38-0.79, P=0.001) for occurrence of carotid plaques, independently of other risk markers and statin treatment. Associations between apo-B100 peptide autoantibodies and cardiovascular risk factors were generally weak but subjects with impaired glucose tolerance had higher levels of IgM against MDA-p210.

Conclusion

The present study demonstrates that high levels of IgM against MDA-p210 are associated with less severe carotid disease in women. These findings provide additional support for a role of immune responses against oxidized LDL in cardiovascular disease.     

TLR4 Expression by Liver Resident Cells Mediates the Development of Glucose Intolerance and Insulin Resistance in Experimental Periodontitis

Background

Results from epidemiological studies indicate a close association between periodontitis and type 2 diabetes mellitus. However, the mechanism linking periodontitis to glucose intolerance (GI) and insulin resistance (IR) is unknown. We therefore tested the hypothesis that periodontitis induces the development of GI/IR through a liver Toll-like receptor 4 (TLR4) dependent mechanism.

Methods

TLR4 chimeric mice were developed by bone marrow transplantation using green fluorescent protein expressing TLR4WT mouse (GFPWT) as donor and TLR4 WT or TLR4-/- as recipient mice (GFPWT:WT and GFPWT:KO chimeras respectively). These chimeras were subjected to experimental chronic periodontitis induced by repeated applications of LPS to the gingival sulci for 18 weeks. The levels of GI/IR were monitored and plasma cytokines and LPS were determined at 18 weeks when differences in glucose tolerance were most apparent. Cytokine gene expression was measured in liver tissue by qPCR.

Results

Alveolar bone loss was significantly greater in GFPWT:WT chimeras treated with LPS compared with chimeras treated with PBS or GFPWT:KO chimeras. However, the degree of gingival inflammation was similar between GFPWT:WT and GFPWT:KO mice with LPS application. Severe GI/IR occurred in GFPWT:WT chimeras but not in the GFPWT:KO chimeras that were subjected to 18 weeks of LPS. Serum LPS was detected only in animals to which LPS was applied and the level was similar in GFPWT:WT and GFPWT:KO mice at the 18 week time point. Surprisingly, there was no significant difference in the plasma levels of IL1β, IL6 and TNFα at 18 weeks in spite of the severe GI/IR in the GFPWT:WT chimeras with LPS application. Also, no difference in the expression of TNFα or IL6 mRNA was detected in the liver of GFPWT:WT vs GFPWT:KO mice. In contrast, liver IL1β expression was significantly greater in GFPWT:WT chimeras compared to GFPWT:KO chimeras treated with LPS.

Conclusion

We observed that GFPWT:WT, but not GFPWT:KO chimeras, treated with LPS developed GI/IR despite similar degrees of gingival inflammation, circulating cytokine levels, and LPS concentrations. We conclude that LPS from periodontitis sites has a pivotal role in triggering the development of GI/IR through a mechanism that involves TLR4 expression by resident macrophages/Kupffer cells in the liver.     

Pericoronary Adipose Tissue as Storage and Supply Site for Oxidized Low-Density Lipoprotein in Human Coronary Plaques

Objectives

It is generally believed that low-density lipoprotein enters the vascular wall from its lumen and oxidized (oxLDL), after which it plays an important role in atherosclerosis. Because voluminous epicardial adipose tissue is a risk factor for coronary events, there is a possibility that the pericoronary adipose tissue (PCAT), which is a part of epicardial adipose tissue, acts as a risk factor by supplying oxLDL to the coronary arterial wall. The present study was performed whether PCAT stores and supplies oxLDL to the coronary wall.

Methods

Localization of oxLDL in PCAT and its relation to plaque morphology were examined by immunohistochemical techniques in 27 epicardial coronary arteries excised from 9 human autopsy cases.

Results

OxLDL deposited in all PCAT of the studied cases. The percent (%) incidence of oxLDL in the intima of 25 normal segment, 19 white plaques, 15 yellow plaques without necrotic core (NC) and 10 yellow plaques with NC, was 32, 84, 93 (p<0.05 vs normal segments and yellow plaques with NC), and 30, respectively. OxLDL deposited either in dotted or diffuse pattern. Double immunohistochemical staining revealed that the dotted oxLDL was that contained in CD68(+)-macrophages. The oxLDL-containing macrophages were observed in the interstitial space but not inside of the vasa vasorum, and they traversed PCAT, adventitia, external and internal elastic laminae, suggesting their migration towards the intima. Diffuse oxLDL deposits were observed in 17 preparations, the majority of which were co-localized with the vasa vasorum in outer or in both inner and outer halves of intima, and rarely in the inner half alone.

Conclusions

The results suggested that PCAT is a supply source of oxLDL to coronary intima and acts as a risk factor for coronary events, that oxLDL increasingly deposits in the intima with plaque growth and decreases after plaque maturation, and therefore molecular therapies targeting the PCAT before plaque growth could be effective in preventing human coronary atherosclerosis.     

Production of IL-8, VEGF and Elastase by Circulating and Intraplaque Neutrophils in Patients with Carotid Atherosclerosis

Objectives

Polymorphonuclear neutrophils (PMN) in atherosclerotic plaques have been identified only recently, and their contribution to plaque development is not yet fully understood. In this study, production of elastase, interleukin (IL)-8 and vascular endothelial growth factor (VEGF) by PMN was investigated in subjects with carotid stenosis undergoing carotid endarterectomy (CEA).

Methods

The study enrolled 50 patients (Pts) and 10 healthy subjects (HS). Circulating PMN (cPMN) isolated from venous blood (in both Pts and HS) and from plaques (pPMN, in Pts) were cultured, alone or with 0.1 μM fMLP. Elastase, IL-8 and VEGF mRNA were analyzed by real-time PCR. In CEA specimens, PMN were localized by immunohistochemistry.

Results

In both Pts cPMN and pPMN, IL-8 mRNA was higher at rest but lower after fMLP (P<0.01 vs HS), and VEGF mRNA was higher both at rest and after fMLP (P<0.01 vs HS), while elastase mRNA was not significantly different. On the contrary, protein production was always higher in cPMN of HS with respect to values measured in cells of Pts. In CEA specimens, CD66b+ cells localized to areas with massive plaque formation close to neovessels. Pts with soft and mix plaques, as defined by computed tomography, did not differ in cPMN or pPMN IL-8, VEGF or elastase mRNA, or in intraplaque CD66b+ cell density. However, Pts with soft plaques had higher white blood cell count due to increased PMN.

Conclusions

In Pts with carotid plaques, both circulating and intraplaque PMN produce IL-8, VEGF and elastase, which are crucial for plaque development and progression. These findings suggest mechanistic explanations to the reported correlation between PMN count and cardiovascular mortality in carotid ATH.     

Carotid Atherosclerotic Disease Predicts Cardiovascular Events in Hemodialysis Patients: A Prospective Study

Background

To evaluate the predictive value of carotid atherosclerotic disease (CAD) and intima-media thickness (IMT) on incident cardiovascular disease and mortality in hemodialysis patients.

Methods

Multicenter, observational, prospective study including 110 patients, followed-up to 6 years. Carotid doppler ultrasonographic findings were classified in 4 degrees of severity: 1) IMT <0.9 mm, 2) IMT >0.9 mm, 3) carotid plaque with stenosis <50% and 4) plaque with stenosis >50%. The associations between IMT and CAD and cardiovascular events, total and cardiovascular mortality were assessed.

Results

83% of the patients had atherosclerotic plaques (CAD degrees 3-4). During follow-up, 29.1% of patients experienced cardiovascular events, and 28.2% died, 38.7% of cardiovascular origin. The presence of plaques was associated with cardiovascular events (p = 0.03) while calcified plaques were associated with both cardiovascular events (p = 0.01), cardiovascular mortality (p = 0.03) and non-significantly with overall mortality (p = 0.08) in the survival analysis. Carotid IMT was not associated with outcomes. Cardiovascular events correlated with CAD severity (HR 2.27, 95% CI 1.13-4.54), age (HR 1.04, 1.01-1.06), previous cardiovascular disease (HR 1.75, 1.05-4.42), dyslipidemia (HR 2.25, 1.11-4.53), lipoprotein (a) (HR 1.01, 1.00-1.02), troponin I (HR 3.89, 1.07-14.18), fibrinogen levels (HR 1.38, 0.98-1.94) and antiplatelet therapy (HR 2.14, 1.04-4.4). In an age-adjusted multivariate model, cardiovascular events were independently associated with previous coronary artery disease (HR 3.29, 1.52-7.15) and lipoprotein (a) (HR 1.01, 1.00-1.02).

Conclusions

The presence of carotid plaques and, especially, calcified plaques, are predictors of new cardiovascular events and cardiovascular mortality in hemodialysis patients, while IMT was not. The prognostic value of calcified plaques should be confirmed in future studies.     

Homocysteine and Carotid Plaque Stability: A Cross-Sectional Study in Chinese Adults

Background and Purpose

This study aimed to explore the possible association of plasma total homocysteine with carotid plaque stability.

Methods

A cross-sectional study was conducted from 2010 to 2011. A stratified random sample of 2,919 Chinese participants aged 40 years or older was enrolled. Plasma total homocysteine levels were measured and carotid plaques were evaluated by ultrasonography. Logistic regression model was used to analyze the association of homocysteine levels to the progression of carotid plaque development, while adjusting for demographics and vascular risk factors.

Results

The mean level of plasma homocysteine in the subjects was 14.9 µmol/l. Along with increase in homocysteine level, the risk of advanced carotid plaque elevated (odds ratio = 1.28; 95% confidence interval = 1.09–1.51) after adjusting for age, sex, and other potential confounders. Stratified by sex, higher homocysteine level was strongly associated with advanced carotid plaque in men (OR = 1.41; 95% confidence interval = 1.17–1.70), but not in women.

Conclusion

The findings suggest that plasma level of homocysteine may be associated with advanced carotid plaque, which constitutes high risks of stroke, in male Chinese adults.     

Targeted Inhibitory Effect of Lenti-SM22alpha-p27-EGFP Recombinant Lentiviral Vectors on Proliferation of Vascular Smooth Muscle Cells without Compromising Re-Endothelialization in a Rat Carotid Artery Balloon Injury Model

Aims

In-stent restenosis remains a serious problem after the implantation of drug-eluting stents, which is attributable to neointima formation and re-endothelialization. Here, we tried to find a new method which aims at selectively inhibiting proliferation of vascular smooth muscle cells (VSMC) proliferation without inhibition of re-endothelialization.

Methods and Results

We used the smooth muscle-specific SM22alpha promoter in a recombinant lentiviral vector to drive overexpression of cell-cycle inhibitor, p27, in VSMCs. p27 effectively inhibited VSMC proliferation mediated by cell cycle arrest at the G0/G1 checkpoint. The SM22alpha-p27 lentiviral vector inhibited VSMC proliferation more effectively than paclitaxel. Rats infected with Lenti-SM22alpha-p27 had a significantly lower intima/media (I/M) ratio and also showed inhibition of restenosis on day 28 after balloon injury. Moreover, the repair of injured endothelium, and re-endothelialization of the carotid artery wall, was not affected by the smooth muscle cell-specific expression of p27.

Conclusion

A recombinant lentiviral vector carrying the SM22alpha promoter was used to effectively infect and selectively overexpress p27 protein in VSMCs, leading to inhibition of intimal hyperplasia without compromising endothelial repair.     

Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector

Background

The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.

Methods/Principal Findings

We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.

Conclusions/Significance

This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.