CD31 Mimetic Coating Enhances Flow Diverting Stent Integration into the Arterial Wall Promoting Aneurysm Healing.

BACKGROUND AND PURPOSE: Beyond aneurysmal occlusion, metallic flow diverters (FDs) can induce an adverse endovascular reaction due to the foreignness of metal devices, hampering FD endothelialization across the aneurysm neck, and arterial healing of intracranial aneurysms. Here, we evaluated the potential benefits of an FD coating mimicking CD31, a coreceptor critically involved in endothelial function and endovascular homeostasis, on the endothelialization of FDs implanted in vivo. METHODS: Nitinol FD (Silk Vista Baby) and flat disks were dip-coated with a CD31-mimetic peptide via an intermediate layer of polydopamine. Disks were used to assess the reaction of endothelial cells and blood elements in vitro. An aneurysm rabbit model was used to compare in vivo effects on the arterial wall of CD31-mimetic-coated (CD31-mimetic, n=6), polydopamine-coated (polydopamine, n=6), and uncoated FDs (bare, n=5) at 4 weeks post-FD implantation. In addition, long-term safety was assessed at 12 weeks. RESULTS: In vitro, CD31-mimetic coated disks displayed reduced adhesion of blood elements while favoring endothelial cell attachment and confluence, compared to bare and polydopamine disks. Strikingly, in vivo, the neoarterial wall formed over the CD31-mimetic-FD struts at the aneurysm neck was characteristic of an arterial tunica media, with continuous differentiated endothelium covering a significantly thicker layer of collagen and smooth muscle cells as compared to the controls. The rates of angiographic complete occlusion and covered branch arterial patency were similar in all 3 groups. CONCLUSIONS: CD31-mimetic coating favors the colonization of metallic endovascular devices with endothelial cells displaying a physiological phenotype while preventing the adhesion of platelets and leukocytes. These biological properties lead to a rapid and improved endothelialization of the neoarterial wall at the aneurysm neck. CD31-mimetic coating could therefore represent a valuable strategy for FD biocompatibility improvement and aneurysm healing.

Oral microbiota and atherothrombotic carotid plaque vulnerability in periodontitis patients. A cross-sectional study.

BACKGROUND: An increased risk of atherothrombotic vascular events has been reported in periodontitis patients. Periodontitis is associated with dysbiotic subgingival biofilms and bacteremia. OBJECTIVE: We hypothesized (a) that the oral microbiome is associated with the carotid microbiome and (b) that periodontitis could contribute to plaque vulnerability. The aim of this study was to determine the associations between periodontitis, the carotid microbiome, and the local innate immune response in carotid atherothrombotic plaques vulnerable to rupture. METHODS: In this cross-sectional study, 45 patients admitted for carotid endarterectomy underwent a preoperative periodontal examination. The volume of intraplaque hemorrhage reflected by the hemoglobin level released in carotid-conditioned media was considered as a criterion of carotid plaque vulnerability. Levels of antibodies against periodontal bacteria were determined in sera. The signature of the oral microbiota was assessed by microbial whole-genome sequencing, nested PCR, and immunostaining in carotid plaque samples. Markers of neutrophil recruitment (leukotriene B4), neutrophil activation (myeloperoxidase, defensins), and cytokines were measured in carotid-conditioned media and/or plasma. RESULTS: All patients exhibited periodontitis. One hundred and forty-four bacterial genera were detected in the carotid microbiome. While Streptococcus was found in 84% of the carotid samples, periodontitis-associated genera were detected in 21%. P. gingivalis DNA and gingipains were also identified in carotid samples. There were significant inverse correlations between periodontal attachment loss/serum anti-P. gingivalis Immunoglobulin A and cytokine inhibiting neutrophils (all P < .01). There were also significant positive correlations between lipopolysaccharides, myeloperoxidase/human neutrophil peptides1-3, and hemoglobin levels (all P < .01). CONCLUSIONS: In patients at risk of stroke, the carotid plaque microbiome was highly diverse and compatible with an oral origin. Periodontitis was significantly associated with neutrophil activation markers and plaque vulnerability to rupture.

Periodontitis and cardiovascular diseases: Consensus report.

BACKGROUND: In Europe cardiovascular disease (CVD) is responsible for 3.9 million deaths (45% of deaths), being ischaemic heart disease, stroke, hypertension (leading to heart failure) the major cause of these CVD related deaths. Periodontitis is also a chronic non-communicable disease (NCD) with a high prevalence, being severe periodontitis, affecting 11.2% of the world's population, the sixth most common human disease. MATERIAL AND METHODS: There is now a significant body of evidence to support independent associations between severe periodontitis and several NCDs, in particular CVD. In 2012 a joint workshop was held between the European Federation of Periodontology (EFP) and the American Academy of Periodontology to review the literature relating periodontitis and systemic diseases, including CVD. In the last five years important new scientific information has emerged providing important emerging evidence to support these associations RESULTS AND CONCLUSIONS: The present review reports the proceedings of the workshop jointly organised by the EFP and the World Heart Federation (WHF), which has updated the existing epidemiological evidence for significant associations between periodontitis and CVD, the mechanistic links and the impact of periodontal therapy on cardiovascular and surrogate outcomes. This review has also focused on the potential risk and complications of periodontal therapy in patients on anti thrombotic therapy and has made recommendations for dentists, physicians and for patients visiting both the dental and medical practices.

An Experimental Study of Paclitaxel Embolisation During Drug Coated Balloon Angioplasty.

OBJECTIVE: Drug coated balloons (DCB) improve the patency of femoropopliteal angioplasty but their use in infrapopliteal lesions is debateable as paclitaxel (PTX) particle embolisation has been suspected in some trials. The aim of this study was to compare experimentally five DCBs in terms of distal embolism of PTX. METHODS: Twenty-five New Zealand rabbits were divided into five groups according to the DCB used: Lutonix (Bard), In.Pact (Medtronic), Passeo-18 Lux (Biotronik), Ranger (Boston Scientific), and Stellarex (Spectranetics) (n = 5 in each group). After ligation of the right common iliac artery, a 4 × 40 mm DCB was inflated in the infrarenal aorta for 180 seconds. Rabbits were euthanised two hours after inflation of the DCB. The infrarenal aorta, a blood sample and three left hind leg muscles (tensor fasciae latae [TFL], vastus lateralis [VL], and tibialis anterior [TA] muscles) were harvested for blind measurement of PTX concentrations and histological analysis (PTX emboli count). RESULTS: In the TA muscle (the most distal), concentrations of PTX were significantly lower for the Ranger (0.067 ng/mg) than for the Lutonix (0.342 ng/mg; p = .008), In.Pact (0.370 ng/mg; p = .012), and Passeo-18-Lux (0.160 ng/mg; p = .021) DCBs. Similarly, concentrations of PTX were significantly lower for the Passeo-18-Lux than for the In.Pact (p = .028). Concentrations of PTX were not significantly different between DCBs in the TFL and VL muscles. Concentrations of PTX were found to be significantly higher in the plasma and lower in the aorta and on the DCBs after use of Lutonix compared with the four other DCBs. Histological analysis revealed evidence of embolised PTX crystals in small arterioles of all muscle tissue samples without any significant difference between the DCBs. CONCLUSIONS: This study suggests some differences regarding distal embolisation profiles between the five assessed DCBs. Although clinical implications remain to be demonstrated, the present results may have implications when choosing a DCB, especially in a critical limb ischaemia setting.

Elastase inhibitor AZD9668 treatment prevented progression of experimental abdominal aortic aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a particular form of arterial disease characterized by the dilation of the aortic wall and the presence of an intraluminal thrombus linked to a high proteolytic activity. The aim of this study was to investigate the effect of an elastase inhibitor (AZD9668 from AstraZeneca) on aneurysm progression. METHODS: For this purpose, we have used a rat model of elastase perfusion followed by repeated injection of Porphyromonas gingivalis (Pg), a weak periodontal pathogen recently reported to enhance AAA thrombus formation. Pg (1.10(7) colony-forming units) was injected through the jugular vein once a week for 4 weeks, and AZD9668, incorporated in the food, was delivered concomitantly. RESULTS: Our results show a beneficial effect of AZD9668 treatment on AAA progression and composition. The increased AAA diameter induced by Pg was significantly reduced by AZD9668 treatment. Histologic analyses allowed us to observe the persistence of a neutrophil-rich luminal thrombus associated with calcifications in Pg-injected rats and the presence of a healing process in the Pg/AZD9668-treated group. The enhanced concentrations of markers of neutrophil activation, cell-free DNA, and myeloperoxidase and elastase activity in Pg-injected rats were significantly reduced both in the conditioned medium of AAA tissue samples and in plasma of rats injected with Pg and treated with AZD9668. CONCLUSIONS: AZD9668 treatment could therefore constitute a new therapeutic tool for treatment of AAA.

Fucoidan interferes with Porphyromonas gingivalis-induced aneurysm enlargement by decreasing neutrophil activation.

PURPOSE: Neutrophils have been shown to be involved in all stages of human and experimental abdominal aortic aneurysm (AAA) development. The initial processes of neutrophil rolling and trapping in the intraluminal thrombus (ILT) are mediated mainly by P-selectin expressed by activated platelets. In the present study, we propose to evaluate the beneficial effect of fucoidan, a competitive binding agent of P-selectin, on aneurysmal growth in a rat model of aortic aneurysm with neutrophil enrichment of the ILT induced by repeated episodes of weak bacteremia. METHODS: Sixty Lewis rats with experimental AAAs, developed from decellularized aortic xenografts, were divided into four groups. Two groups were used as controls: group fucoidan control (FC) was treated with 200 mg of fucoidan (F) delivered by 2 mL, 4-week osmotic pumps placed intraperitoneally before closing the abdomen, and group C received saline instead of fucoidan. Two more groups were injected weekly with Porphyromonas gingivalis (P. gingivalis [Pg]): group F+Pg received 200 mg of intraperitoneal fucoidan and group Pg received saline. AAAs were harvested after 4 weeks and peripheral blood was sampled at that time. Cell-free DNA (cf-DNA) and myeloperoxydase (MPO) antigen concentrations were determined in plasma and in AAA-conditioned media. Histology and P-selectin immunostaining were performed on AAA tissue samples. RESULTS: Comparing rats injected with Pg, those receiving fucoidan presented reduced aneurysmal diameter. Histologic analysis of AAAs showed that fucoidan reduced the ILT thickness in Pg-injected rats, with fewer trapped neutrophils, and with signs of a healing process, as observed in control group C. Immunohistological analysis revealed a substantial decrease in P-selectin immunostaining at the luminal surface of aneurysms in fucoidan-treated rats compared to the other groups, suggesting an interaction between fucoidan and P-selectin. A significant decrease in MPO concentrations in both plasma and conditioned medium was induced by fucoidan treatment in Pg-injected rats, reflecting a pacification of the ILT biological activity. This effect was associated with a reduction in neutrophil activation and apoptosis, reflected by a significant decrease in cf-DNA concentration in both plasma and conditioned medium of fucoidan-treated rats. CONCLUSIONS: Our results suggest that fucoidan has a beneficial effect on experimental aneurysmal degeneration by decreasing neutrophil activation in the ILT enhanced by weak pathogen contamination. This effect seems to be related to its interaction with P-selectin, which may decrease the trapping of neutrophils into the ILT. Fucoidan could represent a therapeutic option in AAAs to decrease the neutrophil activation involved in the degenerative process of aneurysmal expansion and rupture.

Quantifying the evolutionary dynamics of language.

Human language is based on grammatical rules. Cultural evolution allows these rules to change over time. Rules compete with each other: as new rules rise to prominence, old ones die away. To quantify the dynamics of language evolution, we studied the regularization of English verbs over the past 1,200 years. Although an elaborate system of productive conjugations existed in English's proto-Germanic ancestor, Modern English uses the dental suffix, '-ed', to signify past tense. Here we describe the emergence of this linguistic rule amidst the evolutionary decay of its exceptions, known to us as irregular verbs. We have generated a data set of verbs whose conjugations have been evolving for more than a millennium, tracking inflectional changes to 177 Old-English irregular verbs. Of these irregular verbs, 145 remained irregular in Middle English and 98 are still irregular today. We study how the rate of regularization depends on the frequency of word usage. The half-life of an irregular verb scales as the square root of its usage frequency: a verb that is 100 times less frequent regularizes 10 times as fast. Our study provides a quantitative analysis of the regularization process by which ancestral forms gradually yield to an emerging linguistic rule.

Innovative application of nested PCR for detection of Porphyromonas gingivalis in human highly calcified atherothrombotic plaques.

Atherothrombosis, leading to stroke and myocardial infarction, is responsible for most of the deaths in the world. An increased risk of atherothrombotic vascular events has been reported in patients with periodontitis. Periodontitis is a chronic multifactorial inflammatory disease, which involves a dysbiotic microbiota, and leads to a progressive destruction of the tooth-supporting apparatus. Transcient periodontal pathogen blood translocation, mainly bacteremia, has been associated with the severity of gingival inflammation. The identification of periodontal bacteria within atherothrombotic plaques is challenging and unpredictable. This review aims to summarize existing molecular technics for identifying periodontal microbiota in human atherothrombotic samples. A secondary objective is to describe a protocol for the identification of Porphyromonas gingivalis from highly calcified, atherothrombotic human samples that is based on our experience in translational cardiovascular research. Compared to direct real-time PCR, our protocol based on nested PCR has increased the detection of Porphyromonas gingivalis by 22.2% with good specificity.

Effect of blocking platelet activation with AZD6140 on development of abdominal aortic aneurysm in a rat aneurysmal model.

BACKGROUND: Platelet activation and thrombus renewal are keys to intraluminal thrombus formation and progression of abdominal aortic aneurysms (AAA). This study explored the ability of AZD6140, a P2Y(12) receptor antagonist, to inhibit platelet activation and prevent aneurysm development in a rat experimental model of AAA. METHOD: Aortic aneurysms were induced by implanting a segment of sodium dodecyl sulfate-decellularized guinea pig aorta in rat aortas. One day later, rats were randomized to AZD6140 (10 mg/kg twice daily by mouth) or diluent (n = 23 per group) for either 10 (n = 18) or 42 days (n = 28). Adenosine diphosphate (ADP)-mediated platelet aggregation, aneurysm expansion, intraluminal thrombus formation, inflammatory infiltration, matrix metalloproteinase-9 (MMP-9) expression, and smooth muscle cell colonization were measured. RESULTS: AZD6140 inhibited ADP-induced platelet aggregation in vivo for 12 hours, justifying twice-daily administration in rats. The spontaneous increase in aortic diameter shown in the aneurysmal model (2.22 +/- 0.56 mm at day 10 vs 5.21 +/- 1.22 mm at day 42) was reduced with AZD6140 (3.61 +/- 1.46 mm at day 42, P < .01). This beneficial effect was associated with a significant reduction of thrombus development, platelet CD41 expression (P < .05), and leukocyte infiltration of the mural thrombus at days 10 and 42 (P < .01). MMP-9 expression correlated with mural thrombus area and was significantly reduced by AZD6140 (P < .05). AZD6140 limited elastic fiber degradation (P < .05) and enhanced progressive colonization of the thrombus by smooth muscle cells at day 42 (P < .01). CONCLUSIONS: These data suggest that inhibition of platelet activation limits intraluminal thrombus biologic activities, thereby impairing aneurysm development.

Can Periodontitis Influence the Progression of Abdominal Aortic Aneurysm? A Systematic Review.

There is some evidence that periodontitis increases the risk of atherothrombosis. Abdominal aortic aneurysm (AAA) is a cardiovascular disease with specific risk factors and physiopathological mechanisms that can lead to rupture in the absence of treatment. The aim of the present systematic review was to explore the influence of periodontitis on the progression of AAAs as a specific disease. A systematic search in PubMed/MEDLINE and Embase databases was performed. Human and animal studies exploring the influence of periodontal pathogens on the progression of AAA were considered for inclusion. After systematic screening, 5 articles were included in the review. Due to the heterogeneity of the selected studies, a meta-analysis could not be performed. The descriptive analyses of the studies emphasized that periodontal pathogens or their by-products contribute to systemic and local innate immunity likely to be associated with AAA physiopathology. Periodontitis seems to play a role in the development and progression of AAA. The present systematic review suggests that the presence of periodontal bacteria in the bloodstream or in situ in the vascular lesion is a risk associated with aneurysmal disease progression.

Intraplaque hemorrhage, a potential consequence of periodontal bacteria gathering in human carotid atherothrombosis.

Periodontal diseases are multifactorial inflammatory diseases, caused by a bacterial biofilm involving both innate and adaptative immunity, characterized by the destruction of tooth-supporting tissues. In the context of periodontitis, the spread of weak pathogenic bacteria into the bloodstream has been described. These bacteria will preferentially localize to existing clot within the circulation. Atherothrombosis of the carotid arteries is a local pathology and a common cause of cerebral infarction. Intraplaque hemorrhages render the lesion more prone to clinical complications such as stroke. The main objective of this study is to explore the biological relationship between carotid intraplaque hemorrhage and periodontal diseases. This study included consecutive patients with symptomatic or asymptomatic carotid stenosis, admitted for endarterectomy surgical procedure (n=41). In conditioned media of the carotid samples collected, markers of neutrophil activation (myeloperoxidase or MPO, DNA-MPO complexes) and hemoglobin were quantified. To investigate the presence of DNA from periodontal bacteria in atherosclerotic plaque, PCR analysis using specific primers was performed. Our preliminary results indicate an association between neutrophil activation and intraplaque hemorrhages, reflected by the release of MPO (p<0,01) and MPO-DNA complexes (p<0,05). Presence of DNA from periodontitis-associated bacteria was found in 32/41 (78%) atheromatous plaque samples. More specifically, DNA from Pg, Tf, Pi, Aa was found in 46%, 24%, 34% and 68% of the samples, respectively. Hemoglobin levels were higher in conditioned media in carotid samples where the bacteria were found, but this was not statistically significant. Our data confirm the relationship between intraplaque hemorrhage and neutrophil activation. In addition, the presence of periodontal bacteria DNA in carotid atheromatous plaque, may contribute to this activation. Further analysis is needed to fully explore the raw data and specimens.

High-density lipoprotein therapy inhibits Porphyromonas gingivalis-induced abdominal aortic aneurysm progression.

Clinical and experimental studies have highlighted the potential implication of periondontal bacteria contamination in the pathogenesis of abdominal aortic aneurysms (AAA). In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDLs) display multiple functions, including anti-inflammatory and lipopolysaccharide scavenging properties. Low plasma levels of HDL-cholesterol have been reported in AAA patients. We tested the effect of a HDL therapy in Sprague-Dawley rat model of AAA, obtained by intraluminal elastase infusion followed by repeated injections of Porphyromonas gingivalis (Pg). HDLs, isolated by ultracentrifugation of plasma from healthy human volunteers, were co-injected intravenously (10 mg/kg) with Pg (1.107 Colony Forming Unit) one, eight and 15 days after elastase perfusion. Rats were sacrificed one week after the last injection. Our results show that Pg injections promote the formation of a persistent neutrophil-rich thrombus associated with increased aortic diameter in this AAA model. HDLs significantly reduced the increased AAA diameter induced by Pg. Histology showed the onset of a healing process in the Pg/HDL group. HDL injections also reduced neutrophil activation in Pg-injected rats associated with decreased cytokine levels in conditioned media and plasma. Scintigraphic analysis showed an intense uptake of 99mTc-HDL by the AAA suggesting that HDLs could exert their beneficial effect by acting directly on the thrombus components. HDL supplementation may therefore constitute a new therapeutic tool for AAA treatment.

Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation.

OBJECTIVE: Epidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens. METHODS: Carotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium. RESULTS: Intraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers. CONCLUSION: This study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques.

Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation. Proof of concept in rats.

BACKGROUND: Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. METHODS AND FINDINGS: Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. CONCLUSIONS: Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression.

Comparative studies on the mechanisms of action of four polysaccharides on arterial restenosis.

Percutaneous coronary interventions play a major role in the management of patients affected by coronary artery diseases. However, their efficiency is impaired by restenosis, defined as a reduction of the vessel lumen, occurring a few months after the procedure. A low-molecular-weight fraction of fucoidan, a vegetal heparin-like sulphated polysaccharide, was recently shown to greatly reduce in-stent restenosis after angioplasty in rabbits. To better understand the in vivo anti-restenotic effects of this polymer, we used fractions of fucoidan and compared to heparin and dextran of different sizes. We carried out in vitro growth inhibition experiments on vascular smooth muscle cells, performed an in vivo pharmacokinetic study, and locally delivered fluorescently-labeled polysaccharides in rabbit iliac arteries after angioplasty with a non-occlusive catheter. The results indicated that (i) preparation of well-characterized fractions from natural fucoidan is compulsory for in vitro and in vivo studies, (ii) antiproliferative activity of sulphated polysaccharides on cultured smooth muscle cells is not a major predictive factor for the reduction of restenosis in vivo and (iii) pharmacokinetic parameters and binding of low-molecular-weight fucoidan on angioplasty-induced injured vascular walls are important local and general factors controlling its mechanisms of action.

The oral cavity and age: a site of chronic inflammation?

BACKGROUND: Aging may be accompanied by a low grade chronic up-regulation of inflammatory mediators. A variety of endogenous locally released mediators as well as inflammatory cells have been reported in the human oral cavity. The aim of this investigation was to determine the presence of different classes of inflammatory mediators in human saliva and correlate the levels with age. METHODOLOGY AND PRINCIPAL FINDINGS: Unstimulated whole buccal salivary samples were obtained in the morning from 94 healthy volunteers within 30 minutes after waking. None of the participants had taken aspirin in the week prior to the saliva collection. Lysozyme activity, eicosanoid levels (prostaglandin E(2) and leukotriene B(4)) and MMP-9 activity were measured. The antimicrobial activity (lysozyme activity) was not correlated with age whereas PGE(2) levels were markedly correlated with age (r = 0.29; P<0.05; n = 56). Saliva from healthy subjects (< or =40 years) compared with data derived from older volunteers (>40 years) demonstrated a significant increase in the mean values for PGE(2) and MMP-9 activity with age. In addition, significant correlations were observed between LTB(4) and PGE(2) (r = 0.28; P<0.05; n = 56) and between LTB(4) levels and MMP-9 activity in smokers (r = 0.78; P<0.001; n = 15). CONCLUSIONS/SIGNIFICANCE: The presence of significant levels and activity of inflammatory mediators in saliva suggests that the oral cavity of healthy subjects may be in a constant low state of inflammation associated with age.

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications.

BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.