Effect of culture conditions on endothelial cell growth and responsiveness

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions

Lipoproteins inhibit platelet aggregation and arachidonic acid metabolism in experimental hypercholesterolaemia

1. Human plasma contains unidentified components that inhibit arachidonic acid (AA) metabolism. In the present study, we investigated whether plasma from rabbits fed a normal or high-cholesterol diet for 16 weeks also inhibits AA metabolism. Specifically, we studied the effects of plasma on platelet aggregation and on the production of AA metabolites, tri-hydroxyeicosatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B(2). 2. Haematological and lipid profiles were altered by a high-cholesterol diet. Platelets from hypercholesterolaemic rabbits showed enhanced aggregatory sensitivity to AA and platelet-activating factor. However, plasma from hypercholesterolaemic and control rabbits, when added to the incubation mixture, significantly inhibited platelet aggregation and eicosanoid production. 3. High- and low-density lipoprotein (HDL and LDL, respectively) concentrations increased several-fold in plasma with cholesterol feeding. When added directly to the incubation mixture, both HDL and LDL inhibited platelet aggregation, as well as AA metabolism. 4. Haptoglobin, albumin and Cohn\u27s fraction IV, but not globulins, exhibited antiplatelet and anti-AA metabolism activities. Their concentrations in plasma were not affected by cholesterol feeding. 5. We conclude that LDL and HDL account for at least some of the inhibition of AA metabolism produced by plasma

Reduced platelet hyper-reactivity and platelet-leukocyte aggregation after periodontal therapy

Background: Platelets from untreated periodontitis patients are hyper-reactive and form more platelet-leukocyte complexes compared to cells from individuals without periodontitis. It is not known whether the improvement of the periodontal condition achievable by therapy has beneficial effects on the platelet function. We aimed to assess the effects of periodontal therapy on platelet reactivity. Methods: Patients with periodontitis (n=25) but unaffected by any other medical condition or medication were included and donated blood before and after periodontal therapy. Reactivity to ADP or oral bacteria was assessed by flow cytometric analysis of membrane markers (binding of PAC-1, P-selectin, CD63) and platelet-leukocyte complex formation. Reactivity values were expressed as ratio between the stimulated and unstimulated sample. Plasma levels of soluble (s) P-selectin were determined by enzyme-linked immunosorbent assay (ELISA). Results: Binding of PAC-1, the expression of P-selectin and CD63 in response to the oral bacterium P. gingivalis were lower at recall (1.4±1.1, 1.5±1.2, and 1.0±0.1) than at baseline (2.7±4.1, P=0.026, 6.0±12.5, P=0.045, and 2.7±6.7, P=0.042, respectively). Formation of platelet-leukocyte complexes in response to P. gingivalis was also reduced at recall compared to baseline (1.2±0.7 vs. 11.4±50.5, P=0.045). sP-selectin levels were significantly increased post-therapy. Conclusions: In periodontitis patients, the improvement of the periodontal condition is paralleled by a reduction in platelet hyper-reactivity. We suggest that periodontal therapy, as an intervention for improved oral health, can facilitate the management of thrombotic risk, and on the long term can contribute to the prevention of cardiovascular events in patients at risk. Trial registration: Current Controlled Trials identifier ISRCTN36043780. Retrospectively registered 25 September 2013

Small-Interfering RNA–Eluting Surfaces as a Novel Concept for Intravascular Local Gene Silencing

New drug-eluting stent (DES) methods have recently been demonstrated to improve outcomes of intravascular interventions. A novel technique is the design of gene-silencing stents that elute specific small-interfering RNAs (siRNAs) for better vascular wall regeneration. Although siRNAs used to alter gene expression have surpassed expectations in in vitro experiments, the functional and local delivery of siRNAs is still the major obstacle for the in vivo application of RNA interference. In this preliminary in vitro study we investigated a surface-immobilized siRNA delivery technique that would be readily adaptable for local intravascular applications in vivo. The transfection potency of gelatin coatings consisting of a specific siRNA complexed with polyethylenimine (PEI) was examined in primary human endothelial cells by flow cytometry and quantitative real-time polymerase chain reaction. Several media conditions, such as the presence or absence of serum during cultivation, were investigated. Furthermore, different siRNA and PEI amounts, as well as nitrogen/phosphate ratios, were tested for their transfection efficiency. Gelatin coatings consisting of PEI and siRNA against an exemplary endothelial adhesion molecule receptor achieved a significant knockdown of around 70%. The transfection efficiency of the coatings was not influenced by the presence of serum. The results of this preliminary study support the expectation that this novel coating may be favorable for local in vivo gene silencing (for example, when immobilized on stents or balloons for percutanous transluminal coronary angioplasty). However, further animal experiments are needed to confirm the translation into clinical practice. This intriguing technology leads the way to more sophisticated and individualized coatings for the post-DES era, toward silencing of genes involved in the pathway of intimal hyperplasia