Rüdiger Blindt

Efficient Transfection Method for Primary Cells

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.

Coronary artery stents in multislice computed tomography: in vitro artifact evaluation.

Rationale and ObjectiveThe aim of this study was to systematically compare the ability to assess the coronary artery lumen in the presence of coronary artery stents in multislice spiral CT (MSCT). MethodsTen different coronary artery stents were examined with 4- and 16-detector row MSCT scanners. For image reconstruction, a standard and a dedicated convolution kernel for coronary artery stent visualization were used. Images were analyzed regarding lumen visibility, intraluminal attenuation, and artifacts outside the stent lumen. Results were compared using repeated-measure analysis of variance. ResultsDepending on stent type, scanner hardware, and convolution kernel, artificial lumen narrowing ranged from 20% to 100%. The convolution kernel had the most significant influence on the visibility of the stent lumen. Artificial lumen narrowing and intraluminal attenuation changes decreased significantly using the dedicated convolution kernel. In general, most severe artifacts were caused by gold or gold-coated stents. ConclusionsIndependent of the scanner hardware or dedicated convolution kernels, routine evaluation of most coronary artery stents is not yet feasible using MSCT.

Characterization of Differential Gene Expression in Quiescent and Invasive Human Arterial Smooth Muscle Cells

Proliferation, migration and invasion of smooth muscle cells (SMCs) are essential pathogenic processes in the development of a broad spectrum of cardiovascular disorders, like arteriosclerosis, restenosis after percutaneous transluminal angioplasty and stent implantation as well as transplant vessel disease. As an in vitro model mimicking these processes, the Boyden chamber was employed to characterize the diverging migratory and invasive potentials of proliferating and nonproliferating human arterial SMCs (haSMCs). Using this model, differential gene expression of both phenotypes was analyzed by a cDNA array system (Clontech human cardiovascular array). With these arrays, 558 cardiovascular-associated genes could be compared. Further, gene expression was exactly quantified by real-time RT-PCR. Protein expression was analyzed by ELISA and Western blotting. In total, 47 genes were differentially expressed more than 1.5 times. Most of the differentially regulated genes in this study were associated with the extracellular matrix (ECM) and cell motility. In detail, the respective groups were matrix-organizing proteins, ECM proteins, cell adhesion proteins, extracellular communication and cytoskeleton motility proteins. Genes known to be differentially regulated during haSMC migration and invasion, like TIMP 2, TIMP 3, and MMP 3, were confirmed by the array data. Reduced expression of several cytoskeletal proteins, like vimentin, fibronectin, cytokeratins and β1 integrin, was shown in the invasive phenotype. Further, angio-associated protein, alpha E-catenin and atrial brain natriuretic peptide receptor were downregulated whereas TFPI 2 was strongly upregulated in invasive haSMCs. In conclusion, several relevant potential candidate genes for the quiescent and the invasive SMC phenotype were identified and genes already known to be differentially regulated by previous analysis were confirmed.

Sirolimus- and paclitaxel-eluting stents in comparison with balloon angioplasty for treatment of in-stent restenosis.

This study evaluated the acute and follow‐up effectiveness of sirolimus‐eluting stents (SESs) and nonpolymer‐based paclitaxel‐eluting stents (PESs) in comparison will balloon angioplasty for treatment of complex in‐stent restenosis (ISR) lesions. Drug‐eluting stents have been demonstrated to be highly effective for treatment of de novo lesions. The use of drug‐eluting stents for treatment of complex ISR is less well defined. Eighty one lesions with in‐stent restenosis (lesion length < 30 mm in a native coronary artery) were treated with either PTCA alone (n = 26 lesions in 25 patients), PES (n = 27 lesions in 24 patients; Achieve, Cook; 3,1 μg paclitaxel/mm2 nonpolymer‐based coating), SES (n = 28 lesions in 28 patients; Cypher, Cordis; 140 μg sirolimus/cm2 metal surface area). Nine‐month MACE rates were 32%, 8%, and 14% (all due to repeated revascularization procedures, except one death in the SES group) in the PTCA, PES, and SES group, respectively. Postintervention minimal lumen diameter in stent was significantly greater in the SES and the PES group in comparison with the PTCA group (2.37 ± 0.26, 2.54 ± 0.42, 1.78 ± 0.23 mm; P < 0.001). At 6‐month angiographic follow‐up, late loss in stent was 0.77 ± 0.45, 0.43 ± 0.53, and 0.29 ± 0.52 mm for the PTCA, PES, and SES group, respectively (P = 0.005). In‐lesion restenosis rate was 61% for the PTCA group, 20% for the PES group, and 13% for the SES group (P = 0.042). The implantation of SES as well as nonpolymer PES proved to be effective for treatment of ISR. The combination of improved acute gain and reduced late loss results in a significantly improved angiographic follow‐up result in comparison with PTCA. Catheter Cardiovasc Interv 2005;64:28–34.

Activation of IP and EP3 receptors alters cAMP-dependent cell migration

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G(s)-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP(3) receptors with opposite (i.e., G(i)) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP(3) receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP(3) receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP(3)). Chemotaxis of CHO cells transfected with G(s)-coupled IP receptors was concentration-dependently inhibited by iloprost (2-100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP(3) receptors showed a significant, concentration dependent (1-100 nM) increase of cell migration in presence of the selective EP(3) agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G(s)-mediated increase of intracellular cAMP. EP(3) receptors conversely stimulate CHO migration.

Superiority of sirolimus eluting stent compared with intracoronary β radiation for treatment of in-stent restenosis: a matched comparison

Objective: To compare acute and follow up clinical and angiographic results after treatment of in-stent restenosis (ISR) by sirolimus eluting stents (SES) with results obtained after intracoronary radiation therapy (IRT). Design: Matched pair analysis. Methods: 62 consecutive ISR lesions (< 30 mm lesion length, reference diameter < 3.5 mm) in 62 patients were treated with SES. From a database of 174 lesions (n  =  141 patients) treated for ISR by intracoronary β radiation, 62 lesions (62 patients) were pair matched with the SES group for diabetes mellitus, lesion length, vessel size, and pattern of ISR. Six month angiographic and 12 month clinical follow up results were obtained. Results: Baseline clinical and angiographic characteristics were similar between the groups (not significant). SES implantation resulted in significantly lower postprocedural in-lesion diameter stenosis than did IRT (mean (SD) 14.2 (9.5)% v 21.1 (10.6)%, p  =  0.001), significantly higher minimum lumen diameter at follow up (1.91 (0.58) v 1.55 (0.72) mm, p  =  0.005), and a higher net gain (1.16 (0.55) v 0.77 (0.70) mm, p  =  0.002). Angiographic binary in-lesion restenosis rate at six months was 11% in the SES group and 29% in the IRT group (p  =  0.046). In 16 ISR lesions SES were used after failed IRT and in 46 lesions for first time ISR. In-lesion late loss was higher after use of SES for failed IRT than after use of SES for first time ISR (0.61 (0.67) mm v 0.24 (0.41) mm, p  =  0.018). In a multivariate analysis prior failed IRT was the only independent predictor for recurrent restenosis after SES for ISR (p  =  0.052, odds ratio 5.8). Six patients (10%) in the SES group and 17 patients (27%) in the IRT group underwent target lesion revascularisation during the 12 months of follow up (p  =  0.022). Conclusions: In this non-randomised matched cohort SES achieved acute and follow up results superior to IRT for treatment of ISR even if cases of failed IRT are included. Failed IRT is a predictor of impaired SES effectiveness.

Effect of Statin Therapy Before Q-Wave Myocardial Infarction on Myocardial Perfusion

Recent studies emphasized the non-lipid-lowering effects of hydroxymethylglutaryl coenzyme A reductase inhibitors on endothelial function, inflammation, and platelet activation in patients with stable atherosclerosis. This study sought to evaluate the impact of statin pretreatment in patients with acute myocardial infarction (AMI) on level of systemic inflammation and myocardial perfusion. A total of 253 consecutive patients undergoing primary angioplasty on a native vessel within 12 hours of AMI were divided into a group with statin pretreatment (n = 86) and control patients (n = 167). Angiographic myocardial blush grade (MBG) after revascularization of the infarct-related artery was determined to evaluate myocardial perfusion. Statin pretreatment was associated with a lower frequency of increased C-reactive protein (>or=5 mg/L) on admission compared with the control group (48% vs 64%; p = 0.019). The frequency of normal perfusion (MBG 3) was higher in the statin-pretreatment group than the control group (45% vs 26%, respectively; p <0.001). Statin pretreatment was an independent predictor of normal myocardial perfusion (MBG 3; odds ratio 2.53, 95% confidence interval 1.15 to 9.53, p = 0.022) in addition to age <or=70 years and C-reactive protein <5 mg/L. In conclusion, statin pretreatment in patients with AMI was associated with decreased systemic inflammation and better perfusion after primary angioplasty of the infarct-related artery.

Upregulation of the cytoskeletal-associated protein Moesin in the neointima of coronary arteries after balloon angioplasty: a new marker of smooth muscle cell migration?

Migrating cells like coronary smooth muscle cells in restenosis change their cell shape and form cellular protrusions called filopodia. A prerequisite for filopodia formation is the rearrangement of the actin cytoskeleton. An essential role of the 78-kDa protein Moesin is described for Rho- and Rac-dependent assembly of actin filaments. In vivo Moesin is not observed in mature smooth muscle cells. The objective of this study was to demonstrate that Moesin is upregulated in migrating coronary smooth muscle cells during restenosis development. In vivo expression of Moesin was upregulated in neointimal coronary smooth muscle cells of dilated porcine coronary arteries compared to the undilated left circumflex coronary artery of the same swine. Concordant to these results Moesin expression was upregulated in migrating and invading human arterial smooth muscle cells in vitro analyzed by FACS, Western blotting and RT-PCR. In addition, the invasive potential of Moesin-positive Mel Im cells transfected with Moesin sense DNA increased by 28% as compared to mock-transfected control, whereas antisense transfected cells had a decreased invasive potential of 32%. Transfection of Moesin-negative HepG2 with Moesin sense cDNA increased the invasive potential by 43%. Finally, transfection of human arterial smooth muscle cells with Moesin sense cDNA caused an increased invasive potential of 30%. Transfection of haSMCs with antisense cDNA decreased the invasive potential by 37% in comparison to mock-transfected control. These results demonstrate for the first time an upregulation of Moesin expression in coronary smooth muscle cells of the neointima after arterial injury. The increased migrative and invasive potential of cells transfected with Moesin confirmed the functional role of Moesin in cell migration. This indicates an important role of Moesin during restenosis development.

Development and characterization of a coronary polylactic acid stent prototype generated by selective laser melting

In-stent restenosis is still an important issue and stent thrombosis is an unresolved risk after coronary intervention. Biodegradable stents would provide initial scaffolding of the stenosed segment and disappear subsequently. The additive manufacturing technology Selective Laser Melting (SLM) enables rapid, parallel, and raw material saving generation of complex 3- dimensional structures with extensive geometric freedom and is currently in use in orthopedic or dental applications. Here, SLM process parameters were adapted for poly-l-lactid acid (PLLA) and PLLA-co-poly-ε-caprolactone (PCL) powders to generate degradable coronary stent prototypes. Biocompatibility of both polymers was evidenced by assessment of cell morphology and of metabolic and adhesive activity at direct and indirect contact with human coronary artery smooth muscle cells, umbilical vein endothelial cells, and endothelial progenitor cells. γ-sterilization was demonstrated to guarantee safety of SLM-processed parts. From PLLA and PCL, stent prototypes were successfully generated and post-processing by spray- and dip-coating proved to thoroughly smoothen stent surfaces. In conclusion, for the first time, biodegradable polymers and the SLM technique were combined for the manufacturing of customized biodegradable coronary artery stent prototypes. SLM is advocated for the development of biodegradable coronary PLLA and PCL stents, potentially optimized for future bifurcation applications.

Blockade of Angio-Associated Migratory Cell Protein Inhibits Smooth Muscle Cell Migration and Neointima Formation in Accelerated Atherosclerosis

OBJECTIVES The aim of this study was to elucidate the role of angio-associated migratory cell protein (AAMP) for the migration of vascular smooth muscle cells (SMCs) and for the development of neointimal hyperplasia after vascular injury. BACKGROUND Although AAMP has been shown to participate in angiogenesis and cancerogenesis and is predominantly expressed in cells with a migratory phenotype, involvement of AAMP during neointima (NI) formation after arterial injury has not been analyzed previously. METHODS The AAMP content in SMCs was examined using 2-photon laser-scanning microscopy and subcellular fractioning. Migratory potential of SMCs transiently transfected with AAMP expression vectors, transfected with small interfering ribonucleic acid (siRNA), or treated with antirecombinant angio-associated migratory cell protein-antibody (anti-rAAMP-ab) was examined using transwell migration chamber assays. Expression of AAMP was determined in the atherogenic apolipoprotein E knockout (apoE(-/-)) mouse model and in the porcine coronary restenosis model by immunohistochemistry and by Western blot. ApoE(-/-) mice were treated intraperitoneally with anti-rAAMP-ab, and wire-injured carotid arteries were examined. RESULTS Angio-associated migratory cell protein is localized in the membrane of SMCs, and its expression is enhanced in NI-derived SMCs. The AAMP overexpression increases, while both treatment with anti-rAAMP-ab and transfection with siRNA decreases SMC migration. Knockdown of AAMP decreases RhoA activity in the membrane fraction of SMCs. The AAMP expression by SMCs is enhanced in both animal models. Anti-rAAMP-ab reduces neointimal SMC density at 1 week and NI formation at 4 weeks in apoE(-/-) mice without affecting proliferation of SMCs. CONCLUSIONS These data reveal an important functional role of AAMP in the migration of SMCs, identifying AAMP as a potential target to limit lesion formation after injury.