Association of Matrix Metalloproteinase Levels with Collagen Degradation in the Context of Abdominal Aortic Aneurysm.

OBJECTIVE/BACKGROUND: Matrix metalloproteinases (MMPs) have already been identified as key players in the pathogenesis of abdominal aortic aneurysm (AAA). However, the current data remain inconclusive. In this study, the expression of MMPs at mRNA and protein levels were investigated in relation to the degradation of collagen I and collagen III. METHODS: Tissue samples were obtained from 40 patients with AAA undergoing open aortic repair, and from five healthy controls during kidney transplantation. Expression of MMPs 1, 2, 3, 7, 8, 9, and 12, and tissue inhibitor of metalloproteinase (TIMP)1, and TIMP2 were measured at the mRNA level using quantitative reverse transcription polymerase chain reaction. At the protein level, MMPs, collagen I, and collagen III, and their degradation products carboxy-terminal collagen cross-links (CTX)-I and CTX-III, were quantified via enzyme linked immunosorbent assay. In addition, immunohistochemistry and gelatine zymography were performed. RESULTS: In AAA, significantly enhanced mRNA expression was observed for MMPs 3, 9, and 12 compared with controls (p ≤ .001). MMPs 3, 9, and 12 correlated significantly with macrophages (p = .007, p = .018, and p = .015, respectively), and synthetic smooth muscle cells with MMPs 1, 2, and 9 (p = .020, p = .018, and p = .027, respectively). At the protein level, MMPs 8, 9, and 12 were significantly elevated in AAA (p = .006, p = .0004, and p < .001, respectively). No significant correlation between mRNA and protein was observed for any MMP. AAA contained significantly reduced intact collagen I (twofold; p = .002), whereas collagen III was increased (4.6 fold; p < .001). Regarding degraded collagen I and III relative to intact collagens, observations were inverse (1.4 fold increase for CTX-1 [p < .001]; fivefold decrease for CTX-III [p = .004]). MMPs 8, 9, and 12 correlated with collagen I (p = .019, p < .001, and p = 0.003, respectively), collagen III (p = .015, p < .001, and p < .001, respectively), and degraded collagen I (p = .012, p = .049, and p = .001, respectively). CONCLUSION: No significant relationship was found between mRNA and protein and MMP levels. MMPs 9 and 12 were overexpressed in AAA at the mRNA and protein level, and MMP-8 at the protein level. MMP-2 was detected in synthetic SMCs. Collagen I and III showed inverse behaviour in AAA. In particular, MMPs 8, 9, and 12 appear to be associated with collagen I, collagen III, and their degradation products.

CXCR4: A Potential Marker for Inflammatory Activity in Abdominal Aortic Aneurysm Wall.

OBJECTIVES: The aim of the study was to evaluate the potential role of chemokine receptor CXCR4 and its ligand CXCL12 in the pathogenesis of abdominal aortic aneurysm (AAA). METHODS: AAA tissue specimens were obtained from the anterior or lateral aneurysm sac of patients (n = 32, 26 males, 6 females; 66.8 ± 11.2 years, diameter 64.4 ± 17.0 mm), who underwent elective open surgical repair. Twelve non-aneurysmal aortic specimens from transplant donors served as controls. Expression analysis of CXCR4 and CXCL12 at mRNA and protein level was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Immunohistochemical staining of corresponding histological sections for CD3 (T-cells), CD20 (B-cells), and CD68 (macrophages) was performed to determine the cellular localization of CXCR4 and CXCL12. Data were analyzed with SPSS 20.0 using Mann-Whitney U test and Spearman's rank correlation coefficient. RESULTS: Gene expression of CXCR4 and CXCL12 was 9.6 and 4.6 fold higher in AAA than in non-aneurysmal aorta (p = .0004 and p < .0001, respectively). Likewise, the protein level of CXCR4 was increased 3.2 fold in AAA wall compared with non-aneurysmal aortic tissue (p < .0001), although CXCL12 could not be detected. Immunohistochemical analysis revealed that CXCR4 was expressed in B and T lymphocytes and macrophages, and CXCL12 was observed only in plasma cells. CONCLUSIONS: This study confirmed the over expression of CXCR4 in human AAA tissue. CXCR4 was detected both at the mRNA and the protein level and by immunohistochemistry, especially in inflammatory cells. In contrast, CXCL12 expression was observed only at the mRNA level, with the exception of plasma cells. The exact role of CXCR4/CXCL12 in AAA has to be further elucidated.

Interaction of biomechanics with extracellular matrix components in abdominal aortic aneurysm wall.

OBJECTIVE: Little is known about the interactions between extracellular matrix (ECM) proteins and locally acting mechanical conditions and material macroscopic properties in abdominal aortic aneurysm (AAA). In this study, ECM components were investigated with correlation to corresponding biomechanical properties and loads in aneurysmal arterial wall tissue. METHODS: Fifty-four tissue samples from 31 AAA patients (30♂; max. diameter Dmax 5.98 ± 1.42 cm) were excised from the aneurysm sac. Samples were divided for corresponding immunohistological and mechanical analysis. Collagen I and III, total collagen, elastin, and proteoglycans were quantified by computational image analysis of histological staining. Pre-surgical CT data were used for 3D segmentation of the AAA and calculation of mechanical conditions by advanced finite element analysis. AAA wall stiffness and strength were assessed by repeated cyclical, sinusoidal and destructive tensile testing. RESULTS: Amounts of collagen I, III, and total collagen were increased with higher local wall stress (p = .002, .017, .030, respectively) and strain (p = .002, .012, .020, respectively). AAA wall failure tension exhibited a positive correlation with collagen I, total collagen, and proteoglycans (p = .037, .038, .022, respectively). α-Stiffness correlated with collagen I, III, and total collagen (p = .011, .038, and .008), while ß-stiffness correlated only with proteoglycans (p = .028). In contrast, increased thrombus thickness was associated with decreased collagen I, III, and total collagen (p = .003, .020, .015, respectively), and AAA diameter was negatively associated with elastin (p = .006). CONCLUSIONS: The present results indicate that in AAA, increased locally acting biomechanical conditions (stress and strain) involve increased synthesis of collagen and proteoglycans with increased failure tension. These findings confirm the presence of adaptive biological processes to maintain the mechanical stability of AAA wall.

C-type natriuretic peptide and its receptors in atherosclerotic plaques of the carotid artery of clinically asymptomatic patients.

OBJECTIVES: C-type natriuretic peptide (CNP) has anti-inflammatory, anti-proliferative and anti-migratory properties. No data exist on the presence of CNP in human atherosclerotic plaques of the carotid artery. Therefore, this study aimed to analyse qualitatively the distribution pattern and characteristics of CNP and its receptors in both, early and advanced human carotid plaques, as well as in stable and unstable lesions. In addition, the aim of this study was to evaluate CNP and its receptors as possible biomarkers to predict plaque stability in advanced lesions. METHODS: Advanced carotid artery plaques of 40 asymptomatic patients (20 histologically stable and 20 histologically unstable) and early arteriosclerotic lesions of three patients were analysed. RESULTS: Serum level of CNP was similar in patients with stable and unstable plaques (196 ± 19 pg ml(-1) vs. 198 ± 25 pg ml(-1), p = 0.948). Expression level of natriuretic peptide receptor 3 (NPR3) was significantly higher in unstable plaques compared to stable plaques (5.6 ± 1.8% vs. 1.7 ± 0.5%, p = 0.045). Expression levels of CNP and NPR2 were higher in unstable plaques but the differences were not statistically significant. The distribution pattern of CNP, NPR2 and NPR3 varied qualitatively between early and advanced carotid plaques. No relevant histological differences were observed with respect to plaque stability. CONCLUSIONS: This study shows the presence of CNP and its receptors in atherosclerotic plaques of human carotid artery, with increased expression of NPR3 in histologically unstable plaques. In this study, serum CNP was not associated with histological plaque stability. In future, larger studies are required to further evaluate whether proteins of the CNP axis would be useful as biomarkers.

Thrombus Histology of Basilar Artery Occlusions : Are There Differences to the Anterior Circulation?

BACKGROUND: For patients with acute vessel occlusions of the anterior circulation histopathology of retrieved cerebral thrombi has been reported to be associated to stroke etiology. Due to the relatively small incidence of posterior circulation stroke, exclusive histopathologic analyses are missing for this subgroup. The aim of the study was to investigate thrombus histology for patients with basilar artery occlusions and uncover differences to anterior circulation clots with respect to underlying etiology. METHODS: A total of 59 basilar thrombi were collected during intracranial mechanical recanalization and quantitatively analyzed in terms of their relative fractions of the main constituents, e.g. fibrin/platelets (F/P), red (RBC) and white blood cells (WBC). Data were compared to histopathological analyses of 122 thrombi of the anterior circulation with respect to underlying pathogenesis. RESULTS: The composition of basilar thrombi differed significantly to thrombi of the anterior circulation with an overall higher RBC amount (median fraction in % (interquartile range):0.48 (0.37-0.69) vs. 0.37 (0.28-0.50), p < 0.001) and lower F/P count (0.45 (0.21-0.58) vs. 0.57 (0.44-0.66), p < 0.001). Basilar thrombi composition did not differ between the different etiological stroke subgroups. CONCLUSION: The results depict a differing thrombus composition of basilar thrombi in comparison to anterior circulation clots with an overall higher amount of RBC. This may reflect different pathophysiologic processes between anterior and posterior circulation thrombogenesis, e.g. a larger proportion of appositional thrombus growth in the posterior circulation.

Carotid plaque composition in chronic kidney disease: a retrospective analysis of patients undergoing carotid endarterectomy.

OBJECTIVES: Calcified plaques are suggested to represent atherosclerotic lesions with stabilising properties. However, patients with chronic kidney disease (CKD) frequently have calcified plaques but significant higher prevalence of cardiovascular complications. The aim of our study was therefore to analyse the effect of CKD in patients with advanced carotid stenosis (>70%) on plaque composition, lesion stability and risk of rupture. METHODS: We investigated retrospectively, by histology, carotid plaques of patients with high-grade internal carotid artery stenosis undergoing carotid endarterectomy. Comparison of plaque morphology was performed on 41 patients with CKD with estimated glomerular filtration rate (eGFR) <60 ml min(-1) (according to the Modification of Diet in Renal Disease formula, MDRD-eGFR) and 56 patients with normal renal function. RESULTS: Patients with CKD had significantly higher percentage of total calcification (17% vs. 7%, p<0.001), unstable and ruptured plaques (83% vs. 52%, p=0.001 and 59% vs. 36%, p=0.039, respectively) compared with patients with normal renal function. By contrast, the content of collagenous fibres was significantly reduced in CKD patients (40% vs. 57%, p=0.011). No significant differences were found for neurological symptoms and soft plaque content. CONCLUSION: Our results demonstrate that CKD significantly affects plaque composition in patients with advanced carotid artery stenosis. Enhanced calcification and reduced collagenous plaque may lead to plaque instability and rupture.

Is serum pregnancy-associated plasma protein A really a potential marker of atherosclerotic carotid plaque stability?

OBJECTIVES: The search for markers predicting risk of plaque rupture in carotid atherosclerosis is still ongoing. Previous findings showed that pregnancy-associated plasma protein-A (PAPP-A) levels correlate with an adverse plaque morphology. However, the role of PAPP-A in plaque destabilisation is still uncertain. MATERIAL AND METHODS: Patients with carotid artery stenosis involved in the study were asymptomatic (n=29) and symptomatic (n=37). Carotid plaques were characterised by histology (n=33). Immunohistochemistry (n=17) was used to determine expression of PAPP-A and CD68 within the plaques. Serum levels of PAPP-A were measured by the enzyme-linked immunosorbent assay (ELISA). RESULTS: Circulating PAPP-A levels were significantly higher in patients with unstable versus stable plaques (0.10+/-0.06 vs. 0.07+/-0.04 microg ml(-1), p=0.047) and interestingly, in asymptomatic versus symptomatic patients (0.11+/-0.05 vs. 0.069+/-0.09 microg ml(-1), p=0.025). These differences remained statistically significant after adjustment for age, gender and degree of stenosis (p=0.050). PAPP-A expression in plaques correlated significantly with CD68 positive macrophages, cap-thickness and its serological values (r=+0.291, p<0.001, r=-0.639, p<0.001 and r=0.618, p<0.008, respectively). Furthermore, PAPP-A serum values demonstrated a significant positive predictive value of 68.8% for unstable plaques. CONCLUSION: Our present data confirmed the close relationship between expression of PAPP-A and plaque instability and furthermore correlated significantly with cap thickness. However, the question whether PAPP-A is a useful predictive marker of plaque instability remains unresolved.

[Assessment of generic and disease-specific health-related quality of life instruments in peripheral arterial disease]. / Die Erfassung der allgemeinen und gesundheitsbezogenen Lebensqualität bei Patienten mit peripherer arterieller Verschlusskrankheit--eine Ubersicht.

As chronic diseases are continuously increasing in our aging society, the description and improvement of quality of medical care needs critical examination of the multidimensional subject of "quality of life". Health-related quality of life is currently used as an outcome-criterion in modern medicine. As there is no generally accepted definition of quality of life, various components of the state of health and the patient's behaviour are recorded by questionnaires. The level of subjective well- being is determined by several dimensions such as physical constitution of the patient, state of mind, functional competency in everyday life and the form of interpersonal relationships. Based on these principles various instruments for measuring quality of life are developed. The assessment of the subjective quality of life reflects the increased acceptance of the patient's view. In addition to the common generic instruments such as SF-36, FLZ(M), MLDL, EQ-5D, WHOQOL-100, NHP, SIP, also disease-specific instruments e.g. for peripheral arterial disease are currently used (PAVK-86, CLAU-S, VASCUQOL, SIP(IC), and WIQ). At the moment SF-36 is the best established questionnaire as generic QOL instrument. FLZ(M) takes the individual weighting of items into account, by correlating the importance and the contentment for life. For evaluating the effectivity of medical outcome and the success of therapeutic treatment for patients with vascular disease, the VASCUQOL instrument seems to be the best choice. Simultaneous application of a generic instrument and disease-specific questionnaires displays as well the subjective quality of health as the individual impairment of the patient in a good way. As a consequence using both instruments is superior to the exclusive use of a generic questionnaire.

The Impact of Histological Clot Composition in Embolic Stroke.

PURPOSE: Thrombus composition has been suggested to have a decisive impact on the outcome of patients treated by mechanical thrombectomy because of embolic stroke. The recent development of stent retrievers allows collection and, hence, histopathological analysis of fresh thrombus material. Against this background, the aim of this prospective study was to assess the impact of thrombus composition on mechanical recanalization, clinical outcome and stroke etiology. METHODS: Thirty-four patients suffering from acute ischemic stroke due to occlusion of the distal internal carotid artery/carotid-T, anterior cerebral artery, or middle cerebral arteries were mechanically recanalized, and thrombus material was obtained. Histological thrombus composition was compared with imaging, clinical, and neurointerventional data. RESULTS: The main findings were that a higher percentage of white blood cells (WBCs) in the thrombus was associated with (i) cardioembolic etiology, (ii) extended mechanical recanalization time, and (iii) less favorable recanalization (Thrombolysis in Cerebral Infarction score) and clinical outcome (National Institute of Health Stroke Scale). CONCLUSION: Our results suggest that thrombi with a high WBC fraction are related to more organized thrombi of cardioembolic origin associated with less favorable recanalization and clinical outcome in acute ischemic anterior circulation stroke. WBC-mediated immunological and coagulatory processes may play a key role in thrombus formation and pathogenesis of stroke warranting further investigation.

Alternation of histone and DNA methylation in human atherosclerotic carotid plaques.

Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development.

Quiescence, cell viability, apoptosis and necrosis of smooth muscle cells using different growth inhibitors.

Smooth muscle cells and endothelial cells play an important role in cardiovascular diseases and may therefore be a potential target for gene therapy. Most in vitro experiments are performed using proliferating cell cultures. Nevertheless, non-dividing cells would represent more realistic in vivo conditions for gene therapy. Therefore, a simple method to achieve physiologically quiescence in cell cultures is needed for experiments. Growth to confluence is sufficient for endothelial cells to reach quiescence, in contrast to smooth muscle cells. Alternative techniques were investigated to achieve quiescence for smooth muscle cells. N-acetyl-cysteine, heparin, aphidicolin and serum-free medium are known inhibitors of smooth muscle cell proliferation and were tested for cell viability, necrosis and apoptosis. The inhibition status was evaluated counting cells in a cell counter. Toxicity, necrosis and apoptosis were determined using FACS analysis. Then, smooth muscle cells and endothelial cells were transfected with plasmid containing the beta-galactosidase gene using liposomes. Analysis of gene expression in transfected cells included a quantitative beta-galactosidase assay and X-gal staining. Growth inhibition was achieved with all agents tested. Using N-acetyl-cysteine, only slightly reduced growth rates were observed. Aphidicolin stopped cell growth almost immediately, but demonstrated enhanced toxicity. The amount of apoptotic and necrotic cells was lowest using heparin in the presence of foetal calf serum. Transfection experiments using stationary cultures of smooth muscle cells using heparin or aphidicolin demonstrated 5-10-fold lower transfection rates compared to transfected proliferating cell cultures serving as controls. Transfection experiments using stationary cultures of endothelial cells using growth inhibition through confluence demonstrated 40-fold lower transfection rates than transfected proliferating cell cultures. Transfer efficiency was much lower in endothelial cells compared to smooth muscle cells. In conclusion, quiescent cells simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments based on in vitro findings.

Measuring and modeling patient-specific distributions of material properties in abdominal aortic aneurysm wall.

Both the clinically established diameter criterion and novel approaches of computational finite element (FE) analyses for rupture risk stratification of abdominal aortic aneurysms (AAA) are based on assumptions of population-averaged, uniform material properties for the AAA wall. The presence of inter-patient and intra-patient variations in material properties is known, but has so far not been addressed sufficiently. In order to enable the preoperative estimation of patient-specific AAA wall properties in the future, we investigated the relationship between non-invasively assessable clinical parameters and experimentally measured AAA wall properties. We harvested n = 163 AAA wall specimens (n = 50 patients) during open surgery and recorded the exact excision sites. Specimens were tested for their thickness, elastic properties, and failure loads using uniaxial tensile tests. In addition, 43 non-invasively assessable patient-specific or specimen-specific parameters were obtained from recordings made during surgery and patient charts. Experimental results were correlated with the non-invasively assessable parameters and simple regression models were created to mathematically describe the relationships. Wall thickness was most significantly correlated with the metabolic activity at the excision site assessed by PET/CT (ρ = 0.499, P = 4 × 10(-7)) and to thrombocyte counts from laboratory blood analyses (ρ = 0.445, P = 3 × 10(-9)). Wall thickness was increased in patients suffering from diabetes mellitus, while it was significantly thinner in patients suffering from chronic kidney disease (CKD). Elastic AAA wall properties had significant correlations with the metabolic activity at the excision site (PET/CT), with existent calcifications, and with the diameter of the non-dilated aorta proximal to the AAA. Failure properties (wall strength and failure tension) had correlations with the patient's medical history and with results from laboratory blood analyses. Interestingly, AAA wall failure tension was significantly reduced for patients with CKD and elevated blood levels of potassium and urea, respectively, both of which are associated with kidney disease. This study is a first step to a future preoperative estimation of AAA wall properties. Results can be conveyed to both the diameter criterion and FE analyses to refine rupture risk prediction. The fact that AAA wall from patients suffering from CKD featured reduced failure tension implies an increased AAA rupture risk for this patient group at comparably smaller AAA diameters.

siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing.

BACKGROUND: RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra- and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)-associated liposomes for siRNA complexation and gene silencing. METHODS: Cationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf-lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence-activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh-7 hepatocarcinoma and U-373 glioma cells. c-Jun knockdown in HT-22 cells was evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Cytotoxicity of the siRNA complexes was assessed by Alamar blue, lactate dehydrogenase and MTT assays. RESULTS: Complexation of siRNA with the cationic liposomes in the presence of Tf results in the formation of stable particles and prevents serum-mediated degradation. Confocal microscopy showed fast cellular internalization of the Tf-lipoplexes via endocytosis. In the GFP glioma cells Tf-lipoplexes showed enhanced gene silencing at minimum toxicity in comparison to Tf-free lipoplexes. Targeting luciferase in the hepatocarcinoma cell line resulted in more than 70% reduction of luciferase activity, while in HT-22 cells 50% knockdown of endogenous c-Jun resulted in a significant protection from glutamate-mediated toxicity. CONCLUSIONS: Cationic liposomes associated with Tf form stable siRNA lipoplexes with reduced toxicity and enhanced specific gene knockdown activity compared to conventional lipoplexes. Thus, such formulations may constitute efficient delivery systems for therapeutic siRNA applications.

Evaluation of beta-galactosidase activity in tissue in the presence of blood.

The reporter gene for beta-galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used beta-galactosidase substrates, including o-nitrophenyl-beta-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of beta-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of beta-galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the beta-galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous beta-galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying beta-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency.

Local perivascular application of low amounts of a plasmid encoding for vascular endothelial growth factor (VEGF165) is efficient for therapeutic angiogenesis in pigs.

Clinical trials have demonstrated therapeutic benefit in inducing angiogenesis in chronic occlusive arterial disease. The route of application mostly used was the intramuscular injection of high dosages of plasmid. Therefore, a local perivascular application of low amounts of vascular endothelial growth factor (VEGF) plasmid was used in an interventional occlusion model, and the effect of VEGF on coronary and peripheral occlusions compared in the same animal model. Coronary and peripheral arteries were chronically occluded in Pietrain pigs using a non-surgical, interventional approach. Adventitial delivery of the DNA for VEGF was performed with a needle injection catheter. The DNA was applied as lipoplexes using the novel cationic liposomes DOCSPER. Optimized transfer conditions were used. Angiography, polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were undertaken within a follow-up period of 6 months. Expression of the transfected VEGF gene was observed at 1 and 3 weeks following application. The DNA was detected up to 5 months following application. Around occluded coronary arteries, there was formation of new collaterals and arterial prolongation, whereas surrounding occluded peripheral arteries there was no collateralization but development of new arterial branches was seen. Results demonstrate that the response to VEGF is also sufficient, when minimal amounts of plasmid encoding for VEGF are applied locally into the perivasculature allowing for more safety of this therapy. Comparison of treatment of chronic coronary and peripheral arterial disease revealed differences in angiogenesis following VEGF application during a total follow-up period of almost 6 months which may be related to their different developmental origins. This may have important implications for developing future therapeutic strategies using VEGF in different vessels.

Optimization of nonviral gene transfer of vascular smooth muscle cells in vitro and in vivo.

Gene therapy strategies for the prevention of restenosis postangioplasty are promising. Nonviral gene transfer to the arterial wall in vivo has so far been limited by poor efficiency. This study aimed to optimize transfection of primary vascular smooth muscle cells using cationic nonviral formulations based on cholesterol derivates (DC-, DAC-, DCQ-, and Sp-Chol), double-chained amphiphils (LipofectAMINE, DOTMA, DOSGA, DOSPER, and DOCSPER), or heterogeneous reagents (Superfect, Effectene, and Tfx-50). Estimation of transfection efficiencies was performed using galactosidase assays at different ratios of transfection reagent to plasmid DNA with reporter gene. Toxicity was monitored by analyzing cell metabolism. Transfer efficiency and safety were determined in a porcine restenosis model for local gene therapy using morphometry, histology, galactosidase assays, and reverse-transcriptase polymerase chain reaction. The highest in vitro transfection efficiency was achieved using the recently developed DOCSPER liposomes, with transfer rates of at least 20% in vascular smooth muscle cells. Transfer efficiency was further enhanced up to 20% by complexing with poly-L-lysine. Transfection efficiency in vivo in a porcine restenosis model was up to 15% of adventitial cells using DOCSPER versus 0.1% using LipofectAMINE. Toxicity in vivo and in vitro was lowest using DOCSPER. Increased biological effects were demonstrated following optimization of transfer conditions.

Evaluation of endovascular techniques for creating a porcine femoral artery occlusion model.

PURPOSE: To determine the optimal endovascular approach to achieve long-term occlusion of large arteries, while preserving the integrity of periarterial tissue, in an animal model of ischemia. METHODS: Femoral artery occlusions were created in 16 pigs using detachable balloons, coils, or blinded stent-grafts. Feasibility, safety, primary and long-term success, and the degree of neovascularization were determined over a 6-month period by serial angiography and histological analyses. Four animals served as untreated controls. RESULTS: Overall primary success for all occlusion devices was 100%. The 6-month occlusion rate using detachable balloons or coils was 33% and 0%, respectively; however, all arteries occluded with blinded stent-grafts remained obstructed to the end of the study. There was no significant difference in capillary densities and collateralization of periarterial areas when occluded arteries were compared with nonoccluded controls in the same animal. No increase in collateralization was observed following endovascular arterial occlusion. CONCLUSIONS: Percutaneous insertion of blinded stent-grafts easily, safely, and reliably creates long-term arterial occlusion in pigs, which may make this a more appropriate model for studying the effects of angiogenic factors in vivo.

Prevention of Restenosis Using the Gene for Cecropin Complexed with DOCSPER Liposomes under Optimized Conditions.

Up to 30% of patients undergoing coronary angioplasty develop a renarrowing of treated vessels following percutaneous transluminal coronary angioplasty with or without stent implantation, called restenosis. Smooth muscle cell proliferation, among other mechanisms, is an important factor in restenosis leading to neointima formation and consequent arterial lumen narrowing. Cecropins are antimicrobial peptides with antiproliferative properties in mammalian cells which have been shown to suppress neointimal formation. In this investigation, a plasmid carrying the gene for pre-pro-cecropin A, complexed with new generation liposomes optimized for transfer conditions for vascular cells was delivered to the adventitia of arteries in a porcine arterial injury model using a needle injection catheter. Retention of the plasmid in treated arteries was demonstrated for at least 21 days following delivery. Whereas previous experiments using first generation liposomes demonstrated significant but not complete neointima inhibition, the use of new liposomes under optimized conditions resulted in almost total suppression of neointimal proliferation. Thus, in vivo gene transfer of cecropins may be therapeutically applicable in restenosis prevention.