Cholinesterase activities and sepsis-associated encephalopathy in viral versus nonviral sepsis.

PURPOSE: There is evidence that cholinergic imbalance secondary to neuroinflammation plays a role in the pathophysiology of sepsis-associated encephalopathy (SAE). Blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities have been proposed as surrogate parameters for the cholinergic function of the central nervous system. Viral sepsis is associated with systemic inflammation and BChE has been reported to be of prognostic value in a small cohort of COVID-19 patients. Nevertheless, the prognostic value of AChE in patients with viral sepsis remains unclear. METHODS: We investigated the role of AChE and BChE activities as prognostic biomarkers of SAE and mortality in patients with viral vs nonviral sepsis enrolled in two prospective cohort studies. We quantified the AChE and BChE activities in whole blood of patients at two time points in the acute phase of viral sepsis (N = 108) and compared them with the activities in patients with nonviral sepsis (N = 117) and healthy volunteers (N = 81). Patients were observed until discharge from the intensive care unit (ICU). RESULTS: Three days after sepsis onset, the median [interquartile range] levels of AChE and BChE were reduced in both patients with viral sepsis (AChE, 5,105 [4,010-6,250] U·L-1; BChE, 1,943 [1,393-2,468] U·L-1) and nonviral sepsis (AChE, 4,424 [3,630-5,055] U·L-1; BChE, 1,095 [834-1,526] U·L-1) compared with healthy volunteers (AChE, 6,693 [5,401-8,020] U·L-1; BChE, 2,645 [2,198-3,478] U·L-1). Patients with viral sepsis with SAE during their ICU stay had lower AChE activity three days after sepsis onset than patients without SAE (4,249 [3,798-5,351] U·L-1 vs 5,544 [4,124-6,461] U·L-1). Butyrylcholinesterase activity seven days after sepsis onset was lower in patients with viral sepsis who died in the ICU than in surviving patients (1,427 [865-2,181] U·L-1 vs 2,122 [1,571-2,787] U·L-1). CONCLUSION: Cholinesterase activities may be relevant prognostic markers for the occurrence of SAE and mortality in the ICU in patients with viral sepsis. STUDY REGISTRATION: This study constitutes an analysis of data from the ongoing studies ICROS (NCT03620409, first submitted 15 May 2018) and ICROVID (DRKS00024162, first submitted 9 February 2021).

RéSUMé: OBJECTIF: Certaines données probantes soutiennent que le déséquilibre cholinergique secondaire à la neuroinflammation joue un rôle dans la physiopathologie de l'encéphalopathie associée au sepsis (EAS). Les activités de l'acétylcholinestérase (AChE) et de la butyrylcholinestérase (BChE) sanguines ont été proposées comme paramètres de substitution de la fonction cholinergique du système nerveux central. Le sepsis viral est associé à une inflammation systémique et il a été rapporté que la BChE possédait une valeur pronostique dans une petite cohorte atteinte de COVID-19. Néanmoins, la valeur pronostique de l'AChE chez les patient·es atteint·es de sepsis viral reste incertaine. MéTHODE: Nous avons étudié le rôle des activités de l'AChE et de la BChE en tant que biomarqueurs pronostiques de l'EAS et de la mortalité chez les patient·es atteint·es de sepsis viral vs non viral recruté·es dans deux études de cohorte prospectives. Nous avons quantifié les activités de l'AChE et de la BChE dans le sang total de patient·es à deux moments de la phase aiguë du sepsis viral (N = 108) et les avons comparées aux activités chez les patient·es atteint·es de sepsis non viral (N = 117) et chez des volontaires sain·es (N = 81). Les patient·es ont été observé·es jusqu'à leur sortie de l'unité de soins intensifs (USI). RéSULTATS: Trois jours après l'apparition du sepsis, les taux médians [écart interquartile] d'AChE et BChE étaient réduits tant chez la patientèle atteinte de sepsis viral (AChE, 5105 [4010­6250] U·L−1; BChE, 1943 [1393­2468] U·L−1) et de sepsis non viral (AChE, 4424 [3630­5055] U·L−1; BChE, 1095 [834­1526] U·L−1) par rapport aux volontaires sain·es (AChE, 6693 [5401­8020] U·L−1; BChE, 2645 [2198­3478] U·L−1). Les patient·es atteint·es de sepsis viral avec EAS pendant leur séjour aux soins intensifs avaient une activité AChE plus faible trois jours après l'apparition du sepsis que les personnes sans EAS (4249 [3798­5351] U·L−1 vs 5544 [4124­6461] U·L−1). L'activité de la butyrylcholinestérase sept jours après l'apparition du sepsis était plus faible chez les patient·es atteint·es de sepsis viral décédé·es à l'USI que chez les personnes ayant survécu (1427 [865­2181] U·L-1 vs 2122 [1571­2787] U·L-1). CONCLUSION: Les activités des cholinestérases pourraient constituer des marqueurs pronostiques pertinents pour la survenue d'EAS et la mortalité en soins intensifs chez la patientèle atteinte de sepsis viral. ENREGISTREMENT DE L'éTUDE: Cette étude constitue une analyse des données des études en cours ICROS (NCT03620409, première soumission le 15 mai 2018) et ICROVID (DRKS00024162, première soumission le 9 février 2021).

Safety and efficacy of SITAgliptin plus GRanulocyte-colony-stimulating factor in patients suffering from Acute Myocardial Infarction (SITAGRAMI-Trial)--rationale, design and first interim analysis.

AIMS: Our pre-clinical studies demonstrated that G-CSF based stem cell mobilization in combination with genetic or pharmaceutical CD26/DPP-IV inhibition after acute myocardial infarction leads to improved cardiac homing of stem cells, enhanced heart function and increased survival. Thereupon, we initiated a phase III, multi-centre, randomised, placebo-controlled efficacy and safety study (n=100) analyzing the effect of combined application of G-CSF and Sitagliptin, which is a clinically admitted, anti-diabetic DPP-IV-inhibitor, after acute myocardial infarction ("SITAGRAMI-Trial"; EudraCT Number: 2007-003941-34). METHODS: The primary objective of the study is to assess myocardial regeneration by improved myocardial homing of mobilized stem cells, as measured by cardiac function using MRI analysis. In this paper, we report on the study design and a planned first interim-analysis on safety issues without unblinding. RESULTS: During the first 6 weeks of follow-up, only two major adverse cardiac events occurred (one de novo stenosis and one instent-restenosis) in the first 36 patients. Presumably, they were not related to any study medication. No other side effects like headache, bone pain, hypoglycaemias etc. were observed. Furthermore, no myocardial infarction or death occurred in any patient. Thus, the rate of serious adverse events lay within the expected range. CONCLUSIONS: Our data demonstrate that the combined application of Sitagliptin and G-CSF seems to be safe on the short term and feasible after acute myocardial infarction and may represent a new therapeutic option in future.

G-CSF in patients suffering from late revascularised ST elevation myocardial infarction: final 1-year-results of the G-CSF-STEMI Trial.

AIMS: The aims of this trial were to investigate the effect of granulocyte colony-stimulating factor (G-CSF) on left-ventricular ejection fraction and event-free survival in patients suffering from sub-acute myocardial infarction (STEMI). METHODS: We enrolled 44 patients suffering from sub-acute STEMI with late revascularization achieved by percutaneous coronary intervention (PCI). Patients were randomized to receive either G-CSF (Filgrastim) at a dose of 10 µg/kg body weight/day subcutaneously or placebo. Changes of global and regional cardiac function from baseline (1 week after PCI) over 1 and 3 months to 12 months of follow-up were analyzed by magnetic resonance imaging. RESULTS: Ejection fraction improved in G-CSF treated patients from 41.1±11.9% to 47.1±11.9% (3 months) and decreased slightly to 45.7±15.1% after 1 year. Ejection fraction also improved in the placebo group from 43.8±9.0% to 49.5±11.8% (3 months) and decreased slightly to 42.9±15.4% after 1 year (1 year MRI follow-up was performed in 23 out initial 44 patients). There was no significant difference between the two groups at any time point. Other parameters such as infarct size, myocardial perfusion, left ventricular end-diastolic and end-systolic volumes were not different between the two groups. Event-free survival of such as death, (re) myocardial infarction or acute coronary syndromes, coronary artery bypass grafting and target lesion revascularization was not significantly different between both groups. CONCLUSIONS: G-CSF administration after sub-acute STEMI is feasible and safe but does not improve myocardial function or survival when used as a single substance.

Stem cell mobilisation for myocardial repair.

BACKGROUND: The idea that autologous bone marrow derived stem cells (BMCs) can transdifferentiate into cardiomyocytes or vascular cells has been challenged in several scientific reports. OBJECTIVE/METHODS: This review summarises conditions for stem cell mobilisation, their use for therapeutic approaches to prevent ischaemic cardiomyopathy after acute myocardial infarction and current clinical trials. Mechanisms for mobilisation and homing of BMCs are discussed. RESULTS/CONCLUSIONS: The improvement in cardiac function after migration of autologous BMCs to the heart can be explained by their paracrine effects, inducing angiogenesis and preventing ischaemic myocardium from apoptosis. These effects may explain why the number of circulating BMCs is directly correlated with cardiovascular risk and life expectancy. Exercise and hormones are physiological stimuli for the mobilisation of BMCs, whereas cardiovascular risk factors severely reduce their number and functions. Current cardiovascular medications increase the amounts of autologous BMCs.

G-CSF in patients suffering from late revascularized ST elevation myocardial infarction: analysis on the timing of G-CSF administration.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) improves myocardial function after infarction in vivo. Placebo-controlled clinical studies failed to show beneficial effects on myocardial function. Recent data demonstrate that the time point of treatment initiation may be crucial for the efficacy of G-CSF. We investigated the influence of the timing of G-CSF treatment on myocardial function and perfusion in a subgroup study of the G-CSF-ST Elevation Myocardial Infarction trial. MATERIALS AND METHODS: Patients with late revascularized myocardial infarction (n = 44) were treated with either G-CSF or placebo over 5 days after successful percutaneous coronary intervention (PCI). Of the G-CSF group, 13 patients had received G-CSF early treatment started within 24 hours after PCI (mean: 16 +/- 6 hours). In 10 patients, G-CSF was initiated late (>24 hours after PCI, mean: 49 +/- 26 hours). Global and regional myocardial function and perfusion were assessed from baseline to 3 months after PCI using magnetic resonance imaging in 37 patients who completed magnetic resonance follow-up. RESULTS: G-CSF was safe when used early or late after PCI. Early G-CSF administration resulted in significantly improved perfusion at rest 1 month after PCI when compared to placebo (Up-slope, signal intensity 1.2 [0.4-1.8] vs 0.6 [0.1-1.3], p = 0.03). Timing of G-CSF had no influence on global and regional function. CONCLUSION: This post-hoc analysis indicates that timing of G-CSF after myocardial infarction does not improve myocardial function but myocardial perfusion if the cytokine is given early. This urges the need to investigate alternative dosage regimens or combination with novel therapeutics promoting mobilization and homing.

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo.

The potential of two small poly-L-lysines (sPLLs), low molecular weight sPLL (LMW-L) containing 7-30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer (GT) with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N(5)-carbamoyl-spermine)-propane] in vascular smooth muscle cells (SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA (50-100 nm). At high ionic strength, large complexes (>1 microm) were observed without any significant differences in particle size between lipoplexes (DOCSPER/DNA) and lipopolyplexes (DOCSPER/sPLL/DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/sPLL/DNA formulations enhanced GT in vitro up to 5-fold, in a porcine model using local periadventitial application up to 1.5-fold. Both sPLLs significantly increased liposome-mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery.

Circulation of CD34+ progenitor cell populations in patients with idiopathic dilated and ischaemic cardiomyopathy (DCM and ICM).

AIMS: This study aimed at analysing the endogenous stem cell circulation in patients suffering from idiopathic dilated cardiomyopathy (DCM) and ischaemic cardiomyopathy (ICM). METHODS AND RESULTS: Cytokines in peripheral blood were analysed using enzyme-linked immunosorbent assay and circulating CD34(+) stem cell populations (CD34(+)CD133(+), CD34(+)CD31(+), CD34(+)CXCR-4(+)) were measured by flow cytometry in DCM patients (n = 25), ICM patients (n = 15), and controls (n = 10). Explanted DCM (n = 5), ICM (n = 4) and normal hearts (n = 5) were analysed for the expression of several homing factors [stromal cell-derived factor-1 (SDF-1), Stem cell factor (SCF), HIF-1a, vascular cell adhesion molecule (VCAM), and Hepatocyte growth factor] by quantitative real-time polymerase chain reaction (PCR). SDF-1 was significantly elevated and positively correlated with brain natriuretic peptide (BNP) in peripheral blood of DCM and ICM patients showing the same New York heart association- (NYHA) class. In DCM patients circulating CD34(+) cell populations were significantly increased in comparison to ICM patients and controls. mRNA of SDF-1, SCF, HIF-1a, and VCAM related to glyceraldehyde-3-phosphate dehydrogenase was significantly upregulated in ICM hearts when compared with DCM hearts and controls. CONCLUSION: Myocardial homing factors are upregulated in ICM when compared with DCM hearts. Reduced homing of stem cells might therefore explain the increased number of CD34(+) cells in DCM patients. These findings may open a new insight into the pathology and the treatment of idiopathic DCM.

Stem cell therapy after myocardial infarction: ready for clinical application?

The discovery of stem cells capable of generating angiogenic or contractile cells and structures might offer new treatment options for patients suffering from heart disease. In particular, embryonic stem cells are considered to have great potential for regenerative medicine and tissue engineering. Studies suggest that delivery or mobilization of stem and progenitor cells might improve tissue perfusion and contractile performance of the damaged heart; however, the underlying mechanisms are poorly understood. Fusion or trans-differentiation into cardiomyocytes or vascular cells are considered rare events of cellular engraftment, and adult stem cells are now considered as 'regenerator cells', acting via paracrine effects of cytokines, or by activation of resident stent cells, thereby supporting the myocardial healing mechanisms after injury. Administration of autologous hematopoietic stem cells or mobilization of endogenous stem cells has been shown to be safe after myocardial infarction or cardiomyopathies, whereas skeletal myoblasts are considered to be hazardous due to the occurrence of life-threatening arrhythmias. This review focuses on the use of adult human stem cells for treating myocardial infarction and cardiomyopathy, and discusses recent preliminary efficacy data, which suggest that 'regenerator cells' might have the potential to improve myocardial perfusion and contractile performance in patients suffering from myocardial infarction, severe ischemic heart disease and chronic heart failure.

Autologous bone marrow stem cell mobilization induced by granulocyte colony-stimulating factor after subacute ST-segment elevation myocardial infarction undergoing late revascularization: final results from the G-CSF-STEMI (Granulocyte Colony-Stimulating Factor ST-Segment Elevation Myocardial Infarction) trial.

OBJECTIVES: The purpose of this investigator-driven, prospective, randomized, double-blinded, placebo-controlled phase II study was to compare the effects of granulocyte colony-stimulating factor (G-CSF) on the improvement of myocardial function in patients undergoing delayed percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI). BACKGROUND: Experimental and early clinical studies suggest that transplantation of stem cells improves cardiac regeneration and neovascularization after acute myocardial infarction. Most investigators have utilized either a direct injection or intracoronary infusion of bone marrow-derived cells, but early cytokine-mediated mobilization of stem cells has been reported to show similar improvement in cardiac function. METHODS: Forty-four patients with late revascularized subacute STEMI were treated either with G-CSF or placebo over 5 days after successful PCI. Primary end points were change of global and regional myocardial function from baseline (1 week after PCI) to 3 months after PCI assessed by magnetic resonance imaging (MRI). Secondary end points consisted of characterization of mobilized stem cell populations, assessment of safety parameters up to 12 months including 6-month angiography, as well as myocardial perfusion assessed by MRI. RESULTS: Global myocardial function from baseline (1 week after PCI) to 3 months improved in both groups, but G-CSF was not superior to placebo (Delta(ejection fraction) 6.2 +/- 9.0 vs. 5.3 +/- 9.8%, p = 0.77). A slight but non-significant improvement of regional function occurred in both groups. Granulocyte colony-stimulating factor resulted in mobilization of endothelial progenitor cell populations and was well tolerated with a similar rate of target lesion revascularization from in-stent restenosis. In both groups major adverse cardiovascular events occurred in a comparable frequency. Granulocyte colony-stimulating factor resulted in significant improvement of myocardial perfusion 1 week and 1 month after PCI. CONCLUSIONS: Granulocyte colony-stimulating factor treatment after PCI in subacute STEMI is feasible and relatively safe. However, patients do not benefit from G-CSF when PCI is performed late. Granulocyte colony-stimulating factor results in improved myocardial perfusion of the infarcted area, which may reflect enhanced neovascularization.

Chronic perivascular inoculation with Chlamydophila pneumoniae results in plaque formation in vivo.

Hypercholesterolemic and normocholesterolemic rabbit models of chronic arterial Chlamydophila (Chlamydia) pneumoniae (CPN) inoculation were established and the role of both viable and inactivated bacteria was investigated in atherogenesis. A total of 29 rabbits were randomized to four groups. Groups A and B were fed a cholesterol-enriched diet, and groups C and D were fed a normal diet. Arterial segments of group A and C animals were inoculated in vivo using viable CPN chronically using repeated perivascular applications. Contralateral arteries were treated using heat-inactivated CPN. Group B and D animals were treated with repeated perivascular injections of bacterial lipopolysaccharide (LPS) and saline (control). Additional hypercholesterolemic rabbits were treated by repeated injections using viable and inactivated CPN, each controlled by saline injections. To compare the effects of this chronic inoculation model, additional animals received single injections of either viable CPN, inactivated CPN, LPS, or saline. Vascular tissues (n=162 treated arteries of 29 rabbits) were analyzed using morphometry at histology. CPN was detected by fluorescence-immunohistochemistry and nested polymerase chain reaction. Only in hypercholesterolemic, but not in normocholesterolemic rabbits, chronic perivascular infection of all bacterial components, viable and heat-inactivated CPN, as well as LPS resulted in a significant increase in atheromatous lesion formation (lesion area index: 0.23+/-0.08, 0.25+/-0.09, and 0.15+/-0.05) when compared to controls (lesion area index 0.01+/-0.01, P=0.002). CPN persisted in atheromatous lesions and vascular tissues. Single perivascular infection using CPN or inactivated CPN was not able to induce lesion formation (lesion area index: 0.03+/-0.03, 0.03+/-0.02 vs 0.03+/-0.02 after single saline inoculation, P=0.965). In conclusion, chronic vascular infection with CPN or CPN components acts as a cofactor requiring other major atherogenic stimuli, rather than as a causative agent.

Phase-sensitive inversion-recovery MR imaging in the detection of myocardial infarction.

PURPOSE: To prospectively determine if phase-sensitive inversion-recovery (IR) magnetic resonance (MR) imaging eliminates the need to find the precise inversion time (TI) to null the signal of normal myocardium to achieve high contrast between infarcted and normal myocardium. MATERIALS AND METHODS: Informed consent was obtained from each patient for this prospective MR imaging research study, which was approved by the institutional review board. Twenty patients (16 men; four women; mean age, 56 years +/- 12.3) who experienced Q-wave myocardial infarction 2 weeks earlier were examined with a 1.5-T MR system 10 minutes after administration of 0.1 mmol per kilogram of body weight gadobenate dimeglumine. To determine the optimal TI, a TI scout sequence was used. A segmented two-dimensional IR turbo fast low-angle shot (FLASH) sequence and a segmented two-dimensional IR true fast imaging with steady-state precession (FISP) sequence that produces both phase-sensitive and magnitude-reconstructed images were used at TI values of 200-600 msec (TI values were varied in 100-msec steps) and at optimal TI (mean value, 330 msec). Contrast-to-noise ratios (CNRs) of normal and infarcted myocardium and the area of infarcted myocardium were determined. Magnitude-reconstructed IR turbo FLASH images were compared with magnitude-reconstructed and phase-sensitive IR true FISP images. Two-tailed unpaired sample Student t test was used to compare CNRs, and two-tailed paired-sample Student t test was used to compare area of infarction. RESULTS: Mean CNR of images acquired with IR turbo FLASH and IR true FISP (phase-sensitive and magnitude-reconstructed images) at optimal TI (mean value, 330 msec) were 6.6, 6.2, and 6.1, respectively. For a TI of 200 msec, CNR values were -4.3, -4.0, and 7.2, respectively; for TI of 600 msec, CNR values were 3.1, 3.3, and 4.3, respectively. Area of infarcted myocardium was underestimated on magnitude-reconstruction images (P = .002-.03) for short TI values (ie, 200 msec) for both sequences and for a TI of 300 msec for IR true FISP but not on phase-sensitive reconstructed IR true FISP images when compared with IR turbo FLASH images obtained at optimal TI. CONCLUSION: Phase-sensitive image reconstruction results in reduced need for precise choice of TI and more consistent image quality.

Hypereosinophilic syndrome associated with heterozygous factor V gene mutation: an unusual combination resulting in an acute coronary syndrome and recurrent cerebral stroke-a case report.

The authors report the first case of a patient with a hypereosinophilic syndrome associated with heterozygous factor V gene mutation, resulting in an acute coronary syndrome and recurrent cerebral stroke despite effective anticoagulation. A 40-year-old man presented with an acute coronary syndrome accompanied by a brachiofacial right-sided hemiparesis and dysarthria. Diagnosis of a hypereosinophilic syndrome was established by blood testing, myocardial biopsy, and bone marrow analysis. Eosinophilic infiltration was present in the myocardium, accompanied by proliferation in the endomyocardium and a pneumonic infiltrate. Although effective anticoagulation with heparin was administered, a recurrent stroke occurred while blood eosinophils were being normalized by corticosteroid treatment. A coexisting heterozygosity of the factor V mutation was demonstrated, and it is hypothesized that this might have contributed to the recurrent thromboembolic episodes. In patients with hypereosinophilic syndrome and recurrent thromboembolic episodes, other thrombophilic diseases, including factor V mutations, should be considered, and long-term coagulation should be contemplated.

Recurrent perivascular inflammation induced by lipopolysaccharide (endotoxin) results in the formation of atheromatous lesions in vivo.

Bacteria and viruses are suspected to induce arteriosclerosis; however, most investigators have focused on coincidences rather than causal relationships. The aim of this work was to establish a rabbit model in which the vessel reaction to local perivascular injection of defined bacterial products can be analyzed. A total of 23 rabbits were randomized to four groups. Groups A and B were fed a normal diet, groups C and D were fed a cholesterol-enriched diet. Groups A and C were treated with a single perivascular injection of bacterial lipopolysaccharide (LPS, endotoxin) placed next to auricular, carotid and femoral arteries, and sodium chloride placed next to the contralateral arteries (control). Group B and D animals were treated with repeated perivascular injections over 90 days. Vascular tissues (n=116 treated segments of 23 rabbits) were analyzed using morphometry at histology, and using immunohistochemistry to detect macrophages, lymphocytes and vascular smooth muscle cells. LPS treatment resulted in transient focal intima thickening. After single LPS application, no increase in atheromatous lesion formation was observed in comparison with controls (group C, lesion area index 0.031+/-0.012 vs 0.015+/-0.006, P=1.0). Repeated LPS application resulted in significant atheromatous lesion formation compared with saline control (group D, lesion area index 0.148+/-0.049 vs 0.008+/-0.006, P=0.003) in hypercholesterolemic rabbits. Repeated LPS inflammation in normocholesterolemic did not lead to atheromatous lesion formation (intima media ratio 0.04+/-0.01 vs 0.04+/-0.007, P=1.0). Single perivascular administration of low-dose bacterial LPS resulted in transient focal intimal thickening, while significant increase in lesion formation occurred after repeated LPS application in cholesterol-fed animals. In conclusion, this animal model will allow the assessment of the impact of defined dosages of different bacterial pathogens onto the vascular wall in the context of atherogenesis. The atheromatous lesion-promoting effect of repeated perivascular administration of LPS supports the hypothesis that bacterial pathogens may be involved in atherogenesis.

Comparison of surgical versus endovascular occlusion models in pig femoral arteries.

PURPOSE: To compare an endovascular technique with a well established surgical approach to achieve long-term occlusions of large porcine arteries while preserving the integrity of periarterial tissue. METHODS: The femoral arteries in 11 pigs were occluded using surgical techniques on one side and blinded stent-grafts in the contralateral vessel. Feasibility, safety, primary and long-term success, and the extent of vascularization were determined over a 3-month period by conventional angiography and histological analysis. A subgroup of animals (n=5) was treated with a locally administered plasmid coding for vascular endothelial growth factor (pVEGF165) to compare both occlusion techniques under conditions of collateral growth induction. RESULTS: The primary and long-term success rates for both occlusion models were 100%. Surgical occlusion of arteries resulted in a significant amount of scar dehiscence and local groin infection compared to the endograft-occluded side. There was no significant difference in capillary densities and collateralization of periarterial areas in a comparison of the occlusion technique: the cross-sectional area of the superficial femoral artery (SFA) was 300 +/- 24 mm2 for endovascular occlusion versus 320 +/- 23 mm2 for surgical occlusion (p=0.559). In the profunda femoris artery, respective values were 418 +/- 35 and 448 +/- 18 mm2 (p=0.474). The local delivery of pVEGF165 resulted in a significant increase in collateral growth in both occlusion models with comparable neovascularization: cross-sectional SFA area increased from 310 +/- 16 to 428 +/- 13 mm2 (p<0.0001); in the PFA, the area increased from 422 +/- 19 to 658 +/- 49 mm2 (p<0.0001). CONCLUSIONS: Endovascular arterial occlusions using blinded stent-grafts allow easy and safe creation of long-term occlusions. Previously described collateralization following surgical occlusions was not observed, indicating that those collaterals may be associated with wound healing rather than ischemia. The occlusion of arteries using blinded stent-grafts in pigs may therefore be an appropriate model for assessing the effects of angiogenic factors in vivo.

Vascular endothelial growth factor response in porcine coronary and peripheral arteries using nonsurgical occlusion model, local delivery, and liposome-mediated gene transfer.

Angiogenesis and arteriogenesis play an important role in advanced vascular occlusive diseases. Whether angiogenesis or arteriogenesis predominate depends on the preexisting collateral vessel network, the type and location of occlusion, and different developmental origin of the arteries. Angiogenesis and arteriogenesis were investigated following vascular endothelial growth factor (VEGF) treatment in different arteries important in occlusive arterial diseases using a newly developed porcine arterial occlusion model. Porcine coronary and peripheral arteries were occluded interventionally using blinded stent grafts. Gene transfer was performed using a needle injection catheter and cationic lipid DOCSPER as gene carrier. DNA and gene expression in arterial tissue was examined using polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR. Vessel development was determined by angiography, immunohistochemistry, and measurement of capillary density. The transfected gene and its expression were found 3 months following application. In tissue adjacent to coronary arteries, there was significantly enhanced capillary density but no increase in angiographic score. In contrast, tissue surrounding peripheral arteries demonstrated no enhancement of capillary density but an enhancement in angiographic score. These results demonstrate differential responses to VEGF treatment in coronary and peripheral arteries resulting predominantly in either angiogenesis or arteriogenesis. Further investigation of VEGF signaling pathway is necessary for better understanding of the processes of vascular development, which may have potential impact on the design of cardiovascular therapeutics.

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model.

Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.

Vascular endothelial growth factor (VEGF165) and its influence on angiogenesis versus arteriogenesis in different vascular beds.

PURPOSE: To use local gene delivery to determine any district-specific influence of vascular endothelial growth factor (VEGF(165)) on angiogenesis and arteriogenesis in arteries of distinct developmental origin. METHODS: Coronary and peripheral arteries were chronically occluded in 30 Pietrain pigs using a percutaneous approach and blinded stent-graft. DNA was delivered to the adventitia in dosages corresponding to 10% of the body weight-adapted amount used in clinical trials. The coronary arteries in 12 animals and the peripheral arteries in 12 animals were treated or used as controls (no occlusion or occlusion with transfection of the beta-galactosidase gene). Six additional animals were sacrificed at 1 or 3 weeks for expression analyses, while the other 24 animals were sacrificed at 5 months for expression analysis and histology. Angiography, polymerase chain reaction analyses, and immunohistochemistry were performed. RESULTS: Expression of the VEGF gene was observed at 1 and 3 weeks following application, while transfected DNA was detected up to 5 months. New collaterals formed around occluded coronary arteries (2.63 +/- 0.69 fold, p<0.05 versus 1.24 +/- 0.40 fold for peripheral arteries), and angiographic arterial area increase was more pronounced in coronary (2.49 +/- 0.59 fold, p<0.05) than peripheral arteries (1.49 +/- 0.05 fold). There was no collateralization surrounding occluded peripheral arteries, but new arterial branches were seen (2.0 +/- 0.28, p<0.05 versus 1.07 +/- 0.31 for coronary). CONCLUSIONS: The response to VEGF, whether it is predominantly angiogenesis or arteriogenesis, is dependent on the target vessel. These observed differences in the behavior of arteries may be related to their differing developmental origins, which may have important implications for future therapeutic strategies using VEGF in different vessels.