Janet Chamberlain

Ultrasound Enhances Reporter Gene Expression After Transfection of Vascular Cells In Vitro

BACKGROUND Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. METHODS AND RESULTS Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. CONCLUSIONS Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.

Apoptosis and Cell Proliferation After Porcine Coronary Angioplasty

BACKGROUND Angioplasty initiates a number of responses in the vessel wall including cellular migration, proliferation, and matrix accumulation, all of which contribute to neointima formation and restenosis. Cellular homeostasis within a tissue depends on the balance between cell proliferation and apoptosis. METHODS AND RESULTS Profiles of apoptosis and proliferation were therefore examined in a porcine PTCA injury model over a 28-day period. Forty-two arteries from 21 pigs, harvested at the site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days after PTCA, were examined (n=3 animals per time point). Uninjured arteries were used as controls. Apoptosis was demonstrated by the terminal uridine nick-end labeling (TUNEL) method, transmission electron microscopy (TEM), and DNA fragmentation. Cells traversing the cell cycle were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Apoptosis was not detected in control vessels at all time points nor at 28 days after PTCA. Apoptotic cells were identified at all early time points with a peak at 6 hours (5.1+/-0.26%; compared to uninjured artery, P<0.001) and confirmed by characteristic DNA ladders and TEM findings. Regional analysis showed apoptosis within the media, adventitia, and neointima peaked at 18 hours, 6 hours, and 7 days after PTCA, respectively. In comparison, PCNA staining peaked at 3 days after PTCA (7.16+/-0.29%; compared to 1.78+/-0.08% PCNA-positive cells in the uninjured artery, P<0.001). Profiles of apoptosis and cell proliferation after PTCA were discordant in all layers of the artery except the neointima. These profiles also differed between traumatized and nontraumatized regions of the arterial wall. Immunostaining with cell-type specific markers and TEM analysis revealed that apoptotic cells included vascular smooth muscle cells (VSMCs), inflammatory cells, and adventitial fibroblasts. CONCLUSIONS These results suggest that the profile of apoptosis and proliferation after PTCA is regional and cell specific, and attempts to modulate either of these events for therapeutic benefit requires recognition of these differences.

Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding

Background Atherosclerosis is an inflammatory process that develops in individuals with known risk factors that include hypertension and hyperlipidaemia, influenced by diet. However, the interplay between diet, inflammatory mechanisms and vascular risk factors requires further research. We hypothesised that interleukin-1 (IL-1) signaling in the vessel wall would raise arterial blood pressure and promote atheroma. Methodology/Principal Findings Apoe−/− and Apoe−/−/IL-1R1−/− mice were fed high fat diets for 8 weeks, and their blood pressure and atherosclerosis development measured. Apoe−/−/IL-R1−/− mice had a reduced blood pressure and significantly less atheroma than Apoe−/− mice. Selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation also reduced plaque burden (p<0.05). This was associated with an IL-1 mediated loss of endothelium-dependent relaxation and an increase in vessel wall Nox 4. Inhibition of IL-1 restored endothelium-dependent vasodilatation and reduced levels of arterial oxidative stress. Conclusions/Significance The IL-1 cytokine system links atherogenic environmental stimuli with arterial inflammation, oxidative stress, increased blood pressure and atherosclerosis. This is the first demonstration that inhibition of a single cytokine can block the rise in blood pressure in response to an environmental stimulus. IL-1 inhibition may have profound beneficial effects on atherogenesis in man.

Platelet P2Y12 Receptor Influences the Vessel Wall Response to Arterial Injury and Thrombosis

Background— Platelets are believed to play an important role in atherogenesis and the vessel response to vascular injury. The P2Y12 receptor (P2Y12) plays a central role in amplifying platelet aggregation, dense granule and &agr;-granule secretion, P-selectin expression, microparticle formation, and procoagulant membrane changes, regardless of the activating stimulus. We hypothesized that P2Y12 deficiency might reduce the vessel wall response to vascular injury as well as thrombosis in murine vascular injury models. Methods and Results— P2Y12-deficient (−/−) mice and littermate controls (+/+) were bred on a C57 BL/6 background. In vivo murine models of arterial injury were employed alone and in combination with bone marrow transplantation to investigate the role of P2Y12 in the vessel wall response to arterial injury and thrombosis. At 21 days after ferric chloride injury, neointima formation in P2Y12−/− arteries was significantly less than that observed in control strain arteries (P<0.025). In agreement with this, the intima-media ratio was significantly greater in femoral wire-injured arteries from P2Y12+/+ compared with P2Y12−/− animals (P<0.05). Bone marrow transplantation was used to examine the importance of vessel wall P2Y12 versus platelet P2Y12. Analysis of arterial sections from chimeric animals at 21 days after injury revealed a smaller intima-media ratio in −/− to +/+ animals than in the positive (+/+ to +/+) control group (P<0.01). Conclusions— These data demonstrate a role for platelet P2Y12 in the vessel wall response to arterial injury and thrombosis. This illustrates the manner in which platelets may contribute to atherogenesis and restenosis.

Inhibition of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses experimental pulmonary hypertension.

Genetic deletion of TRAIL or antibody blockade prevents the development of pulmonary arterial hypertension and can reverse vascular remodeling in established disease.

TRAIL attenuates the development of atherosclerosis in apolipoprotein E deficient mice

TRAIL (tumour necrosis factor-related apoptosis inducing ligand) is most often reported to induce apoptosis in tumour cells. It is expressed in artery walls but its role and regulation in vascular pathologies is little studied. We aimed to measure the effect of genetic deletion of TRAIL on atherosclerosis in a mouse model. TRAIL was mainly expressed in endothelium, smooth muscle cells and macrophages within plaques. The absence of TRAIL in chow and in fat-fed mice led to greater lesion coverage in aortae (8 weeks, % area ± SEM), n = 7–8, 1.24 ± 0.2 (no TRAIL, chow diet) vs. 0.42 ± 0.1, p < 0.01 and 3.4 ± 0.8 (no TRAIL, Western diet) vs. 0.94 ± 0.2, p < 0.01 and larger, smooth muscle cell rich lesions at aortic roots than control mice (8 weeks, mean lesion area/total cross sectional area ± SEM, n = 7–8, 0.17 ± 0.01 (no TRAIL, chow diet) vs. 0.135 ± 0.006, p < 0.05 and 0.36 ± 0.03 (no TRAIL, Western diet) vs. 0.23 ± 0.02, p < 0.05) particularly at early time points. The larger early lesions appeared to be as a result of increased smooth muscle cells in lesions of TRAIL deficient, pro-atherosclerotic animals. We conclude that TRAIL attenuates plaque size at early stages of atherosclerosis.

Streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein Ibα

Bacterial infection contributes to diverse noninfectious diseases and worsens outcome after stroke. Streptococcus pneumoniae, the most common infection in patients at risk of stroke, is a major cause of prolonged hospitalization and death of stroke patients, but how infection impacts clinical outcome is not known.

Neutrophil Elastase Promotes Interleukin-1β Secretion from Human Coronary Endothelium

Background: The mechanism of IL-1 release from endothelial cells is not fully known. Results: Neutrophil elastase causes secretion of bioactive IL-1 from endothelial cells via microvesicles. Conclusion: A mechanistic link between IL-1 secretion from endothelial cells and neutrophil elastase in atherosclerotic plaques is revealed. Significance: Neutrophil elastase could be a potential target for preventing atherosclerosis. The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1β. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1β in the endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1β remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential “trigger” for IL-1β processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1β from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1β in ECs and causes significant secretion of bioactive IL-1β via extracellular vesicles. This release was attenuated significantly by inhibition of neutrophil elastase but not caspase-1. Transient increases in intracellular Ca2+ levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1β was detected within LAMP-1-positive multivesicular bodies. The released vesicles contained bioactive IL-1β. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1β. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1β secretion from ECs. This could reveal additional potential anti-IL-1β therapeutic targets and provide further insights into the inflammatory process by which vascular disease develops.

Effect of Selective or Combined Inhibition of Integrins αIIbβ3 and αvβ3 on Thrombosis and Neointima After Oversized Porcine Coronary Angioplasty

Background—Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin αIIbβ3 and integrin αvβ3 in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective αIIbβ3 antagonist (lamifiban), a selective αvβ3 antagonist (VO514), and a combined αIIbβ3/αvβ3 antagonist (G3580). Methods and Results—In vitro, both αvβ3 inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective αIIbβ3 inhibition had no effect. Intravenous infusions of either αIIbβ3 inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective αvβ3 inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were adm...

Dietary Phosphate Modulates Atherogenesis and Insulin Resistance in Apolipoprotein E Knockout Mice—Brief Report

Objective— Epidemiological studies link higher serum phosphate and the phosphatonin fibroblast growth factor 23 with cardiovascular events and atheroma, and they link lower serum phosphate with insulin resistance and the metabolic syndrome. We investigated whether manipulating dietary phosphate influences atherogenesis or insulin sensitivity in mice. Methods and Results— Apolipoprotein E knockout mice were fed an atherogenic diet with low (0.2%), standard (0.6%), or high (1.6%) phosphate content. Serum phosphate and fibroblast growth factor 23 significantly increased with increasing dietary phosphate intake, but lipid profile and blood pressure were unaffected. After 20 weeks, mice on the higher phosphate diet had significantly more atheroma at the aortic sinus (42±1.9% versus 30±1.5% for high versus low phosphate, P<0.01). Compared with standard and high-phosphate diet groups, mice on a low-phosphate diet had more adipose tissue and a 4-fold increase in insulin resistance measured by homeostatic model assessment (43.7±9.3 versus 8.9±0.7 for low versus high phosphate, P<0.005). Conclusion— A high-phosphate diet accelerates atherogenesis in apolipoprotein E−/− mice, whereas low phosphate intake induces insulin resistance. These data indicate for the first time that controlling dietary phosphate intake may influence development of both atherosclerosis and the metabolic syndrome.