Richard Baffour

Transendocardial delivery of autologous Bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia

OBJECTIVES We tested the hypothesis that intramyocardial injection of autologous bone marrow (ABM) promotes collateral development in ischemic porcine myocardium. We also defined, in vitro, whether bone marrow (BM) cells secrete vascular endothelial growth factor (VEGF) and macrophage chemoattractant protein-1 (MCP-1). BACKGROUND The natural processes leading to collateral development are extremely complex, requiring multiple growth factors interacting in concert and in sequence. Because optimal angiogenesis may, therefore, require multiple angiogenic factors, we thought that injection of BM, which contains cells that secrete numerous angiogenic factors, might provide optimal therapeutic angiogenesis. METHODS Bone marrow was cultured four weeks in vitro. Conditioned medium was assayed for VEGF and MCP-1 and was added to cultured pig aortic endothelial cells (PAEC) to assess proliferation. Four weeks after left circumflex ameroid implantation, freshly aspirated ABM (n = 7) or heparinized saline (n = 7) was injected transendocardially into the ischemic zone (0.2 ml/injection at 12 sites). Echocardiography to assess myocardial thickening and microspheres to assess perfusion were performed at rest and during stress. RESULTS Vascular endothelial growth factor and MCP-1 concentrations increased in a time-related manner. The conditioned medium enhanced, in a dose-related manner, PAEC proliferation. Collateral flow (ischemic/normal zone X 100) improved in ABM-treated pigs (ABM: 98 +/- 14 vs. 83 +/- 12 at rest, p = 0.001; 89 +/- 18 vs. 78 +/- 12 during adenosine, p = 0.025; controls: 92 +/- 10 vs. 89 +/- 9 at rest, p = 0.49; 78 +/- 11 vs. 77 +/- 5 during adenosine, p = 0.75). Similarly, contractility increased in ABM-treated pigs (ABM: 83 +/- 21 vs. 60 +/- 32 at rest, p = 0.04; 91 +/- 44 vs. 36 +/- 43 during pacing, p = 0.056; controls: 69 +/- 48 vs. 64 +/- 46 at rest, p = 0.74; 65 +/- 56 vs. 37 +/- 56 during pacing, p = 0.23). CONCLUSIONS Bone marrow cells secrete angiogenic factors that induce endothelial cell proliferation and, when injected transendocardially, augment collateral perfusion and myocardial function in ischemic myocardium.

Catheter-based autologous bone marrow myocardial injection in no-option patients with advanced coronary artery disease: a feasibility study.

OBJECTIVES We conducted a pilot study to evaluate the feasibility of transendocardial delivery of autologous bone marrow (ABM) strategy in patients with severe symptomatic chronic myocardial ischemia not amenable to conventional revascularization. BACKGROUND Transendocardial injection of ABM cells appears to enhance perfusion of ischemic porcine myocardium. METHODS Ten patients underwent transendocardial injection of freshly aspirated and filtered unfractionated ABM using left ventricular electromechanical guidance. Twelve injections of 0.2 ml each were successfully delivered into ischemic noninfarcted myocardium pre-identified by single-photon emission computed tomography perfusion imaging. RESULTS Autologous bone marrow injection was successful in all patients and was associated with no serious adverse effects; in particular, there was no arrhythmia, evidence of infection, myocardial inflammation, or increased scar formation. Two patients were readmitted for recurrent chest pain. At three months, Canadian Cardiovascular Society angina score significantly improved (3.1 +/- 0.3 vs. 2.0 +/- 0.94, p = 0.001), as well as stress-induced ischemia occurring within the injected territories (2.1 +/- 0.8 vs. 1.6 +/- 0.8, p < 0.001). Treadmill exercise duration, available in nine patients, increased, but the change was not significant (391 +/- 155 vs. 485 +/- 198, p = 0.11). CONCLUSIONS This study provides preliminary clinical data indicating feasibility of catheter-based transendocardial delivery of ABM to ischemic myocardium.

Safety and efficacy of bioabsorbable magnesium alloy stents in porcine coronary arteries

Objective: We aimed to determine the safety and efficacy of biobasorbable magnesium alloy stents in porcine coronary arteries. Bioabsorbable magnesium stents carry the potential to overcome the limitations posed by permanent metallic stents such as chronic inflammation, late stent thrombosis, prolonged antiplatelet therapy, and artifacts when imaged by multislice‐computed tomography or magnetic resonance imaging. Methods: Magnesium alloy stents or stainless steel stents were randomly deployed in coronary arteries of domestic or minipigs. Domestic pigs were sacrificed at 3 days (n = 2) or 28 days, and minipigs at 3 months. Results: At 3 days, magnesium alloy stents were intact, but started to show signs of degradation by 28 days. There was no evidence of stent particle embolization, thrombosis, excess inflammation, or fibrin deposition. At 28 days and 3 months, neointimal area was significantly less in magnesium alloy stent segments (2.44 ± 0.88 mm2 and 1.16 ± 0.19 mm2) as compared with the stainless steel stent segments (5.03 ± 1.5 mm2 and 1.72 ± 0.68 mm2, P < 0.001 and 0.02). Quantitative coronary analysis indicates that percentage area stenosis and percentage diameter stenosis in magnesium alloy stent segments improved significantly at 3 months as compared to 28 days. Despite decreased neointimal hyperplasia, lumen area of the magnesium alloy stented vessels did not improve significantly. Conclusion: Magnesium alloy stents are safe and are associated with less neointima formation; however, reduced neointima did not result in larger lumen.

Akt Controls Vascular Smooth Muscle Cell Proliferation In Vitro and In Vivo by Delaying G1/S Exit

Abstract— Constitutive activation of serine/threonine kinase Akt causes uncontrolled cell-cycle progression in different cell types and in malignancy. To investigate how Akt activation modulates cell-cycle progression in vascular smooth muscle cells (SMCs) in vitro and in the intact animal, we inhibited Akt-dependent signaling by adenovirus-mediated transfection of a dominant-negative Akt mutant (AA-Akt). We observed reduced proliferation rate (P <0.01), DNA synthesis (P <0.01), and a significant arrest in G1/S exit (P <0.01) both in vitro in response to serum stimulation and in vivo after vascular injury. In vivo transfection of the balloon-injured vessel with AA-Akt reduced SMC proliferation, resulting in decreased neointima compared with control virus (P <0.01). These effects were at least in part modulated, both in vitro and in vivo, by increased p21Cip1 expression, as demonstrated by lack of effect of AA-Akt on cell proliferation in p21−/− mouse SMCs. In conclusion, this study demonstrates that Akt-dependent signaling enhances cell-cycle progression of nontransformed SMCs in vitro and in response to vascular injury in the intact animal. These results suggest a role for Akt signaling in modulating the response of normal tissues to stress and the response of the arterial wall to acute and possibly repetitive injuries that ultimately contribute to restenosis and atherosclerosis.

Lack of correlation between angiographic grading of collateral and myocardial perfusion and function: implications for the assessment of angiogenic response.

BackgroundAngiographic assessment of apparent collaterals (AAC) has been used to quantify the angiogenic response to interventions designed to enhance myocardial perfusion and function in ischemic myocardium. However, the accuracy with which AAC reflects actual myocardial blood flow (MBF) and regional contractility has not been established. ObjectiveTo examine the relationships between myocardial tissue perfusion, AAC grade and myocardial function in a porcine model of chronic myocardial ischemia. MethodsAAC (with results visually graded as 0–3) was performed 4 weeks after placement of an ameroid constrictor around the left circumflex artery in pigs (n  = 27). Fluorescent microspheres were used to quantify regional endocardial, epicardial, and transmural MBF, and echocardiography was used to assess percentage thickening of myocardium (PTM) at rest and under stress (pacing). ResultsThere was no significant correlation between AAC grading and endocardial, epicardial or transmural MBF. MBF but not AAC grade was correlated to PTM at rest according to the formula PTM = 0.06 + 0.42MBFtransmural (r  = 0.39, P  = 0.047). ConclusionResults of simple AAC are not correlated with myocardial perfusion and function and probably should not be used as a primary endpoint in clinical studies designed to enhance myocardial perfusion in ischemic regions.

Peroxisome proliferator-activated receptor gamma ligand pioglitazone alters neointimal composition in a balloon-denuded and radiated hypercholesterolemic rabbit.

Peroxisome proliferator-activated receptor (PPAR)-γ activation suppresses inflammatory response, monocyte recruitment, and vascular cell proliferation. Because inflammation, deregulated growth, and migration of monocytes and vascular smooth muscle cells (VSMC) play important roles in the development of neointima, we tested the effect of pioglitazone, a high-affinity ligand, for PPAR-γ on neointima formation in the iliac arteries of a balloon-denuded and radiated hypercholesterolemic rabbit. Rabbits were fed a 1.0% cholesterol diet for 7 days followed by denudation of endothelial layer and continued on a 0.15% cholesterol diet. On day 32, animals were divided into 2 groups. One group received a 0.15% cholesterol diet (n = 7) and the other group received a 0.15% cholesterol diet supplemented with 400 mg of pioglitazone per kilogram. On day 35, the balloon-denuded area was radiated. Four weeks after radiation, animals were sacrificed and arterial segments were processed for morphometry and immunohistochemistry. Data analysis showed that the pioglitazone group had smaller neointima (0.85 ± 0.36 vs. 1.41 ± 0.56, P < 0.05), with more cells positive for VSMC (23.07 ± 6.16 vs. 18.33 ± 5.19, P = 0.04), less for monocytes (16.01 ± 5.33 vs. 21.29 ± 4.33, P < 0.05), and fewer cells expressing metalloproteinase (MMP)-1 and MMP-9 (3.69 ± 0.47 vs. 4.82 ± 0.93, P < 0.05 and 3.24 ± 0.71 vs. 4.29 ± 0.74, P < 0.05, respectively). Pioglitazone reduced neointimal area and modified its composition in a balloon-denuded and radiated hypercholesterolemic rabbit model.

Bone marrow and bone marrow derived mononuclear stem cells therapy for the chronically ischemic myocardium

Bone marrow stem cells have been shown to differentiate into various phenotypes including cardiomyocytes, vascular endothelial cells and smooth muscle. Bone marrow stem cells are mobilized and home in to areas of injured myocardium where they are involved in tissue repair. In addition, bone marrow secretes multiple growth factors, which are essential for angiogenesis and arteriogenesis. In some patients, these processes are not enough to avert clinical symptoms of ischemic disease. Therefore, in vivo administration of an adequate number of stem cells would be a significant therapeutic advance. Unfractionated bone marrow derived mononuclear stem cells, which contain both hematopoietic and nonhematopoietic cells may be more appropriate for cell therapy. Studies in animal models suggest that implantation of different types of stem cells improve angiogenesis and arteriogenesis, tissue perfusion as well as left ventricular function. Several unanswered questions remain. For example, the optimal delivery approach, dosage and timing of the administration of cell therapy as well as durability of improvements need to be studied. Early clinical studies have demonstrated safety and feasibility of various cell therapies in ischemic disease. Randomized, double blind and placebo-controlled clinical trials need to be completed to determine the effectiveness of stem cell.

Effect of Clopidogrel on Neointimal Formation and Inflammation in Balloon-Denuded and Radiated Hypercholesterolemic Rabbit Iliac Arteries

BACKGROUND Platelet-derived peptide and nonpeptide growth factors are known to play pivotal roles in neointimal proliferation. Along with its antiplatelet activity of reducing P-selectin and hs-CRP, clopidogrel has also been shown to have anti-inflammatory properties. The aim of this study is to find out by modulating inflammation if clopidogrel can affect neointima formation in balloon-denuded iliac arteries of hypercholesterolemic rabbits. METHODS AND RESULTS Rabbits were fed with 1% cholesterol diet with (n = 20) or without (n = 20) clopidogrel (10 mg/kg body weight) for 7 days followed by balloon-denudation of endothelial layer in both the iliac arteries and continued on 0.15% cholesterol diet with or without clopidogrel. Four weeks later, the denuded area in both iliac arteries was radiated (n = 11, cholesterol-only group; n = 9, clopidogrel group) or sham treated (n = 10 from each group). Four weeks after radiation, animals were sacrificed and arterial segments were processed for morphometry. In the sham-treated clopidogrel group, neointimal area, percent stenosis, and macrophage score were 39% (P = 0.01), 32% (P = 0.02), and 50% (P = 0.02) smaller, respectively, when compared to the cholesterol-only group (0.48 +/- 0.18, 32.42 +/- 13.04, and 1.5 +/- 0.83). There were no differences in the radiated group (0.89 +/- 0.32, 50.34 +/- 13.00, and 1.88 +/- 1.27 vs. 0.93 +/- 0.38, 59.41 +/- 11.41, and 2.00 +/- 0.74, respectively). CONCLUSION This study demonstrates that clopidogrel reduces inflammation and neointimal formation in balloon-denuded iliac arteries of hypercholesterolemic rabbits.

Laser Myocardial Revascularization Modulates Expression of Angiogenic, Neuronal, and Inflammatory Cytokines in a Porcine Model of Chronic Myocardial Ischemia

Abstract  Background: Controversy exists whether transmyocardial laser revascularization (TMR) is associated with angiogenesis or neuromodulation and whether these are time‐dependent phenomena. Accordingly, we performed a time‐course analysis of the expression of angiogenic and neuronal factors following experimental percutaneous TMR. Methods and Results: Five weeks after placing ameroid constrictors on the circumflex coronary artery, 16 pigs underwent left ventricular mapping guided TMR using Ho:YAG laser (2 J × 1 pulse) at 30 sites directed at the ischemic zones and 11 animals were ischemic controls. Histology and immunostaining were obtained at 1 and 2 weeks (4 TMR and 3 controls at each time point) and at 4 weeks (8 TMR and 5 controls) for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), nerve growth factor (βNGF), substance P (SP), and monocyte chemoattractant protein‐1 (MCP‐1). Immunoreactivity was scored using a digital image analysis system. Factor VIII staining was used for blood vessel counting. Enhanced regional expression of VEGF, bFGF and MCP‐1 in the TMR group was noted at 1 and 2 weeks with a threefold increase at 4 weeks following TMR compared to controls. βNGF expression in the TMR group was enhanced at 1 and 2 weeks with subsequent decline at 4 weeks to the controls level. SP expression was not significantly different between groups at all time points. There was a twofold increase in the number of blood vessels in the TMR group at 4 weeks, which was not apparent earlier. Conclusions: These immunohistological findings suggest that cytokines expression compatible with angiogenesis and neuromodulation occurs early after TMR. Up‐regulation of angiogenic and inflammatory cytokines may be more sustained than neuromodulation.

In vivo comparison of a polymer‐free Biolimus A9‐eluting stent with a biodegradable polymer‐based Biolimus A9 eluting stent and a bare metal stent in balloon denuded and radiated hypercholesterolemic rabbit iliac arteries

Objectives: To evaluate the effect of a polymer‐free Biolimus A9‐eluting stent [BioFreedom™ (BF)], compared with that of a biodegradable polymer‐based Biolimus A9‐eluting stent [BioMatrix Flex™ (BMF)] and a bare metal stent (BMS) in balloon denuded and radiated hypercholesterolemic rabbit iliac arteries. Methods: Rabbits were fed with 1% cholesterol diet (n = 14) for 14 days, both iliac arteries were balloon denuded and radiated, and then rabbits were switched to 0.15% cholesterol diet. After 4 weeks, BF (n = 8), BMF (n = 8), and BMS (n = 8) were deployed in denuded and radiated areas. Four weeks later animals were euthanized, arterial segments were processed for morphometry. Results: The neointimal area in vessels implanted with BF stents was significantly less than that seen in vessels implanted with BMS (0.90 mm2 ± 0.14 vs. 1.29 mm2 ± 0.23, P <0.01). Percent fibrin and fibrin score were higher with BMF stents compared to BMS (P <0.03 and <0.04) and giant cell number was significantly higher with both BMF and BF stents (P < 0.01 for both). Percent endothelialization was significantly higher and % uncovered struts were lower with BMS compared to either BMF or BF stents (P < 0.05 for both). Conclusion: This study demonstrates that compared to BMS, BF stents significantly decreased neointimal hyperplasia.