Pamela I. Rogers

Radiolabeled Cell Distribution After Intramyocardial, Intracoronary, and Interstitial Retrograde Coronary Venous Delivery Implications for Current Clinical Trials

Background—Several clinical studies are evaluating the therapeutic potential of delivery of various progenitor cells for treatment of injured hearts. However, the actual fate of delivered cells has not been thoroughly assessed for any cell type. We evaluated the short-term fate of peripheral blood mononuclear cells (PBMNCs) after intramyocardial (IM), intracoronary (IC), and interstitial retrograde coronary venous (IRV) delivery in an ischemic swine model. Methods and Results—Myocardial ischemia was created by 45 minutes of balloon occlusion of the left anterior descending coronary artery. Six days later, 107 111indium-oxine–labeled human PBMNCs were delivered by IC (n=5), IM (n=6), or IRV (n=5) injection. The distribution of injected cells was assessed by γ-emission counting of harvested organs. For each delivery method, a significant fraction of delivered cells exited the heart into the pulmonary circulation, with 26±3% (IM), 47±1% (IC), and 43±3% (IRV) of cells found localized in the lungs. Within the myocardium, significantly more cells were retained after IM injection (11±3%) compared with IC (2.6±0.3%) (P<0.05) delivery. IRV delivery efficiency (3.2±1%) trended lower than IM infusion for PBMNCs, but this difference did not reach significance. The IM technique displayed the greatest variability in delivery efficiency by comparison with the other techniques. Conclusions—The majority of delivered cells is not retained in the heart for each delivery modality. The clinical implications of these findings are potentially significant, because cells with proangiogenic or other therapeutic effects could conceivably have effects in other organs to which they are not primarily targeted but to which they are distributed. Also, we found that although IM injection was more efficient, it was less consistent in the delivery of PBMNCs compared with IC and IRV techniques.

Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing.

Background: The authors examined the efficacy of adipose stem cells, when supplied either alone or in platelet-rich fibrin gels, to improve wound healing. Methods: A porcine full-thickness wound model was used to compare six topical treatments: platelet-poor plasma; platelet-rich plasma; autologous adipose stem cells plus platelet-poor plasma; autologous adipose stem cells plus platelet-rich plasma; allogeneic adipose stem cells containing green fluorescent protein plus platelet-poor plasma; and saline (control). One week after isolation, adipose stem cells were applied to full-thickness wounds on the paraspinal and thoracic regions of three pigs (44 wounds per pig; each treatment was applied to eight separate wounds). Each wound was monitored over 21 days for closure, cosmesis, and histopathology. Results: There was no significant difference in the reepithelialization rate, but treatments containing adipose stem cells demonstrated increased microvessel densities (31.75 ± 5.73 vessels/cm2 versus 7.93 ± 3.61 vessels/cm2) compared with groups without adipose stem cells. Wound cosmesis was improved in the adipose stem cell plus platelet-rich plasma group compared with other treatment groups (p < 0.05). Vascular endothelial growth factor levels detected in matrices containing adipose stem cells were approximately 7-fold higher compared with platelet-rich plasma or platelet-poor plasma (p < 0.05). Localization of transgenic green fluorescent protein plus adipose stem cells indicated incorporation near neovasculature. Conclusions: In normal healing wounds, adipose stem cells appear to enhance the healing process only when provided in a fibrin gel vehicle containing a number of complementary wound-healing trophic factors. Perivascular adipose stem cell localization suggests a function in enhancing blood supply through providing physical and paracrine support to newly forming vessels.

Intrapericardial Paclitaxel Delivery Inhibits Neointimal Proliferation and Promotes Arterial Enlargement After Porcine Coronary Overstretch

BackgroundCatheter-based intrapericardial (IPC) delivery of therapeutic agents has recently been demonstrated. Paclitaxel is known to inhibit vascular smooth muscle cell proliferation. This study examined the effect of IPC instillation of paclitaxel on neointimal proliferation after balloon overstretch of porcine coronary arteries. Methods and ResultsOverstretch injury of coronary arteries was followed by IPC administration of micellar paclitaxel at low dose (LD, 10 mg; n=6) or high dose (HD, 50 mg; n=7) or of control micelles (50 mg, n=5). Animals were euthanized 28 days after balloon dilation. Arterial injury indices were no different among the groups. The neointimal area, maximal intimal thickness, and adventitial thickness were significantly reduced in both LD (0.47±0.04 mm2, 0.43±0.03 mm, and 0.35±0.02 mm, respectively) and HD (0.51±0.06 mm2, 0.42±0.03 mm, and 0.38±0.03 mm, respectively) paclitaxel groups compared with the control group (0.79±0.07 mm2, 0.56±0.02 mm, and 0.47±0.02 mm, respectively;P <0.001). Meanwhile, the vessel circumference measured at the external elastic lamina of paclitaxel-treated vessels was significantly larger than the control circumference. Apoptotic cells were found in the neointima. The apoptotic cell percentage was not different between the control (1.72%) and LD (2.31%) groups but was higher in the HD group (7.07%, P <0.0001 versus control and LD groups). Immunostaining for matrix metalloproteinase-2 revealed concurrent reduction in the HD group compared with the control and LD groups. ConclusionsIPC space delivery of a single dose of paclitaxel significantly reduces vessel narrowing in this balloon-overstretch model. This effect is mediated by reduction of neointimal mass as well as positive vascular remodeling.

Widespread regional myocardial transfection by plasmid encoding Del‐1 following retrograde coronary venous delivery

This study quantifies myocardial transfection following percutaneous retrograde coronary venous delivery (RCVD) of a plasmid encoding human Del‐1. RCVD of Del‐1, GFP plasmid, or marker dye was conducted in 14 pigs. After selective cannulation of a coronary vein, a delivery site was confirmed by contrast injection and myocardial blush. Ten milliliters of plasmid hDel‐1 or GFP was administered. Animals were euthanized 3 and 7 days post‐RCVD. hDel‐1 gene expression was evaluated by quantitative RT‐PCR. An average myocardial expression of 4.5 × 105 copies hDel‐1/μg total RNA was observed within the approximately 5 × 5 cm2 target tissue of the left ventricle. GFP expression was detected by fluorescent microscopy. hDel‐1 protein expression was confirmed by immunohistochemistry. Regionalized myocardial expression was found in all pigs. hDel‐1 RNA was not found in distant tissues except in the three pigs with prominent venovenous washout (PVW). These levels were 3 to 4 log unites lower than those found in myocardium. Single retrograde coronary venous administration resulted in efficient regional myocyte transfection of hDel‐1 and GFP. This method may be useful and clinically feasible for myocardial angiogenesis therapy. Cathet Cardiovasc Intervent 2003;58:207–211.

Development of a Porcine Delayed Wound-Healing Model and Its Use in Testing a Novel Cell-Based Therapy

PURPOSE A delayed full-thickness wound-healing model was developed and used for examining the capacity of adipose-derived stem cells (ASCs), either alone or in platelet-rich fibrin gels, to promote healing. METHODS AND MATERIALS Four pigs received electron beam radiation to the dorsal skin surface. Five weeks after radiation, subcutaneous fat was harvested from nonirradiated areas and processed to yield ASCs. Two weeks later, 28 to 30 full-thickness 1.5-cm(2) wounds were made in irradiated and nonirradiated skin. Wounds were treated with either saline solution, ASCs in saline solution, platelet-rich plasma (PRP) fibrin gel, ASCs in PRP, or non-autologous green fluorescence protein-labeled ASCs. RESULTS The single radiation dose produced a significant loss of dermal microvasculature density (75%) by 7 weeks. There was a significant difference in the rate of healing between irradiated and nonirradiated skin treated with saline solution. The ASCs in PRP-treated wounds exhibited a significant 11.2% improvement in wound healing compared with saline solution. Enhancement was dependent on the combination of ASCs and PRP, because neither ASCs nor PRP alone had an effect. CONCLUSIONS We have created a model that simulates the clinically relevant late radiation effects of delayed wound healing. Using this model, we showed that a combination of ASCs and PRP improves the healing rates of perfusion-depleted tissues, possibly through enhancing local levels of growth factors.

Serial kinematic analysis of the canine hindlimb joints after deafferentation and anterior cruciate ligament transection

OBJECTIVE AND DESIGN Transection of the anterior cruciate ligament 2 weeks after ipsilateral hindlimb deafferentation leads to osteoarthritis of the knee joint within 3 weeks. We analyzed the gait of six dogs that underwent this procedure in order to identify kinematic changes that could account for this rapid joint degeneration. All animals were video taped, 1, 3, 6, 9 and 13 weeks after surgery while they trotted on a treadmill. RESULTS In each dog, extension of the hip, knee and ankle joints of the unstable limb was increased, and the yield phase of the unstable knee was delayed or attenuated. When killed, five of six dogs showed a large full-thickness cartilage ulcer on the distal and/or anterior surface of the medial femoral condyle of the unstable knee; in the sixth dog, a smaller ulcer was observed. However, the severity of pathology in each individual was not obviously related to difference among the dogs in postoperative joint kinematics. CONCLUSIONS These data, and results of prior studies in humans and dogs, suggest that knee hyperextension resulting from limb deafferentation, and knee instability resulting from anterior cruciate ligament transection, operate in concert to create a mechanical environment (i.e., increased tibiofemoral separation and changes in the loading of articular surfaces) that results in rapid joint breakdown.

Intracoronary and retrograde coronary venous myocardial delivery of adipose-derived stem cells in swine infarction lead to transient myocardial trapping with predominant pulmonary redistribution.

Objectives: To examine the comparative fate of adipose‐derived stem cells (ASCs) as well as their impact on coronary microcirculation following either retrograde coronary venous (RCV) or arterial delivery. Background: Local delivery of ASCs to the heart has been proposed as a practical approach to limiting the extent of myocardial infarction. Mouse models of mesenchymal stem cell effects on the heart have also demonstrated significant benefits from systemic (intravenous) delivery, prompting a question about the advantage of local delivery. There has been no study addressing the extent of myocardial vs. systemic disposition of ASCs in large animal models following local delivery to the myocardium. Methods: In an initial experiment, dose‐dependent effects of ASC delivery on coronary circulation in normal swine were evaluated to establish a tolerable ASC dosing range for intracoronary (IC) delivery. In a set of subsequent experiments, an anterior acute myocardial infarction (AMI) was created by balloon occlusion of the proximal left anterior descending (LAD) artery, followed by either IC or RCV infusion of 107 111Indium‐labeled autologous ASCs 6 days following AMI. Indices of microcirculatory resistance (IMR) and coronary flow reserve (CFR) were measured before sacrifices to collect tissues for analysis at 1 or 24 hr after cell delivery. Results: IC delivery of porcine ASCs to normal myocardium was well tolerated up to a cumulative dose of 14 × 106 cells (approximately 0.5 × 106 cells/kg). There was evidence suggesting microcirculatory trapping of ASC: at unit doses of 50 × 106 ASCs, IMR and CFR were found to be persistently altered in the target LAD distribution at 7 days following delivery, whereas at 10 × 106 ASCs, only CFR was altered. In the context of recent MI, a significantly higher percentage of ASCs was retained at 1 hr with IC delivery compared with RCV delivery (57.2 ± 12.7% vs. 17.9 ± 1.6%, P = 0.037) but this initial difference was not apparent at 24 hr (22.6 ± 5.5% vs. 18.7 ± 8.6%; P = 0.722). In both approaches, most ASC redistributed to the pulmonary circulation by 24 hr postdelivery. There were no significant differences in CFR or IMR following ASC delivery to infarcted tissue by either route. Conclusions: Selective intravascular delivery of ASC by coronary arterial and venous routes leads to similarly limited myocardial cell retention with predominant redistribution of cells to the lungs. IC arterial delivery of ASC leads to only transiently greater myocardial retention, which is accompanied by obstruction of normal regions of coronary microcirculation at higher doses. The predominant intrapulmonary localization of cells following local delivery via both methods prompts the notion that systemic delivery of ASC might provide similarly beneficial outcomes while avoiding risks of inadvertent microcirculatory compromise.

Multimodality Imaging Methods for Assessing Retinoblastoma Orthotopic Xenograft Growth and Development

Genomic studies of the pediatric ocular tumor retinoblastoma are paving the way for development of targeted therapies. Robust model systems such as orthotopic xenografts are necessary for testing such therapeutics. One system involves bioluminescence imaging of luciferase-expressing human retinoblastoma cells injected into the vitreous of newborn rat eyes. Although used for several drug studies, the spatial and temporal development of tumors in this model has not been documented. Here, we present a new model to allow analysis of average luciferin flux () through the tumor, a more biologically relevant parameter than peak bioluminescence as traditionally measured. Moreover, we monitored the spatial development of xenografts in the living eye. We engineered Y79 retinoblastoma cells to express a lentivirally-delivered enhanced green fluorescent protein-luciferase fusion protein. In intravitreal xenografts, we assayed bioluminescence and computed , as well as documented tumor growth by intraocular optical coherence tomography (OCT), brightfield, and fluorescence imaging. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated in orthotopic xenografts. By OCT, xenografts were dense and highly vascularized, with well-defined edges. Small tumors preferentially sat atop the optic nerve head; this morphology was confirmed on histological examination. In vivo, in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of modeling and in vivo intraocular imaging allows both quantitative and high-resolution, non-invasive spatial analysis of this retinoblastoma model. This technique will be applied to other cell lines and experimental therapeutic trials in the future.

LifeShirt ® acquisition system to monitor ECG from ambulatory swine and the implementation of an arrhythmia detection algorithm

A wearable cardiopulmonary monitoring system, a LifeShirt®, was used to acquire continuous electrocardiograms (ECGs) from ambulatory swine. The animals received intracoronary injections of autologous mesenchymal stem cells, and the LifeShirt® was used for long-duration ECG monitoring in pre-defined periods post cell infusion. The system used here was developed for measurements from non-human primates and canines; however, we demonstrated that it could be used to non-invasively measure ECGs from swine without creating undue stress or restricting movement. A MATLAB-based analysis algorithm was developed to automatically detect premature ventricular contractions (PVCs) that arose 8-10 hours after cell delivery with spontaneous resolution 2-3 days post-infusion. Template based cross-correlation was used to detect the PVCs and identify regions of consecutive ventricular rhythm. The final algorithm was highly specific and sensitive when tested on records from the MIT-BIH arrhythmia database. The algorithm was subsequently used to automatically identify and quantify PVCs from over 200 hours of ECG data obtained from nine ambulatory swine.

Intravenous xenogeneic transplantation of human adipose-derived stem cells improves left ventricular function and microvascular integrity in swine myocardial infarction model

The potential for beneficial effects of adipose‐derived stem cells (ASCs) on myocardial perfusion and left ventricular dysfunction in myocardial ischemia (MI) has not been tested following intravenous delivery.