Joseph B. Delcarpio

Cardiomyocyte Transplantation in a Porcine Myocardial Infarction Model

Transplantation of cardiomyocytes into the heart is a potential treatment for replacing damaged cardiac muscle. To investigate the feasibility and efficiency of this technique, either a cardiac-derived cell line (HL-1 cells), or normal fetal or neonatal pig cardiomyocytes were grafted into a porcine model of myocardial infarction. The myocardial infarction was created by the placement of an embolization coil in the distal portion of the left anterior descending artery in Yorkshire pigs (n = 9). Four to 5 wk after creation of an infarct, the three preparations of cardiomyocytes were grafted, at 1 x 10(6) cells/20 microL into normal and into the middle of the infarcted myocardium. The hearts were harvested and processed for histologic examinations 4 to 5 wk after the cell grafts. Histologic evaluation of the graft sites demonstrated that HL-1 cells and fetal pig cardiomyocytes formed stable grafts within the normal myocardium without any detrimental effect including arrhythmia. In addition, a marked increase in angiogenesis was observed both within the grafts and adjacent host myocardium. Electron microscopy studies demonstrated that fetal pig cardiomyocytes and the host myocardial cells were coupled with adherens-type junctions and gap junctions. Histologic examination of graft sites from infarct tissue failed to show the presence of grafted HL-1 cells, fetal, or neonatal pig cardiomyocytes. Cardiomyocyte transplantation may provide the potential means for cell-mediated gene therapy for introduction of therapeutic molecules into the heart.

Myoblast transplantation in the porcine model: A potential technique for myocardial repair

The use of transgenic cells transplanted in syngeneic rodents has shown modest success, but allogeneic and xenogeneic transplants have not been uniformly successful. To assess the feasibility of xenogeneic and allogeneic myoblast transplantation, we subjected seven adult swine to transplantation of murine atrial tumor cells (xenogeneic), neonatal porcine myocytes (allogeneic), and human fetal cardiomyocytes into the left ventricular wall. After general anesthesia, isolated cells were injected along the anterior and posterior walls of the porcine left ventricle. All the animals were immuno-suppressed and observed for 1 month after injection, at which time they were killed and analyzed. This report will present results primarily concerned with the success of human cell transfers. In all injected sites examined, the transplanted cells thrived within the host myocardium with no significant rejection. Transplant cells formed close associations with host myocytes that resembled nascent intercalated disks on electron microscopy. These cells also contained myofibrils and other cell architecture resembling the transplanted cell lines. Additionally, these cells appeared to produce an angiogenic influence resulting in the proliferation of the surrounding microvasculature. We believe that these findings indicate successful xenogeneic and allogeneic myoblast cell transplantation in a large animal model. These experiments set the stage for future studies to assess the ability of these cells to form a syncytium, contract, and potentially repair failed myocardium.

Morphological characterization of cardiomyocytes isolated from a transplantable cardiac tumor derived from transgenic mouse atria (AT-1 cells).

We have developed a transplantable tumor lineage derived from transgenic mouse atrial cardiomyocytes that express the SV40 large T oncogene and have named these cardiomyocytes AT-1 cells. In this study, the transplantable tumors, freshly isolated tumor cardiomyocytes, and cultured tumor cardiomyocytes were examined using phase-contrast microscopy, autoradiography, and electron microscopy. The vast majority of the subcutaneous tumor cells, greater than 90% of the cellular mass of the tumor, exhibited sarcomeric banding. Ultrastructural characteristics typical of in vivo atrial cardiac muscle cells, including well-organized myofibrils, gap junctions, and atrial-specific cytoplasmic granules, were observed in in situ and in freshly isolated AT-1 cells. Those cells that did not contain some form of organized myofibrils were primarily vascular elements, such as endothelial cells. Labeling with [3H]thymidine indicated that greater than 90% of cultured AT-1 cells were synthesizing DNA; furthermore, many cells could be seen undergoing cell division. Electron microscopy revealed that the cultured AT-1 cardiomyocytes contained all of the above-described characteristics, including a well-developed transverse tubular system.

Effect of basic fibroblast growth factor on angiogenesis in the infarcted porcine heart.

Abstract Administration of growth factors is emerging as a new therapeutic approach for the enhancement of collateral vessel formation in the ischemic heart. We have investigated the effects of intramyocardial delivery of FGF-2 in the presence and absence of heparin on angiogenesis in a porcine model of myocardial infarction. Yorkshire pigs were subjected to myocardial infarction by the placement of an embolization coil in the left anterior descending artery (n=5). Four or five weeks after creation of an infarct, FGF-2 (10μg) alone or in complex with heparin, heparan sulfate, or heparin agarose beads was injected either into the normal myocardium or along the infarct border area. Histologic evaluation of each injection site was performed 4 to 5 weeks post‐injection. The effect of FGF‐2 on angiogenesis was evaluated by determining the number of capillaries (diameter < 20μm)and arterioles (<20μm with tunica media) in each area observed. The number of capillaries were not affected by the treatment of FGF‐2 both in normal myocardium and infarct border area. However, in the normal myocardium, the number of arterioles were increased with the treatment of FGF‐2 alone (85±59 %, P < 0.04), FGF‐2 plus heparin (281 ±193 %, P < 0.004) and FGF‐2‐coated heparin beads (241 ± 141%, P < 0.01), as compared to control. Delivery of FGF‐2 into the infarct border area, also increased the number of arterioles when FGF‐2 was given with heparin (736 ± 154 %, P < 0.001) or heparin beads (700 ± 109 %, P < 0.001), as compared to control. FGF‐2 administered with heparin was the most effective method of enhancing angiogenesis as compared to FGF‐2 alone, FGF‐2 plus heparan sulfate, or FGF‐2 coated heparin agarose beads.

Breast cancer increases initiation of angiogenesis without accelerating neovessel growth rate

BACKGROUND Recurrence and mortality rates in patients with breast cancer correlate with the degree of tumor angiogenesis (angiogenic index). We have developed a novel angiogenesis model by using disks of fresh human placental vein that initiate an angiogenic response and exhibit linear radial capillary growth in culture. We hypothesized that the addition of human breast cancer cells to this human placental vein angiogenesis model would increase the incidence of angiogenesis and accelerate the rate of neovessel growth compared with vein disk cultured without tumor cells. METHODS To test this hypothesis, vein explants from seven human placentas were incorporated into clots of 0.3% fibrin in Medium 199 and fetal bovine serum with or without 1.5 x 10(5) T-47D (n = 6 placentas) or MCF-7 (n = 1 placenta) breast cancer cells. Statistical differences between the experimental (with breast cancer cells) and control (no added cells) cultures were determined by repeated measures ANOVA. RESULTS The proportion of disks exhibiting neovessel growth (initiation) by day 12 was significantly increased in the presence of T-47D cells (p < 0.05 at day 12, p < 0.001 at day 15). No statistical difference was seen in rates of neovessel growth (millimeters per day). Similar results were seen with MCF-7 cells. CONCLUSIONS Tumor enhancement of angiogenesis may occur by increased initiation of the angiogenic response. Subsequent vessel growth rates may be tumor independent. We predict that effective antiangiogenic therapies will block a tumors ability to augment angiogenesis initiation rather than subsequent neovessel growth.

Sarcoplasmic reticulum and intermediate filament organization in cultured neonatal cardiac muscle cells

SummaryCardiac muscle cells from 3-day-old rat neonates were cultured for periods of 2 to 56 days. In order to facilitate ultrastructural studies on the organization of the sarcoplasmic reticulum, the cells were prepared for transmission electron microscopy according to a regimen including postfixation in reduced osmium ferrocyanide. The nonjunctional sarcoplasmic reticulum (NJSR) was organized as a loose, fenestrated sleeve around the exterior of bundles of myofilaments and was particularly prominent at the level of the Z line. The only recognizable junctional elements of the sarcoplasmic reticulum were in a peripheral location. Reduced osmium ferrocyanide was also useful in distinguishing intermediate (10nm) filaments, since it understained Z substance, which often obscured these structures. Intermediate filaments were arranged both at the Z line and the intercalated disc, in parallel strands, approximately at right angles to the myofilaments.

CULTURE AND CHARACTERIZATION OF FETAL HUMAN ATRIAL AND VENTRICULAR CARDIAC MUSCLE CELLS

SummaryAtrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses.

In for the long haul: sustaining and rebuilding educational operations after Hurricane Katrina.

In 2005, Hurricane Katrina and the subsequent levee breaks left 80% of New Orleans under water for weeks. Within 4 short weeks, the Louisiana State University Health Sciences Center at New Orleans had relocated its campus temporarily to Baton Rouge and resumed operations. Many lessons were learned in the first year of recovery and disseminated to the field regarding emergency and disaster preparedness and response. As we approach the second anniversary of the nations most devastating natural disaster, we reflect on the long haul of rebuilding and offer new insights and lessons for sustaining operations and enhancing long-term rebuilding efforts.

Changes in undergraduate medical education, admissions, and student outcomes at LSU School of Medicine: three years after Katrina.

M has been written about the effects of Hurricane Katrina on the Louisiana State University Health Sciences Center (LSUHSC) School of Medicine in New Orleans1–4 and the importance of disaster planning for all medical schools. As we approach Katrina’s third anniversary, it is useful to reflect on the storm’s longer term effects on the school. This brief report summarizes the current status of our admissions process and undergraduate medical education curriculum. Pre-Katrina (July 2005) and immediate post-Katrina (July 2006) measures are used as benchmarks for comparison. The article also examines outcomes for the students most seriously affected by Katrina: the medical school classes of 2006, 2007, 2008, and 2009. Outcomes on the United States Medical Licensing Exams (USMLE) and in the National Resident Match Program (NRMP) remained essentially unchanged. This suggests that we were able to minimize the storm’s overall impact on medical student education. Additionally, we believe that new clinical rotations have actually strengthened medical student education as a whole. The school is well positioned to provide quality medical education in New Orleans’ changing demographic and health care environment.

Multiple SR-T tubule junctions in a single insect flight muscle fiber

The membranes of mesothoracic dorsoventral flight muscle of Libellua needhami and Erythemis simplicicolis (Odonata: Libellulidae) were studied by transmission electron microscopy using uranyl acetate en bloc staining. Intact fibers were studied by conventional scanning electron microscopy procedures. Dyadic, triadic, and tetradic transverse tubule (TT)—sarcoplasmic reticulum (SR) junctions as well as plasma membrane—SR junctions are described for the first time in the same insect muscle fiber. Triadic (SR—TT—SR) and, rarely tetradic (TT—SR—TT—SR) junctions were limited to regions between adjacent myofibrils and near the ends of fibers. Dyadic (TT—SR) junctions are found between myofibrils or within indentations of slablike mitochondria. Interspecific variation in SR—TT junctional assemblies from previous similar studies is reviewed and summarized and a three-dimensional model based on SEM and TEM observations is presented.