Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated ...NaYF(4) upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.
Abstract With the emergence of cell transplant as an attractive treatment modality for various diseases, there is a parallel need to track the fate of these cells to assess their therapeutic ...effectiveness. Here, we report the use of upconversion fluorescent nanoparticles, silica/NaYF4 :Yb,Er, to dynamically track live myoblast cells in vitro and in a living mouse model of cryoinjured hind limb. Nanoparticles loaded into cells were confirmed for its intracellular uptake by confocal imaging, spectrophotometry and inductively coupled plasma analysis. Loaded nanoparticles demonstrated absolute resistance to photobleaching and were applied for dynamic imaging to real time track in vitro cell migratory activity for a continuous 5 h duration using a time-lapse confocal microscope. Direct observation on the direction, speed and cell–cell interaction of migrating cells was clearly visualized. In vivo confocal imaging of nanoparticle-loaded cells intravenously injected into a mouse tail vein showed them flowing in the ear blood vessels. Nanoparticle-loaded cells were also unambiguously identified with superior contrast against a negligible background at least 1300 μm deep in a fully vascularized living tissue upon intramuscular injection. Spatiotemporal migratory activity of the transplanted cells within the three-dimensional living tissue was captured for at least 7 days post-delivery. Direct in vivo visualization of cell dynamics in the native tissue was unobtrusively followed over a 4 h time course and revealed subtle migratory activity of the transplanted cells. With these unique optical properties, we present silica/NaYF4 :Yb,Er nanoparticles as a new fluorescent live cell tracker probe for superior in vitro and in vivo dynamic imaging.
Abstract A regulated promoter system to control gene expression is desirable for safe and efficacious over-expression of therapeutic transgene. Combined with skeletal myoblast (SkMs), we report the ...efficacy of hypoxia-regulated VEGF gene delivery for myocardial repair during acute myocardial infarction (AMI). A hypoxia-regulated VEGF plasmid (pHRE-VEGF) was developed. After optimization, ∼30% SkMs were transfected using polyethyleneimine (PEI) nanoparticles. The peak VEGF expression was higher in pHRE-VEGF transfected SkMs (VEGF SkMs) under hypoxia (151.34 ± 8.59 ng/ml) than that with normoxia (16.92 ± 2.74 ng/ml). The efficacy of hypoxia-regulated gene expression system was assessed in a rabbit model of AMI. The animals were grouped to receive basal M199 without cells (group-1) or containing non-transfected SkMs (group-2) orVEGF SkMs (group-3). In group-4,VEGF SkMs were injected into normal heart to serve as normoxia control. Improved SkM survival was observed in group-3 and -4 ( p < 0.05 vs group-2) at day-3 and 7 after transplantation. Blood vessel density was 20.1 ± 1.3 in group-3 which was significantly higher than any other groups ( p < 0.05) at 2 weeks after treatment. Improved blood flow (ml/min/g) in the left ventricle (LV) anterior wall was observed in group-3 (1.28 ± 0.09, p < 0.05) as compared with group-1 (0.76 ± 0.05) and group-2 (0.96 ± 0.06), and similar to group-4 (1.26 ± 0.05). LV ejection fraction was best preserved in group-3 (58.4 ± 1.75%) which was insignificantly different from group-4 (61.1 ± 1.8%), and group-2 (52.8 ± 1.4%), but significantly improved compared with group-1 (44.7 ± 2.2%, p < 0.05). The study demonstrates that nanoparticle based delivery of hypoxia-regulated VEGF transgene combined with SkMs during AMI effectively preserves LV regional blood flow and contractile function of the heart.
We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF165) gene transfer into human skeletal myoblasts (HSM) for cell based ...delivery to the infarcted myocardium.
Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF165 (phVEGF165) carried by PEI (PEI-phVEGF165) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1=DMEM injection, group-2= HSM transplantation, group-3= PEI-phVEGF165-transfected HSM (PEI-phVEGF165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF165 for 18 days with >90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (x400) by immunostaining for PECAM-1 in group-3 was significantly higher (P=0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P=0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44+/-4.92%) as compared with group-1 (P=0.004).
PEI nanoparticle mediated hVEGF165 gene transfer into HSM is feasible and safe. It may serve as a novel and efficient alternative for angiomyogenesis in cardiac repair.
Myocardial infarction results in the death of cardiomyocytes, which are replaced by scar tissue. Cardiomyocytes cannot regenerate because they are terminally differentiated. Mesenchymal cells are ...pluripotent cells, which have the potential to differentiate to specialized tissues under appropriate stimuli. The aim of this study was to direct differentiation of the adult mesenchymal stem cells isolated from fatty tissue into cardiomyocytes using 5-azacytidine.
Adult mesenchymal stem cells were isolated from the fatty tissue of New Zealand White rabbits and cultured in RPMI medium. Second-passaged mesenchymal cells were treated with various concentrations of 5-azacytidine and incubated for different intervals of time. The cells were plated in six-well dishes at 500, 5,000, and 50,000 cells/well. These cells were treated with 1-, 3-, 6-, 9-, and 12-μmol/L concentrations of 5-azacytidine and incubated for 12, 24, 48, and 72 hours. Later, the medium was replaced with fresh medium and incubated in a CO
2 incubator. The medium was changed once at 3 to 4 days. At 2 months, the cells were fixed with 0.4% glutaraldehyde for 2 hours and later washed with phosphate-buffered saline. The transformed cells were subjected to immunostaining for the myosin heavy chain, α actinin, and troponin-I.
After treatment with 5-azacytidine, the adult mesenchymal stem cells were transformed into cardiomyocytes. At 1 week, some cells showed binucleation and extended cytoplasmic processes with adjacent cells. At 2 weeks, 20% to 30% of the cells increased in size and formed a ball-like appearance. At 3 weeks, these cells began to beat spontaneously in culture when observed under phase contrast microscope. Immunostaining of the transformed cells for myosin heavy chain, α actinin, and troponin-I was positive. The differentiated cells maintained the phenotype and did not dedifferentiate up to 2 months after treatment with 5-azacytidine.
These observations confirm that adult mesenchymal stem cells isolated from fatty tissue can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.
Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka ...α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.
The real promise of a stem cell-based approach for cardiac regeneration and repair lies in the promotion of myogenesis and angiogenesis at the site of the cell graft to achieve both structural and ...functional benefits. Despite all of the progress and promise in this field, many unanswered questions remain; the answers to these questions will provide the much-needed breakthrough to harness the real benefits of cell therapy for the heart in the clinical perspective. One of the major issues is the choice of donor cell type for transplantation. Multiple cell types with varying potentials have been assessed for their ability to repopulate the infarcted myocardium; however, only the adult stem cells, that is, skeletal myoblasts (SkM) and bone marrow-derived stem cells (BMC), have been translated from the laboratory bench to clinical use. Which of these two cell types will provide the best option for clinical application in heart cell therapy remains arguable. With results pouring in from the long-term follow-ups of previously conducted phase I clinical studies, and with the onset of phase II clinical trials involving larger population of patients, transplantation of stem cells as a sole therapy without an adjunct conventional revascularization procedure will provide a deeper insight into the effectiveness of this approach. The present article discusses the pros and cons of using SkM and BMC individually or in combination for cardiac repair, and critically analyzes the progress made with each cell type.
Abstract We aim to investigate the feasibility and efficacy of cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (hVEGF165 ) gene transfer into ...human skeletal myoblasts (hSkM) for cardiac repair. The feasibility and efficacy of CD liposome for gene transfer with hSkM was characterized using plasmid carrying enhanced green fluorescent protein (pEGFP). Based on the optimized transfection procedure, hSkM were transfected with CD lipoplexes carrying plasmid-hVEGF165 (CD–phVEGF165 ). The genetically modified hSkM were transplanted into rat heart model of acute myocardial infarction. Flow cytometry revealed that about 7.99% hSkM could be transfected with pEGFP. Based on the optimized transfection condition, transfected hSkM expressed hVEGF165 up to day-18 (1.7 ± 0.1 ng/ml) with peak at day-2 (13.1 ± 0.52 ng/ml) with >85% cell viability. Animal studies revealed that reduced apoptosis, improved angiogenesis with blood flow in group-3 animal's heart were achieved as compared to group-1 and 2. Ejection fraction was best recovered in group-3 animals. The study demonstrates that though gene transfection efficiency using CD liposome mediated hVEGF165 gene transfer with hSkM was low; hVEGF165 gene expression efficiency was sufficient to induce neovascularization, improve blood flow and injured heart function.
Patent bypass grafts are fundamental to successful coronary artery bypass grafting. Intraoperative flow measurement through newly constructed grafts is a test of patency. We studied the use of ...transit-time flow measurement to determine its ability to detect technical errors in grafts, to measure the mean flow norms for Asian patients, and to compare arterial and vein grafts.
From January 1, 2001, to June 30, 2002, 116 patients underwent isolated primary coronary artery bypass grafting. Sixty-seven patients underwent conventional coronary artery bypass grafting and 49 patients underwent off-pump coronary artery bypass grafting. There were 125 arterial and 197 vein grafts. Transit-time flow measurement was carried out on all completed grafts. Graft patency was assessed using flow curves, mean flow, and pulsatility index. Average of mean flows was calculated to determine mean flow norms. Arterial and vein grafts were compared by statistical analysis between the variables mean flow and pulsatility index.
In 6 patients with seven grafts, intraoperative graft assessment detected technical errors, which were corrected. Average mean flow was 37.4 +/- 23.5 mL/min for left anterior descending coronary artery-to-left internal mammary artery grafts, and values ranging from 21.2 to 36.0 mL/min for the rest. There were no statistically significant differences in mean flow or pulsatility index between arterial and vein grafts.
Transit-time flow measurement enables technical problems to be diagnosed accurately, allowing prompt revision of grafts. It should be mandatory in coronary artery bypass grafting to improve surgical outcomes.
This study investigated the potential of human skeletal myoblast carrying human VEGF(165) for angiomyogenesis for cardiac repair. A porcine heart model of chronic infarction was created in 18 female ...swine by coronary artery ligation. The animals were randomized into: group 1, DMEM injected ( n=6), group 2, myoblast transplanted ( n=5) and group 3, VEGF(165) myoblast transplanted ( n=7). Three weeks later 5 ml DMEM containing 3x10(8) myoblast carrying exogenous genes were injected into 20 sites in left ventricle intramyocardially in groups 2 and 3. Group 1 animals were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted between 6-12 weeks after transplantation for histological studies. Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density at low power field (x100) was 57.13+/-4.20 in group 3 which was significantly higher than the other groups. Regional blood flow was significantly improved 6 and 12 weeks after transplantation, which was 2.41+/-0.11 and 3.39+/-0.11 ml(-1) min(-1) g(-1), respectively, in group 3. Left ventricular ejection fraction increased from 31.25+/-4.09% to 43.0+/-2.68% at 6 weeks in group 3. Human myoblasts are potential transgene carriers for the myocardium, in addition to strengthening the weakened myocardium through myogenesis.
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