Purpose:To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning. Materials and Methods:Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 ϫ 10 6 transduced (n ϭ 5) and control nontransduced (n ϭ 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphatebuffered saline (PBS) (n ϭ 4) or matrigel (n ϭ 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed.

Modulation of the cytotoxicity and mutagenicity of 4-hydroxyestradiol (4-OHE2), an oxidative metabolite of estrogen, by antioxidants was assessed in human MCF7 cells and TK-6 lymphoblast cells. The cytotoxicity of the catecholic estrogens was potentiated by depletion of intracellular glutathione and was independent of oxygen concentration. Agents such as the nitroxide Tempol can facilitate the oxidation of the semiquinone to the Q and enhanced 4-OHE2 cytotoxicity. Conversely, reducing agents such as ascorbate, cysteine, and 1,4-dihydroxytetramethylpiperidine (THP) protected against cytotoxicity and decreased mutation induction, presumably by reducing the semiquinone to the hydroquinone. Our results support the proposition that oxidation of the semiquinone to the corresponding Q is crucial in eliciting the deleterious effects of catecholic estrogens. Furthermore, because the deleterious effects of 4-OHE 2 were abrogated by dietary and synthetic antioxidants, our results would support the chemopreventive use of diets rich in reducing substances (vitamins and added synthetic antioxidants) as a means of decreasing the risks associated with estrogen exposure and developing of breast cancer.

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Background Despite ongoing clinical trials, the optimal time for delivery of bone marrow mononuclear cells (BMCs) following myocardial infarction (MI) is unclear. We compared the viability and effects of transplanted BMCs on cardiac function in the acute and sub-acute inflammatory phases of MI. Methods and Results The time-course of acute inflammatory cell infiltration was quantified by FACS analysis of enzymatically digested hearts of FVB mice (n=12) following LAD ligation. Mac-1+Gr-1high neutrophil infiltration peaked at day 4. BMCs were harvested from transgenic FVB mice expressing firefly luciferase (Fluc) and green fluorescent protein (GFP). Afterwards, 2.5×106 BMCs were injected into the left ventricle of wild-type FVB mice either immediately (Acute BMC) or 7 days (Sub-acute BMC) after MI, or after a sham procedure (n=8 per group). In vivo bioluminescence imaging (BLI) showed an early signal increase in both BMC groups at day 7, followed by a non-significant trend (P=0.203) towards improved BMC survival in the Sub-acute BMC group that persisted until the BLI signal reached background levels after 42 days. Compared to controls (MI + saline injection), echocardiography showed a significant preservation of fractional shortening at 4 weeks (Acute BMC vs saline; P<0.01) and 6 weeks (both BMC groups vs saline; P<0.05), but no significant differences between the two BMC groups. FACS analysis of BMC injected hearts at day 7 revealed that GFP+ BMCs expressed hematopoietic (CD45, Mac-1, Gr-1), minimal progenitor (Sca-1, c-kit), and no endothelial (CD133, Flk-1) or cardiac (Trop-T) cell markers. Conclusion Timing of BMC delivery has minimal effects on intramyocardial retention and preservation of cardiac function. In general, there is poor long-term engraftment and BMCs tend to adopt inflammatory cell phenotypes.

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Background Autologous bone marrow mononuclear cell (BMMC) therapy has shown promise for improving cardiac function post myocardial infarction. However, the efficiency of such therapy for diabetic patients remains unknown. Methods BMMCs were harvested from type II diabetic male BKS.Cg-m+/+Leprdb/J mice or C57BLKS/J (non-diabetic control) mice and were isolated using Ficoll-based separation as seen in clinical trials. Cell characterization was performed by flow cytometry. Cell viability was determined by apoptosis and proliferation assays. Female BKS.Cg-m+/+Leprdb/J mice underwent left anterior descending (LAD) artery ligation and were randomized into 3 groups receiving 2.5×106 diabetic BMMCs (n=8), 2.5×106 control BMMCs (n=8), or PBS (n=6), respectively. Echocardiography and invasive hemodynamic measurements were used to assess cardiac function at week 5. Post-mortem cell survival was quantified by Taqman RT-PCR for male Sry gene. Results BKS.Cg-m+/+Leprdb/J BMMCs showed a significantly lower mononuclear fraction (using flow cytometry) and a significantly lower proliferation rate compared with C57BLKS/J BMMCs. Echocardiography and invasive hemodynamic measurements showed significant in vivo improvement in fractional shorting (40.1±1.2% vs. 30.3±1.9%; P=0.001) and cardiac output (4,166±393 vs. 2,246±462 μl/min; P=0.016) for mice treated with control BMMCs injection compared to those treated with diabetic BMMCs, respectively. This difference could not be attributed to difference in cell engraftment as Taqman RT-PCR showed no significant difference in cell survival in infarcted hearts between the two groups. Conclusions Diabetic BMMCs are significantly impaired in their ability to improve cardiac function following myocardial infarction as compared to control BMMCs. The findings here could have significant clinical implication regarding autologous BMMC therapy in diabetic patients.

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