SUMMARY Sudden unexplained death in epilepsy (SUDEP) is the most common cause of premature mortality in epilepsy and was linked to mutations in ion channels; however, genes within the channel protein interactome might also represent pathogenic candidates. Here we show that mice with partial deficiency of Sentrin/SUMO-specific protease 2 (SENP2) develop spontaneous seizures and sudden death. SENP2 is highly enriched in the hippocampus, often the focus of epileptic seizures. SENP2 deficiency results in hyper-SUMOylation of multiple potassium channels known to regulate neuronal excitability. We demonstrate that the depolarizing M-current conducted by Kv7 channel is significantly diminished in SENP2-deficient hippocampal CA3 neurons, primarily responsible for neuronal hyperexcitability. Following seizures, SENP2-deficient mice develop atrioventricular conduction blocks and cardiac asystole. Both seizures and cardiac conduction blocks can be prevented by retigabine, a Kv7 channel opener. Thus, we uncover a disease-causing role for hyper-SUMOylation in the nervous system and establish an animal model for SUDEP.

Rationale: Human CD34؉ cells have been used in clinical trials for treatment of myocardial infarction (MI). However, it is unknown how long the CD34 ؉ cells persist in hearts, whether the improvement in cardiac function is sustained, or what are the underlying mechanisms. Objective: We sought to track the fate of injected human CD34؉ cells in the hearts of severe combined immune deficiency (SCID) mice after experimental MI and to determine the mechanisms of action. Methods and Results: We used multimodality molecular imaging to track the fate of injected human CD34؉ cells in the hearts of SCID mice after experimental MI, and used selective antibody blocking to determine the mechanisms of action. Bioluminescence imaging showed that injected CD34؉ cells survived in the hearts for longer than 12 months. The PET signal from the injected cells was detected in the wall of the left ventricle. Cardiac MRI showed that left ventricular ejection fraction was significantly improved in the treated mice compared to the control mice for up to 52 weeks (P<0.05). Furthermore, treatment with anti-␣4␤1 showed that generation of human-derived cardiomyocytes was inhibited, whereas anti-vascular endothelial growth factor (VEGF) treatment blocked the production of human-derived endothelial cells. However, the improvement in cardiac function was abolished only in the anti-VEGF, but not anti-␣4␤1, treated group. Conclusions: Angiogenesis and/or paracrine effect, but not myogenesis, is responsible for functional improvement following CD34 ؉ cells therapy.

Promoting the paracrine effects of human mesenchymal stem cell (hMSC) therapy may contribute to improvements in patient outcomes. Here we develop an innovative strategy to enhance the paracrine effects of hMSCs. In a mouse hindlimb ischaemia model, we examine the effects of hMSCs in which a novel triple-catalytic enzyme is introduced to stably produce prostacyclin (PGI2-hMSCs). We show that PGI2-hMSCs facilitate perfusion recovery and enhance running capability as compared with control hMSCs or iloprost (a stable PGI2 analogue). Transplanted PGI2-hMSCs do not incorporate long term into host tissue, but rather they mediate host regeneration and muscle mass gain in a paracrine manner. Mechanistically, this involves long noncoding RNA H19 in promoting PGI2-hMSC-associated survival and proliferation of host progenitor cells under hypoxic conditions. Together, our data reveal the novel ability of PGI2-hMSCs to stimulate host regenerative processes and improve physical function by regulating long noncoding RNA in resident progenitor cells.

Background Mesenchymal stem cells (MSCs) can differentiate into endothelial cells in vivo. However, it is unknown if the differentiated MSCs persist in vivo and if this potential persistence contributes to functional improvement following experimental myocardial infarction (EMI). Methods and Results We generated a lentivector encoding two distinct reporter genes, one driven by a constitutive murine stem cell virus promoter and the other, by an endothelial specific Tie-2 promoter. The endothelial specificity of the lentivector was validated by its expression in endothelial cells, but not in human MSCs (hMSCs). The lentivirus-transduced hMSCs were injected into peri-infarct areas of the hearts of severe combined immune-deficient mice. Persistence of injected cells was tracked by bioluminescence imaging (BLI) and verified by immunohistochemical staining (IHS). The BLI signal from the endothelial-specific reporter revealed that hMSCs differentiated into endothelial cells 48 hours following injection. However, both the constitutive and the endothelial-specific BLI signals disappeared by day 50. Nonetheless, the improvement in left ventricle ejection fraction with hMSC therapy persisted for up to 6 months. IHS showed that hMSC-derived endothelial cells integrated into endogenous CD31+ vessels. Furthermore, hMSC-transplanted hearts had more CD31+ vessels and a lesser degree of cardiac fibrosis compared to the controls at 6 months. Conclusions hMSCs differentiated into endothelial cells and integrated into blood vessels after EMI. The differentiated hMSCs only lasted for up to 50 days in vivo, but improvement in cardiac function persisted for up to 6 months. Increased angiogenesis and decreased fibrosis were associated with cardiac functional improvement following hMSC transplantation.

Introduction We have showed that the transplanted human peripheral blood CD34 + cells can transform into smooth muscle, endothelial, and myocardial cells in infarcted hearts in mice. However, the fate of the transplanted cells in vivo is unknown. Hypothesis Molecular imaging is of substantial value for non-invasively tracking the fate of transplanted stem cells and for developing stem cell transplantation toward a clinically accepted therapy. Objectives To develop and validate molecular imaging techniques for reliably tracking the fate of transplanted stem cells in ischemic hearts in vivo and to follow the tempo-spatial dynamics of their transformation and function. Methods Using SCID mice transplanted with the CD34 + cells after myocardial infarction as a model, the CD34 + cells were transduced with 6 lentivectors bearing a fusion reporter gene driven by 6 different promoters. The reporter protein consists of herpes simplex virus thymidine kinase, green fluorescence protein, and luciferase, allowing us to monitor in vivo the fate of transplanted cells with bioluminescence imaging (BLI) and positron emission tomography (PET). In order to assess cardiac function, the mice were scanned with a 7.0-T MRI. Results The virus containing human ubiquitin promoter produced stable transduction efficiency 30%. Thereafter, 10 6 transduced CD34 + cells were injected into peri-infarct area per mouse heart. At day 7, the BLI signals in hearts were 1.4 ± 0.12x10 5 photons(p)/s/cm 2 /sr (n=6). At day 14, the PET signals were 0.05 ± 0.001 injection dose (ID) %/g. Both signals increased progressively to week 4, then remained in plateaus of 1.8 ± 0.15x10 5 p/s/cm 2 /sr and 1.38 ± 0.002 ID%/g, respectively until week 6, thereafter declining gradually, but were still as high as 9 ± 0.11x10 4 p/s/cm 2 /sr and 0.92 ± 0.001 ID%/g at week 16, indicating the transplanted CD34 + cells survive and function in the host. Co-register MRI/PET showed the concordance of anatomic and functional consequences after the transplantation of the CD34 + cells. Conclusion Our results demonstrate the feasibility of using molecular imaging to reliably track human CD34 + cell transplanted into infarcted hearts in mice, thus identifying PET/MRI imaging as a means to trace stem cell transplants in injured hearts.