The Kcna1 model of SUDEP exhibits progressive respiratory dysfunction, which suggests a potential increased susceptibility for respiratory failure during severe seizures that may result in sudden death.

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in-vitro. The cultured bone marrow derived cells were CD11b−CD34−CD44+CD45−CD90+ and showed mesodermal lineage differentiation which are characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth media (EGM) with and without high concentrations of VEGF (EGM+VEGF; 50ng/ml). The endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE-cadherin, PECAM-1, CD34, VEGF-R1 and VEGF-R2 after 10days of stimulation. The cells cultured in EGM+VEGF media demonstrated higher amount of DiI-AcLDL positive cells, and enhanced presence of vWF (90%), VE-Cadherin (60%) and PECAM-1 (48%) positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2 fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM+VEGF media when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in-vitro inducing conditions. The differentiated cells would provide new options for re-endothelialization following interventional procedures and tissue engineering.

Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)‐mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP‐9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP‐9, MMP‐1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP‐9 activity and mRNA transcripts for MMP‐9, MMP‐1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP‐9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF‐treated VSMCs of both groups. Additionally, the magnitude in decreased MMP‐9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP‐9 gene with siRNA in EGF‐treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP‐9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.

BackgroundAnti-PD-1 immune checkpoint blockade is approved for first-line treatment of recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), but few patients respond. Statin drugs (HMG-CoA reductase inhibitors) are associated with superior survival in several cancer types, including HNSCC. Emerging data suggest that manipulation of cholesterol may enhance some aspects of antitumor immunity.MethodsWe used syngeneic murine models (mouse oral cancer, MOC1 and TC-1) to investigate our hypothesis that a subset of statin drugs would enhance antitumor immunity and delay tumor growth.ResultsUsing an ex vivo coculture assay of murine cancer cells and tumor infiltrating lymphocytes, we discovered that all seven statin drugs inhibited tumor cell proliferation. Simvastatin and lovastatin also enhanced T-cell killing of tumor cells. In mice, daily oral simvastatin or lovastatin enhanced tumor control and extended survival when combined with PD-1 blockade, with rejection of MOC1 tumors in 30% of mice treated with lovastatin plus anti-PD-1. Results from flow cytometry of tumors and tumor-draining lymph nodes suggested T cell activation and shifts from M2 to M1 macrophage predominance as potential mechanisms of combination therapy.ConclusionsThese results suggest that statins deserve further study as well-tolerated, inexpensive drugs that may enhance responses to PD-1 checkpoint blockade and other immunotherapies for HNSCC.

Previously, we and others have shown that the regular intake of green tea polyphenols (GTPs) reduces ultraviolet B (UVB) radiation-induced skin cancer by targeting multiple signaling pathways, including DNA damage, DNA repair, immunosuppression, and inflammation. Here, we determine the effect of GTPs on UVB-induced epigenetic changes, emphasizing DNA hypermethylation in UV-exposed skin and tumors and their association with miR-29, a key regulator of DNA methyltransferases (DNMTs). Skin cancer was induced in SKH-1 hairless mice following repeated exposures of UVB radiation (180 mJ/cm2, three times/week, 24 weeks) with or without GTPs supplementation (0.2%) in drinking water. Regular intake of GTPs inhibited tumor growth by hindering the cascade of DNA hypermethylation events. GTPs supplementation significantly blocked UVB-induced DNA hypermethylation in the skin (up to 35%; p < 0.0001) and in tumors (up to 50%; p < 0.0001). Experimental results showed that the levels of DNA hypermethylation were higher in GTPs-treated mice than in the control group. The expressions of miR-29a, miR-29b, and miR-29c were markedly decreased in UV-induced skin tumors, and GTPs administration blocked UVB-induced miR-29s depletion. Furthermore, these observations were verified using the in vitro approach in human skin cancer cells (A431) followed by treatment with GTPs or mimics of miR-29c. Increased levels of miR-29 were observed in GTPs-treated A431 cells, resulting in increased TET activity and decreased DNA hypermethylation. In conclusion, UVB-mediated miR-29 depletion promotes DNA hypermethylation and leads to enhanced tumor growth by silencing tumor suppressors. Regular intake of GTPs rescued UVB-induced miR-29 depletion and prevented tumor growth by maintaining reduced DNA hypermethylation and activating tumor suppressors. Our observations suggest that miR-based strategies and regular consumption of GTPs could minimize the risk of UVB-induced skin cancers and contribute to better management of NMSCs.