Abstract-MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke. (Circ Res. 2007;100:1579-1588.)Key Words: microRNAs Ⅲ vascular smooth muscle cells Ⅲ proliferation Ⅲ apoptosis Ⅲ neointimal formation M icroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs of Ϸ22 nucleotides and constitute a novel class of gene regulators. [1][2][3] Although the first miRNA, lin-4, was discovered in 1993, 4,5 their presence in vertebrates was confirmed only in 2001. 6 MiRNAs are initially transcribed by RNA polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts. 7 The primiRNAs are processed by the RNase III enzymes, Drosha and Dicer, to generate 18-to 24-nucleotide mature miRNAs. The mature miRNAs negatively regulate gene expression in 1 of 2 ways that depend on the degree of complementarity between the miRNA and its target. MiRNAs that bind to 3ЈUTR of mRNA with imperfect complementarity block protein translation. In contrast, miRNAs that bind to mRNA with perfect complementarity induce targeted mRNA cleavage. Currently, more than 400 miRNAs have been cloned and sequenced in human, and the estimated number of miRNA genes is as high as 1000 in the human genome. 8,9 As a group, miRNAs are estimated to regulate 30% genes of the human genome. 10 Analogous to the first RNA revolution in the 1980s with Cech discovering the enzymatic activity of RNA, 11 this recent discovery of RNAi and miRNA may represent the second RNA revolution. 12 Small interfering RNAs (siRNAs) are another class of small noncoding RNAs that have similar mechanism for gene expression regulation. However, they are different from each other. 5,13 The chief difference lies in their origins....

Limited microRNAs (miRNAs, miRs) have been reported to be necessary for exercise-induced cardiac growth and essential for protection against pathological cardiac remodeling. Here we determined members of the miR-17-92 cluster and their passenger miRNAs expressions in two distinct murine exercise models and found that miR-17-3p was increased in both. miR-17-3p promoted cardiomyocyte hypertrophy, proliferation, and survival. TIMP-3 was identified as a direct target gene of miR-17-3p whereas PTEN was indirectly inhibited by miR-17-3p. Inhibition of miR-17-3p in vivo attenuated exercise-induced cardiac growth including cardiomyocyte hypertrophy and expression of markers of myocyte proliferation. Importantly, mice injected with miR-17-3p agomir were protected from adverse remodeling after cardiac ischemia/reperfusion injury. Collectively, these data suggest that miR-17-3p contributes to exercise-induced cardiac growth and protects against adverse ventricular remodeling. miR-17-3p may represent a novel therapeutic target to promote functional recovery after cardiac ischemia/reperfusion.

We are developing a high performance double lumen cannula (DLC) for a minimally invasive, ambulatory and percutaneous paracorporeal artificial lung (PAL). The Wang-Zwische (W-Z) DLC was designed for percutaneous insertion into the Internal Jugular (IJ) vein with a drainage lumen open to both the superior vena cava (SVC) and the inferior vena cava (IVC) maximizing venous drainage. A separate collapsible but nondistensible membrane infusion lumen open to the right atrium (RA) achieves minimal recirculation allowing for total gas exchange. The W-Z DLC prototypes are made by a proprietary dip molding process with the "molded in" flat wire spiral stainless steel spring resulting in a flexible yet kink resistant thin wall (0.1 mm) outer cannula with one piece construction. With the ultra thin membrane infusion lumen collapsed, an introducer shaft fits tightly within the drainage lumen to facilitate insertion with placement at the SVC-RA-IVC junction. The W-Z DLC prototypes were tested while connected to a compact pump-gas exchanger circuit in three sheep (2 acute and one 15 day performance study). Insertion was simple, using standard percutaneous insertion techniques. Recirculation was as low as 2%. The 15 day performance study demonstrated our prototype 26 Fr W-Z DLC can achieve 2 L/min blood flow with minimal recirculation. The W-Z DLC design minimizes recirculation rate, maximizes flow lumen cross-sectional area, and maximizes achievable blood flow to enhance gas exchange performance allowing for one site percutaneous venovenous support.

Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies have demonstrated activity in early phase clinical trials. While T cells bearing stably expressed CARs are efficacious and have potential long-term persistence, temporary expression of a CAR via RNA electroporation is also potentially efficacious in preclinical models. Temporary CAR expression using RNA presents a method of testing CARs clinically with additional safety where there may be concerns about possible chronic "on-target, off-tumor" toxic effects, as the degradation of RNA ensures complete removal of the CAR over time without relying on suicide induction systems. CD19-directed RNA CAR T cells were tested in vivo for efficacy and comparison to lentiviral vector (LV)-generated stable CAR T cells. We tested the hypothesis that multiple infusions of RNA CAR T cells preceded by lymphodepleting chemotherapy could mediate improved survival and sustained antitumor responses in a robust leukemia xenograft model. The saturation strategy using rationally designed multiple infusions of RNA CARs based on multiple model iterations approached the efficacy of a stable LV expression method. Two-color imaging revealed that relapse was a locoregional phenomenon in both the temporary and the stable expression models. In marked contrast to stably expressed CARs with retroviral or LV technology, the efficacy of RNA CARs appears independent of the costimulatory signaling endodomains likely because they more influence proliferation and persistence rather than short-term efficacy. The efficacy of the RNA CAR infusions may approach that of stably expressed CARs, offer theoretically safer initial clinical testing in addition to suicide systems, and allow for rapid and effective iterative preclinical modeling for the testing of new targets.