Background-Myocardial ischemia and reperfusion-induced tissue injury involve a robust inflammatory response, but the proximal events in reperfusion injury remain incompletely defined. Toll-like receptor 4 (TLR4) is a proximal signaling receptor in innate immune responses to lipopolysaccharide of Gram-negative pathogens. TLR4 is also expressed in the heart and vasculature, but a role for TLR4 in the myocardial response to injury separate from microbial pathogens has not been examined. This study assessed the role of TLR4 in myocardial infarction and inflammation in a murine model of ischemia-reperfusion injury. Methods and Results-Myocardial ischemia-reperfusion (MIR) was performed on 2 strains of TLR4-deficient mice (C57/BL10 ScCr and C3H/HeJ) and controls (C57/BL10 ScSn and C3H/OuJ). Mice were subjected to 1 hour of coronary ligation, followed by 24 hours of reperfusion. TLR4-deficient mice sustained significantly smaller infarctions compared with control mice given similar areas at risk. Fewer neutrophils infiltrated the myocardium of TLR4-deficient Cr mice after MIR, indicated by less myeloperoxidase activity and fewer CD45/GR1-positive cells. The myocardium of TLR4-deficient Cr mice contained fewer lipid peroxides and less complement deposition compared with control mice after MIR. Serum levels of interleukin-12, interferon-␥, and endotoxin were not increased after ischemia-reperfusion. Neutrophil trafficking in the peritoneum was similar in all strains after injection of thioglycollate. Conclusions-TLR4-deficient mice sustain smaller infarctions and exhibit less inflammation after myocardial ischemiareperfusion injury. The data suggest that in addition to its role in innate immune responses, TLR4 serves a proinflammatory role in murine myocardial ischemia-reperfusion injury.

BackgroundSmall caliber vascular prostheses are not clinically available because synthetic vascular prostheses lack endothelial cells which modulate platelet activation, leukocyte adhesion, thrombosis, and the regulation of vasomotor tone by the production of vasoactive substances. We developed a novel method to create scaffold-free tubular tissue from multicellular spheroids (MCS) using a “Bio-3D printer”-based system. This system enables the creation of pre-designed three-dimensional structures using a computer controlled robotics system. With this system, we created a tubular structure and studied its biological features.Methods and ResultsUsing a “Bio-3D printer,” we made scaffold-free tubular tissues (inner diameter of 1.5 mm) from a total of 500 MCSs (2.5× 104 cells per one MCS) composed of human umbilical vein endothelial cells (40%), human aortic smooth muscle cells (10%), and normal human dermal fibroblasts (50%). The tubular tissues were cultured in a perfusion system and implanted into the abdominal aortas of F344 nude rats. We assessed the flow by ultrasonography and performed histological examinations on the second (n = 5) and fifth (n = 5) day after implantation. All grafts were patent and remodeling of the tubular tissues (enlargement of the lumen area and thinning of the wall) was observed. A layer of endothelial cells was confirmed five days after implantation.ConclusionsThe scaffold-free tubular tissues made of MCS using a Bio-3D printer underwent remodeling and endothelialization. Further studies are warranted to elucidate the underlying mechanism of endothelialization and its function, as well as the long-term results.

Background — Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Methods and Results — Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1β protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. Conclusions — This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel “pre-event” gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.