MicroRNAs (miRNAs) are a group of small non-coding RNAs, which regulate gene expression at the stage of protein translation. These small molecules have been shown to be involved in cancer pathogenesis, apoptosis and cell growth, thereby functioning as either tumor suppressor genes or oncogenes. However, expression alterations and roles of these miRNAs in pancreatic cancer are largely unknown. We hypothesize that pancreatic cancer may have a unique miRNA profile, which may play a critical role in pancreatic cancer development, progression, diagnosis and prognosis.

Introduction. In vitro differentiation of endothelial cells has potential applications in vascular tissue engineering and cell-based therapy for many diseases. The purpose of this study was to develop a new strategy that utilizes cytokines and LPS to accelerate the process of endothelial cell differentiation. Methods. CD14-positive monocytes were purified from human peripheral blood mononuclear cells (PBMC) by MACS separator. The purified cells were cultured in EBM2 endothelial cell-culture medium supplemented with GM-CSF and IL-4 with or without LPS for 4 days. The cells were then continuously cultured in EBM2 medium for additional 10 days (a total 14 days culture). Monocyte, dendritic cell, macrophage, endothelial cell, as well as smooth muscle cell markers were analyzed by real-time PCR, flow cytometry, and immunofluorescence staining. Results. In the LPS-treated group, most of suspended CD14-positive monocytes were attached and distinguishably changed in morphology to endothelial-like cells (ELCs) as fast as 4 days, but no morphology change was observed in the absence of LPS treatment. At day 8, the ELCs expressed high levels of endothelial cell markers CD31 (67.7%), vWF (93.9%), and VEGF receptor 1 (94.9%), but no smooth muscle cell markers were detected. At day 14, the ELCs grew to form cobblestone morphology and formed outgrowth colonies, which are typical endothelial cell growth characteristics. Moreover, there was gradually decreased expression of CD14 during the course of the differentiation. There were nearly no dendritic cell maturation marker CD83 (1.5%) and macrophage marker CD68 (2.1%). Importantly, eNOS expression on ELC was substantially increased from 4 to 86%. Furthermore, ELCs showed endothelial functional characteristics of uptaking acetylated LDL and binding ulex-lectin. Conclusions. These data demonstrate that LPS may trigger the switch of CD14-positive monocytes to endothelial cell differentiation in the endothelial culture medium. After the switch, cells continue to fully differentiate into endothelial cells in a relative short period of 14 days.

Introduction: Establishing thrombosis resistant surface is crucial to develop successful tissue engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anticoagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Methods: Porcine carotid arteries were obtained from a local abattoir. Cellular components of the arteries were completely removed with a newly developed method of decellularization. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulfate salt and heparin-EDC. Bound heparin contents were measured by a quantitative colorimetric assay of toludine blue staining. Anti-coagulation property of bound heparin was determined with a clotting time assay using both human and pig fresh blood. In vivo platelet deposition of the vessel was carried out with a baboon model of the femoral arteriovenous external shunt and 111Indium labeling of platelets. Results: The average content of heparin in treated vessels was 35.6 ± 11.6 mg/g tissue (ranging from 31 to 42 mg/g tissue), which represented 6.21 ± 2.03 UPS heparin/cm2 tissue (ranging from 5 to 8 UPS/cm2 tissue). The stability of heparin linkage was tested by incubating the heparin linked vessels either in PBS at 37°C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Furthermore, several vessels were stored in 70% alcohol for more than 2 years and the contents of bound heparin was not reduced. Standardized small pieces (3.3 × 3.3 mm2) of non-heparin bound vessels were clotted in both human and pig fresh blood within 13 min, while all heparin bound vessels did not form clot during 1 hour observation. There were 1.38 ± 0.07 × 109, and 0.64 ± 0.11 × 109 baboon platelets deposited on the control and heparin linked vessels, respectively, at 60 min. Conclusions: These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue engineered biological vascular grafts which could use for human coronary or low extremity artery bypasses.

Introduction: Although HIV Protease inhibitors significantly reduce the viral load, they may be associated with increased risk of cardiovascular disease. The aim of this study was to investigate the effects of HIV protease inhibitor ritonavir on endothelial cell function. Methods: Porcine carotid arteries were perfusion cultured for 24 hours without or with a clinically relevant dose of 15 μM of ritonavir. Subsequently, vessels were pre-contracted with norepinephrine followed by relaxation with graded doses of acetylcholine. Vessel diameters were determined with video imaging. Rings of vessels were cultured without or with ritonavir for 24 hours and subsequently basal and NADPH induced superoxide levels were determined using lucigenin enhanced chemiluminescence. Immunohistochemical staining for nitrotyrosine was also used to demonstrate oxidative stress. Results: Vessel contraction was reduced from 19 ± 3 % in control vessels (n = 12) to 9.9 ± 2.7% in ritonavir treated vessels (n = 11, P < 0.05). Endothelial dependent relaxation was also significantly reduced by 72%, 62%, and 65% in response to 10−9, 10−7, and 10−5 M concentrations of acetylcholine, respectively, in ritonavir treated vessels compared to controls (P < 0.005). There was a significant 56% increase in basal superoxide production (n = 7, P < 0.02) and a significant 40% increase in superoxide production (n = 8, P < 0.01) with addition of NADPH in vessel rings treated with ritonavir compared to control vessels. Diphenyleneiodonium Chloride (NAD(P)H oxidase inhibitor) and Tiron (cell permeable superoxide scavenger) significantly reduced superoxide production in ritonavir treated vessels. Nitrotyrosine immunoreactivity was increased in ritonavir treated vessels compared to controls. Conclusions: These data demonstrate that HIV protease inhibitor ritonavir causes a significant reduction in endothelium-dependent vasorelaxation in cultured porcine carotid arteries. Ritonavir also significantly increases superoxide levels, which may be resulted from increased NAD(P)H oxidase activity.