Impact of omega-3 fatty acids on expression of angiogenic cytokines and angiogenesis by adipose-derived stem cells.

BACKGROUND AND AIMS: Human adipose-tissue derived stem cells (ADSC) are interesting novel targets in tissue engineering and regenerative medicine with pronounced angiogenic capacities. Furthermore, omega-3 fatty acids have been described to mediate cardioprotective effects, but their role in angiogenesis and vascular regeneration is not well-understood. Here, we analyzed the impact of different omega-3 fatty acids on angiogenesis by ADSCs. METHODS: Stem cells were cultured as monolayers or in 3D models, in spheroids embedded in collagen matrix or in co-cultures with human umbilical vein endothelial cells (HUVECs) in the Matrigel™ assay. The angiogenic properties of ADSCs were assessed by their sprouting and paracrine activities, gene expression by RT-PCR, Western blot, and enzyme immunoassay. RESULTS: Stimulation of undifferentiated ADSCs with docosahexaenoic acid (DHA) strongly upregulated angiopoietin-1 mRNA levels up to 4.6 ± 0.3 fold. Furthermore, Il-6 and Il-8 mRNAs were increased 4.2 ± 0.5 fold and 7.1 ± 1.1 fold, respectively. On the other hand, addition of DHA significantly decreased the cumulative sprout length by 2.7 ± 0.8 fold and reduced the total number of sprouts by 2.3 ± 0.9 fold in the in vitro angiogenesis assay. Moreover, excretion of IL-8 into the medium rapidly increased up to 1.7 ± 0.3 fold in response to treatment of ADSCs with DHA. Finally, protein kinase C inhibitor RO-31-8220 abrogated DHA-mediated up-regulation of angiopoietin-1 without significantly affecting ADSCs cell viability. CONCLUSION: In conclusion, ADSCs might regulate the formation and function of microvascular networks.

Selection of a DNA aptamer against norovirus capsid protein VP1.

The genetically and antigenically diverse group of noroviruses is the major cause of human viral epidemic gastroenteritis worldwide. Virus detection and control are thus crucial topics when aiming at containing and preventing the resulting large and often persisting outbreaks. Aptamers provide a promising alternative to antibodies concerning their ability to bind and thus detect and influence bio-active molecules. These small, single-stranded oligonucleotides are able to bind to a multitude of possible target molecules with high affinity. For a specific target the highest affinity aptamers are found by screening a randomized library. In this work a DNA aptamer capable of binding to the norovirus genotype II.4 capsid protein VP1 was found. The general approach is thereby not limited to norovirus capsid, but could be extended to almost any kind of biologically relevant molecule. The development of the library enrichment was further computationally analyzed in order to describe the enrichment during screening. This is the basis for a later extensive characterization of both target and aptamers that could lead to insights regarding the functional coherence of both partners. An abstract model describing this coherence could be utilized to generate a target-specific library, from which future aptamer screening runs could benefit.

Patterned biochemical functionalization improves aptamer-based detection of unlabeled thrombin in a sandwich assay.

Here we propose a platform for the detection of unlabeled human α-thrombin down to the picomolar range in a fluorescence-based aptamer assay. In this concept, thrombin is captured between two different thrombin binding aptamers, TBA1 (15mer) and TBA2 (29mer), each labeled with a specific fluorescent dye. One aptamer is attached to the surface, the second one is in solution and recognizes surface-captured thrombin. To improve the limit of detection and the comparability of measurements, we employed and compared two approaches to pattern the chip substrate-microcontact printing of organosilanes onto bare glass slides, and controlled printing of the capture aptamer TBA1 in arrays onto functionalized glass substrates using a nanoplotter device. The parallel presence of functionalized and control areas acts as an internal reference. We demonstrate that both techniques enable the detection of thrombin concentrations in a wide range from 0.02 to 200 nM with a detection limit at 20 pM. Finally, the developed method could be transferred to any substrate to probe different targets that have two distinct possible receptors without the need for direct target labeling.