Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

INTRODUCTION: Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. CONCLUSION: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Expression of vimentin, TPI and MAT2A in human dermal microvascular endothelial cells during angiogenesis in vitro.

INTRODUCTION: In vitro assays of angiogenesis face immense problems considering their reproducibility based on the inhomogeneous characters of endothelial cells (ECs). It is necessary to detect influencing factors, which affect the angiogenic potency of ECs. OBJECTIVE: This study aimed to analyse expression profiles of vimentin (VIM), triosephosphate isomerase (TPI) and adenosylmethionine synthetase isoform type-2 (MAT2A) during the whole angiogenic cascade in vitro. Furthermore, the impact of knocking down vimentin (VIM) on angiogenesis in vitro was evaluated, while monitoring TPI and MAT2A expression. METHODS: A long-term cultivation and angiogenic stimulation of human dermal microvascular ECs was performed. Cells were characterized via VEGFR-1 and VEGFR-2 expression and a shRNA-mediated knockdown of VIM was performed. The process of angiogenesis in vitro was quantified via morphological staging and mRNA-and protein-levels of all proteins were analysed. RESULTS: While native cells ran through the angiogenic cascade chronologically, knockdown cells only entered beginning stages of angiogenesis and died eventually. Cell cultures showing a higher VEGFR-1 expression survived exclusively and displayed an upregulation of MAT2A and TPI expression. Native cells highly expressed VIM in early stages, MAT2A mainly in the beginning and TPI during the course of angiogenesis in vitro. CONCLUSION: VIM knockdown led to a deceleration of angiogenesis in vitro and knockdown cells displayed expressional changes in TPI and MAT2A. Cell populations with a higher number of stalk cells emerged as being more stable against manipulations and native expression profiles provided an indication of VIM and MAT2A being relevant predominantly in beginning stages and TPI during the whole angiogenic cascade in vitro.

HVC interneurons are not renewed in adult male zebra finches.

Adult neurogenesis is a widespread phenomenon in many species, from invertebrates to humans. In songbirds, the telencephalic region, high vocal center (HVC), continuously integrates new neurons in adulthood. This nucleus consists of a heterogenous population of inhibitory interneurons (HVC(IN)) and two populations of projection neurons that send axons towards either the robust nucleus of the arcopallium (HVC(RA)) or the striatal nucleus area X (HVC(X)). New HVC neurons were initially inferred to be interneurons, because they lacked retrograde labelling from the HVC's targets. Later studies using different tracers demonstrated that HVC(RA) are replaced but HVC(X) are not. Whether interneurons are also renewed became an open question. As the HVC's neuronal populations display different physiological properties and functions, we asked whether adult HVC indeed recruits two neuronal populations or whether only the HVC(RA) undergo renewal in adult male zebra finches. We show that one month after being born in the lateral ventricle, 42% of the newborn HVC neurons were retrogradely labelled by tracer injections into the RA. However, the remaining 58% were not immunoreactive for the neurotransmitter GABA, nor for the calcium-binding proteins, parvalbumin (PA), calbindin (CB) and calretinin (CR) that characterize different classes of HVC(IN). We further established that simultaneous application of parvalbumin, calbindin and calretinin antibodies to HVC revealed approximately the same fraction of HVC neurons, i.e. 10%, as could be detected by GABA immunoreactivity. This implies that the sum of HVC(IN) expressing the different calcium-binding proteins constitute all inhibitory HVC(IN). Together these results strongly suggest that only HVC(RA) are recruited into the adult HVC.