How Necessary is the Vasculature in the Life of Neural Stem and Progenitor Cells? Evidence from Evolution, Development and the Adult Nervous System.

Augmenting evidence suggests that such is the functional dependance of neural stem cells (NSCs) on the vasculature that they normally reside in "perivascular niches". Two examples are the "neurovascular" and the "oligovascular" niches of the adult brain, which comprise specialized microenvironments where NSCs or oligodendrocyte progenitor cells survive and remain mitotically active in close proximity to blood vessels (BVs). The often observed co-ordination of angiogenesis and neurogenesis led to these processes being described as "coupled". Here, we adopt an evo-devo approach to argue that some stages in the life of a NSC, such as specification and commitment, are independent of the vasculature, while stages such as proliferation and migration are largely dependent on BVs. We also explore available evidence on the possible involvement of the vasculature in other phenomena such as the diversification of NSCs during evolution and we provide original data on the senescence of NSCs in the subependymal zone stem cell niche. Finally, we will comment on the other side of the story; that is, on how much the vasculature is dependent on NSCs and their progeny.IK was supported by Action Medical Research, UK (GN2291).This is the final version of the article. It first appeared from Frontiers via http://dx.doi.org/10.3389/fncel.2016.00035

Isolation of neural stem and oligodendrocyte progenitor cells from the brain of live rats

Summary Postnatal brain neural stem and progenitor cells (NSPCs) cluster in anatomically inaccessible stem cell niches, such as the subependymal zone (SEZ). Here, we describe a method for the isolation of NSPCs from live animals, which we term “milking.” The intracerebroventricular injection of a release cocktail, containing neuraminidase, integrin-β1-blocking antibody, and fibroblast growth factor 2, induces the controlled flow of NSPCs in the cerebrospinal fluid, where they are collected via liquid biopsies. Isolated cells retain key in vivo self-renewal properties and their cell-type profile reflects the cell composition of their source area, while the function of the niche is sustained even 8 months post-milking. By changing the target area more caudally, we also isolate oligodendrocyte progenitor cells (OPCs) from the corpus callosum. This novel approach for sampling NSPCs and OPCs paves the way for performing longitudinal studies in experimental animals, for more in vivo relevant cell culture assays, and for future clinical neuro-regenerative applications

Biochemical and cellular in vitro and in vivo approach of sulfated hyaluronan’s anticancer effect in breast cancer

Breast cancer is one of the most prevalent malignancies worldwide, with disease progression largely depending on the expression of estrogen receptors (ERs), while the expression of extracellular matrix (ECM) molecules also plays a critical role. Hyaluronan (HA), a major component of the ECM, is involved in a plethora of physiological and pathological processes. Unlike other glycosaminoglycans, HA does not bear any substitutions to its structure naturally. However, recent studies highlight the use of chemically modified HA derivatives, such as sulfated hyaluronan (sHA), in applications including bio-scaffold manufacturing, wound healing, osteogenesis, and disease targeting. In the present PhD thesis, the effects of sHA on breast cancer cells of different ER status were studied. To this end, high-sulfated sHA was synthesized and its in vitro activity was evaluated on the breast cancer cell lines MCF-7, MDA-MB-231, Hs578T, and the stably transfected shERβ MDA-MB-231. Subsequently, three-dimensional in vitro cell culture models were developed and the effect of sHA was investigated under conditions that better reflect the tumor microenvironment. Moreover, the effect of sHA was studied in vivo in immunodeficient mice. Finally, the mechanism of action was investigated by studying signaling pathways, in addition to in silico molecular docking studies to predict possible interactions with HA receptors and degradation enzymes. According to the results, sHA reduces proliferation, migration, and invasion in the aggressive breast cancer cells, while increasing cell adhesion. Additionally, changes in cell morphology towards a more epithelial phenotype are observed through the use of scanning electron microscopy. These changes are further corroborated by alterations in the expression of important ECM molecules, as well as markers of cancer progression or suppression at the gene and protein level. Moreover, sHA reduces the tumorigenic capacity of breast cancer cells in immunodeficient mice. Overall, sHA demonstrates more potent antitumor activity compared to HA of the same molecular weight in both conventional and 3D cell cultures. This activity is primarily mediated through its main receptor, CD44, and partially attributed to competition with endogenously produced HA. In conclusion, the present thesis highlights the suppressive role of sHA in breast cancer, particularly in triple-negative breast cancer, thus marking it as a promising biomolecule in the development of innovative therapeutic approaches.Ο καρκίνος του μαστού αποτελεί μία από τις επικρατέστερες κακοήθειες παγκοσμίως, με την εξέλιξη της νόσου να εξαρτάται σε μεγάλο βαθμό από την έκφραση των οιστρογονοϋποδοχέων (ERs), ενώ σημαίνοντα ρόλο παίζει και η έκφραση μορίων του εξωκυττάριου χώρου (ECM). Το υαλουρονικό (ΗΑ) αποτελεί ένα κύριο συστατικό του ECM και εμπλέκεται σε πλήθος φυσιολογικών και παθολογικών διεργασιών. Σε αντίθεση με τις υπόλοιπες γλυκοζαμινογλυκάνες, το ΗΑ δε φέρει υποκαταστάσεις στη δομή του φυσικά. Εντούτοις, πρόσφατες μελέτες αναδεικνύουν τη χρήση χημικά τροποποιημένων παραγώγων του ΗΑ, όπως το θειωμένο υαλουρονικό (sHA), σε εφαρμογές όπως η κατασκευή βιο-ικριωμάτων, η επούλωση πληγών, η οστεογένεση, καθώς και στη στόχευση ασθενειών. Στην παρούσα διδακτορική διατριβή μελετήθηκε η δράση του sHA σε κύτταρα καρκίνου του μαστού διαφορετικής έκφρασης ERs. Για το σκοπό αυτό, συντέθηκε sΗΑ υψηλού βαθμού θείωσης και μελετήθηκε η in vitro δράση του στις καρκινικές σειρές MCF-7, MDA-MB-231, Hs578T, καθώς και στη σταθερά διαμολυσμένη σειρά shERβ MDA-MB-231. Παράλληλα, αναπτύχθηκαν τρισδιάστατα μοντέλα in vitro κυτταρικών καλλιεργειών και διερευνήθηκε η επίδραση του sHA σε καταστάσεις που προσομοιάζουν εγγύτερα το μικροπεριβάλλον του όγκου. Ακολούθως, μελετήθηκε η επίδραση του sHA in vivo σε μοντέλα ανοσοκατεσταλμένων μυών. Τέλος, διερευνήθηκε ο πιθανός μηχανισμός δράσης του sHA μέσω μελέτης σηματοδοτικών μονοπατιών και πραγματοποιήθηκαν in silico μελέτες μοριακής πρόσδεσης ώστε να προβλεφθούν οι πιθανές αλληλεπιδράσεις με υποδοχείς και ένζυμα αποικοδόμησης του ΗΑ. Σύμφωνα με τα αποτελέσματα, το sHA μειώνει τον πολλαπλασιασμό, τη μετανάστευση και τη διήθηση στα πιο επιθετικά καρκινικά κύτταρα μαστού, ενώ αυξάνει την κυτταρική προσκόλληση. Επιπλέον, με χρήση ηλεκτρονικής μικροσκοπίας σάρωσης παρατηρούνται αλλαγές στη μορφολογία των κυττάρων προς έναν πιο επιθηλιακό φαινότυπο. Οι αλλαγές αυτές επιβεβαιώνονται και από μεταβολές στην έκφραση σημαντικών μορίων του ECM καθώς και δεικτών προώθησης ή καταστολής του καρκίνου σε γονιδιακό και πρωτεϊνικό επίπεδο. Παράλληλα, το sHA μειώνει την ογκογενετική ικανότητα των καρκινικών κυττάρων μαστού σε ανοσοκατεσταλμένους μύες. Συνολικά, το sHA παρουσιάζει αντικαρκινική δράση ισχυρότερη του ΗΑ ίδιου μοριακού βάρους, τόσο σε συμβατικές όσο και σε τρισδιάστατες κυτταρικές καλλιέργειες, με τη δράση αυτή να μεσολαβείται μέσω του κύριου υποδοχέα του, CD44, και να οφείλεται εν μέρει σε ανταγωνισμό με το ενδογενώς παραγόμενο HA. Συμπερασματικά, η παρούσα διατριβή αναδεικνύει την κατασταλτική δράση του sHA στον καρκίνο του μαστού, ιδιαίτερα στις πιο επιθετικές μορφές όπως ο μη ορμονοεξαρτώμενος τριπλά αρνητικός καρκίνος του μαστού, και το καθιστά ένα πολλά υποσχόμενο βιομόριο στην ανάπτυξη καινοτόμων θεραπευτικών προσεγγίσεων

Sulfated Hyaluronan Modulates the Functional Properties and Matrix Effectors Expression of Breast Cancer Cells with Different Estrogen Receptor Status

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan (GAG) that plays a pivotal role in breast cancer. While HA is the only GAG not normally substituted with sulfate groups, sulfated hyaluronan (sHA) has previously been used in studies with promising antitumor results. The aim of the present study was to evaluate the effects sHA fragments have on breast cancer cells with different estrogen receptor (ER) status. To this end, ERα-positive MCF-7, and ERβ-positive MDA-MB-231 cells were treated with non-sulfated HA or sHA fragments of 50 kDa. The functional properties of the breast cancer cells and the expression of key matrix effectors were investigated. According to the results, sHA attenuates cell proliferation, migration, and invasion, while increasing adhesion on collagen type I. Furthermore, sHA modulates the expression of epithelial-to-mesenchymal transition (EMT) markers, such as e-cadherin and snail2/slug. Additionally, sHA downregulates matrix remodeling enzymes such as the matrix metalloproteinases MT1-MMP, MMP2, and MMP9. Notably, sHA exhibits a stronger effect on the breast cancer cell properties compared to the non-sulfated counterpart, dependent also on the type of cancer cell type. Consequently, a deeper understanding of the mechanism by which sHA facilitate these processes could contribute to the development of novel therapeutic strategies

Studying the Effects of Glycosaminoglycans in Cell Morphological Aspect with Scanning Electron Microscopy

Glycosaminoglycans, the building blocks of proteoglycans, play a central role in the extracellular matrix and regulate a number of cellular processes. Therefore, any imbalance in their levels can lead to significant changes in cell behavior and phenotype. Additionally, glycosaminoglycans and their derivatives can be deployed as therapeutic agents in pathological conditions. Since cell morphology is a critical indicator of specialized cellular functions, its study can provide valuable insight. Scanning electron microscopy is a high-resolution imaging technique that makes for an ideal tool to observe the cellular appearance in 2D and 3D cultures under different conditions and/or substrates. In this chapter we provide a step-by-step protocol to study the influence of exogenously added glycosaminoglycans in the morphology of cells using scanning electron microscopy

pH-Sensitive Gold Nanorods for Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Delivery and DNA-Binding Studies

A facile experimental protocol for the synthesis of poly(ethylene glycol)-modified (PEGylated) gold nanorods (AuNRs@PEG) is presented as well as an effective drug loading procedure using the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP). The interaction of AuNRs@PEG and drug-loaded AuNRs (AuNRs@PEG@NAP) with calf-thymus DNA was studied at a diverse temperature revealing different interaction modes; AuNRs@PEG may interact via groove-binding and AuNRs@PEG@NAP may intercalate to DNA-bases. The cleavage activity of the gold nanoparticles for supercoiled circular pBR322 plasmid DNA was studied by gel electrophoresis while their affinity for human and bovine serum albumins was also evaluated. Drug-release studies revealed a pH-sensitive behavior with a release up to a maximum of 24% and 33% NAP within the first 180 min at pH = 4.2 and 6.8, respectively. The cytotoxicity of AuNRs@PEG and AuNRs@PEG@NAP was evaluated against MCF-7 and MDA-MB-231 breast cancer cell lines. The development of AuNRs as an efficient non-steroidal anti-inflammatory drugs (NSAIDs) delivery system for chemotherapy is still in its infancy. The present work can shed light and inspire other research groups to work in this direction

New Analogs of Polyamine Toxins from Spiders and Wasps: Liquid Phase Fragment Synthesis and Evaluation of Antiproliferative Activity

Polyamine toxins (PATs) are conjugates of polyamines (PAs) with lipophilic carboxylic acids, which have been recently shown to present antiproliferative activity. Ten analogs of the spider PATs Agel 416, HO-416b, and JSTX-3 and the wasp PAT PhTX-433 were synthesized with changes in the lipophilic head group and/or the PA chain, and their antiproliferative activity was evaluated on MCF-7 and MDA-MB-231 breast cancer cells, using Agel 416 and HO-416b as reference compounds. All five analogs of PhTX-433 were of very low activity on both cell lines, whereas the two analogs of JSTX-3 were highly active only on the MCF-7 cell line with IC50 values of 2.63–2.81 μΜ. Of the remaining three Agel 416 or HO-416b analogs, only the one with the spermidine chain was highly active on both cells with IC50 values of 3.15–12.6 μM. The two most potent compounds in this series, Agel 416 and HO-416b, with IC50 values of 0.09–3.98 μΜ for both cell lines, were found to have a very weak cytotoxic effect on the MCF-12A normal breast cells. The present study points out that the structure of both the head group and the PA chain determine the strength of the antiproliferative activity of PATs and their selectivity towards different cells

Isolation of neural stem and oligodendrocyte progenitor cells from the brain of live rats.

Postnatal brain neural stem and progenitor cells (NSPCs) cluster in anatomically inaccessible stem cell niches, such as the subependymal zone (SEZ). Here, we describe a method for the isolation of NSPCs from live animals, which we term "milking." The intracerebroventricular injection of a release cocktail, containing neuraminidase, integrin-β1-blocking antibody, and fibroblast growth factor 2, induces the controlled flow of NSPCs in the cerebrospinal fluid, where they are collected via liquid biopsies. Isolated cells retain key in vivo self-renewal properties and their cell-type profile reflects the cell composition of their source area, while the function of the niche is sustained even 8 months post-milking. By changing the target area more caudally, we also isolate oligodendrocyte progenitor cells (OPCs) from the corpus callosum. This novel approach for sampling NSPCs and OPCs paves the way for performing longitudinal studies in experimental animals, for more in vivo relevant cell culture assays, and for future clinical neuro-regenerative applications