'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

Human Wound Healing Ex Vivo Model with Focus on Molecular Markers.

Wound healing is a complex, multifactorial process that is divided in sequential and overlapping phases in order to restore the skin barrier. For the study of wound healing, different in vivo, in vitro, and ex vivo models have been used in the past. Here we describe in detail the methodology of the human skin punch-in-a-punch ex vivo wound healing model.

Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish.

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Regulation of vascular endothelial growth factor receptor function in angiogenesis by numb and numb-like.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling is a major regulator of physiological and pathological angiogenesis. VEGF receptor activity is strongly controlled by endocytosis, which can terminate or enhance signal transduction in the angiogenic endothelium, but the exact molecular regulation of these processes remains incompletely understood. We have therefore examined the function of Numb family clathrin-associated sorting proteins in angiogenesis. APPROACH AND RESULTS: We show that Numb proteins are expressed by endothelial cells during retinal angiogenesis in mice. Inducible inactivation of the Numb/Numbl genes in the postnatal endothelium led to impaired vessel growth, reduced endothelial proliferation and sprouting, and decreased VEGF receptor activation. Biochemistry and cell biology experiments established that Numb can interact with VEGFR2 and VEGFR3 and controls VEGF receptor activation in response to ligand stimulation. Experiments in cultured endothelial cells showed that Numb proteins counteract VEGF receptor degradation and promote VEGFR2 recycling back to the plasma membrane. CONCLUSIONS: Numb proteins control VEGF receptor endocytosis, signaling, and recycling in endothelial cells, which promotes the angiogenic growth of blood vessels.

Integrin ß1 controls VE-cadherin localization and blood vessel stability.

Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin ß1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that ß1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin ß1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin ß1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.

Spatial regulation of VEGF receptor endocytosis in angiogenesis.

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.