pCdk5 and p35 cluster with integrin beta-1 and talin into the lamellipodias.

<p>(A) Panel shows p35 clusters with talin and integin beta-1 into lamellipodia during cell movement. Red arrows in merge-magnification show dots of integrin and talin co-localisation. (B) Panel shows pCdk5 forming microvesicles with integrin beta-1 and talin at the lamellipodia in spreading cell. pCdk5 (C) and p35 (D) co-localized with talin in late spreading cells at focal tips (arrows in magnification). Immunofluorescent confocal microscopical analysis. Ci and Di show negative control for the immunoco-staining IgG-488 (green), IgG-568 (red), IgG-460 (blue) in Ci; and for the co-staining IgG-488 (green), IgG-568 (red), and DAPI (blue) in Di. Objective x40/x65. Original magnification in merge, x 240. Bars, 25 µm. Each experiment was performed in triplicate.</p

CIP-vector peptide rescues in vitro angiogenesis in hypoxia.

<p>(A) Panel reports a representative image of the effect of CIP–vector peptide on hBMECs cell migration during hypoxia (ii), versus controls in normoxia (iv), detected by phase contrast time-lapse analysis. (B,C) Representative contrast phase microscopy images of the effect of CIP–vector peptide on hBMECs tubule formation (B) and spheroid cell sprouting (C) in normoxic (i) and hypoxic conditions (ii). As Cdk5-wt CIP insertion significantly promoted cell migration during hypoxia, (D). (E) In CIP transfectants, the number of complete closed rings was significantly increased during normoxia (Bi), and maintained during hypoxia (Bii). Compared to the controls and Cdk5-wt, CIP insertion promoted the formation of spheroid–sprouts, which appeared with thicker lumens (C) and increased in number (F) in both hypoxic or normoxic conditions. Calculated results vs. controls and EV are reported in graphs: D, number of migrated cells in wound area during hypoxia, determined by Analyser_TM software; E, number of closed capillary ring structures on Matrigel® assay; F, number of cell sprouts in spheroid assay. Data are expressed as mean ±SD of biological triplicates. For all figures * P <0.05, and ° P <0.01. P value calculated using the Student t test. The phase contrast images of migration assay have been acquired as z-stacks and with automated focusing. Bars, 20µm; bars panel C, 50 µm. Bars, panel A 20 µm; panel B, 50 µm; 20 µm in panel C. Each experiment was performed in triplicate.</p

Cdk5 kinase activity is required for in vitro hBMECs angiogenesis.

<p>Figure shows phase contrast images of in vivo time course analysis of cell spreading (A), cell migration (B), and the formation of tubule like structure in roscovitine (50 µM) treated (+) and untreated (-) cells (G). Graphs C and D report the computational analysis of the characterization of cell typologies during the progression of different phases of cell spreading in controls (C) and roscovitine treated cells (D), determined using Analyser_TM software. Compared to the controls (C), roscovitine (D) inhibited cell elongation and increased the time dependent cell distribution between early and late spreading. Graphs E and F show the number of living and dividing cells presents in the wounded monolayer area in controls (E) and with roscovitine treatment (F) determined using Analyser_TM software. (G) Representative time-lapse images showing the progression of tubule formation throughout the time course, in controls and in treated cells. Graphs H and I, show the relative quantitative evaluation of growth area in controls (H) and treated cells (I), determined by Analyser_TM software. (I) Roscovitine reduced the growth area after 10h, mainly destabilizing neo-forming cell junctions and integrating cell networks. (L) Representative images showing the inhibitory effect of roscovitine on sprout formation, compared to controls (-) and FGF-2 stimulated cells (positive control). The phase contrast images have been acquired as z-stacks and with automated focusing. Bars, 20 µm. Original magnification in L, x 200. Each experiment was performed in triplicate.</p

CIP-vector peptide preserves p35/actin intracellular co-localization during hypoxia.

<p>(A) Images show the effect of hypoxia on p35 (left panel, Ai) and Cdk5 (right panel, Bi) intracellular distribution with actin filaments and fibres in hBMECs. Under hypoxia, p35 (Ai) lost its canonical filament-like structure organization (in merge, upper left panel) and localization at actin fibre tips, showing a more diffuse cytoplasmic distribution, while Cdk5 (Bi) maintained its co-localization with actin fibre tips (arrows in merge, upper right panel) (ii). Compared to the controls CIP insertion protected p35 (Aii) and Cdk5 (Bii) intracellular localization with actin filaments and fibres from the effects of hypoxia, this was associated with increased p35 protein contents (C) and increased p35/p25 protein ratio (D). Calculated results are reported in graph D; optical density (OD) of p35/p25 protein ratio in control untransfected cells and in CIP transfectants determined in normoxic and hypoxic conditions. Typical images from n=2 independent experiments. Immunofluorescence confocal analysis; bars, 20 µm. Original magnification, x 100. Each experiment was performed in triplicate.</p

Cdk5 and MEF2C promote proper spatial organization of brain endothelial cells.

<p>Figure shows the effect of MEF2C knock down, using siRNAs on angiogenesis in control cells (CT) and Cdk5-wt transfectants (wt). Scrambled siRNA has been used as negative controls. (A) Western blot analysis showing the effect of siRNA on MEF2C protein levels in wt cells. (B) siRNA reduced MEF2C protein expression by approximately 80%. (C) Tubule network (24h) was inhibited in control cells, while appeared more structured in Cdk5-wt transfectants. Nonetheless, the number of complete closed rings (D) was significantly reduced in both controls and Cdk5-wt transfectants as the spheroid sprout length and thickness (E) or sprouts number (F). Calculated results are reported in graphs: B, MEF2C protein levels (as ration on GAPDH OD) in wt and siMEF2C, or scrambled wt transfected cells; D, number of closed capillary ring structure determined on Matrigel® assay; F, number of cell sprouts determined in spheroid assay. Data are expressed as mean ± SD of biological triplicates. For all figures * P <0.05 vs controls. P value calculated using the Student t test. Phase contrast images; bars, 20 µm; bars in panel C, 50 µm. Experiments were performed in triplicate.</p

Cyto-architectural properties of Cdk5, activated Cdk5 (pCdk5) and p35.

<p>(A) Representative image showing Cdk5 localization at the tip of actin fibre (arrow) in early spreading (Ai). (B) Panel showing pCdk5 (pTyr_(Ser15)) localization with actin tips (arrows) and fibre in early (Bi) and late (Bii) spreading and in moving (Biii) cells. (c) p35 co-localized with actin stress fibres and was distributed from the nucleus (Ci) as the cell spreads culminating in filament tips (arrows in merge) in moving (Ciii) and late (Cii) spreading cells. Immunofluorescence confocal microscope analysis. Objective 65x. Original magnification in merge, x 240. Bars, 25 µm. Original magnification in Ai merge, x 290. Original magnification in merge in panel A, x300. Each experiment was performed in triplicate.</p

Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p[subscript (Tyr15)]Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition