Mesenchymal Stem Cells Loaded with p5, Derived from CDK5 Activator p35, Inhibit Calcium-Induced CDK5 Activation in Endothelial Cells

Abstract

The potential use of stem cells as therapeutics in disease has gained momentum over the last few years and recently phase-I clinical trials have shown favourable results in treatment of a small cohort of acute stroke patients. Similarly, they have been used in preclinical models drug-loaded for the effective treatment of solid tumours. Here we have characterized uptake and release of a novel p5-cyclin-dependent kinase 5 (CDK5) inhibitory peptide by mesenchymal stem cells and showed release levels capable of blocking aberrant cyclin-dependent kinase 5 (CDK5) signaling pathways, through phosphorylation of cyclin-dependent kinase 5 (CDK5) and p53. These pathways represent the major acute mechanism stimulating apoptosis after stroke and hence its modulation could benefit patient recovery. This work indicates a potential use for drug-loaded stem cells as delivery vehicles for stroke therapeutics and in addition as anticancer receptacles particularly, if a targeting and/or holding mechanism can be defined.

Figure 1

The p5 internalization and release by hADMSCs. (a) The internalization of p5 was analyzed by fluorescence microscopy in hADMSCs primed 1, 2, 4, 8, or 24 hours with 3 μg/mL p5 (green). Cells were also observed after 24 hours priming on Days 2, 3, 4, 5, 10, and 14. (b) The internalization of p5 was analyzed by fluorescence microscopy in BAECs primed with different doses of p5 (green) for 24 hours. The intensity of p5 staining in cytoplasm was correlated with the dosages of p5. (c) Subconfluent cultures (2 × 10⁵) of adherent hADMSCs were exposed to 12 μg/mL biotinylated p5 for 24 hours. After several washes and trypsinization, p5-primed hADMSCs were further cultured and their conditioned medium (CM) was collected every 24 hours. The internalization of p5 was analyzed by fluorescence microscopy in BAECs treated with p5-primed hADMSCs CM for 24 hours. Scale bar: 100 μm. (d) The fluorescein staining intensities of p5 in hADMSCs in (a) were quantified in the bar graph as means ± SD (error bars). Significance (denoted as ∗) was calculated compared to the untreated control group using Student's t-test (with p < 0.01, n = 6). (e) The fluorescein staining intensities of p5 in BAECs in (b) were quantified in the bar graph as means ± SD (error bars). (f) The fluorescein staining intensities of p5 in BAECs in (c) were quantified in the bar graph as means ± SD (error bars). Significance (denoted as ∗) was calculated compared to the untreated control group using Student's t-test (with p < 0.01, n = 6).

Figure 2

LC/MS with Q-TOF analysis of p5. (a) The total ion chromatogram (TIC) of 200 ng/mL p5. (b) Positive ESI mass spectra (1046.1700 m/z corresponding to [M]³⁺ in excellent agreement with the expected value) of 20.70 ± 0.05 minutes of 200 ng/mL p5. The area of peak was circled. The average peak area for 200 ng/mL p5 from 6 tests was 5563.63 counts. (c) Positive ESI mass spectra (1046.1700 m/z) of 20.70 ± 0.05 minutes of 100 ng/mL p5. The area of peak was circled. The average peak area for 100 ng/mL p5 from 6 tests was 2430.55 counts. The ESI-MS spectra showed no peak for 50 ng/mL p5 (data not shown).

Figure 3

The activation of CDK5 by calpain in cultured BAECs. (a) The cultured BAECs were treated with 5 μM calcium ionophore A23187 and 2.5 mM CaCl₂ for 0, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 4 hours. At the end of the treatment, the cells were collected, suspended in SDS-PAGE sample buffer, and boiled for 5 minutes. Each sample was run on a 10% SDS-PAGE gel and blotted with anti-p35, anti-phospho-CDK5 (pTyr¹⁵), anti-phospho-ERK1/2, anti-phospho-p53 (pSer¹⁵), anti-active caspase-3, anti-CDK5, anti-ERK1/2, anti-p53, anti-procaspase-3, and anti-tubulin antibodies. The first lane was loaded with the extract from untreated BAECs as control. The cultured BAECs treated with 10 ng/mL FGF2 for 10 minutes were used as positive control for the phosphorylation of ERK1/2 (lane 8). Blots shown are one of at least three independent experiments performed. (b) Densitometric analysis of immunoblots quantification. Data are means ± SD (error bars). Significance (denoted as ∗) was calculated compared to the untreated control (lane 1) using Student's t-test (with p < 0.05, n = 3).

Figure 4

p5 and CM from p5-primed hADMSCs inhibited CDK5 and p53 activation by calpain in cultured BAECs. (a) The cultured BAECs were treated with p5 (lanes 5–7) or conditioned medium (CM) from p5-primed hADMSCs collected on Day 1 (lanes 8–10) for 1 hour and then further treated with 5 μM calcium ionophore A23187 and 2.5 mM CaCl₂ for 10 minutes. The 3 μg/mL p5 treatment alone (lane 1), CM from untreated hADMSCs (lane 2) and untreated BAECs (lane 3) were used as control. The p35 cleavage and the phosphorylation of CDK5 and p53 were suppressed by p5 and CM from p5-primed hADMSCs. (b) Densitometric analysis of immunoblots quantification. Data are means ± SD (error bars). Significance (denoted as ∗) was calculated compared to cells treated with 5 μM calcium ionophore A23187 and 2.5 mM CaCl₂ (lane 4) using Student's t-test (with p < 0.05).

Figure 5

p5 and conditioned medium (CM) from p5-primed hADMSCs protected BAECs from calpain-induced cytotoxicity. The cultured BAECs were treated with p5 or CM from 12 μg/mL p5-primed hADMSCs collected on Day 1 for 1 hour and then further treated with 5 μM calcium ionophore A23187 and 2.5 mM CaCl₂ for 10 minutes (a) or 2 hours (b). The cell proliferation was monitored for 3 consecutive days using the alamarBlue assay. Data are means ± SD (error bars). Significance (denoted as ∗) was calculated compared to the control using Student's t-test (with p < 0.05, n = 3).