Apoptotic MNC-secretomes in experimental stroke

<p>Mixed Model Analysis (SAS output)</p> <p>The data were analyzed using linear mixed models for the neuroscore on treatment group and time-point with the factor animal included as a random effect. The MIXED procedure in SAS 9.3 was used to perform the calculations. The raw output contains information on the model specifications, the estimated error variance and random effects variance, the estimated regression coefficients, the covariance structure of the model coefficients and type III F-tests for the hypotheses of no effect of either fixed effect or their interactions. An interaction plot was drawn using the GLM procedure. This plot shows the individual observations and their sample mean values in each group and for each time-point. The group labels 0,1,2 and 3 in the raw output refer to the treatment group in setting 1, the control group in setting 1, the treatment group in setting 2 and the control group in setting 2, respectively.</p> <p>Original Western blots to Figure 5 </p> <p>Expression of proteins involved in cytoprotective pathways in human Astrocytes and Schwann Cells </p> <p>Astrocytes (page 1) or Schwann Cells (page 2) were stimulated with hMNCapo sec, control medium (served as control to treatment) or positive control (control to the measured protein). Original blots for all measured proteins are given in this raw data set (pages 1 and 2). For each blot, lanes (1), (2), and (3) correspond to the groups medium control [(1)=control to treatment], human apoptotic MNC-secretomes [(2)=treatment] and positive control [(3)=recombinant protein].<br>Bands in each blot are shown for phosphorylated CREB, total-CREB, phosphorylated Erk1/2, total-Erk 1/2, phosphorylated HSP27, total-HSP27, phosphorylated cJun, total-cJun, phosphorylated Akt, and total-Akt. The molecular weight (kDa) for each protein can be seen under each blot.<br>Ponceau staining was used as loading control for each group (1), (2), and (3) and suggest equal loading.</p> <p>Original Western blots to Figure 6 </p> <p>Expression of Phosphorylated CREB in Astrocytes and Neurons after stimulation with the active compound hMNCapo sec or control medium: </p> <p>Cultured human Astrocytes and Neurons were incubated with hMNCapo sec or control (cell culture-) medium at indicated concentrations. Original blots can be seen here.<br>Ponceau staining shows equal loading.</p> <p> </p

Kaplan-Meier plots showing the lung-metastasis free survival, recurrence free survival and overall survival after metastasectomy dependent on stromal Hsp27 (A), stromal α-SMA (B) and tumor Hsp27 (C) scoring.

<p>Kaplan-Meier plots showing the lung-metastasis free survival, recurrence free survival and overall survival after metastasectomy dependent on stromal Hsp27 (A), stromal α-SMA (B) and tumor Hsp27 (C) scoring.</p

Representative images showing pulmonary metastases with low, intermediate and high intensity of positive tumor stroma stained for Hsp27 and α-SMA.

<p>Stromal fibroblasts were further identified by vimentin staining. (DAB substrate, same tumor specimen per row, 200x magnification).</p

Total Hsp27 (A), phospho-Hsp27 (B) and IL-8 (C) were measured in serum samples of healthy volunteers, patients with CRC lung metastases before and 3 months after metastasectomy (each n = 10; whiskers indicate standard deviation).

<p>Total Hsp27 (A), phospho-Hsp27 (B) and IL-8 (C) were measured in serum samples of healthy volunteers, patients with CRC lung metastases before and 3 months after metastasectomy (each n = 10; whiskers indicate standard deviation).</p

The degree of Hsp27 expression score in the tumor stroma correlated significantly with the expression of stromal α-SMA (A).

<p>CD31-positive microvessels surrounded by tumor stroma next to tumor cells (asterisk) (B). MVD was significantly increased in primary tumors and metastases with strong stromal Hsp27 expression (C). Immunofluorescence showed a co-expression of stromal Hsp27 and α-SMA especially in PM (400x magnification) (D).</p

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Background<p>Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs.</p>Methods<p>Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 10⁶) or intramyocardially (group IM, 9.88 ± 1.44 × 10⁶). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image.</p>Results<p>AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm², p = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p = 0.006), with significant exponential decay between MMP-2 and CXCR4 (r = 0.679, p < 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%, p = 0.046) at the 1-month follow up.</p>Conclusion<p>Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.</p

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Background<p>Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs.</p>Methods<p>Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 10⁶) or intramyocardially (group IM, 9.88 ± 1.44 × 10⁶). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image.</p>Results<p>AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm², p = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p = 0.006), with significant exponential decay between MMP-2 and CXCR4 (r = 0.679, p < 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%, p = 0.046) at the 1-month follow up.</p>Conclusion<p>Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.</p

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Background<p>Intracoronary (IC) injection of mesenchymal stem cells (MSCs) results in a prompt decrease of absolute myocardial blood flow (AMF) with late and incomplete recovery of myocardial tissue perfusion. Here, we investigated the effect of decreased AMF on oxidative stress marker matrix metalloproteinase-2 (MMP-2) and its influence on the fate and homing and paracrine character of MSCs after IC or intramyocardial cell delivery in a closed-chest reperfused myocardial infarction (MI) model in pigs.</p>Methods<p>Porcine MSCs were transiently transfected with Ad-Luc and Ad-green fluorescent protein (GFP). One week after MI, the GFP-Luc-MSCs were injected either IC (group IC, 11.00 ± 1.07 × 10⁶) or intramyocardially (group IM, 9.88 ± 1.44 × 10⁶). AMF was measured before, immediately after, and 24 h post GFP-Luc-MSC delivery. In vitro bioluminescence signal was used to identify tissue samples containing GFP-Luc-MSCs. Myocardial tissue MMP-2 and CXCR4 receptor expression (index of homing signal) were measured in bioluminescence positive and negative infarcted and border, and non-ischemic myocardial areas 1-day post cell transfer. At 7-day follow-up, myocardial homing (cadherin, CXCR4, and stromal derived factor-1alpha) and angiogenic [fibroblast growth factor 2 (FGF2) and VEGF] were quantified by ELISA of homogenized myocardial tissues from the bioluminescence positive and negative infarcted and border, and non-ischemic myocardium. Biodistribution of the implanted cells was quantified by using Luciferase assay and confirmed by fluorescence immunochemistry. Global left ventricular ejection fraction (LVEF) was measured at baseline and 1-month post cell therapy using magnet resonance image.</p>Results<p>AMF decreased immediately after IC cell delivery, while no change in tissue perfusion was found in the IM group (42.6 ± 11.7 vs. 56.9 ± 16.7 ml/min, p = 0.018). IC delivery led to a significant increase in myocardial MMP-2 64 kD expression (448 ± 88 vs. 315 ± 54 intensity × mm², p = 0.021), and decreased expression of CXCR4 (592 ± 50 vs. 714 ± 54 pg/tissue/ml, p = 0.006), with significant exponential decay between MMP-2 and CXCR4 (r = 0.679, p < 0.001). FGF2 and VEGF of the bioluminescence infarcted and border zone of homogenized tissues were significantly elevated in the IM goups as compared to IC group. LVEF increase was significantly higher in IM group (0.8 ± 8.4 vs 5.3 ± 5.2%, p = 0.046) at the 1-month follow up.</p>Conclusion<p>Intracoronary stem cell delivery decreased AMF, with consequent increase in myocardial expression of MMP-2 and reduced CXCR4 expression with lower level of myocardial homing and angiogenic factor release as compared to IM cell delivery.</p