Regulators of the visual cycle are expressed at increased levels in the RPE of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice.

<p>The expression levels of genes encoding visual cycle regulators (<i>Lrat</i>, <i>Rpe65</i>, <i>Rdh5</i>, <i>Rgr</i>), transcription factors implicated in the homeostasis of the RPE (<i>Sox9</i>, <i>Otx2</i>), and house-keeping genes (<i>Alas1</i>, <i>B2m</i>) were determined by quantitative RT-PCR analysis of RNAs from the RPE of <i>Atg7</i>^<i>f/f</i> (n = 5) and <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> (n = 4) mice. Expression levels (a.u., arbitrary units) are shown relative to the expression of the house-keeping gene <i>B2m</i>. Error bars indicate standard deviations. *<i>p</i><0.05, considered statistically significant (two-tailed t-test). n.s., not significant.</p

<i>Tyrosinase-Cre</i>-Mediated Deletion of the Autophagy Gene <i>Atg7</i> Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

<div><p>Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene <i>Atg7</i>. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to <i>Sqstm1</i>, genes involved in retinal regeneration, i.e. <i>Lrat</i>, <i>Rdh5</i>, <i>Rgr</i>, and <i>Rpe65</i>, were expressed at higher mRNA levels. Sequencing of the <i>Rpe65</i> gene showed that <i>Atg7</i>^<i>f/f</i> and <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> mice is, at least partly, mediated by upregulation of <i>Rpe65</i> transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by <i>Atg7</i>-dependent autophagy in <i>Atg7</i>^<i>f/f</i> mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms.</p></div

Deletion of Atg7 leads to the accumulation of p62 and RPE65 in the RPE.

<p>Double-immunofluorescence labelling of p62 (green) (A, B) and RPE65 (red) (C, D) in <i>Atg7</i>^<i>f/f</i> (A, C, E) and <i>Atg7</i>^<i>f/f</i> <i>Tyr-Cre</i> (B, D, F) eyes. Nuclear DNA was labelled with Hoechst 33258 (blue). Panels E and F show merged images. ONL, outer nuclear layer; PRL, photoreceptor layer; RPE, retinal pigment epithelium; Ch, choroid. Scale bars: 50 μm.</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