Cytoprotective and proangiogenic activity of ex-vivo netrin-1 transgene overexpression protects the heart against ischemia/reperfusion injury

Abstract

In continuation of a previous work that transgene expression of sonic hedgehog promoted neo-vascularization via netrin-1 release, the current study was aimed at assessing the anti-apoptotic and pro-angiogenic role of netrin-1 transgene overexpression in the ischemic myocardium. pLP-Adeno-X ViralTrak vectors containing netrin-1 cDNA amplified from rat mesenchymal stem cells (Ad-netrin) or without a therapeutic gene (Ad-null) were constructed and transfected into HEK-293 cells to produce Ad-netrin and Ad-null vectors. Sca-1(+)-like cells were isolated and propagated in vitro and were successfully transduced with Ad-netrin transduced Sca-1(+) cells ((Net)Sca-1(+)) and Ad-null transduced Sca-1(+) cells ((Null)Sca-1(+)). Overexpression of netrin-1 in (Net)Sca-1(+) was confirmed by reverse transcription-polymerase chain reaction and western blot. Neonatal cardiomyocytes and rat endothelial cells expressed netrin-1 specific receptor Uncoordinated-5b and the conditioned medium from (Net)Sca-1(+) cells was protective for both the cell types against oxidant stress. For in vivo studies, the rat model of myocardial ischemia/reperfusion injury was developed in female Wistar rats by left anterior descending coronary artery occlusion for 45 min followed by reperfusion. The animals were grouped to receive 70 μL of Dulbecco's modified Eagle's medium without cells (group-1), containing 2×10(6) (Null)Sca-1(+) cells (group-2) and (Net)Sca-1(+) cells (group-3). (Net)Sca-1(+) cells significantly reduced ischemia/reperfusion injury in the heart and preserved the global heart function in group-3 (P<0.05 vs. groups-1 and group-2). Ex-vivo netrin-1 overexpression in the heart increased NOS activity in the heart. Blood vessel density was significantly higher in group-3 (P<0.05 vs. controls). We concluded that netrin-1 decreased apoptosis in cardiomyocytes and endothelial cells via activation of Akt. Netrin-1 transgene expression was proangiogenic and effectively reduced ischemia/reperfusion injury to preserve global heart function.

FIG. 1.

Construction of Ad-netrin-1 vector and UNC5b expression on different cell types. (A) Construction of Ad-Netrin-1 vector with GFP reporter gene. (B) Reverse transcription–polymerase chain reaction (RT-PCR) showing distribution of netrin-1 receptor UNC5b in; lane-1=^NullSca-1⁺ cells, lane-2=^NetSca-1⁺; lane-3=neonatal cardiomyocytes; lane-4=HUVEC; lane-5=rat endothelial cells; and lane-6=heart as a positive control. ^NetSca-1⁺, Ad-netrin transduced Sca-1⁺ cells; ^NullSca-1⁺, Ad-null transduced Sca-1⁺ cells. UNC5, uncoordinated-5.

FIG. 2.

In vitro characterization of ^NetSca-1⁺-like cells. (A) Fluorescence microscopic images of Sca-1⁺ cells successfully transduced with Ad-null (red) and Ad-netrin (green) 48 h after transduction with the respective viral vector. Overexpression of netrin-1 transgene was confirmed by (B) RT-PCR. (C) Western blot showed significantly higher netrin-1 protein expression in ^NetSca-1⁺ cells as compared with ^NullSca-1⁺ and ^NatSca-1⁺ cells. (D) Transwell migration of rat endothelial cells (DAPI labeled; blue fluorescence) in response to conditioned medium from ^NetSca-1+ (^NetCM) was significantly higher as compared with the conditioned medium obtained from ^NullSca-1+ cells (^NullCM) and basal DMEM, using VEGF as a positive control. (E) Tube formation assay on matrigel using HUVECs treated with ^NetCM showed significantly higher number of branch points per microscopic field (200×) as compared with ^NativeCM and ^NullCM controls (P<0.01 vs. controls). VEGF treated cells were used as positive control. HUVECs, human umbilical vein endothelial cells; DMEM, Dulbecco's modified Eagle's medium. Color images available online at www.liebertpub.com/scd

FIG. 3.

Netrin-1 is cytoprotective for cardiomyocytes and endothelial cells. (A) TUNEL staining and (B) LDH release from the cardiomyocytes treated with 100 μM H₂O₂ for 4 h. The number of TUNEL⁺ cells (A; red fluorescence) and release of LDH were significantly reduced in the cardiomyocytes pretreated with 100 ng/mL recombinant netrin-1 as compared with the cardiomyocytes without netrin-1 pretreatment (P<0.01 vs. cardiomyocytes without netrin-1 treatment) after exposure to 100 μM H₂O₂. Netrin-1 treatment alone was well tolerated by the cells without any cytotoxicity as indicated by insignificantly lower TUNEL positivity and LDH release. (C) LDH release assay on rat endothelial cells treated with 100 μM H₂O₂ for 4 h. LDH release was significantly reduced in rat endothelial cells pretreated with 100 ng/mL recombinant netrin-1 as compared with the rat endothelial cells without netrin-1 pretreatment (P<0.01 vs. endothelial cells without netrin-1 pretreatment). Netrin-1 treatment alone was well tolerated by the cells without any cytotoxicity as indicated by insignificantly lower LDH release. (D, E) Western blot showed significantly higher phosphorylation of Akt in netrin-1 treated (D) cardiomyocytes and (E) rat endothelial cells as compared with the respective native cells without netrin-1 treatment. Phosphorylation of Akt was abrogated by pretreatment of both cardiomyocytes and rat endothelial cells with 40 μM wortmannin. LDH, lactate dehydrogenase. Color images available online at www.liebertpub.com/scd

FIG. 4.

Cardioprotective effects of transgenic netrin-1 expression. (A, B) quantitative (q)RT-PCR and western blot studies showing significantly higher expression of netrin-1 transgene in the left ventricle on day 4 after transplantation of ^NetSca-1⁺ (P<0.05 vs. ^NullSca-1⁺ injected control animal hearts). (C) qRT-PCR for Nos2 and Nos3 expression in the experimental animal hearts on day 4 after transplantation of ^NullSca-1⁺ cells (group-2) and ^NetSca-1⁺ cells (group-3). (D) Nitric oxide assay showing significantly higher Nos activity in ^NetSca-1⁺ transplanted animal hearts on day 4 after treatment (P<0.05 vs. ^NullSca-1⁺ transplanted group-2). (E) TUNEL on histological sections from different treatment groups of animals (red) counter stained for immmunodetection of the muscle architecture using cardiac actin (green). DAPI was used to visualize the nuclei. The number of TUNEL+ endothelial cells was significantly higher in groups 1 and 2 as compared with group 3. (F, G) Mason trichrome staining of the rat heart tissue samples from the three animal groups after 6 weeks of their respective treatment. Myocardial protection was markedly improved in the heart tissues in group-3 ^NetSca-1⁺ cell transplanted animal hearts as compared with ^NullSca-1⁺ transplanted animal group 2 and DMEM injected group-1 animal hearts.

FIG. 5.

(A) Fluorescence immunostaining of the rat heart tissue sections from different treatment groups of animals for expression of vonWillebrand Factor-VIII for blood vessel density determination. (B) Blood vessel density was significantly higher in group-3 animal hearts in both infarct and peri-infarct regions as compared with groups 1 and 2. (C) Transthoracic echocardiography was performed to assess global heart function. Both LV ejection fraction and fractional shortening were significantly preserved in groups 2 and 3 as compared with group-1. LV, left ventricle; vWF, vonWillebrand Factor.