Sirt3 is essential for apelin-induced angiogenesis in post-myocardial infarction of diabetes

Heart failure following myocardial infarction (MI) is the leading cause of death in diabetic patients. Angiogenesis contributes to cardiac repair and functional recovery in post-MI. Our previous study shows that apelin (APLN) increases Sirtuin 3 (Sirt3) expression and ameliorates diabetic cardiomyopathy. In this study, we further investigated the direct role of Sirt3 in APLN-induced angiogenesis in post-MI model of diabetes. Wild-type (WT) and Sirt3 knockout (Sirt3KO) mice were induced into diabetes by i.p. streptozotocin (STZ). STZ mice were then subjected to MI followed by immediate intramyocardial injection with adenovirus-apelin (Ad-APLN). Our studies showed that Sirt3 expression was significantly reduced in the hearts of STZ mice. Ad-APLN treatment resulted in up-regulation of Sirt3, angiopoietins/Tie-2 and VEGF/VEGFR2 expression together with increased myocardial vascular densities in WT-STZ+MI mice, but these alterations were not observed in Sirt3KO-STZ+MI mice. In vitro, overexpression of APLN increased Sirt3 expression and angiogenesis in endothelial progenitor cells (EPC) from WT mice, but not in EPC from Sirt3KO mice. APLN gene therapy increases angiogenesis and improves cardiac functional recovery in diabetic hearts via up-regulation of Sirt3 pathway.


Introduction
Myocardial angiogenesis is a process of forming new vessels to provide oxygen and nutrient supply to the ischaemic area of myocardial infarction (MI), which is a key adaptive mechanism to restore blood perfusion and a key determinant of infarct size expansion in post-MI [1,2]. Improvement of angiogenesis is being considered as an innovative therapeutic approach for the treatment of ischaemic heart disease [3,4]. Diabetes mellitus (DM) is characterized by hyperglycaemia, which leads to extensive cardiovascular complications including impairment of angiogenesis [5,6]. Coronary artery disease (CAD) is one of the major complications of DM [6]. Epidemiological studies revealed that myocardial ischaemia/infarction is the leading cause of morbidity and mortality in the patients with DM [7,8]. A populationbased study also showed that the incidence of MI in diabetic patients is significantly higher than non-diabetic patients [9]. Previously, we have shown that DM impairs myocardial angiogenesis via disruption of angiopoietins/Tie-2 and suppression of VEGF expression [10,11]. DM-associated impairment of angiogenesis contributes to the exacerbation of ischaemic injury and heart failure of diabetes [10,12,13]. Therefore, it is urgent to develop new agents for the treatment of impaired myocardial angiogenesis and post-MI heart failure in DM.
Apelin (APLN) is a bio-activated peptide with a potent angiogenic activity [14]. APLN exerts its biological effect via binding to the APLN receptor (APJ). APLN has been indicated as a key regulator of angiogenesis in different tissues [15,16]. A recent study shows that deficiency of APLN exacerbates MI adverse remodelling and ischaemia-reperfusion injury [17]. Our previous study shows that treatment with APLN promotes myocardial angiogenesis and improves cardiac function in post-MI mice [18]. Our recent study further demonstrated that treatment with bone marrow cells overexpressing APLN enhances myocardial angiogenesis and functional recovery, accompanied by increased Sirt3 levels in the ischaemic heart [19]. These findings indicate that Sirt3 may have a critical role for APLN-mediated cardiac protection in post-MI. Sirt3 is a member of a highly conserved family of protein deacetylases, which is closely associated with the prolonged lifespan of human [20]. Sirt3 has been attracted much attention because it regulates cardiomyocyte apoptosis, survival and cardiac hypertrophy [21][22][23]. So far, the direct link between APLN and Sirt3 in the regulation of myocardial angiogenesis in post-MI diabetes has not been reported.
This study was designed to evaluate the direct functional role of Sirt3 in APLN-mediated angiogenesis in diabetic mouse model. Wildtype (WT) and Sirt3 knockout (Sirt3 KO) mice were treated with streptozotocin (STZ) to induce hyperglycaemic DM model followed by MI by ligation of left anterior descendant artery (LAD). Using this ischaemic STZ mouse model, we have examined the effects of APLN gene therapy on the ischaemia-induced angiogenesis in diabetic mice. Moreover, we have explored the potential mechanisms by which Sirt3 regulates APLN-induced myocardial angiogenesis in diabetes.

Echocardiography
Transthoracic echocardiograms were performed on STZ mice at 2 weeks after LAD ligation using a Vevo770 Imaging System (Visual-Sonics Inc, Canada). Left ventricle ejection fraction (EF) and fractional shortening (FS) were recorded along with LV cavity dimensions (enddiastolic and end-systolic).

Endothelial progenitor cells treatment and transfection
Endothelial progenitor cells (EPC) was isolated and cultured from bone marrow of WT and Sirt3KO mice as described previously [12,13,24]. Two EPC markers, IB4 and CD34, were used for EPC identification by immunohistochemistry. To mimic in vivo hyperglycaemic conditions of DM model, EPC were exposed to high glucose (30 mmol/l) for 24 hrs, and followed by transfection with Ad-APLN and Ad-b-gal (1 9 10 9 PFU) in serum-free medium.

EPC tube formation
Endothelial progenitor cells (4.5 9 10 4 cells/well) were seeded on the layer of polymerized Matrigel (BD Biosciences, Bedford, MA, USA), followed by incubation for 6 hrs. Tube formation was quantified by measuring the long axis of each tube in five random fields per well using image-analysis software (Image J, NIH).

Western blot analysis
Hearts were harvested and homogenized in lysis buffer for Western analysis. The membranes were blotted with APLN, VEGFR2, Tie-2 (1:1000; Cell Signaling, MA, USA), VEGF, Ang-2 and Ang-1 (1:1000; Sigma-Aldrich) antibodies. Akt and eNOS phosphorylation were measured by phosphorylated Akt and eNOS antibodies followed by total Akt and eNOS antibodies. The membranes were then washed and incubated with a secondary antibody coupled to horseradish peroxidase and densitometric analysis was carried out using image acquisition and analysis software (TINA 2.0).

Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay
The apoptotic cells in heart tissue and cultured cells were detected by in situ DeadEndTM Colorimetric Apoptosis Detection System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The sections were counterstained with DAPI. Apoptosis was indexed by counting TUNEL + cells per 100 nuclei per section. vWF was co-stained with TUNEL.

APLN gene therapy on Sirt3 expression in WT-STZ+MI and Sirt3KO-STZ+MI mice
We first examined Sirt3 levels in the hearts of SIZ mice. Our Western blot analysis showed that Sirt3 expression was significantly reduced in STZ mice (Fig. 1A). Intramyocardial injection with Ad-APLN resulted in a significant increase in APLN expression in the hearts of WT-STZ and Sirt3KO-STZ mice (Fig. 1B). This was accompanied by a significant increase in Sirt3 expression in the hearts of WT-STZ mice, but not in Sirt3KO-STZ mice (Fig. 1C).

APLN gene therapy increases expression of angiogenic growth factors and neovascularization in STZ+MI mice
In WT-STZ+MI mice, APLN gene therapy significantly increased expression of Ang-1, Ang-2 and Tie-2 in the ischaemic hearts ( Fig. 2A-C). Overexpression of APLN led to significant increases in VEGF and VEGFR2 expression in WT-STZ+MI mouse hearts ( Fig. 2D and E). Moreover, Akt and eNOS phosphorylation was significantly increased in WT-STZ+MI mice treated with Ad-APLN compared to WT-STZ+MI received Ad-b-gal ( Fig. 2F and G). In Sirt3KO-STZ+MI mice, Ad-APLN treatment has little effect on the expression of angiopoietins/Tie-2 and VEGF/VEGFR2 as well as levels of Akt/eNOS phosphorylation ( Fig. 2A-G). CXCR-4 and SDF-1a expression was also significantly up-regulated by Ad-APLN treatment in post-MI STZ mice ( Fig. 3A and B). Knockout of Sirt3 in STZ+MI mice further blunted APLN-induced CXCR-4 and SDF-1a expression ( Fig. 3A and B). Ad-APLN treatment significantly increased capillary (IB4 and vWF) density and arteriole density in the border zone of ischaemia as compared to WT-STZ+MI mice treated with Ad-b-gal (Fig. 3C-F). However, APLN gene therapy did not increase capillary and arteriole densities in Sirt3KO-STZ+MI mice (Fig. 3C-F).

APLN gene therapy attenuates myocardial apoptosis in post-MI STZ mice
Sirt3KO-STZ+MI mice had a significant higher cleaved caspase-3 expression than WT-STZ+MI mice. Myocardial cleaved caspase-3 expression was significantly suppressed by Ad-APLN treatment when compared with Ad-b-gal treatment in WT-STZ mice (Fig. 4A). Knockout of Sirt3 in STZ mice significantly attenuated APLN gene therapymediated suppression of cleaved caspase-3 expression. As shown in Figure 4B and C, the number of TUNEL + cells in the ischaemic border zone was significantly higher in Sirt3KO-STZ+MI mice than WT-STZ+MI mice. In comparison with WT-STZ + Ad-b-gal mice, WT-STZ mice received Ad-APLN treatment showed a significant reduction in TUNEL + cells. In contrast, TUNEL + cells were similar between Sirt3-KO+Ad-APLN and Sirt3KO+Ad-b-gal mice ( Fig. 4B and C). Surprisingly, TUNEL + cells were not co-localized with endothelial marker vWF (Fig. 4D); suggested cardiomyocytes, but not EC, were apoptosis in the border zone of ischaemic heart.

APLN-mediated improvement of cardiac function is dependent on Sirt3
The echocardiography parameters are shown in Table 1. There was no significant difference in cardiac functional recovery between WT-STZ and Sirt3KO-STZ after MI. The EF% and FS% were significantly improved in WT-STZ + Ad-APLN mice as compared to WT-STZ + Ad-b-gal mice. However, there were no significant differences in

Overexpression of APLN increases expression of angiogenic growth factors and angiogenesis in EPC, but not EPC from Sirt3KO mice
To mimic STZ hyperglycaemic condition in vivo, cultured EPCs were exposed to high glucose (30 mmol/l) for 24 hrs before transfection with Ad-APLN. APLN expression was dramatically up-regulated in both WT-EPC and Sirt3KO-EPC by Ad-APLN transfection (Fig. 6A).
Overexpression of APLN resulted in a significant increase in Sirt3 expression in WT-EPC. The expression of Sirt3 was not detected in Sirt3KO-EPC (Fig. 6B). Ad-APLN treatment further significantly increased expression of Ang-1, Ang-2 and Tie-2 compared to Ad-b-gal treatment in WT-EPC (Fig. 6C-E). Ad-APLN treatment also led to a significant increase in expression of CXCR4 and SDF1a in WT-EPC. Knockout of Sirt3 in EPC completely abolished APLN-induced expression of angiopoietins/Tie-2 expression as well as CXCR-4 and SDF1a expression ( Fig. 6F and G).
Overexpression of APLN further significantly enhanced tube formation and cell proliferation in WT-EPC, but did not in Sirt3KO-EPC ( Fig. 7A-C). Moreover, knockout of Sirt3 abolished APLN-mediated suppression of apoptosis in EPC ( Fig. 7D and E). Knockout of Sirt3 in EPC significantly blunted APLN-induced suppression of apoptosis under high glucose conditions ( Fig. 7D and E).

Discussion
Our data demonstrated that overexpression of APLN resulted in a significant up-regulation of Sirt3 and angiogenic growth factor ‡SIRT3KO-STZ+IS+Ad-APLN, P < 0.05. ds, diameter systolic; dd, diameter diastolic; vs, volume systolic; vd, volume diastolic; SV, stroke volume; EF, Ejection Fraction; FS, Fractional Shortening; aws, systolic anterior wall thickness; awd, diastolic anterior wall thickness; pws, systolic posterior wall thickness; pwd, systolic posterior wall thickness. Our present study provides direct evidence that Sirt3 has a critical role in apelin-induced myocardial angiogenesis in ischaemic heart of diabetes. Sirt3 is expressed abundantly in the heart and has been reported to play a protective role in heart. Sirt3 protects cells from stress-mediated death by deacetylation of Ku70 [25] and blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant action in mice [26]. Although several recent studies indicate the involvement of Sirt1 in blood vessel formation [27,28], the functional role of Sirt3 in the regulation of angiogenesis especially in ischaemic diabetes has not been reported. APLN has been shown to induce endothelial cells sprouting in an autocrine or paracrine manner, thus contributing to vascular morphogenesis [14,29]. Angiogenesis is mainly regulated by the interplay between VEGF/VEGFR2 and angiopoietins/Tie-2 system. The APLN/APJ and Angs/Tie2 system interaction has been shown to be involved in the vessel calibre size and blood vessel maturation [30]. Ang-1/Tie2 interaction can activate endothelial cells (ECs) to produce endogenous APLN, which contributes to the lumen enlargement of tube-like structures by promoting proliferation and aggregation/assembly of ECs [30]. Our present study revealed that APLN gene therapy increased Angs/Tie-2 and VEGF/VEGFR2 expression together with a significant increase in capillary and arteriole densities in post-MI STZ mice. Interestingly, knockout of Sirt3 completely abolished APLN gene therapy-mediated enhancement of angiogenic factor expression, which resulted in poorer neovascularization in vivo. Knockout of Sirt3 further blunted APLN-mediated cardiac function recovery in STZ+MI mice. Based upon these data, we proposed that Sirt3 regulates APLN-mediated angiogenesis in ischaemic heart, at least in part, via up-regulation of Angs/Tie-2 and VEGF/VEGFR2 system. The mobilization of bone marrow-derived EPC has been shown to promote new vessel formation and ameliorates ischaemic injury [31,32]. Both animal and clinical studies showed that augment of EPC mobilization improved angiogenesis in ischaemic area of post-MI [33,34]. CXCR4 and SDF-1a are two key regulators for the mobilization of EPC from bone marrow to the ischaemic heart [35,36]. Our previous study has indicated that APLN promotes cardiac repair by increasing BM-derived vascular progenitor cell homing and stimulating angiogenesis in post-MI mouse heart [18]. Our recent study further demonstrated that APLN-overexpressed bone marrow EPC improves cardiac angiogenesis and functional recovery in post-MI mice via activation of Sirt3 signalling pathway [19]. Consistent with our previous studies, our in vitro data showed that overexpression of APLN up-regulated CXCR4 and SDF-1a and enhanced angiogenic growth factor expression in EPC. APLN also significantly increased EPC proliferation and tube formation ability. Knockout of Sirt3 in EPC abolished APLNinduced angiogenic growth factor expression and angiogenesis in vitro. Moreover, APLN gene therapy increased CXCR4 and SDF-1a expression in WT-STZ post-MI, but not in Sirt3KO-STZ mice. These data suggest that Sirt3 may also be involved in APLN-mediated mobilization of EPC to ischaemic heart via SDF-1a/CXCR4 axis in post-MI STZ mice.

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Apelin has direct role in cardiomyocyte survival and cardiac contractility. Consistent with these, our co-localization of vWF and TUNEL data suggested a potential involvement of apelin gene therapy on cardiomyocyte apoptosis, but not EC in ischaemic heart of diabetes. Some limitations should be pointed out in this study. Sirt3 is known to improve cardiac metabolisms, limit the cardiac fibrosis and cardiac hypertrophy. In addition, inflammation has an important role in improving the cardiac function more than angiogenesis in post-MI. All these effects, no doubt, contribute to the post-MI function recovery of diabetic hearts. We also recognized that a recent study indicates double-edged role of the CXCL12/CXCR4 axis in experimental MI [37]. In this study, we did not evaluate these effects.
In summary, our study provides direct evidence that overexpression of APLN enhances angiogenesis and improves post-MI cardiac function of STZ mice via up-regulation of Sirt3. Our data suggest that modification of Sirt3 with APLN could be used as a novel therapy strategy for the treatment of diabetes-associated impairment of angiogenesis after MI.