Involvement of phosphoinositide 3-kinase gamma in angiogenesis and healing of experimental myocardial infarction in mice

Abstract

Rationale: Phosphoinositide 3-kinase (PI3K)gamma is expressed in hematopoietic cells, endothelial cells (ECs), and cardiomyocytes and regulates different cellular functions relevant to inflammation, tissue remodeling and cicatrization. Recently, PI3Kgamma inhibitors have been indicated for the treatment of chronic inflammatory/autoimmune diseases and atherosclerosis.

Objective: We aimed to determine PI3Kgamma contribution to the angiogenic capacity of ECs and the effect of PI3Kgamma inhibition on healing of myocardial infarction (MI).

Methods and results: Human umbilical ECs were treated with a selective PI3Kgamma inhibitor, AS605240, or a pan-phosphoinositide 3-kinases inhibitor, LY294002. Both inhibitory treatments and small interfering RNA-mediated PI3Kgamma knockdown strongly impaired ECs angiogenic capacity, because of suppression of the PI3K/Akt and mitogen-activated protein kinase pathways. Constitutive activation of Akt rescued the angiogenic defect. Reparative angiogenesis was studied in vivo in a model of MI. AS605240 did not affect MI-induced PI3Kgamma upregulation, whereas it suppressed Akt activation and downstream signaling. AS605240 strongly reduced inflammation, enhanced cardiomyocyte apoptosis, and impaired survival and proliferation of ECs in peri-infarct zone, which resulted in defective reparative neovascularization. As a consequence, AS605240-treated MI hearts showed increased infarct size and impaired recovery of left ventricular function. Similarly, PI3Kgamma-deficient mice showed impaired reparative neovascularization, enhanced cardiomyocyte apoptosis and marked deterioration of cardiac function following MI. Mice expressing catalytically inactive PI3Kgamma also failed to mount a proper neovascularization, although cardiac dysfunction was similar to wild-type controls.

Conclusions: PI3Kgamma expression and catalytic activity are involved at different levels in reparative neovascularization and healing of MI.

Figure 1

PI3Kγ inhibition impairs angiogenesis. A, Bar graph shows the effects of AS (1 μmol/L) or LY (15 μmol/L) on serum-induced EC proliferation as assessed by measurement of 5-bromodeoxyuridine incorporation. B, Bar graph and microphotographs illustrate gap closure (percentage) measured 24 hours after scratch in DMSO-, AS-, or LY-treated HUVECs. C, Endothelial network formation in Matrigel-based angiogenesis assay: both number of branches/view field (i) and network total length/view field (mm) (ii) were counted. D, Bar graph shows Caspase-3/7 activity assessed by measuring the luminescent signal generated by DEVD cleavage in extracts of HUVECs that were serum-starved and exposed to hypoxia (0.2% O₂, 5% CO₂) in the presence of DMSO, AS, or LY for 18 hour (n=3 for each assay). *P<0.05, **P<0.01, ***P<0.001 vs DMSO; ###P<0.001 vs AS. Scale bars: 500 μm.

Figure 2

Silencing of PI3Kγ impairs angiogenesis. A, HUVECs were transduced with 100 multiplicities of infection of Ad.scrambled or Ad.siRNAγ overnight. Both adenoviral constructs carried GFP allowing straightforward assessment of infection efficiency. Top, GFP expression 3 days after infection. Bottom, Immunoblot analysis of PI3K isoforms expression in HUVECs harvested 3 days after infection. GAPDH is shown as loading control. The silencing approach does not affect PI3Kα or PI3Kβ expression. PI3Kγ is selectively and effectively downregulated. Assays were performed as in Figure 1, 3 days after adenoviral transduction. Three independent infections were performed for both Ad.siRNAγ and Ad.scrambled. Results indicate that PI3Kγ is essential to cell proliferation (B), migration (C), tube-like network formation (D), and survival (E) of ECs (n=3 for each experiment). *P<0.05, **P<0.01, ***P<0.001 vs scrambled; ###P<0.001 vs Ad.siRNAγ.

Figure 3

PI3K/Akt signaling suppression on PI3Kγ inhibition accounts for defective angiogenesis. A and B, HUVECs were incubated in EC basal medium with neither growth factors nor serum for 3 hours. During the last 30 minutes before stimulation, cells were pre-treated with DMSO, AS (1 μmol/L), or LY (15 μmol/L), followed by 10 minutes stimulation with complete medium containing growth factors and serum. A similar procedure was carried out on Ad.scrambled and Ad.siRNAγ HUVECs 3 days after transduction. Immunoblot analysis and averaged densitometric data show the effects of PI3K inhibition on phosphorylated (p)Akt (i), pGSK3β (ii), peNOS (iii), and pErk1/2 (iv) levels. C, Immunoblot analysis confirming the activation of Akt and downstream effectors achieved by infection with Ad.MyrAkt (hemagglutinin [HA]-tagged, top immunoblot image). D, i and ii, Bar graphs show the effects of the restoration of Akt activity by Ad.MyrAkt in Matrigel-based angiogenesis assay. In C and D, Ad.Null-HUVECs are shown for comparison. n=4. *P<0.05, **P<0.01, ***P<0.001 vs DMSO (A) or Ad.scrambled (B); #P<0.05, ##P<0.01, ###P<0.001 vs AS (A), Ad.siRNAγ (B), or Ad.Null AS/LY (D).

Figure 4

Effects of PI3Kγ inhibition on cell signaling in the infarcted heart. Immunoblot analyses (A, B, D, and F) of PI3Kγ/Akt signaling pathway and Akt relative kinase activity (C) in protein extracts from LV of DMSO- or AS-treated mice 3 days post-MI or sham (S). Bar graphs show fold changes calculated from the ratio between either phosphorylated and total protein content (B, D, and E) or total protein content and GAPDH, used as loading control (A and F). In all graphs, fold changes are calculated toward DMSO-treated Sham. n=4 hearts in each group. *P<0.05, **P<0.01, ***P<0.001 vs DMSO- or AS-treated sham; ##P<0.01, ###P<0.001 vs DMSO-treated MI.

Figure 5

PI3Kγ is crucial for reparative neovascularization. A, Immunofluorescence images show the vascularization of PI zone of DMSO- or AS-treated hearts 14 days post-MI. ECs are stained by lectin IB₄ (red), α-smooth muscle actin (α-SMA, green) identifies arterioles, and nuclei are stained by DAPI (blue). Scale bars: 20 μm. Bar graphs show the capillary density (B) or arteriole density (C and D) of R and PI zone of DMSO- or AS-treated hearts (i) and of WT, KD, or KO hearts (ii). n=4 to 8 hearts per group. *P<0.05, **P<0.01, ***P<0.001 vs DMSO-treated R or WT R; #P<0.05, ##P<0.01, ###P<0.001 vs DMSO-treated PI or WT PI; $P<0.05 vs KD PI; $$P<0.01 vs WT/KD R.

Figure 6

Targeting of PI3Kγ inhibits EC proliferation in infarcted hearts. Representative immunofluorescence images (A) show the fraction of proliferating ECs in PI zone of DMSO- or AS-treated hearts (a and b, respectively) 3 days post-MI. Proliferating ECs (arrowheads) are identified by lectin IB₄ (red) and positivity for proliferating-cell nuclear antigen (PCNA) (light blue). Nuclei are stained by DAPI (blue). Scale bars: left, 100 μm; right, 20 μm. I indicates infarct; unspecific lectin IB₄ binding delimiting infarcted area. B, Bar graph illustrates the number of proliferating ECs in PI zone of DMSO- or AS-treated mice (i) or of WT, KD and KO mice (ii). n=4 to 6 mice in each group. **P<0.01 vs DMSO; ***P<0.001 vs WT; ##P<0.01 vs KD; ns, not significant vs WT.

Figure 7

PI3Kγ is crucial for EC and cardiomyocyte survival in vivo. Representative immunofluorescence images (A) and bar graphs (B) show the fraction of apoptotic ECs (i) and cardiomyocytes (iii) in PI zone of DMSO- and AS-treated hearts 3 days after MI. Apoptotic ECs and cardiomyocytes are positive to TUNEL reaction (light magenta). ECs are stained by lectin IB₄ (green) and nuclei by DAPI (blue). n=6 DMSO- and 4 AS-treated hearts. Scale bars: left, 100 μm, right, 20 μm. I indicates infarct; unspecific lectin IB₄ binding delimiting infarcted area. The number of apoptotic ECs (B, ii) and cardiomyocytes (B, iv) was also evaluated in PI zone of WT, KD, and KO hearts. n=4 for each group. C, HL-1 were serum starved and exposed to hypoxia (0.2% O₂, 5% CO₂) in the presence of DMSO or AS for 18 hours. Bar graph shows caspase-3/7 activity assessed as in Figure 1D; n=3. *P<0.05, **P<0.01, ***P<0.001 vs DMSO or WT; #P<0.05, ###P<0.001 vs KD.