Senescent impairment in synergistic cytokine pathways that provide rapid cardioprotection in the rat heart

Abstract

Pretreatment of rodent hearts with platelet-derived growth factor (PDGF)-AB decreases myocardial injury after coronary occlusion. However, PDGF-AB cardioprotection is diminished in older animals, suggesting that downstream elements mediating and/or synergizing the actions of PDGF-AB may be limited in aging cardiac vasculature. In vitro PDGF-AB induced vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 expression in 4-mo-old rat cardiac endothelial cells, but not in 24-mo-old heart cells. In vivo injection of young hearts with PDGF-AB increased densities of microvessels staining for VEGF and its receptor, Flk-1, and Ang-2 and its receptor, Tie-2, as well as PDGF receptor (PDGFR)-alpha. In older hearts, PDGF-AB-mediated induction was primarily limited to PDGFR-alpha. Studies in a murine cardiac transplantation model demonstrated that synergist interactions of PDGF-AB plus VEGF plus Ang-2 (PVA) provided an immediate restoration of senescent cardiac vascular function. Moreover, PVA injection in young rat hearts, but not PDGF-AB alone or other cytokine combinations, at the time of coronary occlusion suppressed acute myocardial cell death by >50%. However, PVA also reduced the extent of myocardial infarction with an age-associated cardioprotective benefit (4-mo-old with 45% reduction vs. 24-mo-old with 24%; P < 0.05). These studies showed that synergistic cytokine pathways augmenting the actions of PDGF-AB are limited in older hearts, suggesting that strategies based on these interactions may provide age-dependent clinical cardiovascular benefit.

Figure 1.

PDGF-AB induction of cardiac microvascular endothelial proangiogenic cytokines. (A) Real-time RT-PCR of VEGF and Ang-2 in PDGF-AB treated 4- and 24-mo-old rat CMECs in vitro. (B) Representative immunohistochemical staining of VEGF or Ang-2 in cardiac capillaries in rat hearts injected with vehicle (PBS) or PDGF-AB 24 h before euthanization (n = 3, each). Bar = 10 μm. (C) Graph shows the densities of VEGF and Ang-2 positive capillaries in PDGF-AB– or PBS-treated hearts. *, P < 0.05; **, P < 0.005. Treatment versus control.

Figure 2.

Age-associated induction of cytokine receptors by intramyocardial injection of PDGF-AB. Representative immunohistochemical staining of PDGFR-α, Flk-1, and Tie-2 in cardiac capillaries in rat hearts injected with vehicle (PBS) or PDGF-AB 24 h before euthanization in 4- and 24-mo-old hearts (n = 3, each). Bar = 10 μm. Graph shows the densities of PDGFR-α, Flk-1, and Tie-2 positive capillaries (mm²) in PDGF-AB– or PBS-treated hearts. *, P < 0.05. PDGF-AB versus PBS.

Figure 3.

Cytokine synergism in restoration of senescent cardiac allografts. (A) Representative examples of neonatal cardiac transplants into 18-mo-old senescent hosts with pretreatment of VEGF (100 ng) or VEGF (100 ng) and PDGF-AB (3 ng). The arrowhead (left) indicates the site of necrotic loss of both allograft and host pinnal tissue beyond the transplant site in the majority of the VEGF-pretreated transplants. The arrow (right) indicates viable/intact cardiac transplants in the host pinnal tissue. (B) Dose response curve of PDGF-AB pretreatment in the restoration of cardiac allograft viability in 18-mo-old mice (n ≥ 20, per group). 10 ng and 100 ng of PDGF-AB restored a significant number of allografts compared with 0- and 1-ng doses. P < 0.005. (C) Assessment of VEGF and Ang-2 pretreatment and combined VEGF and PDGF-AB (n ≥ 20, per group). *, P < 0.05. PDGF-AB (3 ng) versus PBS. **, P < 0.01. Treatment versus PDGF-AB (3 ng) or PBS. (D) Assessment of cytokine combinations in the peri-transplantation of pinnal tissue at the time of cardiac allograft transplantation (n > 10, per group). *, P < 0.05. PDGF-AB plus VEGF plus Ang-2 or VEGF plus Ang-2 versus PBS and all other treatment groups.

Figure 4.

Synergistic suppression of cardiac apoptosis after coronary occlusion. TUNEL labeling of rat cardiac sections 24 h after injection with combinations of PDGF-AB, VEGF, and Ang-2 at the time of LAD ligation. (A) Reconstructed cross-section micrograph from lower magnification (4×) images for measurement of apoptotic area (percentage of left ventricular area). (B) High magnification (40×) TUNEL staining images used in the quantification of apoptotic cell density. Bar = 10 μm. (C) A graph is shown plotting mean apoptotic risk area size (white) and cell density (TUNEL⁺; black) in the sections of rat anterior left ventricular wall injected with PBS, PDGF-AB, PDGF-AB plus VEGF (PV), PDGF-AB plus Ang-2 (PA), VEGF plus Ang-2 (VA), or PDGF-AB plus VEGF and Ang-2 (PVA) (n = 3, each group) at the time of LAD ligation. *, P < 0.01; **, P < 0.005 PVA versus PBS and all other cytokine groups.

Figure 5.

Age-associated cardioprotection by combined cytokine injection at the time of coronary occlusion. (A) Representative Masson's trichrome staining of rat hearts harvested 2 wk after peri-occlusion injections of PBS, PDGF-AB (P), or PDGF-AB plus VEGF and Ang-2 (PVA) and LAD ligation (n ≥ 5, each). Infarction area (blue stain) at the mid–papillary muscle level was significantly smaller in PVA peri-injection heart section than PBS- or PDGF-AB–injected sections. However, there is significant difference between 4- and 24-mo-old rats with PVA treatment. (B) Graph shows average myocardial infarction size (percentage of left ventricular area). *, P < 0.05. 4-mo-old PVA versus 4-mo-old PBS, 4-mo-old PDGF-AB, and 24-mo-old PVA. **, P < 0.05. 24-mo-old PVA versus 24-mo-old PBS and 24-mo-old PDGF-AB.