Effects of late administration of pentoxifylline and tocotrienols in an image-guided rat model of localized heart irradiation

Abstract

Radiation-induced heart disease (RIHD) is a long-term side effect of radiotherapy of intrathoracic, chest wall and breast tumors when radiation fields encompass all or part of the heart. Previous studies have shown that pentoxifylline (PTX) in combination with α-tocopherol reduced manifestations of RIHD in rat models of local heart irradiation. The relative contribution of PTX and α-tocopherol to these beneficial effects are not known. This study examined the effects of PTX alone or in combination with tocotrienols, forms of vitamin E with potential potent radiation mitigation properties. Rats received localized X-irradiation of the heart with an image-guided irradiation technique. At 3 months after irradiation rats received oral treatment with vehicle, PTX, or PTX in combination with a tocotrienol-enriched formulation. At 6 months after irradiation, PTX-treated rats showed arrhythmia in 5 out of 14 animals. PTX alone or in combination with tocotrienols did not alter cardiac radiation fibrosis, left ventricular protein expression of the endothelial markers von Willebrand factor and neuregulin-1, or phosphorylation of the signal mediators Akt, Erk1/2, or PKCα. On the other hand, tocotrienols reduced cardiac numbers of mast cells and macrophages, but enhanced the expression of tissue factor. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation.

Figure 1. Cardiac inflammatory cell numbers at 6 months after irradiation.

Radiation caused an increase in mast cell numbers and CD68 positive cells, as shown by representative micrographs (A) and total cell counts (B). TSB reduced the effects of radiation on cardiac mast cell number. PTX enhanced the effects of radiation on the number of CD68 positive cells. Average ± SEM, n=8-9. *Significant difference with sham-irradiation (p<0.05), ^† Significant difference with 21 Gy (p<0.05). Scale bar: 100 μm.

Figure 2. Cardiac expression of vWf at 6 months after irradiation.

In sham-irradiated hearts, vWF immunoreactivity was only found on the endothelium of larger arteries, while in irradiated hearts vWF was also found in endothelial cells of capillaries and arterioles, and in some hearts in the extracellular matrix (A). Areas immunoreactive for vWf were not altered by PTX or TSB (B). Average ± SEM, n=8-9. *Significant difference with sham-irradiation (p<0.05). Scale bar: 100 μm.

Figure 3. Left ventricular protein levels of Nrg-1 and TF at 6 months after irradiation.

Radiation caused a significant increase in Nrg-1 and TF expression (A). PTX did not alter Nrg-1 or TF (B), while PTX in combination with TSB enhanced TF expression (C). Average ± SEM, n=4-5. *Significant difference with sham-irradiation (p<0.05), ^† Significant difference with 21 Gy (p<0.05). Scans of the Western-Blot films are shown in Figure S2.

Figure 4. Left ventricular protein levels of total and phosphorylated Akt, Erk1/2, and PKCα at 6 months.

Radiation caused a significant reduction in the phosphorylated form of all three signaling molecules. Scans of the Western-blot films are shown in Figure S4. Average ± SEM, n=6. *Significant difference with sham-irradiation (p<0.05).

Figure 5. Effects of PTX and TSB on left ventricular Akt, Erk1/2, and PKCα in irradiated animals.

PTX alone reduced the levels of phosphorylated Erk1/2 and phosphorylated Akt, but did not significantly change the ratio of phosphorylated to total Erk1/2 or Akt. PTX in combination with TSB increased protein levels of total Akt, but did not change the ratio of phosphorylated to total Akt. PTX and TSB had no effect on PKCα. Scans of the Western-blot films are shown in Figure S5. Average ± SEM, n=6. ^†Significant difference with 21 Gy (p<0.05).

Figure 6. Analysis of collagen deposition at 6 months after irradiation.

Radiation caused increases in interstitial collagen, as measured by interstitial areas staining positive with Sirius Red. Interstitial collagen areas were not altered by PTX or TSB. Average ± SEM, n=8-9. *Significant difference with sham-irradiation (p<0.05).