Vascular endothelial growth factor mRNA and protein do not change in parallel during non-inflammatory skeletal muscle ischaemia in rat

Abstract

Impaired blood flow is thought to induce a pro-angiogenic environment due to local hypoxia, yet prolonged mild ischaemia induces only modest capillary growth. We compared the expression of vascular endothelial growth factor (VEGF) mRNA and protein with capillary to fibre ratio (C: F) and muscle blood flow in extensor digitorum longus of rats that had undergone unilateral ligation of the common iliac artery. Resting blood flow during the first two weeks after ligation (3, 7 and 14 days) was decreased by approximately 60% but recovered partially after 5 weeks (36% reduction). Functional hyperaemia (9-fold increase in blood flow during contractions) was eliminated in the first week after ligation, with a moderate recovery seen after 14 and 35 days. Muscle histology confirmed the absence of tissue necrosis or inflammation. Both VEGF mRNA (60%, P<0.05) and protein levels (700%, P<0.01) increased during the initial phase of ischaemia (at 1 and 3 days), well before any overt angiogenesis, and both declined towards control levels by 7 days. A secondary increase in VEGF protein (by 60% at 14 days, P<0.05) preceded the 20% increase in C: F seen after 5 weeks, but occurred while VEGF transcript levels continued to decline (to 50% of control at 35 days, P<0.05). Thus, evaluation of neither VEGF mRNA nor protein is an adequate index of angiogenic potential in response to ischaemia. We conclude that VEGF alone is insufficient to induce angiogenesis in ischaemic conditions, and that effective angiotherapy requires intervention aimed at other cytokines.

Figure 1. Effect of arterial ligation on VEGF expression by Northern analysis in ischaemic and non-ischaemic rat EDL versusβ-actin internal control for various times post-surgery (A), and quantification of VEGF expression normalized against non-ischaemic muscles (B)

Each bar represents mean ±s.e.m. (n = 6), *P < 0.05 versus control.

Figure 2. Effect of arterial ligation on VEGF protein levels by Western analysis in ischaemic and non-ischaemic rat EDL versusβ-actin internal control for various times post-surgery (A), and quantification of VEGF levels normalized against non-ischaemic muscles (B)

Each bar represents mean ±s.e.m. (n = 6), *P < 0.05 versus control.

Figure 3. Representative cryosections of EDL muscles stained for alkaline phosphatase and showing maintenance of tissue integrity

A and B, control, ligated 1 week, C and D, control, ligated 2 weeks; E and F, both ligated 5 weeks (scale bars = 50 μm).

Figure 4. A comparison of the time course of changes in VEGF mRNA and protein levels with that of VEGF-R2 levels and cell proliferation associated with capillaries

The schematic illustrates the essentially simultaneous increase in VEGF expression and production, subsequent increases in angiogenic receptor expression and proliferative angiogenesis, and finally the coincidence of increased capillarity with a second wave of VEGF protein and improved tissue oxygenation.

Figure 5. Blood flow in tibialis anterior and extensor digitorum longus (combined samples) in control muscles (n = 14), and 3 (n = 10), 7 (n = 10), 14 (n = 7) and 35 days (n = 7) after ligation, at rest and at the end of 5 min isometric contractions at 4 Hz

*P < 0.05 versus control.