Effects of hypoxia on angiogenesis and angiogenic factors in crucian carp brain

Abstract

Most vertebrates depend on an uninterrupted supply of oxygen to maintain the energy production. Some species, like the crucian carp (Carassius carassius) can survive hypoxia for an extended period. This fish is an expert in acquiring the little oxygen that exists in hypoxic water, and can boost its anaerobic ATP production by up-regulating the glycolysis and convert lactate to ethanol. Angiogenesis, the formation of new blood vessels, is induced by hypoxia and controlled through several factors. One of the most important hypoxia driven transcription factors, the hypoxia-inducible factor (HIF)-a, has been found to play a major role in coordinating many adaptive responses to hypoxia. It is continuously synthesized and degraded in normoxic conditions, but accumulates at low oxygen tension. Several factors control the expression of HIF-a, among them the factor-inhibiting HIF-1 (FIH-1). One mechanism by which HIF-a mediates increased oxygen delivery is through inducing the expression of vascular endothelial growth factor (VEGF), which in turn stimulates the formation of blood vessels. In this thesis mRNA levels of HIF-1a, HIF-2a, FIH, VEGFA, VEGFC and VEGFD were quantified by real-time RT-PCR in brain tissue from the crucian carp exposed to hypoxia. The effect of hypoxia on vascular density in crucian carp brain was also studied. A significant increase in mRNA levels was seen in both VEGFA and VEGFC, while no change was seen in VEGFD. VEGFA increased 6-fold and constituted the largest part of the quantified VEGF expression. Different splice isoforms of VEGFA have previously been characterized in mammals and fish, and two of these splice isoforms were cloned in the crucian carp, VEGFA121 and VEGFA165. A trend towards a reduction in VEGFD expression was observed in hypoxia, which may indicate that it does not have the same function in angiogenesis in crucian carp as VEGFA and VEGFC. The expression level of FIH increased 4-fold in hypoxia. No changes in mRNA levels were seen in either HIF-1a or HIF-2a, but a trend towards reduction was seen in HIF-1a. The increase seen in VEGFA and FIH indicates that the fishes have been hypoxic. This might imply that the expression of HIF-a also increases, but in order to verify this protein analyses would have to be performed to verify this.

Regarding blood vessel density two parameteres were measured, blood vessel surface area per unit volume and blood vessel length per unit volume. A significant decrease was seen in both of these parameters. The increase in VEGFA expression along with a reduction in blood vessel density can indicate that the hypoxic crucian carp does not require additional blood vessels during hypoxia, but instead suppresses global HIF-a effects, which may work well for local regulation of vascularization, but not for adapting the whole organism to hypoxia.