FGF–1 and FGF–2

The fibroblast growth factor (FGF) family and the mediating receptors have extensively been studied (for reviews see ^124-126). FGF–1 and FGF–2 were shown to induce angiogenesis in vitro and in vivo ^127,128.

In contrast to VEGF expression, the response of FGF–1 and/or FGF–2 expression to physical activity remains to be determined. It was found that chronic electrical stimulation of rat skeletal muscle for five days leads to elevated mRNA levels for FGF–1, FGF–2, FGF receptor 1 (FGFR1), and FGF receptor 4 (FGFR4) ¹²⁹. After longer stimulation FGF–1 and FGFR4 mRNAs decreased to normal levels, whereas FGFR1 mRNA returned to normal levels after ten days but increased again after 20 days of stimulation. FGF–2 expression remained elevated over the whole experimental period. When satellite cell cultures were stimulated FGF–1 and FGF–2 expressions also increased but to a lesser extend in comparison to intact skeletal muscle. FGF–1 and FGF–2 proteins increased in electrically stimulated rabbit skeletal muscle. These changes were concomitant with increased mitotic activity, fast to slow skeletal muscle fiber conversion and increased capillarity ¹³⁰.

A short-term exercise program of rats did not cause increased FGF–2 expression ¹¹⁷ when compared to resting levels. A single bout of submaximal exercise also didn’t lead to a response of FGF–2 expression ¹²³. There was no FGF–2 response to a single exercise bout in rats under normoxic conditions or

after a chronic hypoxia period of eight weeks ¹¹⁵. However, it was shown earlier by the same group that the same exercise stimulus leads to increased FGF–2 expression in the rat ¹⁰⁹. In humans, these investigators reported no measurable FGF–2 expression after a single endurance exercise bout ¹¹⁰. Similarly, Gustafson and coworkers did not observe increased FGF–2 mRNA after a single exercise bout in the human ¹¹¹.

A short-term exercise program in rats with bilateral femoral artery occlusion led to increased capillarity in the gastrocnemius muscle when compared to sedentary control rats ¹³¹. The increase in capillarity was caused by endothelial cell proliferation. However, FGF–2 content of the gastrocnemius muscle was not altered by exercise, suggesting that other endothelial cell mitogens are responsible for the observed changes.

Overload of a particular skeletal muscle by removal of the synergist has been reported to cause increased capillary-to-fiber ratio ¹³². This change was not associated by an increase in FGF–2 expression. No FGF–2 immunoreactivity was observed in capillaries, whereas it was present in larger vessels and nerves.

There was no difference in maximal muscle blood flow between overloaded muscle and muscle of unoperated control rats, suggesting that neither FGF–2 nor increased blood flow are responsible for the increase in capillarity.

The role of FGFs in exercise induced angiogenesis remains to be clarified.

Electrical stimulation of rat skeletal muscle appears to induce expression of FGF–1 and FGF–2 mRNAs and to increase their protein levels. However, most of the endurance exercise studies in both rats and humans did not lead to a significant response of FGF expression. It has to be mentioned that most of these investigations concentrated on the expression levels while paying no attention to the protein levels.

It has been questioned whether FGF–1 and FGF–2 are secreted via the endoplasmic reticulum since they lack the conventional leader sequences.

Cellular damage and stretching as well as a contraction based mechanism have been suggested to cause the release of FGFs from cells in vivo ^133-137. Thus, a possible influence of physical activity on the release of intracellular FGFs can not be excluded and further investigations are needed.

Recently, Maciag and colleagues started to resolve the puzzle of FGF–1 release.

The release of FGF–1 may proceed through a novel release/export pathway.

They found that FGF–1, but not FGF–2, is released as a latent homodimer by a transcription- and translation-dependent mechanism in response to a variety of

cellular stresses including heat shock ¹³⁸, hypoxia ¹³⁹, and serum starvation ¹⁴⁰. The disruption of communication between the endoplasmic reticulum and Golgi apparatus by brefeldin A does not prevent the release of FGF–1 from 3T3 cells ¹³⁸, which confirms that FGF–1 release may occur through a nonconventional pathway. FGF–1 is released in vitro complex sensitive to reducing agents and denaturants. The complex contains the p40 extravesicular domain of the calcium-binding protein p65 synaptotagmin 1 ¹⁴¹. The expression of synaptotagmin 1 is essential for the release of FGF–1 in response to stress ¹⁴². Furthermore, it was demonstrated that FGF–1 isolated from tissues as a high molecular weight aggregate exists as a component of a noncovalent heparin-binding complex with p40 synaptotagmin 1 and S100A13, a member of the S100 gene family of calcium-binding proteins ¹⁴³. The precursor form of interleukin 1α was shown to block the release of FGF–1 suggesting that their release pathways may be mechanistically linked ¹⁴⁴. The anti-allergic drug amlexanox, which binds S100A13, is able to inhibit the release of FGF–1 and p40 synaptotagmin 1 in response to temperature stress ¹⁴⁵. Amlexanox also causes the reversible disassembly of actin stress fibers indicating that the actin cytoskeleton also plays a role in the regulation of FGF–1 release. The expression of S100A13 facilitates the release of FGF–1 into the extracellular compartment in response to temperature stress in vitro ¹⁴⁶. Interestingly, the expression of S100A13 was demonstrated to reverse the sensitivity of FGF–1 release to inhibitors of transcription and translation ¹⁴⁶. S100A13 may also induce the formation of a noncovalent FGF–1 homodimer, which is essential for FGF–1 release ¹⁴⁶. The most recent study of Maciag and colleagues showed that copper induces the formation of a multiprotein aggregate between S100A13, FGF–1, and p40 synaptotagmin 1 ¹⁴⁷. When copper was bound by a copper chelator, the heat shock-induced release of FGF–1 and S100A13 was repressed in a dose-dependent manner. However, the mechanism by which the multiprotein complex is finally released to the extracellular space remains to be determined. Maciag and colleagues anticipate that phosphatidylserine flipping from the inner leaflet to the outer leaflet of the plasma membrane might be involved in this mechanism since S100A13, FGF–1, and p40 synaptotagmin 1 are phosphatidylserine-binding proteins ¹⁴⁶.

Since FGF receptors are not restricted to endothelial cells and biological effects of FGFs on other cell types have been shown, skeletal muscle adaptations in response to physical activity beyond angiogenesis remain to be determined.