Two distinct types of the inhibition of vasculogenesis by different species of charged particles

Abstract

Background: Charged particle radiation is known to be more biologically effective than photon radiation. One example of this is the inhibition of the formation of human blood vessels. This effect is an important factor influencing human health and is relevant to space travel as well as to cancer radiotherapy. We have previously shown that ion particles with a high energy deposition, or linear energy transfer (LET) are more than four times more effective at disrupting mature vessel tissue models than particles with a lower LET. For vasculogenesis however, the relative biological effectiveness between particles is the same. This unexpected result prompted us to investigate whether the inhibition of vasculogenesis was occurring by distinct mechanisms.

Methods: Using 3-Dimensional human vessel models, we developed assays that determine at what stage angiogenesis is inhibited. Vessel morphology, the presence of motile tip structures, and changes in the matrix architecture were assessed. To confirm that the mechanisms are distinct, stimulation of Protein Kinase C (PKC) with phorbol ester (PMA) was employed to selectively restore vessel formation in cultures where early motile tip activity was inhibited.

Results: Endothelial cells in 3-D culture exposed to low LET protons failed to make connections with other cells but eventually developed a central lumen. Conversely, cells exposed to high LET Fe charged particles extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons indicated that motility at the extending tips of endothelial cells is inhibited by low LET but not high LET particles. Actin-rich protrusive structures that contain bundled microtubules showed a 65% decrease when exposed to low LET particles but not high LET particles, with commensurate changes in the matrix architecture. Stimulation of PKC with PMA restored tip motility and capillary formation in low but not high LET particle treated cultures.

Conclusion: Low LET charged particles inhibit the early stages of vasculogenesis when tip cells have motile protrusive structures and are creating pioneer guidance tunnels through the matrix. High LET charged particles do not affect the early stages of vasculogenesis but they do affect the later stages when the endothelial cells migrate to form tubes.

Figure 1

Exposure to protons and Fe ions results in distinct morphologies of mature 3-Dimensional vessel models. 24 hours after either HUVEC or HBMEC were seeded into matrices they were exposed to 1Gy of each type of particle radiation and then cultured for a further 5 days until vessel structures had formed. Fixed cultures were stained for all protein material (DTAF – green) and nuclei (Propidium Iodide – red and imaging as yellow). Images are 10 slices 2 μm apart projected onto a single plane. A. Control HUVEC culture shows vessels with lumens that have formed a connecting network. B. HUVEC cultures exposed to 1 Gy Fe ions formed a network but vessels are often thinner without lumens (arrow). C. HUVEC cultures exposed to 1 Gy protons fail to form a network and vessels terminate in a dead end (arrow) Bar = 100 μm. D-F. HBMEC at a higher magnification. D. Control cultures form a network. E. cultures exposed to 1 Gy Fe ions formed a network but vessels are often thinner without lumens (arrow). F. Cultures exposed to 1 Gy protons fail to form a network and vessels terminate in a dead end (arrow). G. Quantitation based on length of vessel per cell shows a similar overall effect. Bar = 50 μm.

Figure 2

High-energy protons but not high-energy Fe ions reduce the frequency of specialized motile tips of endothelial cells extending into the matrix. A and B low magnification 3-D images (projected onto a single plane) of HBMEC 24 hours after plating and 2 hours after, sham exposure A and exposure to 1Gy of protons B. Cultures are stained for microtubules (green) and actin (red). Cells appear similar although microtubules are more concentrated in un-irradiated cultures. Bar = 25 μm. C and D Higher magnification in controls C reveals motile tips - streamlined cellular processes with motile actin structures such as filopodia (arrowhead) and bundled microtubules (arrow). In cultures exposed to 1 Gy of protons D cellular processes are less streamlined with unbundled microtubules (arrowhead). In cultures exposed to Fe ions E cellular processes contain bundled microtubules (arrow) as in the controls. Bar = 10 μm. F. Line scans measuring pixel brightness (grey value) along the horizontal lines shown in C-E demonstrate the determination of bundled microtubules (Materials and Methods), a single peak denotes bundled microtubules whereas multiple peaks seen after proton exposure reveals unbundled microtubules. G. Quantitation of processes with bundled microtubules shows that motile tips are inhibited 4-fold by protons but not by 1Gy of Fe ions.

Figure 3

Distinct modifications of the matrix architecture. 24 hours after plating 3- Dimensional cultures of HBMEC were either unexposed A or exposed to 1Gy Fe ions B and 1 Gy protons C, then cultured for a further 5 days before fixation. Two-photon microscopy was used to visualize cell material stained with DTAF and collagen was visualized by Second Harmonic Generation microscopy (as shown). Control cultures have branched vessels with compressed bright staining collagen deposits surrounding them. Cultures exposed to Fe ions B display narrow guidance tunnels (arrow) surrounding narrow processes. Cultures exposed to protons C display shorter widened tunnels lined with compressed bright staining collagen deposits (Arrowhead). Bar = 50 μm.

Figure 4

Rescue of vessel formation at 5 days in endothelial cultures treated with high-energy protons and Fe ions. HBMEC were allowed to begin vessel development for 24 hours. Cultures were then treated with PMA 15 minutes before irradiation. After maturation 5 days later, cultures were fixed and stained for all proteins (green) and for nuclei (red). A. Un-irradiated controls show robust vessel formation. B. 1 Gy Protons reduce vessel formation. Cellular processes extended short distances into the gel matrix and terminated in a dead end (arrow). C. 1 Gy protons and 30nM PMA, vessel growth is restored. D. Fe ions and 30nM PMA vessel growth is not restored. Cellular processes make connections but remain thin without lumens (arrow) similar to Fe ions without PMA (Figure 1). Bar = 50 μm. E. Quantitation of vessels under all conditions (length of vessel with lumen per cell). PMA rescues vessel formation inhibited by protons but not Fe ions.

Figure 5

Rescue of motile tips in endothelial cultures 2h post irradiation with high-energy protons and Fe ions. HBMEC were allowed to begin vessel development for 24 hours. Cultures were then treated with PMA 15 minutes before irradiation. After 2 hours, cultures were fixed and stained for microtubules (green) and actin (red). In the extending cellular processes of control cultures A, F-actin was localized more closely to the microtubules and also present as filopodia (arrowhead). Microtubules were bundled (arrow). In the extending cellular processes of cultures irradiated with 1 Gy protons B, actin filaments showed fewer motile structures such as filopodia and a tendency to have spread away from the microtubules (arrowhead). microtubules were unbundled (arrow). Cultures treated with 1Gy Fe ions C resembled control cultures with bundled microtubules (arrow). D In the extending cellular processes of cultures treated with 1 Gy protons in the presence of 30 nM PMA, microtubules were bundled (arrow) PMA restored the motile tip morphology. Bar = 25 μm. E. Quantitation of motile tips under all conditions (Materials and Methods). PMA rescues motile tips formation inhibited by protons. Motile tips are not inhibited by Fe ions.