We have shown here that low LET protons and high LET Fe ions inhibit the formation of model human brain capillaries by different mechanisms. In the case of protons, the inhibition involves the regulation of PKC-dependent motile tips leading to a failure of cellular processes to migrate through the matrix, form guidance tunnels, and meet up with other cell processes. In the case of Fe ions, inhibition does not involve the blockage of motile tip activity since these structures are not affected and cellular processes succeed in making guidance tunnels and connections. Instead, the cells fail to form widened tunnels in the matrix and lumen-containing tubular structures at the later stages of vasculogenesis.
An examination of the final vessel morphology, cytoskeletal arrangements, and matrix architecture, together with rescue of the motile tip phenotype by PMA, have efficiently distinguished between the inhibition of early and later stages of vasculogenesis by particles of different LET’s.
The notion that low LET protons inhibited the motility of tip cells penetrating the matrix is confirmed by the changes in the actin and microtubule skeletons, which play a major role in the formation of angiogenic sprouts reviewed in . Unirradiated controls showed motile actin structures (filopodia) and bundled microtubules whereas proton-irradiated cells lost these features. The distal tip no longer had the characteristics ideally suited to cells that penetrate and grow through other tissues, that of spear-shaped and streamlined protrusions containing bundled microtubules. To our knowledge, this is the first report of bundled microtubules in motile tip cells although they are remarkably similar to the bundled microtubules in other cells that grow through tissue, such as rapidly growing axonal growth cones  and cancer cells making an epithelial/mesenchyme transition . Cells exposed to Fe ions displayed motile tip features and were able to form a network although later stages were inhibited.
Inhibition of tip cell activity is confirmed by visualization of the collagen matrix. A fundamental mechanism in vessel tube formation is the matrix type 1-metalloproteinase dependent creation of a network of guidance tunnels, which serve as conduits for later events of endothelial cell migration and tube remodeling . SHG microscopy has been used to show that only the wider mature vessels have increases in collagen density around the perimeter of tunnels . The matrix protein in these areas is anisotropically altered suggesting that collagen was displaced or compacted during tube and lumen formation. Our observations with SHG show that high LET Fe ions halt vessel development after guidance tunnel formation but before tubulogenesis. Narrow guidance tunnels with or without cell processes were evident while wider collagen lined tunnels were absent. Low LET protons inhibit vessel formation during guidance tunnel stage but then tubulogenesis continues even though there is a much reduced guidance tunnel network. Narrow guidance tunnels were absent while wider collagen lined tunnels were present.
The difference between the two types of inhibition was further confirmed by the selective rescue of vessel phenotype, and of tip motility by the use of PMA to stimulate PKC immediately before irradiation. Protein Kinase C isomers of different types are known to be second messengers involved in angiogenesis (reviewed in 15) and studies have implicated Protein Kinase C in vessel formation and the effects of radiation [21, 22]. However, further studies are required to show that inhibition of PKC is the cause of the effect of protons. Although the use of PMA here does not give any new information on the role of specific PKC’s in vasculogenesis, it has proved useful for distinguishing between inhibition of the early stages by low LET ions and the later stages by high LET ions. It also reveals that the effect is transient. Development of vessels is resumed after further culture (including PMA treatment) since wider tubes with lumens are eventually formed even though the extent of the network has been limited.
Although most studies on radiation and the vasculature have been carried out using sources that produce low LET electrons (gamma photons and X-rays), a comparison with studies on low LET particles reviewed in  show that several biological responses of protons including angiogenesis, are different or even opposite. Our own observations show that protons are at least 8 times more effective than gamma rays at inhibiting vasculogenesis . Furthermore, protons have been shown to down-regulate the expression of pro-angiogenic factors like VEGF, in addition to invasion, in endothelial cells and fibroblasts . This is one possible mechanism whereby protons could be inhibiting vasculogenesis in the present study. However, the mechanistic basis for the difference in low LET proton response versus low LET electron response remains a puzzle.
The effect of high LET Fe ions was more insidious and longer lasting. The initial motility during the first 24 hours after irradiation appeared to be unaffected while later development was inhibited. Therefore, the adverse effects of these heavy ions lasted much longer than those of the low LET protons. Although there are relatively few studies on the effects of high LET ion particles on vessel formation, they support the notion that these particles inhibit vessel formation. The effect of 290 MeV carbon ions (LET 110 KeV/μm) was examined on developing vessel models  indicating sensitivity to heavy ions, with a low dose (10 cGy) of carbon ions inhibiting vessel formation in addition to cell migration. In vivo, the effects of Fe particles on mouse hippocampal microvessels was examined , and it was found that a dose as low as 50 cGy resulted in loss of endothelial cells 1 year after irradiation. The mechanism for heavy ion inhibition is also not well known. A tip cell with average morphology exposed to a dose of 75 cGy is estimated to get approximately 42 Fe ion particle traversals compared to 25000 traversals by high LET protons. For protons, the energy deposition is spread over the cell more evenly and this may facilitate global effects on cellular signaling. For Fe ions, a few sites receive much more concentrated, and therefore locally damaging, energy depositions. Apoptosis is unlikely since we have previously shown that doses of Fe ions greater than 1 Gy and doses of protons greater than 2 Gy are necessary to induce apoptosis as detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay . One possible mechanism is the downstream effects of DNA damage. Heavy ions are known to cause complex DNA damage , which is persistent in these HUVEC 3-D cultures . Other possibilities are, mitochondrial damage and apoptosis signaling that occurs prior to DNA fragmentation.
In the space environment, humans will be exposed to a variety of ion particles with a range of LET’s.. Recently, measurements of energetic particle radiation were made on the Mars Science Laboratory spacecraft, containing the Curiosity rover. For a short round trip (360 days) the total dose of heavy particles was found to be 17.2 cGy and a dose equivalent of 662 mSieverts, with additional variable contributions from solar particle events . These daily doses are lower than those that will inhibit vasculogenesis, although the existence of reduced vasculature in mice one year after exposure , suggests that damage might be accumulative.
Solar particle event (SPE) dose-rates, can vary between 0 and 100 mGy/h in a spacecraft or up to 500 mGy/h for an astronaut exposed outside the vehicle in deep space or on the Moon’s surface . In this case, doses high enough to inhibit vasculogenesis could be achieved even in deeper tissues like bone marrow . SPE’s contain protons of mixed energy and therefore mixed LET’s, some, could be low enough to inhibit the early stages of angiogenesis and others high enough to inhibit the later stages. We are currently investigating the LET ranges for each type of inhibition to determine the contribution of each type of radiation in the space environment. The existence of distinct mechanisms of the inhibition of vasculogenesis according to LET, raises the possibility that normal angiogenic repair could be inhibited by two different species of particles, and that these effects could be additive or even synergistic. Furthermore, if the radiation also damages the endothelial barrier, thereby creating the need for more angiogenic repair, the harmful effects of space radiation to the vasculature could be further compounded.
For particle radiotherapy, the inhibition of tumor vasculature would be desirable. The results shown here raise the possibility that mixed particle therapies with defined LET ranges might target different stages of angiogenesis and therefore be more effective at inhibiting tumor vessel growth. Also, a combination of specific anti-angiogenic drugs and particle radiation of specific LETs could efficiently target selected stages of angogenesis.