A determination of the proteins secreted by micro-vessels in response to low doses of specific charged particles would be highly useful in determining the risks of space radiation in causing cardiovascular and other neurodegenerative pathologies during long-term space flight.

It would also provide a useful database of proteins that are potentially released from the endothelial cells of the human vasculature in response to space radiation. Such a database would be useful to other researchers investigating other specific degenerative disorders.

Mature micro-vessel tissue models lose structure after exposure to heavy ions detectible at a dose as low as 1.25cGy time (low LET (linear energy transfer) ions are not effective on the mature structures up to 4 Gy)). For developing micro-vessels -- angiogenesis, the growth of new vessels, is inhibited by light ions (3 KeV/n or below) and heavy ions (8 KeV/n or above) each detectible at a dose of 1.25cGy although the mechanisms of inhibition are distinct. Even more striking, the combined effect of each ion affecting the early stages of angiogenesis by light ions and later stages by heavy ions has a synergistic effect that is detectible as low as 0.6 cGy.

Hypothesis & Specific Aims

We hypothesized that low doses of space radiation directly target the endothelium causing the secretion of bioactive proteins eventually leading to increased risk to the health of the space crew during long-range missions.

Aim 1. Identify the proteins secreted by the endothelial cells in human 3D micro-vessel tissue models in response to space radiation. Carry out proteomics analysis and follow up with ELISA to determine the secreted proteins from developing micro-vessels after exposure to a mixture of low and high LET particle radiation.

Aim 1a. Identify the proteins secreted in response to gamma radiation. The timing of beam time requests at the NASA Space Radiation Laboratory (NSRL) dictates that an initial period without beam access is likely. This time will be used to examine gamma radiation for a reference radiation and for a crucial development of the assay before NSRL runs.

Aim 1b. Identify the Proteins secreted by mature micro-vessels in response to a low dose of high LET charged particles. To determine the proteins secreted by mature micro-vessels, which are the majority of micro-vessels in the body at any one time.

Aim 1c. Identify the Proteins secreted by developing micro-vessels in response to a low dose of a space -relevant mixture of low and high LET charged particles. To determine the proteins secreted by developing vessels that are still not connected.

Rationale: Human 3D microvessel models are much closer to microvessels in vivo than 2D monolayers. The fluence estimates are low enough to indicate that the effect is through a bystander response mediated by secreted bioactive proteins. A database of proteins secreted by endothelial cells is the research product of this proposal and is of practical use to the NASA Human Research Program (HRP) program for a number of reasons.

1) Create a resource for NASA researchers to examine the changes in secreted proteins important to various specific pathologies. 2) Form the basis of future databases that would include other associated cell types. 3) Provide information relevant to proteins secreted into the blood in vivo (mice and astronauts). 4) Provide mechanistic insights into our own research on the specific effects of radiation on mature vessels and developing vessels. 5) Provide research leads on the synergistic effects of mixed heavy and light ions.

By understanding the effect of space radiation on the secretion of bioactive proteins from the endothelium, we will gain the knowledge to understand potential pathologies and develop interventional strategies to block or repair the deleterious effects of space travel.

Preliminary data: Existing data indicates that the effect of different charged particles on human 3D microvessel models show that both mature and developing microvessels lose structure and function after exposure to very low doses of various charged particles. Data on developing particles is now published (Wuu et al. 2020). The low fluence of these doses indicates a bystander effect where the response is transmitted through secretion of factors by the target cell to other cells. In the case of endothelial cells this would include secretion into either the surrounding tissue or into the blood stream. Therefore, it is highly likely that low doses of charged particles induce the secretion of bioactive proteins from the microvasculature.

Changes to Specific Aims: After consultation with NASA, a change in the specific Aims was suggested. This mainly concerned substituting the high and low LET charged particles with the newly available simplified Galactic Cosmic Radiation simulation (sim GCR sim). The sim GCR sim is a mixture of ions, energy, and doses determined by a NASA consensus formula that consists of 5 ions: protons at 1000 MeV, 28Si at 600 MeV/n, 4He at 250 MeV/n, 16O at 350 MeV/n, 56Fe at 600 MeV/n, and protons at 250 MeV, in the following proportions: Protons 100 MeV at 34.8%, Si at 1.1%, He at 18%, O at 5.8%, 56Fe at 1%, and Protons 250 MeV at 39.3%. This mixture is the simplified version of the full GCR simulation and represents the proportions of ions found in space and thus translates to exploratory class missions. Thus, the simGCRsim supersedes individual ions and would be more relevant for use in this study. Due to budget limitations Gamma irradiated samples were eliminated from the study. Proteomic analysis was successful in both developing and mature microvessel cultures and the change in secreted proteins was not the same for each. Notably, protein disulfide-isomerases were differentially expressed in mature vasculature cultures but were unchanged in developing microvessels. Ongoing analysis should reveal more insights into the changes in protein revealed in this study. Data will be submitted to the NASA database.

Summary mature microvessels

§ Identifications were returned 1,687 proteins with a 1% false discovery rate (predicted error).

§ More than 200 secreted proteins were detected.

§ A variety of proteins were differentially expressed in 50 cGy radiation treated including Platelet endothelial cell adhesion molecule (known to be radiation induced), Protein disulfide-isomerase A1, A3, A4, A6, 40S ribosomal protein S8, Gamma-glutamyl hydrolase, Endoplasmin, Peptidyl-prolyl cis-trans isomerase B, HLA class I histocompatibility antigen, Cw-18 alpha chain, Prothymosin alpha, Vesicular integral-membrane protein VIP36.

§ Chaperone proteins and ribosomal proteins were enriched in the 50 cGy secretome.

Summary developing microvessels

§ Identifications were returned 1,291 proteins with a 1% false discovery rate (predicted error).

§ About 200 secreted proteins were detected.

§ A variety of proteins were differentially expressed in 50 cGy radiation treated including Neuroblast differentiation-associated protein AHNAK, ATP-citrate synthase, ATP-dependent 6-phosphofructokinase, platelet type, 40S and 60S ribosomal protein, Trifunctional purine biosynthetic protein adenosine-3, Alpha-centractin.

§ A variety of proteins were enriched in the 50 cGy secretome including translational protein, ribosomal protein, actin-binding cytoskeletal protein, chaperones, and vesicle coat proteins.

References (see also Bibliography section)

Yen-Ruh Wuu, Burong Hu, Hazeem Okunola, Amber M. Paul, Elizabeth A. Blaber, Margareth Cheng-Campbell, Afshin Beheshti* and Peter Grabham*. LET dependent low dose and synergistic inhibition of human angiogenesis by charged particles: Validation of miRNAs that drive inhibition. IScience 23, 22020.

The specific purpose of the research proposed here is to determine the proteins that are potentially released into the blood stream by the lining of the human vasculature in response to exposure to space radiation. This would create a useful database for radiobiology studies and comparisons with the proteins secreted in astronaut blood. Such proteins have the potential to cause pathological processes such as inflammation, they are also spread around the body in the blood, and are important factors in many pathologies. The microvasculature permeates all tissues at the microscopic level so the whole body is a target for charged particles. A single heavy ion particle would be expected to traverse many microvessels as it passes through the body causing a more widespread response. Studies on the effect of different charged particles on human 3D microvessel models shows that both developing and mature microvessels lose structure and function after exposure to very low doses of various charged particles. Mature microvessels lose structure detectible as low as 1.25 cGy. Angiogenesis, the growth of new vessels, is inhibited by light ions and heavy ions detectible at 1.25 cGy. Even more striking, the combined effect of each ion has a synergistic effect detectible as low as 0.6 cGy. The low fluence of these doses indicates a bystander effect where the response is transmitted to other cells and such a mechanism would involve the secretion of molecules by the target cell. We propose to use proteomics and other techniques to determine the proteins secreted by the human microvessel models. A database of these proteins secreted by human tissue models would not only be of great use to a number of researchers investigating a diverse number of pathologies related to space radiation but also provide insights into the mechanisms of the vascular response to charged particles.

Amended Aims—November 2019

Additional aims were included for the determination of relative biological effectiveness (RBEs) for the 5 ion galactic cosmic radiation (GCR) simulations.

Aim 1. Create RBEs for the vascular damage endpoints.

Aim 1a Carry out Gamma radiation studies at Brookhaven National Laboratory (BNL) for the determination of reference RBEs.

Aim 1b Carry out charged particle studies at BNL for the determination of RBEs.

Aim 2. Identify the proteins secreted by the endothelial cells during angiogenesis and in mature human 3 D micro-vessel tissue models in response to radiation.

Aim 2a Development of the proteomics assay.

Aim 2b Identify the proteins secreted by the endothelial cells during angiogenesis and in mature human 3 D micro-vessel tissue models in response to gamma radiation.

Aim 2c Identify the proteins secreted by the endothelial cells during angiogenesis and in mature human 3 D micro-vessel tissue models in response to the Simplified GCR sim.

1) By the summer of 2019 Aims 1a and b had been partially carried out for Mature vessels.

2) Aim 1a and 1b for developing microvessels (angiogenesis) were not yet completed. The new protocol requested by NASA that entails remaining at Brookhaven for an extra day does not appear to be compatible with microvessel growth. The reasons for this are unknown but we continued to trouble shoot the assay through the summer.

3) By the Fall run (19C – the last possible run for the current time limit) culture problems had been resolved. In the 2 visits for this run we have completed one dose response for angiogenesis in addition to proteomics samples and the samples are now being processed.

4) Work on the sample preparation for proteomics studies has been carried out and, as expected, a scale up of the cultures was necessary. A larger gel matrix proved to be less stable and detached from the flasks.

5) A goal of 5 samples for each condition (20 total) was set by the Proteomics expert CoInvestigator Dr. Brown. These goals were reached by the end of the Fall (19C) run. Samples will be processed and delivered to Dr. Brown.