Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA's globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique "fingerprint" or "biomarker" vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Characterization of Epstein-Barr virus reactivation in a modeled spaceflight system.

Epstein-Barr virus (EBV) is the causative agent of mononucleosis and is also associated with several malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and nasopharyngeal carcinoma, among others. EBV reactivates during spaceflight, with EBV shedding in saliva increasing to levels ten times those observed pre-and post-flight. Although stress has been shown to increase reactivation of EBV, other factors such as radiation and microgravity have been hypothesized to contribute to reactivation in space. We used a modeled spaceflight environment to evaluate the influence of radiation and microgravity on EBV reactivation. BJAB (EBV-negative) and Raji (EBV-positive) cell lines were assessed for viability/apoptosis, viral antigen and reactive oxygen species expression, and DNA damage and repair. EBV-infected cells did not experience decreased viability and increased apoptosis due to modeled spaceflight, whereas an EBV-negative cell line did, suggesting that EBV infection provided protection against apoptosis and cell death. Radiation was the major contributor to EBV ZEBRA upregulation. Combining modeled microgravity and radiation increased DNA damage and reactive oxygen species while modeled microgravity alone decreased DNA repair in Raji cells. Additionally, EBV-infected cells had increased DNA damage compared to EBV-negative cells. Since EBV-infected cells do not undergo apoptosis as readily as uninfected cells, it is possible that virus-infected cells in EBV seropositive individuals may have an increased risk to accumulate DNA damage during spaceflight. More studies are warranted to investigate this possibility.

Retinal non-visual photoreception in space.

BACKGROUND: Circadian rhythm disruption occurs during spaceflight, leading to crew health and performance decrements. Spaceflight-related retinal changes, including oxidative stress and neuronal loss, have been previously reported in mice. METHODS: Animal tissue from experiments aboard shuttle missions STS-133 (BALB/cJ mice, albino strain) and STS-135 (C57BL mice, pigmented strain), along with ground controls, was examined to determine survival of intrinsically photosensitive retinal ganglion cells (ipRGC) and melanopsin expression in retinas of mice exposed to the spaceflight environment. Real-time qPCR (RTqPCT) and microarray approaches were used to analyze Opn4 (melanopsin) gene expression, while immunohistologic studies were conducted to detect melanopsin localization in the retina. RESULTS: Opn4 expression was decreased in albino BALB/cJ mice exposed to spaceflight, as measured by RTqPCR, but not in C57BL mice samples as analyzed by microarray. Opn4 expression returned to control levels at 7 d postreturn in the BALB/cJ samples. Melanopsin positive RGCs were found in the expected proportion in all samples, except for the BALB/cJ samples at 1 d after flight, where virtually no immunoreactive cells were found. DISCUSSION: Spaceflight environmental factors may affect the nonvisual function of the retina, mediated by a reduction in melanopsin expression and ipRGC survival, contributing to circadian disruption.

Opposite response of blood vessels in the retina to 6° head-down tilt and long-duration microgravity.

The Spaceflight Associated Neuro-ocular Syndrome (SANS), associated with the headward fluid shifts incurred in microgravity during long-duration missions, remains a high-priority health and performance risk for human space exploration. To help characterize the pathophysiology of SANS, NASA's VESsel GENeration Analysis (VESGEN) software was used to map and quantify vascular adaptations in the retina before and after 70 days of bed rest at 6-degree Head-Down Tilt (HDT), a well-studied microgravity analog. Results were compared to the retinal vascular response of astronauts following 6-month missions to the International Space Station (ISS). By mixed effects modeling, the trends of vascular response were opposite. Vascular density decreased significantly in the 16 retinas of eight astronauts and in contrast, increased slightly in the ten retinas of five subjects after HDT (although with limited significance). The one astronaut retina diagnosed with SANS displayed the greatest vascular loss. Results suggest that microgravity is a major variable in the retinal mediation of fluid shifts that is not reproduced in this HDT bed rest model.

Dendro[C(60)]fullerene DF-1 provides radioprotection to radiosensitive mammalian cells.

In this study, the ability of the C(60) fullerene derivative DF-1 to protect radiosensitive cells from the effects of high doses of gamma irradiation was examined. Earlier reports of DF-1's lack of toxicity in these cells were confirmed, and DF-1 was also observed to protect both human lymphocytes and rat intestinal crypt cells against radiation-induced cell death. We determined that DF-1 protected both cell types against radiation-induced DNA damage, as measured by inhibition of micronucleus formation. DF-1 also reduced the levels of reactive oxygen species in the crypt cells, a unique capability of fullerenes because of their enhanced reactivity toward electron-rich species. The ability of DF-1 to protect against the cytotoxic effects of radiation was comparable to that of amifostine, another ROS-scavenging radioprotector. Interestingly, localization of fluorescently labeled DF-1 in fibroblast was observed throughout the cell. Taken together, these results suggest that DF-1 provides powerful protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, and cell death.

The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase.

The checkpoint protein Rad9/Rad1/Hus1 heterotrimer (the 9-1-1 complex) is structurally similar to the proliferating cell nuclear antigen sliding clamp and has been proposed to sense DNA damage that leads to cell cycle arrest or apoptosis. Human (h) NEIL1 DNA glycosylase, an ortholog of bacterial Nei/Fpg, is involved in repairing oxidatively damaged DNA bases. In this study, we show that hNEIL1 interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. Residues 290-350 of hNEIL1 are important for the 9-1-1 association. A significant fraction of the hNEIL1 nuclear foci co-localize with hRad9 foci in hydrogen peroxide treated cells. Human NEIL1 DNA glycosylase activity is significantly stimulated by hHus1, hRad1, hRad9 separately and the 9-1-1 complex. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of base excision repair.

Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1).

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8-13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.

High dietary iron increases oxidative stress and radiosensitivity in the rat retina and vasculature after exposure to fractionated gamma radiation.

Radiation exposure in combination with other space environmental factors including microgravity, nutritional status, and deconditioning is a concern for long-duration space exploration missions. Astronauts experience altered iron homeostasis due to adaptations to microgravity and an iron-rich food system. Iron intake reaches three to six times the recommended daily allowance due to the use of fortified foods on the International Space Station. Iron is associated with certain optic neuropathies and can potentiate oxidative stress. This study examined the response of eye and vascular tissue to gamma radiation exposure (3 Gy fractionated at 37.5 cGy per day every other day for 8 fractions) in rats fed an adequate-iron diet or a high-iron diet. Twelve-week-old Sprague-Dawley rats were assigned to one of four experimental groups: adequate-iron diet/no radiation (CON), high-iron diet/no radiation (IRON), adequate-iron diet/radiation (RAD), and high-iron diet/radiation (IRON+RAD). Animals were maintained on the corresponding iron diet for 2 weeks before radiation exposure. As previously published, the high-iron diet resulted in elevated blood and liver iron levels. Dietary iron overload altered the radiation response observed in serum analytes, as evidenced by a significant increase in catalase levels and smaller decrease in glutathione peroxidase and total antioxidant capacity levels. 8-OHdG immunostaining, showed increased intensity in the retina after radiation exposure. Gene expression profiles of retinal and aortic vascular samples suggested an interaction between the response to radiation and high dietary iron. This study suggests that the combination of gamma radiation and high dietary iron has deleterious effects on retinal and vascular health and physiology.

Cell killing and radiosensitizing effects of atorvastatin in PC3 prostate cancer cells.

Recent studies have indicated that autophagy may be one of the important pathways induced by ionizing radiation. Atorvastatin (statin), an inhibitor of 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase, may exhibit anticancer effects as an autophagy inducer. In our study, the cell killing and radiosensitizing effects of statin were analyzed in PC3 cell line. Activation of the autophagy pathway was analyzed using the GFP-LC3 assay and western blot to determine LC3-II expression. The radiosensitivity of PC3 cells was determined using the clonal survival assay, TUNEL assay, and the Annexin V apoptosis assay. The expression profiles of autophagy related genes were analyzed using a pathway specific real-time polymerase chain reaction (PCR) array. Autophagic response was induced in PC3 cells after exposure to statin and/or gamma rays. Inhibition of the autophagic process using small interfering RNAs (siRNA) targeting Atg7 and/or Atg12 significantly reduced radiosensitivity of PC3 cells. Statin also exhibited a significant apoptosis-inducing effect in PC3 cells, which can be partially suppressed by Atg7 siRNA. Cells treated with statin and gamma irradiation showed significantly reduced colony forming efficiency and increased number of Annexin V positive early apoptotic cells. Analysis of autophagy and its regulatory gene profile showed that the expressions of 22 genes out of 86 genes assessed were significantly altered in the cells exposed to combined treatment or statin alone. The data indicate that activation of the autophagy pathway may be responsible for apoptosis inducing effect of statin. Furthermore, combined treatment with radiation and autophagic inducer, such as statin, may be synergistic in inducing cell death of PC3 cells.

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.

Interaction of the human DNA glycosylase NEIL1 with proliferating cell nuclear antigen. The potential for replication-associated repair of oxidized bases in mammalian genomes.

NEIL1 and NEIL2 compose a family of DNA glycosylases that is distinct from that of the other two DNA glycosylases, OGG1 and NTH1, all of which are involved in repair of oxidized bases in mammalian genomes. That the NEIL proteins, unlike OGG1 and NTH1, are able to excise base lesions from single-stranded DNA regions suggests their preferential involvement in repair during replication and/or transcription. Previous studies showing S phase-specific activation of NEIL1, but not NEIL2, suggested NEIL1 involvement in the repair of replicating DNA. Here, we show that human NEIL1 stably interacts both in vivo and in vitro with proliferating cell nuclear antigen (PCNA), the sliding clamp for DNA replication. PCNA stimulates NEIL1 activity in excising the oxidized base 5-hydroxyuracil from single-stranded DNA sequences including fork structures. PCNA enhances NEIL1 loading on the substrate. In contrast, although present in the NEIL2 immunocomplex, PCNA does not stimulate NEIL2. NEIL1 interacts with PCNA via a domain that is located in a region near the C terminus, dispensable for base excision activity. The interacting sequence in NEIL1, which lacks the canonical PCNA-binding motif, includes a sequence conserved in DNA polymerase delta and implicated in its PCNA binding. Mammalian two-hybrid analysis confirmed PCNA interaction with NEIL1. The G127A mutation in PCNA reduces its stimulatory activity, suggesting that the interdomain connector loop, a common binding interface of PCNA, is involved in NEIL1 binding. These results strongly support in vivo function of NEIL1 in preferential repair of oxidized bases in DNA prior to replication.

Physical and functional interaction between human oxidized base-specific DNA glycosylase NEIL1 and flap endonuclease 1.

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.