Gene Expression Changes in a Prefinal Health Stage of Lethally Irradiated Male and Female Rhesus Macaques.

Radiation-induced gene expression (GE) changes can be used for early and high-throughput biodosimetry within the first three days postirradiation. However, is the method applicable in situations such as the Alexander Litvinenko case or the Goiania accident, where diagnosis occurred in a prefinal health stage? We aimed to characterize gene expression changes in a prefinal health stage of lethally irradiated male and female rhesus macaques. Peripheral blood was drawn pre-exposure and at the prefinal stage of male and female animals, which did not survive whole-body exposure with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study comprising altogether 142 irradiated rhesus macaques of whom 60 animals and blood samples (15 samples for both time points and sexes) were used for this analysis. We evaluated GE on 34 genes widely used in biodosimetry and prediction of the hematological acute radiation syndrome severity (H-ARS) employing quantitative real-time polymerase chain reaction (qRT-PCR). These genes were run in duplicate and triplicate and altogether 96 measurements per time point and sex could be performed. In addition, 18S ribosomal RNA (rRNA) was measured to depict the ribosome/transcriptome status as well as for normalization purposes and 16S rRNA was evaluated as a surrogate for bacteremia. Mean differential gene expression (DGE) was calculated for each gene and sex including all replicate measurements and using pre-exposure samples as the reference. From 34 genes, altogether 27 genes appeared expressed. Pre-exposure samples revealed no signs of bacteremia and 18S rRNA GE was in the normal range in all 30 samples. Regarding prefinal samples, 46.7% and 40% of animals appeared infected in females and males, respectively, and for almost all males this was associated with out of normal range 18S rRNA values. The total number of detectable GE measurements was sixfold (females) and 15-fold (males) reduced in prefinal relative to pre-exposure samples and about tenfold lower in 80% of prefinal compared to pre-exposure samples (P < 0.0001). An overall 11-fold (median) downregulation in prefinal compared to pre-exposure samples was identified for most of the 27 genes and even FDXR appeared 4-14-fold downregulated in contrast to a pronounced up-regulation according to cited work. This pattern of overall downregulation of almost all genes and the rapid reduction of detectable genes at a prefinal stage was found in uninfected animals with normal range 18S rRNA as well. In conclusion, in a prefinal stage after lethal radiation exposure, the ribosome/transcriptome status remains present (based on normal range 18S rRNA values) in 60-67% of animals, but the whole transcriptome activity in general appears silenced and cannot be used for biodosimetry purposes, but probably as an indicator for an emerging prefinal health stage.

Predicting the Radiation Sensitivity of Male and Female Rhesus Macaques Using Gene Expression.

Radiosensitivity differs in humans and likely among closely-related primates. Reasons for variation in radiosensitivity are not well known. We examined preirradiation gene expression in peripheral blood among male and female rhesus macaques which did or did not survive (up to 60 days) after whole-body irradiation with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study in 142 irradiated rhesus macaques. Animals were untreated (placebo), or treated using recombinant human IL-12, G-CSF or combination of the two. We evaluated gene expression in a two-phase study design where phase I was a whole genome screen [next generation sequencing (NGS)] for mRNAs (RNA-seq) using five RNA samples from untreated male and female animals per group of survivor and non-survivor (total n = 20). Differential gene expression (DGE) was defined as a statistically significant and ≥2-fold up- or downregulation of mRNA species and was calculated between groups of survivors and non-survivors (reference) and by gender. Altogether 659 genes were identified, but the overlapping number of differentially expressed genes (DGE) observed in both genders was small (n = 36). Fifty-eight candidate mRNAs were chosen for independent validation in phase II using the remaining samples (n = 122) evaluated with qRT-PCR. Among the 58 candidates, 16 were of significance or borderline significance (t test) by DGE. Univariate and multivariate logistic regression analysis and receiver operating characteristic (ROC) curve analysis further refined and identified the most outstanding validated genes and gene combinations. For untreated male macaques, we identified EPX (P = 0.005, ROC=1.0), IGF2BP1 (P = 0.05, ROC=0.74) and the combination of EPX with SLC22A4 (P = 0.03, ROC=0.85) which appeared most predictive for the clinical outcome for treated and combined (untreated and treated) male macaque groups, respectively. For untreated, treated and both combined female macaque groups the same gene (MBOAT4, P = 0.0004, ROC = 0.81) was most predictive. Based on the probability function of the ROC curves, up to 74% of preirradiation RNA measurements predicted survival with a positive and negative predictive value ranging between 85-100% and associated odds ratios reflecting a 2-3-fold elevated risk for surviving per unit change (cycle threshold value) in gene expression. In conclusion, we identified gender-dependent genes and gene combinations in preirradiation blood samples for survival prediction after irradiation in rhesus macaques.

Mathematical model of radiation effects on thrombopoiesis in rhesus macaques and humans.

A mathematical model that describes the effects of acute radiation exposure on thrombopoiesis in primates and humans is presented. Thrombopoiesis is a complex multistage dynamic process with potential differences between species. Due to known differences in cellular radiosensitivities, nadir times, and cytopenia durations, direct extrapolation from rhesus to human platelet dynamics is unrealistic. Developing mathematical models of thrombopoiesis for both humans and primates allows for the comparison of the system's response across species. Thus, data obtained in primate experiments can be extrapolated to predictions in humans. Parameter values for rhesus macaques and humans were obtained either from direct experimental measurements or through optimization procedures using dynamic data on platelet counts following radiation exposure. Model simulations accurately predict trends observed in platelet dynamics: at low radiation doses platelet counts decline after a time lag, and nadir depth is dose dependent. The models were validated using data that was not used during the parameterization process. In particular, additional experimental data was used for rhesus, and accident and platelet donor data was used for humans. The model aims to simulate the average response in rhesus and humans following irradiation. Variation in platelet dynamics due to individual variability can be modeled using Monte Carlo simulations in which parameter values are sampled from distributions. This model provides insight into the time course of the physiological effects of radiation exposure, information which could be valuable for disaster planning and survivability analysis and help in drug development of radiation medical countermeasures.

Neuroendocrine cells along the digestive tract express neuropilin-2.

Neuropilin-2 (np-2) is a receptor for semaphorin-3F (sema-3F) and semaphorin-3C (sema-3C). These semaphorins repel tips of growing axons that express np-2. In addition, np-2 functions as a receptor for heparin binding forms of the angiogenic factor vascular endothelial growth factor (VEGF) such as VEGF145 and VEGF165. We report that np-2 is strongly expressed in neuroendocrine cells located all along the human digestive tract. Confocal fluorescent microscopy revealed that np-2 is concentrated in vesicle-like structures located near the nucleus at the basolateral side of these cells. In the colon, the np-2-expressing subpopulation of neuroendocrine cell is almost identical with the serotonin-producing subpopulation of neuroendocrine cells. Gastrointestinal carcinoid tumors are digestive tract tumors that develop from neuroendocrine cells. Interestingly, most of the carcinoid tumors derived from the colon and the appendix did not contain np-2-producing cells. However, some carcinoid tumors derived from the small intestine and stomach did express low levels of np-2 in isolated foci of cells. By contrast, strong serotonin and chromogranin-A expression was observed in all of the carcinoid tumors that were examined. These results suggest that loss of np-2 expression may accompany tumor progression in carcinoid tumors.

The contribution of proangiogenic factors to the progression of malignant disease: role of vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor is a major inducer of angiogenesis and a vascular permeability inducing factor. Its expression is upregulated in many types of tumors and it is thought to be a major inducer of tumor angiogenesis. This article focuses on the role of vascular endothelial growth factor in tumor progression and on current efforts aimed at the inhibition of tumor progression through the inhibition of vascular endothelial growth factor activity.

Vascular endothelial growth factor receptor-1 and neuropilin-2 form complexes.

The products of the neuropilin-1 (Np-1) and neuropilin-2 (Np-2) genes are receptors for factors belonging to the class 3 semaphorin family and participate in the guidance of growing axons to their targets. In the presence of heparin-like molecules, both receptors also function as receptors for the heparin-binding 165-amino acid isoform of vascular endothelial growth factor (VEGF(165)). Both receptors are unable to bind to the 121-amino acid isoform of vascular endothelial growth factor (VEGF(121)), which lacks a heparin-binding domain. Interestingly, complexes corresponding in size to (125)I-VEGF(121).neuropilin complexes are formed when (125)I-VEGF(121) is bound and cross-linked to porcine aortic endothelial cells co-expressing VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to represent complexes of (125)I-VEGF(121) with a truncated form of VEGFR-1, presumably formed as a result of the presence of Np-1 or Np-2 in the cells, because such truncated forms could not be detected with anti-VEGFR-1 antibodies. Antibodies directed against VEGFR-1 co-immunoprecipitated the (125)I-VEGF(121).Np-2 sized cross-linked complex along with (125)I-VEGF(121).VEGFR-1 complexes from cells expressing both VEGFR-1 and Np-2 but not from control cells, indicating that VEGFR-1 and Np-2 associate with each other. To perform the reciprocal experiment we have expressed in porcine aortic endothelial cells a Np-2 receptor containing an in-frame myc epitope at the C terminus. Surprisingly, the myc-tagged Np-2 receptor lost most of its VEGF(165) binding capacity but not its semaphorin-3F binding ability. Nevertheless, when Np-2myc was co-expressed in cells with VEGFR-1, it partially regained its VEGF(165) binding ability. Antibodies directed against the myc epitope co-immunoprecipitated (125)I-VEGF(165).Np-2myc and (125)I- VEGF(165).VEGFR-1 complexes from cells co-expressing VEGFR-1 and Np-2myc, indicating again that VEGFR-1 associates with Np-2. Our experiments therefore indicate that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such complexes may be part of a cell membrane-associated signaling complex.

Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected].

Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.