Abstract
BACKGROUND:
Angiogenin is a small protein encoded by the ANG gene. It is activated by tissue hypoxia, and is known to be a potent stimulator of angiogenesis. The role of angiogenic factors in the pathogenesis of HIE is poorly understood, yet, angiogenin may be part of the molecular mechanisms underlying HIE.
OBJECTIVE:
Our objective was to explore the predictive value of angiogenin as a biochemical marker in early hypoxic ischemic encephalopathy staging.
STUDY DESIGN:
We prospectively studied 36 full term HIE neonates and 20 non- asphyxia neonates. Cord blood samples from all subjects immediately at delivery were withdrawn. Neurological examination and grading of HIE were performed during the first day of life.
RESULTS:
Concentrations of cord blood angiogenin were increased in infants with asphyxia when compared txht o controls (P = 0). Within the asphyxia group, the median cord blood angiogenin was significantly higher in stage III encephalopathy patient compared to stage I and stage II (p = 0). There was a negative correlation between pH, HCo3 level and angiogenin in stage II and stage III.
CONCLUSION:
Angiogenin helps in assessing the severity of HIE in neonates and is promising marker predicting the stage of hypoxia-ischemia so treatment may be initiated earlier.
Introduction
Despite the great improvements in perinatology, hypoxic ischemic encephalopathy (HIE) still has a high impact on neonatal mortality and neurological morbidity [1]. HIE is a syndrome occurring in the 1st day of life and manifesting as a cluster of disturbed neurologic functions. It occurs in 1% to 1.5% of live births in developed countries [2]. Based on severity, HIE can be staged by the widely used Sarnat and Sarnat scoring system into 3 stages, but unfortunately staging of HIE is assigned when HIE has been evolved [3]. Following ischemia, significant cellular death of the brain takes place, which carries the possibility of significant research potentials. Therefore, researchers work on numerous biochemical markers aiming at early diagnosis of perinatal asphyxia so as to initiate effective treatment as soon as possible [4].
Angiogenesis, the de novo development of new vessels, is thought to be crucial for the normal brain physiology. Angiogenin (ANG) is a potent angiogenesis inducer belonging to the ribonuclease A (RNaseA) superfamily which provoke neovascularization, thus promoting in a variety of physiological and pathological states. Human ANG is a small protein with a molecular weight of 14.1 kDa and shows 33% sequence identity with bovine pancreatic RNase A. ANG is up regulated by hypoxia in human cell lines [5]. Furthermore, ANG expression correlates with hypoxia inducible-factor (HIF-1a) and basic-helix-loop-helix transcription factor E40 (BHLHE40), a target gene of HIF-1a, in breast cancer [6]. Functionally important regions of the protein include the catalytic residues, a nuclear localization sequence, a putative receptor-binding region and a region important for immunomodulation. Angiogenin is widely expressed in the developing nervous system both in the brain and in the spinal cord, predominantly in the neurons [7]. To the best of our knowledge, there is limited information about brain biomarkers in neonates with HIE. Moreover, only few among these markers are specific to different stages of HIE and could thus possibly be used in its staging.
The objective of the current study was, therefore, to test the hypothesis that brain tissue, under low oxygen tension, releases ANG that could affect microvascular brain density, and consequently providing a link between the HIE staging, and hypoxia-induced secondary brain insult, on one hand and serum concentration of angiogenin on the other.
Patients and methods
This is a prospective study that included 56 appropriate-for-gestational-age full term infants delivered in the Gynecology and Obstetrics department of Ain Shams University maternity Hospital in Cairo. The study has been approved by the Research Committee at Ain Shams University in Cairo, Egypt. Parental consents were obtained for all subjects. Thirty six infants suffered from perinatal asphyxia and 20 control infants of comparable age and sex.
To determine the probability of neonatal HIE, we assessed our patients for features suggestive of hypoxic and/or ischemic injury during the perinatal and/or intrapartum period that included [8]:
Fetal umbilical artery acidemia: pH less than 7.00 and/or base deficit ≤12 mmol/L. Apgar score of ≤ to 5 at 10 minutes Clinical signs consistent with mild, moderate or severe encephalopathy according Sarnat and Sarnat HIE clinical staging system.
Infants were excluded from the study if any of the following conditions was pertinent: a) onset of multisystem organ failure, b) conditions related to infections such as, history of maternal fever and rupture of amniotic membranes ≥18 hours, c) congenital anomalies and any inherited metabolic disease, d) twin pregnancy, and e) maternal conditions with possible impact on Angiogenin such as liver diseases, diabetes and different forms of hypertension [9].
Length of gestation was estimated using the date of the last menstrual period, early antenatal ultrasound when available and the new Ballard scoring system was applied [10].
Sampling
Umbilical cord blood of all recruited patients in the study was sampled at delivery. Blood samples were collected and delivered in sterile tubes after being allowed to clot, the tubes were centrifuged at 1000 rpm at room temperature to obtain serum. Hemolytic samples were excluded from analysis. Serum was stored at –70°C prior to analysis. Other laboratory investigations as CBC. Kidney functions, ABG etc. … were done to assess multiorgan dysfunction.
Angiogenin assays
We used Human Angiogenin Immunoassay (R&D Systems, Abingdon, Oxon, UK) based on quantitative colorimetric sandwich ELISA tests, according to the manufacturer’s instructions. All samples, standards and controls were assayed in duplicate. The optical density was determined of each well using micro plate reader set to 450 nm. The duplicate readings were averaged for each standard and sample and the average zero standard optical density was subtracted. A standard curve was created by blotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and drawing the best fit curve through the points on the graph.
Statistical analysis
Data was processed using IBM SPSS statistics (V. 23.0, IBM Corp., USA). Parametric quantitative data were expressed as Mean±SD while Median Percentiles for quantitative non-parametric measures and both number and percentage for categorized data. Statistical significance between two continuous variables was determined using student’s t test; the Wilcoxon Rank Sum test for non-parametric two in dependent groups comparison. Comparison between more than 2 patient groups for parametric data using Analysis of Variance (ANOVA), Comparison between more than 2 patient groups for non-parametric data using Kruskall Wallis test. Chi-square test to study the association between each 2 variables or comparison between 2 independent groups as regards the categorized data. The probability of error at 0.05 was considered significant, while at 0.01 and 0.001 are highly significant.
Results
Demographic data of recruited patients are presented in Table 1. Concentrations of angiogenin were significantly increased in infants with hypoxia group when compared to non-hypoxia group (Table 1). The median cord blood angiogenin was significantly higher in stage III patients as compared to stage I and stage II (Table 2). In the logistic regression model, Angiogenin concentrations were inversely correlated with PH and HCO3) (Figs. 1 and 2 respectively). Within the asphyxia group, the most frequently affected system was the respiratory system, 50% were ventilated. Multi organ dysfunction was seen in 90% of Grade III patients which was proportionate to the mortality (Table 3).
Demographic and laboratory data of the study group
Demographic and laboratory data of the study group
*Wilcoxon Rank Sum Test # Student t Test. Analysis of variance (ANOVA test), TLC: total leukocytic count×109/L, HB: hemoglobin: gm%, HcT: hematocrite %, PLT: platelet count×109/L, pH: H+ ion concentration, HCO3: carbonate.
Comparison of Angiogenin (pg/ml) levels among different HIE stage
*Kruskall Wallis test comparison.
Morbidity and mortality among HIE stages
Linear regression of pH correlation to angiogenin level.
Linear regression of HCO3 level correlation to angiogenin.
To the best of our knowledge this is the first study correlating the expression of human angiogenin to the degree of hypoxic-ischemic encephalopathy in full term newborns. We demonstrated in this study that angiogenin is specifically most increased in infants who later developed hypoxic ischemic encephalopathy. Angiogenin negatively correlated with PH and HCO3 in cord blood. Moreover, angiogenin is mostly increased in infants with stage III hypoxic ischemic encephalopathy.
HIE is characterized by the progressive damage that it causes to all organ systems and particularly to the brain which cannot tolerate oxygen deficiency for long periods. Following the hypoxic-ischemic insult, it can take up to 72 hours for neurological manifestations to appear [1]. Biochemical markers of angiogenesis have been besieged as a novel therapeutic strategy in adult stroke. Recently, many studies have reviewed the expression of angiogenesis markers resulting from birth asphyxia in newborn [11]. The human angiogenin (hANG) is highly expressed in the developing fetal nervous system of the murine orthologue both in the brain and in the spinal cord predominantly ranging from 15 to 30 weeks’ gestation [12, 13]. Strong staining of angiogenin in motor neurons of prenatal spinal cords suggests a physiological role of angiogenin in early development and in maintaining the integrity of spinal cord vasculature supporting motor neurons physiological health [14, 15]. This supports the hypothesis that angiogenin abnormalities may affect the nervous system both directly, through motor neuron function, and indirectly, through aberrant endothelial cells angiogenesis [16]. Both growth and survival activities of ANG seem to be mediated by the same receptor. Growth signals stimulate nuclear translocation of ANG, whereas stress signals direct ANG to stress granules. Both pathways are mediated by a cell surface receptor that remains to be identified. Nuclear ANG stimulates rRNA transcription, enabling ribosome biogenesis and therefore cell growth and proliferation. Under stress conditions, ANG is not translocated into the nucleus but is rather accumulated in such cytoplasmic compartments as stress granules where it mediates the production of tiRNA, reprograming protein translation and promoting survival [17]. Under stress conditions, angiogenin could act as a neuro-protective mediator that targets endothelial cells, cancer cells, and motor neurons. These cells differentially respond to ANG probably due to the difference in ANG receptor expression [18]. Studies on the cerebral ischemia in post stroke rats found that ANG-1 and ANG-2, and other angiogenic factors may regulate new vessel formation to promote blood flow, restrain neuron apoptosis in ischemic penumbra and enhance nerve function to recover. Induction of angiogenic processes through exogenous administration of angiogenin may be, therefore, important for neuronal regeneration [19]. However, a major concern precluding the use of angiogenin is the fact that this growth factor is implicated in inducing significant vascular permeability and blood brain barrier leakage [20, 21]. Angiogenin expression during cerebral ischemia synergies expression of other angiogenic factors promoting edema and tissue damage as an inflammatory responses to hypoxia and downregulation of its expression decreases VEGF rRNA transcription leading to blockage of the angiogenic activity of VEGF after cerebral ischemic stroke in rats [22, 23].
In our study, higher angiogenin values in grade III encephalopathy denotes progressive neuropathology. The newborn developing brain harbors neuronal and glial precursor cells, which are in different stages of proliferation, migration and maturation. This indicates high metabolic demands and oxygen requirements. Hence, the rapid angiogenesis induced by angiogenic factors as ANG can be ascribed to hypoxia of affected brain region in an effort to maintain oxygen delivery to the brain, the vascular density is increased resulting in smaller intercapillary distances, which will finally restore tissue oxygenation [2]. Therefore we suggested that increased ANG in cases with stage III encephalopathy may be an indicator of higher levels of angiogenesis and capillary remodeling that are more severe in comparison to cases with mild and moderate HIE. The levels of angiogenin are inversely correlated to Apgar and PH. The role of micro-environmental stresses especially lactic acidosis and the relationship between cord blood acidemia and angiogenin have been relatively little explored [24]. The strength of this study resides in the uniform timing of blood sampling, and the classification of patient according to follow up to decide clinically, and to correlate laboratory findings to the clinical correlates of the asphyxial insult that has occurred. Hence, we could exclude the factor of postnatal age or time as a variable that could affect angiogenin expression. Assay of the angiogenin in different degrees of severity was tested highlighting the ongoing neurovascular process following hypoxia. Further studies are needed to explore the correlation between expression of angiogenin and central nervous system compartment with the long-term neurodevelopmental outcomes.
In conclusion, angiogenin is increased in cord blood of neonates following birth asphyxia and it is a promising marker in determining the stage of hypoxia before clinical manifestations occur so treatment can be initiated earlier. Such studies will help in determining whether a therapeutic role of angiogenin or its inhibitors can help HIE infants.