The RI in the CRA of ODD patients was significantly higher than in the controls (p < 0.001). Women with ODD presented with significantly lower values of PSV and EDV than men . Arterial hypertension, cigarette smoking and location of ODD in the left or right eye did not exert a significant effect on the blood flow in the CRA. Blood flow in the CRA is considerably disturbed in patients with ODD. This disturbance might result from mechanical stenosis of the vascular wall caused by the drusen. Color Doppler ultrasound is a useful method in the assessment of retrobulbar hemodynamics in ODD.To evaluate retrobulbar hemodynamics in patients with optic disc drusen (ODD) by color Doppler imaging. A prospective study included 52 patients with bilateral ODD and 50 age-matched controls. Blood flow in the central retinal artery (CRA) in both eyes was evaluated. Peak-systolic velocity (PSV), end-diastolic velocity (EDV), and the resistivity index (RI) were measured. Mean values of the blood flow Doppler parameters were compared between the patients with ODD and the controls. Both PSV and EDV in the CRA were significantly lower in patients with ODD than in the controls ( Although the pathogenesis of ODD is yet to be understood, according to the most accepted theory, they form due to disturbance in the normal axonal metabolism and a resultant gradual calcification of mitochondria. This results in a rupture of axonal membranes and extrusion of mitochondria into the extracellular space whereby they undergo further calcification and fuse into larger complexes, forming the drusen5.Optic disc drusen (ODD) are hyaline-like deposits localized in the prelaminar portion of the optic nerve head. The prevalence of the ODD is estimated at 0.3–2.0%; in 66–92% of the cases, the drusen are found bilaterally10. The most common clinical manifestations of ODD are visual field defects, according to various reports, detected in 24–88% of eyes with the drusen14. Studies examining the thickness of the retinal nerve fiber layer (RNFL) demonstrated that the changes in the visual field are associated with ganglion cell axon damage. Furthermore, patients with ODD were shown to present with thinning of the peripapillary RNFL, especially in the superior and inferior quadrant, similar to that observed in glaucomatous eyes17.A loss of vision occurs very rarely in patients with ODD and are usually associated with the development of vascular complications4; (2) direct compression of the drusen on the optic nerve fibers in the prelaminar part of the scleral canal18; and (3) ischemia of the optic nerve head caused by vascular abnormalities19. It is still unclear whether the presence of calcified drusen in the proximity of normal ocular vessels may directly affect retrobulbar hemodynamics. Similarly, little is known about the consequences of the retrobulbar hemodynamics impairment to the optic nerve functioning. The review of literature showed that only one previously published study used Doppler imaging to determine ocular blood flow parameters in patients with ODD20. However, retrospective character of this study and small sample size justify further research in this matter.Current evidence suggests that optic nerve damage in patients with ODD may result from disturbances in the axonal flow. These disturbances may be a consequence of (1) improper axonal metabolism in the ganglion cellsIn this study, we used color Doppler ultrasonography to verify whether the presence of ODD was associated with blood flow disturbances in the central retinal artery (CRA) and to determine a character of the retrobulbar hemodynamic disorders. To the best of our knowledge, this was the first a large prospective study which compare the blood flow parameters in the ocular vessels of patients with ODD and age-matched controls.We examined prospectively all adult patients with anomalous optic disc appearance, admitted to the Department of Ophthalmology, Medical University of Bialystok, between May 2010 and January 2018. All study patients underwent basic ophthalmic examination and visual field testing with Medmont Automated Perimeter . ODD was diagnosed based on the appearance of the optic disc  during indirect slit-lamp biomicroscopy with Volk lens. If the appearance of the optic disc was atypical, the ultimate diagnosis was confirmed with B-scan ultrasonography , optical coherence tomography , and fluorescein angiography with evaluation of autofluorescence . Moreover, basic laboratory parameters, such as complete blood count, coagulation parameters and blood glucose levels, were determined in all the study patients.The patients were excluded from the study if they had (1) unilateral drusen, (2) intraocular pressure (IOP) greater than 21 mmHg, (3) evidence of other ocular diseases, not associated with ODD, on ophthalmic examination, (4) refractive error >  ± 3.0 diopters, (5) history of intraocular surgeries or laser treatment, (6) systemic conditions, such as diabetes mellitus, cardiovascular diseases and respiratory disorders, and/or (7) blood clotting disturbances. Arterial hypertension was not an exclusion criterion from the study.The control group was comprised of 50 age- and sex-matched persons, 20 men and 30 women with a mean age of 43.2 years. To be eligible for the study, the controls need to present with good general health; arterial hypertension was the only concomitant disease which did not constitute an exclusion criterion from the control group. All controls had normal visual acuity and did not show any abnormalities on basic ophthalmic examination and perimetry.Color Doppler imaging (CDI) was preceded by the measurement of IOP and blood pressure (BP). All tests were carried out at the same time of the day, between 9:00 AM and 11:00 AM. IOP was determined by means of Goldmann applanation tonometry and corrected for pachymetry value determined using specular microscope, and BP was measured by sphygmomanometry in a sitting position. The list of analyzed parameters included systolic blood pressure , diastolic blood pressure , pulse pressure index (PPI = [SBP-DBP]/SBP) and ocular perfusion pressure . OPP was calculated from the following formula: 2/3 [DBP + 1/3(SBP-DBP)] – IOP.Blood flow was examined by an experienced investigator (AU) using an ultrasound unit with a 7.5-MHz linear probe . Blood flow in the CRA was analyzed in both eyes of each subject. The assessment of the eye’s vascular bed was preceded by the examination of the carotid artery (CA) and ophthalmic artery (OA), to exclude blood flow disturbances in these vessels. The examination was carried out with the patient in a recumbent position, after a small amount of ultrasound gel was applied onto the closed eyelids, with the probe positioned gently not to compress the eyeball. The examined vessel was identified based on its position, the direction of blood flow  and relation to the eyeball and optic nerve. The CRA was visualized as a fine artery situated in the central part of the optic nerve. Its spectrum was presented together with the spectrum of blood flow in the central retinal vein, which runs within 10–15 mm of the CRA. The blood flow parameters in the CRA were examined at the standard position, approximately 3–6 mm behind the posterior wall of the eyeball at the Doppler angle’s range of 0–20º. This enabled us to attain the maximum Doppler shift values.The list of analyzed flow parameters included peak-systolic velocity , end-diastolic velocity , and the resistivity index (RI). PSV was defined as the highest value of blood flow during cardiac systole, EDV as the lowest value of blood flow at the end of cardiac diastole, and RI as the quotient of the difference between peak systolic and peak diastolic velocity over peak systolic velocity [RI = (PSV-EDV)/PSV].p < 0.1) between the patients with ODD and the controls was estimated on a univariate logistic regression analysis. Odds ratios (ORs) for the occurrence of ODD were calculated during the analysis, along with their 95% confidence intervals (CIs). All variables which turned out to be significant or nearly significant (p < 0.1) determinants of ODD on the univariate analysis, were included in a single multivariate logistic regression model. Moreover, a receiver operating characteristic (ROC) analysis was carried out to determine the cut-off values of the hemodynamic variables that distinguished optimally between the patients with ODD and the controls. The following characteristics were calculated for each hemodynamic parameter: (1) sensitivity, i.e. the proportion of the patients with ODD, in whom the values of the analyzed variable were above the proposed cut-off value, and (2) specificity, i.e. the proportion of the controls in whom the values of the analyzed variable were below the proposed cut-off value. The accuracy of the analyzed variables was confirmed based on the values of Z-statistic. Moreover, the areas under the ROC curves (AUCs) were calculated, along with their 95% CIs. All calculations were carried out with Statistica 10 package , with the threshold of statistical significance set at p ≤ 0.05.Normal distribution of continuous variables was verified with the Kolmogorov–Smirnov test. Statistical characteristics of continuous variables were presented as arithmetic means, standard deviations (SD), medians and interquartile ranges (IQR). Statistical characteristics of qualitative and discrete variables were presented as numbers and percentages. Student t-test was used for between-group comparisons of normally distributed variables, whereas the variables with distributions other than normal were compared with the Mann–Whitney U-test. The distributions of qualitative and discrete variables were compared with the chi-squared test or the Fisher exact test. The power of the variables the values of which differed significantly or at a threshold of statistical significance  were enrolled in the study.Visual acuity impairment (< 6/6 on the Snellen chart) was observed in 7 (13.5%) patients with ODD. The impairment was bilateral in two patients and unilateral in the remaining five. The presence of peripapillary subretinal neovascular membrane was observed in two of these subjects. Thirty-seven (71%) patients had visual field defects in one (n = 21) or both eyes (n = 16).The patients with ODD presented with significantly lower values of PSV and EDV in the CRA and significantly higher values of RI than the controls Fig. . No statUnivariate logistic regression analysis confirmed that lower values of PSV and EDV in the CRA and higher values of the RI were significant determinants of ODD. However, none of these parameters was identified as an independent determinant of ODD on multivariate logistic regression analysis.ROC analysis identified the cut-off values for PSV and EDV in the CRA and RI that most accurately distinguished between the patients with ODD and the controls Table . The mosCompared with male patients, women with ODD presented with significantly lower values of PSV and EDV in the CRA. Patient sex did not exert a significant effect on the values of RI Table . The par20 concluded that both patients with ODD (16 eyes) and glaucomatous patients (20 eyes) had lower systolic and diastolic flow velocities in the CRA. The authors did not find statistically significant between-group differences in RI values.The aim of the present study was to compare the retrobulbar hemodynamics in patients with ODD and healthy controls. The study demonstrated that the presence of ODD was associated with substantial disturbances of blood flow in the CRA. To the best of our knowledge, only one previous study analyzed the blood flow in the ocular vessels of patients with ODD. Based on the analysis of a small cohort, Pinto et al.21. Since the flow measurements in the CRA were taken routinely 3–6 mm behind the posterior ocular wall, the stenosis might have occurred at the level of the prelaminar part of the optic nerve and might result from mechanical compression of the CRA by surrounding drusen. Vascular stenosis usually results in worse perfusion, which in turn leads to ischemia in the area supplied by the affected vessel. As mentioned above, Pinto et al.20 did not find significant differences in RI values, which seems surprising given the hereby discussed plausible mechanism of alterations in the CRA flows. This fact was also highlighted by Pinto et al. who mentioned that “the reasons for such results are unclear”20.Our study demonstrated that the blood flow in the CRA of patients with ODD had typical features of a resistance flow with disturbed outflow, i.e. (1) a decrease in PSV along with spectrum flattening (changed proportion of systolic wave height to the time of its duration), and (2) marked decrease in EDV and increase in RI. These findings, especially the significantly higher values of RI in ODD patients than in the controls, are indirect indicators of vascular stenosis located peripherally from the examined site20.In our present study, some blood flow parameters (PSV and EDV) of ODD patients were significantly lower in women than in men. However, patient age, location of ODD in the right or left eye, IOP, smoking, SBP, DBP and presence of concomitant arterial hypertension did not exert a significant effect on the blood flow parameters in the CRA of ODD patients. To the best of our knowledge, this study was the first to analyze the effect of clinicodemographic characteristics on the retrobulbar flow, as this issue has not been addressed by Pinto et al.25. These OCTA findings were shown to correlate positively with the RNFL thickness26. Based on those findings, it has been concluded that enlarged ODD may cause acute or chronic ischemia by compressing optic nerve fibers and surrounding vessels. Hence, identification of disorders in retinal blood flow, whether on Doppler imaging or OCTA, might be an early predictor of future axonal damage in patients with ODD.Stenosis of small retinal vessels in patients with ODD was also documented in previous studies that used optical coherence tomography angiography (OCTA) to assess the retinal microcirculation. OCTA is a technique to visualize microvessels and to measure their width and density based on the erythrocyte flow. However, OCTA cannot be considered an alternative to the evaluation of hemodynamics by Doppler imaging. The results of OCTA-based studies suggest that the presence of ODD is associated with capillary narrowing in the superficial capillary plexus layer, areas of capillary dropout and decreased vascular density on the optic nerve head4; however, we still lack objective data regarding the influence of this atrophy on the ocular blood flow. A few previous studies documented vascular changes in the optic nerve atrophy. Some authors found the evidence for the narrowing of total arteriolar and capillary diameter in patients with the optic nerve atrophy29. Sebag et al.30 analyzed blood flow in peripheral branches of the CRA in cats with experimental optic nerve atrophy. Using laser Doppler methods, those authors demonstrated a 53% decrease in erythrocyte speed in the capillaries of the atrophic optic nerve head. Hence, we cannot exclude that the worsening of hemodynamic conditions in the CRA vascular bed might occur secondarily to the atrophy of the optic nerve fibers.It is still unclear whether the disturbances in the CRA flow may occur secondarily to the optic nerve atrophy and result from reduced metabolic demand in atrophic tissues. Patients with ODD without a doubt show atrophy of the retinal ganglion cell axons31. Also, according to Pino et al.20, the degree of retrobulbar flow impairment in patients with ODD and glaucomatous patients was similar. No effective treatment to prevent progressive injuries of the optic nerve fibers in patients with ODD has been developed thus far. In a pilot study conducted by Schargus and Gramer2, IOP-lowering therapy substantially slowed down the progression of visual field loss in patients with ODD, although the latter have never presented with elevated IOP values. The authors of that study did not fully explain the reasons behind this phenomenon. However, it can be assumed that a decrease in IOP might be associated with an increase in perfusion pressure and resultant enhancement of blood flow through the ocular vessels. Hence, improved retrobulbar hemodynamics associated with a decrease in IOP might exert a positive effect, slowing down the progression of RFNL injuries in patients with ODD, similar as in glaucomatous neuropathy. A better insight into this mechanism could be obtained if the hemodynamic parameters for the vessels supplying the optic nerve were compared between two groups of patients with ODD: those who received an IOP-lowering therapy and those who did not. The results of our present study, as well as the findings reported by Pinto et al.20, imply that color Doppler ultrasonography might be a useful diagnostic instrument in this context.The data gathered during several years of follow-up of patients with ODD suggest that both visual field loss and decrease in RNFL thickness have a progressive character and sometimes may resemble changes associated with glaucomaWe are well aware of the potential limitations of this study. The most significant constraint pertains to the research methodology. Doppler ultrasonography visualizes solely hemodynamic parameters of flow in a vessel, and therefore, it cannot provide any evidence to directly support the theory of CRA stenosis resulting from mechanical compression by ODD. We could only speculate about this mechanism based on a character of blood flow changes in the CRA. Similarly, we were unable to distinguish whether the flow disturbances in the CRA had a primary character or occurred secondarily to the injury of the optic nerve fibers. Another potential limitation might be a selection bias, as our study included solely the patients with bilateral drusen. Therefore, we could not analyze the whole spectrum of hemodynamic changes in retrobulbar circulation associated with the presence of the drusen. Nevertheless, we used this criterion to obtain a large, homogenous patient population. Furthermore, we kept in mind that drusen occur bilaterally in most patients.To summarize, this study provided a novel insight into the optic nerve hemodynamics in ODD. The study demonstrated that patients with bilateral ODD showed considerable disturbances of the CRA blood flow parameters. These disturbances might result from mechanical stenosis of the vessel caused by the drusen or might occur secondarily to the optic nerve atrophy. Further research is needed to verify whether the CRA flow disturbances progress with time and if patients with unilateral and bilateral drusen differ in terms of the retrobulbar hemodynamics.The first author—Iwona Obuchowska has full access to all data in the study and she takes responsibility for the integrity of the data and accuracy of the data analysis.


PurposeThe aim of this study was to evaluate the relationship between Fibroblast Growth Factor-23 (FGF23) serum levels and cardiovascular disease and early graft failure in renal transplant recipients. MethodsThis cross-sectional study was conducted on renal transplant recipients followed by our adult kidney transplant clinic. The patients were divided into two groups according to the mean FGF23 levels (mean FGF23 level=71.2 ± 34.6pg/mL). The patients included in the study were classified as Group 1  and Group 2  and the data was analyzed as a statistical significance between the two groups. The presence of atherosclerosis was determined by a Doppler ultrasound for evaluate the carotid artery intima-media thickness (CA-IMT). Intrarenal Doppler spectra were obtained with same Doppler ultrasound to determine the renal resistivity index (RRI) for evaluate graft renal failure.ResultsA total of 88 kidney transplantation recipients were included in the study. In the multivariate analysis adjusted for age and gender, the eGFR , CA-IMT  and RRI  parameters were statistically significant, while the remaining parameters were not statistically significant. In the group analysis, Ca , CA-IMT  and RRI  were significantly higher in the patients in group 2 than the patients in group 1. ConclusionAccording to our results, FGF23 can be considered as a descriptive biomarker for cardiovascular prognosis and graft function for patients with kidney transplantation. Chronic kidney disease (CKD) is a growing public health problem with a prevalence rate of approximately 8-16% all over the world , 3. In gCardiovascular diseases (CVD) are the leading cause of morbidity and mortality in CKD patients . PatientPatients who have undergone kidney transplantation have also been shown to have increased FGF23 levels, even in the case of normal graft function . Kidney In renal transplant recipients, a good marker for early detection of loss of graft function and early markers of atherosclerosis (an indicator of CVD risk factors) has not yet been found. FGF23 may be an early indicator of both early graft loss and increased cardiovascular risk in these patients. We hypothesized that FGF23 was associated with CVD and early graft failure in renal transplant recipients (RTR).This cross-sectional study was conducted on 88  renal transplant recipients followed by our adult kidney transplant clinic between February 2018- January 2019. The study exclusion criteria were patients under 18 years old who were determined to have a recent cardiovascular event , an episode of active infection, a bone fracture in the past year, active malignancy, active vasculitis or diabetes mellitus, Paget's disease, serum creatinine of >2.5 mg/dL, and a renal transplantation duration of <1 year. Until enrollment, none of the participants were treated with folate, vitamin D, vitamin B12, erythropoietin or biphosphonate. The etiologies of pretransplant renal failure were as follows: hypertensive nephrosclerosis (55.68%), chronic diabetes (18.18%), nephrolithiasis (6%), glomerulonephritis (10.22%), polycystic kidney disease (5.6%), and others or unknown etiology (3.4%). All patients were receiving triple therapy (immunosuppressive therapy) including calcineurin inhibitor (CNI) (cyclosporine or tacrolimus) along with mycophenolate mofetil (MMF)/mycophenolate sodium (MNa) and steroids. During transplantation, induction therapy was carried out with basiliximab or anti-thymocyte globulin. The study protocol was approved by the local scientific ethics committee and informed consent was obtained from all patients.The demographic characteristics, blood pressure values, medications, and smoking history of the patients were recorded. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Fasting blood sample was taken from each patient. C-reactive protein (CRP), total cholesterol, low-density lipoprotein cholesterol, high-density lipoproteins, serum creatinine, triglycerides, albumin, phosphorus, alkaline phosphatase, calcium, and parathyroid hormone were measured for all participants using standard methods. Serum samples and plasma separated within 30 minutes were stored at -80 °C if not analyzed immediately. The levels of FGF23  were calculated using commercial enzyme-linked immunoadsorbent assay (ELISA) kits. The FGF23 measurement range was determined as 15.63-1000 pg/mL and the measurement sensitivity as 9.38 pg/mL. Meanwhile, eGFR was estimated based on the Modification of Diet in Renal Disease formula .Carotid artery intima-media thickness measurementThe presence of atherosclerosis was determined by a Doppler ultrasound (USG) device using a 2-5 mHz linear probe with . Imaging of the carotid artery was carried out when all participants were lying in the supine position with their neck slightly extended and head rotated about 20° from the side being examined, and performed after 15 min of rest in a supine position. The measurements were taken from the right-left distal common carotid artery bifurcation, and three points on the distal walls of the proximal portion of the internal carotid artery. Carotid artery intima-media thickness (CA-IMT) was determined by longitudinal examination from the distance defined as the interface between vascular lumen echogenicity and media/adventitia echogenicity . Both riThe patients were divided into two groups according to the mean FGF23 levels (mean FGF23 level=71.2 ± 34.6pg/mL). The patients included in the study were classified as Group 1  and Group 2  and the data were analyzed as a statistical significance between the two groups.Statıstıcal Analysis Continuous variables were determined as mean ± standard deviation and categorical variables as percentage (%). The Mann-Whitney U test, Student's t test, Pearson chi-square test and Fisher's Exact tests were used to compare the patient characteristics with that of the healthy control group. A p value of <0.05 was considered statistically significant. Statistical analyses were carried out using the SPSS computer software .Our study included 88 kidney transplantation recipients. The mean age of the patients was 44.9 ± 8.7 years. Of the study patients, 54% were male. The duration of dialysis before transplantation was 42.3 ± 16.7 months and the mean duration after transplantation was 61.2 ± 22.5 months. The demographic, clinical and laboratory parameters of the study are shown in Table In the multivariate analysis adjusted for age and gender, eGFR , CA-IMT  and RRI  parameters were statistically significant, while the remaining parameters were not statistically significant (Table The study patients were divided into two groups according to the mean FGF23 71.2 ± 34.6 pg/mL) level. Mean FGF23 level lower than 71 pg/mL were included in group 1 (n=42), the others (mean FGF23 level higher than 71 pg/mL) were included in group 2 (n=46). In the group analysis, eGFR was significantly lower in the patients in group 2 than in the patients in group 1 . Again, in the group analysis, Ca , CA-IMT  and RRI  were significantly higher in the patients in group 2 than the patients in group 1 , CA-IMT were ≥0.9 mm and in two-thirds of subjects (n=60) CA-IMT were < 0.9 mm . The FGFCVDs are still the cause of more than 50% of deaths in renal transplant recipients with functional grafts . Renal tFGF23 is a hormone that is mostly synthesized by osteocytes and regulates phosphate metabolism. It induces phosphaturia by suppressing Na/P co-transporters in the proximal tubule and reduced calcitriol levels . FGF23 lIn renal transplant recipients with good graft functions (eGFR> 30-45 ml/min), high PTH levels are detected in the first year . Since tOur study has some limitations that negatively affected the results. First, our study was a single-center study and conducted with a small sample size. Second, the temporal effects of the correlation between FGF23 graft function and CV events could not be investigated in renal transplant recipients since it was a cross-sectional study. Third, the characteristics of the patients were not compared with that of healthy individuals and CKD patients with similar age and GFR values. Because of the limited number of studies on this subject, further multi-center studies with a large sample size are needed.FGF23 can be considered as a descriptive biomarker for cardiovascular prognosis and graft function for patients with kidney transplantation. Preemptive kidney transplantion should be encouraged for all CKD patients with live donors. CA-IMT measurement should be used more frequently to assess cardiovascular risk parameters for these patients.


Morning blood pressure surge (MBPS) had been known to be associated with hypertensive target organ injury and vascular events. Retinal vein occlusion (RVO) is also known to be related with underlying cardiovascular risk factors. This study investigated the effect of MBPS on patients with RVO. In total, 76 patients with RVO who had undergone systemic cardiovascular examination including a 24-hour ambulatory blood pressure monitoring, carotid artery intima media thickness, and pulse wave velocity were evaluated between January 2015 and February 2019. The MBPS was calculated as follows: mean systolic blood pressure measured over two hours after awakening minus mean systolic blood pressure measured during the one hour that included the lowest sleep blood pressure. Macular edema was significantly more prevalent in the MBPS group compared with the non-MBPS group. After adjusting for confounding factors, multivariate regression analyses revealed that MBPS independently predicted macular edema in patients with RVO . In conclusion, evaluating blood pressure patterns, especially MBPS, using 24-hour ambulatory blood pressure monitoring may be useful for assessing and predicting ophthalmologic outcome and may facilitate better blood pressure control in patients with RVO. RVO includes branch retinal vein occlusion (BRVO), which is caused by venous thrombosis at an arteriovenous crossing where an artery and vein share a vascular sheath or at the optic disc, and central retinal vein occlusion (CRVO), which results from thrombosis of the central retinal vein and a disturbance in the venous outflow, at the lamina cribrosa or posterior to it3. The main complication of RVO is macular edema, which can lead visual impairment4. Macular edema is the swelling or thickening of the macula due to leakage of fluid and blood components from blood vessels and is a major cause of RVO-associated blindness, angiogenesis, and retinal detachment. Therefore, it is associated with poor ophthalmologic outcomes5.Retinal vein occlusion (RVO) is a common retinal vascular disease that leads to vision loss7. In hypertensive patients, nocturnal blood pressure (BP) rise and morning BP surge (MBPS) have been known to be associated with an increased risk of target-organ damage and cardiovascular events12. Interestingly, RVO has been shown to have circadian variation as well, with event onset time peaking in the early morning hours in previous studies14. In this study, we evaluated the effect of morning blood pressure surge on patients with RVO.It has been demonstrated that many systemic cardiovascular risk factors such as hypertension, diabetes, dyslipidemia, and systemic inflammatory diseases are associated with RVO; hypertension is one of the most significant risk factors among themAmong 123 patients who had been diagnosed with RVO, 76 patients who were eligible and underwent 24-hour ambulatory blood pressure monitoring (ABPM) were finally included in the analysis. The mean age was 59.3 ± 10.8 years, and 39 (51.3%) patients were males whereas 37 (48.7%) were females. On 24-hour ABPM, average 24-hour systolic blood pressure (SBP) / diastolic blood pressure (DBP) was 133.4 ± 16.9 mmHg/81.7 ± 11.8 mmHg. Average sleep-trough MBPS was 27.0 ± 14.2 mmHg, and average prewaking MBPS was 6.7 ± 19.3 mmHg. The best cut-off points for predicting macular edema, determined through the receiver operating characteristics (ROC) curve analyses, were 29.3 mmHg for sleep-trough MBPS and 14.4 mmHg for prewaking MBPS  had branch retinal vein occlusion (BRVO) and 20 (26.3%) had central retinal vein occlusion (CRVO). Macular edema was significantly more common in the MBPS group than in the non-MBPS group  Table .Table 2OUnivariate logistic regression analysis showed that MBPS was associated with macular edema . Average daytime SBP and average 24-hour SBP were also associated with macular edema in patients with RVO Table . InteresThe results of this study revealed that there is a greater prevalence of macular edema among patients with higher MBPS than among patients with lower MBPS; it also showed that exaggerated MBPS is a predictor of macular edema in RVO patients, independent of ambulatory BP levels and hypertension-mediated organ damage.3. Because patients with RVO exhibit characteristics of cardiovascular disease, their cardiovascular morbidity and mortality rates are also increased16. Among these cardiovascular co-morbidities, hypertension is most commonly seen in patients with RVO and is a well-known risk factor of RVO6. RVO is also considered an end-organ damage in patients with hypertension and may be associated with arterial stiffness or carotid atherosclerosis. Moreover, differences in diurnal variation of BP in patients with hypertension, abnormal dipping pattern of nocturnal BP, or presence of MBPS are known to be associated with cardiovascular events9. A previous study reported that patients with RVO had more non-dipping patterns of nocturnal BP compared with patients without RVO17.RVO is closely associated with systemic cardiovascular diseases including hypertension, dyslipidemia, diabetes, kidney disease, atherosclerosis, myocardial infarction, and stroke18. In the early morning hours, an abrupt and steep increase in BP occurs, coincident with arousal and awakening from overnight sleep19. Normal MBPS is a physiological phenomenon, but an exaggerated MBPS is a cardiovascular risk and there is a substantial ethnic differences in the degree of MBPS20. There are compelling evidences suggesting a significant association between MBPS and various surrogate markers of target organ damage as well as cardiovascular events21. Kuwajima et al. reported that in 23 elderly hypertensive patients, the SBP change after awaking in the morning was significantly correlated with the left ventricular mass index and the A/E ratio, which represents diastolic function22. Kario K. et al. also showed that early-morning systolic BP surge was significantly associated with organ damage including left ventricular mass index, CIMT, and PWV23. Previous reports also indicated that MBPS is associated with the risk of stroke, independent of the 24-hour BP level in hypertensive patients24. In addition, the recent International Database on Ambulatory Blood Pressure Monitoring in Relation to Cardiovascular Outcome Study clearly confirmed that the risk of MBPS for cardiovascular events is only increased in the top tenth percentile of both sleep-trough surge (≥37 mmHg) and prewaking surge (≥28 mmHg)25. There is no consensus on the definition or on the threshold of pathological morning BP surge. We defined it in two ways: sleep-trough MBPS and prewaking MBPS. In the present study, both sleep-trough MBPS and prewaking MBPS were significantly associated with macular edema in RVO patients; among the cardiovascular risk factors and BP parameters known to be associated with RVO, MBPS is the strongest predictor of macular edema development. This association was particularly strong for BRVO patients. Although BP was a powerful risk factor of RVO and subsequent macular edema events, the association between MBPS and macular edema was independent of BP. Thus, the identification of a “high morning surge” group in RVO patients may have some clinical significance even after assessment of target organ damage.BP shows a diurnal variation, with a decrease during sleep and a surge in the morning27. Macular edema is a clinically important complication in that it causes visual impairment. Although not clearly understood yet, the mechanism of macular edema is known to be associated with the dysfunction of the blood-retinal barrier, inflammation within the vascular wall, and protein and fluid fluxes27. Several inflammatory mediators interact with the vascular wall through complex mechanisms28, which breaks the blood-retinal barrier and increases vascular permeability29, and developed macular edema. Although it is yet to be understood in great detail, MBPS, which is one indicator of BP variability, may be related to the aforementioned complex mechanisms, and may contribute in increasing the risk of developing macular edema in patients with RVO. In this study, arterial stiffness or carotid atherosclerosis was not significantly associated with macular edema; however, there were significant associations between MBPS and macular edema. Even after adjusting for ambulatory BP parameters, MBPS was an independent predictor for macular edema in RVO patients. Therefore, the present data indicate that exaggerated MBPS may contribute to the development of macular edema in RVO patents.Macular edema is a non-specific manifestation associated with retinal vascular disease in general, and a common final pathway of ocular diseases including RVOTo the best of our knowledge, this is the first study designed to evaluate the association between MBPS and macular edema in RVO patients. The level of the MBPS evaluated by 24-hour ABPM may be an important component in the management and treatment of patients with RVO. Additional studies will be needed to characterize BP variability, especially MBPS, in patients with RVO, and clarify its association with treatment response and ocular prognosis.30, and frequently present in CRVO eyes31. Next, we analyzed a relatively small sample size without a control group that was assessed for systemic cardiovascular risk factors without retinal disease. Generalizing our results to a broader group may be limited because we only recruited patients with RVO who underwent cardiovascular risk evaluation without a control group. Further research on a larger sample of RVO patients is needed to confirm the generalizability of our new findings. Finally, the relationship between MBPS and macular edema would be non-linear, given that MBPS within a certain level is physiologic. Although the maximum Youden’s J point would be a reasonable approach to find the optimal cut-off points, the ROC curves analyses for both the sleep-trough and prewaking MBPS did not have sufficiently high C-indexes to provide a confidence in the cut-off points. Since there were no previous reports proposing the optimal cut-off value for the MBPS, further researches are required to determine it.Some limitations of this study should be considered. The first limitation of this study was that it was designed as a retrospective observational study and a single-center experience. We may have overlooked the presence of RVO in asymptomatic patients, because only relatively symptomatic patients generally visit clinics and are diagnosed with RVO by ophthalmologists. Considering the ratio of patients with BRVO to patients with CRVO in this study, the development of macular edema was more common than the known prevalence of macular edema in RVO, which is around 15% in BRVO eyesIn conclusion, morning surge in BP was significantly associated with macular edema in patients with RVO. This study suggests that MBPS could be a new therapeutic target for managing and treating RVO and subsequent macular edema in RVO patients. Therefore, evaluating BP patterns using 24-hour ABPM may be useful in assessing ophthalmologic outcomes and may facilitate the initiation of appropriate treatment. Furthermore, it is necessary to control BP delicately to reduce diurnal variation and to improve the outcome of vascular events.Between January 2015 and February 2019, 123 patients who were diagnosed with RVO and received hospital treatment as well as cardiologic consultation were recruited in this study. They underwent 24-hour ABPM and CIMT and PWV measurements. Patients with RVO diagnosed more than 12 months prior to this study were excluded. Among these, six patients who had anti-phospholipid syndrome, three patients who had severe aortic valve stenosis, one patient who had active cancer, and one patient who underwent percutaneous coronary intervention owing to unstable angina were excluded. In addition, 36 patients who had inadequate 24-hour ABPM data were excluded as well  and we used a 12 × 9 mm 3D wide scan protocol, which comprises 256 B-scans, each comprising 512 A scans , and obtained retinal thickness map to identify macular edema. Macular edema was defined when central macular thickness was >300 μm. Patients who had high refractive errors , other concomitant ocular diseases , history of vitreoretinal surgery, and history of ocular trauma were excluded. In addition, patients with a low-quality OCT image that could not be interpreted due to media opacity such as severe cataract were excluded.RVO diagnoses were confirmed by retina specialists. All patients underwent comprehensive ophthalmologic exams such as determination of best-corrected visual acuity, measurement of intraocular pressure and refractive errors, slit-lamp biomicroscopy, and fundoscopy for both eyes. For detailed retinal exams, fundus photography, optical coherence tomography (OCT), and fluorescein angiography were performed. OCT is a non-invasive imaging modality capable of generating 2], office SBP and DBP, and current smoking status at the time of retinal vascular occlusion diagnosis, as well as traditional cardiovascular risk factors, which included history of diagnosis and treatment of hypertension, diabetes mellitus, dyslipidemia, coronary artery disease, and cerebrovascular attack. Data on antiplatelet drug use were also obtained and 10-year ASCVD risk was calculated from age, history of diabetes, sex, race, smoking status, total cholesterol level, high-density-lipoprotein cholesterol level, SBP, and treatment for hypertension32.Patients’ demographic and clinical characteristics were reviewed using electronic records. The following demographic and clinical characteristics were extracted: age, sex, BMI [kg/mThe following laboratory data were extracted: hemoglobin, total cholesterol, low-density-lipoprotein cholesterol, hemoglobin A1C, D-dimer, lipoprotein A, and homocysteine, which are known to be associated with thromboembolic events.33.Average daytime SBP/DBP was calculated during each patient’s diurnal cycle and average 24-hour and nighttime SBP/DBP were also obtained. The MBPS was calculated in two ways: sleep-trough MBPS, defined as the morning SBP measured over two hours after awakening minus the average of the lowest nocturnal SBP, and prewaking MBPS, defined as the morning SBP measured over two hours after awakening minus the SBP measured over the last two hours before awakening25.ABPM was performed in the non-dominant arm and recorded using an oscillometric device . ABPM was obtained at 15-minute intervals between 6 a.m. and 10 p.m., and was obtained at 30-minute intervals between 10 p.m. and 6 a.m. Patients were instructed to keep their forearms steady during measurements; they were also instructed to maintain their normal daily activities throughout the duration of the study. Awake time was defined as the actual waking time that patient recorded34. The procedure was performed with the patient in a supine position after resting for more than five minutes. Both brachia and ankles were wrapped with a cuff. Blood pressure, pulse volume waveform and heart rate were obtained simultaneously. Mean values of the left and right PWVs, ABI were used in the study analysis.For non-invasive measurement of arterial stiffness, brachial-ankle PWV was measured with an oscillometric sphygmomanometric device  according to the manufacturer’s recommendation35.CIMT of both common carotid arteries were measured with a high-resolution ultrasonography iE33 ultrasound system  equipped with an 11 L linear transducer. An experienced diagnostic medical sonographer recorded the CIMT measurements using semi-automated edge detection software, which calculates the mean CIMT value from a left and a right common carotid artery at end diastole in a 10-mm segment located 10 mm proximal to the carotid bulb. Mean CIMT values of the left and right carotid arteries were used in the study analysis. Carotid plaque was identified as a focal increase in the CIMT greater than 15 mm or greater than 50% of the surrounding wallGiven that physiologic levels of MBPS would not increase the risk of macular edema, the sleep-trough MBPS and prewaking MBPS were dichotomized into binary variables using their respective cut-off values, which were derived from the ROC curve analyses. The best cut-off points were determined at the maximum Youden’s J points of their respective ROC curves.t-test was used to compare normally distributed continuous variables, and the Mann–Whitney U-test was used to compare continuous variables with a skewed distribution.Patients were divided into 2 groups according to the presence of macular edema to compare the baseline characteristics. All categorical data were summarized as frequencies and percentages, and continuous variables were presented as means and standard deviations. The Pearson chi-squared test was used for comparison of categorical variables, and the Fisher’s exact test was used for comparison of categorical variables with 20% or more of the expected cell frequencies ≤5. Student’s Univariate logistic regression analyses were used to evaluate the associations of clinical factors and ABPM parameters including 24-hour SBP, daytime SBP, nighttime SBP and MBPS with the presence of macular edema. Multivariate logistic regression analyses were performed to identify variables that were strongly associated with macular edema in the presence of confounding factors. Covariates of the multivariate logistic regression models included sex, past histories of diabetes, hypertension and dyslipidemia, current smoking, serum low density lipoprotein cholesterol levels, BMI, average PWV, carotid IMT, the binary MBPS and the average SBPs in ABPM.A multivariate logistic regression model including these covariates was reduced to a simpler model through a backward variable selection process (with a cut-off criterion of p > 0.1) to prevent the occurrence of overfitting biases and identify the strong determinants of macular edema. To prevent problems related to multicollinearity, we tested 6 different multivariate logistic regression models, each of which included one of the 2 binary MBPS (sleep-trough and prewaking) and one of the 3 average SBPs in ABPM  with the other covariates, and kept the variance inflation factors <2 in the original multivariate model. The goodness of fit of the multivariate logistic regression models were evaluated using the C-index and Akaike information criterion.All statistical analyses were conducted using the statistical software R-3.5.2 and its packages including “rms”, “ROCR”, “descr” and “tableone”. A p-value < 0.05 was considered significant.Supplementary Information.


The present study aimed to explore the relationship between electrocardiographic (ECG) and pulse wave analysis variables in patients with hypertension (HT) and high normal blood pressure (HNBP). A total of 56 consecutive, middle-aged hypertensive and HNBP patients underwent pulse wave analysis and standard 12-lead ECG. Pulse wave velocity (PWV), heart rate, intrinsic heart rate (IHR), P wave and QT interval durations were as follows: 7.26 ± 0.69 m/s, 69 ± 11 beats/minute, 91 ± 3 beats/minute, 105 ± 22 mm and 409 ± 64 mm, respectively. Significant correlations were obtained between PWV and IHR and P wave duration, respectively, between early vascular aging (EVA) and P wave and QT interval durations, respectively. Linear regression analysis revealed significant associations between ECG and pulse wave analysis variables but multiple regression analysis revealed only IHR as an independent predictor of PWV, even after adjusting for blood pressure variables and therapy. Receiver-operating characteristic (ROC) curve analysis revealed P wave duration (area under curve (AUC) = 0.731; 95% CI: 0.569–0.893) as a predictor of pathological PWV, and P wave and QT interval durations were found as sensitive and specific predictors of EVA. ECG provides information about PWV and EVA in patients with HT and HNBP. IHR and P wave durations are independent predictors of PWV, and P wave and QT interval may predict EVA. Central and Eastern Europe are regions with a high cardiovascular burden . RomaniaArterial stiffness, the consequence of arteriosclerosis and subclinical atherosclerosis, is involved in cardiovascular disease development and, if assessed, can improve risk prediction, especially in hypertensive and intermediate risk patients ,5,6,7,8.Heart rate and electrocardiography, especially repolarization variables, including QT interval duration, can also provide useful information in cardiovascular risk assessment ,11,12,13Chronological age has some limitations when assessing cardiovascular risk . VasculaArterial stiffness is characterized by a decrease of elastin content, as well as by an increased production and accumulation of collagen within the arterial wall . ArteriaConsidering the importance of prophylactic measures in hypertensive patients and the limits of the classical cardiovascular risk factors in predicting cardiovascular events, new, simple, inexpensive surrogate biomarkers are needed. Our objective is to explore the relationship between electrocardiographic (ECG) and pulse wave analysis variables in patients with hypertension and high normal blood pressure. We hypothesized that ECG variables can reveal increased arterial stiffness and early vascular aging in patients with elevated blood pressure values.The study included 56 consecutive hypertensive and high normal blood pressure (HNBP) patients, recruited from the Military Hospital Timisoara in the period of July 2016–March 2017.Patients with essential hypertension and high normal blood pressure, aged between 18 and 55 years, were included. Essential hypertension and high normal blood pressure were diagnosed according to the criteria of the European Society of Cardiology . The mosThe investigations were performed considering the principles outlined in the Declaration of Helsinki . The stuThe patients underwent pulse wave analysis and standard 12-lead electrocardiogram (ECG). Additional data related to diagnosis and therapy of the patients were available from medical records. The investigations were performed independently by different skilled investigators, who were not aware of the patient’s diagnosis and data.Pulse wave analysis, including pulse wave velocity (PWV), augmentation index and pressure , vascular age and central and peripheral blood pressure were assessed using a Mobil-O-Graph , a noninvasive, cuff-based, one site, validated device. Pulse wave velocity is the speed at which the arterial pulse wave propagates along an arterial segment . AugmentStandard ECG was recorded at a paper speed of 25 mm/s. Resting heart rate (HR), P wave (P), PR (PR—the time from the onset of the P wave to the start of the QRS complex) and QT interval duration (QT) were automatically measured. QT was corrected considering heart rate, according to the Bazett formula, resulting in heart rate corrected QT interval (QTc). Intrinsic heart rate (IHR = 118 − (0.57 × age)) and the difference between IHR and resting heart rate (DHR) were also calculated . The ampp < 0.05 was considered statistically significant. PASS 2019  was used to conduct a power analysis before the study to calculate the sample size needed for the present study [Categorical data are given as numbers and percentages, continuous data as means ± standard deviation. Correlations , univariate and multivariate linear regression analysis and receiver-operating characteristic curve (ROC) analysis were used as statistical methods. Analyses were performed using IBM SPSS base edition . Normality testing of dependent variables was performed using Q–Q plots, skewness and kurtosis. A nt study .The study included middle-aged patients (48 ± 6 years) with hypertension and high normal blood pressure, most of them male (57%) . Table 1Normality testing of dependent variables using Q–Q plots, skewness and kurtosis revealed that the data were approximately normally distributed.Correlations were calculated for all ECG and pulse wave analysis variables. Significant correlations were obtained just between pulse wave velocity (PWV) and intrinsic heart rate (IHR) and the difference between IHR and resting heart rate (DHR) . P wave Linear regression analysis revealed significant associations of IHR with PWV and early vascular aging (EVA). A prolonged QTc interval (exceeding 450 ms: QTc450) was significantly associated with EVA and augmentation index (AI). The association EVA-long QTc lost its significance after adjusting for systolic blood pressure (SBP). Significant associations were also found between AI and S wave in V1 .Multiple linear regression analysis revealed only IHR as an independent predictor of PWV, even after adjusting for blood pressure variables and heart rate and blood pressure-lowering therapy . IHR losROC curve analysis revealed P wave duration as a predictor of pathological pulse wave velocity (pPWV) . P wave The present study reveals significant correlations and associations between electrocardiographic biomarkers and pulse wave velocity and early vascular aging, respectively, in middle-aged patients with hypertension and high normal blood pressure. Intrinsic heart rate and P wave duration were found as independent predictors of pulse wave velocity, while QT interval and P wave durations as independent predictors of early vascular aging, confirming our hypothesis that ECG variables can reveal increased arterial stiffness and early vascular aging in patients with elevated blood pressure values.The influence of heart rate on PWV measurements remains controversial, with conflicting results observed in both acute and epidemiological studies ,31,32,33Several other studies reported links between electrocardiographic and pulse wave analysis biomarkers. QTc interval, prolonged in patients with Systemic Lupus Erythematosus, was related to subclinical atherosclerosis, measured by carotid-femoral pulse-wave velocity, after controlling for age and hypertension . A study2, with a high prevalence of overweight (25%) and obesity (25%), which might be associated with a sedentary lifestyle in our study, but P wave duration exceeded 120 ms only in 7 patients (13%). The possible link could also be left atrial volume. A significant relationship between carotid arterial stiffness and left atrial volume was reported previously in patients with untreated hypertension [P wave duration was previously correlated with arterial stiffness variables, related to interatrial block, in overweight patients . Body martension . Ruaengsrtension . A studyrtension . Other frtension . Our stuStudy limitations: arterial stiffness and early vascular aging are surrogate endpoints for cardiovascular events but using them demonstrates the prophylactic character of our study. The cross-sectional study design does not assure causality between the evaluated variables. The sample size is relatively small but the power analysis demonstrated an appropriate number of patients. Another limitation is related to the selection of study participants, including hypertensive patients receiving antihypertensive and heart rate-lowering drugs, which may question the validity of some results. However, the results did not change after controlling for blood pressure and heart rate-lowering therapy in the multiple regression analysis. The study lacks echocardiographic parameters and information about physical activity, which were not available for the patients included in the study but will be the aim of a future study. Mobil-O-Graph estimates pulse waves using an oscillometric method, which differs from the most widely applied applanation tonometry. Previous validation studies in different study populations, including hypertensive patients, revealed acceptable agreement between Mobil-O-Graph derived variables and invasive and noninvasive measurements and a good reproducibility of the technique ,51,52.Strengths of the study lie in the recruitment of real-world, middle-aged participants with grade 1–3 hypertension and high normal blood pressure, with a PWV increased for age in 21.4% of the participants and with both high and low cardiovascular risk, providing insights in terms of heart-vascular coupling rather than substitution of arterial stiffness measurement by ECG technique. As far as we know, our study is the first one reporting a significant relationship between intrinsic heart rate and arterial stiffness, respectively, as well as between P wave duration and EVA. The clinical implications of the present study are related to the prognostic importance of increased arterial stiffness and early arterial aging for cardiovascular risk. Simple, cost-effective, reproducible, quick to determine biomarkers, such as intrinsic heart rate, an index of sinus node function regardless of autonomic control, the duration of the P wave and QT interval can provide valuable, noninvasive information related to large vessels, arterial age and cardiovascular risk stratification in treated hypertensive patients, enabling personalized diagnosis and treatment of each patient, providing further targets for strategies to improve clinical outcomes and enable selection of patients requiring more aggressive therapy. Larger studies are needed in hypertensive patients to confirm the findings of the present study, to demonstrate that aging related functional and structural changes in the heart and artery are simultaneous and to reveal any connection between interstitial fibrosis related to aging associated remodeling of the myocardium, collagen content in the sinus node and arterial wall. Further prospective studies must confirm the synergistic prognostic effect of pulse wave velocity, early vascular aging, intrinsic heart rate, P wave and QT interval durations in hypertensive patients.ECG provides information about pulse wave velocity and early vascular aging in patients with hypertension and high normal blood pressure. IHR and P wave duration are independent predictors of pulse wave velocity in patients with hypertension and high normal blood pressure. P wave and QT interval duration are sensitive and specific predictors of EVA.


No significant associations were found between UA and ABI < 0.9 or baPWV > 1822 cm/s. Multivariate stepwise linear regression analysis showed that a high UA level correlated with high LAD , high observed/predicted LVM  and low LVEF . No significant associations between UA and low ABI and high baPWV were found. Conclusion. A high serum UA level was associated with a high LAD, high observed/predicted LVM and low LVEF in the patients with CKD. A high serum UA level may be correlated with abnormal echocardiographic parameters in patients with stage 3–5 CKD.Background. Hyperuricemia is common in patients with chronic kidney disease (CKD), and this may lead to poor cardiovascular (CV) outcomes. The aim of this cross-sectional study was to assess associations among serum uric acid (UA) and echocardiographic parameters, ankle-brachial index (ABI), and brachial-ankle pulse wave velocity (baPWV) in patients with CKD. Methods. A total of 418 patients with CKD were included. The echocardiographic measurements included left atrial diameter (LAD), left ventricular ejection fraction (LVEF) and the ratio of observed to predict left ventricular mass (LVM). ABI, baPWV and medical records were obtained. Results. Multivariable forward logistic regression analysis showed that a high UA level was significantly associated with LAD > 47 mm (odds ratio [OR], 1.329;  Cardiovascular (CV) abnormalities such as left ventricular hypertrophy (LVH) and left ventricular (LV) dysfunction are common in patients with chronic kidney disease (CKD) due to chronic inflammation, activation of the renin-angiotensin-aldosterone system and changes in hemodynamics . In addiLeft atrial size, which reflects LV diastolic dysfunction, has been associated with various adverse CV events , and lefUric acid (UA) is the end product of purine metabolism in humans, and approximately 70% of UA load is eliminated through the kidneys ,10,11,12The study was conducted at a regional hospital in southern Taiwan, and consecutively enrolled 540 pre-dialysis patients with CKD stages 3 to 5 according to the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines from our Outpatient Department of Internal Medicine from January 2007 to May 2010 . We excl3 − LVIDd3] − 13.6 g [2.7) + 0.64 × stroke work − 18.07 × sex . Stroke work (in gram meters) was estimated as systolic blood pressure × stroke volume product × 0.0144. Inappropriate LVM was defined as when observed/predicted LVM > 128%, i.e., when the observed LVM was >28% of the predicted value [One experienced cardiologist blind to all other data performed all of the echocardiographic examinations using a VIVID 7 system . During the examinations, the patients were placed in the left decubitus position and asked to breathe quietly. Two-dimensional M-mode images were recorded from standardized views. The echocardiographic measurements included LV posterior wall thickness in diastole (LVPWTd), LV internal diameter in diastole (LVIDd), interventricular septal wall thickness in diastole (IVSTd), and left atrial diameter (LAD). LV systolic function was assessed by LV ejection fraction (LVEF). Observed LV mass (LVM) was calculated using the modified Devereux method, i.e., LVM = 1.04 × [(IVSTd + LVIDd + LVPWTd)− 13.6 g . Predict− 13.6 g : predicted value ,20.baPWV and ABI were measured using an ABI-form device that allowed for simultaneous blood pressure measurements in the ankles and arms. Occlusion and monitoring cuffs were placed snugly around both upper arms and lower extremities with the patients placed in the supine position. ABI was calculated as ankle/arm systolic blood pressure using the lowest ankle systolic blood pressure value. baPWV was calculated as transmission distance/transmission time. The lowest of the bilateral ABI values and highest of baPWV values were used for each patient. Peripheral artery occlusive disease was defined as an ABI < 0.9 in either leg.Demographic and medical data including age, sex and comorbidities were obtained from the patients’ medical records or interviews. Fasting blood samples were collected from all patients within 30 days of enrollment, and an autoanalyzer  was used to obtain laboratory data. This autoanalyzer was also used to measure serum creatinine level with the compensated Jaffé method using an isotope-dilution mass spectrometry-traceable calibrator . EstimatThirty patients were randomly selected for evaluation of the reproducibility of LVM. Mean percent error was calculated as the absolute difference divided by the average of the two observations.p value was less than 0.05. Statistical analysis was performed using SPSS version 19.0 for Windows . Data are expressed as percentage, mean ± standard deviation or median (25–75th percentile) for triglycerides. Differences between groups were analyzed using the chi-square test for categorical variables and the independent t-test for continuous variables. Multiple logistic and linear regression analyses were used to identify the factors associated with ABI, baPWV and echocardiographic parameters. A difference was considered significant if the p = 0.017) and observed/predicted LVM > 128% , but not LVEF < 50% , ABI < 0.9  or baPWV > 1822 cm/s .A total of 418 patients with CKD who were not receiving dialysis were included . Sex-specific median UA values were used to stratify the patients into two groups . Comparisons of the clinical characteristics between the two study groups are shown in p = 0.002) was significantly associated with LAD > 47 mm. In addition, diabetes mellitus, low mean artery pressure, high UA level , and the use of diuretics were significantly associated with observed/predicted LVM > 128%. Coronary artery disease, congestive heart failure, high UA level , high fasting glucose, and lower rates of the use of calcium channel blockers and diuretics were significantly correlated with LVEF < 50%. Old age, high fasting glucose, and the use of ACEIs and/or ARBs were significantly associated with ABI < 0.9. Finally, old age, diabetes mellitus, congestive heart failure, high mean artery pressure, and high fasting glucose were significantly associated with baPWV > 1822 cm/s.r = 0.228, p < 0.001) and observed/predicted LVM , and negatively correlated with LVEF .p < 0.001) and low eGFR were significantly correlated with high LAD. We have further analyzed the determinants of the ratio of LAD to body surface area (BSA) using multivariable stepwise linear regression analysis, and still found that high UA level  was significantly correlated with high LAD/BSA. p < 0.001) and the use of diuretics were significantly associated with high observed/predicted LVM. Coronary artery disease, congestive heart failure, high UA level , high fasting glucose, and lower rates of the use of calcium channel blockers and diuretics were significantly associated with a low LVEF. Old age, coronary artery disease, and congestive heart failure were significantly associated with a low ABI. Lastly, old age, diabetes mellitus, congestive heart failure, high mean artery pressure, and high fasting glucose were significantly associated with a high baPWV.Furthermore, young age, congestive heart failure, low mean artery pressure, high UA level , a biomarker of renal failure and CVD, was associated with LVH and LV diastolic dysfunction. The authors suggested that NGAL is involved in the alterations of the cardiac extracellular matrix, and further contributed to ventricular remodeling . AbnormaThere are several limitations to this study. First, as a cross-sectional study, the results may not precisely reflect the cause–effect relationship between UA and echocardiographic parameters. Second, predicted LVM was calculated using age, sex, height and stroke work. Therefore, single blood pressure measurements may have had large impacts on predicted LVM. An average 24 h ambulatory blood pressure measurement may have been more appropriate to calculate LVM than single clinic blood pressure measurements. Third, the use of antihypertensive medications may have influenced LV geometry and functional parameters. For ethical reasons, we did not withhold any drugs during the study. Therefore, to investigate the influence of the use of medications, we included various classes of antihypertensive drugs in the analysis, and still found that UA was significantly associated with abnormal echocardiographic parameters. In addition, some important data were lacking, such as transmitral flow velocity, mineral metabolism biomarkers, and treatment. Lastly, as previously mentioned, further study including data on mineral metabolism is needed.In conclusion, in our patients with CKD stages 3–5, we found significant associations among hyperuricemia and high LAD, high o/p LVM and low LVEF. However, a high serum UA level was not closely associated with ABI or baPWV. A high serum UA level may be correlated with abnormal echocardiographic parameters in patients with stage 3–5 CKD. Further longitudinal studies with adjustments for confounding factors are warranted to clarify the relationship between UA and atherosclerosis and arterial stiffness in patients with CKD.


In the present study the blood expression level of inflammatory response and autoimmunity associated long non-coding RNAs (lncRNAs) were compared in patients with different chronic respiratory diseases and investigated whether they could be used as biomarkers in these diseases.In the discovery cohort, the gene expression level of 84 lncRNAs were measured in the blood of 24 adult patients including healthy controls and patients with asthma and COPD. In the replication cohort the expression of 6 selected lncRNAs were measured in 163 subjects including healthy controls and adults with allergic rhinitis, asthma, COPD and children with asthma. It was evaluated whether these lncRNAs can be used as diagnostic biomarkers for any studied disease. With systems biology analysis the biological functions of the selected lncRNAs were predicted.OIP5-AS1, HNRNPU, RP11-325K4.3, JPX, RP11-282O18.3, MZF1-AS1 were selected for measurement in the replication cohort. Three lncRNAs  expressed significantly higher in healthy children than in adult controls. All the mean expression level of the 6 lncRNAs differed significantly between adult allergic rhinitis patients and controls. RP11-325K4.3, HNRNPU and OIP5-AS1 expressed higher in allergic asthma than in non-allergic asthma. COPD and asthma differed in the expression of RP11-325K4.3 from each other. In examining of the lncRNAs as biomarkers the weighted accuracy (WA) values were especially high in the comparison of healthy controls and patients with allergic rhinitis. OIP5-AS1 and JPX achieved 0.98 and 0.9 WA values, respectively, and the combination of the selected lncRNAs also resulted in a high performance (WA = 0.98). Altogether, OIP5-AS1 had the highest discriminative power in case of three out of six comparisons.In the discovery cohort, the mean expression of 27 lncRNAs showed nominally significant differences in at least one comparison. Differences were detected in the expression of circulating lncRNAs in chronic respiratory diseases. Some of these differences might be utilized as biomarkers and also suggest a possible role of these lncRNAs in the pathomechanism of these diseases. The lncRNAs and the associated pathways are potential therapeutic targets in these diseases, but naturally additional studies are needed for the confirmation of these results. Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and allergic rhinitis cause an enormous burden on the societies and are considered as major non-communicable diseases . Over 1 While rhinitis is characterized by an inflammation of the upper airways, asthma and COPD are featured by an inflammation of the lower airways. Although these are separate disease entities, there is a considerable overlap between them . Often aIn addition, all these diseases, especially asthma and COPD have several endotypes, i.e. different molecular pathomechanisms can lead to similar phenotypes. Presently, there are no or only very few biomarkers for accurate classification of these diseases or to follow-up the responses to the therapies , 15.Numerous studies have confirmed that 70–90% of the human genome is transcribed into RNA but only 1.2% has protein coding ability. Long non-coding RNAs (lncRNAs) are greater than 200 bp in length, building a major part of non-coding RNAs but in the meantime the least characterized . DependiIn the present study, first we measured the gene expression level of 84 inflammatory response and autoimmunity associated lncRNAs in the blood of patients with mild or moderate  1–3) and severe (GINA 4–5) asthma, COPD and control patients (discovery cohort). Then, based on these results and the scientific literature we selected 6 lncRNAs and compared their expression in an expanded population of patients with different chronic respiratory diseases including pediatric and adult asthma, mild and severe asthma, COPD, allergic rhinitis and in corresponding healthy controls. We also compared the expression of these lncRNAs in different subgroups of these diseases and investigated whether they could be used as biomarkers. Finally, we performed a systems biology analysis aiming to predict the biological functions associated with these lncRNAs.www.ginasthma.org), as described previously [https://goldcopd.org) designation. Control subjects were healthy donors. Some characteristics of these subjects are summarized in Table Our research consisted of two stages. In the discovery cohort, 24 adult patients were involved, out of which 6 had mild or moderate asthma GINA 1–3), 6 severe asthma (GINA 4–5), 6 COPD and 6 were healthy controls. Participants with asthma were recruited from Asthma ambulance of National Korányi Institute of TB and Pulmonology and from the Department of Pulmonology of Semmelweis University. Asthmatic subjects were diagnosed based on Global Initiative for Asthma (GINA) guidelines  and 64 mild or moderate asthma (GINA 1–3). Adult patients with allergic rhinitis were selected from patients of five Hungarian allergic outpatient centers with documented ragweed allergy with clinical history for at least 2 years with peak symptoms in August–September. Detailed description of this project, named DesensIT can be found elsewhere . These pSubjects were all Caucasian with about 5% Gypsy origin based on Hungarian statistical databases. Written informed consent was provided by all participants or parent/guardian at the time of recruitment. The study was conducted according to the designations determined in the Declaration of Helsinki and approved by the Hungarian Scientific and Research Ethics Committee of the Medical Research Council .Whole blood samples (2.5 ml) were collected in PAXgene Blood RNA Tube  to avoid rapid RNA degradation and to stabilize the intracellular RNA. Thereafter, PAXgene tubes were carefully inverted 8 to 10 times and stored for 2 h to 3 days at room temperature before long-term storage in freezer (− 20 °C).260/A280 ratio, 1.8–2.2 was accepted as pure.Prior to RNA extraction, after tubes were removed from − 20 °C, they were allowed to thaw and incubated at room temperature for 2 h. RNA purification was carried out according to the protocol in the manual of PAXgene Blood RNA Kit. RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer  and the purity of RNA was determined based on the A2 PreAMP cDNA Sythesis Kit . In the replication study, because of the TaqMan Non-Coding experimental design, High Capacity RNA-to-cDNA Kit  was used.In the discovery cohort, before the reverse transcription, due to the low amount of isolated RNA, amplification was carried out with RT2 lncRNA PCR Array . This panel consists of 84 verified, pre-validated primer pairs specific for the target genes associated with inflammatory response and autoimmunity. The array also contains 5 primer pairs for housekeeping genes , 1 for the detection of human genomic DNA contamination, 3 for reverse transcription control and 3 for positive PCR control. RT2 SYBR Green ROX qPCR Master Mix  was used for real-time PCR reaction on Applied Biosystems 7900HT instrument with 96-Well Block Module.The pre-amplified cDNA was measured with a prefabricated Human RTJPX: Hs0139517_g1; AC016629.8: Hs03678951_m1; HNRNPU: Hs00402532; OIP5-AS1: Hs01587687_g1; RP11-282O18.3: Hs00416786_m1; RP11-325K4.3: Hs01594146_s1) and Gene Expression Assays  with Gene Expression Master Mix . The measurements were performed in duplicate on 384-well plate (ABI 7900HT) in 10 μl of total PCR Reaction Mix volume.In the replication cohort, expression level of 6 lncRNAs and 2 reference genes were detected with TaqMan Non-coding RNA Assays . Normalized RNA expression levels were calculated using the formula 2 package . For thaTo analyze the potential usefulness of the six lncRNAs (chosen for the replication cohort) as diagnostic biomarkers in the studied chronic respiratory diseases, we created Naïve Bayesian classifiers using the e1071 package in R . The modAs the number of patients varied highly in the different subgroups, we assessed the performance of the classification models by computing their weighted accuracy  defined by the following formula:We calculated the confusion matrix for each model by a leave-one-out cross-validation scheme as the following: For a given comparison, we left out one sample and trained the model using all other samples. Next, we predicted the class of the left-out sample using a default cut-off of probability 0.5, and compared the predicted class label with the true one of that sample. Each step of this procedure yielded one element of the confusion matrix based on which we computed the weighted accuracy as described above.We performed a systems biology analysis to identify the putative functional pathways and Gene Ontology terms associated with each of the six lncRNAs (chosen for the replication cohort). The overview of this analysis can be seen in Additional file XIST, which is involved in the inactivation of the X chromosome in women, and NAV2-AS5, which is mainly expressed in the testis. These two genes were excluded from the selection. Interestingly, there was no such gender dependent difference in the expression of JPX, although according to the scientific literature this lncRNA is transcribed within the X-inactivation center and activates the expression of the XIST gene [In the discovery group, using a prefabricated human inflammatory response and autoimmunity array, the expression levels of 84 lncRNAs were measured in the blood of 6 patients with mild or moderate asthma GINA 1–3), 6 with severe asthma (GINA 4–5), 6 with COPD, and in 6 healthy controls  have been indirectly associated with eosinophil asthma [HNRNPU showed increased expression in severe asthma compared with mild asthma, while OIP5-AS1 showed increased expression in COPD compared to asthma . It must be added that during our study the transcript variations of the HNRNPU which are not translated to a protein and were considered as lncRNAs were withdrawn from the database, in this way, we probably investigated the expression of a protein-coding gene. But, for the sake of simplicity, in this paper, we refer to this gene also as an lncRNA.In previous studies two lncRNAs  expressed significantly higher in pediatric controls than in adult controls  were significantly higher than in COPD, three  than in asthma, five  than in non-allergic asthma and one (OIP5-AS1) than in allergic asthma. Adult allergic and non-allergic asthma differed in the expression of three lncRNAs from each other, RP11-325K4.3, HNRNPU and OIP5-AS1 expressed higher in allergic asthma. COPD and asthma differed in the expression of one lncRNA from each other. RP11-325K4.3 expressed significantly higher in the blood of asthmatics than in patients with COPD. The comparisons where significant differences (adjusted P < 0.05) were found can be seen in Additional file The most and largest differences were found between adult allergic rhinitis and control patients Fig. . In thesJPX did not show a gender specific expression.In contrast to the discovery cohort, in this expanded population none of the lncRNAs showed association with asthma severity. No differences were found between pediatric asthma and controls. In the replication cohort similarly to the discovery cohort, Next, we also analyzed whether the expression of these lncRNAs differ in different subgroups of asthma. No differences were found when asthmatic patients were stratified according to their lung functions (FEV1 < 80% vs. FEV1 > 80%), inhaled corticosteroid usage (regular vs. non-regular), severity, and controllability (controlled vs. non-controlled). We also tested whether the expression levels of these lncRNAs correlated with the blood eosinophil or neutrophil levels but found no correlation (data not shown).Next, we investigated, whether these lncRNAs can be used as diagnostic biomarkers for any studied chronic respiratory disease. The results can be seen in Fig. OIP5-AS1 alone, (2) using all six lncRNAs, which is the same model as (3) using all significant lncRNAs with respect to the given comparison). Clearly, these models utilized the high discriminative power of OIP5-AS1. Comparing adult COPD and adult patients with allergic rhinitis, using all five significant lncRNAs also resulted in a high performance (WA = 0.85).Classifying adult allergic rhinitis patients and adult controls, three models achieved a very high performance (WA = 0.98 in case of (1) using In certain cases, combining all six lncRNAs resulted in significantly higher performance than any individual lncRNAs. Comparing adult allergic rhinitis and asthmatic patients, the best model using individual lncRNAs resulted in a WA of 0.53, however, combining all six lncRNAs resulted in a WA of 0.7. Similarly, comparing adult COPD and adult asthmatic patients, the best individual model had a WA of 0.53, and the full model had 0.61, respectively.In other cases, using the combination of those lncRNAs that showed statistically significant expression differences resulted in a slightly higher performance than the full model. Namely, in case of the aforementioned comparison of adult COPD and adult allergic rhinitis patients, and in case of comparing adult allergic asthmatic and non-allergic asthmatic patients .OIP5-AS1 lncRNA had the highest discriminative power in case of three out of the six comparisons. Moreover, comparing adult patients with allergic and adult non-allergic asthmatic patients, the model using the individual OIP5-AS1 had the highest performance of all models .The Finally, we aimed to predict the biological functions associated with the six lncRNAs that were selected for the replication study in order to gain insight into their underlying biological processes , cell activation involved in immune response (FDR = 0.084), the neutrophil degranulation pathway (FDR = 0.035) and the innate immune system pathway (FDR = 0.035). HNRNPU is predicted to have an effect on several FGFR2 related pathways, namely the signaling by FGFR2 in disease pathway (FDR = 0.094), the signaling by FGFR2 IIIA TM pathway (FDR = 0.094) and the FGFR2 mutant receptor activation pathway (FDR = 0.094). MZF1-AS1 is predicted to affect several pathways that regulate cell cycle, cell differentiation/development, proliferation and metabolism, e.g. the PI3K−Akt signaling pathway (FDR = 0.071), the focal adhesion −PI3K−Akt−mTOR−signaling pathway (FDR = 0.071) and the nuclear receptors meta —pathway (FDR = 0.014). RP11-325K4.3 is predicted to affect developmental processes, such as keratinization (FDR = 0.01). RP11-282O18.3 is predicted to influence amino acid metabolism (FDR = 0.063). In case of OIP5-AS1, the method did not identify any biological processes or pathways. However, it was predicted that genes that are annotated with the transport vesicle and the exocytic vesicle cellular components were significantly enriched among its predicted targets (FDR = 0.04).The results can be seen in Fig. OIP5-AS1, with a very high potency to discriminate patients with severe pollen allergy from non-allergic patients.In the present study we measured the expression of inflammatory response and autoimmunity associated lncRNAs in the blood of patients with different chronic respiratory diseases. We detected several differences and identified an lncRNA, TUG1, MALAT1, NEAT1 and MEG3, all of them were found to be associated with asthma in different studies [GAS5 and its antisense GAS5-AS1 in certain comparisons. These lncRNAs were not selected for the replication study, because the differences were not exceedingly significant  and at the time of the selection no data about their roles were available in the scientific literature. But, the fact that in a later study the expression of GAS5 was found to be higher in asthmatics, and knock-down of GAS5 significantly decreased airway hyperresponsiveness in asthmatic rats, together with our results indicate their possible roles in asthma [In the stage I or discovery study, a smaller number of patients with chronic respiratory diseases and controls were screened with 84 lncRNAs. According to the results of the measurements and data from the scientific literature, 6 lncRNAs were selected for testing on an expanded population. During our study, several studies have been published where the expression of lncRNAs were tested in different chronic respiratory diseases, mainly in asthma, and several differences were found. Some of them were also measured in our stage I study. In our discovery cohort we did not find differences in any comparison in the expression of  studies –33. Althn asthma .OIP5-AS1, HNRNPU and JPX are the best studied. Using a combination of three biological networks we also carried out a bioinformatic analysis to predict the biological function and the associated GO terms of these six lncRNAs.The expression of the selected lncRNAs were measured in an expanded population. The largest differences were found between controls and patients with allergic rhinitis. The expression of all selected lncRNAs were significantly higher in patients with allergic rhinitis. Among these, OIP5-AS1 is a conserved gene acting as a sponge for multiple cellular RNAs and microRNAs, regulating mitosis, maintaining cell proliferation, and functioning as an oncogene in several cancers [OIP5-AS1 by binding to miR-200b, also regulates indirectly the expression of ACE2, the receptor for COVID-19, but its implication in the infection has not yet been studied [OIP5-AS1. In our study its mean expression level was significantly higher in all diseases, where allergy was involved, e.g. in allergic rhinitis vs. COPD or in allergic asthma vs. non-allergic asthma, but its highest level was measured in allergic rhinitis. cancers –38. Inte studied . It was  studied , 29, butHNRNPU gene is more complicated. It has several aliases in the databases, and earlier it was determined that there are several transcripts from its genome locus, including those that are not translated into a protein , but recently these have been withdrawn from the databases [HNRNPU gene is probably a protein-coding gene. The function of the protein, however, is similar to several lncRNAs, namely it binds nucleic acids, participates in the formation of ribonucleoprotein complexes in the nucleus with heterogeneous nuclear RNA and plays important role in three-dimensional genome organization. As we have measured gene expression (i.e. RNA), we think that the characteristics of an RNA whether it is translated into a protein or not, cannot influence its possible use as a biomarker, thus we assume that involving this protein-coding gene in the evaluations did not cause bias in our results. HNRNPU was found to be implicated in several processes, including regulation of the innate immunity, proliferation and several diseases like cancers and eosinophilic asthma [HNRNPU was associated with several FGFR2 related pathways.The situation with the atabases . In thisc asthma , 41–45. JPX is that it serves as a molecular switch in the X chromosome inactivation in females, but studies also show that it is implicated in different cancers and can act as an oncogene in certain cases while as a tumor suppressor in others [JPX is predicted to influence several immune-related processes, such as immune effector process, cell activation involved in immune response, the neutrophil degranulation pathway and the innate immune system pathway. The lncRNA MZF1‐AS1 was identified as a transcriptional regulator of proline synthesis and neuroblastoma progression and was associated with several pathways that regulate cell cycle, cell differentiation/development, proliferation and metabolism [The best-known role of n others , 46. JPXtabolism .RP11-325K4.3 and RP11-282O18.3 until now no publications have been found. Our bioinformatic analysis predicted that RP11-325K4.3 was associated with developmental processes, while RP11-282O18.3 with amino acid metabolism.In respect of HNRNPU, RP11-325K4.3, JPX) showed significantly higher expression in children than in adults. As HNRNPU and JPX are both implicated in cell proliferation, their increased blood levels in children suggest that they might have roles in their development. The function of RP11-325K4.3 has not yet been clarified, but its increased level in children also confirms its possible role in developmental processes found in our bioinformatic analysis. It is also noteworthy, that this was the only lncRNA that showed significant difference between adult asthma and COPD. The expression of the investigated genes, however, did not differ between asthmatic children and controls.Some of the studied genes (According to a study where ageing-associated changes in the expression of lncRNAs in adult human tissues were investigated (between 20 and 79 years of age) no lncRNA was identified in the blood that showed age-dependent expression . It suggRP11 − 325K4.3 in COPD vs. asthma (adjusted P = 0.0092) and COPD vs. allergic rhinitis (adjusted P = 0.0002)), still its discriminative power, due to its high variance, was low (weighted accuracy (WA) = 0.49 and 0.52, respectively). In these cases, the given lncRNA is not suitable for being a circulating blood biomarker, but these differences suggest that it might have a role in the pathomechanism of one of these diseases or their endotypes. In some cases, however, the lncRNAs alone or in combinations achieved very high performances. The WA values were especially high in the comparison of healthy adult controls and adult patients with allergic rhinitis. OIP5-AS1 and JPX achieved 0.98 and 0.9 WA values, respectively, and the combination of the selected lncRNAs also resulted in a high performance (WA = 0.98). The WA values were also high in the comparison of COPD and allergic rhinitis , although 30% of the COPD patients also had allergic rhinitis. The WA value was not very high in comparison of allergic vs. non-allergic asthma  but because there is still no solid biomarker in the differential diagnosis of these two endotypes, an additional biomarker might be worth testing [Perhaps, the most interesting finding of this study is the large significant differences between healthy controls and allergic rhinitis patients in the expression of the selected circulating lncRNAs. Until now no paper has been published about the human blood levels of lncRNAs in allergic rhinitis. We also tested whether these lncRNAs are suitable as biomarkers. Those comparisons were analyzed where at least one significant difference was found. For the evaluations the Naïve Bayesian classifiers were used. The selected lncRNAs were tested individually and in combinations. In some cases, the expression levels of the lncRNAs showed highly significant differences between two groups , there is still no objective biomarker in allergen specific immunotherapy (AIT) which is able to track how patients respond to the therapy. Presently, the evaluation of clinical improvement is based on changes in subjective clinical and immunological parameters. Different algorithms have been developed for calculating adjusted symptom and medication scores, but none of them is universally accepted . NaturalHNRNPU-AS1). In the present study, only 84 selected lncRNAs were involved. Methods with higher capacity (e.g. RNA-seq) additional lncRNAs with larger potentials might be identified. In some groups, the number of study subjects were low. Moreover, in these diseases a lot of additional endotypes exist that were not tested in the present study. Additional, larger studies with more patients with verified, diverse endotypes are needed to utilize the biomarker potential of these lncRNAs and to get better understanding of their roles in these diseases.Some limitations of the study must also be mentioned. The estimated number of lncRNAs in the human genome is more than 50,000 [Differences were detected in the expression of circulating lncRNAs in chronic respiratory diseases. Some of these differences might be utilized as biomarkers and also suggest a possible role of these lncRNAs in the pathomechanism of these diseases. With a systems biology analysis, novel functions of some of the lncRNAs were predicted. The lncRNAs and the associated pathways are potential therapeutic targets in these diseases, but naturally additional studies are needed for the confirmation of these results.Additional file 1: Overview of the systems biology analysis to identify the functional pathways and Gene Ontology terms of the 6 selected lncRNAs. A. Construction of a meta-network consisting of two types of meta-nodes, namely lncRNAs and genes; and four meta-edges, namely (1) the tissue-specific transcriptional similarity of lncRNAs, (2) the tissue-specific transcriptional similarity between lncRNAs and genes, (3) the experimentally validated lncRNA-target gene pairs connecting lncRNAs and genes, and (4) protein-protein interaction of genes. B. The heterogeneous lncRNA-gene network induced by the meta-network. Diamond-shaped nodes represent lncRNAs, and circular nodes represent genes. Edges represent functional connection between the corresponding nodes consistent with the meta-edges. C. A random walk with restart network propagation algorithm is initiated from each of the six lncRNAs to quantitatively prioritize the genes that are expected to be functionally relevant with respect to a particular lncRNA. The color of the nodes represent the amount of propagated information in that node . D. Schematic representation of gene set enrichment analysis on the propagated gene scores.Additional file 2: Heatmap of the relative expression of the lncRNAs in each sample of the discovery cohort. Color codes above the heatmap: blue: severe allergic asthma; red: mild allergic asthma; green: COPD; yellow: control; brown: non-allergic mild asthma; black: non-allergic severe asthma.Additional file 3: Heatmap of the log2FC values in comparison of the blood expression of 84 lncRNAs of the study subjects in the discovery cohort.Additional file 4: Heatmap of the sex adjusted –log10P values in comparison of the blood expression of 84 lncRNAs of the study subjects in the discovery cohort.Additional file 5: Log2FC and P-values in the comparison of different groups in the discovery cohort in respect of the mean blood expression levels of altogether 84 lncRNAs.Additional file 6: Log2FC and adjusted P-values in the comparison of different groups in the replication cohort in respect of the mean blood expression levels of the selected lncRNAs. P-values <0.05 are highlighted.Additional file 7: Heatmap of the adjusted –log10P values in comparison of the blood expression of the 6 selected lncRNAs of the study subjects in the replication cohort.Additional file 8: Comparison of the selected lncRNAs. Left: Comparison of the blood expressions of the 6 selected lncRNAs between adult and childhood controls. The adjusted P values are given for each comparison. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 9:  Comparison of the selected lncRNAs. Left: Comparison of the blood expression of the 6 selected lncRNAs between groups. The adjusted P-values are given for each comparison. Only those comparisons are depicted, where at least one significant difference was found. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 10: Comparison of the selected lncRNAs. Left: Comparison of the blood expression of the 6 selected lncRNAs between groups. The adjusted P-values are given for each comparison. Only those comparisons are depicted, where at least one significant difference was found. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 11: Comparison of the selected lncRNAs. Left: Comparison of the blood expression of the 6 selected lncRNAs between groups. The adjusted P-values are given for each comparison. Only those comparisons are depicted, where at least one significant difference was found. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 12: Comparison of the selected lncRNAs. Left: Comparison of the blood expression of the 6 selected lncRNAs between groups. The adjusted P-values are given for each comparison. Only those comparisons are depicted, where at least one significant difference was found. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 13: Comparison of the selected lncRNAs. Left: Comparison of the blood expression of the 6 selected lncRNAs between groups. The adjusted P-values are given for each comparison. Only those comparisons are depicted, where at least one significant difference was found. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 14: Comparison of the selected lncRNAs. Left: Comparison of the blood expression of the 6 selected lncRNAs between groups. The adjusted P-values are given for each comparison. Only those comparisons are depicted, where at least one significant difference was found. Right: Principal component analysis bi-plot showing the scores of the samples (colored circles) and the loadings of the variables (i.e. the six selected lncRNA as grey arrows) along the first two principal components.Additional file 15. Detailed description of the systems biology analysis used for the prediction of lncRNA functions