The present review discusses the epidemiology, pathophysiology, and current medical management in patients with HF developing in the context of CKD.The function of the kidney is tightly linked to the function of the heart. Dysfunction/disease of the kidney may initiate, accentuate, or precipitate of the cardiac dysfunction/disease and vice versa, contributing to a negative spiral. Further, the reciprocal association between the heart and the kidney may occur on top of other entities, usually diabetes, hypertension, and atherosclerosis, simultaneously affecting the two organs. Chronic kidney disease (CKD) can influence cardiac function through altered hemodynamics and salt and water retention, leading to venous congestion and therefore, not surprisingly, to heart failure (HF). Management of HF in CKD is challenging due to several factors, including complex interplays between these two conditions, the effect of kidney dysfunction on the metabolism of HF medications, the effect of HF medications on kidney function, and the high risk for anemia and hyperkalemia. As a result, in most HF trials, patients with severe renal impairment (i.e., eGFR 30 mL/min/1.73 m Patients with chronic kidney disease (CKD) frequently suffer from heart failure (HF) ,2. Manag2 body surface area (BSA) is indicative of CKD, even when evidence of kidney damage such as albuminuria is absent. In patients with eGFR 60 mL/min/1.73 m2 or more, kidney damage reflected by the occurrence of albuminuria  must be documented before the diagnosis of CKD can be made. A decrease in eGFR or damage of the kidney must be present for more than 3 months for the diagnosis of CKD .,26.25,26Finally, both CKD and HF are frequently accompanied by anemia, which is due to a constellation of diverse factors, including relative erythropoietin deficiency, uremia-induced inhibitors of erythropoiesis, short erythrocyte survival, and disturbed iron homeostasis . The dec2), belittling CKD patients [In most CV trials, CKD patients are excluded based on diverse criteria  from the Swedish Heart Failure Registry, β-blockers use was accompanied by lower morbidity and mortality [β-blockers have proved beneficial in CKD patients with HF. In a retrospective cohort of elderly CKD patients with HF, β-blockers reduced all-cause mortality, even those with an eGFR 30 mL/min per 1.73 m or less . Likewisortality . As neitortality . FurtherIn mild or moderate CKD, ACEi/ARB reduce blood pressure, retard eGFR decline, attenuate proteinuria, and delay progression to CKD stages 4 or 5 ,49. HoweACEi and ARB differ in their structure and mechanism(s) of action, exhibiting diverse pharmacokinetic properties. The pharmacokinetics of ACEi are poorly characterized owing to several factors interfering with their analysis, whereas the pharmacokinetics of ARB vary the least with renal dysfunction ,53. SurpRegarding HF management, both ACEi and ARB reduce morbidity and mortality ,55,56. AIn summary, the choice between ACEi and ARB in patients with CKD and HF should be individualized based on the clinical setting, availability, and healthcare provider expertise . ACEi/ARAldosterone attaches to the mineralocorticoid receptor (MR) at the distal nephron epithelium and augments sodium reabsorption and potassium secretion . InhibitPatients treated with ACEi or ARB may exhibit the so-called aldosterone breakthrough, a rise in plasma aldosterone, the prevalence of which in CKD patients can reach 50% . The incSeveral studies, including the Randomized Aldactone Evaluation Study (RALES) , the ComFinerenone, a novel selective MRA, possesses chemical and physical properties providing balanced renal and cardiac drug delivery . Fineren2 or less.In summary, the non-steroidal MRAs (spironolactone and eplerenone) are currently the agents of choice in CKD patients with HF, although finerenone is a very promising agent with attractive properties. However, there is lack of evidence regarding the safety and efficacy of MRA in patients with eGFR 30 mL/min/1.73 mSacubitril/valsartan attenuates the neurohormonal overactivity in HF by inhibiting both the neutral endopeptidase neprilysin (sacubitril) and the angiotensin II type 1 receptors , therefore improving the neurohormonal balance more than RASi . 2; follow-up: 12 months), although sacubitril/valsartan and irbesartan had similar effects on renal function and albuminuria, sacubitril/valsartan proved superior to irbesartan in lowering blood pressure and cardiac biomarkers [Sacubitril/valsartan has proved beneficial in CKD patients. In the United Kingdom Heart and Renal Protection-III (UK HARP-III) trial  with 3460 patients with CKD and HF compared sacubitril/valsartan with irbesartan, valsartan, and enalapril . SacubitIn summary, sacubitril/valsartan is extremely useful in lessening cardiac events and ameliorating renal impairment in CKD patients with HF.The SGLT-2i were initially considered glucose-lowering agents. However, due to their pleiotropic effects, the use of SGLT-2i has expanded far beyond T2DM and nowadays includes CKD and HF with or without diabetes. 2 and the need for tubular reabsorption and increasing cortical oxygenation, which, in association with the diminished tubular glucotoxicity, protect tubular function and maintain GFR. Further, it seems that SGLT-2i imitate systemic hypoxia and prompt erythropoiesis, lessen volume retention and blood pressure, and preserve CV function, presumably by conquering diuretic and natriuretic-peptide resistance and hampering Na+-H+ exchangers and sympathetic tone [The mechanisms underpinning the renal and cardiovascular benefits of SGLT-2i are multiple, with some related and some unrelated to the hypoglycemic effect of these agents . SGLT-2itic tone .2 and albuminuria more than 300 to 5000 mg/g), which was stopped early, as canagliflozin reduced by 30% the primary outcome  [The first trial assessing the SGLT-2i effect on renal dysfunction was the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) study (4401 diabetic patients with eGFR 30 to less than 90 mL/min per 1·73 m causes) .2 and urinary albumin-to-creatinine ratio 200 to 5000 mg/g) reported that dapagliflozin reduced the risk of the primary endpoint  by 44% compared with placebo regardless of diabetes status or CKD cause [2, or eGFR of 45 but less than 90 mL/min/1.73 m2 plus urinary albumin-to-creatinine ratio of 200 mg/g or more), in which empagliflozin reduced the primary endpoint  compared with placebo [The renoprotective effects of SGLT-2i have also been documented in patients without diabetes. The Dapagliflozin and Prevention of Adverse Outcomes in Chronic Kidney Disease (DAPA-CKD) trial  , Empagli2 or less [Based on the above, the benefits of SGLT-2i are consistent across many CKD with coexistent HF subgroups regardless of the T2DM status . In addi or less . One of  or less .Diuretics are of prime importance in the management of volume overload and comprise several classes . Loop diWater and sodium retention, often resulting from an unjustified interruption or reduction of doses by the patients themselves, lead to venous congestion associated with dyspnea on the one hand and worsening renal function on the other. Diuretics cause decongestion but may worsen renal function since the optimal stopping point for decongestive therapy and avoidance of hypovolemia remain major challenges in HF management. Larger doses of diuretics are required in patients with CKD and HF, as diuretic resistance is frequent in this context . Many mePractical approaches to overcome diuretic resistance include readjustment of the diuretic dose; addition of other categories of diuretics (preferably thiazides), always with caution regarding electrolyte disturbances; change from furosemide to torasemide due to its long action; and discontinuation of anti-inflammatory drugs . The recLoop diuretics are indicated to alleviate congestion in symptomatic patients, and when this is achieved, the dose has to be down-titrated to the lowest dose that will keep the patient euvolemic. Higher stages of CKD may require higher doses of loop diuretics to reach decongestion or euvolemia, as tubular delivery of diuretics decreases ,103. It Worsening renal function (WRF) is usually defined as a 20–30% decrease in eGFR or an increase in creatinine of 0.3 mg/dL or more 15–30 days after an intervention and occurs in approximately 25% of the cases ,108. The(A) After RAASi initiation, rise in creatinine up to 50% and a rise in potassium to 5.5 mmol/L or less are allowable;(B) A rise in creatinine 50–100% requires a systematic evaluation of the clinical context  and adjustment of medications . Close monitoring of the patient is also required;2 necessitates discontinuation of RAASi and ARNI and seeking the advice of a nephrologist;(C) A rise of potassium more than 5.5 mmol/L or a rise in creatinine more than 100% or a creatinine level more than 310 mol/L (3.5 mg/dL) or eGFR less than 20 mL/min/1.73 m2, acutely decompensated HF, severe ionic disorders , hypokalemia (potassium level less than 3 mEq/L) or hyperkalemia (potassium level more than 6.0 mEq/L)), severe dehydration with symptomatic hypotension, cardiogenic shock, hemodynamic instability accompanied by urinary tract disorders (obstruction/infection), or ineffectiveness of 48 h outpatient treatment require urgent hospital referral.(D) A rise in creatinine more than 100% or underlying CKD with an acute decrease in eGFR to less than 20 mL/min/1.73 mHyperkalemia, defined as a serum potassium level > 5.0 to 5.5 mmol/L, is uncommon in health due to the renal excretion of the dietary potassium, but it is one of the most prevalent electrolyte disorders in CKD, adversely affecting outcomes . The preTimely detection of moderate or severe hyperkalemia is of major importance to avoid fatal cardiac arrhythmias and paralysis of the muscles. Treatment of hyperkalemia entails the eradication of reversible etiology (diet and medications) and implementation of therapies that act rapidly by shifting potassium into cells and mitigate the effects of hyperkalemia on cardiac membrane and measures augmenting the removal of potassium from the organism . Hyperkalemia with electrocardiographic (ECG) changes or with a potassium level more than 6.5 mEq/L (even in the absence of ECG changes) should be treated as a medical emergency .Treatment must be started with calcium gluconate for cardiomyocyte membrane stabilization, followed by insulin injection and β-agonist administration. During intercurrent illness, stopping RAASi should be discouraged, but if potassium levels exceed 6.0 mEq/L, or creatinine rise exceeds 30%, RAASi should be temporarily discontinued . In the The constellation of CKD, HF, and anemia  occurs fPatients with advanced CKD and iron deficiency should be treated with iron supplementation, as first demonstrated in the Proactive IV irOn Therapy in hemodiALysis (PIVOTAL), which demonstrated that high-dose intravenous iron was superior to the low-dose regimen and resulted in lower doses of ESA being administered . SimilarA turning point in the management of anemia both in CKD and HF has been treatment with SGLT-2i. These agents, which favorably affect prognosis both in CKD and HF, increase hemoglobin by approximately 0.6–0.7 g/dL, an effect that has been linked to a rise in EPO and an expansion in red blood cell mass that alleviate anemia . The extCKD affects millions of individuals globally, and for the majority of CKD, patients the risk of developing CVD is higher than the risk of progression to advanced or end-stage kidney disease. Further, CV mortality in CKD patients is markedly higher than in the general population, and LV ventricular hypertrophy as well as HF contribute to the grave prognosis. The etiology of CV complications and especially HF in CKD is multifactorial. SGLT-2i have revolutionized treatment of CKD complicated by HF . The ben


Nitric oxide (NO) is a short-lived gas molecule which has been studied for its role as a signaling molecule in the vasculature and later, in a broader view, as a cellular messenger in many other biological processes such as immunity and inflammation, cell survival, apoptosis, and aging. Fractional exhaled nitric oxide (FeNO) is a convenient, easy-to-obtain, and non-invasive method for assessing active, mainly Th2-driven, airway inflammation, which is sensitive to treatment with standard anti-inflammatory therapy. Consequently, FeNO serves as a valued tool to aid the diagnosis and monitoring of several asthma phenotypes. More recently, FeNO has been evaluated in several other respiratory and/or immunological conditions, including allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, atopic dermatitis, eosinophilic esophagitis, and food allergy. In this review, we aim to provide an extensive overview of the current state of knowledge about FeNO as a biomarker in type 2 inflammation, outlining past and recent data on the application of its measurement in patients affected by a broad variety of atopic/allergic disorders. Nitric oxide (NO) is a short-lived gas molecule that has been studied since the late 80s for its role as a signaling molecule in the vasculature and later, in a broader view, as a cellular messenger in many other biological processes such as immunity and inflammation, cell survival, apoptosis, and aging ,2. NO isNO detected by a non-invasive method in exhaled air, fractional exhaled nitric oxide (FeNO), has gained increasing importance as a biomarker of type 2 inflammation , providiThe arrival of biological drugs has helped to gain further insight into NO physiopathology, and, in turn, the assessment of FeNO is a useful, non-invasive, and easy-to-obtain tool in monitoring conventional and biologic therapy and predicting the outcome of a wide spectrum of type 2-mediated diseases. In this review, we aim to provide an overview of the current state of knowledge about FeNO as a biomarker in type 2 inflammation, outlining past and recent data on the application of its measurement in patients affected by a broad variety of atopic/allergic disorders including bronchial asthma and cough variant asthma (CVA), allergic rhinitis (AR), chronic rhinosinusitis (CRS) with/without nasal polyps (CRSwNP/CRSsNP), atopic dermatitis (AD), eosinophilic esophagitis (EoE), and food allergy.NO is produced by the nitric oxide synthase (NOS) . There aiNOS is physiologically expressed in bronchial epithelial cells, ensuring a basal NO production mediated by IFNγ through JAK/STAT-1 signaling. In chronic airway inflammation, including asthmatic patients’ airways, iNOS is expressed by several cells, including macrophages, neutrophils, epithelial, endothelial, and vascular smooth muscle cells, which use L-arginine and oxygen as a substrate . In paraNO plays a role in both physiological and pathological conditions. For instance, in the nervous system, this molecule is involved in synaptic plasticity and the signaling within neurons , but it In inflammatory processes, NO production increases by participating in immune defense against infectious pathogens and causing noxious effects by increasing oxidative stress . A basalNO plays a pivotal role in Th2 inflammation , stimulaHowever, eosinophil-mediated inflammation is involved in many conditions, such as upper and lower airway disease , allergiBased on the prevalent type of airway inflammation, asthma has been broadly categorized into two endotypes: T2-high and T2-low. T2 airway inflammation in patients with asthma, although occurring along a continuum, is characterized by a specific set of cytokines, including IL-4, IL-5, IL-13, and the so-called alarmins, IL-25 and IL-33, and thymic stromal lymphopoietin (TSLP), secreted by activated epithelial cells. The T2 inflammatory response is frequently associated with atopy ) , eosinopIn approximately 50–70% of asthmatic patients, a T2 inflammatory response represents the pathogenetic mechanism responsible for the disease [Both innate and adaptive immune responses are involved in the inflammation of type 2 asthma. Innate lymphoid cells (ILC2s) and Th2 cells are critical in orchestrating the type 2 inflammatory response. When exposed to pollutants or viral and fungal infections, ILC2s are stimulated by alarmins secreted by epithelial cells. Allergens, on the other hand, are the activators of Th2 cells. Once stimulated, ILC2 and Th2 produce type 2 cytokines, including IL-4, IL-5, and IL-13, responsible for the main alterations present in asthma. IL-4 and IL-13 cause class switching of B cells to produce IgE. IL-13 is responsible for hyperplasia of both globular and smooth cells. IL-5 is the key cytokine involved in the differentiation, maturation, and survival of eosinophils . EosinopT2-high asthma phenotypes include both early- and late-onset asthma, frequent exacerbations, atopy, eosinophilia, and increased levels of FeNO.FeNO levels, compared to healthy controls, are elevated in T2 high asthma patients  and otheIn asthma, FeNO originates predominantly from the lower airways , and a rFeNO has been used, in combination with clinical history, spirometry, and other biomarkers, to support asthma diagnosis, as a surrogate marker of airway eosinophilic inflammation, as a marker to predict response to inhaled corticosteroids, and to establish the optimal dose for asthma control. In addition, FeNO can assist in the choice of treatment with biological drugs ,55. In pFeNO has a 90% specificity and 85% sensitivity in the diagnosis of asthma . The higHowever, FeNO and eosinophilia, although related, represent two distinct biomarkers resulting from two different T2 cytokine pathways: IL-4 and IL-13, which are implied in IgE synthesis and iNOS induction and thus NO production, and IL-5, which is involved in the activation, development, and recruitment of eosinophils. The different effects on FeNO and eosinophilia of biological drugs acting on different cytokines support this view. Indeed, in asthmatic patients treated with an IL-4 receptor inhibitor (dupilumab)  or an ILCompared to other T2 biomarkers, FeNO has great potential in clinical practice because its determination is simple, non-invasive, and can be easily repeated.The American Thoracic Society recommends FeNO to support the initial diagnosis of asthma. These guidelines define high FeNO levels in adults as >50 ppb (>35 ppb in children) and low levels as <25 ppb (<20 ppb in children) . Values FeNO provides prognostic information because increased FeNO levels are associated with disease activity and increased risk of exacerbations. Patients with baseline FeNO of 50 ppb or higher have a 1.54 times higher exacerbation rate than patients with FeNO of less than 25 ppb. Patients with baseline FeNO of 25 to <50 ppb hvae a 1.33 times higher exacerbation rate than patients with FeNO of less than 25 ppb .The simultaneous increase in both FeNO and blood eosinophil counts could be associated with a higher prevalence of uncontrolled asthma. Indeed, Bertolini et al., in a real-world cross-sectional study, showed that elevated FeNO values were present in younger patients with lower body mass index, higher blood eosinophil counts, higher reversibility, and worse airflow obstruction and who were atopic. Furthermore, such patients often presented with persistent rhinitis and chronic rhino-sinusitis with or without nasal polyps .CVA is a specific and atypical asthma phenotype and the most common cause of chronic cough. About 33.3% of patients with chronic cough are caused by CVA . CVA is A subset of subjects with CVA (approximately 30%), especially those with higher airway responsiveness, higher sputum eosinophils, and atopy, can progress into classic asthma in the absence of appropriate treatment for asthma .Small airways dysfunction (SAD) is a clinically relevant characteristic of CVA . Feng-JiAlthough different inflammatory phenotypes in CVA have been described ,73, simiFeNO, being a marker of eosinophilic airway inflammation in both the central and small airways has the potential as a clinical marker in screening and diagnosing CVA in clinical practice ,77. FeNOFeNO levels in patients with CVA are lower than those observed in patients with classic asthma, indicating greater chronic eosinophilic airway inflammation in the latter than in the former .Compared with other small airway pulmonary function tests such as MMEF/MEF50, FeNO shows increased sensitivity and specificity in the diagnosis of CVA, and it is negatively correlated with both MMEF and MEF50 values. In addition, combining FeNO with MMEF/MEF50 increases the diagnostic accuracy of CVA in children , resultiThe diagnostic accuracy of FeNO in detecting CVA in chronic cough patients with AR was higher than in those with chronic cough without AR .FeNO can be used as a predictor for airway eosinophilic inflammation but also as a predictor for steroid responsiveness in patients with chronic cough. The value of 34.5 ppb of FeNO has been suggested to distinguish between patients with chronic cough who respond to corticosteroid treatment and non-responders (with 85% sensitivity and about 90% specificity) .Airway inflammation in COPD is driven by type 1 immune response; however, in a subset of COPD patients, a T2 inflammatory response plays a significant role both in the stable state and during exacerbation of the disease . EosinopThe reasons for T2 inflammation only in a subset of COPD are poorly understood but may be due to the presence of atopy and/or asthma. An increase in Th2 and ILC2 cells has been reported in the lungs of subjects with COPD, and the epithelial cells of COPD patients produce alarmins that can activate them . PulmonaFeNO levels in COPD patients are higher than those observed in healthy non-smokers; however, they are not as high as those observed in untreated asthma .In COPD subjects, high FeNO values, as compared to those with low values, may suggest eosinophilic inflammation and the presence of some asthmatic features , airway Increased FeNO levels have been reported during the exacerbated phases of COPD , and in FeNO monitoring in COPD has a less defined role as compared to asthma. However, FeNO might also reflect in COPD the presence of airway eosinophilia and predict the response to corticosteroids. In addition, FeNO could play a role as a potential prognostic biomarker in COPD. Indeed, its level increased in patients with greater disease severity and during acute exacerbations  and in tAs compared to healthy subjects, a greater intra-individual FeNO variation in COPD patients during stable clinical conditions has been reported . In addiIn conclusion, in COPD, although FeNO values show greater variability, its use, particularly when combined with other T2-type inflammatory biomarkers, could improve COPD phenotypic discrimination or help to select candidates for personalized therapies, particularly in the context of biological therapy.Upper and lower airways represent a continuous anatomo-physiological entity, and a high percentage up to 38%) of patients with AR also have asthma % of pati. Indeed,Patients with AR frequently express elevated levels of FeNO as an index of subclinical bronchial inflammation with potential prognostic implications . Chen etThese results taken together suggest that FeNO is rather a measure of allergy than of the severity of the disease in AR, but it could gain a value in evaluating the therapeutic effect of certain drugs in these patients.CRS is a chronic, heterogeneous, and multifactorial inflammatory disease of the upper airways, whose prevalence in the general adult population is about 10–15% . In CRS,CRS, like asthma, is a phenotypically heterogeneous disease. Depending on the presence or absence of nasal polyps, CRS can be classified into two types: CRS with (CRSwNP) and without nasal polyps (CRSsNP). In addition, from the histological point of view of CRSwNP, depending on the presence or absence of eosinophils infiltrating the nasal mucosa, two distinct histo-phenotypes have been reported: the eosinophilic-CRSwNP (ECRSwNP) and the non-eosinophilic-CRSwNP (NECRSwNP) . The infLower airway morbidity is highly prevalent in patients with CRSwNP , being pIn patients with CRSwNP, type 2 inflammation affects not only the nasal mucosa but also the bronchi and alveoli, even in the absence of asthmatic comorbidity . CRSwNP Asthmatic patients affected by ECRSwNP, as compared with those with NECRSwNP, have a higher rate of nasal polyps’ recurrence, higher levels of FeNO, and more severe asthma .CRSwNP is more commonly associated with severe asthma than mild-to-moderate asthma, suggesting that the presence of nasal polyps in CRS asthmatic patients may be a risk factor for asthma severity .Nasal polyps in CRS patients are a major determinant of increased FeNO levels . Non-astHigher FeNO levels have also been reported by Kobayashi et al. in well-controlled asthmatic patients if they were affected by ECRSwNP. Therefore, in asthmatic patients, particularly those with severe disease, presenting with high FeNO levels, the co-presence of ECRSwNP should be strongly suspected . The incp < 0.05). A ROC curve analysis showed that a FeNO level of 27 ppb had a good ability to distinguish patients with asthmatic T2-CRSwNP , while the optimal cut-off values for FeNO and blood eosinophil percentage for diagnosing asthmatic T2-CRSwNP were 68 ppb and 5.6%, respectively  [FeNO is a useful marker for screening asthmatic T2-CRSwNP prior to biopsy or asthma evaluation and may assist in selecting a proper treatment. As reported by Park et al., FeNO levels and the blood eosinophil percentage were significantly higher in asthmatic patients with histological T2-CRSwNP as compared to those in patients with non-T2-CRSwNP ( = 86.1) .In subjects with CRSwNP, high levels of FeNO have been associated with a better clinical response to dupilumab, which has been the first biological agent approved for the treatment of CRSwNP .Among eosinophilic gastrointestinal disorders (EGIDs), EoE is the most frequent and the most prevalent cause of chronic esophagitis, after gastroesophageal reflux disease (GERD), which is often a cause of misdiagnosis . EoE is Symptoms of EoE are related to esophageal dysfunction . The golThe pathogenic pathway underlying EoE is an antigen-driven Th2 immune response. Ingested allergens, especially food allergens, interacting with esophageal epithelial cells stimulate them to release alarmins , thereby activating a T2-mediated immune response . The relH2, and IL-17RB, able to produce the highest levels of IL-5 and IL-13, has been isolated from EoE tissue [A subset of the Th2 cell population expressing HPGDS, CRTE tissue .Atopy is a major risk factor in EoE as most patients have a family history of bronchial asthma, AR, or atopy, and about 75% of patients with EoE have at least one coexisting type 2 inflammatory disease such as asthma, CRS, AR, and AD .The study of FeNO as a non-invasive biomarker to support diagnosis and to monitor disease activity in EoE has yielded disappointing and sometimes conflicting results.Z = 3.33, p < 0.001) and had a statistically significant, although weak, correlation with the number of esophageal eosinophils  [In a paediatric cohort of 134 children with suspected EoE, evaluated with endoscopic biopsy, Kaur et al. confirmed the diagnosis of EoE in 45 of them. In these patients, after adjusting for atopic diseases, FeNO levels were higher as compared to control subjects (< 0.005) .In a prospective multicenter study in non-asthmatic EoE patients, Leung and colleagues evaluated the effect of topical fluticasone treatment on FeNO levels. The results obtained, while showing a statistically significant difference in FeNO levels and esophageal eosinophil counts before and after treatment, were not able to anticipate the clinical or histological response to treatment .Lanz et al., in a paediatric population composed of 55 subjects, investigated the role of FeNO in the assessment of EoE. The histopathologic diagnosis of EoE was confirmed in 18 subjects. In subjects with EoE, FeNO levels were normal in nine and elevated in the remaining nine. Based on the results obtained, the authors indicate that a normal FeNO level may be useful to exclude EoE with high specificity (>87%) and NPV (78%); however, FeNO is not suitable for the detection of subjects with EoE .The ability of FeNO to reflect the level of esophageal eosinophilic inflammation was evaluated in a study by Jonhson et al. In that study, it was found that FeNO was poorly suited to distinguish between patients with esophageal eosinophils greater than or less than 15/hpf. However, the subset of patients with active disease and FeNO > 40 ppb could be identified with a high degree of certainty .In another prospective study of 128 children undergoing endoscopy for suspected EoE, FeNO levels were statistically higher in active EoE than in control subjects and subjects with EoE in remission. However, the FeNO range was broad and poorly correlated with absolute eosinophil count. A panel of biomarkers including FeNO, plasma amino acids, and polyamine spermine had AUC of 0.90 and 0.87 when differentiating active EoE from controls and active EoE from EoE in remission, respectively . PresentWith a prevalence of 3% in adults and 20% in children, AD is one of the most frequent allergic conditions affecting the skin . PathogeA recent study by Galiniak and Rachel  demonstrAll of this evidence taken together indicates that currently, FeNO assessment is not a reliable tool in patients with concomitant mild or moderate AD and respiratory disease. ,144.Food allergy likely affects nearly 5% of adults and 8% of children, with growing evidence of an increase in prevalence .Among food allergies, a peanut allergy can be a life threatening event and accounts for approximately two-thirds of all fatal food-induced anaphylaxis . ClinicaFeNO has been shown to be elevated in children with a peanut allergy who have “outgrown” their asthma . In addiFeNO was increased also in patients with asthma and sensitization to other food allergens, with or without sensitization to airborne allergens . In thisAs a bedside test that can be used in children, it has potential for further research into mechanisms of anaphylaxis in food allergies and potentially assists in predicting an imminent anaphylactic reaction in some patients.In conclusion, eosinophils-mediated inflammation is involved in many conditions including upper and lower airway disease, allergies, and gastrointestinal disorders. In this view, the use of FeNO as a marker of eosinophilic inflammation and type 2 diseases has largely been approved . On the


Background: Pre-eclampsia is a major public health issue. Current screening methods are based on maternal characteristics and medical history, but complex predictive models combining different clinical and biochemical markers have been proposed. However, although their accuracy is high, the implementation of these models in clinical practice is not always feasible, especially in low- and middle-resource settings. CA-125 is a tumoral marker, accessible and cheap, with proven potential as a severity marker in the third trimester of pregnancy in pre-eclamptic women. Assessment of its use as a first-trimester marker is necessary. Methods: This observational study involved fifty pregnant women between 11 and 14 weeks of pregnancy. Clinical and biochemical markers (PAPP-A), known for their value in pre-eclampsia screening, were recorded for every patient as well as first-trimester value of CA-125 and third-trimester data regarding blood pressure and pregnancy outcome. Results: No statistical correlation between CA-125 and first-trimester markers was observed except with PAPP-A, with which it exhibited a positive correlation. Additionally, no correlation was made between it and third-trimester blood pressure or pregnancy outcomes. Conclusions: CA-125 first-trimester values do not represent a valuable marker for pre-eclampsia screening. Further research on identifying an accessible and cheap marker to improve pre-eclampsia screening in low- and middle-income settings is needed. Currently, the most accepted pathophysiological theory regarding the development of pre-eclampsia suggests the presence of a two-stage process . The fir2, nulliparity family history of pre-eclampsia, or interpregnancy interval greater than 10 years [The current screening approach for pre-eclampsia is based on the use of a combination of maternal characteristics and medical history to identify groups of high-risk women . The Nat10 years . However10 years .Several different biochemical markers have been proposed as additional factors to the current approach to improve its detection rates. The most used markers are PAPP-A, PlGF and sFLT-1. CA-125 is a transmembrane glycoprotein and a member of the mucins group. Mucins are glycosylated in different manners and have functions dependent on the tissue in which they are found . SeveralA valuable screening marker must identify asymptomatic individuals at sufficient risk for disease to justify therapeutic intervention to prevent or minimise the impact of the screened disease. Although the current screening approach can be applied in any clinical setting, its value is limited. The proposed clinical and biochemical markers and their combinations have demonstrated an increased detection rate, but none of the improved models have undergone clinical validation . MoreoveBased on the existing research, CA-125 has the potential to be used as a first-trimester marker in pre-eclampsia screening. As a simple and affordable investigation, screening for pre-eclampsia in low-resource settings could be significantly improved if a predictive value could be demonstrated. Currently, there is no article published assessing its predictive value in the first trimester. This study was designed to analyse the relation between first-trimester CA-125 values and other known clinical and biochemical markers for pre-eclampsia as well as third-trimester blood pressure.This observational study is part of a larger research project on CA-125 in the context of pre-eclampsia. It was led at a tertiary maternity facility of the Clinical Emergency City Hospital of Timisoara in the Obstetrics–Gynaecology Clinic between January 2022 and January 2023. The study population included pregnant women who required antenatal care in our hospital. The criteria for inclusion were pregnant patients between 11 + 0 and 14 + 6 weeks of gestation who were willing to attend complete follow-ups and provide the required data. The criteria for exclusion were refusal of inclusion in the study, refusal to attend complete follow-ups, chronic hypertension, chronic renal or hepatic disease, diabetes mellitus, a diagnosed gynaecologic pathology (ovarian or uterine disease), multiple pregnancies, diagnosed foetal anomalies, and pregnancies achieved by ART.In the planned period for the study, fifty pregnant women were selected for inclusion, with five patients being lost at follow-up or from second-trimester spontaneous abortions. Using a standard interview, social and demographic characteristics, obstetric history, family history, and clinical data were obtained from every enrolled patient. Each patient’s PAPP-A, first-trimester mean arterial pressure, and uterine artery pulsatility index (UtA-PI) were converted in MoM. Blood samples for serum CA-125 were collected for every patient. The samples were processed using the ECLIA method (electrochemiluminescent immunoassay). The entire study population was given follow-up care until delivery. The hospital database was accessed for records of delivery, maternal, and neonatal outcomes.For defining pregnancy-induced hypertension, two measurements of blood pressure obtained at least 4 h apart with values over 140 mmHg for systolic blood pressure, over 90 mmHg for diastolic blood pressure, or both and with appropriately sized cuffs were used. For further grouping of the hypertension group, criteria proposed by the American College of Obstetricians and Gynaecologists were used . Pre-eclEthical approval for the current study was obtained from the Ethics Committee of the University of Medicine and Pharmacy, “Victor Babes,” Timisoara with No. 80/07.12.2020. All enrolled patients provided informed consent.https://www.jamovi.org) was used for statistical analysis. (Accessed on 23 August 2022) Descriptive statistics were applied for all appropriate data, and the normality of distribution was tested for all quantitative data. A t-test was performed to compare the two groups, and a one-way ANOVA test was applied for the parametric variables of multiple groups associated with the Tukey post-hoc test. Pearson’s correlation coefficient (Pearson’s r) was used to establish the correlation between different variables. A p-value of <0.05 was considered significant.Jamovi software . A total of 18% (n = 9) of the pregnant patients were smokers. Additionally, 20% (n = 10) of the pregnant patients took low-dose aspirin for pre-eclampsia prophylaxis. Indications were a history of pregnancy-related hypertension or age-related (over 35 years old) and nulliparity.The mean PAPP-A value (MoM) was 1.41. A total of 12% (n = 6) of patients had a low level of PAPP-A (<0.4 MoM). The mean MAP was 86.3 mmHg (±10.2) or 1.01 MoM (±0.11). The mean UtAPI was 1.18 (±0.44) MoM. The mean CA-125 value was 20.2 UI/L (±11.8). When analysing correlations of the CA-125 first-trimester values with other parameters and markers used for pre-eclampsia first-trimester screening, a positive correlation was observed with PAPP-A values (MoM) . Additiop-value < 0.001. The mean gestational age at birth was 38.3 weeks (±2.01), and the mean birth weight was 3223 g (±517). There were no statistical differences between patients with normal blood pressure and patients with high blood pressure regarding these parameters. When comparing first-trimester CA-125 values with third-trimester clinical parameters, no correlation was observed  had high blood pressure, with five patients diagnosed with gestational hypertension and one with mild pre-eclampsia. The mean MAP among patients with normal blood pressure was 96 mmHg (±8.1), while the mean MAP among patients with high blood pressure was 113 mmHg (±3.97) with a observed .p-value = 0.34.The mean first-trimester CA-125 value of the patients with normal blood pressure in the third trimester was 16.6 UI/L (±8.78), while the mean first-trimester CA-125 value of the patients with third-trimester high blood pressure was 20.7 UI/L (±12.2). However, no statistical difference was observed with a Our study investigated the probability of a correlation between CA-125 first-trimester levels and other first-trimester clinical or biochemical markers used in pre-eclampsia screening as well as with clinical third-trimester parameters, allowing us to identify a possible predictive marker that would improve the screening of pre-eclampsia.Currently a few recognised markers are used for pre-eclampsia screening. PAPP-A (pregnancy-associated plasma protein A) is a regulatory protein secreted primarily by the syncytiotrophoblast that is essential for the bioavailability of the insulin-like growth factor used for normal foetal development. Initially used as an aneuploidy marker in first-trimester screening, it was later associated with unfavourable pregnancy outcomes such as intrauterine growth restriction, intrauterine foetal demise, premature birth, and pre-eclampsia ,20. UsedSeveral studies have associated a high CA-125 value in the third trimester with severe pregnancy outcomes in pre-eclamptic patients . The staPre-eclampsia represents one of the most researched topics in modern obstetrics. However, no cure has been found. Thus, its management is focused mainly on screening, prevention, early diagnosis, and reducing maternal and foetal morbidity and mortality to a minimum . CurrentIt is well known that pre-eclampsia is a public health issue with a huge cost impact. Although the current prophylaxis method, aspirin, is quite cheap, effective screening models to establish the high-risk population that would benefit from it are complex and include some expensive investigations. The highest pre-eclampsia-related morbidity and mortality rates are reported in low- and middle-resource countries where most of the proposed screening methods are hard to implement successfully. Pre-eclampsia screening with basic tools, such as medical history, blood pressure measurements, or proteinuria dipstick detection, are the most appropriate and cost-effective methods. However, due to low detection rates, identifying additional cheap markers could significantly improve pre-eclampsia screening. Until identifying such markers, an alternative strategy that could be feasible even for low-resource areas is universal aspirin treatment. Arguments for this management method are both financial and medical. Aspirin has a good safety profile, with no increase in haemorrhagic complications . It has Based on the encouraging results reported in the third-trimester assessment with CA-125 as a severity marker for pre-eclampsia, this study analysed its potential use in the first trimester. The dynamics of CA-125 levels in pregnancy, including in the first trimester and with influencing factors, have been studied in several papers. Reported means of first-trimester values in healthy pregnant patients vary largely between 19.0 U/mL and 74.3 U/mL ,40,41. IUnfortunately, the results of this research show no correlation between CA-125 levels and some of the most used screening markers, except for PAPP-A, with which a positive correlation was observed. Moreover, first-trimester CA-125 values showed no correlation with blood pressure values measured in the third trimester for the normotensive pregnant patients or the pregnant patients with high-blood pressure. Additionally, no correlation was observed with pregnancy outcomes, such as gestational age at birth or birth weight.Significant limitations of this study are caused by the sample size of the study group, which is low as a result of hospital-based enrolment. Additionally, the absence of other studies focusing on the first trimester, the need for standardisation in CA-125 measurement, and the different biological and genetic characteristics of the populations studied in the third trimester impose caution in interpreting the results.Pre-eclampsia remains a major public health issue due to its high maternal and foetal morbidity and mortality rates despite huge advances in knowledge over the last few decades. Current screening methods are either easily accessible for clinical practice but have low detection rates or have high accuracy but are extremely complex and expensive. CA-125 is a cheap and accessible marker, and its value in third-trimester screening has been proven. However, its first-trimester values do not correlate with other recognised pre-eclampsia screening markers or with third-trimester blood pressure values or pregnancy outcomes. Further research focusing on new methods that are accessible and cheap is needed, as improving the screening of pre-eclampsia, especially in low- and middle-income countries, is a topic that must be treated with high priority.


Background and Objectives: The global prevalence of chronic kidney disease (CKD) is on the rise, posing important challenges for healthcare systems. Thus, the search for new factors potentially involved in the pathogenesis, progression and complications of early CKD remains urgent. Marinobufagenin (MBG) is a natriuretic endogenous cardiotonic steroid, and increased circulating levels of it may accelerate kidney damage. In this study, we explored the possible clinical significance of measuring urinary marinobufagenin (uMBG) in patients with non-advanced CKD. Materials and Methods: One hundred and eight adult CKD patients  were enrolled in this cross-sectional study. uMBG was measured together with a series of clinical, anthropometric, laboratory and instrumental analyses. Twenty-five healthy matched subjects served as controls for the uMBG measurement. Results: The uMBG values were lower in the patients with CKD as compared to those of the controls (0.37 [IQR: 0.25–0.45] vs. 0.64 [0.46–0.78] nmol/L. p = 0.004), and a significant trend in eGFR levels was noticed across the decreasing uMBG tertiles (p = 0.03). Regarding the correlation analyses, the uMBG values remained robustly associated with the eGFR in multivariate models employing either uMBG or eGFR as the dependent variable . Besides the eGFR, the independent predictors of uMBG values in this population were the use of statins , the presence of diabetes  and urine sodium . Conclusions: Reduced uMBG excretion may reflect impaired renal clearance, which may contribute to the detrimental effects attributed to this hormone due to systemic accumulation. Future studies are needed to clarify the biological mechanisms placing uMBG at the crossroad of sodium intake and the presence of diabetes in CKD-suffering individuals and to verify whether a statin treatment may somewhat limit the detrimental effects of MBG in the presence of impaired renal function. Nowadays, the prevalence of chronic kidney disease (CKD) has reached pandemic proportions, posing a significant burden across health care systems at the global level . CKD por+/K+-ATPase membrane pump, triggering the activation of specific ionic intracellular pathways  or frequency percentage, as appropriate. Differences between groups were assessed using the unpaired t-test for normally distributed values, the Mann–Whitney U test for non-parametric values and the chi-square test, followed by a Fisher’s exact test for frequency distributions.p value. All results are considered to be significant for p values ≤ 0.05.Differences in clinical parameters across the tertiles of uMBG were assessed using ANOVA for continuous covariates and chi-square tests for categorical data (p for trend). Pearson (R) and the Spearman (Rho) correlation coefficients were employed to test the correlations between variables, as appropriate. Before testing the correlations, all the values showing a skewed distribution were log transformed to better approximate normal distributions. Multiple regression analyses were performed to assess the independent relationships first by employing fully adjusted models including all univariate correlates of uMBG values, respectively. The variables included in the equations  were not tested in the models, including the corresponding formulas to avoid co-linearity. Stratified models were also built by removing single covariates to explore possible confounding. Data are expressed as partial correlation coefficients (β) and 2. The CKD etiology was diabetic kidney disease in half of the population, nephroangiosclerosis in 16.7%, glomerulonephritides in 12.9%, interstitial diseases in 7.4% and rare diseases, including ADPKD, in the remaining 3.7%. The prevalence of cardiovascular comorbidities spanned from 91.6% (hypertension) to 5.6% (history of stroke). Diabetes was present in 54.6% of the individuals. Almost all the patients were undergoing combined anti-hypertensive therapy, which frequently included an RAS blocker (82.4%). Roughly one half of the patients underwent diuretic therapy, while the majority were on statins (64.8%) and hypouricemic agents (74.1%).The final study population consisted of 108 adult patients  who met the inclusion/exclusion criteria for being eligible to participate. Mean eGFR (CKD-Epi) was 40.54 ± 17 mL/min/1.73 mp = 0.004 (p = 0.01), eGFR (p = 0.03), hemoglobin (p = 0.03), proteinuria (p = 0.04), urine sodium (p = 0.03) and urine potassium (p = 0.04). The patients with higher uMBG excretion were also younger (p = 0.01), more frequently diabetics (p = 0.004), less likely to be on statin therapy (p = 0.001) and displayed lower levels of urea (p = 0.05) and iPTH (p = 0.04).The uMBG values in the CKD population were significantly lower compared with those measured in the healthy controls (0.37 [IQR: 0.25–0.45] vs. 0.64 [0.46–0.78] nmol/L.  = 0.004 ). In theThe complete information regarding the characteristics, co-morbidities and active therapy for all the CKD patients and individuals stratified for uMBG tertiles are displayed in p = 0.008), eGFR , hemoglobin , proteinuria , urine potassium , urine sodium  and the presence of diabetes , while the inverse predictors were age  and the use of statins . In a fully adjusted multivariate model including all the univariate correlates, only the use of statins , the presence of diabetes , eGFR  and urine sodium  remained significantly associated with uMBG excretion, while the correlations with BMI, urine potassium, age, hemoglobin and proteinuria were lost. Of note, forcing diuretic use (any type) in the model did not affect the relationship between uMBG and urine sodium. The fully adjusted model held considerable power, explaining the 33% of the total uMBG variability in this cohort. Interestingly, the correlations with urine potassium  and hemoglobin  reattained statistical significance in an exploratory model excluding eGFR , suggesting a likely confounding effect of residual renal function on such univariate relationships. By the same token, significant associations between uMBG and, respectively, BMI  and proteinuria  re-emerged only in a separate model excluding diabetes , thereby indicating a significant modulating effect of this disorder. In univariate analyses, uMBG was directly associated with BMI  accounts for more than one half of CKD cases in the present cohort, an observation which reflects epidemiological data that rank diabetes as the leading cause of CKD in the developed world .Yet, desTwo more findings from our study are worth mentioning.First, the uMBG values in the CKD individuals paralleled the urinary sodium excretion findings, and such a close relationship remained apparently unaffected by possible confounders, including the use of diuretics. This observation pairs well with previous experimental  and clinp = 0.001). Despite the fact that the impact of possible unknown confounders on this relationship cannot be excluded, taken together, these observations may indicate that a statin treatment, by negatively modulating cholesterol metabolism, could also impact upon the biosynthesis or peripheral metabolism of MBG, eventually leading to reduced urinary elimination. In line with this hypothesis, in previous experimental evidence, HMG CoA reductase inhibitors were found to interfere with the sarcolemmal Na+/K+ pump function and to exert an anti-arrhythmogenic effect by modulating the expression and activity of endogenous cardiotonic steroids [Second, and no less important, the clear influence of statin treatment on uMBG excretion has emerged, as revealed by the significant, decreasing trend in the percentage of statin users across the growing categories of uMBG excretion. In the fully adjusted multivariate regression model, such an inverse association was apparently not even affected by diabetes, ranking as the strongest correlation among all those reported (β = −0.326; steroids ,26.We acknowledge some key limitations of our study. First, despite the study being powered enough to perform large multivariate adjustments without model overfitting occurring, the observational design cannot rule out the presence of a selection bias or residual confounding, and this prevents us from raising definite assumptions about the biological mechanisms underlying the relationships found between uMBG and the different clinical variables. Last, given the lack of a longitudinal phase, we cannot evaluate whether fluctuations in uMBG over time pair with the changes in renal function, thereby limiting the potential applicability of this substance as a biomarker of kidney damage.Individuals with mild-to-moderate CKD display reduced urinary marinobufagenin levels, which parallel the severity of renal impairment. Future studies on larger, heterogeneous populations are advocated to generalize our findings and to shed light on the biological mechanisms underlying the complex interplay between uMBG excretion, diabetes, sodium intake and the use of statins in the presence of impaired renal function.


We administered fludarabine to a 58-year-old female patient with myeloma-associated severe renal impairment as part of lymphodepletion before Idecabtagen vicleucel infusion. Fludarabine was administered in reduced dose (15mg/m2) and cyclophosphamide with a dose of 300mg/m2 followed by hemodialysis over six hours using a larger filter (FX-100). The therapy was well tolerated with excellent CAR-T cell expansion and complete remission which is ongoing now beyond 12 months.Acute kidney injury and chronic kidney disease is common in multiple myeloma. Fludarabine which is part of lymphodepletion before CAR-T cell therapy is renally eliminated and its use is not recommended for patients with severe renal impairment defined as a glomerular filtration rate below 30ml/min/1.73m Below 3efficacy . In this2) and progressive disease with elevated kappa light chains in blood and urine as well as new osteolytic lesions and new extramedullary manifestations in a CT and MRI scan. Another cycle of isatuximab/carfilzomib/dexamethasone was initiated but failed to achieve a response. Free kappa light chains increased to 15105.56 with a kappa/lambda ratio of 18881.95. Eventually, chemotherapy with cyclophosphamide/etoposide/dexamethasone was initiated. After chemotherapy serum kappa light chains decreased to 8732.90 (kappa/lambda ratio 6237.79) and kidney function improved slightly but GFR stayed below 30ml/min/1.73m2. Following quality release of ide-cel we administered fludarabine and cyclophosphamide using the dose adjustment and dialysis strategy previously published in the context of simultaneous allogeneic and kidney transplantation . We decided to apply full dose of cyclophosphamide at least 12h before dialysis. Cyclophosphamide serum concentration is elevated in patients harboring kidney disease but it is partly removed during hemodialysis . At initial diagnosis she presented with multiple osteolysis in lumbar spine and pelvis along with 60% of plasma cells in bone marrow biopsy. Cytogenetically her disease was classified as high risk due to 4;14 translocation, R-ISS stage was stage II. As first line therapy she received an induction regimen with cyclophosphamide/bortezomib/dexamethasone followed by high dose melphalan. Thereafter, under lenalidomide maintenance she stayed in complete remission for 2 years and three months until disease progression. The patient then received local radiotherapy of the left iliac bone and daratumumab/Bortezomib/dexamethasone but progressed after only two months. Next, Carfilzomib/Dexamethasone was administered, and the patient stayed in partial remission for three months before further disease progression again, leading to CAR-T cell indication. Due to further progression on bridging therapy with 2 cycles of elotuzumab/pomalidomide/dexamethasone treatment was changed to isatuximab/carfilzomib/dexamethasone and two cycles were administered prior to admission for CAR-T cell therapy. At this time, she presented with acute kidney injury , two patients received standard dialysis. One of the patients died from fludarabine neurotoxicity, so the investigators decided to perform fludarabine pharmacokinetic analysis, which did not show any difference to patients receiving fludarabine with normal kidney function .Hematological toxicity was present in our patient with grade IV neutropenia and thrombocytopenia and grade III anemia. In patients with normal kidney function grade III or IV neutropenia was observed in 89%, anemia in 60%, and thrombocytopenia in 52% .In our patient, grade III thrombocytopenia was still present on day 60 after CAR-T infusion, while the patient did not need transfusion or granulocyte-colony stimulating factor anymore. In Sidana et al., >/= grade III thrombocytopenia was observed in 73% in the group of patients with GFR < 30 ml/min on day 60 after CAR-T infusion, while >/= grade III anemia and neutropenia were still present in 36%. On day 90 after CAR-T infusion, Sidana et al. still observed >/= grade III anemia and thrombocytopenia in 10 and 30%, respectively, while our patient no longer demonstrated grade III cytopenias .In future studies, it would be useful to do fludarabine pharmacokinetics and, to determine the optimal target exposition of fludarabine in terms of toxicity and efficacy.2. For the first time, we performed lymphodepletion with fludarabine and cyclophosphamide followed by long hemodialysis over 6 hours using a larger filter (FX 100). The patient showed excellent CAR-T cell expansion and complete remission of her disease for at least 12 months without severe toxicity events. Further investigations addressing pharmacokinetics of fludarabine under hemodialysis and examining therapy response and toxicity in a larger cohort of patients should be initiated.CAR-T cell therapy is an important therapy option for patients with relapsed and refractory multiple myeloma and should also be offered to patients with severe renal impairment and eGFR below 30ml/min/1.73mThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants' legal guardians/next of kin in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.FM: Conceptualization, Writing – original draft, Writing – review & editing. CL: Writing – review & editing. DJ: Writing – review & editing. MM: Writing – review & editing. AD: Writing – review & editing. NK: Supervision, Writing – review & editing. FA: Conceptualization, Writing – original draft, Writing – review & editing. Written informed consent was obtained from the patient in accordance with approval of the local ethics committee (PV7081).


Introduction: The conversion of dietary inorganic nitrate (NO3-) to nitric oxide (NO) is a non-canonical pathway that plays an important role in NO biology, especially under pathological conditions. Inorganic NO3- supplementation is a proven method for controlling mild hypertension. Recent reports have suggested that another gaseous transmitter, hydrogen sulfide (H2S), influences NO biosynthesis and metabolism. Here, data are presented from an open-label clinical trial examining the effect of an encapsulated formulation (Vascanox® HP) that combines dietary sources of inorganic NO3- and S-allylcysteine (SAC), a source of H2S from garlic, on NO bioavailability and blood pressure in subjects experiencing elevated blood pressure or mild hypertension.Methods: An open-label clinical trial was conducted among patients with hypertension. Participants took Vascanox® for four weeks. Blood pressure was measured at baseline, two weeks, and four weeks. Salivary nitrite (NO2-), a surrogate of NO bioavailability, and NO3- were assessed prior to and two, six, and 24 hours after dosing on the first day of the study and prior to and two hours after dosing at subsequent study visits using saliva NO test strips. Changes in study outcomes over time were evaluated via analysis of variance (ANOVA) and paired t-tests.Results: Twelve participants completed the clinical trial. Vascanox® HP decreased systolic blood pressure by ~11 mmHg (p < 0.001) at two weeks and persisted beyond four weeks with daily supplementation. It also decreased the diastolic blood pressure of hypertensive subjects but not normotensive ones. The magnitude of the decrease was 11 mmHg (p < 0.01) at four weeks of study. Measurements of salivary concentrations of NO2- revealed high peak levels (743 uM) at two hours post-administration and a slow decay to elevated levels (348 uM) at 24 hours. NO2- salivary concentrations, a surrogate biomarker of NO bioavailability, remained above baseline for the duration of the study.Conclusions: Vascanox® HP was shown to be a safe, effective, quick-acting, and long-lasting dietary supplement for controlling mild hypertension. Elevated blood pressure (BP) in the absence of an underlying pathology (or primary hypertension) is the most common medical diagnosis in the US and a major risk factor for cardiovascular disease . AccordiPrimary hypertension is associated with a decline in vasodilation due to a dysfunctional endothelium that synthesizes decreased levels of nitric oxide (NO) . NO mediNO is produced endogenously as a by-product of the oxidation of L-arginine to L-citrulline, a reaction that is catalyzed by the endothelial nitric oxide synthase (eNOS). Endogenous NO has a short half-life (t1/2 = 1-2 ms) and is rapidly oxidized to form nitrite (NO2-) with a half-life of 20-45 minutes and then nitrate (NO3-) with a half-life of five to eight hours ,8. The cRothia mucilaginosa, Haemophilus parainfluenzae, Neisseria flavescens, and Neisseria subflava [NO3-, formed endogenously by oxidation of NO, is also supplemented by the ingestion of nitrate-rich foods , which serve as an exogenous source of NO -11. The subflava -10. SeveRecent literature suggests that another gaseous transmitter, hydrogen sulfide (H2S), plays an essential role in maintaining NO homeostasis and proper vasodilation ,14. It hDietary sources of H2S include garlic as a rich source ,16. S-alHere, we present data from a small, open-label study that aims to begin to evaluate the durability of an encapsulated H2S-NO donor supplement on the systolic and diastolic BP of human subjects with elevated BP. The encapsulated formulation combines natural reductants and polyphenols with dietary sources of NO3- and H2S and may represent an increment alternative to current encapsulated NO3- supplementation by simultaneously restoring NO and H2S bioavailability in the vasculature.Study populationThe study was conducted in accordance with the Declaration of Helsinki and approved by the Salus Institutional Review Board  affiliated with the Association for the Accreditation of Human Research Protection Programs (AAHRPP) for studies involving humans. The Salus IRB approval # is HTI-005. Informed consent was obtained from all subjects involved in the study at the Hypertensive Institute, Nashville, TN under the supervision of Dr. Mark Houston, MD. Written informed consent has been obtained from the patient(s) to publish this paper.ClinicalTrials.gov , under the following title: Open Label Clinical Trial of Vascanox® HP on Nitric Oxide and Blood Pressure  on Blood Pressure and Nitric Oxide Levels: An Open-Label Clinical Trial Among Hypertensive Adults).This study is registered with The eligibility criteria for participation were: (a) elevated BP at baseline (>120-140/85-90 mmHg), (b) the ability to provide informed consent, (c) the absence of any significant cardiac or other medical history, and (d) no medication changes in the preceding six months. The study recruited 12 participants in an outpatient setting: six participants with normotensive diastolic BP (≤80 mmHg) at baseline and six participants with elevated diastolic BP (>80 mmHg) at baseline. The male-to-female ratio was 1:2. The participants aged between 52 and 73 years.Product formulationVascanox HP® is a proprietary formulation that combines dietary nitrates in the form of beetroot extract with a source of hydrogen sulfide (black garlic extract), known reductants (vitamin C), various berry extracts , and other vitamins and essential metals. A serving dose of two capsules of Vascanox® contains 242 mg of nitrates, which is equal to 109% of the acceptable daily intake (ADI).Study protocolThe study participants were instructed to self-administer two capsules of Vascanox HP® each morning. They were evaluated in the clinic at baseline, at two weeks, and again at four weeks of study. The study lasted four weeks. During evaluation, the BP was measured in the branchial artery three times, five minutes apart. Salivary levels of NO were assessed using commercially available FDA-registered strips  prior to and two hours after dosing. NO test strips measure nitrite, which is used as an established surrogate marker for NO and nitrate . SalivarStatistical analysisDescriptive statistics were performed to characterize the study sample. Analysis of variance (ANOVA) and paired t-tests were conducted to determine changes in study outcomes across time points. Subgroup analyses were performed among the subsamples with systolic hypertension and diastolic hypertension. Statistical significance was defined as p < 0.05. Statistical analyses were performed with SAS version 9.4.1 . Additional analysis was performed using GraphPad Prism 9.0 .Study population and designThe study population of 12 subjects consisted of 33% males (four subjects) and 77% females (eight subjects) between the age of 52-73 years. Systolic blood pressure (SBP) ranged between 127 and 141  mmHg and was defined as elevated blood pressure in all subjects. Diastolic blood pressure (DBP) ranged between 62 and 94 mmHg for the 12 subjects where normotensive blood pressure was defined below 80 mmHg for DBP consisting of six subjects (50% of the study group) with a range of 62-74  mmHg and six subjects (50% of the study group) were defined as elevated DBP with a range of 80-94 (84 ± 5) mmHg.The study design is summarized in Figure Salivary nitric oxide levelsMeasurements of salivary nitrite NO2-) and nitrate (NO3-) levels at the start of the clinical trial showed that Vascanox HP® administration led to a rapid increase in salivary NO2-  mmHg at baseline, 71.1 ± 6.4 (60-80) mmHg at two weeks, and 68.3 ± 6.1 (57-74) mmHg at four weeks. For normotensive subjects, the differences were statistically significant.This open-label clinical trial evaluated the safety and efficacy of Vascanox® HP, a proprietary formulation designed to restore the bioavailability of gaseous transmitters NO and hydrogen disulfide in individuals with elevated BP or mild hypertension. The clinical trial raised no safety issues and demonstrated that short-term supplementation with Vascanox® HP leads to statistically significant decreases of 10-11 mmHg in both SBP and DBP. Interestingly, participants who exhibited elevated SBP but not DBP experienced a decrease only in their SBP. This reinforces the safety of Vascanox® HP as a dietary supplement and may be suggestive that there is crosstalk between the canonical (eNOS) and non-canonical (dietary nitrate) pathways of NO synthesis with the latter becoming important when the former is dysfunctional.Since the identification of dietary nitrates as a source of NO, numerous clinical trials have been performed, which examined the effect of nitrate supplementation on SBP and DBP. In early clinical trials, nitrates were administered as inorganic salts or beetroot juice to healthy volunteers, whereas later trials explored combinations of concentrated beetroot powder, sodium nitrite, and various phytochemicals given to patients diagnosed with elevated BP -25. In 2More recent clinical trials on dietary supplements formulated with nitrate or nitrite decreased SBP and DBP in mostly prehypertensive subjects. A complex formulation by Berkeley Life®, consisting of encapsulated beetroot extract, thiamine nitrate, potassium nitrate, and ascorbic acid, showed a decrease of 6.3 mmHg in SBP and 4.7 mmHg in DBP when given daily to individuals with baseline BP of >120/80 mmHg for 12 weeks . The souAnother supplement, Neo40®, was evaluated on the acute effects of a formulation containing sodium nitrite with a reported decrease of 4 mmHg in SBP and 5 mmHg in DBP, respectively, within 20 minutes . Neo40® A significant decrease in SBP and DBP was reported by Baik et al., who used a garlic extract fermented to contain nitrite . In thatTo our knowledge, Vascanox® HP is the first commercial dietary supplement that, inspired by recent advances in the field of NO biology, combines dietary sources of NO and H2S. Unlike Neo40®, whose nitrite contents per daily serving exceed the established ADI of 0.07 mg/kg/day by 10-fold, Vascanox® HP contains no nitrite and relies on commensal oral bacteria for the reduction of NO3- to NO2-, a necessary and required step in the well-established enterosalivary loop ,10. Its Despite its reduced dietary nitrate content and absence of added nitrite salts, Vascanox® HP supplementation demonstrated efficacy in reducing BP. We attributed that effect to the ability of its complex formulation to generate and sustain elevated levels of NO. As suggested in Figure LimitationsThe study was limited by the small number of participants and the open-label design. A placebo effect influencing the degree of BP reduction in the study subjects cannot be excluded. Furthermore, the clinical trial did not include measurements of the circulating concentrations of NO and sulfides, which would have been helpful in determining peak NO levels in the blood, the duration of its bioavailability, and the possible effect of the inclusion of black garlic as an ingredient in Vascanox® HP. The study would also have benefited from experiments examining NO release in gastric simulants. Finally, the study tested sub-chronic supplementation and did not report the development of resistance in the participants with the possible exception of a single recruit. It is apparent that a larger number of participants and longer supplementation are needed to exclude the possibility that a certain percentage of subjects will develop resistance to Vascanox® HP in future studies.This open-label clinical trial evaluated the safety and efficacy of Vascanox® HP, a proprietary formulation designed to restore the bioavailability of gaseous transmitters NO and hydrogen disulfide in individuals with elevated BP or mild hypertension. The clinical trial raised no safety issues and demonstrated that short-term supplementation with Vascanox® HP leads to statistically significant decreases of 10-11 mmHg in both SBP and DBP. Interestingly, participants who exhibited elevated SBP but not DBP experienced a decrease only in their SBP. This reinforces the safety of Vascanox® HP as a dietary supplement and may be suggestive that there is crosstalk between the canonical (eNOS) and non-canonical (dietary nitrate) pathways of NO synthesis with the latter becoming important when the former is dysfunctional.


This study presents a cardiovascular simulator that mimics the human cardiovascular system's physiological structure and properties to reproduce the human blood pressure waveform. Systolic, diastolic blood pressures, and its waveform are key indicators of cardiovascular health. The blood pressure waveform is closely related to the pulse wave velocity and the overlap of the forward and reflected pressure waves. The presented cardiovascular simulator includes an artificial aorta made of biomimetic silicone. The artificial aorta has the same shape and stiffness as the human standard and is encased with a compliance chamber. The compliance chamber prevents distortion of the blood pressure waveform from strain-softening by applying extravascular pressure. The blood pressure waveform reproduced by the simulator has a pressure range of 80–120 mmHg, a pulse wave velocity of 6.58 m/s, and an augmentation index of 13.3%. These values are in the middle of the human standard range, and the reproduced blood pressure waveform is similar to that of humans. The errors from the human standard values are less than 1 mmHg for blood pressure, 0.05 m/s for pulse wave velocity, and 3% for augmentation index. The changes in blood pressure waveform according to cardiovascular parameters, including heart rate, stroke volume, and peripheral resistance, were evaluated. The same pressure ranges and trends as in humans were observed for systolic and diastolic blood pressures according to cardiovascular parameters. Therefore, the importance of cardiovascular health monitoring is increasing. Today, health sensors are gradually being distributed in the form of wearable devices such as smartwatches, and pulse measurements using photo-plethysmography (PPG) or electrocardiogram (ECG) to monitor cardiovascular health conditions4. Cardio-vascular pulse waves are monitored for various healthcare purposes, such as exercise monitoring and arrhythmia diagnosis. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) mean the maximum and minimum BPs, respectively, the most important and frequently monitored health indicators for cardiovascular health management. However, BP is correlated with various cardiovascular parameters such as the stiffness of the vessel wall, pulse wave velocity (PWV), vessel diameter, and cardiovascular diseases5. Therefore, to study the causes and effects of various cardiovascular diseases systematically, studies on the BP waveform have been conducted8. It is known that the number of hypertensive patients increases with age, and one of the causes is increasing vascular stiffness with age. In both men and women, the PWV increases proportional to their ages, since the backward reflected pulse wave from the aortic bifurcation (AB), where the abdominal aorta diverges into the left and right iliac arteries, arrives earlier. As a result, the proportion of the backward reflected wave added to the forward propagating wave increases to raise the SBP, leading to hypertension and degenerative cardiovascular disease11. From clinical statistics, exercise load, heart rate (HR), stroke volume (SV), and muscle stiffness were suggested as the causes of changing BP12. How HR and SV change the mean arterial pressure (MAP) and pulse pressure  was studied through pacemaker and drug dosing14. However, in clinical studies, the control of cardiovascular parameters and corresponding data collection are almost impossible due to mostly the difficulty of invasive measurements and the bias of sample collections.To reproduce the blood pressure (BP) waveform and investigate the mechanism of the BP waveform generation, we developed a dedicated hardware simulator that replicates the physiological structure and physical properties of the human cardiovascular system. Before the pandemic distorted the global causes of death, according to WHO statistics, 17.9 million people worldwide died from cardiovascular diseases in 2019, accounting for 32% of all deaths in a year24. For this purpose, a series of cardiovascular simulators were developed, such as the phantom of the left ventricle-valve-aorta28, the pneumatic-based brachial pulse wave generator for calibration of a BP monitor30, the cardiovascular surgery model31, or the pulse wave generator for durability testing of artificial valves and stents were presented36. In these previous studies, only the structural shape and basic layout of the human cardiovascular system were mimicked. To reproduce the human BP waveform determined by the PWV and wave propagating and reflection mechanism, the structure of the blood vessel wall and its physical properties must be implemented close to human. To reproduce the physical properties of actual blood vessels, studies on the fabrication and characteristics of artificial blood vessels were conducted40. Rather than the entire blood vessel, only physical properties were implemented by stacking silicone layers of various stiffness in the form of sheets, or only PWV reproduction in a short straight section was studied41. These existing cardiovascular simulators and artificial blood vessel studies use different geometries and properties from those of the human cardiovascular system. Therefore, the overlap of the forward and reflected waves and PWV, i.e., the main parameters that determine the BP wave, are dissimilar to those of the human body. It only creates a periodically pulsating pressure waveform, which is different from the human BP waveform. Therefore, its use is limited to surgical training or durability evaluation of artificial organs in a pulsating environment, and it is impossible to study the generation mechanism and characteristics of BP waveforms of human in a precise way.Therefore, previous studies have been conducted using various cardiovascular simulators that mimic human body physiology to study the relationship between cardiovascular system and BP waveformFigure 5. The central artery BP waveform is formed by the overlap of the forward propagating wave and the backward reflected wave. Constructive interference occurs in the section where the two waves overlap. Therefore, the BP is augmented higher than the forward wave, and the increased pressure is called augmentation pressure (AP). The SBP is the highest value in the BP waveform augmented by AP. The augmentation index (AIx), the ratio of PP to AP, dependent on BP waveform, ranges from 11 to 16% in the human standard42. After the pressure change due to the systole ends, diastole begins, and a sudden pressure change due to the occlusion of the aortic valve appears. A slight increase in BP or an inflection point may appear due to the valve occlusion, a dicrotic notch. In overall, the BP waveform is critically affected by the reflected wave arrival time and amplitude, which determines the overlap5. The position where the forward wave is reflected from the aorta is the AB where the abdominal aorta divides into the bifurcated femoral artery. As shown in Fig. 5. Therefore, the region where the augmentation occurs during systole is widened, the systolic peak is raised, i.e., SBP is increased.The central aortic BP waveform refers to the pressure signal measured from the aortic arch (AA), which is directed upward just after the left ventricle. Its canonical waveform and characteristics are shown in Fig. L between measurement points by the pulse transit time . The aPWV is the average value of the PWV from the aortic root to the AB and the cfPWV is the average value of the PWV from the carotid artery to the femoral artery, which about 20% larger than aPWV because it includes the femoral artery, which has a small diameter and large PWV9.The right-hand side of Fig. 48. The increase in PWV with aging applies equally to the clinically measured cfPWV and the estimated aPWV from the central aortic BP waveform.The PWV is an effective indicator of 'vascular age' because the aging of blood vessels induces change in stiffness, which is directly related to PWV. As described in Eq. , the sti5. In the young-aged (19) BP waveform, two distinct peaks of forward and reflected waves are observed. In the middle-aged (42) BP waveform, which is the human standard BP waveform with SBP of 120 mmHg and BDP of 80 mmHg, AP is partially observed during systole due to the overlap of forward and reflected waves. In the old-aged (83) BP waveform, AP is observed in almost the entire area of systole due to the overlap of forward and reflected waves in wider region. As mentioned in the previous section, the stiffness of the vessel wall increases with aging, which leads to increase in PWV. With aging, the rupture and regeneration of elastic fibers of the inner layer are repeated, fibrosis and hardening proceed, and the composition ratio of the collagen fiber of the outer layer rises, resulting in increase of stiffness5. Clinically measured cfPWV values are 5–7 m/s for the young-aged, 6–9 m/s for the middle-aged, and 9–12 m/s for the old-aged51. The increased PWV with aging shortens the reflected wave's return time from AB, widens the overlapping region with the forward wave, and increases AP and SBP in turn. In other words, one of major causes of hypertension with aging is the increase in vascular stiffness and corresponding PWV.Cardiovascular parameters include passive factors that depend on blood vessel, such as stiffness, diameter, and thickness; and, active factors that depend on cardiovascular conditions, such as HR, SV, and PR. The clinically observed trend of blood pressure change according to the cardiovascular parameters is shown in Fig. 14. Millasseau studied the changes in BP by controlling HR using drugs and pacemakers separately. The reason for comparing the two HR control methods is that HR and SV cannot be controlled independently but changes simultaneously. Cardiac output (CO), i.e., the amount of blood pumped out per minute, is defined by the product of HR and SV. The CO is related to the amount of oxygen required depending on psychological state, exercise, and hormones, etc. When an adrenaline-like drug is dosed, the amount of required oxygen in the body is increased to have a large CO, i.e., HR and SV increase simultaneously. In this case, it is observed clinically that the SBP increases, and the DBP is maintained. On the other hand, when the HR is artificially controlled using a pacemaker, the amount of oxygen required in the human body does not change, so the CO remains constant, thus the SV decreases as the HR increases. In this case, it is observed clinically that the SBP is maintained and the DBP increased, which shows a different behavior compared to the case of drug dosing. This contradictory tendencies of BP change are evidence that multiple parameters influence human BP in a complex way. Both HR control methods increased MAP but showed opposite changes in PP.The center of Fig. MAP is an area average for one period of the BP waveform, and is defined as Eq.  in a sim57. To control the PR, in case of injection level of the vasodilator increases, the vessel diameter increases, and the PR decreases. As PR decreases, a decrease in MAP and an increase in PP are observed. This is estimated to be caused by a decrease in TPR and a decrease in the ratio of PR among TPR, but there are limitations in clinical studies because other cardiovascular variables, such as HR, were not considered.PR means the hydrodynamic resistance acting in the circulation of blood in the cardiovascular system. The total peripheral resistance (TPR) is the sum of the PR of each blood vessel in the entire cardiovascular system, and it is defined as the ratio of MAP to CO. The TPR is an indicator of the pressure required to circulate blood with rate valued in CO [entclass1pt{minima3, and the viscosity is around 3–4 cP5. In large-diameter aortas, the effect of inertia is significant, while the impact of viscosity is negligible47. Therefore, in this study, distilled water was used as the fluid inside the simulator since it has a 1000 kg/m3 density similar to blood and a 1 cP of viscosity has the same order of magnitude as that of blood.Figure During systole, the ventricular pump contracts, generating flow and forward pressure waves. The forward pressure wave propagates through the artificial aorta, overlaps with the reflected wave returning from the AB, and generates a BP waveform. Blood flow through the AB reaches the reservoir. During diastole, the ventricular pump expands, the aortic valve prevents backflow, and fluid moves from the reservoir through the atrium and into the ventricle. The cardiovascular simulator in this study controls HR and SV by changing the input function to the pump and controls PR by changing the PR ball valves occlusion ratios. In addition, the internal and external pressures of the artificial aorta are controlled using the reservoir and the compliance chamber with a connected pneumatic pump. To observe the blood pressure waveform according to the cardiovascular parameters, the pressure is measured with pressure sensors at ventricle and multiple points in the artificial aorta, compliance chamber, and reservoir. NI-9234 DAQ  was used to acquire the pressure sensor data, the sampling rate was 8 kHz, and a 30 Hz low-pass filter was applied.5. The shape of the artificial aorta is shown on the left-hand side of Fig. 58.The cardiovascular simulator includes the artificial aorta that mimics the physiological structure and physical properties of the human aorta. The artificial aorta was designed based on the 3D scanned shape and dimensions of the actual human central aorta with 1:1 scale, and some of peripheral structures, such as the renal artery and the right subclavian artery, were simplified51. Also, since these physical quantities change dynamically due to the IVP pulsating between the SBP and DBP, this study used pulse-averaged physical quantities which are averaged values on time within a single pulse. These values have already considered the effects of interactions with surrounding tissues and IVP-induced changes. The transmural pressure (TP), which is the difference between IVP and EVP, is similar to DBP and acts as an IVP at 80 mmHg based on negligible EVP in the body. TP determines the initial diameter and stiffness with expansion and strain-hardening. However, in this study, since the artificial aorta was already manufactured based on the modified diameter and stiffness due to IVP, pulse-averaged TP needs to be maintained at zero. To reproduce the human standard PWV and BP waveform, the artificial aorta was fabricated using SortaClear 12 biomimetic silicone manufactured by Smooth-On, Inc. (USA). To measure the stiffness of silicone used in the artificial aorta, a dog-bone specimen for tensile testing and a cylinder specimen for compression testing were fabricated simultaneously with the artificial aorta. The tensile and compression tests were performed using a push–pull gauge . The tensile and compression test results of SortaClear 12 silicone used to fabricate the artificial aorta are shown on the right-hand side of Fig. 60.In this study, the human standard BP waveform was mimicked as shown in Fig. 59. This is one of the reasons why blood vessels do not rupture even when high blood pressure is present in the body. As blood pressure increases, the stiffness of blood vessels increases rapidly due to strain-hardening properties, and the diameter of blood vessels does not increase as much as the stiffness, leading to the observation of rapid PWV45. On the other hand, in the case of biomimetic silicone, as shown on the red line in the right-hand side of Fig. 62. Thus, silicone has a similar stiffness to human blood vessels up to 10% tensile and compressive strain, but expands as the IVP increases, and there is a risk of over-expansion and rupture due to excessive strain-softening effect. The stiffness softening of silicone must be suppressed to reproduce PWV and BP waveforms similar to human. In this study, a specially designed compliance chamber was introduced to maintain the TP of the artificial aorta at zero and prevent strain-softening effects.However, biomimetic silicones, including SortaClear 12, have some different physical properties and behavior from those of human blood vessels. Human blood vessels have a complex structure with inner wall of elastin and muscle cells and an outer wall of collagen, as shown on the left-hand side of Fig. 41. However, applying the composite layers to mimic the complex blood vessel structures is one of challenging tasks due to many of design variables in nonlinear relations.The compliance chamber in this study was designed to control the EVP of the artificial aorta. It is used to prevent strain-softening, along with a reservoir that controls the IVP of the artificial aorta. The strain-softening of silicone changes the stiffness and makes it impossible to reproduce the desired PWV and even BP waveforms similar to human. When reproducing the BP waveform in the simulator, the IVP of the artificial aorta is raised using a reservoir to move the pressure to the same as the DBP of a human. In the case of the human blood vessel, this increase in IVP would lead to an increase in diameter and a rapid increase in stiffness due to strain-hardening, resulting in an increase in PWV. However, in the artificial aorta, the biomimetic silicone with strain-softening behavior causes a rapid increase in diameter and a decrease in stiffness, which leads to a rapid decrease in PWV. This distorts the BP waveform, and severe stiffness decreases can lead to rupture of the artificial aorta. To mimic the strain-hardening of human blood vessel walls, composite layers in which silicone or fiber meshes with different stiffnesses are multi-layered have been proposedIn this study, instead of developing the multi-layered tube, the outside of the artificial aorta is pressurized with air by the compliance chamber to compensate the strain-softening effect of the biomimetic silicone. The compliance chamber uses air, which has a negligible density compared to water, for EVP control. This minimizes contact, interaction, or loading with the artificial aorta because the changes caused by IVP and interactions with surrounding tissues have already been considered in manufacturing the artificial aorta. The compliance chamber seals the entire exterior of the artificial aorta, and the working principle is on the right-hand side of Fig. 5. Figure 63. The MAP of the reproduced central aortic BP waveform is also 93.3 mmHg same as the human standard5.Figure As shown in Fig. 42. Precise PWV and BP level reproduction enables reproduction of BP waveforms similar to humans. In the following section, based on the BP waveforms with 120 mmHg SBP and 80 mmHg DBP reproduced by the cardiovascular simulator, changes of the BP waveform according to cardiovascular parameters are investigated. By controlling cardiovascular parameters such as HR, SV, PR, IVP, and EVP, the change in BP waveform is observed and compared with human characteristics under the similar changes of cardiovascular parameters.Therefore, the cardiovascular simulator in this study successfully reproduced the SBP, DBP, and PWV of the human standard. PP was 40 mmHg, and AP, the pressure difference between the inflection point and the peak, was 5.3 mmHg, so AIx was calculated as 13.3%. This value is in the middle of the human standard AIx range (11–16%)Figure By simultaneous control of HR and SV, the change of the BP waveform was observed. Figure Figure The cardiovascular simulator in this study includes the dedicated compliance chamber. In the artificial aorta, the reservoir increases BP to the same level as humans by applying IVP, and the compliance chamber prevents strain-softening by applying EVP to suppress expansion by air pressure. Using a cardiovascular simulator, through the control of IVP and EVP, the change in BP waveform according to TP was observed. Figure However, if TP is greater than zero, the IVP generated by the reservoir is greater than the EVP generated by the compliance chamber. Figure 51. These values are in the middle of the human standard range, and the reproduced blood pressure waveform is similar to that of humans. The errors from the human standard values are less than 1 mmHg for BP, less than 0.05 m/s for PWV, and less than 3% for AIx. The simulator reproduced not only the standard BP waveform but also the BP waveform changes according to cardiovascular parameters such as HR, SV, and PR observed in humans. In contrast to the human body, where HR and SV cannot be independently controlled, the simulator in this study allows for independent or simultaneous control of HR and SV. HR was adjusted at 10 BPM intervals from 60 to 100 BPM, and SV was adjusted at 5 mL intervals from 60 to 80 mL. Individual parameter control experiments showed that HR was involved in MAP, and SV was involved in PP. With simultaneous control of HR and SV, the simulator reproduced the changes in BP under conditions, which are the increase in CO due to drug administration and the maintenance of CO due to pacemaker stimulation14. In addition, by controlling the valve occlusion area at the end of the femoral artery, the simulator reproduced the changes in BP waveform according to PR. The occlusion area of the PR valve was increased from 5 to 25% in 5% increments. A decrease in PR resulted in a decrease in MAP and PP, which is similar to the change observed in humans when PR is decreased by administering vasodilators that increase the cross-sectional area of capillaries57. The simulator uses a compliance chamber to apply the same EVP as the IVP, which sets TP to 0 and suppresses the expansion of the artificial aorta to prevent changes in stiffness and PWV and distortion of the BP waveform due to strain-softening. As a result of the experiment, with zero TP condition, it was observed that a constant PWV and a BP waveform without distortion were maintained even when the DBP level was controlled from 40 to 120 mmHg. On the other hand, with positive TP condition up to 20 mmHg, the EVP given by the compliance chamber is insufficient, and the artificial aorta expands, strain-softening occurs, resulting in a decrease in stiffness and PWV and distortion of the BP waveform.The presented cardiovascular simulator can reproduce a human BP waveform with a SBP of 120 mmHg and a DBP of 80 mmHg under the condition of 75 BPM HR and 70 mL SV pulsation. The measured PWV of 6.58 m/s, and AIx of 13.3%, which is similar to the clinical value of middle-aged28. Some others implemented an aorta structure but used materials like acrylic, PVC, or soft silicone but not in the physiological range36. However, these methods cannot reproduce PWV, the critical physical property involved in generating blood pressure in humans. There have been studies attempting to manufacture artificial blood vessels that have similar physical properties to those of humans. However, due to the complex manufacturing process that involves mixing or multi-layering various materials, the resulting artificial vessels are limited in shape to either a plate or a cylindrical shape41. It is difficult to replicate the size and structure of a human aorta using these artificial vessels. In contrast, the simulator in this study implemented an artificial aorta that mimics the shape and stiffness of a human, and it is possible to reproduce a BP waveform with the same magnitude and shape as a human by maintaining the stiffness through a compliance chamber. The simulator is able to reproduce not only the magnitude and shape of the BP waveform but also the changes observed with variations in cardiovascular parameters because it reproduced the process of BP waveform generation through the superposition of forward and reflected waves. The simulator demonstrated and investigated with physically explainable mechanisms, the changes in BP waves according to the controllable cardiovascular parameters that are difficult to control in clinical studies. Expanding the design of the simulator can lead to more clinical and practical applications. Currently, the artificial aorta of the simulator is manufactured with middle-aged stiffness to reproduce the standard central BP waveform, but artificial aortas with various stiffnesses are required to study the change of BP waveform according to aging. Diversification of the artificial aorta according to stiffness will be the basis for reproducing aging and vascular diseases such as aortic aneurysm and arteriosclerosis and their symptoms. In addition, expanding the simulator to other arteries can enable the measurement of blood pressure in the brachial artery and the generation of PPG in the radial artery. Also, in contrast to the aorta, PWV and BP waveforms in small-diameter arteries are influenced by viscosity, which can be useful in studying conditions related to blood health, such as hyperlipidemia47. This would enable the simulator to be a preclinical testing platform for developing smart wearable devices or medical instruments.In this study, the simulator features an artificial aorta with a 1:1 scale shape and similar vascular stiffness to reproduce PWV and generate the BP waveform. Previous studies on simulators for blood pressure waveform reproduction have used different methods for generating blood pressure. Some simulators only implement the BP ventricle and atrium without the aorta and adjust the input volume-time curve65.In this paper, the presented cardiovascular simulator reproduces the human BP waveform and behaviors according to cardiovascular parameters in same pressure range and trends. The cardiovascular simulator in this study is applicable to supplement missing links in clinical data to collect a massive dataset and analyze the mechanism of cardiovascular diseases and symptoms. However, the artificial aorta of the simulator has a limitation in that it cannot reproduce the BP waveform of the brachial or radial artery, which is frequently measured and applied. In the future, by replacing the artificial aorta with properties of young and old-aged, it can be expanded to study BP waves according to age. In addition, it can be applied to developing BP measuring instruments and wearable devices, e.g. cuffs and smartwatches by extending the vascular system of the brachial artery and radial artery from the artificial aorta in the future works