Sex-dependent expression of galectin-1, a cardioprotective ß-galactoside-binding lectin, in human calcific aortic stenosis.

We aimed to analyze sex-related differences in galectin-1 (Gal-1), a ß-galactoside-binding lectin, in aortic stenosis (AS) and its association with the inflammatory and fibrocalcific progression of AS. Gal-1 was determined in serum and aortic valves (AVs) from control and AS donors by western blot and immunohistochemistry. Differences were validated by ELISA and qPCR in AS samples. In vitro experiments were conducted in primary cultured valve interstitial cells (VICs). Serum Gal-1 was not different neither between control and AS nor between men and women. There was no association between circulating and valvular Gal-1 levels. The expression of Gal-1 in stenotic AVs was higher in men than women, even after adjusting for confounding factors, and was associated with inflammation, oxidative stress, extracellular matrix remodeling, fibrosis, and osteogenesis. Gal-1 (LGALS1) mRNA was enhanced within fibrocalcific areas of stenotic AVs, especially in men. Secretion of Gal-1 was up-regulated over a time course of 2, 4, and 8 days in men's calcifying VICs, only peaking at day 4 in women's VICs. In vitro, Gal-1 was associated with similar mechanisms to those in our clinical cohort. ß-estradiol significantly up-regulated the activity of an LGALS1 promoter vector and the secretion of Gal-1, only in women's VICs. Supplementation with rGal-1 prevented the effects elicited by calcific challenge including the metabolic shift to glycolysis. In conclusion, Gal-1 is up-regulated in stenotic AVs and VICs from men in association with inflammation, oxidative stress, matrix remodeling, and osteogenesis. Estrogens can regulate Gal-1 expression with potential implications in post-menopause women. Exogenous rGal-1 can diminish calcific phenotypes in both women and men.

Albumin Redox Modifications Promote Cell Calcification Reflecting the Impact of Oxidative Status on Aortic Valve Disease and Atherosclerosis.

Calcific aortic valve disease (CAVD) and coronary artery disease (CAD) are related cardiovascular diseases in which common mechanisms lead to tissue calcification. Oxidative stress plays a key role in these diseases and there is also evidence that the redox state of serum albumin exerts a significant influence on these conditions. To further explore this issue, we used multimarker scores (OxyScore and AntioxyScore) to assess the global oxidative status in patients with CAVD, with and without CAD, also evaluating their plasma thiol levels. In addition, valvular interstitial cells were treated with reduced, oxidized, and native albumin to study how this protein and its modifications affect cell calcification. The differences we found suggest that oxidative status is distinct in CAVD and CAD, with differences in redox markers and thiol levels. Importantly, the in vitro interstitial cell model revealed that modified albumin affects cell calcification, accelerating this process. Hence, we show here the importance of the redox system in the development of CAVD, emphasizing the relevance of multimarker scores, while also offering evidence of how the redox state of albumin influences vascular calcification. These data highlight the relevance of understanding the overall redox processes involved in these diseases, opening the door to new studies on antioxidants as potential therapies for these patients.

Sex-specific role of galectin-3 in aortic stenosis.

BACKGROUND: Aortic stenosis (AS) is characterized by inflammation, fibrosis, osteogenesis and angiogenesis. Men and women develop these mechanisms differently. Galectin-3 (Gal-3) is a pro-inflammatory and pro-osteogenic lectin in AS. In this work, we aim to analyse a potential sex-differential role of Gal-3 in AS. METHODS: 226 patients (61.50% men) with severe AS undergoing surgical aortic valve (AV) replacement were recruited. In AVs, Gal-3 expression and its relationship with inflammatory, osteogenic and angiogenic markers was assessed. Valve interstitial cells (VICs) were primary cultured to perform in vitro experiments. RESULTS: Proteomic analysis revealed that intracellular Gal-3 was over-expressed in VICs of male AS patients. Gal-3 secretion was also higher in men's VICs as compared to women's. In human AVs, Gal-3 protein levels were significantly higher in men, with stronger immunostaining in VICs with myofibroblastic phenotype and valve endothelial cells. Gal-3 levels in AVs were positively correlated with inflammatory markers in both sexes. Gal-3 expression was also positively correlated with osteogenic markers mainly in men AVs, and with angiogenic molecules only in this sex. In vitro, Gal-3 treatment induced expression of inflammatory, osteogenic and angiogenic markers in male's VICs, while it only upregulated inflammatory and osteogenic molecules in women-derived cells. Gal-3 blockade with pharmacological inhibitors (modified citrus pectin and G3P-01) prevented the upregulation of inflammatory, osteogenic and angiogenic molecules. CONCLUSIONS: Gal-3 plays a sex-differential role in the setting of AS, and it could be a new sex-specific therapeutic target controlling pathological features of AS in VICs.

Aortic stenosis (AS) is a condition that affects the aortic valves (AVs) of the heart and leads to death if untreated. Males and females show clear differences in the onset of AS, both clinically and in valve deterioration. In this study we identified galectin-3 (Gal-3) as a molecule involved in the development of AS alterations with different effects in men and women. We analyzed AVs of 226 patients (139 male and 87 female) with severe AS who underwent surgical AV replacement to study the association of Gal-3 with markers of mechanisms related to AS, such as inflammation, calcification and blood vessels formation. We performed experiments in valvular interstitial cells (VICs) to evaluate the impact of Gal-3 in these cells and its potential use as a therapeutic target. Our results showed that Gal-3 was more expressed in AVs and VICs of men over women. In AVs, Gal-3 levels were associated with inflammatory markers either in male and female, while they correlated with osteogenic markers mainly in men and with angiogenic only in male. The treatment of VICs with Gal-3 produced increased levels of inflammatory and osteogenic molecules by cells of both sexes, but of angiogenic markers only in male's. Pharmacological inhibition of Gal-3 prevented the increase of these pathological markers in VICs. Overall, our study indicates that Gal-3 is a molecule implicated in the setting of AS in a sex-differential way and its targeting may lead to a new sex-specific therapeutic option for AS treatment.

Influence of diabetes mellitus on the pathological profile of aortic stenosis: a sex-based approach.

BACKGROUND: Diabetes mellitus (DM) accelerates the progression of aortic stenosis (AS), but how their underlying molecular mechanisms interact is not clear. Moreover, whether DM contributes to clinically relevant sex-differences in AS is unknown. In this work we aim to characterize the sex-specific profile of major pathological mechanisms fundamental to aortic valve (AV) degeneration in AS patients with or without concomitant DM. METHODS: 283 patients with severe AS undergoing surgical valve replacement (27.6% DM, 59.4% men) were recruited. Expression of pathological markers related to AS were thoroughly assessed in AVs and valve interstitial cells (VICs) according to sex and presence of DM. Complementary in vitro experiments in VICs in the presence of high-glucose levels (25 mM) for 24, 48 and 72 h were performed. RESULTS: Oxidative stress and metabolic dysfunction markers were increased in AVs from diabetic AS patients compared to non-diabetic patients in both sexes. However, disbalanced oxidative stress and enhanced inflammation were more predominant in AVs from male AS diabetic patients. Osteogenic markers were exclusively increased in the AVs of diabetic women. Basal characterization of VICs confirmed that oxidative stress, inflammation, calcification, and metabolic alteration profiles were increased in diabetic VICs with sex-specific differences. VICs cultured in hyperglycemic-like conditions triggered inflammatory responses in men, whereas in women rapid and higher production of pro-osteogenic molecules. CONCLUSIONS: DM produces sex-specific pathological phenotypes in AV of AS patients. Importantly, women with diabetes are more prone to develop AV calcification. DM should be considered as a risk factor in AS especially in women.

Association of Klotho with Coronary Artery Disease in Subjects with Type 2 Diabetes Mellitus and Preserved Kidney Function: A Case-Control Study.

Circulating Klotho levels are significantly reduced in subjects with type 2 diabetes mellitus (T2DM) and in kidney disease patients. In this work, the relationship between Klotho levels and the coronary artery disease (CAD) burden in subjects with T2DM and preserved kidney function was analyzed. For this, we performed a cross-sectional case-control study involving 133 subjects with T2DM and 200 age-, sex- and CAD-incidence-matched, non-diabetic patients undergoing non-emergency diagnostic coronary angiography. All of them were non-albuminuric and with normal glomerular filtration rates. The concentrations of serum Klotho, fibroblast growth factor 23, and inflammatory markers were also measured. As expected, the serum Klotho concentration was lower in the T2DM group (12.3% lower, p = 0.04). However, within the group of patients with T2DM, those subjects with CAD presented significantly higher Klotho levels than those without significant coronary stenosis (314.5 (6.15-562.81) vs. 458.97 (275.2-667.2) pg/mL; p = 0.02). Multiple regression analysis revealed that serum Klotho was positively related with stenosis values exclusively in subjects with T2DM (adjusted R2 = 0.153, p < 0.01). Moreover, logistic regression analysis showed that Klotho was positively associated with the presence of significant CAD in the group of T2DM patients (OR: 1.001; p = 0.041). Our data suggest that higher levels of circulating Klotho in subjects with T2DM and preserved kidney function are associated with the presence of significant CAD.

Associations between Inflammation, Hemoglobin Levels, and Coronary Artery Disease in Non-Albuminuric Subjects with and without Type 2 Diabetes Mellitus.

In this cross-sectional study, we evaluated the associations of inflammation and hemoglobin with coronary artery disease (CAD) in subjects with type 2 diabetes mellitus (T2DM) and preserved kidney function. We recruited 638 participants-254 with T2DM-subjected to coronary angiography with no known cardiovascular disease, normal glomerular filtration rates, and without albuminuria. The hemoglobin and serum levels of inflammatory markers, including high-sensitivity C-reactive protein (hs-CRP), were measured. Multivariable analyses showed that inflammatory markers were not related to the severity of the stenosis in the group of subjects with diabetes. Conversely, inflammatory cytokines and albuminuria were directly related to the percentage of stenosis in subjects without T2DM (R2 = 0.038, p < 0.001). Patients with diabetes presented lower hemoglobin levels, particularly in those who also had significant CAD (14.4 [13.6-15.1] vs. 13.6 [12.2-14.8] g/dL, p = 0.03). Similarly, hemoglobin levels and albuminuria were inversely related to the severity of stenosis exclusively in subjects with diabetes, even after adjusting for multiple confounding factors (R2 = 0.081, p < 0.001). We conclude that reductions in hemoglobin levels in subjects with T2DM and normoalbuminuria may constitute a more relevant risk factor for CAD than inflammation.

Los niveles de ST2 soluble están relacionados con la fibrosis miocárdica de sustitución en la estenosis aórtica grave / Soluble ST2 levels are related to replacement myocardial fibrosis in severe aortic stenosis

Introducción y objetivos: Los pacientes con estenosis aórtica presentan remodelado del ventrículo izquierdo (VI) y fibrosis miocárdica de sustitución (FMS). Se desconoce si sST2 se asocia con la FMS medida por resonancia magnética y con el sexo. Métodos: Se incluyó a 79 pacientes consecutivos (73,0 [68,0-78,0] años; 61% varones) con estenosis aórtica grave aislada tratados con sustitución valvular. Se identificaron y cuantificaron la FMS mediante realce tardío post-gadolinio (RTG) y se valoró el sST2 sérico. Resultados: La FMS se asoció con sST2 elevado, hipertrofia y dilatación del VI y menor fracción de eyección del VI. Todos los pacientes con disfunción del VI tenían FMS. sST2 ≥ 28,2 ng/ml se asoció con FMS y mayor hipertrofia del VI. La masa de RTG se correlacionó con el remodelado del VI y sST2. Los niveles de sST2 fueron mayores en pacientes con fibrosis intramiocárdica frente a subendocárdica. El análisis multivariante evidenció que solo la fracción de eyección y sST2 se asociaban con la FMS. Los varones presentaron mayores niveles de FMS y sST2. En varones la FMS correlacionó con mayor dilatación e hipertrofia ventricular, y con la masa de RTG. Conclusiones: El sST2 es un factor independiente de FMS en la estenosisi aórtica grave aislada. sST2 ≥ 28,2 ng/ml predice la FMS y se relaciona con mayor hipertrofia del VI. La expresión de sST2 y asociaciones clínicas deben ser sexo-específicas.(AU)

Introduction and objectives: Patients with aortic stenosis (AS) exhibit left ventricular (LV) remodeling and replacement myocardial fibrosis (RMF). Whether sST2 is associated with RMF measured by cardiac magnetic resonance and with sex remains unknown. Methods: We recruited 79 consecutive patients (73.0 [68.0-78.0] years; 61% men) with severe isolated AS underdoing valve replacement. RMF was identified and quantified by late gadolinium enhancement (LGE). Serum sST2 levels were determined. Results: RMF was associated with higher circulating sST2 levels, LV hypertrophy and dilation, and lower LV ejection fraction. All patients with LV dysfunction had RMF. Circulating levels of sST2 ≥ 28.8 ng/mL were associated with RMF and greater LV hypertrophy. LGE mass was correlated with LV remodeling and sST2. Of note, sST2 levels were also associated with the RMF pattern, being higher in midwall than in subendocardial fibrosis. Multivariate analyses showed that only LV ejection fraction and sST2 levels were associated with RMF. Moreover, men had higher levels of sST2 and RMF. RMF was associated with higher LV dilation and hypertrophy only in men and was correlated with LGE mass. Conclusions: SST2 was an independent factor for RMF in patients with severe isolated AS. The presence of RMF was predicted by sST2 ≥ 28.2 ng/mL, and was associated with greater LV hypertrophy. sST2 expression and clinical associations may be sex-specific.(AU)

Klotho inversely relates with carotid intima- media thickness in atherosclerotic patients with normal renal function (eGFR ≥60 mL/min/1.73m2): a proof-of-concept study.

Introduction: Klotho protein is predominantly expressed in the kidneys and has also been detected in vascular tissue and peripheral blood circulating cells to a lesser extent. Carotid artery intima-media thickness (CIMT) burden, a marker of subclinical atherosclerosis, has been associated with reductions in circulating Klotho levels in chronic kidney disease patients, who show reduced levels of this protein at all stages of the disease. However, the contribution of serum Klotho and its expression levels in peripheral blood circulating cells and in the carotid artery wall on the CIMT in the absence of kidney impairment has not yet been evaluated. Methods: We conducted a single-center study in 35 atherosclerotic patients with preserved kidney function (eGFR≥60 mL/min/1.73m2) subjected to elective carotid surgery. Serum levels of Klotho and cytokines TNFa, IL6 and IL10 were determined by ELISA and transcripts encoding for Klotho (KL), TNF, IL6 and IL10 from vascular segments were measured by qRT-PCR. Klotho protein expression in the intima-media and adventitia areas was analyzed using immunohistochemistry. Results: APatients with higher values of CIMT showed reduced Klotho levels in serum (430.8 [357.7-592.9] vs. 667.8 [632.5-712.9] pg/mL; p<0.001), mRNA expression in blood circulating cells and carotid artery wall (2.92 [2.06-4.8] vs. 3.69 [2.42-7.13] log.a.u., p=0.015; 0.41 [0.16-0.59] vs. 0.79 [0.37-1.4] log.a.u., p=0.013, respectively) and immunoreactivity in the intimal-medial area of the carotids (4.23 [4.15-4.27] vs. 4.49 [4.28-4.63] log µm2 p=0.008). CIMT was inversely related with Klotho levels in serum (r= -0.717, p<0.001), blood mRNA expression (r=-0.426, p=0.011), and with carotid artery mRNA and immunoreactivity levels (r= -0.45, p=0.07; r= -0.455, p= 0.006, respectively). Multivariate analysis showed that serum Klotho, together with the gene expression levels of tumor necrosis factor TNFa in blood circulating cells, were independent determinants of CIMT values (adjusted R2 = 0.593, p<0.001). Discussion: The results of this study in subjects with eGFR≥60mL/min/1.73m2 show that patients with carotid artery atherosclerosis and higher values of CIMT present reduced soluble Klotho levels, as well as decreased KL mRNA expression in peripheral blood circulating cells and Klotho protein levels in the intima-media of the carotid artery wall.

Soluble ST2 levels are related to replacement myocardial fibrosis in severe aortic stenosis.

INTRODUCTION AND OBJECTIVES: Patients with aortic stenosis (AS) exhibit left ventricular (LV) remodeling and replacement myocardial fibrosis (RMF). Whether sST2 is associated with RMF measured by cardiac magnetic resonance and with sex remains unknown. METHODS: We recruited 79 consecutive patients (73.0 [68.0-78.0] years; 61% men) with severe isolated AS underdoing valve replacement. RMF was identified and quantified by late gadolinium enhancement (LGE). Serum sST2 levels were determined. RESULTS: RMF was associated with higher circulating sST2 levels, LV hypertrophy and dilation, and lower LV ejection fraction. All patients with LV dysfunction had RMF. Circulating levels of sST2 ≥ 28.8 ng/mL were associated with RMF and greater LV hypertrophy. LGE mass was correlated with LV remodeling and sST2. Of note, sST2 levels were also associated with the RMF pattern, being higher in midwall than in subendocardial fibrosis. Multivariate analyses showed that only LV ejection fraction and sST2 levels were associated with RMF. Moreover, men had higher levels of sST2 and RMF. RMF was associated with higher LV dilation and hypertrophy only in men and was correlated with LGE mass. CONCLUSIONS: SST2 was an independent factor for RMF in patients with severe isolated AS. The presence of RMF was predicted by sST2 ≥ 28.2 ng/mL, and was associated with greater LV hypertrophy. sST2 expression and clinical associations may be sex-specific.

Sex-dependent expression of neutrophil gelatinase-associated lipocalin in aortic stenosis.

BACKGROUND: Accumulating evidence suggest the existence of sex-related differences in the pathogenesis of aortic stenosis (AS) with inflammation, oxidative stress, fibrosis and calcification being over-represented in men. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in a myriad of tissues and cell types, and it is associated with acute and chronic pathological processes comprising inflammation, fibrosis or calcification. Sex-dependent signatures have been evidenced for NGAL which expression has been associated predominantly in males to metabolic and cardiovascular disorders. We aimed to analyse sex-related differences of NGAL in AS and its role in the inflammatory and fibrocalcific progression of AS. METHODS AND RESULTS: 220 (60.45% men) patients with severe AS elective for surgical aortic valve (AV) replacement were recruited. Immunohistochemistry revealed higher expression of NGAL in calcific areas of AVs and that was validated by qPCR in in 65 (60% men) donors. Valve interstitial cells (VICs) were a source of NGAL in these samples. Proteome profiler analyses evidenced higher expression of NGAL in men compared to women, and that was further validated by ELISA. NGAL expression in the AV was correlated with inflammation, oxidative stress, and osteogenic markers, as well as calcium score. The expression of NGAL, both intracellular and secreted (sNGAL), was significantly deregulated only in calcifying male-derived VICs. Depletion of intracellular NGAL in calcifying male-derived VICs was associated with pro-inflammatory profiles, dysbalanced matrix remodelling and pro-osteogenic profiles. Conversely, exogenous NGAL mediated inflammatory and dysbalanced matrix remodelling in calcifying VICs, and all that was prevented by the pharmacological blockade of NGAL. CONCLUSIONS: Owing to the over-expression of NGAL, the AV from men may be endowed with higher expression of inflammatory, oxidative stress, matrix remodelling and osteogenic markers supporting the progression of calcific AS phenotypes. The expression of NGAL in the VIC emerges as a potential therapeutic checkpoint, with its effects being potentially reverted by the pharmacological blockade of extracellular NGAL.

Repurposing drugs for highly prevalent diseases: pentoxifylline, an old drug and a new opportunity for diabetic kidney disease.

Diabetic kidney disease is one of the most frequent complications in patients with diabetes and constitutes a major cause of end-stage kidney disease. The prevalence of diabetic kidney disease continues to increase as a result of the growing epidemic of diabetes and obesity. Therefore, there is mounting urgency to design and optimize novel strategies and drugs that delay the progression of this pathology and contain this trend. The new approaches should go beyond the current therapy focussed on the control of traditional risk factors such as hyperglycaemia and hypertension. In this scenario, drug repurposing constitutes an economic and feasible approach based on the discovery of useful activities for old drugs. Pentoxifylline is a nonselective phosphodiesterase inhibitor currently indicated for peripheral artery disease. Clinical trials and meta-analyses have shown renoprotection secondary to anti-inflammatory and antifibrotic effects in diabetic patients treated with this old known drug, which makes pentoxifylline a candidate for repurposing in diabetic kidney disease.

Characterization of the sex-specific pattern of angiogenesis and lymphangiogenesis in aortic stenosis.

Objective: We aim to analyze sex-related differences in angiogenesis and lymphangiogenesis in aortic valves (AVs) and valve interstitial cells (VICs) from aortic stenosis (AS) patients. Approach and Results: Totally 230 patients (59% men) with severe AS undergoing surgical valve replacement were recruited. The density of total neovessels was higher in AVs from men as compared to women. Both small and medium neovessels were more abundant in men's AVs. Accordingly, male AVs exhibited higher CD31 and VE-cadherin expressions. The levels of the pro-angiogenic markers, such as vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)1, VEGFR2, insulin-like growth factor-binding protein-2 (IGFBP-2), interleukin (IL)-8, chemerin, and fibroblast growth factor (FGF)-7, were increased in AVs from men. Transforming growth factor-ß expression was higher in male AVs. The expression of antiangiogenic molecules thrombospondin (Tsp)-1, endostatin, and CD36 was upregulated in male AVs, although the levels of Tsp-2, IL-4, IL-12p70, and chondromodulin-1 were similar between both sexes. The number of lymphatic vessels and the expression of the lymphangiogenic markers Lyve-1 and D2-40 was higher in men's AV as well as VEGF-C, VEGF-D, and VEGFR3. Multivariate analyses adjusted for confounders further validated the sex-dependent expression of these targets. VICs isolated from men's AVs secreted higher amounts of the pro-angiogenic factors, VEGF-A, VEGFR1, IGFBP-2, and FGF-7, as well as the pro-lymphangiogenic factors, VEGF-C, VEGF-D, and VEGFR3, than women without changes in antiangiogenic markers. Conclusion: Our data show that aberrant angiogenic and lymphangiogenic cues are over-represented in male AVs. Importantly, the VIC is a relevant source of multiple morphogens involved in angiogenesis and lymphangiogenesis likely endowing the AV of men with the predominant calcific AS phenotypes.

Targeting Fatty Acid-Binding Protein 4 Improves Pathologic Features of Aortic Stenosis.

Aortic stenosis (AS) is a fibrocalcific disease of the aortic valves (AVs). Sex-differences in AS pathophysiology have recently been described. High levels of fatty acid-binding protein 4 (FAPB4) in atherosclerotic plaques have been associated with increased local inflammation, endothelial dysfunction, and plaque vulnerability. FABP4 pharmacological blockade has been shown to be effective for the treatment of atherosclerosis by modulating metabolic and inflammatory pathways. We aimed to analyze the sex-specific expression of FABP4 in AS and its potential role as a therapeutic target. A total of 226 patients (61.5% men) with severe AS undergoing surgical AV replacement were recruited. The FABP4 levels were increased in the AVs of AS patients compared to the control subjects, showing greater expression in the fibrocalcific regions. Male AVs exhibited higher levels of FABP4 compared to females, correlating with markers of inflammation (IL-6, Rantes), apoptosis (Bax, caspase-3, Bcl-2), and calcification (IL-8, BMP-2 and BMP-4). VICs derived from AS patients showed the basal expression of FABP4 in vitro. Osteogenic media induced upregulation of intracellular and secreted FABP4 levels in male VICs after 7 days, along with increased levels of inflammatory, pro-apoptotic, and osteogenic markers. Treatment with BMS309403, a specific inhibitor of FABP4, prevented from all of these changes. Thus, we propose FABP4 as a new sex-specific pharmacological therapeutic target in AS.

Sex-Related Signaling of Aldosterone/Mineralocorticoid Receptor Pathway in Calcific Aortic Stenosis.

BACKGROUND: There are sex differences in the pathophysiology of aortic valve (AV) calcification in patients with aortic stenosis, although the molecular and cellular mechanisms have not been elucidated. Aldosterone (Aldo) promotes proteoglycan synthesis in valve interstitial cells (VICs) from mitral valves via the mineralocorticoid receptor (MR). We investigated the influence of sex in the role of Aldo/MR pathway in AV alterations in patients with aortic stenosis. METHODS AND RESULTS: MR was expressed by primary aortic VICs and in AVs from patients with aortic stenosis. MR expression positively correlated with VIC activation markers in AVs from both sexes. However, MR expression was positively associated with molecules involved in AV calcification only in AV from men. Aldo enhanced VIC activation markers in cells from men and women. Interestingly, Aldo increased the expression of calcification markers only in VICs isolated from men. In female VICs, Aldo enhanced fibrotic molecules. MR antagonism (spironolactone) blocked all the above effects. Cytokine arrays showed ICAM (intercellular adhesion molecule)-1 and osteopontin to be specifically increased by Aldo in male VICs. In AVs from men, MR expression positively associated with both ICAM-1 (intercellular adhesion molecule-1) and osteopontin. Only in female VICs, estradiol treatment blocked Aldo-induced VICs activation, inflammation, and fibrosis. CONCLUSIONS: These findings demonstrate that the Aldo/MR pathway could play a role in early stages of aortic stenosis by promoting VICs activation, fibrosis, and ulterior calcification. Importantly, Aldo/MR pathway is involved in fibrosis in women and in early AV calcification only in men. Accordingly, MR antagonism emerges as a new sex-specific pharmacological treatment to prevent AV alterations.

Klotho expression in peripheral blood circulating cells is associated with vascular and systemic inflammation in atherosclerotic vascular disease.

Cardiovascular disease is the leading cause of death worldwide. New therapeutic strategies are aimed to modulate the athero-inflammatory process that partially orchestrates underlying vascular damage. Peripheral blood circulating cells include different immune cells with a central role in the development of the atherogenic inflammatory response. The anti-aging protein α-Klotho has been related to protective effects against CVD. KL is expressed in monocytes, macrophages, and lymphocytes where it exerts anti-inflammatory effects. In this work, we analyse the relationships of the levels of inflammatory markers with the expression of the KL gene in PBCCs and with the serum levels of soluble KL in atherosclerotic vascular disease. For this, we conducted a cross-sectional single-center case-control study including a study group of 76 CVD patients and a control group of 16 cadaveric organ donors without medical antecedent or study indicating CVD. Vascular artery fragments and whole blood and serum samples were obtained during elective or organ retrieval surgery. Serum levels of sKL, TNFα and IL10, and gene expression levels of KL, TNF, IL10, NFKB1, DNMT1, and DNMT3A in PBCCs were measured. In these cells, we also determined KL promoter methylation percentage. Histological and immunohistochemical analyses were employed to visualize atherosclerotic lesions and to measure IL10 and TNFα levels in vascular fragments. Patients with CVD presented higher values of proinflammatory markers both at systemic and in the vasculature and in the PBCCs, compared to the control group. In PBCCs, CVD patients also presented lower gene expression levels of KL gene (56.4% difference, P < 0.001), higher gene expression levels of DNMT1 and DNMT3A (P < 0.0001, for both) and a higher methylation status of in the promoter region of KL (34.1 ± 4.1% vs. 14.6 ± 3.4%, P < 0.01). In PBCCs and vasculature, KL gene expression correlated inversely with pro-inflammatory markers and directly with anti-inflammatory markers. sKL serum levels presented similar associations with the expression levels of pro- and anti-inflammatory markers in PBCCs. The differences in KL expression levels in PBCCs and in serum sKL levels with respect to control group was even greater in those CVD patients with macroscopically observable atheromatous plaques. We conclude that promoter methylation-mediated downregulation of KL gene expression in PBCCs is associated with the pro-inflammatory status in atherosclerotic vascular disease.

Sodium-glucose co-transporter-2 inhibitors increase Klotho in patients with diabetic kidney disease: A clinical and experimental study.

Sodium-glucose co-transporter-2 inhibitors (SGLT2i) provide cardiorenal protection. However, the molecular mechanisms remain poorly understood. We explored the impact of SGLT2i on Klotho, a kidney-derived protein with antiaging, renal-protective and heart-protective properties. A real world prospective observational study addressed the impact of initiating SGLT2i (canagliflozin, dapagliflozin, empagliflozin) or dipeptidyl peptidase-4 inhibitors (DPP4i) in patients with early diabetic kidney disease (DKD). Serum and urinary soluble Klotho, albuminuria and serum and urinary tumor necrosis factor-alpha (TNFa) were measured. The effect of SGLT2i on Klotho mRNA and protein was explored in vitro in kidney proximal tubular cells stressed with high glucose concentrations to simulate the diabetic milieu, albumin to simulate albuminuria, and the inflammatory cytokine TWEAK to simulate the inflammatory environment in DKD. Baseline urinary Klotho was negatively associated with albuminuria (r - 0.45, P < 0.001) and urinary TNFa (r - 0.40, P < 0.01). Both DPP4i and SGLT2i reduced HbA1c similarly, but only SGLT2i decreased eGFR, albuminuria and urinary TNFa and increased (P < 0.001) serum (5.2 %) and urinary Klotho (38.9 %). Changes in urinary TNFa (ß - 0.53, P = 0.001) and albuminuria (ß - 0.31, P < 0.05) were independently associated with changes in urinary Klotho (adjusted R2 = 0.54, P < 0.001). Studies in renal tubular cells demonstrated that high glucose, albumin and TWEAK decreased Klotho mRNA expression and protein levels, an effect similarly prevented by SGLT2i. SGLT2i increase Klotho availability in type 2 diabetic patients with poorly controlled diabetes and early DKD, as well as in stressed tubular cells. This effect on Klotho may contribute to the kidney and heart protection afforded by SGLT2i.

Factores asociados a mortalidad en pacientes con Covid-19 en un hospital público / Factors associated with mortality in patients with Covid-19 in a public hospital

El objetivo del estudio fue determinar los factores asociados a mortalidad hospitalaria en pacientes con enfermedad por coronavirus 2019 (COVID-19). Se realizó un estudio observacional, analítico, de casos y controles. Se incluyeron pacientes hospitalizados con diagnóstico de la COVID-19 por prueba serológica y/o prueba molecular entre el marzo y agosto del 2020. Para el análisis estadístico se empleó la prueba de Mann Whitney y para el análisis de los factores asociados se utilizó regresión logística. La significancia de p-valor fue < 0,05. Se incluyó 814 pacientes, 556 (68,3%) fueron varones y 246 (30,2%) mayores de 60 años. La presencia de alguna comorbilidad se evidenció en 29,6 % (241 pacientes); 35,8% (292) fallecieron. La mediana de la edad de los fallecidos fue mayor en comparación a los sobrevivientes (59 vs 49; p>0,01). Las comorbilidades asociadas a la COVID-19 fueron: la obesidad (OR= 2,14; IC 95%: 1,38 ­ 3,32) y la hipertensión arterial (OR=1,86; IC 95%: 1,06-3,24). Asimismo, niveles de saturación de oxígeno menor al 85% al ingreso al hospital (OR= 3,58; IC 95%: 2,82- 4,53); la edad mayor a 60 años (OR=1,96; IC 95%: 1,54- 2,50) y el sexo masculino (OR= 1,64; IC95; 1,12-2,39) fueron asociados a mayor mortalidad. Finalmente, los factores asociados a mortalidad hospitalaria fueron saturación de oxígeno menor al 85% al ingreso al hospital, mayor de 60 años de edad, obesidad e hipertensión arterial(AU)

The objective of the study was to determine the factors associated with hospital mortality in patients with coronavirus disease 2019 (COVID-19). An observational, analytical, case-control study was conducted. Hospitalized patients diagnosed with COVID-19 by serological test and/or molecular test between March and August 2020 were included. The Mann Whitney test was used for statistical analysis and logistic regression was used for the analysis of associated factors. The significance of p-value was <0.05. A total of 814 patients were included, 556 (68.3%) were men and 246 (30.2%) were older than 60 years. The presence of some comorbidity was evidenced in 29.6% (241 patients); 35.8% (292) died. The median age of the deceased was higher compared to the survivors (59 vs. 49; p>0.01). The comorbidities associated with COVID-19 were: obesity (OR= 2.14; 95% CI: 1.38-3.32) and arterial hypertension (OR=1.86; 95% CI: 1.06- 3.24). Likewise, oxygen saturation levels less than 85% at hospital admission (OR= 3.58; 95% CI: 2.82-4.53); age over 60 years (OR=1.96; 95% CI: 1.54-2.50) and male gender (OR= 1.64; 95% CI: 1.12-2.39) were associated with greater mortality. Finally, the factors associated with hospital mortality were oxygen saturation less than 85% at hospital admission, older than 60 years of age, obesity, and arterial hypertension(AU)

Klotho as a biomarker of subclinical atherosclerosis in patients with moderate to severe chronic kidney disease.

Chronic kidney disease (CKD) has been associated with a higher risk of cardiovascular disease (CVD). CKD patients present a decrease in the levels of the protein Klotho that accompanies the decrease in kidney function. This protein has been related to protective effects against CVD. However, it is unclear whether circulating Klotho, and its expression in peripheral blood cells (PBCs) are also associated with subclinical atherosclerosis in CKD. The present study aimed to study the relationship between Klotho and subclinical atherosclerosis in a population of patients with moderate to severe CKD. We determined the serum levels and gene expression in PBCs levels of Klotho and three inflammatory cytokines in 103 patients with CKD and investigated their relationship with two surrogate markers of subclinical atherosclerotis: ankle-brachial index (ABI) and carotid intima-media thickness (CIMT). Patients with subclinical atherosclerosis presented lower serum and PBCs expression levels of Klotho. Both variables were associated with the presence of subclinical atherosclerosis, being directly related with ABI and inversely with CIMT (P < 0.0001 for both). Multiple regression analysis demonstrated that both variables were significant determinants for ABI (adjusted R2 = 0.511, P < 0.0001) and CIMT (adjusted R2 = 0.445, P < 0.0001), independently of traditional and emergent cardiovascular risk factors. Moreover, both constituted protective factors against subclinical atherosclerosis [OR: 0.993 (P = 0.002) and 0.231 (P = 0.025), respectively]. Receiver operating characteristic analysis pointed to the utility of serum Klotho (area under the curve [AUC]: 0.817, 95% CI: 0.736-0.898, P < 0.001) and its gene expression in PBCs (AUC: 0.742, 95% CI: 0.647-0.836, P < 0.001) to distinguish subclinical atherosclerosis. The reductions in serum and PBCs expression levels of Klotho in CKD patients are independently associated with the presence of for subclinical atherosclerosis. Further research exploring whether therapeutic approaches to maintain or elevate Klotho could reduce the impact of CVD in CKD patients is warranted.

Pathophysiological Implications of Imbalances in Fibroblast Growth Factor 23 in the Development of Diabetes.

Observational studies have associated the increase in fibroblast growth factor (FGF) 23 levels, the main regulator of phosphate levels, with the onset of diabetes. These studies open the debate on the plausible existence of undescribed diabetogenic mechanisms derived from chronic supraphysiological levels of FGF23, a prevalent condition in chronic kidney disease (CKD) and end-stage renal disease (ESRD) patients. These maladaptive and diabetogenic responses to FGF23 may occur at different levels, including a direct effect on the pancreatic ß cells, and an indirect effect derived from the stimulation of the synthesis of pro-inflammatory factors. Both mechanisms could be mediated by the binding of FGF23 to noncanonical receptor complexes with the subsequent overactivation of signaling pathways that leads to harmful effects. The canonical binding of FGF23 to the receptor complex formed by the receptor FGFR1c and the coreceptor αKlotho activates Ras/MAPK/ERK signaling. However, supraphysiological concentrations of FGF23 favor non-αKlotho-dependent binding of this molecule to other FGFRs, which could generate an undesired overactivation of the PLCγ/CN/NFAT pathway, as observed in cardiomyocytes and hepatocytes. Moreover, the decrease in αKlotho expression may constitute a contributing factor to the appearance of these effects by promoting the nonspecific activation of the PLCγ/CN/NFAT to the detriment of the αKlotho-dependent Ras/MAPK/ERK pathway. The description of these mechanisms would allow the development of new therapeutic targets susceptible to be modified by dietary changes or by pharmacological intervention.

Extracellular water/total body water ratio as predictor of mortality in hemodialysis patients.

BACKGROUND: Overhydration is a predictor of mortality in hemodialysis (HD) patients. Bioimpedance spectroscopy (BIS) is used to determine the body composition. Extracellular Water/Total Body Water (ECW/TBW) ratio has been proposed to predict mortality. METHODS: Multicenter, prospective, observational, proof-of-concept study to estimate the impact of ECW/TBW in global and cardiovascular mortality and the relationship with cardiovascular biomarkers. The study included 60 patients (mean age, 71.8 ± 11.4 years; mean time on HD, 52.3 ± 30.8 months) with a median follow-up of 30.5 months (IQ range, 17.2-34 months). RESULTS: Post-dialysis ECW/TBW was directly associated with NT-proBNP and cTnT. During the study 28 patients died, most of them (43%) due to cardiovascular events. Compared to the survivors, these subjects had a higher post-dialysis ECW/TBW ratio (p = 0.006), while for cardiovascular mortality the only significant difference was a higher pre-dialysis ECW/TBW. The ability of post-dialysis ECW/TBW ratio to predict all-cause mortality had an area under the ROC curve (AUC) of 0.71 (CI 95%, 0.57-0.81; p = 0.002), with a cutoff point of 0.5023. For cardiovascular mortality the AUC was 0.66 (CI 95%, 0.52-0.77; p = 0.045), with a cutoff point of 0.4713. CONCLUSIONS: The post-dialysis ECW/TBW ratio measured by BIS can be a predictor of all-cause and cardiovascular mortality.