Natalia López-Andrés

The study uncovers a relevant role of glycolysis and its side-branch route of hexosamine biosynthesis by regulating sugar-driven posttranslational modifications that are critical for the inflammatory response and key steps of leukocyte adhesion to human aortic valve cells. These events are essential for the recruitment of cells of the monocytic lineage to aortic valve leaflets in the initial stages of calcific aortic valve disease.

BACKGROUND: Patients with chronic kidney disease (CKD) are at high risk of cardiovascular complications. We have shown that Ngal (neutrophil gelatinase–associated lipocalin)/lcn2 is involved in aldosterone-induced cardiac remodeling and inflammation. Here, we investigated the role of Ngal in the progression of cardiorenal syndrome. METHODS: CKD was induced in rats via 5/6 nephrectomy in wild-type and Ngal knockout rats. Cardiorenal functions were assessed 3 months after subtotal nephrectomy or sham operation. Cardiac fibroblasts were isolated from wild-type rats and incubated with or without rNgal (recombinant Ngal) and Gal-3 (galectin-3). RESULTS: Cardiac perfusion was less impaired in CKD Ngal knockout than in CKD wild type. Left ventricle interstitial fibrosis was more severe in CKD wild type than in sham but was blunted in CKD Ngal knockout rats. Levels of Gal-3, Col1 (collagen 1), Ccl2 (C-C motif chemokine ligand 2), and IL-6 (interleukin-6) were high in cardiac fibroblasts incubated with rNgal. A similar pattern was observed in cells treated with recombinant Gal-3. Both Ngal and Gal-3 induced activation of the Tlr4 (toll-like receptor 4)-Myd88 (myeloid differentiation primary response 88) pathway. The effects of rNgal were blunted by concomitant treatment with Gal-3 or Tlr4 inhibitors, suggesting that Gal-3 contributes to Ngal-induced cardiac fibrosis and inflammation by activating the Tlr4-Myd88 pathway. In both MEDIA-DHF (Metabolic Road to Diastolic Heart Failure) and BIOSTAT-CHF (Biology Study to Tailored Treatment in Chronic Heart Failure) cohorts, elevated levels of Ngal and Gal-3 were associated with advanced diastolic dysfunction and adverse clinical outcomes, particularly among patients with impaired renal function. CONCLUSIONS: In CKD rats, Ngal was involved in cardiac remodeling via a Gal-3/Tlr4-dependent pathway, increasing inflammation and fibrosis, and correlated to cardiac outcomes in the MEDIA-DHF and BIOSTAT-CHF cohorts.

Background: Aortic stenosis is the most common valvular heart disease, and its progression is accelerated in chronic kidney disease (CKD), where multiple circulating abnormalities worsen its pathophysiology. This study examined the impact of CKD‐related aldosterone excess, focusing on mineralocorticoid receptor signaling and its crosstalk with the innate immune pathway mediated by Toll‐like receptor 4 (TLR4) in aortic valve (AV) interstitial cells and AV tissue. Methods: For the study, we conducted in vivo studies using a CKD rat model challenged with high phosphate, followed by ex vivo analysis of AVs. In parallel, primary rat and human valve interstitial cells were used in vitro to evaluate inflammatory, fibrotic, and osteogenic responses to aldosterone and to identify the key signaling pathways involved. Human AVs from patients with aortic stenosis were also analyzed. Quantitative polymerase chain reaction, western blot, and ELISA were used to characterize these pathways. Results: Mineralocorticoid receptor activation emerged as a central driver of aortic stenosis progression in CKD, promoting valve interstitial cell differentiation, inflammation, fibrosis, and calcification through activation of the TLR4–myeloid differentiation primary response 88 innate immunity pathway in response to aldosterone. Moreover, chemerin was identified as a downstream effector of the mineralocorticoid receptor that activates TLR4, further enhancing remodeling and calcification in AVs and valve interstitial cells. These results were corroborated in human stenotic AV samples from patients with CKD. Conclusions: Collectively, the study defines a novel mineralocorticoid receptor–TLR4–chemerin axis that mediates aldosterone‐driven inflammatory and calcific processes in CKD, in both a rat model and in vitro systems.

Mitral valve disease (MVD) is the most common valvulopathy and a frequent cause of heart failure and death. However, data regarding the molecular basis are scarce. We aimed to thoroughly explore and compare the circulating, molecular, and histopathological profiles in the main subtypes of primary chronic MVD. In total, 300 patients with chronic primary MVD undergoing mitral valve (MV) replacement were enrolled and classified in the four main etiologic subtypes: calcific mitral valve disease (CMVD, n = 81), rheumatic disease (RHVD, n = 114), Barlow’s disease (BD, n = 70), and fibroelastic deficiency (FED, n = 35). Discovery studies were performed using Olink Proteomics® technology in 80 serums from MVD patients (n = 20/etiology). Histopathologic study, ELISA, and zymography were performed on resected MVs to analyze extracellular matrix (ECM) composition and remodeling, inflammation, and calcification. Serum proteomics identified markers exclusively overexpressed in each MVD etiologic subtype. Further enrichment analyses revealed specific etiology-dependent pathways involving calcification, inflammation, or ECM remodeling. Such etiology-dependent differences were mirrored in the excised MVs both architecturally and compositionally. CMVD and RHVD valves had a marked ECM disorganization with increased collagen deposition and presence of nodular and diffuse calcification, respectively. Moreover, RHVD valves exhibited high levels of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, and matrix degradation enzymes. BD valves showed enhanced inflammatory markers and infiltrates. Moreover, BD and FED valves presented marked proteoglycans deposition. Our study identified circulating markers involved in calcification, inflammation, and ECM remodeling, which may be associated with specific MVD etiologies. The differences in serum markers seem to mirror histopathologic and molecular alterations in the MVs with potential applications into the clinic as diagnosis biomarkers. Identification of underlying molecular mechanisms of each etiology is essential to discover new specific therapeutic targets.

Inflammation and metabolism reprogramming are hallmarks of calcific aortic valve disease (CAVD). Recent studies link inflammation to hyperglycolysis and calcification in valve interstitial cells (VICs). The metabolism of valve endothelial cells (VECs) has received less attention despite both resident valve cells are exposed to alike inflammatory clues involved in the biosynthesis of pathologically relevant glycoproteins during the early stages of CAVD. On this basis, we investigated the outcomes of glucose metabolism rewiring on glycoprotein maturation and immune cell adhesion in human resident valve cells. Real-time metabolic analysis revealed that basal VECs are more glycolytic than VICs. Also, VECs and VICs exposed to inflammatory stimuli exhibited a distinct rewiring, with VECs shifting to a more energetic metabolism, despite a similar upregulation of glycolytic genes. Blunting glucose metabolism in VICs and VECs inhibited inflammatory routes canonically associated with glycolysis, and the expression of proteins associated to the inflammatory response like interleukin-6 and cyclooxigenase-2. Moreover, Western blot and adhesion assays revealed that glycolysis is necessary for the expression and post-translational modifications of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and the ensuing process of monocyte-VECs adhesion. Notably, inhibition of the hexosamine biosynthetic pathway using DON and of N-glycosylation by tunicamycin, further disrupted adhesion molecule maturation and monocyte-VECs adhesion. In conclusion, glycolysis and its side-branch route the hexosamine biosynthesis pathway are necessary for nutrient-driven post-translational modifications of inflammatory proteins in inflamed valve cells and the subsequent process of monocyte-VECs adhesion that plays a key role in the initial stages of CAVD pathogenesis.

Low birth weight (LBW) is a risk factor for type 2 diabetes (T2D). We hypothesized that 4 weeks of carbohydrate overfeeding (COF) with +25% energy would unmask key T2D perturbations among 22 nonobese LBW men, including five with screen-detected metabolic dysfunction–associated steatotic liver disease (MASLD), compared with 21 healthy control participants with normal birth weight (NBW). Body weight, lean and fat mass, and hepatic fat content increased to the same extent in both groups during COF, whereas fasting glucose and insulin resistance increased significantly more in LBW compared with NBW participants. The differential COF responses were most pronounced in LBW participants without MASLD, including increased resting energy expenditure. Plasma adiponectin was lower, whereas fibroblast growth factor 21 levels increased more during COF in LBW participants. Subcutaneous adipose tissue (SAT) density was lower in LBW participants and decreased during COF in both groups. Serum alanine, phosphatidylcholines, and triglycerides increased significantly more in LBW participants during COF. Multiomics analysis of SAT RNA sequencing, serum lipidomics, and metabolomics uncovered impaired peroxisome proliferator–activated receptor signaling as well as aberrant collagen and extracellular matrix regulation in LBW participants. The results document differential and MASLD-independent metabolic perturbations in LBW participants during COF. Article Highlights: Individuals with low birth weight (LBW) are at increased risk of type 2 diabetes. Four weeks of carbohydrate overfeeding (COF) was associated with differential elevations in fasting glucose, lipids, alanine, insulin resistance, and resting energy expenditure in LBW participants versus control participants. Multiomics analyses indicated reduced peroxisome proliferator–activated receptor signaling, as well as differential expression of genes involved in collagen and extracellular matrix metabolism in LBW participants during COF. Interestingly, the COF perturbations in LBW participants became more pronounced when excluding five LBW men with screen-detected metabolic dysfunction–associated steatotic liver disease. The findings support the notion of unhealthy subcutaneous adipose tissue expandability potentially underlying a reduced metabolic buffering capacity in nonobese LBW men.

INTRODUCTION: Aortic stenosis (AS) is the most prevalent valve heart disease. Renal failure increases the risk of AS and many circulating factors released during chronic kidney disease (CKD) participate to AS pathophysiology. We assessed in this study the role of increased aldosterone levels occurring in CKD as well as MR signaling and its interplay with Neutrophil Gelatinase-Associated Lipocalin (NGAL) in aortic valve interstitial cells (VICs) pathophysiology. METHODS: We conducted in vivo studies using a CKD rat model, including both wild-type (WT) and NGAL knockout (KO-NGAL) animals, for subsequent ex vivo analysis of aortic valves. In parallel, primary rat VICs were used in vitro to assess osteogenic, fibrotic, and inflammatory responses to aldosterone, as well as to identify the key signaling pathways involved. qPCR, Western blot, and ELISA were employed to characterize these pathways. RESULTS: Our findings demonstrate that MR signaling plays a central role in AS progression during CKD, as well as in VIC differentiation, inflammation, fibrosis, and calcification, mediated via the TLR4–MyD88 innate immunity pathway in aldosterone-induced responses. Furthermore, NGAL was shown to act downstream of MR to activate TLR4, promoting additional remodeling and calcification in aortic valves and VICs. These results were further validated in human samples from CKD patients. CONCLUSION: Overall, this study identifies a novel signaling pathway in AS pathophysiology in the CKD rat model and in vitro systems, highlighting for the first time the interplay between MR, NGAL, and TLR4 in driving the pathological processes underlying AS in CKD. GRAPHICAL https://BioRender.com/j7vufds

Background: Patients with chronic kidney disease (CKD) are at high risk of cardiovascular (CV) complications. Neutrophil gelatinase-associated lipocalin (NGAL) is a well established marker of kidney injury, but recent evidence suggests that NGAL might also play an active in the progression of the cardiorenal syndrome. Methods: CKD was induced in rats via 5/6 nephrectomy in wild-type (WT) and Ngal-knockout (KO) rats. Cardiorenal functions were assessed three months after subtotal nephrectomy or sham operation. Cardiac fibroblasts (CFs) were incubated with or without recombinant Ngal and galectin-3 (Gal-3). Results: Cardiac function, including diastolic hemodynamics and perfusion, was less impaired in CKD Ngal KO than in CKD WT. Cardiac fibrosis was more severe in CKD WT than sham, but was blunted in CKD Ngal KO rats. Levels of Gal-3, collagen I, MCP-1 and IL-6 were elevated in cardiac fibroblasts incubated with recombinant Ngal. A similar pattern was observed in cells treated with recombinant Gal-3. Both Ngal and Gal-3 induced activation of the Tlr4-Myd88 pathway. Using Gal-3 or Tlr4 inhibitors, we showed that Gal-3 contributes to Ngal-induced cardiac fibrosis and inflammation by activating the Tlr4-Myd88 pathway. In patients with heart failure with preserved ejection fraction (HFpEF) (MEDIA-DHF and BIOSTAT-CHF cohorts), elevated levels of NGAL and Gal-3 were associated with pulmonary artery systolic pressure, a marker of advanced diastolic dysfunction and adverse clinical outcomes, particularly among patients with impaired renal function. Conclusion: In non-diabetic CKD rats, Ngal was involved in the progression of diastolic dysfunction via a Gal-3/Tlr4-dependent pathway increasing inflammation and fibrosis.