Knosalla, C. | Search bioRxiv

bioRxiv2026

Sphingolipid-Driven Lipotoxicity Promotes Calcific Aortic Valve Disease Through ER-Stress-NF-κB-EndMT Signaling and Is Therapeutically Targetable Through Serine Palmitoyltransferase Inhibition

Background: Calcific aortic valve disease (CAVD) lacks disease-modifying therapy. LDL-cholesterol lowering does not attenuate valvular calcification, suggesting cholesterol-independent drivers. Thus, we tested whether ceramide accumulation drives CAVD and whether inhibiting sphingolipid de novo synthesis is therapeutic. Methods: We integrated lipidomics and proteomics of calcified and non-calcified human aortic valves with matched plasma. Mechanistic effects of ceramides were tested in primary human valvular interstitial cells (VICs). Therapeutic efficacy of serine palmitoyltransferase (SPT) inhibition with Myriocin was evaluated in ApoE-/- mice with PCSK9DY-AAV-driven hyperlipidemia on Western diet using preventive treatment and therapeutic initiation after echocardiographically verified gradient elevation. Myriocin was additionally tested in a normolipidemic wild-type aortic valve wire-injury model. Hemodynamics, valve structure, lipid composition, and cell-state transitions were assessed by echocardiography, histology, lipidomics, and single-cell RNA sequencing. Results: Calcified human valves showed enrichment of long-chain ceramides linked to NF-{kappa}B, endothelial-to-mesenchymal transition (EndMT), and ER-stress/unfolded protein response (UPR) programs, whereas matched plasma sphingolipids were not elevated. In hVICs, long-chain C18 ceramide induced GRP78/BiP, CHOP, and ATF4, activated NF-{kappa}B, and promoted dose-dependent calcification. In vivo, SPT inhibition with Myriocin lowered valvular ceramides, reduced leaflet inflammation and mineralization, and improved cusp separation and transvalvular hemodynamics without lowering plasma LDL-cholesterol. In a therapeutic protocol initiated after gradient elevation, Myriocin halted further hemodynamic progression, providing the first evidence that de novo sphingolipid synthesis can be targeted after established CAVD onset in vivo. Myriocin also attenuated injury-induced stenosis in normolipidemic mice. Single-cell RNA sequencing showed reduced NF-{kappa}B/EndMT and ER-stress signatures and shifted VIC states away from osteogenic phenotypes. Conclusions: Human valve lipidomics and human VIC experiments identify ceramide-driven lipotoxicity as a driver of an ER-stress-NF-{kappa}B-EndMT axis in CAVD. In vivo, de novo sphingolipid synthesis is an actionable, LDL-independent target capable of modifying established hemodynamic disease progression, supporting SPT inhibition as a tractable strategy for medical CAVD therapy.

molecular biology↗

bioRxiv2026

Lung microvascular rarefaction impairs pulmonary gas exchange and exacerbates heart failure with preserved ejection fraction

BackgroundDyspnea and exercise intolerance are the primary clinical symptoms of heart failure. Heart failure patients experience frequent hypoxemic episodes, yet underlying mechanisms and relevance remain poorly understood. In a cohort of heart failure patients and multiple animal models, we identify pulmonary capillary rarefaction driven by excessive autophagy in endothelial cells as a novel mechanism of hypoxemia and cardiac disease progression. MethodsA cohort of heart failure with preserved ejection fraction (HFpEF) patients was analyzed for parameters of left ventricular (LV) dysfunction and pulmonary gas exchange. Morphological and cellular mechanisms of impaired pulmonary oxygenation were assessed in three animal models of heart failure, namely two HFpEF models, SU5416-treated ZSF1 obese rats and high fat diet/L-NAME treated mice, and in rats subjected to aortic banding. Lung microvascular rarefaction was quantified by micro-computed tomography, stereology, flow cytometry and dye efflux. Cellular mechanisms of capillary loss were analyzed by single-cell transcriptomics, electron microscopy and immunofluorescence, and in mice with endothelial-specific deletion of the autophagy gene Atg7 (Atg7EN-KO). ResultsIn 234 HFpEF patients, advancing NYHA class was associated with progressive worsening of arterial oxygen saturation at rest and during exercise and a reduced lung diffusing capacity. Impaired gas diffusion correlated with indices of LV diastolic dysfunction. Impaired oxygenation and reduced exercise capacity were similarly evident in animal models of left heart disease, which showed a distinct loss of pulmonary microvessels and capillaries. Lung microvascular endothelial cells in HFpEF showed characteristics of increased autophagic flux and apoptosis. Relative to their wild type HFpEF controls, Atg7EN-KO mice had less capillary loss, restored normoxemia, improved exercise tolerance, and mitigated LV diastolic dysfunction. Additional studies in HFpEF mice corroborated the functional relevance of impaired gas exchange for the progression of left heart disease by demonstrating that additional hypoxia aggravated, whereas moderate hyperoxia improved LV function. ConclusionOur findings identify pulmonary microvascular rarefaction as a novel pathomechanism in heart failure that i) contributes to dyspnea and exercise intolerance, ii) impairs pulmonary gas exchange and iii) accelerates LV disease progression. Strategies targeting this axis such as moderate oxygen therapy may mitigate cardiopulmonary morbidity in heart failure. Clinical Trial RegistrationRegistered in the DRKS (Deutsches Register fur klinische Studien) as trial# DRKS00032974 at https://drks.de/search/en/trial/DRKS00032974.

physiology↗