Circulation Research

Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme important for the reduction of toxic lipid peroxide products. Previous studies revealed the importance of mouse Gpx4 in protecting cardiomyocytes from ferroptosis and, subsequently, the development of cardiovascular disease. In this paper, we investigate the transcriptional consequences of cardiac-specific deletion of Gpx4 in mice and compare this response with that observed in human cardiomyopathy. The findings in this study highlight the importance of GPX4 in maintaining both structural and functional stability of the heart and identify key pathway changes resulting from excessive ferroptosis in cardiac tissue. By overlapping common transcriptional programs perturbed in this animal model and human cardiomyopathy, our findings identify putative mechanisms through which ferroptosis contributes to the development and progression of heart disease. These studies may help guide future cardiovascular therapeutics targeting ferroptosis-dependent pathways.

Smooth muscle (SM) contraction is well known to be regulated by the reversible phosphorylation of the myosin regulatory light chain. However, SM force generation and relaxation are often uncoupled from myosin phosphorylation levels (e.g. the latch-state), indicating that additional regulatory mechanisms must be at play. The precise effects of the actin binding protein caldesmon (CaD) on SM force production and relaxation remain ambiguous, largely due to contradictory findings in experiments performed at the tissue level. To date, there are no studies that have measured the effects of CaD on force and relaxation at the molecular level. Here, we use a laser-trap assay to measure the force produced by SM myosin molecules in the presence and absence of CaD. Measurements were performed before and during myosin dephosphorylation, thus simulating SM contraction and relaxation in-vitro. We demonstrate that CaD inhibits force generation, most likely through competitive inhibition of actomyosin binding while simultaneously introducing a resistive load via tethering of actin and myosin. We also establish CaD as a potentiator of relaxation, increasing force decay rate during myosin dephosphorylation. Finally, we show that CaD directly modulates the dependence of myosin-actin mechanics on myosin phosphorylation levels. These findings refine our understanding of SM regulation, highlighting CaD not merely as a passive structural stabilizer, but as a critical regulatory component of force development and relaxation. Ultimately, understanding these mechanical functions offers new perspectives on pathophysiologies involving SM, such as asthma, hypertension, and gastrointestinal disorders, potentially guiding targeted therapeutic strategies. SIGNIFICANCE STATEMENTSmooth muscle (SM) is responsible for controlling the internal diameter of blood vessels and viscera. Understanding the precise regulation of SM relaxation by actin-binding proteins remains a fundamental lacuna in physiology. Using a molecular mechanics chamber to manipulate the biochemical milieu during active measurements, we demonstrate, for the first time at the molecular level, that caldesmon (CaD) acts as a mechanical modulator that inhibits force generation and accelerates relaxation of SM myosin ensembles. Our results provide a molecular basis for resolving previous contradictory findings reported in tissue-level experiments. Ultimately, understanding the role of contractile and regulatory proteins of SM will provide the basis for understanding SM disorders, such as hypertension and asthma, and guide the development of targeted therapeutic strategies.

Genetic cardiomyopathies consist of a heterogeneous group of myocardial disorders caused by variants that disrupt key regulators of cardiac structure and function. Variants in PLN, encoding phospholamban (PLN), the main inhibitor of the sarco/endoplasmic reticulum Ca{superscript 2}-ATPase 2a (SERCA2a), have been linked to both dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Among these, the PLN Arg14del (R14del) variant is the most prevalent. PLN R14del cardiomyopathy is characterized by the accumulation of large perinuclear PLN aggregates in cardiomyocytes of end-stage heart failure tissue. However, the mechanisms driving PLN aggregate formation and their role in disease progression remain unresolved. Using a humanized plna R14del zebrafish model, left ventricular tissue from end-stage PLN R14del cardiomyopathy patients and pharmacological modeling in wild type (WT) cardiac slices, we demonstrate that previously described PLN aggregates represent accumulated sarcoplasmic reticulum (SR)-derived PLN-containing vesicles that form due to impaired SERCA2a activity and increased cytosolic Ca{superscript 2} levels. Furthermore, these SR-derived vesicles often localize adjacent to lysosomes. Interestingly, Ca2+ dysregulation in plna R14del hearts leads to reduced lysosomal function, resulting in SR-derived vesicle accumulation at the microtubule organizing center (MTOC). This perinuclear accumulation induces microtubule aster formation and subsequent cellular disorganization, including sarcomere misalignment and nuclear deformation. Strikingly, reactivation of lysosomal function through fasting reduces SR-derived vesicle accumulation, restores microtubule integrity, and rescues cellular organization in plna R14del zebrafish hearts. Together, these findings identify impaired lysosomal clearance of SR-derived vesicles and the resulting microtubule disorganization as key pathological mechanisms driving PLN R14del cardiomyopathy. Additionally, our results highlight lysosomal reactivation as a promising potential therapeutic strategy to halt or reverse PLN R14del cardiomyopathy progression. Main findingsO_LIPLN aggregates in PLN R14del cardiomyopathy represent SR-derived vesicles formed due to Ca{superscript 2} dysregulation. C_LIO_LIThese SR-derived vesicles often localize perinuclearly at the microtubule organizing center (MTOC), where they are positioned adjacent to lysosomes. C_LIO_LICa2+ dysregulation leads to lysosomal dysfunction which drives vesicle accumulation responsible for microtubule remodeling and pathological cellular rearrangements. C_LIO_LILysosomal reactivation restores vesicle clearance and rescues cardiomyocyte structure. C_LI

BACKGROUNDExcessive trabeculations and myocardial crypts are recurrent features across cardiomyopathies, yet their developmental origins and clinical significance remain poorly defined. To reveal the link between cardiac morphogenesis and disease, we generated humanized mouse models carrying patient-derived MYBPC3 frameshift mutations associated with overlapping hypertrophic cardiomyopathy (HCM) and left ventricular non-compaction (LVNC). METHODSWe applied CRISPR-Cas9 to introduce distinct MYBPC3 frameshift alleles into the mouse genome and performed comprehensive phenotypic and transcriptomic profiling from fetal life through adulthood. RESULTSAdult homozygous Mybpc3 frameshift mutant mice like humans displayed hallmark HCM; however, without LVNC. Fetal and neonatal mutant hearts exhibited markedly enlarged ventricular trabeculae and crypts that progressed postnatally into the observed adult hypertrophy. Transcriptomic analysis revealed stage-specific dysregulation of oxidative metabolism, nonsense-mediated decay (NMD), and cell cycle pathways, peaking at postnatal days 1 and 7, indicating that these stages represent critical time points in disease onset. The persistent NMD signature, also observed in phenotype-negative heterozygotes, suggests a compensatory stress response. Enlarged trabeculae exhibited 2-fold increased trabecular cardiomyocyte proliferation, reversing the normal compact-trabecular proliferative gradient and leading to impaired ventricular compaction in neonates. Hey2CreERT2 lineage tracing demonstrated invasion of Hey2+ compact cardiomyocytes into the trabeculae and ectopic trabecular expression of the Prdm16 transcription factor, indicating defective ventricular wall patterning and maturation. Postnatally, Hey2+-derived cardiomyocytes became restricted to the outer/compact myocardium in mutants, while the inner/trabecular myocardium underwent accelerated hypertrophy concurrent with Prdm16 downregulation. Mice with a Mybpc3 missense variant also exhibited Hey2+ myocardial lineage expansion into trabeculae but no increased proliferation, implicating additional mechanisms beyond Hey2 regulation. Postnatal Prdm16 restoration, via transgenic expression in Mybpc3-null mice effectively attenuated hypertrophy, establishing a causal link between Mybpc3 loss, Prdm16 decline, and pathological remodeling. CONCLUSIONSMybpc3 governs ventricular wall maturation by regulating cardiomyocyte proliferation, patterning, and maturation, partly via Prdm16. Disruption of these developmental programs precedes and drives adult HCM, highlighting a developmental role for sarcomeric proteins, and revealing postnatal Prdm16 modulation as an antihypertrophic therapeutic strategy.

Pathologic cardiac hypertrophy requires increased protein synthesis, but the mechanosensors that link membrane stretch to translational control remain poorly understood. Polycystin-1 (PC1), encoded by PKD1, has been proposed as a cardiac mechanosensor, with its C-terminal tail (PC1-CT) promoting hypertrophy in rodent cardiomyocytes. However, its subcellular localization and downstream signaling remain incompletely defined, especially in human cardiomyocytes. Here, we examined endogenous PC1 C-terminus localization and the effects of adenoviral PC1-CT overexpression in human iPSC-derived ventricular cardiomyocytes (hiPSC-CMs) and adult mouse ventricular myocytes. Immunofluorescence revealed a striking striated pattern for both endogenous PC1 C-terminus (detected with a PC1-CT antibody) and the overexpressed PC1-CT fragment. In hiPSC-CMs, the PC1 C-terminus localized between the -actinin bands. In contrast, in adult cardiomyocytes, the overexpressed protein colocalized with -actinin and desmin, suggesting that PC1-CT sarcomeric distribution depends on cardiomyocyte maturation. We performed RNA-seq to assess transcriptional responses downstream of PC1-CT overexpression in hiPSC-CMs relative to LacZ controls. Gene Set Enrichment Analysis (GSEA) revealed enrichment of gene sets related to ribosome biogenesis, RNA processing, and protein synthesis, while classical hypertrophic markers remained unchanged. Pathway analysis suggested increased PI3K activity. PC1-CT overexpression increased phosphorylation of Akt, ERK, S6K1, and ribosomal protein S6 without altering 4EBP1 phosphorylation, suggesting preferential activation of the mTOR-S6K1-S6 branch. Pharmacological studies showed that pan-PI3K inhibition abolished S6 phosphorylation, whereas MEK blockade did not affect it; pertussis toxin and PI3K{gamma}-selective inhibitors also did not affect S6, suggesting a Gi/o-independent PI3K/Akt signaling driving mTOR-S6K1-S6 activation. Collectively, these data identify a sarcomere-associated pool of PC1-CT that engages PI3K-Akt-mTOR-S6K1-S6 signaling to enhance transcriptional programs related to ribosome biogenesis and protein synthesis, without activating a canonical hypertrophic gene program. These findings reveal a mechanistic link between PC1-CT and cardiomyocyte growth.

BACKGROUNDMyocarditis is an inflammatory cardiac disease in which Th17-driven immune responses contribute to progression toward dilated cardiomyopathy and heart failure. Current therapies mainly rely on corticosteroids but lack specificity, while the role of miR-721, synthesized by Th17 cells, remains largely unexplored in disease pathogenesis. METHODSWe characterized the presence of mmu-miR-721 and its human homolog hsa-RNA-Chr8:96 in extracellular vesicles (EVs) secreted by Th17 cells from IL-17eGFP mice with experimental autoimmune myocarditis (EAM) and myocarditis patients. MxCre-Ppargfl/fl mice and luciferase reporter assays were used to validate the target genes of miR-721 and hsa-RNA-Chr8:96, respectively. The functional role of miR-721 in EAM was investigated by lentiviral vectors overexpression and inhibition using miRNA sponge molecules. Th17 responses and heart inflammation were assessed and echocardiography was performed after in vivo blockade of mmu-miR-721 in EAM mice. RESULTSBoth mmu-miR-721 and hsa-RNA-Chr8:96 were encapsulated in EVs and secreted by Th17 cells of mice and patients with myocarditis. Overexpression of mmu-miR-721 in draining-lymph node cells from EAM mice inhibited Pparg transcription, leading to increased ROR{gamma}t and IL-17 expression and promoting Th17 differentiation. In contrast, in the absence of Pparg, a target of miR-721, no differences in ROR{gamma}t expression were observed, indicating that miR-721 promotes Th17 responses through repression of Pparg. Human PPARG was validated as a target gene of hsa-RNA-Chr8:96 and its overexpression in peripheral blood leukocytes downregulated PPARG mRNA levels, suggesting similar pathways involved in human pathology. In vivo blockade of mmu-miR-721 increased Pparg expression, reducing ROR{gamma}t and IL-17 activation in T cells and leading to decreased leukocyte infiltration in the heart and improved cardiac function. CONCLUSIONSmiR-721 is released by Th17 cells in EVs and promotes Th17 responses during myocarditis through repression of PPAR{gamma}, identifying this miRNA as both a mechanistic driver of disease and a potential therapeutic target. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/713340v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1a69953org.highwire.dtl.DTLVardef@9c36bdorg.highwire.dtl.DTLVardef@1cdce4dorg.highwire.dtl.DTLVardef@a34715_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and significanceO_ST_ABSWhat is known?C_ST_ABSO_LImiR-721 and its human homolog are upregulated in the plasma of mice and humans with myocarditis C_LIO_LITh17 cells synthesize miR-721 C_LIO_LIMmu-miR-721 targets Pparg mRNA C_LI What new information does this article contribute?O_LImiR-721 is sorted into extracellular vesicles in the context of acute myocarditis C_LIO_LImiR-721 enhances Th17 differentiation via the Pparg/Rorc double inhibitory axis. C_LIO_LIHsa-RNA-Chr8:96 targets human PPARG mRNA for degradation, inhibiting its expression C_LIO_LIBlockade of miR-721 dampens acute myocarditis development in vivo C_LI This study reveals a novel miRNA-based therapeutic strategy to inhibit Th17 responses and treat myocarditis. Using the experimental autoimmune myocarditis model, the authors unravel the mechanisms by which mmu-miR-721 can enhance Th17 responses and show how targeting this regulatory molecule could ameliorate the progression of the disease. Remarkably, this regulatory axis is suggested to be present in humans as well, since PPARG gene is validated as a target gene for hsa-RNA-Chr8:96. These findings highlights the potential of miR-721 not only as a diagnostic tool but also as a cell-specific therapeutic target to control Th17 responses in the clinical setting.

Cardiac fibrosis driven by persistent myofibroblast activation is a major contributor to adverse ventricular remodeling and heart failure. Bromodomain and extra-terminal domain (BET) inhibition reduces fibrosis and hypertrophy in preclinical models, but direct targeting of the BET co-activator BRD4 is limited by family homology and potential systemic toxicity. Sertad4 (SERTA domain containing protein 4) is a BRD4-dependent gene induced in activated cardiac fibroblasts, yet its role in cardiac pathology is unknown. Here, we examined Sertad4 expression and function in human heart failure and in murine myocardial infarction (MI). SERTAD4 protein was increased in left ventricular tissue from heart failure patients compared with non-failing controls. In Sertad4/LacZ reporter mice, MI triggered strong Sertad4 activation localized to the infarct scar and border zone, with minimal expression in remote myocardium; single-nucleus RNA sequencing further demonstrated that Sertad4 expression is predominantly fibroblast-restricted and significantly upregulated after MI. To test causality, we subjected global Sertad4 knockout mice to 28-day left anterior descending coronary artery ligation. Sertad4 deletion attenuated post-MI remodeling, reduced hypertrophy and ventricular dilation, and preserved systolic function. Consistent with improved structure and function, knockout hearts exhibited reduced cardiomyocyte cross-sectional area and decreased expression of fibrosis and hypertrophy associated genes. Together, these findings identify Sertad4 as a fibroblast enriched regulator of pathological remodeling and suggest that targeting Sertad4 may offer a more cell type-selective alternative to direct BET/BRD4 inhibition for limiting cardiac fibrosis and progression to heart failure

BackgroundCoronary autoregulation is the ability of the normal heart to maintain constant coronary blood flow (CBF) over a wide range of coronary driving pressures (CDP). Despite being vital for survival, the mechanism of coronary autoregulation is unknown. We hypothesized that GPR39, present in vascular smooth muscle cells, together with its endogenous agonist 15-hydroxyeicosatetraenoic acid (15-HETE) orchestrate coronary autoregulation. MethodsWe created coronary stenoses of varying degrees in open-chest, anesthetized dogs where we measured CBF and CDP. In a subset of animals, coronary venous blood was sampled for eicosanoid, adenosine, endothelin-1, polyunsaturated fatty acids, and prostaglandins levels. Stenoses were recreated during intravenous administration of VC108, a specific GPR39 antagonist and systemic, pulmonary, and coronary hemodynamics measured. ResultsGPR39 was identified in coronary arterioles by immunohistochemistry and in heart tissue by western blot. In-vivo, 15-HETE correlated linearly with CDP over the autoregulatory range (r2=0.47, p=0.0024). Apart from 6-keto PGF1 no other metabolite had any relation with CDP. Prior to administration of VC108, CBF did not change within the autoregulatory range. VC108 had no effect of systemic and pulmonary hemodynamics but increased CBF (p=0.02 versus vehicle) by decreasing coronary microvascular resistance (p=0.01 versus vehicle), indicating that GPR39 participates in control of normal coronary vascular tone. With VC108, coronary autoregulation was abolished and CBF became CDP dependent (r2=0.96, p=0.004). ConclusionGPR39 and its endogenous agonist 15-HETE together orchestrate coronary autoregulation when CDP is reduced. These novel findings provide a mechanism for coronary autoregulation and could direct pharmacological treatment of various coronary syndromes in humans.

Vascular endothelial (VE)-cadherin is essential for maintaining endothelial junctional barrier integrity. The Angiopoietin-1 (Ang-1)/Tie2 axis induced Akt1 activation is crucial for maintaining endothelial junctional barrier by inhibiting FoxO1 and suppressing expression of Angiopoietin-2 (Ang-2), a Tie2 antagonist. Systemic inflammatory conditions such as sepsis, Akt1 expression is reduced, whereas FoxO1-dependent Ang-2 expression is increased, resulting in endothelial barrier dysfunction. We previously showed that the TLR4/FoxO1 axis induces the ubiquitin E3 ligase CHFR, which promotes endothelial barrier disruption by targeting VE-cadherin for ubiquitylation and degradation. However, little is known about Akt1 expression during vascular inflammation. Here, we identified FoxO1-dependent CHFR expression as a key mechanism driving K48-linked polyubiquitylation and proteasomal degradation of Akt1 in endothelial cells (EC). LPS-induced K48-linked ubiquitylation of Akt1 was prevented in CHFR-depleted human EC and in endothelial-specific Chfr knockout (Chfr{Delta}EC) mice. Accordingly, CHFR depletion increased Akt1 and VE-cadherin expression in both human lung EC and Chfr{Delta}EC mice. Chfr{Delta}EC mouse lungs also exhibited elevated Ang-1 and Tie2 expression, and Ang-1 stimulation induced sustained Akt1 phosphorylation in CHFR-deficient EC. Moreover, CHFR depletion prevented LPS-induced expression of FoxO1 and Ang-2 in EC. Mechanistically, CHFR interacted with phosphorylated Akt1 and mediated its ubiquitylation at lysine residues K30, K39, K154, and K268. Expression of a ubiquitylation-deficient Akt1 mutant prevented LPS-induced VE-cadherin degradation and vascular injury. Collectively, these findings identify CHFR as a critical regulator of endothelial inflammatory responses by controlling Akt1 stability and VE-cadherin expression during inflammation.

BACKGROUNDThe vascular endothelium is a dynamic tissue central to vascular homeostasis and disease, with endothelial cells (ECs) exhibiting plasticity that drives adaptive remodeling. Reelin, a secreted extracellular matrix glycoprotein critical for neuronal migration via ApoER2/VLDLR-DAB1 signaling, may also modulate vascular function and inflammation. However, its direct role in EC biology remains unclear. We investigated Reelin as a context-dependent signaling modulator in ECs, assessing its engagement of non-canonical pathways and regulation of endothelial plasticity relevant to cardiovascular pathology. METHODSHuman endothelial cells were stimulated with recombinant Reelin and analyzed by immunoblotting, immunofluorescence, and functional assays. Time-course studies assessed signaling, including phosphorylation of FAK, AKT, and DAB1 by Western blotting, while wound-healing assays quantified endothelial migratory capacity in vitro systems. RESULTSReelin rapidly robustly activated noncanonical signaling in endothelial cells, increasing FAK and AKT phosphorylation in a time-dependent manner consistent with cytoskeletal remodeling. Canonical DAB1 activation was limited. Functionally, Reelin enhanced migration, upregulated Endoglin/CD105, and induced a remodeling-associated phenotype. Reelin silencing altered endothelial phenotype, clearly indicating a role in homeostasis. Signaling was independent of VEGFR2 interaction. Overall, Reelin preferentially engages FAK/AKT pathways to drive partial phenotypic modulation without full endothelial-to-mesenchymal transition. CONCLUSIONWe show that Reelin is a previously unrecognized regulator of endothelial signaling and plasticity, acting via non-canonical FAK- and AKT-dependent pathways. By partially and dynamically modulating endothelial phenotype, Reelin promotes a remodeling-permissive state without triggering full mesenchymal transition. These findings identify Reelin as a novel modulator of endothelial function with potential implications for vascular remodeling and cardiovascular disease. What Are the Clinical Implications?Our findings identify Reelin as a modulator of endothelial signaling with a clear bias toward non-canonical FAK- and AKT-dependent pathways that regulate endothelial plasticity and remodeling. This signaling profile is highly relevant to vascular diseases in which endothelial dysfunction is driven by maladaptive cytoskeletal reorganization, altered migration, and persistent activation rather than complete loss of endothelial identity. The ability of Reelin to promote partial and dynamically regulated phenotypic modulation suggests that it may operate at early and potentially reversible stages of vascular pathology. In this context, dysregulated Reelin signaling could contribute to pathological vascular remodeling, including processes underlying atherosclerosis, fibrosis, and microvascular dysfunction. These results also raise the possibility that circulating or locally produced Reelin may serve as an indicator of endothelial activation state, providing a novel biomarker for vascular disease progression. Importantly, the identification of a signaling bias toward FAK- and AKT-dependent pathways highlights potential therapeutic targets downstream of Reelin that could be selectively modulated to limit maladaptive endothelial remodeling while preserving essential endothelial functions. Collectively, this study positions Reelin signaling as a previously unrecognized and potentially actionable pathway in the regulation of endothelial behavior, with direct implications for the development of targeted strategies aimed at preventing or attenuating cardiovascular disease progression

BackgroundPersistent neurohormonal activation is a key driver of maladaptive remodeling and disease progression in heart failure (HF). Sodium-glucose cotransporter 2 inhibitors (SGLT2is) confer robust renoprotective effects in HF; however, the extent to which these benefits involve modulation of renal neurohormonal activity remains unclear. We hypothesized that SGLT2i-mediated renoprotection in HF is associated with attenuation of excessive renal neurohormonal activation. MethodsMale rats with myocardial infarction-induced HF and sham controls were fed standard chow or chow containing empagliflozin (EMPA, 300 mg/kg) for four weeks, followed by assessment of renal inflammatory and neurohormonal markers. Parallel in vitro studies in THP-1 macrophages and HK-2 proximal tubule cells evaluated the direct effects of EMPA on norepinephrine (NE)-dependent tubular inflammatory signaling. ResultsHF rats displayed higher renal cortical renin gene expression and angiotensin II concentrations, which remained unaffected by EMPA. Conversely, EMPA normalized the elevated urinary NE excretion and renal cortical NE content observed in HF rats. Given the inflammatory role of sympathetic hyperactivity, we assessed renal macrophage polarization. EMPA-treated HF rats showed reduced expression of pro-inflammatory markers (Tnf, Ccr2, Nos2, Il-6) and increased expression of markers associated with a reparative macrophage profile (Arg1, Mrc1, CD163), supported by higher CD206 macrophages in kidney sections. While EMPA did not directly alter THP-1 macrophage activation in vitro, it significantly reduced NE-induced SGLT2 expression and interleukin-6 (IL-6) release by HK-2 human proximal tubule epithelial cells. ConclusionThese findings support a model in which SGLT2 inhibitors confer renoprotection in HF by suppressing renal sympathetic hyperactivity, independently of the intrarenal renin-angiotensin system, thereby disrupting a maladaptive renal neuro-epithelial-immune axis and promoting a reparative macrophage phenotype. CLINICAL PERSPECTIVE Whats new?O_LIThis study identifies a renal neuro-epithelial-immune axis underlying empagliflozin-mediated renoprotection in heart failure. C_LIO_LIEmpagliflozin reduces renal cortical and urinary norepinephrine levels in heart failure without altering intrarenal renin-angiotensin system activity, revealing a distinct neurohumoral target of SGLT2 inhibition. C_LIO_LIThis sympatholytic effect is associated with a shift in renal macrophages toward a reparative (M2) phenotype, without changes in total macrophage abundance. C_LIO_LIEmpagliflozin blocks norepinephrine-induced SGLT2 upregulation, limiting proximal tubular glucose reabsorption and IL-6 production, and linking sympathetic signaling to renal inflammation. C_LI What are the clinical implications?O_LIOur findings provide a mechanistic basis for the additive cardiorenal benefits of SGLT2 inhibitors in heart failure, beyond conventional RAS-directed therapies. C_LIO_LITargeting renal sympathetic-driven inflammation may help preserve kidney function and attenuate the progression of cardiorenal syndrome. C_LIO_LISuppression of a renal neuroinflammatory pathway may help explain the early and sustained benefits of SGLT2 inhibitors across heart failure phenotypes, including nondiabetic patients. C_LI

ABSTRACT/SUMMARYVascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-{beta} (sPDGFR{beta}) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFR{beta} expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFR{beta} might influence PDGF-BB signaling, we exposed TESCl-derived vascular networks to excess PDGF-BB with or without a sPDGFR{beta} mimetic. PDGF-BB alone reduced full-length PDGFR{beta} levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFR{beta} mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFR{beta} isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development.

Vascular dysfunction and coagulopathy are hallmarks of severe COVID-19. How SARS-CoV-2 infection drives endothelial dysfunction, despite the virus not infecting or replicating in endothelial cells, remains controversial. Here, we used an in vitro co-culture model of the human pulmonary epithelial-endothelial cell barrier to investigate which inflammatory mediators drive endothelial dysfunction during SARS-CoV-2 infection. SARS-CoV-2 infection of primary human bronchial epithelial cells increased adjacent endothelial cell expression of the leukocyte adhesion marker ICAM-1, disrupted endothelial VE-cadherin junctions, promoted endothelial cell death, and promoted platelet adherence to gaps in the endothelial monolayers. Dexamethasone treatment rescued these dysregulated endothelial phenotypes in infected co-cultures, confirming that inflammatory signalling was the primary driver of SARS-CoV-2-induced endothelial dysfunction. Specifically, epithelial-derived TNF and IL-1{beta} promoted endothelial dysfunction, as inhibition of TNF or IL-1R signalling blocked SARS-CoV-2-induced endothelial dysfunction in co-cultures. SARS-CoV-2-infected wild-type mice, but not TNF, IL-1{beta}, or TNF/IL-1{beta}- deficient mice, displayed increased endothelial ICAM-1 expression, while an anti-IL-1{beta} monoclonal antibody prevented SARS-CoV-2-induced ICAM-1 expression and fibrin deposition in aged K18-ACE2 mice. Our data indicate that TNF and IL-1{beta} are the specific cytokines that drive multiple aspects of endothelial dysfunction during acute SARS-CoV-2 infection, and that inhibiting their signalling pathways may provide therapeutic benefit in preventing vascular complications of COVID-19.

Preeclampsia (PE), or gestational hypertension, affects around 5% of pregnancies and leads to approximately 70,000 maternal and 500,000 fetal deaths per year worldwide, with increased cardiovascular and metabolic disease in survivors. PE is associated with elevated circulating levels of the alternative splice isoform of VEGF receptor 1 (sFlt1), defects in placental vasculature, kidney damage and, in severe disease, fetal growth restriction. Current mouse models induce PE via direct expression of sFlt1 or elevation of blood pressure, which bypass the natural risk factors for human disease, such as age, obesity, hypertension and diabetes. These risk factors have in common reduced expression of Kruppel-like factors 2 and 4 (KLF2/4), the endothelial transcription factors that protect against cardiovascular disease. We now report that inducible deletion of KLF4 in maternal endothelium (KLF4iECKO) results in gestational hypertension, elevated sFlt1, defective placental vasculature, kidney damage and fetal growth restriction. KLF4iECKO may thus serve as a mouse PE model suitable for mechanistic analysis and screening of treatments that address upstream risk factors.

While previous studies have suggested that leptin regulates cardiovascular function independently of body weight, the specific leptin receptor (Lepr)-expressing neurons that mediate these distinct effects remain unknown. We found that genes located in blood pressure (BP)-associated genome-wide association study loci were regulated by leptin in Lepr and glucagon-like peptide-1 receptor (Glp1r)-expressing (LeprGlp1r) neurons. Ablating Lepr from these cells decreased BP despite causing hyperphagic obesity. Single-cell and spatial transcriptomics revealed that LeprGlp1r neurons segregate into two distinct subpopulations of cells located in the arcuate nucleus (ARC) and dorsomedial hypothalamic nucleus (DMH). Activating ARC LeprGlp1r neurons suppressed food intake without impacting energy expenditure or cardiovascular function. Conversely, DMH LeprGlp1r neurons increased energy utilization and BP without altering food intake. Our results identify distinct LeprGlp1r neuron subpopulations that dissociate the control of food intake from outputs related to sympathetic tone, including BP, suggesting the potential therapeutic utility of targeting of these subpopulations independently.

BACKGROUNDMono-ADP ribosylation is a post-translational modification that regulates various cellular physiological processes, including cell cycle progression, genomic stability, transcription, and cellular protein turnover. PARP16 is an endoplasmic reticulum (ER)-localized mono-ADP-ribosyltransferase that has been shown to regulate the unfolded protein response and maintain ER homeostasis under stress conditions. Despite its established role in ER stress signaling, the functional significance of PARP16 in cardiac pathophysiology, particularly in cardiac hypertrophy and heart failure, remains poorly understood. In this study, we aim to investigate the role of PARP16 in cardiac hypertrophy and heart failure using in vitro and mouse model systems. METHODSWe analysed PARP16 expression in human heart failure samples as well as in heart failure-based mouse models. We evaluated gene expression by RT-PCR, immunoblotting, and confocal microscopy to understand the role of PARP16 in heart failure under phenylephrine- or isoproterenol-treated conditions. We also investigated the role of PARP16 in regulating cardiac function in genetically engineered mouse models, including whole-body PARP16 knockout, cardiac-specific PARP16 knockout, inducible cardiac-specific PARP16 knockout, and cardiac-specific PARP16 Transgenic mice. We performed echocardiography to assess cardiac function. We also used an in vitro primary cardiomyocyte system to knock down and overexpress PARP16. We performed RNA sequencing and mass spectrometry, followed by molecular docking, molecular dynamics simulation, immunoprecipitation, and luciferase assay to characterise the molecular mechanism by which PARP16 regulates cardiac function. RESULTSHuman heart failure samples showed reduced PARP16 expression. PARP16 expression was also significantly reduced in models of heart failure, including the hearts of isoproterenol-treated C57B/L6 mice and phenylephrine-treated primary cardiomyocytes. PARP16-deficient NRCMs showed signs of pathological remodelling. Whole-body, cardiac-specific, and inducible cardiac-specific PARP16 KO mice exhibited cardiac remodelling and dysfunction. In contrast, cardiac-specific PARP16-overexpressing mice were protected from iso-induced cardiac hypertrophy. Mechanistically, several hypertrophic signalling pathway genes are dysregulated in PARP16 knockout mouse hearts concomitant with upregulated NFAT1 transcriptional activity and nuclear translocation. PARP16 binds to and catalytically downregulates NFAT activity, thereby maintaining cardiac function. Mass spectrometry analysis showed that PARP16 is involved in ADP-ribosylation of NFAT1 at E398 and T533. Pharmacological inhibition of NFAT activation attenuates structural and functional abnormalities associated with PARP16 deficiency. CONCLUSIONSPARP16 binds to and inhibits NFAT1 activity to regulate cardiac function in mice, and its downregulation may activate NFAT1 signalling, leading to hypertrophy. In this manner, PARP16 plays a critical role in cardiac hypertrophy and failure and may serve as a potential therapeutic target for the treatment of heart failure.

BackgroundSex-related differences in cardiovascular disease suggest the presence of intrinsic vasoprotective mechanisms, with estrogen recognized as an important modulator of endothelial function. Building on existing evidence, the present study provides mechanistic insights into how estrogen and nitric oxide (NO) signaling regulate selective pathways of oxLDL uptake, mitochondrial dynamics, and inflammatory responses during early atherogenesis. MethodsWe combined an in vitro endothelial cell-macrophage co-culture model with in vivo studies in low-density lipoprotein receptor-knockout (LDLr-/-) mice to investigate the role of estrogen in early atherosclerotic processes. Human aortic endothelial cells (HAECs) were exposed to oxidized low-density lipoprotein (oxLDL) in the presence or absence of 17{beta}-estradiol (E2) and the nitric oxide (NO*) donor S-nitroso-N-acetylcysteine (SNAC). Key outcomes included oxLDL uptake, mitochondrial oxidative stress, mitochondrial dynamics, and inflammatory signaling. In vivo, male and female LDLr-/- mice were exposed to a short-term high-fat diet with or without SNAC treatment. Plasma lipid levels, blood pressure, aortic lesion formation, and cardiac remodeling were evaluated. ResultsE2 reduced oxLDL uptake and oxidative stress, effects recapitulated by SNAC; however, these responses involved distinct entry pathways, with E2 preferentially modulating lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) dependent uptake and SNAC targeting caveolae-associated mechanisms. In parallel, both E2 and SNAC reduced Scavenger Receptor Class B Type 1 (SR-B1) expression, suggesting an additional modulation on oxLDL transcytosis via this mechanism. Endothelial cells exposed to oxLDL exhibited altered mitochondrial regulatory proteins, including superoxide dismutase 2 (SOD-2), dynamin-related protein 1 (Drp-1), and optic atrophy protein 1 (OPA-1). Despite reducing oxidative stress, E2 increased the expression of adhesion molecules and enhanced monocyte adhesion in response to oxLDL exposure, particularly when combined with SNAC. Strikingly, E2 also modulated macrophage responses, increasing interleukin receptor antagonist (IL-1ra) expression and reducing GDF15, macrophage inhibitory factor (MIF), macrophage inflammatory protein 3 alfa (MIP-3), and matrix metalloproteinase 9 (MMP-9) levels, consistent with a less pro-inflammatory macrophage profile. In vivo, HFD increased plasma lipid levels and atherosclerotic lesion area in LDLr-/- mice, whereas SNAC partially attenuated these effects without affecting plasma lipid levels. In vivo, female LDLr-/- mice developed approximately 50% smaller aortic lesions than males, despite comparable or higher plasma lipid levels. A dyslipidemia led to increased blood pressure and a hypertensive phenotype in both males and females. SNAC treatment reduced lesion burden in both sexes and prevented diet-induced hypertension in females. ConclusionEstrogen limits early atherogenic injury by reducing endothelial uptake of oxLDL, preserving mitochondrial homeostasis, and modulating inflammatory signaling. Together, the E2 and NO pathways regulate early atherosclerosis through distinct yet complementary mechanisms, offering a potential framework for vascular-protective strategies.

Von Willebrand factor (VWF) is an essential contributor to hemostasis through its interaction with the platelet glycoprotein (GP) Ib receptor. VWF is cleaved by ADAMTS13 to limit its prothrombotic properties. Failure to do so can result in platelet-VWF complexes that occlude the microcirculation, as seen in thrombotic thrombocytopenic purpura (TTP). In this setting, plasmin becomes active to cleave VWF, forming a distinct plasmin-generated cleavage product of VWF (cVWF) that is detectable during acute attacks in patients with TTP and following therapeutic plasminogen activation in a mouse model of TTP. However, it remains unclear whether plasmin-mediated proteolysis of VWF alone accounts for the breakdown of platelet-VWF complexes. Using ristocetin-induced platelet agglutinations, we show that plasmin cleavage of VWF does not impair its platelet-binding capacity, whereas plasmin-mediated cleavage of GPIb reduces the ability of platelets released from agglutinates to bind VWF. Furthermore, platelets in suspension are relatively resistant to plasmin cleavage. We therefore propose that VWF binding may enhance GPIb cleavage by recruiting plasmin(ogen) to the platelet surface. In a TTP mouse model, plasminogen activation led to a VWF-dependent reduction in GPIb detectability, although to a lesser extent than observed in vitro. In patients with acute TTP, soluble GPIb levels were elevated, indicating increased GPIb shedding during attacks of thrombotic microangiopathy, although the extent to which this is plasmin-mediated remains unclear. Together, our findings demonstrate that plasmin cleavage of GPIb drives the disruption of ristocetin-induced agglutinates, while its contribution to the breakdown of platelet-VWF complexes in vivo appears limited.

BackgroundIndividuals with JAK2V617F-mutant myeloproliferative neoplasms or clonal hematopoiesis of indeterminate potential have a markedly increased risk of cardiovascular disease, yet the mechanisms by which mutant blood cells drive vascular and cardiac dysfunction remain incompletely understood. Although the thrombopoietin (TPO) receptor MPL is central to hematopoiesis and is expressed in vascular endothelial cells (ECs), its role in JAK2V617F-associated cardiovascular complications is unknown. Methods and ResultsWe generated chimeric mice with JAK2V617F-mutant blood cells and wild-type endothelium by bone marrow transplantation and challenged them with a high-fat/high-cholesterol diet to model cardiometabolic stress. These mice developed a distinct cardiovascular phenotype characterized by microvascular disease, increased left ventricular mass, and relatively preserved left ventricular ejection fraction. Histological analysis revealed coronary arteriole stenosis, perivascular fibrosis, reduced microvascular density, and endocardial injury, without evidence of epicardial coronary stenosis or myocardium infarction. Single-cell RNA sequencing revealed activation of inflammatory, stress-response, and endothelial-to-mesenchymal transition gene signatures in ECs, most prominently within the endocardial ECs. Immunohistochemistry identified MPL expression predominantly in endocardial ECs. TPO/MPL signaling was upregulated in endocardial ECs in mice with JAK2V617F-mutant hematopoiesis, and treatment with an anti-MPL neutralizing antibody markedly improved cardiac pathology, restored endocardial integrity, and increased coronary microvascular density despite persistent systemic inflammation. ConclusionsJAK2V617F-mutant hematopoiesis induces coronary microvascular dysfunction. Endocardial ECs represent a key cellular target under cardiometabolic stress, and endocardial MPL signaling constitutes a potential targetable pathway in JAK2V617F-associated cardiovascular disease. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/715884v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1b0c2d7org.highwire.dtl.DTLVardef@1c7da20org.highwire.dtl.DTLVardef@1c19af9org.highwire.dtl.DTLVardef@1a588b3_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIJAK2V617F-mutant hematopoiesis induces cardiac microvascular disease C_LIO_LIMPL is expressed in endocardial ECs and MPL inhibition restores endocardial integrity and improves cardiac microvascular function C_LI

BackgroundCerebral ischemia-reperfusion injury (CIRI) is a major determinant of poor outcome after recanalization therapy in acute ischemic stroke. Microglial functional heterogeneity underpins neuroinflammation, yet the molecular mechanisms governing microglial phenotypic transitions remain incompletely understood. Metabolite-driven post-translational modifications (PTMs) have emerged as key regulators of microglial metabolism and inflammation, but whether PTM regulatory enzymes form co-expression modules that define microglial states is unknown. MethodsWe analyzed single-cell RNA-seq datasets from five GEO studies (GSE174574, GSE227651, GSE245386, GSE267240, GSE319237) covering tMCAO reperfusion and permanent ischemia models. Microglia were purified using double filtration (P2ry12/Tmem119/Cx3cr1+, Cd68/Adgre1/Ly6c-). PTM enzyme co-expression modules were identified by non-negative matrix factorization (NMF). Spatiotemporal dynamics were assessed by module projection across timepoints (Sham, 1d, 3d, 7d) and pseudotime analysis. Independent validation was performed in an additional tMCAO dataset (GSE245386). Sex differences were explored in a mixed-sex permanent ischemia dataset (GSE267240). ResultsThree robust PTM enzyme co-expression modules were identified: Metabolic stress-associated (M1), Pro-inflammatory-associated (M2), and Reparative-associated (M3). M1 was enriched in TCA cycle enzymes, M2 in inflammatory pathways (leukocyte activation, chemotaxis), and M3 in vascular development and translation. Module proportions and scores showed dynamic transitions: M1 decreased after reperfusion, M2 peaked at day 1-3, and M3 slightly increased at day 7. Independent validation in GSE245386 yielded high module conservation (cosine similarity = 0.874). Sex-specific differences in module distribution were observed in permanent ischemia ({chi}2 = 14.98, p = 0.00056). ConclusionsPTM enzyme co-expression modules delineate metabolic, pro-inflammatory, and reparative microglial states in CIRI with distinct spatiotemporal dynamics. This transcriptional framework supports the "PTM enzyme code" hypothesis and provides stage-specific targets for stroke therapy.