BACKGROUND:Pulmonary hypertension (PH) is a serious complication of heart failure with preserved ejection fraction (HFpEF), for which no targeted therapies are currently available. Endothelial dysfunction plays a crucial role in PH associated with HFpEF (PH-HFpEF), yet its molecular drivers remain poorly defined. METHODS:Transcriptome profiling uncovered endothelial characteristics of PH-HFpEF. Endothelial-specific GPRASP1 (GPCR [G protein-coupled receptor]-associated sorting protein 1 deletion in mice was conducted to investigate its participation in PH-HFpEF pathology. Multimodal metabolomics, isotope tracing, proteomics, and mechanistic biochemical assays were used to map downstream pathways and identify druggable mediators. RESULTS:GPRASP1 was significantly lowered in pulmonary endothelial cells of PH-HFpEF models. Endothelial Gprasp1 knockout mice exhibited major PH-HFpEF features, including pulmonary vascular remodeling, elevated pulmonary pressure, diastolic dysfunction, and abnormal glucose/lipid metabolism. GPRASP1 loss impaired tricarboxylic acid cycle activity by stabilizing ASNS (asparagine synthetase), preferentially shifting aspartate toward asparagine synthesis over oxaloacetate production. This metabolic reprogramming led to adenosine triphosphate depletion, reactive oxygen species accumulation, endothelial nitric oxide synthase uncoupling, and nitric oxide deficiency. We discovered that, beyond its classic role in GPCR sorting, GPRASP1 functioned as a noncanonical adaptor protein that scaffolded the E3 ubiquitin ligase PRKN (Parkin) and ASNS, promoting PRKN-dependent K48-linked ubiquitination and proteasomal degradation of ASNS via its C-terminal domain. In parallel, under mitochondrial stress, GPRASP1 strengthened PRKN interactions with MFN1/2, enhanced their K63-linked ubiquitination, and facilitated PRKN-mediated mitophagy. Restoration of GPRASP1 expression or pharmacological inhibition of ASNS activity with olopatadine normalized aspartate utilization, improved mitochondrial bioenergetics, rescued endothelial function, and attenuated cardiopulmonary pathology in PH-HFpEF models. CONCLUSIONS:Our findings unlocked a noncanonical role of GPRASP1 in preserving pulmonary endothelial homeostasis and delineated a novel GPRASP1-PRKN-ASNS axis that connected proteostasis with endothelial metabolic integrity, highlighting aspartate metabolism as a targetable vulnerability in cardiopulmonary disease.

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and has been linked to cardiovascular disease. Apparent treatment-resistant hypertension (aTRH) is a severe, age-associated form of hypertension with poor response to therapy. Here we show that CHIP is enriched in patients with aTRH and is independently associated with poorer treatment response and adverse cardiac remodeling. In a multicenter discovery cohort and two community-based validation cohorts, CHIP was detected in 23

BACKGROUND:Cerebrovascular malformations are a pivotal cause of hemorrhage and neurological disability, orchestrated largely by aberrant vascular homeostasis. However, a malformed angiogenic regulation pattern remains elusive. METHODS:Single-cell transcriptome analysis uncovered the endothelial features of human cerebral cavernous malformations and brain arteriovenous malformations, 2 typical cerebrovascular malformation diseases. Endothelial AR (androgen receptor, a steroid receptor in the nuclear receptor superfamily) overexpression was conducted to investigate its involvement in tip cell formation. ARD-2585, an AR degrader, was applied to mouse models of cerebral cavernous malformations (endothelial-specific Pdcd10 knockout mice) and brain arteriovenous malformations (endothelial-specific KrasG12D mutant [KrasG12D] mice) to evaluate its vascular rescue potential. RESULTS:We profiled single-cell transcriptomes of 13 human cerebrovascular malformation samples (10 cerebral cavernous malformations and 3 brain arteriovenous malformations) and 13 control brain samples, identifying a crucial pathological tip cell population in lesions. Integrative bioinformatics revealed a systemic endothelial regulatory network, with AR as a key regulator of this aberrant state. AR expression was elevated in endothelial cells from both human cerebrovascular malformations and Pdcd10 knockout/KrasG12D mice, correlating with suppressed Dll4 (delta-like canonical Notch ligand 4)-Notch signaling. AR overexpression augmented endothelial tube formation, filopodia extension, and polarization in vitro and fostered sex-independent vascular sprouting in vivo. High levels of AR facilitated proangiogenic gene transcription by recruiting coactivators EP300 (EP300 lysine acetyltransferase)/CBP (CREB binding lysine acetyltransferase), augmenting histone H3 lysine 18/histone H3 lysine 27 acetylation, and boosting chromatin accessibility, potentially independent of androgen. Notably, ARD-2585 treatment effectively normalized vascular anomalies and alleviated cerebral hemorrhage in Pdcd10 knockout and KrasG12D mice. CONCLUSIONS:We delineated a novel androgen-independent AR-mediated endothelial sprouting pattern in malformed cerebrovasculature, highlighting a promising foundation for developing interventions targeting tip cells in angiogenic diseases.

BACKGROUND:Cerebrovascular malformations are a pivotal cause of hemorrhage and neurological disability alongside lacking effective medication. Thyroid hormones (THs), including thyroxine and triiodothyronine, are essential for vascular development, yet whether they participate in malformed cerebrovascular pathology remains elusive. METHODS:Single-cell transcriptome analysis characterized human cerebral cavernous malformations and brain arteriovenous malformations, 2 typical cerebrovascular malformation diseases. Adeno-associated virus-mediated Dio2 (iodothyronine deiodinase 2; an enzyme that converts thyroxine to active triiodothyronine) overexpression/knockdown or triiodothyronine/methimazole (an antithyroid drug) treatment was applied to mouse models of cerebral cavernous malformations (endothelial-specific Pdcd10 knockout mice, Pdcd10 endothelial-specific knockout [KO]) and brain arteriovenous malformations (endothelial-specific KrasG12D mutant mice, KrasG12D) to evaluate the involvement of DIO2 and TH signaling in cerebrovascular malformations. RESULTS:TH signaling was markedly activated in fibroblasts of human cerebral cavernous malformation and arteriovenous malformation single-cell samples, accompanied by elevated DIO2 expression. Similar DIO2 upregulation was observed in cerebrovascular fibroblasts of Pdcd10 KO/KrasG12D mice and patient brain sections. Exogenous Dio2 or triiodothyronine replenishment effectively reduced brain hemorrhage, excessive ECM (extracellular matrix) remodeling, and vascular leakage in juvenile and adult male and female Pdcd10 KO/KrasG12D mice. In contrast, Dio2 silencing or TH inhibition deteriorated vascular anomalies. Mechanistically, transcription factor FOXK1 (forkhead box K1) was determined to interact with the DIO2 promoter region. The activation of fibroblast PI3K (phosphoinositide 3-kinase)-Akt (protein kinase B)-mTOR (mammalian target of rapamycin) signaling in Pdcd10 KO/KrasG12D mice triggered Foxk1 nuclear translocation to promote Dio2 transcription. Triiodothyronine treatment mitigated inflammatory infiltration, normalized mitochondrial morphology, and restored mitochondrial biogenesis in malformed brain vessels by activating the Pgc1a (peroxisome proliferator-activated receptor gamma coactivator 1-alpha)-Sod2 (superoxide dismutase 2)/Prdx3 (peroxiredoxin 3)/Gpx1 (glutathione peroxidase 1) axis to reduce reactive oxygen species accumulation. We also determined that the vascular repair effects of triiodothyronine were Pgc1a-dependent. CONCLUSIONS:We delineate a novel DIO2-mediated adaption in malformed cerebrovasculature and conclude that targeting TH signaling may represent a potential therapy for cerebrovascular disorders.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease, often caused by sarcomere gene mutations, though many sporadic cases remain genetically unexplained. Here we show that the somatic variant NAP1L1 p.D349E was involved in cardiac hypertrophy in sporadic HCM patients. Through next generation sequencing, we found that somatic variant NAP1L1 p.D349E was recurrent in the cardiomyocytes of gene-elusive sporadic HCM patients. Subsequent in vivo and in vitro functional analysis confirmed that NAP1L1 p.D349E contributes to HCM by triggering an innate immunity response. This mutation destabilizes nucleosome formation, causing DNA to leak into the cytoplasm. This leakage activates a key immune pathway, cGAS-STING, which leads to the release of inflammatory molecules and promotes heart muscle thickening. Our findings reveal a new mechanism driving HCM and suggest that somatic variants could be important in understanding and management of HCM.

Metabolic syndrome combines major risk factors for cardiovascular disease, making deeper insight into its pathogenesis important. We here explore the mechanistic basis of metabolic syndrome by recruiting an essential patient cohort and performing extensive gene expression profiling. The mitochondrial fatty acid metabolism enzyme acyl-CoA synthetase medium-chain family member 3 (ACSM3 ) was identified to be significantly lower expressed in the peripheral blood of metabolic syndrome patients. In line, hepatic ACSM3 expression was decreased in mice with metabolic syndrome. Furthermore, Acsm3 knockout mice showed glucose and lipid metabolic abnormalities, and hepatic accumulation of the ACSM3 fatty acid substrate lauric acid. Acsm3 depletion markedly decreased mitochondrial function and stimulated signaling via the p38 MAPK pathway cascade. Consistently, Acsm3 knockout mouse exhibited abnormal mitochondrial morphology, decreased ATP contents, and enhanced ROS levels in their livers. Mechanistically, Acsm3 deficiency, and lauric acid accumulation activated nuclear receptor Hnf4α-p38 MAPK signaling. In line, the p38 inhibitor Adezmapimod effectively rescued the Acsm3 depletion phenotype. Together, these findings show that disease-associated loss of ACSM3 facilitates mitochondrial dysfunction via a lauric acid-HNF4a-p38 MAPK axis, suggesting a novel therapeutic vulnerability in systemic metabolic dysfunction.

Arteriovenous malformations (AVMs) are fast-flow vascular malformations and refer to important causes of intracerebral haemorrhage in young adults. Getting deep insight into the genetic pathogenesis of AVMs is necessary. Herein, we identified two vital missense variants of G protein-coupled receptor (GPCR) associated sorting protein 1 (GPRASP1) in AVM patients for the first time and congruously determined to be loss-of-function variants in endothelial cells. GPRASP1 loss-of-function caused endothelial dysfunction in vitro and in vivo. Endothelial Gprasp1 knockout mice suffered a high probability of cerebral haemorrhage, AVMs and exhibited vascular anomalies in multiple organs. GPR4 was identified to be an effective GPCR binding with GPRASP1 to develop endothelial disorders. GPRASP1 deletion activated GPR4/cAMP/MAPK signalling to disturb endothelial functions, thus contributing to vascular anomalies. Mechanistically, GPRASP1 promoted GPR4 degradation. GPRASP1 enabled GPR4 K63-linked ubiquitination, enhancing the binding of GPR4 and RABGEF1 to activate RAB5 for conversions from endocytic vesicles to endosomes, and subsequently increasing the interactions of GPR4 and ESCRT members to package GPR4 into multivesicular bodies or late endosomes for lysosome degradation. Notably, the GPR4 antagonist NE 52-QQ57 and JNK inhibitor SP600125 effectively rescued the vascular phenotype caused by endothelial Gprasp1 deletion. Our findings provided novel insights into the roles of GPRASP1 in AVMs and hinted at new therapeutic strategies.

Cavernous malformations (CMs) invading the central nervous system occur in ~0.16-0.4% of the general population, often resulting in hemorrhages and focal neurological deficits. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of CMs in humans. Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from twelve human CM samples and three control samples. A total of 112,670 high-quality cells were clustered into 11 major cell types, which shared a number of common features in CMs harboring different genetic mutations. A new EC subpopulation marked with PLVAP was uniquely identified in lesions. The cellular ligand‒receptor network revealed that the PLVAP-positive EC subcluster was the strongest contributor to the ANGPT and VEGF signaling pathways in all cell types. The PI3K/AKT/mTOR pathway was strongly activated in the PLVAP-positive subcluster even in non-PIK3CA mutation carriers. Moreover, endothelial-to-mesenchymal transition (EndMT) cells were identified for the first time in CMs at the single-cell level, which was accompanied by strong immune activation. The transcription factor SPI1 was predicted to be a novel key driver of EndMT, which was confirmed by in vitro and in vivo studies. A specific fibroblast-like phenotype was more prevalent in lesion smooth muscle cells, hinting at the role of vessel reconstructions and repairs in CMs, and we also confirmed that TWIST1 could induce SMC phenotypic switching in vitro and in vivo. Our results provide novel insights into the pathomechanism decryption and further precise therapy of CMs.

Insulin resistance is associated with many pathological conditions, and an in-depth understanding of the mechanisms involved is necessary to improve insulin sensitivity. Here, we show that ZFYVE28 expression is decreased in insulin-sensitive obese individuals but increased in insulin-resistant individuals. Insulin signaling inhibits ZFYVE28 expression by inhibiting NOTCH1 via the RAS/ERK pathway, whereas ZFYVE28 expression is elevated due to impaired insulin signaling in insulin resistance. While Zfyve28 overexpression impairs insulin sensitivity and causes lipid accumulation, Zfyve28 knockout in mice can significantly improve insulin sensitivity and other indicators associated with insulin resistance. Mechanistically, ZFYVE28 colocalizes with early endosomes via the FYVE domain, which inhibits the generation of recycling endosomes but promotes the conversion of early to late endosomes, ultimately promoting phosphorylated insulin receptor degradation. This effect disappears with deletion of the FYVE domain. Overall, in this study, we reveal that ZFYVE28 is involved in insulin resistance by promoting phosphorylated insulin receptor degradation, and ZFYVE28 may be a potential therapeutic target to improve insulin sensitivity.

White adipose tissue browning can promote lipid burning to increase energy expenditure and improve adiposity. Here, we show that Slc35d3 expression is significantly lower in adipose tissues of obese mice. While adipocyte-specific Slc35d3 knockin is protected against diet-induced obesity, adipocyte-specific Slc35d3 knockout inhibits white adipose tissue browning and causes decreased energy expenditure and impaired insulin sensitivity in mice. Mechanistically, we confirm that SLC35D3 interacts with the NOTCH1 extracellular domain, which leads to the accumulation of NOTCH1 in the endoplasmic reticulum and thus inhibits the NOTCH1 signaling pathway. In addition, knockdown of Notch1 in mouse inguinal white adipose tissue mediated by orthotopic injection of AAV8-adiponectin-shNotch1 shows considerable improvement in obesity and glucolipid metabolism, which is more pronounced in adipocyte-specific Slc35d3 knockout mice than in knockin mice. Overall, in this study, we reveal that SLC35D3 is involved in obesity via NOTCH1 signaling, and low adipose SLC35D3 expression in obesity might be a therapeutic target for obesity and associated metabolic disorders.