BACKGROUND: As the innermost lining of blood vessels, endothelial cells regulate barrier function, maintain vascular tone, and limit inflammation for vessel health. Involved in various fundamental endothelial processes,...BACKGROUND: As the innermost lining of blood vessels, endothelial cells regulate barrier function, maintain vascular tone, and limit inflammation for vessel health. Involved in various fundamental endothelial processes, RhoA (Ras homolog family member A)/ROCK (Rho-associated coiled-coil containing protein kinase) signaling has been identified as a major contributor to age-associated hypertension and vascular disease; RhoA/ROCK hyperactivation promotes vessel permeability and impairs nitric oxide (NO) production. Recent studies identify Sun1 (Sad1 and UNC84 domain-containing protein 1), a key component of the linker of nucleoskeleton and cytoskeleton complex, as a major repressor of RhoA/ROCK activity. Our latest studies identify endothelial loss of Nup93 (nucleoporin93), a component of the nuclear pore complex, as a hallmark of vascular aging. Insinuating a role for nuclear envelope components in vessel homeostasis, the role of Nup93 in RhoA/ROCK signal regulation, however, remains entirely unknown. METHODS: Targeted Nup93 knockdown approaches were used in primary human endothelial cells to assess the role of Nup93 in endothelial RhoA/ROCK signaling and downstream readouts, including endothelial barrier function, cellular stiffness, and eNOS (endothelial NO synthase) function. Rescue studies include the use of pharmacological ROCK inhibitors and lentiviral-mediated Sun1 restoration methods. We additionally measure barrier function and vessel reactivity in our novel inducible endothelial-specific Nup93 mouse model. RESULTS: Targeted loss of endothelial Nup93 significantly increases RhoA/ROCK activity for consequent endothelial permeability and decreased NO bioavailability both in vitro and in vivo. Mechanistically, we find that loss of Nup93 drastically reduces endothelial Sun1 levels for a concomitant increase in RhoA activity. Indeed, restoring Sun1 protein levels in Nup93-deficient endothelial cells mitigates RhoA activity, thereby rescuing both endothelial barrier function and eNOS expression. CONCLUSIONS: Taken together, we demonstrate endothelial Nup93 as a novel regulator of vascular permeability and NO-dependent vessel reactivity, contributing to the growing importance of nuclear membrane components in endothelial cell and vascular biology.
Arterioscler Thromb Vasc Biol
· 2026 Jun · PMID 42273731
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Phosphoregulatory events underlie vascular responses to environmental and pathological stimuli, regulate hemostasis and thrombosis, and drive vascular remodeling in health and disease. Consequently, defining phosphorylat...Phosphoregulatory events underlie vascular responses to environmental and pathological stimuli, regulate hemostasis and thrombosis, and drive vascular remodeling in health and disease. Consequently, defining phosphorylation-dependent signaling networks in vascular physiology and pathology has enabled biomarker discovery and informed therapeutic interventions. Advances in mass spectrometry-based phosphoproteomics have enabled accurate and high-resolution mapping of dynamic phosphorylation events at the systems level, providing comprehensive mechanistic insights into vascular signaling and pathology. In this review, we contextualize phosphorylation in the vascular niche and summarize the current state of phosphoproteomics in vascular research, highlighting experimental design considerations, technological advances, quantification strategies, and data analysis approaches to uncover biological insights from large-scale phosphoproteomic data sets. Finally, we discuss recent discoveries in vascular signaling and disease, along with current challenges and emerging directions for applying phosphoproteomics to critical questions in vascular biology.
BACKGROUND: Preeclampsia is the most common complication of pregnancy, significantly affecting maternal and fetal health, and is characterized by placental and systemic endotheliopathy. Patients with preeclampsia have el...BACKGROUND: Preeclampsia is the most common complication of pregnancy, significantly affecting maternal and fetal health, and is characterized by placental and systemic endotheliopathy. Patients with preeclampsia have elevated levels of VWF (von Willebrand Factor), which is associated with poor clinical outcomes. However, whether VWF serves merely as a marker for endotheliopathy or contributes to the pathogenesis of preeclampsia remains poorly understood. METHODS: We investigated the role of hyperadhesive VWF in the development of preeclampsia by studying patients, evaluating the phenotype of mouse models, and performing in vitro experiments. RESULTS: We show that patients develop VWF- and fibrin-rich thrombosis in the placenta and have significantly elevated levels of VWF adhesive activity and placenta-derived extracellular vesicles. In mouse models, pregnant wild-type mice infused with hyperadhesive VWF alone, or in combination with placenta-derived extracellular vesicles, developed a preeclampsia-like condition, which was reduced by the VWF-cleaving metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif 13). Furthermore, -deficient mice with high baseline VWF (ADAMTS13) developed a preeclampsia-like condition spontaneously, with VWF adhesive activity increasing 5.6-fold and placenta-derived extracellular vesicles increasing 2.9-fold during late pregnancy. VWF became hyperadhesive during pregnancy by undergoing conformational changes and promoted preeclampsia-associated endotheliopathy by enhancing the interaction of placenta-derived extracellular vesicles with endothelial cells. CONCLUSIONS: This study demonstrates that hyperadhesive VWF plays a causal role in preeclampsia and is a potential therapeutic target.
BACKGROUND: RIP2 (receptor-interacting protein kinase 2) is an essential mediator of inflammation and innate immunity downstream of PRRs (pattern recognition receptors). Platelets express RIP2, while its role in platelet...BACKGROUND: RIP2 (receptor-interacting protein kinase 2) is an essential mediator of inflammation and innate immunity downstream of PRRs (pattern recognition receptors). Platelets express RIP2, while its role in platelet activation, thrombosis, and myocardial infarction, and whether these effects are mediated through the PRR pathway, is unknown. METHODS: In vitro assays of platelet aggregation, dense and α-granule secretion, spreading, and clot retraction, along with an ex vivo microfluidic whole blood perfusion assay, and in vivo models of FeCl-induced mesenteric arteriolar thrombosis and ischemia/reperfusion myocardial infarction, were used to assess the impact of RIP2 deficiency on platelet function. Immunoprecipitation followed by liquid chromatography-tandem mass spectrometry was performed to elucidate the mechanism by which RIP2 limits dense granule secretion in platelets. Furthermore, RIP2 expression levels in platelets from healthy donors and patients with coronary artery disease were measured by Western blotting to evaluate their clinical relevance. RESULTS: Here, we show that RIP2 deficiency enhances platelet dense granule secretion in response to GPIb (platelet glycoprotein Ib) and GPVI (platelet glycoprotein VI) activation; platelet aggregation in whole blood and adhesion under arterial shear are also increased. Consistently, RIP2 inhibitor WEHI-345 potentiates human platelet dense granule secretion and inhibits RIP2 phosphorylation induced by thrombin and collagen. These phenotypes are translated into shorter bleeding time and accelerated FeCl-induced arterial thrombosis. Importantly, platelets from patients with coronary artery disease and mice with atherosclerosis express lower RIP2, and RIP2 deficiency worsens myocardial infarction and cardiac function in a mouse ischemia/reperfusion model. Mechanistically, we found that platelets express DOCK8 (dedicator of cytogenesis protein 8), which is sequestered by p-RIP2 (phosphorylated RIP2), causing inhibition of Cdc42 (cell division cycle 42) activation and subsequent dense granule release. CONCLUSIONS: RIP2 restrains platelet activation and thrombosis, thereby mitigating myocardial infarction. We identify a novel PRR-independent pathway, p-RIP2-DOCK8-Cdc42, which suppresses dense granule release downstream of GPIb and GPVI. Targeting the platelet RIP2 pathway may offer a therapeutic strategy against atherothrombotic diseases from early atherosclerosis to arterial thrombosis and myocardial infarction.
BACKGROUND: Individuals with Loeys-Dietz syndrome 3 are at risk for thoracic aortic dissection even if their aortas are not substantially dilated. A pathogenic variant of SMAD3 is a genetic trigger, but the cellular prog...BACKGROUND: Individuals with Loeys-Dietz syndrome 3 are at risk for thoracic aortic dissection even if their aortas are not substantially dilated. A pathogenic variant of SMAD3 is a genetic trigger, but the cellular programs that underlie the heightened aortic risk remain poorly understood. METHODS: We ascertained the cardiovascular profile of 57 individuals in a 4-generation family with a missense variant in SMAD3 (p.Arg287Gln). Aortic medial architectures were quantified from aortas harvested during thoracic aortic replacement surgery, with 2-dimensional and 3-dimensional profiling of the elastin-microfibril network. TGFß-dependent regulatory networks were investigated by whole-genome RNA sequencing of ascending aortic smooth muscle cells (SMCs) and bioinformatics-informed chromatin immunoprecipitation. RESULTS: Cardiovascular phenotypes of family members harboring the SMAD3 variant included thoracic aortopathy, peripheral artery aneurysms and tortuosity, cerebrovascular disease, coronary artery dissections, and mitral valve prolapse. The median age at the first aortic event was 36.5 years. Ascending aortas were less dilated than the aortic root, with relatively intact lamellar structure and SMC content. However, early pathology was evident, with SMCs separated from elastic lamellae by a thin layer of glycosaminoglycans. Confocal reconstructions revealed that elastic fibers were poorly decorated by microfibrils, with reduced fibrillin-1, fibrillin-2, microfibril associated glycoprotein-2, fibulin-1 and fibulin-2. RNA sequencing of Loeys-Dietz syndrome 3 SMCs revealed reduced microfibril gene expression. Furthermore, PRRX2 (paired related homeobox 2) was found to be downregulated in Loeys-Dietz syndrome 3 SMCs in culture and in situ, occupies gene promoter sites for all 5 microfibrils, and mediates their expression in SMCs. SMAD3 overexpression rescued both and microfibril expression. CONCLUSIONS: These findings identify SMCs delaminating from unsheathed elastic fibers as an incipient pathological program in Loeys-Dietz syndrome 3 aortas. They also uncover a SMAD3-PRRX2-microfibril axis that, being perturbed, could explain this disconnection phenomenon and contribute to risk.
Lainé A, Begué-Racapé Z, Lindenbaum P
… +12 more, Caillaud A, Marrec M, Simonet F, Girardeau A, Vignard V, Goudal A, Boisseau P, Wargny M, Le May C, Di Filippo M, Cariou B, Rimbert A
BACKGROUND: FUT8 (fucosyltransferase 8) adds core fucose to the innermost -acetylglucosamine of -glycans on surface receptors, affecting ligand binding and downstream signaling in various cellular contexts. Here, we exam...BACKGROUND: FUT8 (fucosyltransferase 8) adds core fucose to the innermost -acetylglucosamine of -glycans on surface receptors, affecting ligand binding and downstream signaling in various cellular contexts. Here, we examine the role of FUT8-catalyzed core fucosylation in platelet activity in vivo, and we evaluate the potential antithrombotic strategy of FUT8 inhibition. METHODS: To investigate whether the extracellular domains of platelet adhesion receptors are core fucosylated, we used genetic, biochemical, glycomics, and glycoproteomics approaches. To investigate the impact of core fucosylation on platelet receptors for their ligand affinity, platelet activation, and downstream signaling, we used binding assays, platelet aggregometry, and phospho-specific effector antibodies. To investigate the biological effect of genetic and pharmacological inhibition of FUT8, we used murine thrombosis models. RESULTS: We identified core fucosylated -glycans in the extracellular domains of platelet adhesion receptors, such as GPVI (glycoprotein VI) and integrin αβ. Core fucosylation at platelet GPVI or αβ N-glycans enhanced affinity and binding to type I collagen or fibrinogen, leading to greater platelet activation and downstream signaling. In 2 murine thrombosis models, platelet-specific deletion inhibited platelet activation and thrombus formation. Furthermore, FUT8 inhibition with FDW028 led to reduced platelet aggregation and activation, protecting mice from lethal thrombosis. CONCLUSIONS: This study provides genetic and pharmacological evidence that FUT8-mediated core fucosylation of platelet receptors promotes platelet activation and thrombus formation. Our results suggest that FUT8 in platelets may be a therapeutic target for thrombosis-related cardiovascular diseases.
Li L, Zhang Y, Truong B
… +7 more, Yu Z, Gupta RM, Tian H, Natarajan P, Haas DM, Liu N, Honigberg MC
Arterioscler Thromb Vasc Biol
· 2026 Jul · PMID 42237906
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BACKGROUND: The hypertensive disorders of pregnancy (HDPs), that is, preeclampsia and gestational hypertension, are characterized by endothelial dysfunction in pregnancy and are epidemiologically and genetically associat...BACKGROUND: The hypertensive disorders of pregnancy (HDPs), that is, preeclampsia and gestational hypertension, are characterized by endothelial dysfunction in pregnancy and are epidemiologically and genetically associated with risk for coronary artery disease (CAD). A recent study suggested that endothelial cell (EC)-acting CAD risk variants may identify individuals who benefit from more intensive lipid-lowering treatment. The present study evaluated whether the same EC CAD genetic risk score is also associated with HDPs using comprehensive statistical genetic approaches. METHODS: We examined 35 previously identified EC-acting and 205 non-EC-acting risk variants associated with CAD. First, using the nuMoM2b (Nulliparous Pregnancy Outcomes Study: Monitoring Mothers-to-Be), a prospective, multicenter US pregnancy cohort, we calculated polygenic risk scores (PRS) comprised of either EC-acting or non-EC-acting CAD risk variants in genotyped, unrelated participants across multiple ancestry groups. Scores were adjusted for population genetic structure using 10 principal components, then tested for associations with HDPs using logistic regression models adjusted for age. Second, we tested the genetic association of EC-acting and non-EC-acting CAD variants with preeclampsia and gestational hypertension using 2-sample Mendelian randomization. RESULTS: Among 6782 unrelated nuMoM2b participants (mean [SD] age, 27.0 [5.5] years, 1040 [15.3%] with HDPs in the index pregnancy), participants with higher EC-acting PRS had a higher incidence of HDPs. Each SD of higher EC-acting PRS was associated with 1.09-fold increased odds of HDP (95% CI, 1.02-1.17; =0.008); by contrast, the non-EC-acting PRS was not significantly associated with HDP risk (odds ratio, 1.05 [95% CI, 0.98-1.12]; =0.14). In 2-sample Mendelian randomization, EC-acting CAD variants were strongly associated with both preeclampsia (odds ratio, 1.55 [95% CI, 1.30-1.86]; <0.001) and gestational hypertension (odds ratio, 1.54 [95% CI, 1.29-1.85]; <0.001). CONCLUSIONS: PRS and 2-sample Mendelian randomization approaches suggest enrichment for EC-acting CAD genetic risk variants in women with HDPs. These findings support a shared genetic architecture between HDPs and CAD through endothelial dysfunction.
BACKGROUND: Biophysical interactions between forces exerted by blood and the vascular endothelium influence atherogenesis. The accurate characterization of these relationships, however, is obfuscated by the variety of me...BACKGROUND: Biophysical interactions between forces exerted by blood and the vascular endothelium influence atherogenesis. The accurate characterization of these relationships, however, is obfuscated by the variety of metrics that exist to quantify arterial shear waveforms. Spectral analyses of these hemodynamic waveforms are an underutilized metric that may critically inform endothelial biology. This investigation, therefore, examined associations between shear spectral characteristics and endothelial function. METHODS: Arterial shear rate waveforms were recorded using Doppler ultrasonography from the brachial arteries of healthy humans. Spectral characteristics of the shear rate waveform were enumerated using Fourier transformations. Associations were explored between spectral characteristics and arterial function represented by brachial artery flow-mediated dilation calculated from the same anatomic site as waveform retrieval. Intracellular endothelial adaptations to different shear spectra were measured in vitro using cultured endothelial cells. Feature selection strategies were used to nominate hemodynamic mechanisms that may regulate the spectral characteristics of the brachial artery shear rate waveform. Vascular resistance was suggested as a candidate mechanism and was manipulated in 2 independent participant samples using local forearm heating and cooling and moderate-intensity continuous exercise. RESULTS: Positive associations were found between lower-frequency spectral amplitudes and brachial artery flow-mediated dilation. In cultured endothelial cells, lower-frequency spectra imparted an atheroprotective transcriptome measured by bulk RNA-sequencing. Fluorescence microscopy confirmed atheroprotective morphological and functional changes to endothelial cells exposed to lower-frequency shear. Vascular resistance was suggested as a hemodynamic mechanism that regulates shear rate spectra. Experimental manipulations of vascular resistance demonstrated that reductions in vascular resistance induce a leftward shift in shear spectra. Larger amplitudes of shear spectra were further observed among healthy humans compared with humans with atherosclerotic cardiovascular disease. CONCLUSIONS: Low-frequency characteristics extracted from the arterial shear rate waveform associate with decreases in peripheral vascular resistance and may impart atheroprotection in humans.
BACKGROUND: Clonal hematopoiesis driven by somatic mutations is an emerging cardiovascular risk factor, and the DNA damage response gene is among the most frequently mutated genes. Mutations in are enriched in cancer p...BACKGROUND: Clonal hematopoiesis driven by somatic mutations is an emerging cardiovascular risk factor, and the DNA damage response gene is among the most frequently mutated genes. Mutations in are enriched in cancer patients and survivors, where cytotoxic therapies promote the expansion of mutant clones, a condition termed therapy-related clonal hematopoiesis. Although -mutant clonal hematopoiesis has been associated with increased risk and poorer prognosis of atherosclerotic cardiovascular disease in humans, it remains unclear whether these mutations, or their expansion under cytotoxic stress, causally contribute to atherosclerosis. METHODS: We modeled -mutant clonal hematopoiesis in mice through bone marrow transplantation strategies. Conventional transplantation approaches were used to generate mice with complete or partial hematopoietic reconstitution by cells carrying monoallelic or biallelic gain-of-function mutations. To mimic therapy-related clonal hematopoiesis, we used a nonconditioned adoptive transfer model in which a small fraction of mutant hematopoietic cells was introduced into recipients, followed by fractionated low-dose γ-radiation to promote clonal expansion. All mice were fed a Western diet to induce atherosclerosis. Clonal dynamics, plaque size and characteristics, and macrophage functions were evaluated using flow cytometry, histopathology, and in vitro assays. RESULTS: -mutant cells expanded in blood and bone marrow after low-dose radiation, but not in nonirradiated mice. Across all transplantation strategies, mutations did not affect plasma cholesterol, atherosclerotic plaque size, or composition. In vitro, mutant macrophages showed no alterations in proliferation, cytokine expression, or cholesterol handling, although apoptosis in response to genotoxic stress was modestly reduced (≈20%). CONCLUSIONS: -mutant hematopoietic cells expand predominantly under genotoxic stress and do not promote atherosclerosis in mice under the conditions tested. These findings raise the possibility that the association of mutations with atherosclerotic cardiovascular disease may, at least in part, reflect exposure to DNA damage response-activating stressors that independently promote clonal expansion and atherosclerosis, rather than direct causality.
Franklin MK, Howatt DA, Moorleghen JJ
… +5 more, Sheppard MB, Katsumata Y, Sawada H, Lu HS, Daugherty A
Arterioscler Thromb Vasc Biol
· 2026 Jul · PMID 42206371
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BACKGROUND: Mice harboring a missense variant (C1041G) of have been used extensively for aortopathy research, but do not mimic all facets of the human disease. The role of increased AngII (angiotensin II) or blood press...BACKGROUND: Mice harboring a missense variant (C1041G) of have been used extensively for aortopathy research, but do not mimic all facets of the human disease. The role of increased AngII (angiotensin II) or blood pressure in determining the arterial phenotype of these mice remains incompletely defined. The purpose of this study was to determine whether AngII, either directly or indirectly through increased blood pressure, promotes pathology in the proximal thoracic aorta and beyond. METHODS: littermates were infused with either AngII or norepinephrine via subcutaneously implanted osmotic pumps. Microcomputed tomography was used to visualize vascular pathologies. Maximal arterial dimensions were measured using in situ or microcomputed tomography images. RESULTS: AngII infusion dramatically augmented aortopathy in mice. Aortic dissection was visible within 3 days of AngII infusion. Over 50% of male mice died during AngII infusion, primarily due to aortic rupture in either the thoracic or abdominal regions. Surviving males had increased ascending and suprarenal aortic diameters and developed pathological lesions at the celiac and superior mesenteric branches of the abdominal aorta. Female mice had a much lower incidence of death but had increased ascending aortic and branch diameters. Although norepinephrine infusion also increased systolic blood pressure, it did not affect mortality or enlarge aortic or branch diameters in mice. While microcomputed tomography identified novel pathological changes during AngII infusion, including the development of aortic branch aneurysms in the celiac and superior mesenteric arteries, the maximal diameters of the adjacent suprarenal aorta showed only modest increases in male mice. CONCLUSIONS: AngII exacerbated aortic pathology in mice. It also promoted the development of pathologies at aortic branch points, including the celiac and superior mesenteric arteries.
BACKGROUND: Bacterial communities and their metabolites are increasingly recognized as key contributors to cardiovascular disease, yet their role and mechanistic involvement in abdominal aortic aneurysm (AAA) pathogenesi...BACKGROUND: Bacterial communities and their metabolites are increasingly recognized as key contributors to cardiovascular disease, yet their role and mechanistic involvement in abdominal aortic aneurysm (AAA) pathogenesis remain insufficiently defined. METHODS: Dental plaques from patients with AAA and matched healthy controls were subjected to metagenomic sequencing, and corresponding plasma samples underwent untargeted metabolomic profiling. In vivo, mice were topically exposed in the oral cavity to (Fn) followed by AngII (angiotensin II) infusion to evaluate its impact on AAA progression. A homologous recombination-based deletion strategy was used to confirm the role of Fn in isoleucine biosynthesis. Molecular assays were performed to assess ferroptosis-related signatures and histone acetylation in smooth muscle cells, while chromatin immunoprecipitation-quantitative polymerase chain reaction verified the specific acetylation target. In addition, dietary restriction of isoleucine was introduced in the AAA murine model to explore therapeutic relevance. RESULTS: Patients with AAA showed a marked enrichment of Fn in dental plaque, and topical application of Fn aggravated AngII-induced AAA in mice. Elevated plasma isoleucine concentrations were observed in both human AAA and experimental models. Genetic deletion of in Fn diminished bacterial isoleucine release and mitigated AAA development in mice. Mechanistic analyses revealed that Fn-derived isoleucine promoted ferroptosis in smooth muscle cells through H3K9ac (histone H3 lysine 9 acetylation)-dependent transcriptional activation of (acyl-CoA [coenzyme A] synthetase long-chain family member 4), a core regulator of ferroptosis. Dietary isoleucine restriction in the AngII-induced model reduced H3K9ac, suppressed ferroptosis, and alleviated aneurysmal progression. CONCLUSIONS: Fn-derived isoleucine drives ferroptosis in smooth muscle cells via H3K9ac-mediated activation of ACSL4, delineating a microbiota-metabolite-epigenetic axis in AAA pathogenesis and nominating dental plaque Fn abundance and circulating isoleucine as exploratory biomarker candidates requiring larger, independent validation.
Current antithrombotic therapies (combining aspirin, purinergic P2Y inhibitors, and anticoagulants) target both platelet and coagulation pathways to reduce ischemic events in high-risk cardiovascular patients. Despite ef...Current antithrombotic therapies (combining aspirin, purinergic P2Y inhibitors, and anticoagulants) target both platelet and coagulation pathways to reduce ischemic events in high-risk cardiovascular patients. Despite efficacy, these regimens are limited by increased bleeding risk, as evidenced by multiple clinical trials. Suboptimal pharmacodynamics and nonindividualized treatment approaches complicate the therapeutic success. Pediatric cardiac conditions and severe thrombophilia underlying arterial and immune thrombosis pose special challenges. The dual actions aim at addressing these gaps by especially attenuating arterial platelet-rich thrombi and safety concerns. We highlight the need for improved antithrombotic strategies across diverse patient populations. To potentially overcome the limitations, we present antiplatelet and anticoagulant (APAC), a heparin proteoglycan mimetic integrating APAC effects. APAC, administered locally or intravenously, targets VWF (von Willebrand Factor) and thrombin, effectively inhibiting platelet-fibrin deposition, under high shear rate flow conditions over surfaces of collagen and TF (tissue factor). Preclinical models show that APAC reduces atherosclerotic and stenotic lesions, inflammation, and ischemia-reperfusion injury without bleeding propensity. These findings position APAC as a promising candidate for safe, translational dual antithrombotic. Early clinical trials demonstrated the safety of APAC under both systemic and local administration. A phase 2 clinical study in peripheral arterial disease is underway, and another is planned for arteriovenous fistula patency for end-stage renal patients.
BACKGROUND: Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary vascular remodeling, primarily driven by excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Although noncodin...BACKGROUND: Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary vascular remodeling, primarily driven by excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Although noncoding RNAs, such as circular RNAs, have been implicated in PH, the role of tRNA-derived small RNA remains poorly understood. Among them, i-tRF (internal tRNA-derived fragment)-Asp represents a novel tRNA-derived small RNA whose function in hypoxic PH is unknown. This study aims to investigate whether i-tRF-Asp contributes to PASMC proliferation and vascular remodeling in hypoxic PH. METHODS: To elucidate the functional role of i-tRF-Asp, loss-of-function experiments were conducted in vitro using hypoxic mouse PASMCs, alongside in vivo studies using both SuHx (Sugen5416+hypoxia) induced PH models in both adult male C57BL/6 mice and Sprague-Dawley rats. These models employed antisense oligonucleotide inhibitors and adeno-associated virus-mediated silencing. The underlying mechanisms were further explored via RNA immunoprecipitation, liquid-liquid phase separation assays, cell proliferation assessment, Western blot, alternative splicing profiling, and molecular dynamics simulation. RESULTS: Inhibition of i-tRF-Asp ameliorated hypoxia-induced pulmonary vascular remodeling, primarily through suppressing excessive proliferation of mouse PASMCs. Further analysis indicated that i-tRF-Asp facilitates liquid-liquid phase separation of hnRNPU (heterogeneous nuclear ribonucleoprotein U). This process mediates selective recruitment of (transcription factor 7-like 2) pre-mRNA and promotes alternative splicing, resulting in a pro-proliferative Tcf7l2 transcript variant that drives mouse PASMC proliferation. Furthermore, we have identified that the endonuclease ERN1 (endoribonuclease IRE1) can upregulate the expression of i-tRF-Asp. CONCLUSIONS: The endoribonuclease ERN1 upregulates the expression of i-tRF-Asp, which in turn facilitates mouse PASMC proliferation and pulmonary vascular remodeling. This process is driven by hnRNPU liquid-liquid phase separation-dependent alternative splicing of the long Tcf7l2 transcript isoform.
Abdominal aortic aneurysm (AAA) pathogenesis reflects a convergence of extracellular matrix degradation, thrombosis, and vascular inflammation (thromboinflammation), where platelets and the intraluminal thrombus (ILT) ar...Abdominal aortic aneurysm (AAA) pathogenesis reflects a convergence of extracellular matrix degradation, thrombosis, and vascular inflammation (thromboinflammation), where platelets and the intraluminal thrombus (ILT) are implicated in playing central, stage-dependent roles. Beyond their traditional role in hemostasis, activated platelets are known to localize in ILT, releasing chemokines, proteases, and growth factors, which aid in recruiting leukocytes and may drive aneurysm expansion through matrix breakdown. Although preclinical models and tissue analyses suggest platelet adhesion receptors and chemokine interactions contribute to leukocyte influx and aneurysm progression, the availability of preclinical models that recapitulate ILT is limited, and overall translational evidence remains inconclusive. A crucial question is whether ILT acts as a mere bystander in AAAs or actively contributes to aneurysm growth and rupture. Notably, the only randomized controlled trial to date testing an antiplatelet agent (ticagrelor) demonstrated no effect on AAA growth, raising concerns that platelets may not be an appropriate AAA treatment target. The development of AAA models that consistently manifest ILT will be essential for determining the therapeutic potential of targeting platelet inflammatory functions (eg, glycoprotein VI signaling inhibition). Ultimately, clarifying whether ILT-driven platelet inflammation is a modifiable mechanism or a bystander signal will require ILT-specific models and rigorously powered trials to define safe, hemostasis-sparing antiplatelet strategies for AAA.
BACKGROUND: Mutations in antithrombin () lead to the hereditary antithrombin deficiency. Conventional therapies for hereditary antithrombin deficiency are prophylactic or on-demand oral anticoagulants, which have poor co...BACKGROUND: Mutations in antithrombin () lead to the hereditary antithrombin deficiency. Conventional therapies for hereditary antithrombin deficiency are prophylactic or on-demand oral anticoagulants, which have poor compliance and side effects. This study explored the therapeutic efficacy of adeno-associated virus serotype 8 (AAV8)-mediated human gene (AAV8-hSERPINC1) in AT (antithrombin)-deficiency mice. METHODS: AAV8-hSERPINC1 carrying luciferase was injected into AT mice via tail vein injection at low, medium, and high doses. The biodistribution and expression of the carrier were visualized by in vivo bioimaging technology. Plasma AT levels were serially monitored by ELISA, and an inferior vena cava model was established to evaluate thrombotic propensity. Safety was evaluated by monitoring hepatic, renal, and cardiac function parameters and employing flow cytometry. RESULTS: A dose-dependent increase in AT expression was observed in AT mice after AAV8-hSERPINC1 injection. Compared with the untreated mice, medium-dose treatment restored plasma AT activity and antigen in AT mice to normal levels by week 8, with maintenance within the normal reference range for 40 weeks. In the venous thrombosis model, the rate of thrombosis in mice treated with medium-dose AAV8-hSERPINC1, rivaroxaban, low-molecular-weight-heparin, and wild-type mice were 60%, 60%, 70% and 60%, respectively. After AAV injection, transient elevations in hepatic transaminases and cytokines were observed in mice during a short-term period. CONCLUSIONS: Our study demonstrates that AAV8-hSERPINC1 gene delivery resulted in durable AT expression, sustained blood hypercoagulation state correction, thereby rescuing thrombophilia in AT-deficient male mice. These data support the long-term efficacy and safety of AAV gene therapy for hereditary antithrombin deficiency.