Arteriosclerosis, Thrombosis, and Vascular Biology

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.

BackgroundDecreased hepatic removal of low density lipoproteins (LDL) and increased apolipoprotein B (apoB) production cause hypercholesterolemia, a major causal risk factor of atherosclerotic cardiovascular disease (ASCVD). By a genome-wide siRNA screen, we previously identified subunits of the Coat protein I (COPI) complex to limit LDL uptake into Huh-7 hepatocarcinoma cells. MethodsThese findings were validated by targeted in vitro experiments as well as genetic association studies in humans and three mouse models with mutated or disrupted COPI genes. ResultsSilencing of COPA, COPB1, COPB2, ARCN1, COPG1, and COPZ1 in Huh-7 cells resulted in decreased uptake of LDL and aberrant glycosylation and altered cell surface abundance of the LDL receptor (LDLR) as well as increased apoB secretion and cellular lipid storage. Single nucleotide polymorphisms of ARCN1 were associated with lower ARCN1 expression and higher levels of LDL-cholesterol (LDL-C). Rare variants of COPA and COPG1 were enriched among patients with LDL-C > 5 mmol/L. Patients and mice carrying other rare immunopathogenic missense variants of COPA and COPG1 did not present with elevated plasma levels of LDL-C, while hepatic knockdown of murine Copg1 increased the concentrations of non-HDL-cholesterol in plasma and triglycerides in the liver. ConclusionsThe COPI coatomer regulates LDLR activity and apoB secretion as well as lipid content of liver cells. Loss of function of some variants of COPI genes are associated with higher LDL-C levels.

BackgroundPlatelet activation via G protein-coupled receptors (GPCRs) is central to arterial thrombosis. P2Y12 is a canonical Gi-coupled receptor mediating ADP-dependent platelet activation, yet the role of Regulator of G protein Signaling 6 (RGS6), a modulator of Gi signaling, in platelet function and thrombosis remains unclear. ObjectivesTo determine the role of RGS6 in platelet activation and arterial thrombosis and to define its impact on P2Y12/Gi signaling. MethodsArterial thrombosis was assessed using a FeCl{square}-induced carotid artery injury model in wild-type (WT) and Rgs6-/- mice. Platelet aggregation was measured ex vivo. Signaling pathways were analyzed by Western blot in ADP-stimulated platelets. P2Y12/Gi signaling was further evaluated using a cAMP-responsive luciferase reporter assay in HEK293 cells. ResultsMale Rgs6-/- mice exhibited significantly accelerated thrombosis compared with WT controls. Rgs6-/- platelets showed enhanced ADP-induced aggregation, whereas collagen-induced aggregation was unchanged. In ADP-stimulated platelets, RGS6 deficiency altered signaling kinetics, characterized by delayed Akt phosphorylation and reduced PKA and VASP phosphorylation. In a heterologous cAMP-luciferase assay, RGS6 attenuated P2Y12/Gi-mediated suppression of cAMP. Two-way ANOVA demonstrated significant effects of ADP and RGS6 expression on luciferase activity, with no interaction, indicating that RGS6 modulates signaling magnitude rather than agonist sensitivity. Pharmacologic inhibition of P2Y12 with clopidogrel abolished the genotype-dependent difference in thrombosis in vivo. ConclusionsRGS6 acts as a negative regulator of platelet P2Y12/Gi signaling and thrombus formation. Loss of RGS6 enhances ADP-dependent platelet activation and accelerates arterial thrombosis, establishing RGS6 as an endogenous brake on platelet activation.

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.

The loss of smooth muscle cell (SMC) contractile phenotype contributes to various diseases including atherosclerosis. However, its metabolic basis is not entirely elucidated. Since the transforming growth factor beta (TGF{beta}) signaling is among principal regulators of SMC contractility, we studied metabolic regulation of TGF{beta} signaling in SMCs in vitro and atherosclerotic mouse models and human lesions. We found that TGF{beta} induced Ac-CoA synthetase 2 (ACSS2)-dependent Ac-CoA production, by suppressing pyruvate dehydrogenase kinase 4 (PDK4). This stabilized R-SMADs and TGF{beta} receptor 1, preserving SMC contractile phenotype. SMC-specific PDK4 knockout mimicked the effect of TGF{beta} signaling both metabolically and phenotypically, increasing glucose-derived synthesis of Ac-CoA and SMC contractile phenotype. SMC-specific Pdk4 knockout in ApoE knockout mice reduced atherosclerosis. Furthermore, human specimens demonstrated a strong correlation between PDK4 level and atherosclerosis severity. These findings indicate that continuous TGF{beta} signaling, critical to the maintenance of the normal SMC contractile state and is regulated by PDK4 and carbohydrate metabolism. TeaserReducing PDK4 metabolically restricts aortic plaque growth via TGF{beta}-dependent SMC contractility.

BackgroundCarotid intima-media thickening (IMT) is a major risk factor for cardiovascular disease (CVD). The large ribosomal subunit protein 17 (Rpl17) was recently reported as a CVD-associated gene; however, ribosomal mutations generally are not associated with vascular dysfunction. We have created a novel genetic model of decreased RpL17 in endothelial cells (EC) to determine how changes in endothelial ribosome expression cause IMT. MethodsEC-restricted RpL17 heterozygous mice (Cdh5-Cre; RpL17fl/wt, or Rpl17-Het), were generated and subjected to sham or partial carotid ligation (PCL) surgery of the left artery to induce acute disturbed (d)-flow in vivo. Carotids were harvested on day 14 for quantitative tissue immunostaining. Purified mouse and human EC in vitro were exposed to steady (s)-flow or d-flow using cone viscometry, and collected for flow cytometry, protein expression, electron microscopy, or purification of ribosomes. Human carotid samples from healthy and endarterectomy patients were used for tissue analysis. ResultsCarotids from RpL17-Het mice with PCL-induced d-flow showed increased IMT relative to RpL17-WT controls. In addition, RpL17 protein levels were decreased in regions of d-flow compared to s-flow. Increased levels of ER stress markers were observed by carotid immunostaining, as well as activation of the integrated stress response (ISR) in RpL17-Het EC. Analysis of mRNAs bound to polysomes vs. monosomes in EC-RpL17-Het revealed increased translational efficiency of key regulators of glycolysis, redox, inflammation, matrix, and endothelial-to-mesenchymal transition (EndMT). Metabolic profiling by Seahorse assay showed enhanced anaerobic glycolysis and decreased oxidative respiration in RpL17-Het EC, consistent with the translational efficiency data. Immunostaining of carotids identified upregulated EC inflammation and EndMT. ConclusionsOur data support RpL17 as a key mediator of EC phenotypic modulation that causes IMT in response to d-flow. We show a novel pathway for d-flow-mediated IMT: endoplasmic reticulum stress and activation of the ISR. These changes alter translational efficiency and reprogram EC cell cycle, metabolism, and redox state in the presence of d-flow to cause IMT, a precursor to cardiovascular pathology.

Genome-wide association studies have identified rare and common mutations associated with increased risk of pulmonary arterial hypertension (PAH), but the mechanism by which impaired SOX17 expression increases PAH risk is not known. Notably, SOX17 plays a critical role in endothelial identity during development by suppressing RUNX1 through binding to its promoter and directing stem and progenitor cells toward an endothelial rather than a hematopoietic cell fate. RUNX1 functions as a key regulator of myeloid differentiation, aberrant angiogenesis and adverse cardiac remodeling. Previously, we found that RUNX1 inhibition reverses pulmonary hypertension (PH) in multiple animal models. Here, we hypothesize that impaired expression of SOX17 in PAH leads to endothelial cell (EC) dysfunction by failing to suppress RUNX1. METHODSHuman pulmonary artery endothelial cells (HPAECs) with stable SOX17 CRISPR/Cas9 knockout or RUNX1 overexpression were generated and examined for endothelial and hematopoietic gene expression, proliferation, migration, apoptosis, and angiogenesis. Immortalized lymphoblastoid cell lines (LCLs) from PAH patients with SOX17 mutations and healthy controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into ECs. The effect of RUNX1 inhibition on Sugen/hypoxia-PH was examined in rats, SOX17 enhancer knockout (SOX17enhKO) mice, and Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic mice. SOX17 and RUNX1 expression were analyzed in peripheral blood samples from PAH patients (n=359). RESULTSHPAECs with SOX17 deletion or RUNX1 overexpression exhibited decreased expression of EC markers, enhanced proliferation and migration, defective angiogenesis, and decreased apoptosis. RUNX1 siRNA knockdown or RUNX1 inhibition by Ro5-3335 partially restored the endothelial properties in SOX17 KO HPAECs. ECs differentiated from SOX17 mutant PAH patient iPSCs exhibited upregulated RUNX1 expression and loss of endothelial identity, which was also partially restored by RUNX1 siRNA or Ro5-3335. In addition, SOX17enhKO mice had increased RUNX1 expression and susceptibility to Sugen/hypoxia-induced PH (SuHx-PH). Treatment with RUNX1 inhibitors or inducible endothelial-specific deletion of RUNX1 rescued SuHx-PH susceptibility in SOX17enhKO mice. RUNX1 inhibitors Ro5-3335 and Ro24-7429 also reversed SuHx-PH in wild-type rats. In addition, plasma RUNX1 expression was higher in PAH patients lacking detectable SOX17 expression than in patients with detectable SOX17 expression. CONCLUSIONSImpaired SOX17 expression increases the risk of PAH through insufficient suppression of RUNX1, leading to pulmonary endothelial dysfunction. RUNX1 inhibition mitigates PH associated with SOX17 deficiency and may represent a novel therapeutic strategy for PAH, especially those with rare or common SOX17 mutations.

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

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

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.

BACKGROUNDInactivation of ANGPTL3 (angiopoietin-like protein 3, A3) is a proven therapeutic strategy for lowering plasma lipid levels independently of the LDL receptor (LDLR), yet the optimal approach to inactivate A3 remains unclear. A3 is proteolytically cleaved and circulates as full-length (A3-FL), N-terminal (A3-Nter) and C-terminal (A3-Cter) fragments. The specific contribution of each form of A3, and of its paralog, ANGPTL8 (A8), in modulating circulating levels of ApoB-Containing Lipoproteins (ABCLs) remain poorly defined. Clarifying these relationships will inform next-generation A3-directed therapies. METHODSWe performed liver perfusion studies to directly compare the number and composition of VLDL particles secreted from mice with and without A3. To amplify effects on cholesterol metabolism, we generated Ldlr-/- mice expressing wildtype A3 (A3-WT), A3-FL or A3-Nter, with or without co-expression of A8, and analyzed plasma lipids, circulating A3 and A8 complexes, and intravascular lipase activities. Complementary in vitro assays and structural modeling were used to assess relative endothelial lipase (EL) inhibition by A3 alone or in complex with A8. RESULTSLiver perfusion studies revealed that A3 inactivation does not alter the rates of hepatic secretion of VLDL in wildtype or Ldlr-/- mice. Inactivation of A8 alone lowered plasma LDL-cholesterol (C) levels by [~]20%, an effect dependent upon the expression of both EL and A3. Maximal inhibition of lipoprotein lipase (LPL) required co-expression of A8 plus both A3-FL and A3-Nter, indicating that A3 cleavage, in addition to A8 expression, is essential for maximal LPL inhibition. In contrast, A8 expression, but not A3 cleavage, was required for optimal EL inhibition. CONCLUSIONSA8 acts in concert with A3 to differentially modulate LPL- and EL-mediated lipolysis, which antagonizes hepatic clearance of newly-secreted atherogenic ABCLs. This mechanistic framework refines our understanding of A3-targeted lipid lowering and highlights the therapeutic potential of dual A3- plus A8-directed strategies to treat dyslipidemia and prevent atherosclerotic cardiovascular disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIInactivation of A3 lowers circulating ABCL levels without altering hepatic secretion rates of VLDL-ApoB or -TG. C_LIO_LIProteolytic cleavage of A3 is required for maximal inhibition of LPL. C_LIO_LIInactivation of A8 lowers LDL-C levels through an A3- and EL-dependent, but LDLR-independent, mechanism. C_LI What are the clinical implications?O_LICombining A8 inhibition with A3-inactivating therapies offers a strategy to achieve greater reduction in LDL-C levels and atherosclerotic cardiovascular risk. C_LI

BACKGROUND: MicroRNAs (miRNAs) regulate macrophage plasticity in atherosclerosis (AS). We tested the hypothesis that miR487a-3p and miR6855-3p accelerate AS by promoting macrophage inflammatory responses and metabolic dysregulation. METHODS: miRNA-seq and mRNA-seq were performed on peripheral monocytes from CAD patients and healthy controls. Macrophages in mouse aortas and human coronary arteries were characterized by flow cytometry and immunostaining. AS was evaluated in PCSK9-overexpressing mice with myeloid-specific deficiency of carboxypeptidase E (CPE) or ribonucleotide reductase subunit M2 (RRM2) fed a high-fat diet. RESULTS: miR487a-3p and miR6855-3p were the top differentially expressed miRNAs in peripheral monocytes from CAD patients versus controls. Both miRNAs were lipid-inducible, with transcription driven by ox-LDL via KLF5 and IRF1, respectively, and were secreted extracellularly. Plasma levels of both miRNAs were elevated in CAD patients, correlated positively with blood lipids and Gensini score, and exhibited diagnostic accuracy (AUC 0.83 each). Both miRNAs were predominantly expressed in coronary plaque macrophages, and their abundance correlated with lesion area. Overexpression of either miRNA promoted macrophage pro-inflammatory polarization, lipid metabolic dysregulation, foam cell formation, and endothelial cell apoptosis, whereas miRNA inhibition attenuated these ox-LDL-induced phenotypes. CPE and RRM2 were identified as direct targets of miR487a-3p and miR6855-3p, respectively, by integrating mRNA-seq and TargetScan predictions, with binding confirmed by dual-luciferase assays and miRNA pulldown. CPE or RRM2 overexpression partially reversed miRNA-induced macrophage dysfunction. Conversely, myeloid-specific deletion of Cpe or Rrm2 exacerbated AS in hypercholesterolemic mice. CONCLUSIONS: miR-487a-3p and miR-6855-3p are promising biomarkers for CAD diagnosis and prognosis. Mechanistically, they drive macrophage inflammation and lipid metabolic disruption, identifying them as potential therapeutic targets. Key Words: microRNA; atherosclerosis; macrophage; inflammation; lipid disorders

OBJECTIVEThis study aimed to explore the role and underlying mechanism of microRNA-128 (miR-128) in regulating vascular remodeling in spontaneously hypertensive rats (SHRs), focusing on its targeting of peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) and modulation of the Toll-like receptor 4/nuclear factor-{kappa}B (TLR4/NF-{kappa}B) inflammatory pathway. METHODSAll experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University. In vivo, ten-week-old male SHRs were randomly assigned to three groups: renal denervation (RDN, n=6), sacubitril/valsartan (Sac/Val, n=6), and Sham (n=6). Age-matched Wistar-Kyoto (WKY) rats served as normotensive controls (n=6).Eight weeks after intervention, mesenteric arteries were harvested for histological, functional, and molecular analyses. Serum miR-128 levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of key proteins in the vascular wall were assessed via immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting (WB). Bioinformatics analysis and RNA sequencing (RNA-seq) were employed to identify core genes and signaling pathways associated with hypertension-induced pathological inflammation. RESULTSIn vivo, in the SHR sham-operated group, elevated blood pressure, severe vascular remodeling, and impaired vasodilatory function were observed, accompanied by downregulated miR-128 expression and upregulated TLR4/NF-{kappa}B signaling activity (all p < 0.0001).RDN postoperative, miR-128 expression was significantly restored, which in turn inhibited the TLR4/NF-{kappa}B pathway, reduced the production of pro-inflammatory cytokines (including IL-1{beta}, IL-6, and TNF-), and ameliorated vascular dilation dysfunction in SHRs (all p < 0.0001). Mechanistically, miR-128 negatively regulated the TLR4/NF-{kappa}B signaling pathway while upregulating the expression of PPAR-{gamma} (p < 0.05). CONCLUSIONRDN not only exerts a hypotensive effect but also improves hypertensive vascular remodeling. miR-128 inhibits excessive inflammation in vascular smooth muscle cells and alleviates vascular remodeling in SHRs via the PPAR-{gamma}/TLR4/NF-{kappa}B axis. These findings identify miR-128 as a potential therapeutic target for RDN in the treatment of hypertension, providing a novel regulatory strategy for the precision management of cardiovascular diseases.

Aortic dissection is life-threatening due to continued loss of medial integrity that may culminate in secondary rupture within hours to days. While pre-existing defects or hemodynamic loads compound structural deterioration of the aorta, pathological progression from symptomatic dissection channel to lethal transmural tear is poorly understood. We examined the structure of referent and acutely dissected ascending aortas by microscopy. Elastic, collagen, and cellular components of non-dissected media were intricately interconnected. Medial damage in dissection lesions was traced from ingress to central to peripheral areas. Entry tears broke cleanly through successive laminae leading to cavernous false lumens in which medial structure was destroyed. Nearby laminae with widening between flanking elastic lamellae (termed minor delaminations) were filled with blood and showed severe medial damage. Farther laminae without delamination but containing red blood cells (termed blood extravasation) displayed moderate medial damage. More distant, non-delaminated laminae with accumulation of albumin but not red blood cells (termed plasma extravasation) exhibited mild medial damage. Varying medial hemorrhage with scattered sloughing of laminae was observed along the entire false lumen. We conclude that hydraulic fracturing of residual dissected media by pressurized blood via communications from the false lumen contributes to further structural weakening of the aortic wall.

BackgroundThe formation of a patent vascular lumen is fundamental to circulatory function, a process governed by cytoskeletal dynamics and mechanosensory signalling. Endothelial cilia are present during blood vessel lumen development, but their precise functional role remains poorly understood. Understanding how cilia coordinate with endothelial cytoskeletal and signalling pathways is critical for elucidating mechanisms of vascular morphogenesis. MethodsWe have established a microfluidic system that recapitulates endothelial tube formation under fluid flow, enabling pharmacological and genetic manipulation with real-time visualisation of tube behaviour. Cilia, cytoskeletal dynamics, and lumen development were analysed in vitro, and in vivo. ResultsEarly perfusion in the microfluidic system induced a hierarchical vascular network. Inhibiting Rho-kinase (ROCK) or knocking down ciliary components (IFT88 and RPGRIPL1) suppressed lumen formation. ROCK inhibition or genetic ablation disrupted cilia in endothelial and non-endothelial cells, associated with LIM-kinase inhibition. Crucially, ROCK2 genetic ablation caused endothelial cilia loss, misorientation, and abrogated lumen formation, leading to haemorrhages and compromised vascular integrity in vivo. ConclusionsOur findings unveil a previously unrecognised co-regulation between cilia and ROCK signalling essential in vascular lumen formation.

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.

Pulmonary arterial hypertension is a progressive, fatal disease driven by pathologic vascular remodeling including arterial medial hypertrophy, occlusive neointimal lesion formation, and venous muscularization. Current vasodilatory therapies improve hemodynamics but do not reverse established remodeling. Imatinib mesylate, a tyrosine kinase inhibitor targeting the PDGF-PDGFR signaling axis, has been proposed as an anti-remodeling therapy for pulmonary arterial hypertension and has demonstrated hemodynamic benefit in both preclinical models and clinical trials. However, prior preclinical models lack the neointimal lesions characteristic of human disease, effects on venous remodeling have not been examined, and direct histologic assessment in human trials is precluded by the invasiveness of serial lung biopsy. Here, leveraging the house dust mite mouse model of pulmonary hypertension, which recapitulates medial thickening, neointimal lesion formation, and venous muscularization, we rigorously evaluate the anti-remodeling and hemodynamic effects of imatinib during two defined remodeling stages: neointimal lesion growth and neointimal lesion maintenance. Imatinib treatment significantly reduced right ventricular systolic pressure at both stages. Despite this hemodynamic improvement, quantitative vessel-level analysis of over 1,700 arteries and 1,200 veins revealed no significant effect of imatinib on arterial medial thickness, neointimal lesion growth, neointimal lesion maintenance, or venous muscularization across any vessel size class. These findings dissociate imatinibs hemodynamic benefit from structural vascular remodeling and suggest that imatinib functions primarily as a pulmonary vasodilator rather than an anti-remodeling agent.

BackgroundThoracic aortic aneurysm (TAA) is a life-threatening condition with an unpredictable lisk of rupture. Current clinical parameters have limited ability to accurately predict imminent rupture. Osteopontin (OPN) has been implicated in aortic aneurysm pathology, however, it role as a marker of imminent rupture remains. unclear. We investigated the dynamics of OPN expression dynamics in a mouse model with predictable rupture timing and validated our findings in human TAA. MethodsOne-month-old fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice were used as a TAA model; with wild-type (WT) mice served as controls. Angiotensin II (AngII) was administered to Fbn1mgR/mgR to induce acute aortic rupture. Single-section transcriptome analysis and immunofluorescence staining were performed on ascending aortic tissue at 24 and 72 hours after AngII infusion, with pre-treatment Fbn1mgR/mgR and WT mice serving as controls. To determine conservation in human disease, we reanalyzed publicly available single-cell RNA sequencing data from ascending thoracic aortic aneurysm (ATAA) patients. ResultsAngII infusion induced progressive mortality beginning at 24 hours, with approximately 60% survival at 72 hours and nearly no survival by 8 days in Fbn1mgR/mgR mice. At this pre-rupture time point, OPN showed prominent upregulation at both mRNA and protein levels in ascending aortic tissues compared to controls. Immunofluorescence staining revealed increased OPN expression in the aortic wall, particularly in regions exhibiting structural deterioration. Reanalysis of human ATAA single-cell data showed elevated OPN expression compared to controls, with enrichment in immune cell populations, especially macrophages. Within the macrophage compartment, subcluster analysis identified a stress-responsive subpopulation (MC1) that was markedly expanded and almost exclusively composed of ATAA-derived cells, representing the primary source of OPN upregulation. ConclusionsOPN upregulation represents a conserved molecular signature of the pre-rupture state in TAA across mice and humans. Our mode, which enables predictable rupture timing, allowed the capture of acute pre-rupture molecular changes, suggesting OPN as a potential biomarker for predicting imminent aortic rupture.

BackgroundWildland firefighters experience repeated occupational exposure to wildfire smoke at high particulate matter (PM) concentrations, leading to elevated cardiovascular disease risk and hypertension prevalence. However, the pathophysiological processes linking cumulative smoke inhalation to vascular damage and blood pressure elevation remain poorly characterized. To evaluate these effects under controlled exposure conditions, we used a preclinical exposure model calibrated to match the cumulative PM burden deposited in wildland firefighter airways over 7-14 years of service. Male apolipoprotein E knockout (Apoe-/-) mice underwent whole-body inhalation of Douglas fir smoke or filtered air for 2 hours/day, 5 days/week, for 8 or 16 weeks at target PM concentrations of 40 mg/m3. ResultsProlonged smoke exposure induced sustained elevation of circulating tumor necrosis factor-alpha (TNF-), interleukin-1 beta (IL-1{beta}), and interleukin-6 (IL-6), coupled with diffused nuclear factor kappa B (NF-{kappa}B) activation throughout the aortic wall. Smoke inhalation disrupted endothelial adherens junctions, upregulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and promoted monocyte recruitment to aortic tissues, concurrent with enhanced monocyte chemoattractant protein-1 (MCP-1) expression. Oxidative stress was evidenced by increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit 2 (NOX2) expression, elevated superoxide levels, and endothelial nitric oxide synthase (eNOS) uncoupling in the aorta, leading to lipid peroxidation and accompanied by intimal apoptosis. These inflammatory and oxidative perturbations occurred alongside a pro-fibrotic phenotypic shift characterized by transforming growth factor beta 1 (TGF-{beta}1) upregulation, myofibroblast differentiation, and progressive collagen accumulation in medial and adventitial compartments of the aortic wall. Functionally, smoke exposure progressively impaired aortic cyclic distensibility through combined wall thickening and circumferential tissue stiffening, while severely attenuating endothelium-dependent and nitric oxide (NO)-mediated vasodilation. These functional and structural shifts culminated in elevated systolic and diastolic blood pressures. While endothelial dysfunction reached maximal impairment by 8 weeks, aortic stiffening continued to worsen through 16 weeks of exposure, demonstrating differential temporal progression of vascular damage. ConclusionsThese findings demonstrate that occupationally relevant wildfire smoke exposure produces convergent inflammatory, oxidative, and profibrotic vascular remodeling with progressive loss of arterial compliance and impaired endothelium-dependent vasodilation, underscoring potential vascular targets for cardiovascular health surveillance and risk mitigation in wildland firefighters.

The aorta, normally resilient to hemodynamic stresses, becomes vulnerable to structural failure due to diverse conditions that weaken the wall. We injected fluid into excised specimens of human ascending aorta with pressure monitoring to quantify the impact of clinical and histological factors on mural damage. Two modes of medial injury emerged with distinct pressure tracings. Extravasation was characterized by diffuse infiltration of fluid with widespread damage of smooth muscle cells and collagen fibers but limited separation of elastic lamellae. By contrast, delamination was characterized by marked separation of elastic lamellae along a single plane with damage to cells and fibrillar matrix restricted to adjacent laminae. Aging, aortic dilatation, and family history associated with lower pressures causing delamination, whereas a diagnosis of hypertension associated with higher pressures suggesting resilience to dissection. Collagen fraction adjacent to delamination correlated with higher pressures as did decreased smooth muscle cell density and increased glycosaminoglycan fraction, although several clinical and histological variables were interrelated. Protein cross-linking strengthened and enzymatic digestion of collagen weakened the aortic wall, while acute cell lysis with detergent had no effect. We conclude that increased functional medial collagen has an adaptive protective role in aortic remodeling rather than signifying medial degeneration.