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.

Thoracic aortic aneurysms involving the root and ascending aorta progressively enlarge and can lead to life-threatening acute aortic dissections, collectively termed thoracic aortic disease (TAD). Oxidative stress due to an excess of reactive oxygen species (ROS) has been implicated in TAD pathogenesis by promoting vascular inflammation, extracellular matrix degradation, and smooth muscle cell apoptosis. Cobinamide (Cbi), a potent antioxidant and vitamin B12 analog, has been reported to slow aortic aneurysm growth in TAD mouse models. We evaluated its efficacy in mice administered β-aminopropionitrile (BAPN) to induce TAD, a model in which aneurysms progress to dissections. C57BL/6J mice of both sexes were administered either BAPN alone or BAPN plus Cbi in drinking water from postnatal day 21 (P21). Survival analysis showed that 56% of mice in the BAPN group died from aortic rupture by P49, whereas mortality increased to 91% in the BAPN + Cbi group (P = 0.049). Necropsy confirmed thoracic aortic rupture as the cause of death. At P35, proximal aortic analyses showed that Cbi significantly reduced ROS abundance in the ascending aorta, based on decreased dihydroethidium staining and lower oxidative stress markers, including protein carbonylation and DNA oxidation by 8-hydroxy-deoxyguanosine staining. Cbi did not affect aortic diameters by echocardiography or elastic fiber fragmentation. Cbi was associated with reduced oxidative stress in aortic tissue; it did not prevent aortic dissection and was associated with increased rupture-related mortality in the BAPN model. These findings highlight context-dependent roles of ROS in TAD and the importance of carefully evaluating antioxidant strategies in dissection-prone settings.NEW & NOTEWORTHY Cobinamide reduced oxidative stress in aortas of mice administered BAPN but was associated with worsened fatal aortic dissections, highlighting a context-dependent role of redox signaling in aortic disease and the need for careful evaluation of antioxidant therapies intended to prevent thoracic aortic dissection.

Background:Ascending thoracic aortic dissection (ATAD) is characterized by extensive macrophage (MΦ) accumulation and profound inflammation; however, the mechanisms sustaining pro-inflammatory MΦ activation remain incompletely defined. Emerging evidence indicates that epigenetically generated immune memory drives innate immune cells toward persistent inflammatory states. In this study, we investigated whether epigenetic reprogramming governs MΦ phenotypic fate and contributes to ATAD pathogenesis. Methods:We performed single-cell RNA sequencing of human ascending aortic tissues from controls, patients with ascending thoracic aortic aneurysm (ATAA), and patients with acute ascending thoracic aortic dissection (ATAD). We also performed integrated single-cell RNA sequencing, single-cell ATAC sequencing, and spatial transcriptomics in an angiotensin II (Ang II)-infused mouse model. The role of the STING-IRF3 signaling axis in MΦ epigenetic programming was examined using MΦ- Sting -/- and MΦ- Irf3 -/- mice. Results:In human and mouse aortic tissues, we identified multiple functional MΦ populations including pro-inflammatory, phagocytic/anti-inflammatory, proliferative, and reparative/healing MΦs. Aortic MΦs in both sporadic ATAD patients and Ang II-induced ATAD mice exhibited a pronounced pro-inflammatory bias with enhanced differentiation toward pro-inflammatory MΦs and impaired differentiation toward phagocytic/anti-inflammatory states. Pro-inflammatory MΦs were particularly abundant in dissection sites, whereas phagocytic MΦs were enriched in discrete adventitial niches. Origin analyses revealed a substantial increase in CCR2 ⁺ recruited MΦs within the aortic wall, which preferentially differentiated into pro-inflammatory MΦs. In contrast, LYVE1 ⁺ resident MΦs- predominantly biased toward phagocytic phenotypes-were markedly depleted in ATAD. Single-cell ATAC sequencing identified coordinated chromatin remodeling with increased accessibility at pro-inflammatory gene loci and decreased accessibility at phagocytic gene loci. Among candidate transcriptional regulators identified, IRF family TFs, including IRF3 emerged as unique factors capable of simultaneously promoting pro-inflammatory gene programs while suppressing phagocytic gene expression. Mechanistically, STING-IRF3 signaling orchestrates this biased transcriptional state, likely through coordinated BRG1-dependent chromatin opening at pro-inflammatory gene loci and chromatin closing at phagocytic/anti-inflammatory gene loci. MΦ specific Sting -/- and Irf3 -/- mice exhibited attenuated inflammatory reprogramming and reduced aortic destruction and dissection. Conclusions:These findings identify STING-IRF3-mediated epigenetic programming of MΦs as a fundamental mechanism driving aortic inflammation and ATAD development. Targeting MΦ epigenetic programming may represent a promising therapeutic strategy to prevent aortic dissection. Graphic Abstract:

Aims Abdominal aortic aneurysms (AAA) rupture is a life-threatening event with unclear molecular mechanisms. Our previous work demonstrated elevated levels of the matricellular protein thrombospondin-1 (TSP1, encoded by Thbs1) in human and mouse AAA tissues. Single-cell RNA sequencing analysis identified macrophages, endothelial cells, and smooth muscle cells as the major TSP1-expressing cells in aneurysmal tissues. Global Thbs1 deletion reduces aneurysm formation by inhibiting vascular inflammation. The aim of this study was to investigate how TSP1 deficiency in different cell types affects AAA rupture. Methods and results AAA and rupture were induced by angiotensin II infusion in hypercholesterolemic mice. In global Thbs1 deficient mice, hypercholesterolemia was achieved by crossing them with Apoe knockout mice. To generate cell type-specific TSP1 deficient mice, Thbs1flox/flox mice were crossed with VE-cadherin-Cre, SMMHC-iCreERT2, and Lyz2-Cre mice to target endothelial cells, smooth muscle cells, and myeloid cells, respectively. In these conditional knockout models, hypercholesterolemia was induced via AAV-PCSK9. We found that both global and myeloid-specific Thbs1 deletion increased rupture rate over two-fold, whereas endothelial- or smooth muscle cell-specific deletion had no significant effect. Endothelial-specific Thbs1 deletion reduced aneurysm size in the CaCl₂ model. Single-cell RNA sequencing and histology in myeloid-specific Thbs1 knockout aortas revealed broad suppression of inflammation and extracellular matrix production. Conclusion Myeloid-derived TSP1 plays a critical role in inhibiting aneurysm rupture in mice, likely by promoting matrix repair phenotypes in vascular smooth muscle cells, enhancing vascular wall integrity.

Background: Plasminogen activator inhibitor-1 (PAI-1), the primary regulator of fibrinolysis, is highly abundant in human thoracic aortic aneurysms (TAA). PAI-1 is also increased in the ascending aortas of mice infused with angiotensin II (AngII) prior to overt pathology. The purpose of this study was to determine whether deletion of PAI-1 influenced the development of TAAs during AngII infusion. Methods and Results: Whole-body PAI-1 deficient mice (PAI-1-/-) and wild type littermates (PAI-1+/+) were infused with AngII (1,000 ng/kg/min) for 28 days to induce TAA. Despite the upregulation of PAI-1 in ascending aortas, aortic diameters were not altered by PAI-1 deficiency. However, PAI-1 deficiency augmented grossly visible and histologically evident cardiac fibrosis, predominantly within the epicardium and posterior septum. Ferric iron, indicative of prior hemorrhage, was observed coincidently with cardiac fibrosis in PAI-1-/- mice. To verify the presence of hemorrhage, we infused PAI-1+/+ and -/- mice with AngII for 1 and 7 days. Hemorrhage was observed in PAI-1-/- mice as early as 1 day by in situ imaging and TER-119 staining and was accompanied by myocyte loss defined by troponin I staining. By 7 days, cardiac hemorrhage and myocyte loss were increased in PAI-1-/- mice. The pathologies observed at 1 and 7 days of AngII were pronounced within the epicardium and posterior septum, spatially preceding observed fibrosis at 28 days of AngII. Similar hemorrhagic and fibrotic phenotypes were observed in PAI-1-/- mice infused with a pressor dose of norepinephrine (5.6 mg/kg/day in 0.2% w/v L-ascorbic acid) for 7 and 28 days, respectively. To investigate mechanisms underlying cardiac hemorrhage, PAI-1+/+ and -/- mice were infused with saline or AngII for 7 days. Mid-ventricular tissue was analyzed by bulk RNA sequencing with hemorrhage included as a binary covariate. Differential expression and gene set enrichment analyses identified upregulation of genes related to extracellular matrix organization and proteolysis in AngII-infused PAI-1-/- hearts, including Fn1 , Ecm1 , Lgals3 , and Ctss . These results suggest that PAI-1 deficiency induced transcriptional signatures of wound healing after AngII infusion, even in the absence of coincident hemorrhage. Conclusion: PAI-1 deficiency does not affect TAA formation, but augments cardiac fibrosis in AngII-infused mice. Cardiac hemorrhage, in association with myocyte loss, precedes fibrosis in PAI-1 deficiency.

Background: Elastic fibers are crucial for aortic wall integrity and are severely disrupted in aortic dissection (AD). We demonstrated previously that new elastic fibers are generated in the false lumen wall of AD in humans and mice after long-term β-aminopropionitrile (BAPN) administration. The false lumen wall was predominantly populated by cells expressing smooth muscle cell (SMC) markers. However, the mechanisms by which these elastogenic cells emerge and expand after AD remain unclear. Methods and Results: To explore the molecular basis underlying the expansion of elastogenic cells following AD, we first performed bulk RNA sequencing in normal aortas of control mice and AD samples of mice administered BAPN for 12 weeks. Transcriptomic analyses revealed upregulation of genes implicated in SMC differentiation, along with increased expression of elastic fiber-component genes. Genes associated with TGFβ signaling, including Tgfb1 , Tgfbr2 , and Smad3 , as well as Notch signaling-related genes, such as Notch3 , Jag1 , and Hey1 , were increased in AD samples. In addition, retinoic acid (RA) signaling-related genes, including Aldh1a1 , Rara , and Cyp26a1 , were upregulated. These data indicated active SMC differentiation accompanied by de novo elastic fiber synthesis in AD. Next, spatial transcriptome was performed to determine the molecular features of elastogenic cells. Although cells composing the false lumen wall were enriched for SMC marker genes, Acta2 and Myh11 , their abundance was lower than that observed in resident SMCs. Instead, immature and progenitor cell signatures, such as Cd34 and Thy1 , were prominent in the false lumen wall. Consistent with the bulk RNA sequencing, RA signaling-related genes, Aldh1a1 and Rbp1 , were highly abundant in cells composed of the false lumen. Interestingly, lineage tracing studies found that elastogenic cells did not originate from the resident SMC lineage, supporting differentiation from a non-SMC lineage after AD. Immunostaining revealed that these cells were positive for stem cell antigen 1, markers for adventitial progenitor cells of the aorta. In vitro experiments demonstrated that adventitial progenitor cells spontaneously initiated expression of SMC markers and Eln mRNA. RA stimulation enhanced SMC marker expression and elastic fiber synthesis in cultured progenitor cells. Conclusion: RA mediates the differentiation of adventitial progenitor cells into SMC-like cells and de novo elastic fiber synthesis following AD.

ABSTRACT Following aortic dissection (AD), there is a sustained risk of vascular complications, progressive false lumen aneurysm formation, and rupture. However, no effective therapy exists to prevent these complications, highlighting the need to elucidate the pathophysiology following AD. Elastic fibers are crucial for maintaining aortic wall integrity but are thought to have limited regenerative capacity once disrupted during AD. This study defined that elastic fibers were newly generated in the false lumen wall following AD in humans and mice. In human ADs, new elastic fibers were observed in the false lumen wall 6 months after onset. In mice with descending AD induced by β-aminopropionitrile (BAPN), elastin mRNA was markedly upregulated in the chronic phase following AD, accompanied by elastic fiber formation. These fibers coincided with smooth muscle cell (SMC) markers within the false lumen wall. Of note, lineage tracing studies demonstrated that these cells were not derived from resident SMCs but adventitial progenitor cells. In vitro experiments further demonstrated that adventitial progenitor cells produced elastic fibers while expressing SMC markers. Collectively, these findings suggest that adventitial progenitor cells differentiate into elastogenic SMC-like cells, contributing to false lumen remodeling through de novo elastic fiber formation following AD.

BACKGROUND:Mice harboring a missense variant (C1041G) of Fbn1 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:Fbn1+/+ and Fbn1C1041G/+ 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 Fbn1C1041G/+ mice. Aortic dissection was visible within 3 days of AngII infusion. Over 50% of male Fbn1C1041G/+ 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 Fbn1C1041G/+ 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 Fbn1C1041G/+ mice. CONCLUSIONS:AngII exacerbated aortic pathology in Fbn1C1041G/+ mice. It also promoted the development of pathologies at aortic branch points, including the celiac and superior mesenteric arteries.

Background: Aortic dissection (AD) often leads to a false lumen aneurysm as a life-threatening complication during its late phase. Previous preclinical studies of AD have predominantly focused on the mechanism of disease initiation and the acute phase. Therefore, the pathophysiology underlying false lumen aneurysm formation and stability remains poorly understood. Methods and Results: To determine pathological features of false lumen aneurysm, a time course study was performed using AD induced in mice by administration of β-aminopropionitrile (BAPN, 0.5% wt/vol, drinking water). After 4 weeks of BAPN administration, mice developed a false lumen formation in the descending aorta with minimal dilatation, primarily filled with fresh thrombus. However, after 12 weeks of BAPN administration, the false lumen exhibited significant vascular wall thickening and severe aneurysm formation, accompanied by organized thrombus. Interestingly, the thickened vascular wall of the false lumen contained substantial amounts of disorganized elastic fibers. qPCR revealed a significantly increased abundance of elastin mRNA and other elastic fiber-related genes, such as Fbn1 , Fbln5, and Lox , in AD after 12 weeks of BAPN administration compared to mice with normal aortas. These findings indicate that elastic fibers are newly synthesized in response to AD progression. Importantly, mice that succumbed to false lumen rupture during the study did not exhibit evidence of de novo elastic fiber formation, suggesting that these newly synthesized fibers contribute to stabilization of the false lumen wall. Immunostaining revealed that cells of the false lumen wall were composed predominantly of cells expressing smooth muscle cell (SMC) markers, such as MYH11 and αSMA. However, the lineage-tracing study showed that these cells did not originate from resident SMCs in the native aortic wall. Of note, these cells expressed stem cell antigen 1 (Sca1), a marker for progenitor cells. In situ hybridization showed that Sca1 progenitor cells were coincident with newly synthesized elastin mRNA. Conclusion: Following aortic dissection, Sca1 progenitor cells differentiated into SMCs and synthesized de novo elastic fibers in the false lumen wall, which protected against false lumen rupture.

Background: Atherosclerosis is a multicellular pathology involving dynamic interactions of infiltrated and resident cells in the arterial wall. To understand the contribution and phenotypic plasticity of adventitial fibroblasts to atherogenesis, the spatiotemporal dynamics of fibroblasts were determined during progression of atherosclerosis. Methods: First, fibroblast clusters were analyzed in human coronary artery single-cell RNA-sequencing (scRNA-seq) from datasets (GSE131778). Second, atherosclerosis was induced in male C57BL/6J mice by administration of Ldlr antisense oligonucleotides (ASO) in combination with Western diet feeding and angiotensin II infusion for 4 or 12 weeks to generate early and progressive stages of lesions, respectively. These results were compared to data from mice that did not receive Ldlr ASO, Western diet, or angiotensin II. Aortic roots were harvested for integrated scRNA-seq and spatial transcriptomics analyses to map the phenotypic evolution and spatial distribution of fibroblasts. Since decorin (DCN) is highly abundant in fibroblasts, fibroblast clusters were defined based on the presence of DCN and five other major fibroblast markers ( PI16 , LUM, DPP4, LY6A, and LY6C1 ). Results: DCN was highly enriched in fibroblasts present in human coronary arteries, consistent with its use as a fibroblast marker. scRNA-seq of mouse tissue revealed that Dcn + cells increased progressively during atherosclerotic development. The largest expansion of Dcn + fibroblast clusters was characterized by increased mRNA abundance of extracellular matrix genes ( Col1a1 , Fn1 ) but reduced contractile markers ( Myh11 , Cnn1 ) as lesions progressed. Spatial transcriptomics revealed progressive redistribution with migration of adventitia to intima. While Dcn + cells were confined to the adventitia in the non-atherosclerotic state, this cluster infiltrated the medial layers and accumulated within the intima during progression of atherosclerosis, accompanied by a parallel accumulation of Lyz2 + cells in atherosclerotic lesions. Conclusion: Dcn + cells exhibit phenotypic transition and migrate from the adventitia to the intima during progression of atherosclerosis. We will validate these findings using in vivo Dcn -lineage tracing and complementary in vitro systems.

Background: Fibrillin-1 (FBN1) is essential for the structural integrity of the aortic wall. Our recent study revealed that FBN1 deficiency in smooth muscle cells (SMCs) induces aortic aneurysms, predominantly affecting the distal portion of the ascending aorta and aortic arch. This study aimed to determine whether angiotensin II (AngII) augments aortic pathologies in mice with FBN1 deficiency in SMCs. Methods: Male Sm22α -Cre +/0 ; Fbn1 floxed mice were bred with female Sm22α -Cre 0/0 ; Fbn1 floxed mice to generate SMC-FBN1-deficient mice (SMC-FBN1 -/-: Sm22α- Cre +/0 ; Fbn1 fl/fl ) and their wild-type littermates (SMC-FBN1 +/+: Sm22α -Cre 0/0 ; Fbn1 fl/fl ). Vehicle (saline) or AngII (1 or 0.5 µg/kg/min) was infused into 7-8-week-old mice for 28 days. At study termination, micro-computed tomography was used to visualize aortic pathologies and quantify aortic diameters, lengths, and volumes. Both male and female mice were studied. Results: Infusion of AngII at a rate of 1 µg/kg/min resulted in >70% death due to aortic rupture in either the thoracic or abdominal region of SMC-FBN1 -/- mice of both sexes. Infusion at 0.5 µg/kg/min led to mortality rates of 27% in males and 50% in females. In addition to pathologies in the ascending aorta and aortic arch, prominent pathological features were observed in surviving AngII-infused SMC-FBN1 -/- mice, including: (1) increased total aortic length; (2) pronounced pathological changes in the descending and suprarenal aortic regions, with the infrarenal aorta relatively spared; (3) branch aneurysms involving the celiac and superior mesenteric arteries; and (4) cardiac enlargement, including enlarged hearts or left atria, observed in approximately 64% of males and 25% of females. Conclusions: AngII infusion exacerbates aortic pathology in SMC-FBN1 -/- mice, manifested by aortic rupture, increased aortic tortuosity, widespread aortic aneurysms, aneurysms of the celiac and superior mesenteric arteries, and cardiac enlargement. Keywords: smooth muscle cell; FBN1; extracellular matrix; aortopathy; AngII; mouse

Background:The comparative roles of triglyceride-rich lipoproteins (TRLs) and low-density lipoproteins (LDLs) in abdominal aortic aneurysm (AAA) pathogenesis are unclear. Objectives:To evaluate the putative causal role of TRLs in AAA, quantify the relative effect on AAA risk ("aneurysmogenicity") of TRL vs LDL particles, and prioritize lipid-lowering drug targets for AAA prevention and treatment. Methods:We performed summary-level and individual-level Mendelian randomization (MR) analyses. Genetic variants were selected from 383,983 UK Biobank participants and ranked into 10 sets of variants where set 1 predominantly affected LDL cholesterol (LDL-C) and set 10 predominantly affected TRL cholesterol (TRL-C; and with mixed effects for intermediate variant sets). AAA outcome data were obtained from AAAgen (37,214 cases), FinnGen (4,439 cases), and the VA Million Veteran Program (MVP; 23,848 cases). Multivariable MR was used to assess the independent roles of LDL-C and TRL-C in AAA. For each set of variants, MR or logistic regression was used to estimate AAA odds ratios (ORs) per 10 mg/dL higher apolipoprotein B (apoB). Interaction analyses were conducted between a statin-like LDL-C-lowering variant set (set 3) and a TRL-C-lowering variant set (set 10). Drug-target MR was performed to evaluate lipid-lowering targets relevant to LDL-C- and TRL-C-lowering. Results:Genetically predicted LDL-C and TRL-C concentrations were each associated independently with genetic liability for AAA after mutual adjustment, with 3.0 to 5.5 times stronger associations for TRL-C compared to LDL-C on a per-cholesterol basis. In AAAgen, the AAA OR per 10 mg/dL increased apoB concentrations were 1.10 (95% CI, 1.05-1.14) for variant set 1 (LDL-C-predominant) and 1.89 (95% CI, 1.69-2.11) for variant set 10 (TRL-C-predominant). Using the ratio of log(OR) per 10 mg/dL apoB for set 10 versus set 1 as a conservative estimate of relative aneurysmogenicity, TRLs were approximately 3.2 to 6.9 times more aneurysmogenic than LDLs across the three studies. No evidence of interaction was observed between LDLs and TRLs, indicating additive contribution to AAA risk. Drug-target MR supported strong protective associations for genetically proxied inhibition of TRL-pathway targets, particularly APOC3 and LPL, with AAA risk. Conclusions:TRLs are at least threefold more aneurysmogenic than LDLs on a per-particle basis. Therapeutic strategies targeting TRL-C -especially via APOC3 and LPL-should be prioritized for AAA prevention and treatment.

Introduction: Aortic Dissection (AoD) is the disruption in the layers of the aortic wall with bleeding into the medial layers. Aortic Aneurysm (AA) is a dilation of the aorta. One fatal consequence of AoD and AA is the free rupture of the aortic wall. There is no pharmacological or therapeutic agents to decrease the risk of aortic rupture after AoD. Phosphodiesterase inhibitor (PDEi) 3 and 5 have many uses clinically including vasodilatory small and large vessels, as well as anti-platelet activity in peripheral vessels. Two studies suggest protective effects of cilostazol in abdominal aortic aneurysm (AAA), and one study found sildenafil exacerbated AAA. These agents have not been tested directly in a mouse model of thoracic AoD or AA. This is a gap in knowledge. It is unknown if PDEi’s will be efficacious in attenuating AoD or AA. Methods and Results: A lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), was administered to induce AA and AoD in male C57BL/6J mice at 4 weeks of age. We first performed bulk RNA sequencing using ascending and descending aortas harvested from mice with BAPN administration for 1 week, representing the pre-AoD/AA phase. PDE3 and PDE5 mRNA abundance were significantly increased in both ascending and descending aortas. To determine the impact of pharmacological inhibition of PDE3 and PDE5, either cilostazol (a PDE3 inhibitor) or sildenafil (a PDE5 inhibitor) was administered by diet to BAPN-administered mice for 4 weeks. Mice fed a regular diet were used as controls. Mass spectrometry of plasma samples verified the delivery of cilostazol or sildenafil in mice at 4 weeks of BAPN administration. Despite effective drug delivery, neither cilostazol nor sildenafil affected the incidence of aortic rupture in BAPN-administered mice. In addition, AA formation assessed by the in situ imaging approach was not changed by either drug. Extending the observation period to 12 weeks showed consistent findings, with no significant differences among the groups in the survival rate or aortic diameters in BAPN-treated mice. Conclusion: Although there is interest on the clinical effect of PDE 3 an 5 inhibitors on anerysm diesease. There is no protective or exacerbating effect of phosphodiesterase inhibitors cilostazol and sildenafil on BAPN-induced AoD and AA in mice. Further work is needed to gain mechanistic insight.

Cardiac microvascular endothelial cells (CMECs) dysfunction is a well-recognized mediator of heart failure with preserved ejection fraction (HFpEF), but the underlying mechanism remains unclear. Here we find that scavenger receptor class B type I (SR-B1) is predominantly expressed in CMECs and decreased significantly in HFpEF. Endothelial-specific SR-B1 deficiency exacerbates cardiac pathological remodeling and diastolic dysfunction in HFpEF, which can be prevented by endothelial SR-B1 reconstitution through adeno-associated virus serotype 1 (AAV1)-mediated delivery in endothelial-specific SR-B1-deficient mice. Single-cardiac-endothelial-cell transcriptomics and lineage-tracing system reveal that inflammatory CMECs subcluster activation is responsible for the deteriorating HFpEF progression induced by endothelial SR-B1 loss, rather than endothelial-to-mesenchymal transition. Mechanistically, SR-B1 loss drives increased CXCL10 secretion, which orchestrates CMECs activation and CXCR3-positive T-cell cardiotropism to promote diastolic dysfunction-a process associated with endothelial IRF1 activation. Most importantly, the SR-B1-CXCL10-CXCR3 axis is activated in human HFpEF cardiac tissue, and the elevated CXCL10 level in plasma is independently associated with a higher HFpEF prevalence. This study uncovers that activation of the SR-B1-CXCL10-CXCR3 axis in CMECs aggravates HFpEF pathogenesis through the accumulation of CXCR3-positive T-cells in hearts.

Angiotensin II (AngII) exerts a critical role in thoracic aortic aneurysm (TAA) formation via AngII type 1a receptor (AT1aR). However, the principal cell type mediating this process remains unclear. Our previous study demonstrated that S100A4-lineage cells are present in the aortic wall and involved in AngII-induced vascular remodeling. In the present study, we investigated whether S100A4-lineage cells contribute to AngII-mediated TAA formation through AT1aR. Proteomic, bulk RNA sequencing, and single-cell RNA sequencing data were analyzed to assess changes in S100A4 abundance in response to AngII infusion. Lineage tracing was performed to track S100A4-lineage cells during AngII-mediated TAA formation. Either saline or AngII was infused in mice with genetic deletion of AT1aR in S100A4-lineage cells and their wild-type littermates. AngII infusion increased S100A4 protein and mRNA abundance significantly in the ascending aorta, particularly within smooth muscle cells and fibroblasts. Lineage tracing revealed that S100A4-positive cells were localized to the media and adventitia under basal conditions. Following AngII infusion, S100A4-lineage cells expanded markedly throughout the entire aortic wall and comprised a heterogeneous population including smooth muscle cells and fibroblasts. Deletion of AT1aR in S100A4-lineage cells partially reduced AngII-induced TAA formation. In conclusion, S100A4-lineage cells modestly contribute to AngII-mediated TAA development through AT1aR in mice.

ABSTRACT Angiotensinogen (AGT) deletion in hepatocytes reduces Western diet–induced adiposity and hepatic steatosis in mice maintained under conventional room-temperature (RT) housing. Given the high metabolic activity of mice, this temperature imposes adaptive metabolic responses in this species. Whether this metabolic protection persists independent of increased thermogenic demand remains unclear. In this study, we first determined whether thermoneutral housing (TN, 30 °C) alters Western diet–induced metabolic phenotypes compared with RT housing (20 °C) in wild-type mice. Although body weight did not differ significantly between housing conditions, Western diet–fed mice housed at TN exhibited brown adipose tissue whitening and more pronounced hepatic steatosis than mice housed at RT, confirming that thermoneutrality exacerbated diet-induced metabolic dysfunction. We then housed hepatocyte Agt deficient (hepAGT-/-) mice and wild-type (hepAGT+/+) littermates at TN and fed them Western diet for 12 weeks. Despite enhanced metabolic dysfunction under TN, hepatocyte AGT deletion resulted in reductions in diet-induced body weight gain, fat mass, liver weight, and hepatic triglyceride accumulation. Bulk RNA sequencing of liver revealed hepatocyte AGT deficiency–dependent alterations in lipid-metabolic pathways. Cross-temperature analysis of RT and TN housing identified 35 shared differentially expressed genes, including 27 concordantly downregulated genes enriched in lipid metabolism and transport. Extended Western diet feeding for 24 weeks confirmed sustained reductions in body weight gain, liver weight, and hepatic lipid accumulation in hepAGT-/- mice. These findings demonstrate that hepatocyte AGT deletion provides sustained protection against Western diet–induced metabolic dysfunction under thermoneutral housing, a condition that more closely recapitulates human basal metabolism. NEW & NOTEWORTHY This study investigated hepatocyte angiotensinogen (AGT) biology during Western diet feeding in mice under thermoneutral housing, a condition relevant to human metabolism. By minimizing adaptive thermogenesis induced by standard room temperature housing, thermoneutrality more closely recapitulates human basal metabolic conditions. Under this condition, hepatocyte AGT deletion remains protective against adipo and hepatic lipid accumulation, despite exacerbated Western diet-induced metabolic dysfunction in wild-type mice, demonstrating that this protection persists in a human-relevant thermal environment. GRAPHIC ABSTRACT

Background: Hyperhomocysteinemia (Hhcy), defined as plasma homocysteine concentrations >15μM, is an independent risk factor for early-onset atherosclerosis and ischemic strokes. We have previously demonstrated that complex phenotypic modulation of smooth muscle cells (SMCs) during atherogenesis is regulated in part by cholesterol-induced endoplasmic reticulum (ER) stress and PERK signaling. We also determined that activation of cytosolic stress and heat shock factor 1 (HSF1) leading to increased intracellular cholesterol biosynthesis from activation of HMG-CoA reductase (HMGCR) can augment PERK signaling (HSF1→HMGCR→PERK) and phenotypic modulation, which is responsible for early-onset atherosclerosis associated with missense variants in the gene ACTA2 , that codes for SMC-specific α-actin. Here, we investigated whether HSF1→HMGCR→PERK signaling drives augmented atherosclerosis associated with Hhcy. Methods: Wildtype (WT) and SMC-specific Perk -deficient ( Perk SMC-/- ) HC mice were fed a high fat diet for 12 weeks, with and without Hcy, and atherosclerotic plaque formation was characterized using en face aortic Oil Red O staining and histopathology. Single cell transcriptomics (scRNA-seq) of aortic tissue from SMC lineage-traced mice was pursued, while SMCs explanted from ascending aortas of WT and Perk SMC-/- mice were used to investigate atherosclerosis-associated SMC phenotypic modulation. Results: Hcy supplementation increased atherosclerotic plaque burden in WT mice, and both inhibition of HMGCR by pravastatin, and genetic depletion of Perk from SMCs successfully reversed the augmented plaque burden associated with Hhcy. scRNA-seq indicated that Hcy exposure disrupted protein folding, activated HSF1, and augmented PERK-mediated SMC phenotypic modulation. Cellular studies confirmed that Hcy treatment activated HSF1→HMGCR→PERK signaling, and sensitized WT SMCs to undergo atherosclerosis-associated phenotypic modulation at lower concentrations of cholesterol, and this effect was completely reversed in Perk -deficient SMCs. Conclusion: These data establish a novel mechanism by which high Hcy levels in SMCs predispose to early onset atherosclerosis. Targeting components of HSF1→HMGCR→PERK signaling in SMCs, including the use of statins – which is not currently used commonly in Hhcy patients - could prove effective against augmented atherosclerosis in these patients.

Liver steatosis is a common cause of chronic liver disease. To investigate the molecular basis of hepatic steatosis, low-density lipoprotein receptor-deficient (LDLR -/-) mice were fed a Western diet (WD, 42% of calories from fat) for 5, 14, or 42 days and evaluated against mice fed a normal laboratory diet. Histological analyses revealed that steatosis was detected as early as 14 days of WD feeding. Bulk RNA sequencing demonstrated that WD feeding altered liver transcriptomes related to inflammation and cell adhesion consistent with the progression of liver steatosis. Previous studies determined that hepatocyte-specific deficiency of angiotensinogen (AGT), the unique substrate of the renin-angiotensin system (RAS), alleviates WD-induced hepatic steatosis in mice. However, the effects of hepatic AGT deficiency were not mimicked by pharmacological inhibition of the RAS, and the molecular mechanisms by which AGT deficiency protects against WD-induced steatosis is unknown. Therefore, liver transcriptomes were compared between hepatocyte-specific AGT-deficient mice (hepAGT -/-) and their wild-type littermates (hepAGT +/+) after 14 days of WD feeding. Gene ontology analyses showed that upregulated genes in hepAGT -/- mice were enriched for metabolic processes and downregulated genes were enriched for cell division pathways. The integration analysis of the two RNA sequencing data identified 5 key genes, Smpd3, Dtl, Cdc6, Mki67, and Top2a, which were primarily associated with cell division processes in hepAGT +/+ mice and were suppressed in hepAGT -/- mice. In conclusion, hepatic AGT deficiency downregulated genes related to cell division during the progression of liver steatosis.