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Background: Smooth muscle cell (SMC) phenotypic reprogramming toward a mixed synthetic-proteolytic state is a central feature of aortic root aneurysm in Marfan syndrome (MFS). Previous work identified Klf4 as a potential mediator of SMC plasticity in MFS. Methods: MFS ( Fbn1 C1041G/+ ) mouse strains with an inducible vascular SMC fluorescent reporter ( MFS SMC ) with or without SMC-specific deletion of Klf4 exons 2 to 3 ( MFS SMC-Klf4Δ ) were generated. Simultaneous SMC tracing and Klf4 loss-of-function ( Klf4Δ mice) was induced at 6 weeks of age. Aneurysm growth was assessed via serial echocardiography (4–24 weeks). Twenty-four-week-old mice were assessed via histology, RNA in situ hybridization, and aortic single-cell RNA sequencing. Results: MFS mice demonstrated progressive aortic root dilatation compared with control (WT SMC ) mice regardless of Klf4 genotype ( P MFS SMC-Klf4Δ versus MFS SMC ( P =0.884). Efficient SMC Klf4 deletion was confirmed via lineage-stratified genotyping, RNA in situ hybridization, and immunohistochemistry. Single-cell RNA sequencing of traced SMCs revealed a highly similar pattern of phenotype modulation marked by loss of contractile markers (eg, Myh11, Cnn1 ) and heightened expression of matrix genes (eg, Col1a1, Fn1 ) between Klf4 genotypes. Pseudotemporal quantitation of SMC dedifferentiation confirmed that Klf4 deletion did not alter the global extent of phenotype modulation, but reduced expression of 23 genes during this phenotype transition in MFS SMC-Klf4Δ mice, including multiple chondrogenic genes expressed by only the most severely dedifferentiated SMCs (eg, Cytl1, Tnfrsf11b ). Conclusions: Klf4 is not required to initiate SMC phenotype modulation in MFS aneurysm but may exert regulatory control over chondrogenic genes expressed in highly dedifferentiated SMCs.

Background: Aortic root smooth muscle cells (SMC) develop from both the second heart field (SHF) and neural crest. Disparate responses to disease-causing Fbn1 variants by these lineages are proposed to promote focal aortic root aneurysm formation in Marfan syndrome (MFS), but lineage-stratified SMC analysis in vivo is lacking. Methods: We generated SHF lineage-traced MFS mice and performed integrated multiomic (single-cell RNA and assay for transposase-accessible chromatin sequencing) analysis stratified by embryological origin. SMC subtypes were spatially identified via RNA in situ hybridization. Response to TWIST1 overexpression was determined via lentiviral transduction in human aortic SMCs. Results: Lineage stratification enabled nuanced characterization of aortic root cells. We identified heightened SHF-derived SMC heterogeneity including a subset of Tnnt2 (cardiac troponin T)-expressing cells distinguished by altered proteoglycan expression. MFS aneurysm-associated SMC phenotypic modulation was identified in both SHF-traced and nontraced (neural crest–derived) SMCs; however, transcriptomic responses were distinct between lineages. SHF-derived modulated SMCs overexpressed collagen synthetic genes and small leucine-rich proteoglycans while nontraced SMCs activated chondrogenic genes. These modulated SMCs clustered focally in the aneurysmal aortic root at the region of SHF/neural crest lineage overlap. Integrated RNA-assay for transposase-accessible chromatin analysis identified enriched Twist1 and Smad2/3/4 complex binding motifs in SHF-derived modulated SMCs. TWIST1 overexpression promoted collagen and SLRP gene expression in vitro, suggesting TWIST1 may drive SHF-enriched collagen synthesis in MFS aneurysm. Conclusions: SMCs derived from both SHF and neural crest lineages undergo phenotypic modulation in MFS aneurysm but are defined by subtly distinct transcriptional responses. Enhanced TWIST1 transcription factor activity may contribute to enriched collagen synthetic pathways SHF-derived SMCs in MFS.

BACKGROUND: To delineate the effects of integrin αv signaling in Marfan syndrome (MFS) and examine the potential efficacy of integrin αv blockade as a therapeutic strategy for MFS aneurysms. METHODS: Induced pluripotent stem cells were differentiated into aortic smooth muscle cells (SMCs) of the second heart field (SHF) and neural crest lineages, enabling in vitro modeling of thoracic aortic aneurysm in MFS. Fbn1 C1039G/+ MFS mice treated with integrin αv antagonist (GLPG0187) confirmed the pathological role of integrin αv on aneurysm formation. RESULTS: Induced pluripotent stem cell–derived MFS SHF SMCs overexpress integrin αv relative to MFS neural crest and healthy control SHF cells. Furthermore, downstream targets of integrin αv (FAK [focal adhesion kinase]/Akt Thr308 /mTORC1 [mechanistic target of rapamycin complex 1]) were activated, especially in MFS SHF. Treatment GLPG0187 reduced p-FAK/p-Akt Thr308 /mTORC1 activity in MFS SHF back to control SHF levels. Functionally, MFS SHF SMCs had increased proliferation and migration compared to MFS neural crest and control SMCs, which was then inhibited by GLPG0187 treatment. In the Fbn1 C1039G/+ MFS mouse model, integrin αv, p-Akt Thr308 , and downstream targets of mTORC1 proteins were elevated in the aortic root/ascending segment compared to littermate wild-type control. Mice treated with GLPG0187 (age 6–14 weeks) resulted in reduced aneurysm growth, elastin fragmentation, and normalization of the FAK/Akt Thr308 /mTORC1 pathway. GLPG0187 treatment reduced the amount and severity of SMC modulation assessed by single-cell RNA sequencing. CONCLUSIONS: The integrin αv-FAK-Akt Thr308 signaling pathway is activated in induced pluripotent stem cell SMCs from MFS patients, specifically from the SHF lineage. Mechanistically, this signaling pathway promotes SMC proliferation and migration in vitro. As biological proof of concept, GLPG0187 treatment slowed aneurysm growth and p-Akt Thr308 signaling in Fbn1 C1039G/+ mice. Integrin αv blockade via GLPG0187 may be a promising therapeutic approach to inhibit MFS aneurysmal growth.

OBJECTIVE: To evaluate the effect of perioperative allogeneic packed red blood cell (RBC) transfusion during aortic root replacement. METHOD: We reviewed patients undergoing aortic root replacement at our institution between March 2014 and April 2020. In total, 760 patients underwent aortic root replacement, of whom 442 (58%) received a perioperative RBC transfusion. Propensity score matching was used to account for baseline and operative differences resulting in 159 matched pairs. All-cause mortality was assessed with Kaplan–Meier curves. Data were obtained from our institutional Society of Thoracic Surgeons database and chart review. RESULTS: After propensity score matching, the RBC-transfused and -nontransfused groups were similar for all preoperative characteristics. Cardiopulmonary bypass time, crossclamp time, and lowest operative temperature were similar between the transfused and nontransfused groups (standardized mean difference

Objective: To delineate temporal and spatial dynamics of vascular smooth muscle cell (SMC) transcriptomic changes during aortic aneurysm development in Marfan syndrome (MFS). Approach and Results: We performed single-cell RNA sequencing to study aortic root/ascending aneurysm tissue from Fbn1 C1041G/ + (MFS) mice and healthy controls, identifying all aortic cell types. A distinct cluster of transcriptomically modulated SMCs (modSMCs) was identified in adult Fbn1 C1041G/ + mouse aortic aneurysm tissue only. Comparison with atherosclerotic aortic data (ApoE −/− mice) revealed similar patterns of SMC modulation but identified an MFS-specific gene signature, including plasminogen activator inhibitor-1 ( Serpine1 ) and Kruppel-like factor 4 ( Klf4 ). We identified 481 differentially expressed genes between modSMC and SMC subsets; functional annotation highlighted extracellular matrix modulation, collagen synthesis, adhesion, and proliferation. Pseudotime trajectory analysis of Fbn1 C1041G/ + SMC/modSMC transcriptomes identified genes activated differentially throughout the course of phenotype modulation. While modSMCs were not present in young Fbn1 C1041G/ + mouse aortas despite small aortic aneurysm, multiple early modSMCs marker genes were enriched, suggesting activation of phenotype modulation. modSMCs were not found in nondilated adult Fbn1 C1041G/ + descending thoracic aortas. Single-cell RNA sequencing from human MFS aortic root aneurysm tissue confirmed analogous SMC modulation in clinical disease. Enhanced expression of TGF-β (transforming growth factor beta)-responsive genes correlated with SMC modulation in mouse and human data sets. Conclusions: Dynamic SMC phenotype modulation promotes extracellular matrix substrate modulation and aortic aneurysm progression in MFS. We characterize the disease-specific signature of modSMCs and provide temporal, transcriptomic context to the current understanding of the role TGF-β plays in MFS aortopathy. Collectively, single-cell RNA sequencing implicates TGF-β signaling and Klf4 overexpression as potential upstream drivers of SMC modulation.

Objective: To investigate predictors of acute kidney injury (AKI) following open aortic repair (OAR) requiring suprarenal clamping. Methods: The study included 833 nonhemodialysis patients who had undergone elective OAR (with suprarenal clamping, n=73; with infrarenal clamping, n=760). We evaluated AKI as defined by the criteria of the Kidney Disease Improving Global Outcomes (KDIGO) and compared in-hospital outcomes between the two groups. We also investigated the effects of AKI on outcomes, factors related to post-suprarenal clamping AKI, and efficacy of hypothermic renal perfusion (HRP) in the suprarenal clamping group. Results: For the suprarenal vs. infrarenal clamping group, in-hospital mortality was 0% (0/73) vs. 0.5% (4/760). The incidence of AKI was greater in the suprarenal clamping group (37% vs. 15%, P1,000 mL were associated with post-suprarenal clamping AKI. Renal ischemia time was longer with HRP (n=15) than without HRP (n=58) (median, 51 min vs. 33 min; P=0.011), and HRP did not decrease the incidence of AKI (40% vs. 36%; P=0.78). Conclusion: Prolonged renal ischemia and substantial intraoperative bleeding are associated with postoperative AKI following suprarenal clamping.

Background: Vascular smooth muscle cell (SMC) phenotype modulation produces a mixed proteolytic/collagen synthetic state in Marfan syndrome (MFS) aortic root aneurysm. While the transcriptomic changes associated with this process are established, upstream regulators governing this plasticity are poorly characterized. Methods/Results: We performed concurrent single-cell RNA sequencing (scRNAseq) and assay for transposase-accessible chromatin using sequencing (scATACseq) on aortic root aneurysm tissue from adult Fbn1 C1041G/+ (MFS) mice and littermate controls. MFS/control scRNAseq data analysis identified four SMC subtypes including MFS-specific ‘modulated’ cells (modSMCs) which enriched for extracellular matrix organization and collagen synthesis pathway activation. These SMC clusters were projected onto the MFS/control scATAC dataset via integrative label transfer to study dynamic chromatin accessibility during SMC phenotype modulation. We compared DNA accessibility in modSMCs versus mature SMCs, finding 336 enriched and 29 suppressed peaks, suggesting increased open chromatin during modulation. Using chromVAR, we identified 242 enriched transcription factor motifs overrepresented in modSMCs. Motifs representing central but nonspecific transcription factor families including numerous AP-1 (FOS/JUN/ATF) heterodimers and TEAD family members showed highest enrichment, while enriched TWIST1, HAND2, and SMAD2:SMAD3:SMAD4 complex motifs suggested more specific functions. To functionally validate these findings, we examined TWIST1 as a potential regulator of SMC modulation in vitro via lentiviral overexpression in MFS patient-derived aortic SMCs.In SMCs with forced TWIST1 overexpression, we found heightened promoter region DNA accessibility (via bulk ATACseq) and increased mRNA expression (via RT-PCR) for specific modSMC markers (e.g., COL1A1 and LUM confirming TWIST1 promotes collagen synthesis. Conclusions: Integrated single-cell transcriptomic/epigenomic analysis permits identification of critical upstream regulatory signals promoting disease-specific cell phenotype changes. TWIST1 is a potential driver of SMC modulation and a target for therapeutic agent design in MFS aortic aneurysm.

Introduction: Mutation in the extracellular matrix (ECM) protein fibrillin-1 lead to aortic root aneurysm in Marfan Syndrome (MFS). ECM composition influences vascular smooth muscle cell (SMC) phenotype, contractility, and signaling through mechanotransduction. Induced pluripotent stem cell-derived SMCs (iPSC-SMCs) are emerging as an attractive approach to model patient-specific vascular disease, but the use of iPSC-SMCs to investigate extracellular matrix biology remains limited. Hypothesis: Diseased MFS ECM induces phenotype modulation in healthy Control cells, while Control ECM will rescue contractile phenotype in MFS. Methods: Label-free mass spectroscopy core matrisome proteomic profiling of MFS and Control iPSC-SMCs from ammonia hydroxide decellularized ECM was performed. Decellularized ECMs were repopulated with MFS or Control iPSC-SMCs (N=3) (Figure A) and RNA extracted from whole cell lysate for RNA sequencing after four days of culture. Results: 24 core matrisome proteins were differentially expressed including overexpressed Collagen 1A1 and Tenascin C in MFS and enriched Thrombospondin 1 and Latent TGF-β Binding Protein 2 in Control (Figure B). Control iPSC-SMCs dramatically decreased contractile gene expression ( ACTA2 and CNN1 ) and ECM synthesis ( COL1A1 and FN1 ) when cultured on the diseased MFS ECM compared to the native Control ECM (Figure C). Conversely, the MFS cells expressed equally low contractile genes and the synthetic phenotype was exacerbated when cultured on the Control ECM (Figure D). Conclusions: Control iPSC-SMC mechanotransduction remains intact when interacting with the MFS ECM resulting in phenotype modulation into a MFS-like cell. Irreversible reprogramming of the MFS iPSC-SMC prevents rescue of the contractile phenotype when grown on a healthy ECM, despite the absence of MFS specific matrisome proteins. The decellularized iPSC-SMC ECM system effectively captures cell-ECM interactions and signaling.

Introduction: Vascular smooth muscle cells (SMC) undergo phenotype modulation (silenced contractile genes and activated proteolytic/synthetic genes) in Marfan syndrome (MFS) aortic aneurysm. Aortic root SMCs develop from the second heart field (SHF) proximally and neural crest (NC) distally. We hypothesize that both developmental lineage and hemodynamic factors dictate SMC vulnerability to phenotype change, promoting focal aortic root aneurysm. Methods/Results: Aortic roots from adult MFS and littermate control mice (n=4 each sex) with a knockin fluorescent SHF tracing construct ( Nkx2-5 cre ) were sorted for lineage-resolved single cell RNA sequencing (scRNAseq). Modulated SMCs (modSMC) were enriched in MFS vs. controls (18.1% of all SMCs vs. 2.2%) and originated from both SHF (15.5%) and NC (20.2%) lineages, ruling out complete lineage specificity. NC-derived modSMCs expressed more aggrecan ( Acan ) and sclerostin ( Sost ) while small proteoglycans decorin ( Dcn ) and lumican ( Lum ) were enriched in SHF modSMCs. Insulin-like growth factor binding protein-2 ( Igfbp2 ) was a lineage-independent modSMC marker. RNA in situ hybridization localized Igfbp2 + SMCs within SHF/NC overlap in the aortic root. To study regional SMC propensity for modulation due to hemodynamic stresses, we generated aortic pressure overload via 27-gauge transverse aortic arch constriction (TAC) or sham surgery on healthy Myh11 cre / Rosa tdT (SMC-traced) mice. TAC induced mild root dilation vs . sham (1.93±0.07 mm vs. 1.69±0.09 mm, n=4, p Conclusions: Embryologic origin does not dictate aortic SMC propensity for pathologic phenotype change in MFS but does promote distinct ECM proteoglycan gene activation. Aortic pressure overload incites root SMC modulation, suggesting hemodynamic effects may promote regional susceptibility to SMC-mediated aortic remodeling.

Introduction: Marfan syndrome (MFS) patients classically develop focal aortic root aneurysms. Aortic root smooth muscle cells (SMCs) develop predominately from second heart field (SHF), whereas ascending aorta/arch SMCs originate from cardiac neural crest (CNC). We previously demonstrated induced pluripotent stem cell (iPSC)-derived SHF SMCs overexpress integrin αV (ITGAV). Therefore, we hypothesize that ITGAV promotes SHF SMC proliferation, contributing to thoracic aortic root aneurysm development. Methods: iPSCs from MFS patients (n=2) and healthy control (n=1) were differentiated into SHF and CNC SMCs. ITGAV-pFAK-pAkt Thr308 levels were detected with western blot and ELISA at baseline and following recombinant TGF-β (2ng/ml) and/or an ITGAV antagonist (GLPG0187, 10nM) for 24 hours. Cell proliferation was measured with quantitative BrdU assay. Results: Baseline ITGAV-pFAK-pAkt Thr308 activation were significantly elevated in MFS SHF lines (n=5 experimental replicates) compared to MFS CNC (n=5) or control SHF (n=4, pThr308 levels in MFS SHF. Treatment with GLPG0187 reduced pFAK-pAkt Thr308 in MFS SHF and CNC, confirming ITGAV-mediated signaling promotes pathologic FAK- Akt Thr308 activation. SMCs from MFS SHF (n=6) had increased proliferation compared to other SMCs (p Conclusions: The integrin-FAK-Akt Thr308 signaling pathway is activated in iPSC SMCs from MFS patients specifically from the SHF lineage, which is further exacerbated by TGF-β. Mechanistically, this pathway promotes SMC proliferation in vitro . ITGAV blockade via GLPG0187 may be a promising therapeutic approach for MFS aneurysmal growth.

Introduction: Mutations in TGF-beta (TGF-ß) signaling genes lead to aortic root aneurysm in Loeys Dietz syndrome (LDS). Smooth muscle cells (SMCs) in the proximal aorta develop from two embryologic origins: second heart field (SHF) and neural crest (NC). Induced pluripotent stem cell (iPSC) models simulate these lineages, but direct correlation to clinical disease is lacking. Hypothesis: iPSC-derived SMCs accurately model lineage-specific aortopathy in LDS. Methods: We generated SMC lines from root and ascending aortic surgical tissue and iPSC-derived SMCs through SHF and NC-specific pathways from an LDS patient ( TGFBR1 mutation). Lineage-specific TGF-ß responses were determined by western blot/ELISA. RNA sequencing and RT-PCR identified SMC transcriptomes. Results: Aortic root SMCs showed greater canonical TGF-ß activation (p-SMAD2/3) versus ascending at baseline and with TGF-ß stimulation ( Figure ). Synonymous results were seen in SHF versus NC SMCs from the iPSC pathway. RNAseq identified 1,600 differentially expressed genes between iPSC lineages, including altered TGF-ß receptor and ligand expression profiles. Primary aortic lines validated iPSC data: root SMCs showed enriched TGF-ß receptor 1/2/3 expression (1.7-, 3.9- and 5.9-fold) while ascending SMCs overexpressed TGFB1 and TGFB2 ligands (1.8- and 3.5-fold). Despite discordant TGF-ß activation, SMC contractile gene expression was similar between lineages in aortic and iPSC-SMCs, suggesting alternative downstream effects in LDS aneurysm. Conclusion: iPSC-derived SMCs effectively model lineage-specific aortic root aneurysm pathology, validating this model as a tool for mechanistic testing and therapy discovery.

Background Male patients with Marfan syndrome have a higher risk of aortic events and root dilatation compared with females. The role androgens play during Marfan syndrome aneurysm development in males remains unknown. We hypothesized that androgens potentiate transforming growth factor beta induced Erk (extracellular‐signal‐regulated kinase)/Smad activation, contributing to aneurysm progression in males. Methods and Results Aortic diameters in Fbn1 C1039G/+ and littermate wild‐type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1 C1039G/+ males were treated with (1) flutamide (androgen receptor blocker) or (2) vehicle control from age 6 to 16 weeks and then euthanized. p‐Erk1/2, p‐Smad2, and matrix metalloproteinase (MMP) activity were measured in ascending/aortic root and descending aorta specimens. Fbn1 C1039G/+ male and female ascending/aortic root‐derived smooth muscle cells were utilized in vitro to measure Erk/Smad activation and MMP‐2 activity following dihydrotestosterone, flutamide or transforming growth factor beta 1 treatment. Fbn1 C1039G/+ males have increased aneurysm growth. p‐Erk1/2 and p‐Smad2 were elevated in ascending/aortic root specimens at age 16 weeks. Corresponding with enhanced Erk/Smad signaling, MMP‐2 activity was higher in Fbn1 C1039G/+ males. In vitro smooth muscle cell studies revealed that dihydrotestosterone potentiates transforming growth factor beta‐induced Erk/Smad activation and MMP‐2 activity, which is reversed by flutamide treatment. Finally, in vivo flutamide treatment reduced aneurysm growth via p‐Erk1/2 and p‐Smad2 reduction in Fbn1 C1039G/+ males. Conclusions Fbn1 C1039G/+ males have enhanced aneurysm growth compared with females associated with enhanced p‐Erk1/2 and p‐Smad2 activation. Mechanistically, in vitro smooth muscle cell studies suggested that dihydrotestosterone potentiates transforming growth factor beta induced Erk/Smad activation. As biological proof of concept, flutamide treatment attenuated aneurysm growth and p‐Erk1/2 and p‐Smad2 signaling in Fbn1 C1039G/+ males.

A 66-year-old woman with primary antiphospholipid antibody syndrome (APS) was admitted due to severe dyspnea. Eight months prior to admission, she underwent bioprosthetic mitral valve replacement for mitral valve stenosis and regurgitation. Transthoracic echocardiogram showed thickening bioprosthetic valve leaflets and severe valve stenosis. Emergency reoperation for artificial valve failure was performed. The explanted bioprosthetic valve showed massive thrombus formation. After the operation, she started strict anticoagulant and antiplatelet therapies and was discharged without recurrence of valve thrombosis.

Surgical treatment for distal arch aortic aneurysm is generally invasive, and there is no definitive approach for it. We report 2 cases of men who was admitted for the treatment of aortic aneurysm. First case is a 78-year-old man. Two saccular aneurysm were observed on distal aortic arch and descending aorta by contrast-enhanced computed tomography. Two staged-repair was performed with using the Lupiae vascular graft and thoracic endovascular aortic repair(TEVAR). The postoperative course was uneventful, and he was discharged on day 21 after 1st operation. Second case is a 68-yearold man. Dessecting aneurysm was observed on distal aortic arch and descending aorta 3 months after incidence of type B aortic dissection. Two staged-repair was performed with using the Lupiae vascular graft and TEVAR. However, additional TEVAR was performed for enlargement of descending aorta half a year after 1st operation. Two staged-repair using Lupiae vascular graft and TEVAR was useful for alleviating a burden of a patient and avoiding paraplegia.

A 49-year-old female was admitted to our hospital with a history of fever for 2 weeks and consciousness disorder.Transthoracic echocardiography demonstrated aortic regurgitation with a mobile fibrous band adhering to the right cusp. Infective endocarditis was diagnosed by positive blood culture and echocardiographic findings. Emergent aortic valve surgery was performed because of uncontrollable infection. A destroyed aortic annulus and subvalvular abscess was found during the operation. Removal of abscess tissue and annuloplasty with self-pericardium were successfully performed.

Mesenteric leiomyosarcoma is a rare disease with poor prognosis. Previously, mesenteric leiomyosarcoma was not differentiated from gastrointestinal stromal tumor (GIST), which is the most common mesenchymal tumor of the gastrointestinal tract, and several cases of GIST may have been misclassified as mesenteric leiomyosarcoma. Thus, the actual clinicopathological characteristics of mesenteric leiomyosarcomas remain unclear. We herein describe a case of leiomyosarcoma arising in the descending mesocolon in a patient who developed metachronous liver metastasis. A 76-year-old woman reported a mass in her left upper abdomen. Computed tomography imaging revealed a low-density tumor adjacent to the descending colon. The patient underwent surgery and the tumor was resected along with part of the descending colon. Immunohistochemical differential diagnosis revealed that the tumor was positive for smooth muscle actin and desmin, and negative for CD117 (c-KIT) and S-100, which are characteristic of gastrointestinal leiomyosarcoma. A single liver metastasis developed 24 months after the operation. The patient underwent curative resection of the metastatic lesion. Sixteen months following surgery for the liver metastasis and 40 months after the initial removal of the primary lesion, the patient remains disease-free. The prognosis of leiomyosarcoma remains poor and standardized chemotherapy for this rare disease has not yet been established. Early diagnosis and surgical removal of the tumor is the only potentially curative option for liver metastasis of mesenteric leiomyosarcoma.