Smooth Muscle Cell Klf4 Expression Is Not Required for Phenotype Modulation or Aneurysm Formation in Marfan Syndrome Mice-Brief Report.

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 (Fbn1C1041G/+) mouse strains with an inducible vascular SMC fluorescent reporter (MFSSMC) with or without SMC-specific deletion of Klf4 exons 2 to 3 (MFSSMC-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 (WTSMC) mice regardless of Klf4 genotype (P<0.001), but there was no difference in aneurysm growth in MFSSMC-Klf4Δ versus MFSSMC (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 MFSSMC-Klf4Δmice, 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.

Lineage-Specific Induced Pluripotent Stem Cell-Derived Smooth Muscle Cell Modeling Predicts Integrin Alpha-V Antagonism Reduces Aortic Root Aneurysm Formation in Marfan Syndrome Mice.

BACKGROUND: The role of increased smooth muscle cell (SMC) integrin αv signaling in Marfan syndrome (MFS) aortic aneurysm remains unclear. Herein, we examine the mechanism and potential efficacy of integrin αv blockade as a therapeutic strategy to reduce aneurysm progression in MFS. METHODS: Induced pluripotent stem cells (iPSCs) were differentiated into aortic SMCs of the second heart field (SHF) and neural crest (NC) lineages, enabling in vitro modeling of MFS thoracic aortic aneurysms. The pathological role of integrin αv during aneurysm formation was confirmed by blockade of integrin αv with GLPG0187 in Fbn1C1039G/+ MFS mice. RESULTS: iPSC-derived MFS SHF SMCs overexpress integrin αv relative to MFS NC and healthy control SHF cells. Furthermore, integrin αv downstream targets (FAK [focal adhesion kinase]/AktThr308/mTORC1 [mechanistic target of rapamycin complex 1]) were activated, especially in MFS SHF. Treatment of MFS SHF SMCs with GLPG0187 reduced p-FAK/p-AktThr308/mTORC1 activity back to control SHF levels. Functionally, MFS SHF SMCs had increased proliferation and migration compared to MFS NC SMCs and control SMCs, which normalized with GLPG0187 treatment. In the Fbn1C1039G/+ MFS mouse model, integrin αv, p-AktThr308, 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) had reduced aneurysm growth, elastin fragmentation, and reduction of the FAK/AktThr308/mTORC1 pathway. GLPG0187 treatment reduced the amount and severity of SMC modulation assessed by single-cell RNA sequencing. CONCLUSIONS: The integrin αv-FAK-AktThr308 signaling pathway is activated in iPSC 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-AktThr308 signaling in Fbn1C1039G/+ mice. Integrin αv blockade via GLPG0187 may be a promising therapeutic approach to inhibit MFS aneurysmal growth.

Embryologic Origin Influences Smooth Muscle Cell Phenotypic Modulation Signatures in Murine Marfan Syndrome Aortic Aneurysm.

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.

Single-Cell Transcriptomic Profiling of Vascular Smooth Muscle Cell Phenotype Modulation in Marfan Syndrome Aortic Aneurysm.

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 Fbn1C1041G/+ (MFS) mice and healthy controls, identifying all aortic cell types. A distinct cluster of transcriptomically modulated SMCs (modSMCs) was identified in adult Fbn1C1041G/+ 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 Fbn1C1041G/+ SMC/modSMC transcriptomes identified genes activated differentially throughout the course of phenotype modulation. While modSMCs were not present in young Fbn1C1041G/+ 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 Fbn1C1041G/+ 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-ß (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-ß plays in MFS aortopathy. Collectively, single-cell RNA sequencing implicates TGF-ß signaling and Klf4 overexpression as potential upstream drivers of SMC modulation.

[Bioprosthetic Mitral Valve Thrombosis in Patient with Antiphospholipid Antibody Syndrome;Report of a Case].

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.

[Hybrid Repair of Distal Arch Aortic Aneurysm:Total Aortic Arch Repair with the Lupiae Vascular Graft and Thoracic Endovascular Aortic Repair;Report of Two Cases].

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.

[Successful Surgical Repair of the Aortic Annular Infective Endocarditis with Subvalvular Abscess;Report of a Case].

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.

A Case of Popliteal Venous Aneurysm Diagnosed after Sudden Cardiac Arrest.

A popliteal venous aneurysm (PVA) is a rare vascular disorder. We report a case of PVA discovered through further evaluation of sudden cardiac arrest (CA) caused by a pulmonary embolism (PE). It is well-known that PVA causes PE; however, there are few reports of PVA causing CA. A tangential aneurysmectomy and lateral venorrhaphy were performed. The patient's postoperative course was uneventful. When contrast-enhanced computed tomography is performed to search for the cause of CA, PVA should be considered and thus, screening below the knee is recommended. At 1-year follow-up, there were no complications.

Outcomes of Reoperative Aortic Root Replacement After Previous Acute Type A Dissection Repair.

Limited aortic root repair for acute type A dissection is associated with greater risk of proximal reoperations compared to full aortic root replacement. Surgical outcomes for patients undergoing reoperative root replacement after previous dissection repair are unknown. This study seeks to determine outcomes for these patients to further inform the debate surrounding optimal upfront management of the aortic root in acute dissection. Retrospective record review of all patients who underwent full aortic root replacement after a previous type A dissection repair operation at a tertiary academic referral center from 2004-2020 was performed. Among 57 cases of reoperative root replacement after type A repair, 35 cases included concomitant aortic arch replacements, and 21 cases involved coronary reconstruction (unilateral or bilateral modified Cabrol grafts). There were 3 acute postoperative strokes and 4 operative mortalities (composite 30-day and in-hospital deaths, 7.0%). Mid-term outcomes were equivalent for patients who required arch replacement compared to isolated proximal repairs (81.8% vs 80.6% estimated 5-year survival, median follow-up 5.53 years. Reoperative root replacement after index type A dissection repairs, including those with concomitant aortic arch replacement and/or coronary reconstruction is achievable with acceptable outcomes at an experienced aortic center.

Outcomes after concomitant arch replacement at the time of aortic root surgery.

Background: Contemporary series of aortic arch replacement at the time of aortic root surgery are limited in number of patients and mostly address hemiarch replacement. We describe outcomes after aortic root and concomitant arch replacement, including total arch replacement. Methods: This single-institution retrospective review studied 1196 consecutive patients from May 2004 to September 2020 who underwent first-time aortic root replacement. Patients undergoing surgery for endocarditis were excluded (n = 68, 5.7%). Patients undergoing concomitant root and arch replacement were propensity matched with patients undergoing isolated root surgery based on indication, clinical and operative characteristics, demographics, medical history including connective tissue disorders, and urgency. Multivariable Cox proportional hazards and logistic regression modeling were used to assess the primary outcome of all-cause mortality and the secondary outcomes of prolonged ventilator use, postoperative blood transfusion, and debilitating stroke, adjusted for patient and operative characteristics. Results: Among the 1128 patients who underwent aortic root intervention during the study period, 471 (41.8%) underwent concomitant aortic arch replacement. Most underwent hemiarch replacement (n = 411, 87.4%); 59 patients (12.6%) underwent total arch replacement (with elephant trunk: n = 23, 4.9%; without elephant trunk: n = 36, 7.7%). The mean follow-up time was 4.6 years postprocedure. Operative mortality was 2.2%, and total mortality over the entire study period was 9.2%. Propensity matching generated 348 matches (295 concomitant hemiarch, 53 concomitant total arch). Concomitant hemiarch (hazard ratio, 1.00; 95% confidence interval, 0.54-1.86, P = .99) and total arch replacement (hazard ratio, 1.60, 95% confidence interval, 0.72-3.57, P = .24) were not significantly associated with increased mortality. Rates of stroke were not significantly different among each group: isolated root (n = 11/348, 3.7%), root + hemiarch (n = 17/295, 5.8%), and root + total arch (n = 3/53, 5.7%) replacement (P = .50), nor was the adjusted risk of stroke. Both concomitant arch interventions were associated with prolonged ventilator use and use of postoperative blood transfusions. Conclusions: Hemiarch and total arch replacement are safe to perform at the time of aortic root intervention, with no significant differences in survival or stroke rates, but increased ventilator and blood product use.

Early clinical outcomes and molecular smooth muscle cell phenotyping using a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome.

OBJECTIVES: Patients with Loeys-Dietz syndrome demonstrate a heightened risk of distal thoracic aortic events after valve-sparing aortic root replacement. This study assesses the clinical risks and hemodynamic consequences of a prophylactic aortic arch replacement strategy in Loeys-Dietz syndrome and characterizes smooth muscle cell phenotype in Loeys-Dietz syndrome aneurysmal and normal-sized downstream aorta. METHODS: Patients with genetically confirmed Loeys-Dietz syndrome (n = 8) underwent prophylactic aortic arch replacement during valve-sparing aortic root replacement. Four-dimensional flow magnetic resonance imaging studies were performed in 4 patients with Loeys-Dietz syndrome (valve-sparing aortic root replacement + arch) and compared with patients with contemporary Marfan syndrome (valve-sparing aortic root replacement only, n = 5) and control patients (without aortopathy, n = 5). Aortic tissues from 4 patients with Loeys-Dietz syndrome and 2 organ donors were processed for anatomically segmented single-cell RNA sequencing and histologic assessment. RESULTS: Patients with Loeys-Dietz syndrome valve-sparing aortic root replacement + arch had no deaths, major morbidity, or aortic events in a median of 2 years follow-up. Four-dimensional magnetic resonance imaging demonstrated altered flow parameters in patients with postoperative aortopathy relative to controls, but no clear deleterious changes due to arch replacement. Integrated analysis of aortic single-cell RNA sequencing data (>49,000 cells) identified a continuum of abnormal smooth muscle cell phenotypic modulation in Loeys-Dietz syndrome defined by reduced contractility and enriched extracellular matrix synthesis, adhesion receptors, and transforming growth factor-beta signaling. These modulated smooth muscle cells populated the Loeys-Dietz syndrome tunica media with gradually reduced density from the overtly aneurysmal root to the nondilated arch. CONCLUSIONS: Patients with Loeys-Dietz syndrome demonstrated excellent surgical outcomes without overt downstream flow or shear stress disturbances after concomitant valve-sparing aortic root replacement + arch operations. Abnormal smooth muscle cell-mediated aortic remodeling occurs within the normal diameter, clinically at-risk Loeys-Dietz syndrome arch segment. These initial clinical and pathophysiologic findings support concomitant arch replacement in Loeys-Dietz syndrome.

Blood transfusion in aortic root surgery impairs midterm survival.

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 <0.05). RBC transfusion was associated with more frequent postoperative ventilation greater than 24 hours (36/159 [23%] vs 19/159 [12%]; P = .01), postoperative hemodialysis (9/159 [5.7%] vs 0/159 [0%]; P = .003), reoperation for mediastinal hemorrhage (9/159 [5.7%] vs 0/159 [0%]; P = .003), and longer intensive care unit and hospital length of stay (3 vs 2 days and 8 vs 6 days respectively; P < .001). Thirty-day operative mortality after propensity score matching was similar between the cohorts (1.9%; 3/159 vs 0%; P = .2), and 5-year survival was reduced in the RBC transfusion cohort (90.2% [95% confidence interval, 84.1%-96.7%] vs 97.1% [95% confidence interval, 92.3%-100%] P = .035). Conclusions: Aortic root replacement frequently requires RBC transfusion during and after the operation, but even after matching for observed preoperative and operative characteristics, RBC transfusion is associated with more frequent postoperative complications and reduced midterm survival.

Acute Kidney Injury Following Elective Open Aortic Repair with Suprarenal Clamping.

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%, P<0.001), and the hospital stay for patients with AKI was longer than for those patients without AKI (median, 21 days vs. 16 days; P=0.005). Renal ischemia time and bleeding volume >1,000 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.

Androgens Accentuate TGF-ß Dependent Erk/Smad Activation During Thoracic Aortic Aneurysm Formation in Marfan Syndrome Male Mice.

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 Fbn1C1039G/+ and littermate wild-type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1C1039G/+ 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. Fbn1C1039G/+ 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. Fbn1C1039G/+ 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 Fbn1C1039G/+ 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 Fbn1C1039G/+ males. Conclusions Fbn1C1039G/+ 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 Fbn1C1039G/+ males.

Curative resection for leiomyosarcoma of the descending mesocolon with metachronous liver metastasis: A case report and literature review.

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.