Comprehensive Integration of Multiple Single-Cell Transcriptomic Data Sets Defines Distinct Cell Populations and Their Phenotypic Changes in Murine Atherosclerosis.

BACKGROUND: The application of single-cell transcriptomic (single-cell RNA sequencing) analysis to the study of atherosclerosis has provided unique insights into the molecular and genetic mechanisms that mediate disease risk and pathophysiology. However, nonstandardized methodologies and relatively high costs associated with the technique have limited the size and replication of existing data sets and created disparate or contradictory findings that have fostered misunderstanding and controversy. METHODS: To address these uncertainties, we have performed a conservative integration of multiple published single-cell RNA sequencing data sets into a single meta-analysis, performed extended analysis of native resident vascular cells, and used in situ hybridization to map the disease anatomic location of the identified cluster cells. To investigate the transdifferentiation of smooth muscle cells to macrophage phenotype, we have developed a classifying algorithm based on the quantification of reporter transgene expression. RESULTS: The reporter gene expression tool indicates that within the experimental limits of the examined studies, transdifferentiation of smooth muscle cell to the macrophage lineage is extremely rare. Validated transition smooth muscle cell phenotypes were defined by clustering, and the location of these cells was mapped to lesion anatomy with in situ hybridization. We have also characterized 5 endothelial cell phenotypes and linked these cellular species to different vascular structures and functions. Finally, we have identified a transcriptomically unique cellular phenotype that constitutes the aortic valve. CONCLUSIONS: Taken together, these analyses resolve a number of outstanding issues related to differing results reported with vascular disease single-cell RNA sequencing studies, and significantly extend our understanding of the role of resident vascular cells in anatomy and disease.

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.

Divergent effects of canonical and non-canonical TGF-ß signalling on mixed contractile-synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms.

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF-ß driven via Smad (canonical) and ERK (non-canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF-ß-driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF-ß-dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF-ß-dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a 'mixed' contractile-synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.

Acute Type A Dissection Causing Impending Rupture of Abdominal Aortic Aneurysm Previously Treated with EVAR.

This report describes the rapid expansion of a previously excluded abdominal aortic aneurysm (AAA) following type A aortic dissection repair in a 74-year-old male. Following successful Hemiarch replacement, CT angiography (CTA) showed residual dissection throughout the thoracoabdominal aorta, which had created a proximal endoleak at the prior endovascular stent graft resulting in the rapid growth of the residual AAA sac. Urgent thoracic endovascular aortic repair (TEVAR) did not fully obliterate false lumen flow allowing further unstable expansion of the AAA and abdominal pain. This was ultimately managed with an open replacement of the infrarenal neck with a Dacron interposition graft sewn to the prior EVAR. Postoperative CTA showed resolution of the false lumen communication to the infrarenal AAA and no further endoleak. Open interposition AAA neck replacement is a possible treatment for new-onset endoleak in patients with aortic dissection following prior infrarenal EVAR.

Anatomically specific reactive oxygen species production participates in Marfan syndrome aneurysm formation.

Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-ß antibody, angiotensin II (AngII), anti-TGF-ß antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-ß dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-ß dependent.

Enhanced caspase activity contributes to aortic wall remodeling and early aneurysm development in a murine model of Marfan syndrome.

OBJECTIVE: Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome. APPROACH AND RESULTS: Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs. CONCLUSIONS: Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.

Subhypnotic Intravenous Ketamine Improves Patient Satisfaction With Burn Wound Care: A Quality Improvement Project.

Despite advancements in pain management for burn injuries, analgesia often fails to meet our patients' needs. We hypothesized that low doses of intravenous (IV) ketamine as an adjunct to our current protocol would be safe, improving both nurse and patient satisfaction with analgesia during hydrotherapy. Burn patients admitted who underwent hydrotherapy from June 1, 2021, to June 30, 2023 were surveyed. Ketamine was administered with the standard opioid-midazolam regimen. Demographics, oral morphine equivalents, midazolam, ketamine doses and time of administration, and adverse events were collected. Patient and nurse satisfaction scores were collected. The ketamine and no-ketamine groups were compared. P < .05 was considered significant. Eighty-five hydrotherapies were surveyed, 47 without ketamine, and 38 with ketamine. Demographics, comorbidities, %TBSA, and hospital length of stay were not different. The median amount of ketamine given was 0.79 mg/kg [0.59-1.06]. Patients who received ketamine were more likely to receive midazolam (100% vs 61.7%; P < .001), and both oral and IV opioids (94.7% vs 68.1%; P = .002) prior to hydrotherapy and less likely to receive rescue opioids or midazolam during hydrotherapy. Two patients in the ketamine group had hypertension (defined as SBP > 180) that did not require treatment. Nurses tended to be more satisfied with patient pain control when ketamine was used (10 [8-10] vs 9 [7-10], P = .072). Patient satisfaction was higher in the ketamine group (10 [8.8-10] vs 9 [7-10], P = .006). Utilizing subhypnotic dose of IV ketamine for hydrotherapy is safe and associated with increased patient satisfaction.

Optimizing burn wound procedural pain control, efficiency, and satisfaction through integrated nurse and physician education.

Herein, we report the results of a quality improvement project (QI). Following a review of the burn unit practices, a nursing-led, physician supported educational intervention regarding optimal timing, dosage, and indication for medications used during hydrotherapy, including midazolam and opioids, was implemented. We hypothesized that such intervention would support improvement in both nurse and patient satisfaction with pain control management. Patients undergoing hydrotherapy were surveyed. Demographics, opioid dose prescribed (oral morphine equivalents), midazolam use, timing of administration, and adverse events were collected. Patient pain scores (1-10) before and after hydrotherapy and patient and nurse satisfaction scores (1-10) after hydrotherapy were collected. The pre- and post-education populations were compared. P < 0.05 was considered significant. Post-education, administration of opioids (59.1% v. 0%, p < 0.001) and midazolam (59.1% vs. 10.4%; p < 0.001) prior to hydrotherapy significantly improved, leading to fewer patients requiring rescue opioids during hydrotherapy (25% vs. 74%, p < 0.001). Hydrotherapy duration significantly decreased post-education (19 [13.3-30] min vs. 32 [18-43] min, p = 0.003). Nurses' ratings of their patient's pain control (9 [7.3-10] vs. 7.5 [6-9], p = 0.004) and ease of procedure (10 [9,10] vs. 9 [7.8-10], p = 0.037) significantly improved. Patients' pain management satisfaction rating did not change, but the number of subjects rating their pain management as excellent tended to increase (36.4% vs. 20%, p = 0.077). Nursing led, physician supported, education can improve medication administration prior to and during hydrotherapy, increasing the ease of the procedure as well as staff satisfaction.

Intrinsic Gata4 expression sensitizes the aortic root to dilation in a Loeys-Dietz syndrome mouse model.

Loeys-Dietz syndrome (LDS) is an aneurysm disorder caused by mutations that decrease transforming growth factor-ß (TGF-ß) signaling. Although aneurysms develop throughout the arterial tree, the aortic root is a site of heightened risk. To identify molecular determinants of this vulnerability, we investigated the heterogeneity of vascular smooth muscle cells (VSMCs) in the aorta of Tgfbr1 M318R/+ LDS mice by single cell and spatial transcriptomics. Reduced expression of components of the extracellular matrix-receptor apparatus and upregulation of stress and inflammatory pathways were observed in all LDS VSMCs. However, regardless of genotype, a subset of Gata4-expressing VSMCs predominantly located in the aortic root intrinsically displayed a less differentiated, proinflammatory profile. A similar population was also identified among aortic VSMCs in a human scRNAseq dataset. Postnatal VSMC-specific Gata4 deletion reduced aortic root dilation in LDS mice, suggesting that this factor sensitizes the aortic root to the effects of impaired TGF-ß signaling.

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.

Nuclear Smooth Muscle α-actin in Vascular Smooth Muscle Cell Differentiation.

Missense variants throughout ACTA2, encoding smooth muscle α-actin (αSMA), predispose to adult onset thoracic aortic disease, but variants disrupting arginine 179 (R179) lead to Smooth Muscle Dysfunction Syndrome (SMDS) characterized by childhood-onset diverse vascular diseases. Our data indicate that αSMA localizes to the nucleus in wildtype (WT) smooth muscle cells (SMCs), enriches in the nucleus with SMC differentiation, and associates with chromatin remodeling complexes and SMC contractile gene promotors, and the ACTA2 p.R179 variant decreases nuclear localization of αSMA. SMCs explanted from a SMC-specific conditional knockin mouse model, Acta2SMC-R179/+, are less differentiated than WT SMCs, both in vitro and in vivo, and have global changes in chromatin accessibility. Induced pluripotent stem cells from patients with ACTA2 p.R179 variants fail to fully differentiate from neural crest cells to SMCs, and single cell transcriptomic analyses of an ACTA2 p.R179H patient's aortic tissue shows increased SMC plasticity. Thus, nuclear αSMA participates in SMC differentiation and loss of this nuclear activity occurs with ACTA2 p.R179 pathogenic variants.

Nuclear Smooth Muscle α-actin Participates in Vascular Smooth Muscle Cell Differentiation.

Missense variants throughout ACTA2, encoding smooth muscle α-actin (αSMA), predispose to adult-onset thoracic aortic disease, but variants disrupting arginine 179 (R179) lead to Smooth Muscle Dysfunction Syndrome (SMDS) characterized by diverse childhood-onset vascular diseases. Here we show that αSMA localizes to the nucleus in wildtype (WT) smooth muscle cells (SMCs), enriches in the nucleus with SMC differentiation, and associates with chromatin remodeling complexes and SMC contractile gene promotors. The ACTA2 p.R179 αSMA variant shows decreased nuclear localization. Primary SMCs from Acta2 SMC-R179C/+ mice are less differentiated than WT SMCs in vitro and in vivo and have global changes in chromatin accessibility. Induced pluripotent stem cells from patients with ACTA2 p.R179 variants fail to fully differentiate from neuroectodermal progenitor cells to SMCs, and single-cell transcriptomic analyses of an ACTA2 p.R179H patient's aortic tissue show increased SMC plasticity. Thus, nuclear αSMA participates in SMC differentiation, and loss of this nuclear activity occurs with ACTA2 p.R179 pathogenic variants.

Augmenting Mitochondrial Respiration in Immature Smooth Muscle Cells with an ACTA2 Pathogenic Variant Mitigates Moyamoya-like Cerebrovascular Disease.

ACTA2 pathogenic variants altering arginine 179 cause childhood-onset strokes due to moyamoya disease (MMD)-like occlusion of the distal internal carotid arteries. A smooth muscle cell (SMC)-specific knock-in mouse model (Acta2SMC-R179C/+) inserted the mutation into 67% of aortic SMCs, whereas explanted SMCs were uniformly heterozygous. Acta2R179C/+ SMCs fail to fully differentiate and maintain stem cell-like features, including high glycolytic flux, and increasing oxidative respiration (OXPHOS) with nicotinamide riboside (NR) drives the mutant SMCs to differentiate and decreases migration. Acta2SMC-R179C/+ mice have intraluminal MMD-like occlusive lesions and strokes after carotid artery injury, whereas the similarly treated WT mice have no strokes and patent lumens. Treatment with NR prior to the carotid artery injury attenuates the strokes, MMD-like lumen occlusions, and aberrant vascular remodeling in the Acta2SMC-R179C/+ mice. These data highlight the role of immature SMCs in MMD-associated occlusive disease and demonstrate that altering SMC metabolism to drive quiescence of Acta2R179C/+ SMCs attenuates strokes and aberrant vascular remodeling in the Acta2SMC-R179C/+ mice.