KPT-330 Prevents Aortic Valve Calcification via a Novel C/EBPß Signaling Pathway.

[Figure: see text].

Loss of flow responsive Tie1 results in ImpairedAortic valve remodeling.

The mechanisms regulating endothelial cell response to hemodynamic forces required for heart valve development, especially valve remodeling, remain elusive. Tie1, an endothelial specific receptor tyrosine kinase, is up-regulated by oscillating shear stress and is required for lymphatic valve development. In this study, we demonstrate that valvular endothelial Tie1 is differentially expressed in a dynamic pattern predicted by disturbed flow during valve remodeling. Following valvular endocardial specific deletion of Tie1 in mice, we observed enlarged aortic valve leaflets, decreased valve stiffness and valvular insufficiency. Valve abnormalities were only detected in late gestation and early postnatal mutant animals and worsened with age. The mutant mice developed perturbed extracellular matrix (ECM) deposition and remodeling characterized by increased glycosaminoglycan and decreased collagen content, as well as increased valve interstitial cell expression of Sox9, a transcription factor essential for normal ECM maturation during heart valve development. This study provides the first evidence that Tie1 is involved in modulation of late valve remodeling and suggests that an important Tie1-Sox9 signaling axis exists through which disturbed flows are converted by endocardial cells to paracrine Sox9 signals to modulate normal matrix remodeling of the aortic valve.

A Comprehensive Clinical Genetics Approach to Critical Congenital Heart Disease in Infancy.

OBJECTIVE: To investigate the frequency of genetic diagnoses among infants with critical congenital heart disease (CHD) using a comprehensive cardiovascular genetics approach and to identify genotype-phenotype correlations. STUDY DESIGN: A retrospective chart review of patients evaluated by cardiovascular genetics in a pediatric cardiac intensive care unit from 2010 to 2015 was performed. Infants with CHD who were <1 month of age were included. CHD was classified using structured phenotype definitions. Cardiac and noncardiac phenotypes were tested for associations with abnormal genetic testing using χ1 and Fisher exact tests. RESULTS: Genetic evaluation was completed in 293 infants with CHD, of whom 213 had isolated congenital heart disease (iCHD) and 80 had multiple congenital anomalies. Overall, the yield of abnormal genetic testing was 26%. The multiple congenital anomalies cohort had a greater yield of genetic testing (39%) than the iCHD cohort (20%) (OR 2.7). Using a non-hierarchical CHD classification and excluding 22q11.2 deletion and common aneuploidies, right ventricular obstructive defects were associated with abnormal genetic testing (P = .0005). Extracardiac features associated with abnormal genetic testing included ear, nose, and throat (P = .003) and brain (P = .0001) abnormalities. A diagnosis of small for gestational age or intrauterine growth retardation also was associated with abnormal genetic testing (P = .0061), as was presence of dysmorphic features (P = .0033, OR 3.5). Infants without dysmorphia with iCHD or multiple congenital anomalies had similar frequencies of abnormal genetic testing. CONCLUSIONS: The present study provides evidence to support a comprehensive cardiovascular genetics approach in evaluating infants with critical CHD while also identifying important genotype-phenotype considerations.

Heart Failure in Pediatric Patients With Congenital Heart Disease.

Heart failure (HF) is a complex clinical syndrome resulting from diverse primary and secondary causes and shared pathways of disease progression, correlating with substantial mortality, morbidity, and cost. HF in children is most commonly attributable to coexistent congenital heart disease, with different risks depending on the specific type of malformation. Current management and therapy for HF in children are extrapolated from treatment approaches in adults. This review discusses the causes, epidemiology, and manifestations of HF in children with congenital heart disease and presents the clinical, genetic, and molecular characteristics that are similar or distinct from adult HF. The objective of this review is to provide a framework for understanding rapidly increasing genetic and molecular information in the challenging context of detailed phenotyping. We review clinical and translational research studies of HF in congenital heart disease including at the genome, transcriptome, and epigenetic levels. Unresolved issues and directions for future study are presented.

Familial co-occurrence of congenital heart defects follows distinct patterns.

Aims: Congenital heart defects (CHD) affect almost 1% of all live born children and the number of adults with CHD is increasing. In families where CHD has occurred previously, estimates of recurrence risk, and the type of recurring malformation are important for counselling and clinical decision-making, but the recurrence patterns in families are poorly understood. We aimed to determine recurrence patterns, by investigating the co-occurrences of CHD in 1163 families with known malformations, comprising 3080 individuals with clinically confirmed diagnosis. Methods and results: We calculated rates of concordance and discordance for 41 specific types of malformations, observing a high variability in the rates of concordance and discordance. By calculating odds ratios for each of 1640 pairs of discordant lesions observed between affected family members, we were able to identify 178 pairs of malformations that co-occurred significantly more or less often than expected in families. The data show that distinct groups of cardiac malformations co-occur in families, suggesting influence from underlying developmental mechanisms. Analysis of human and mouse susceptibility genes showed that they were shared in 19% and 20% of pairs of co-occurring discordant malformations, respectively, but none of malformations that rarely co-occur, suggesting that a significant proportion of co-occurring lesions in families is caused by overlapping susceptibility genes. Conclusion: Familial CHD follow specific patterns of recurrence, suggesting a strong influence from genetically regulated developmental mechanisms. Co-occurrence of malformations in families is caused by shared susceptibility genes.

Fetal origins of adult cardiac disease: a novel approach to prevent fetal growth restriction induced cardiac dysfunction using insulin like growth factor.

BACKGROUND: Fetal growth restriction (FGR) is a risk factor for adult cardiovascular disease. Intraplacental gene transfer of human insulin-like growth factor-1 (IGF-1) corrects birth weight in our mouse model of FGR. This study addresses long term effects of FGR on cardiac function and the potential preventive effect of IGF-1. STUDY DESIGN: Laparotomy was performed on pregnant C57BL/6J mice at embryonic day 18 and pups were divided into three groups: Sham operated; FGR (induced by mesenteric uterine artery ligation); treatment (intraplacental injection of IGF-1 after uterine artery ligation). Pups were followed until 32 wk of life. Transthoracic echocardiography was performed starting at 12 wk. RESULTS: Systolic cardiac function was significantly impaired in the FGR group with reduced fractional shortening compared with sham and treatment group starting at week 12 of life (20 ± 4 vs. 31 ± 5 vs. 32 ± 5, respectively, n = 12 for each group; P < 0.001) with no difference between the sham and treatment groups. CONCLUSION: Intraplacental gene transfer of IGF-1 prevents FGR induced cardiac dysfunction. This suggests that in utero therapy may positively impact cardiac remodeling and prevent adult cardiovascular disease.

Valve Endothelial Cell-Derived Tgfß1 Signaling Promotes Nuclear Localization of Sox9 in Interstitial Cells Associated With Attenuated Calcification.

OBJECTIVE: Aortic valve disease, including calcification, affects >2% of the human population and is caused by complex interactions between multiple risk factors, including genetic mutations, the environment, and biomechanics. At present, there are no effective treatments other than surgery, and this is because of the limited understanding of the mechanisms that underlie the condition. Previous work has shown that valve interstitial cells within the aortic valve cusps differentiate toward an osteoblast-like cell and deposit bone-like matrix that leads to leaflet stiffening and calcific aortic valve stenosis. However, the mechanisms that promote pathological phenotypes in valve interstitial cells are unknown. APPROACH AND RESULTS: Using a combination of in vitro and in vivo tools with mouse, porcine, and human tissue, we show that in valve interstitial cells, reduced Sox9 expression and nuclear localization precedes the onset of calcification. In vitro, Sox9 nuclear export and calcific nodule formation is prevented by valve endothelial cells. However, in vivo, loss of Tgfß1 in the endothelium leads to reduced Sox9 expression and calcific aortic valve disease. CONCLUSIONS: Together, these findings suggest that reduced nuclear localization of Sox9 in valve interstitial cells is an early indicator of calcification, and therefore, pharmacological targeting to prevent nuclear export could serve as a novel therapeutic tool in the prevention of calcification and stenosis.

Analysis of TGFBR1*6A variant in individuals evaluated for Marfan syndrome.

Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS) are genetic disorders that affect connective tissue as a result of dysregulated TGF-ß signaling. MFS is most frequently caused by mutations in FBN1 whereas Loeys-Dietz syndrome results from mutations in TGFBR1 or TGFBR2. There is substantial inter- and intra-familial phenotypic variability among these disorders, suggesting the presence of genetic modifiers. Previously, a polymorphism in the TGFßR1 protein termed the TFGBR1*6A allele was found to be overrepresented in patients with MFS and was identified as a low penetrance allele with suggestion as a possible modifier. To further investigate the importance of this variant, a retrospective review of genetic and phenotypic findings was conducted for 335 patients evaluated for suspicion of MFS or related disorders. In patients with a diagnosis of MFS, the presence of the TFGBR1*6A allele was not associated with phenotypic differences. Similarly, careful phenotyping of patients who carried the TFGBR1*6A allele but did not have MFS did not identify an altered frequency of specific connective tissue features. In this small cohort, the results did not reach significance to identify the TFGBR1*6A allele as a major modifier for aortic dilation, ectopia lentis, or systemic features associated with MFS or other connective tissue disorders. © 2016 Wiley Periodicals, Inc.

Clinically relevant variants identified in thoracic aortic aneurysm patients by research exome sequencing.

Thoracic aortic aneurysm (TAA) is a genetically heterogeneous disease involving subclinical and progressive dilation of the thoracic aorta, which can lead to life-threatening complications such as dissection or rupture. Genetic testing is important for risk stratification and identification of at risk family members, and clinically available genetic testing panels have been expanding rapidly. However, when past testing results are normal, there is little evidence to guide decision-making about the indications and timing to pursue additional clinical genetic testing. Results from research based genetic testing can help inform this process. Here we present 10 TAA patients who have a family history of disease and who enrolled in research-based exome testing. Nine of these ten patients had previous clinical genetic testing that did not identify the cause of disease. We sought to determine the number of rare variants in 23 known TAA associated genes identified by research-based exome testing. In total, we found 10 rare variants in six patients. Likely pathogenic variants included a TGFB2 variant in one patient and a SMAD3 variant in another. These variants have been reported previously in individuals with similar phenotypes. Variants of uncertain significance of particular interest included novel variants in MYLK and MFAP5, which were identified in a third patient. In total, clinically reportable rare variants were found in 6/10 (60%) patients, with at least 2/10 (20%) patients having likely pathogenic variants identified. These data indicate that consideration of re-testing is important in TAA patients with previous negative or inconclusive results.

COX2 inhibition reduces aortic valve calcification in vivo.

OBJECTIVE: Calcific aortic valve disease (CAVD) is a significant cause of morbidity and mortality, which affects ≈1% of the US population and is characterized by calcific nodule formation and stenosis of the valve. Klotho-deficient mice were used to study the molecular mechanisms of CAVD as they develop robust aortic valve (AoV) calcification. Through microarray analysis of AoV tissues from klotho-deficient and wild-type mice, increased expression of the gene encoding cyclooxygenase 2 (COX2; Ptgs2) was found. COX2 activity contributes to bone differentiation and homeostasis, thus the contribution of COX2 activity to AoV calcification was assessed. APPROACH AND RESULTS: In klotho-deficient mice, COX2 expression is increased throughout regions of valve calcification and is induced in the valvular interstitial cells before calcification formation. Similarly, COX2 expression is increased in human diseased AoVs. Treatment of cultured porcine aortic valvular interstitial cells with osteogenic media induces bone marker gene expression and calcification in vitro, which is blocked by inhibition of COX2 activity. In vivo, genetic loss of function of COX2 cyclooxygenase activity partially rescues AoV calcification in klotho-deficient mice. Moreover, pharmacological inhibition of COX2 activity in klotho-deficient mice via celecoxib-containing diet reduces AoV calcification and blocks osteogenic gene expression. CONCLUSIONS: COX2 expression is upregulated in CAVD, and its activity contributes to osteogenic gene induction and valve calcification in vitro and in vivo.

MALDI Imaging Mass Spectrometry as a Lipidomic Approach to Heart Valve Research.

BACKGROUND: Valvular disease is characterized in part by lipid deposition, but systematic analysis of the patterns of global lipid expression in healthy and diseased valve tissues are unknown. This is due in part to tissue limitations for lipidomic preparations and technologies for evaluating lipid distribution in tissues. The study aim was to examine the application of matrixassisted laser desorption ionization imaging mass spectrometry (MALDI IMS) to the aortic valve during development and disease, as an approach to detect and map lipids and ultimately better understand valve structure and function. METHODS: Established MALDI IMS strategies were applied to thin tissue sections of heart valves to map lipids to corresponding morphological features. Healthy prenatal and adult ovine aortic valve tissues were evaluated using the developed techniques. Lipid expression levels were compared between prenatal and adult valves using Wilcoxon rank sum testing and area under the receiver operating curves. A classification algorithm was used to determine distinct lipid signatures in adult extracellular matrix (ECM) substructures, including fibrosa and spongiosa layers. Lipid patterns were examined in heart valve tissue from pediatric patients with congenital aortic valve stenosis (CAVS). RESULTS: Lipid levels were decreased in adult ovine aortic valves when compared with prenatal valves. Classification algorithms applied to lipid signatures reported distinct lipid signatures mapping to ECM substructures in the adult aortic valve, but could not distinguish amorphous structures at pre-natal day 5. In CAVS, the in-situ lipid aggregation of distinct lipid species showed unique patterning both concurrent and divergent with ECM disarray. Fatty acid content varied between normal and diseased human aortic valves. CONCLUSIONS: MALDI IMS provides a new and useful approach to evaluate lipid biology in heart valve tissue. These findings define a role for lipid regulation in aortic valve development and demonstrate patterns of lipid deregulation in congenital disease.

CHD associated with syndromic diagnoses: peri-operative risk factors and early outcomes.

CHD is frequently associated with a genetic syndrome. These syndromes often present specific cardiovascular and non-cardiovascular co-morbidities that confer significant peri-operative risks affecting multiple organ systems. Although surgical outcomes have improved over time, these co-morbidities continue to contribute substantially to poor peri-operative mortality and morbidity outcomes. Peri-operative morbidity may have long-standing ramifications on neurodevelopment and overall health. Recognising the cardiovascular and non-cardiovascular risks associated with specific syndromic diagnoses will facilitate expectant management, early detection of clinical problems, and improved outcomes--for example, the development of syndrome-based protocols for peri-operative evaluation and prophylactic actions may improve outcomes for the more frequently encountered syndromes such as 22q11 deletion syndrome.

Clinical Stratification of Pediatric Patients with Idiopathic Thoracic Aortic Aneurysm.

OBJECTIVES: To describe the global phenotypes of pediatric patients with thoracic aortic aneurysm (TAA) who do not have a clinical diagnosis of Marfan syndrome (MFS) or related connective tissue disorders. We hypothesized that the presence of noncardiovascular abnormalities correlate with TAA severity and that medical therapy reduces TAA progression. STUDY DESIGN: This is a retrospective case series of patients with TAA age ≤ 21 years evaluated in a cardiovascular genetics clinic. Patients meeting clinical criteria for MFS or related disorders were excluded. Repeated measures analyses of longitudinal echocardiographic measurements of the aorta were used to test associations between TAA severity and noncardiovascular phenotype and to assess the impact of medical therapy. RESULTS: Sixty-nine patients with TAA at mean age 12.5 ± 5.3 years were included. Noncardiovascular abnormalities, including skeletal (65%) or craniofacial (54%) findings, were frequently observed. Increased rate of aortic root enlargement was associated with ocular (P = .002) and cutaneous (P = .003) abnormalities, and increased rate of ascending aorta enlargement was associated with craniofacial (P < .001) abnormalities. Beta blocker or angiotensin receptor blocker therapy (n = 41) was associated with reduction in the rate of aortic root growth (P = .018). CONCLUSIONS: Children with TAA not satisfying diagnostic criteria for MFS or related disorders frequently have noncardiovascular findings, some of which are associated with TAA progression. Because therapy initiation may reduce risk of progression and long-term complications, comprehensive assessment of noncardiovascular findings may facilitate early risk stratification and improve outcomes.

Exon 3 deletion of ryanodine receptor causes left ventricular noncompaction, worsening catecholaminergic polymorphic ventricular tachycardia, and sudden cardiac arrest.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a highly malignant genetic channelopathy associated with exertional syncope and reproducible polymorphic ventricular tachycardia with exercise. Approximately 65% of patients with CPVT are found to have a disease causing mutation in the RYR2 gene. RYR2 encodes a calcium ion transporter in the sarcomeric reticulum, and is responsible for the calcium induced calcium release that results in ventricular contraction. Recently, exon 3 deletion of RYR2 has been reported to be associated with left ventricular noncompaction. Herein we describe a patient with a novel, de novo deletion of exon 3 in the RYR2 gene that resulted in a severe CPVT phenotype and sudden cardiac arrest (SCA), and the development of left ventricular non-compaction (LVNC) coinciding with worsening arrhythmias. This case is unique in that the patient initially presented with exertional syncope and developed LVNC that coincided with increasingly severe ventricular arrhythmias and multiple episodes of SCA. This case supports the idea that RYR2 deletions cause a severe subtype of CPVT associated with LVNC and suggests LVNC may play a role in exacerbating the arrhythmias of CPVT. Deletion duplication testing should be considered in the context of CPVT and LVNC or SCA.

Aortopathy in the 7q11.23 microduplication syndrome.

The 7q11.23 microduplication syndrome, caused by the reciprocal duplication of the Williams-Beuren syndrome deletion region, is a genomic disorder with an emerging clinical phenotype. Dysmorphic features, congenital anomalies, hypotonia, developmental delay highlighted by variable speech delay, and autistic features are characteristic findings. Congenital heart defects, most commonly patent ductus arteriosus, have been reported in a minority of cases. Included in the duplicated region is elastin (ELN), implicated as the cause of supravalvar aortic stenosis in patients with Williams-Beuren syndrome. Here we present a series of eight pediatric patients and one adult with 7q11.23 microduplication syndrome, all of whom had aortic dilation, the opposite vascular phenotype of the typical supravalvar aortic stenosis found in Williams-Beuren syndrome. The ascending aorta was most commonly involved, while dilation was less frequently identified at the aortic root and sinotubular junction. The findings in these patients support a recommendation for cardiovascular surveillance in patients with 7q11.23 microduplication syndrome.

Heart valve structure and function in development and disease.

The mature heart valves are made up of highly organized extracellular matrix (ECM) and valve interstitial cells (VICs) surrounded by an endothelial cell layer. The ECM of the valves is stratified into elastin-, proteoglycan-, and collagen-rich layers that confer distinct biomechanical properties to the leaflets and supporting structures. Signaling pathways have critical functions in primary valvulogenesis as well as the maintenance of valve structure and function over time. Animal models provide powerful tools to study valve development and disease processes. Valve disease is a significant public health problem, and increasing evidence implicates aberrant developmental mechanisms underlying pathogenesis. Further studies are necessary to determine regulatory pathway interactions underlying valve pathogenesis in order to generate new avenues for novel therapeutics.

Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions.

Diabetes-induced cardiomyopathy is characterized by cardiac remodeling, fibrosis, and endothelial dysfunction, with no treatment options currently available. Hyperglycemic memory by endothelial cells may play the key role in microvascular complications in diabetes, providing a potential target for therapeutic approaches. This study tested the hypothesis that a proangiogenic environment can augment diabetes-induced deficiencies in endothelial cell angiogenic and biomechanical responses. Endothelial responses were quantified for two models of diabetic conditions: 1) an in vitro acute and chronic hyperglycemia where normal cardiac endothelial cells were exposed to high-glucose media, and 2) an in vivo chronic diabetes model where the cells were isolated from rats with type I streptozotocin-induced diabetes. Capillary morphogenesis, VEGF and nitric oxide expression, cell morphology, orientation, proliferation, and apoptosis were determined for cells cultured on Matrigel or proangiogenic nanofiber hydrogel. The effects of biomechanical stimulation were assessed following cell exposure to uniaxial strain. The results demonstrate that diabetes alters cardiac endothelium angiogenic response, with differential effects of acute and chronic exposure to high-glucose conditions, consistent with the concept that endothelial cells may have a long-term "hyperglycemic memory" of the physiological environment in the body. Furthermore, endothelial cell exposure to strain significantly diminishes their angiogenic potential following strain application. Both diabetes and strain-associated deficiencies can be augmented in the proangiogenic nanofiber microenvironment. These findings may contribute to the development of novel approaches to reverse hyperglycemic memory of endothelium and enhance vascularization of the diabetic heart, where improved angiogenic and biomechanical responses can be the key factor to successful therapy.

Advances in the treatment of aortic valve disease: is it time for companion diagnostics?

PURPOSE OF REVIEW: Aortic valve disease (AVD) is a growing public health problem, and the pathogenesis underlying AVD is complex. The lack of durable bioprostheses and pharmacologic therapies remain central needs in care. The purpose of this review is to highlight recent clinical studies that impact the care of children with AVD and is to explore ongoing translational research efforts. RECENT FINDINGS: Clinical studies have evaluated the durability of bioprosthetics and surgical strategies, tested statins during early disease, and identified new predictive biomarkers. Large animal models have demonstrated the effectiveness of a novel bioprosthetic scaffold. Mouse models of latent AVD have advanced our ability to elucidate natural history and perform preclinical studies that test new treatments in the context of early disease. SUMMARY: Current priorities for AVD patients include identifying new pharmacologic treatments and developing durable bioprostheses. Multidisciplinary efforts are needed that bridge pediatric and adult programs, and bring together different types of expertise and leverage network and consortium resources. As our understanding of the underlying complex genetics is better defined, companion diagnostics may transform future clinical trials and ultimately improve the care of patients with AVD by promoting personalized medicine and early intervention.

Insufficient versican cleavage and Smad2 phosphorylation results in bicuspid aortic and pulmonary valves.

Bicuspid or bifoliate aortic valve (BAV) results in two rather than three cusps and occurs in 1-2% of the population placing them at higher risk of developing progressive aortic valve disease. Only NOTCH-1 has been linked to human BAV, and genetically modified mouse models of BAV are limited by low penetrance and additional malformations. Here we report that in the Adamts5(-/-) valves, collagen I, collagen III, and elastin were disrupted in the malformed hinge region that anchors the mature semilunar cusps and where the ADAMTS5 proteoglycan substrate versican, accumulates. ADAMTS5 deficient prevalvular mesenchyme also exhibited a reduction of α-smooth muscle actin and filamin A suggesting versican cleavage may be involved in TGFß signaling. Subsequent evaluation showed a significant decrease of pSmad2 in regions of prevalvular mesenchyme in Adamts5(-/-) valves. To test the hypothesis that ADAMTS5 versican cleavage is required, in part, to elicit Smad2 phosphorylation we further reduced Smad2 in Adamts5(-/-) mice through intergenetic cross. The Adamts5(-/-);Smad2(+/-) mice had highly penetrant BAV and bicuspid pulmonary valve (BPV) malformations as well as increased cusp and hinge size compared to the Adamts5(-/-) and control littermates. These studies demonstrate that semilunar cusp malformations (BAV and BPV) can arise from a failure to remodel the proteoglycan-rich provisional ECM. Specifically, faulty versican clearance due to ADAMTS5 deficiency blocks the initiation of pSmad2 signaling, which is required for excavation of endocardial cushions during aortic and pulmonary valve development. Further studies using the Adamts5(-/-); Smad2(+/-) mice with highly penetrant and isolated BAV, may lead to new pharmacological treatments for valve disease.

Microcephaly is associated with early adverse neurologic outcomes in hypoplastic left heart syndrome.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) is associated with significant mortality and morbidity. Fetal head growth abnormalities have been identified in a subset of HLHS fetuses, but it is unclear whether specific patterns of maladaptive growth affect clinical outcomes. We hypothesized that poor fetal head growth is associated with an increased frequency of adverse clinical outcomes. METHODS: We retrospectively examined a cohort of HLHS patients from midgestation to 1 y of age. Fetal and birth anthropometric measurements were analyzed using the Olsen standard, and clinical outcomes were obtained. RESULTS: A total of 104 HLHS patients were identified over a 12-y period; fetal data were available in 38 cases. HLHS neonates demonstrated a high incidence of microcephaly (12%), small head size (27%), and poor head growth (32%). All-cause mortality was 31% at 30 d and 43% at 1 y. Neurologic outcomes were observed in 12% of patients and were significantly increased with microcephaly (43 vs. 4%; P = 0.02). The average length of hospital stay following stage I palliation was 33.4 ± 33 d, correcting for early death. CONCLUSION: In term nonsyndromic HLHS, fetal and neonatal microcephaly are associated with early adverse neurologic outcomes but not mortality.