Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells.

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

Vascular Ehlers-Danlos Syndrome: A Comprehensive Natural History Study in a Dutch National Cohort of 142 Patients.

BACKGROUND: Vascular Ehlers-Danlos syndrome (vEDS) is a rare connective tissue disorder with a high risk for arterial, bowel, and uterine rupture, caused by heterozygous pathogenic variants in COL3A1. The aim of this cohort study is to provide further insights into the natural history of vEDS and describe genotype-phenotype correlations in a Dutch multicenter cohort to optimize patient care and increase awareness of the disease. METHODS: Individuals with vEDS throughout the Netherlands were included. The phenotype was charted by retrospective analysis of molecular and clinical data, combined with a one-time physical examination. RESULTS: A total of 142 individuals (50% female) participated the study, including 46 index patients (32%). The overall median age at genetic diagnosis was 41.0 years. More than half of the index patients (54.3%) and relatives (53.1%) had a physical appearance highly suggestive of vEDS. In these individuals, major events were not more frequent (P=0.90), but occurred at a younger age (P=0.01). A major event occurred more often and at a younger age in men compared with women (P<0.001 and P=0.004, respectively). Aortic aneurysms (P=0.003) and pneumothoraces (P=0.029) were more frequent in men. Aortic dissection was more frequent in individuals with a COL3A1 variant in the first quarter of the collagen helical domain (P=0.03). CONCLUSIONS: Male sex, type and location of the COL3A1 variant, and physical appearance highly suggestive of vEDS are risk factors for the occurrence and/or early age of onset of major events. This national multicenter cohort study of Dutch individuals with vEDS provides a valuable basis for improving guidelines for the diagnosing, follow-up, and treatment of individuals with vEDS.

Menke-Hennekam syndrome; delineation of domain-specific subtypes with distinct clinical and DNA methylation profiles.

CREB-binding protein (CBP, encoded by CREBBP) and its paralog E1A-associated protein (p300, encoded by EP300) are involved in histone acetylation and transcriptional regulation. Variants that produce a null allele or disrupt the catalytic domain of either protein cause Rubinstein-Taybi syndrome (RSTS), while pathogenic missense and in-frame indel variants in parts of exons 30 and 31 cause phenotypes recently described as Menke-Hennekam syndrome (MKHK). To distinguish MKHK subtypes and define their characteristics, molecular and extended clinical data on 82 individuals (54 unpublished) with variants affecting CBP (n = 71) or p300 (n = 11) (NP_004371.2 residues 1,705-1,875 and NP_001420.2 residues 1,668-1,833, respectively) were summarized. Additionally, genome-wide DNA methylation profiles were assessed in DNA extracted from whole peripheral blood from 54 individuals. Most variants clustered closely around the zinc-binding residues of two zinc-finger domains (ZZ and TAZ2) and within the first α helix of the fourth intrinsically disordered linker (ID4) of CBP/p300. Domain-specific methylation profiles were discerned for the ZZ domain in CBP/p300 (found in nine out of 10 tested individuals) and TAZ2 domain in CBP (in 14 out of 20), while a domain-specific diagnostic episignature was refined for the ID4 domain in CBP/p300 (in 21 out of 21). Phenotypes including intellectual disability of varying degree and distinct physical features were defined for each of the regions. These findings demonstrate existence of at least three MKHK subtypes, which are domain specific (MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4) rather than gene specific (CREBBP/EP300). DNA methylation episignatures enable stratification of molecular pathophysiologic entities within a gene or across a family of paralogous genes.

Doxycycline induces mitochondrial dysfunction in aortic smooth muscle cells.

The antibiotic doxycycline is known to inhibit inflammation and was therefore considered as a therapeutic to prevent abdominal aortic aneurysm (AAA) growth. Yet mitochondrial dysfunction is a key-characteristic of clinical AAA disease. We hypothesize that doxycycline impairs mitochondrial function in the aorta and aortic smooth muscle cells (SMCs). Doxycycline induced mitonuclear imbalance, reduced proliferation and diminished expression of typical contractile smooth muscle cell (SMC) proteins. To understand the underlying mechanism, we studied krüppel-like factor 4 (KLF4). The expression of this transcription factor was enhanced in SMCs after doxycycline treatment. Knockdown of KLF4, however, did not affect the doxycycline-induced SMC phenotypic changes. Then we used the bioenergetics drug elamipretide (SS-31). Doxycycline-induced loss of SMC contractility markers was not rescued, but mitochondrial genes and mitochondrial connectivity improved upon elamipretide. Thus while doxycycline is anti-inflammatory, it also induces mitochondrial dysfunction in aortic SMCs and causes SMC phenotypic switching, potentially contributing to aortic aneurysm pathology. The drug elamipretide helps mitigate the harmful effects of doxycycline on mitochondrial function in aortic SMC, and may be of interest for treatment of aneurysm diseases with pre-existing mitochondrial dysfunction.

Serine phosphorylation of the RhoGEF Trio stabilizes endothelial cell-cell junctions.

The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.

Inadequate detection of the FSHR complicates future research on extragonadal FSHR localization.

Introduction: Recently, follicle stimulating hormone (FSH) through interaction with its receptor (FSHR) has been proposed to play a role in postmenopausal osteoporosis and cardiovascular disease, rather than the loss of estrogen. To explore this hypothesis, unravelling which cells express extragonadal FSHR on protein level is key. Methods: We used two commercial anti-FSHR antibodies and validated them by performing immunohistochemistry on positive (ovary, testis) and negative controls (skin). Results: The monoclonal anti-FSHR antibody could not identify the FSHR in ovary or testis. The polyclonal anti-FSHR antibody stained the granulosa cells (ovary) and Sertoli cells (testis), yet there was equally intense staining of other cells/extracellular matrix. Furthermore, the polyclonal anti-FSHR antibody also stained skin tissue extensively, suggesting that the antibody stains more than just FSHR. Discussion: The findings in this study may add accuracy to literature on extragonadal FSHR localization and warrants attention to the use of inadequate anti-FSHR antibodies to value the potential role of FSH/FSHR in postmenopausal disease.

Growth charts for Marfan syndrome in the Netherlands and analysis of genotype-phenotype relationships.

To optimize care for children with Marfan syndrome (MFS) in the Netherlands, Dutch MFS growth charts were constructed. Additionally, we aimed to investigate the effect of FBN1 variant type (haploinsufficiency [HI]/dominant negative [DN]) on growth, and compare MFS-related height increase across populations. Height and weight data of individuals with MFS aged 0-21 years were retrospectively collected. Generalized Additive Models for Location, Scale and Shape (GAMLSS) was used for growth chart modeling. To investigate genotype-phenotype relationships, FBN1 variant type was included as an independent variable in height-for-age and BMI-for-age models. MFS-related height increase was compared with that of previous MFS growth studies from the United States, Korea, and France. Height and weight data of 389 individuals with MFS were included (210 males). Height-for-age, BMI-for-age, and weight-for-height charts reflected the tall and slender MFS habitus throughout childhood. Mean increase in height of individuals with MFS compared with the general Dutch population was significantly lower than in the other three MFS populations compared to their reference populations. FBN1-HI variants were associated with taller height in both sexes, and decreased BMI in females (p-values <0.05). This Dutch MFS growth study broadens the notion that genetic background and MFS variant type (HI/DN) influence tall and slender stature in MFS.

Endothelial dysfunction in Marfan syndrome mice is restored by resveratrol.

Patients with Marfan syndrome (MFS) develop thoracic aortic aneurysms as the aorta presents excessive elastin breaks, fibrosis, and vascular smooth muscle cell (vSMC) death due to mutations in the FBN1 gene. Despite elaborate vSMC to aortic endothelial cell (EC) signaling, the contribution of ECs to the development of aortic pathology remains largely unresolved. The aim of this study is to investigate the EC properties in Fbn1C1041G/+ MFS mice. Using en face immunofluorescence confocal microscopy, we showed that EC alignment with blood flow was reduced, EC roundness was increased, individual EC surface area was larger, and EC junctional linearity was decreased in aortae of Fbn1C1041G/+ MFS mice. This modified EC phenotype was most prominent in the ascending aorta and occurred before aortic dilatation. To reverse EC morphology, we performed treatment with resveratrol. This restored EC blood flow alignment, junctional linearity, phospho-eNOS expression, and improved the structural integrity of the internal elastic lamina of Fbn1C1041G/+ mice. In conclusion, these experiments identify the involvement of ECs and underlying internal elastic lamina in MFS aortic pathology, which could act as potential target for future MFS pharmacotherapies.

Abnormal aortic hemodynamics are associated with risk factors for aortic complications in patients with marfan syndrome.

Background: It is difficult to assess the risk for aortic dissection beyond the aortic root in patients with Marfan syndrome (MFS). To aid risk assessment in these patients, we investigated aortic flow and wall shear stress (WSS) by 4D flow magnetic resonance imaging (MRI) in patients with MFS and compared the results with healthy volunteers. We hypothesized that MFS patients with a high-risk profile for aortic dissection would show abnormal hemodynamics in aortic regions associated with aortic dissection. Methods: MFS patients (n = 55) and healthy subjects (n = 25), matched for age and sex, prospectively underwent 4D flow MRI. 4D flow maps were constructed to detect elevated (defined as higher than the three-dimensional 95 % confidence interval) and deviant directed (defined as vector angle differences higher than 120°) WSS in MFS patients as compared to the controls. Univariate and multivariate associations with risk factors for aortic dissection in MFS patients were assessed. Results: The maximum incidence for elevated WSS was 20 % (CI 9 %-31 %) and found in the ascending aorta. The maximum for deviant directed WSS was 39 % (CI 26 %-52 %) and found in the inner descending aorta. Significantly more male patients had deviant directed WSS in the inner proximal descending aorta (63 % vs 24 %, p = 0.014). Multivariate analysis showed that deviant directed WSS was associated with male sex (p = 0.019), and a haplo-insufficient FBN1 mutation type (p = 0.040). In 60 % of MFS patients with a previous aortic root replacement surgery, abnormal hemodynamics were found in the ascending aorta. No significant differences between hemodynamics were found in the descending aorta between operated and non-operated patients. Conclusion: Deviant directed WSS in the proximal descending aorta is associated with known risk factors for aortic dissection in MFS patients, namely male sex and a haploinsufficient FBN1 mutation type.

No prominent role for complement C1-esterase inhibitor in Marfan syndrome mice.

Marfan syndrome (MFS) is a connective tissue disorder causing aortic aneurysm formation. Currently, only prophylactic aortic surgery and blood pressure-lowering drugs are available to reduce the risk of aortic rupture. Upon whole genome sequencing of a Marfan family, we identified a complement gene C1R variant (p.Ser152Leu), which is associated with severe aortic patients. Therefore, we assessed the role of complement activation in MFS aortic tissue. Expression of various complement genes and proteins was detected in human and murine MFS aneurysm tissue, which prompted us to study complement inhibition in MFS mice. Treatment of the Fbn1C1041G/+ MFS mice with human plasma-derived C1-esterase inhibitor Cetor® resulted in reduced complement deposition, decreased macrophage influx in the aorta, and lower circulating TNFα levels. However, in line with previous anti-inflammatory treatments, complement inhibition did not change the aortic dilatation rate in this MFS mouse model. Thus, while complement factors/component 3 activation were detected in human/murine MFS aorta, Cetor® had no effect on aortic dilatation in MFS mice, indicating that complement inhibition is not a suitable treatment strategy in MFS.

A cross-sectional study on fatigue, anxiety, and symptoms of depression and their relation with medical status in adult patients with Marfan syndrome. Psychological consequences in Marfan syndrome.

Marfan syndrome (MFS) is a connective tissue disorder affecting the cardiovascular, ocular, and skeletal system, which may be accompanied by psychological features. This study aimed to determine the prevalence of fatigue, anxiety, and symptoms of depression in MFS patients, and to assess the degree to which sociodemographic and clinical variables are associated with fatigue and psychological aspects. The prevalence of fatigue, anxiety, and symptoms of depression were assessed in two cohorts of MFS patients and compared with healthy controls. The checklist individual strength (CIS), and hospital anxiety and depression scale (HADS) questionnaires were utilized. Medical status was assessed (family history of MFS, aortic root dilatation >40 mm, previous aortic surgery, aortic dissection, chronic pain, skeletal involvement, and scoliosis). Severe fatigue was experienced by 37% of the total MFS cohort (n = 155). MFS patients scored significantly higher on the CIS questionnaire, concerning severe fatigue, as compared with the general Dutch population (p < 0.0001). There were no differences in HADS anxiety or depression scores. In older MFS patients, with a more severe cardiovascular phenotype, chronic pain, and a higher unemployment rate, significantly more symptoms of depression were observed, when compared with the general population (p = 0.027) or compared with younger MFS patients (p = 0.026). Multivariate analysis, showed that anxiety was associated with chronic pain (p = 0.022) and symptoms of depression with unemployment (p = 0.024). MFS patients report significantly more severe fatigue as compared with the general population. Since the cause of fatigue is unclear, more research may be needed. Psychological intervention, for example, cognitive behavioral therapy, may contribute to a reduction in psychological symptoms.

Genome-wide methylation patterns in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the Fibrillin-1 gene (FBN1). Here, we undertook the first epigenome-wide association study (EWAS) in patients with MFS aiming at identifying DNA methylation loci associated with MFS phenotypes that may shed light on the disease process. METHODS: The Illumina 450 k DNA-methylation array was used on stored peripheral whole-blood samples of 190 patients with MFS originally included in the COMPARE trial. An unbiased genome-wide approach was used, and methylation of CpG-sites across the entire genome was evaluated. Additionally, we investigated CpG-sites across the FBN1-locus (15q21.1) more closely, since this is the gene defective in MFS. Differentially Methylated Positions (DMPs) and Differentially Methylated Regions (DMRs) were identified through regression analysis. Associations between methylation levels and aortic diameters and presence or absence of 21 clinical features of MFS at baseline were analyzed. Moreover, associations between aortic diameter change, and the occurrence of clinical events (death any cause, type-A or -B dissection/rupture, or aortic surgery) and methylation levels were analyzed. RESULTS: We identified 28 DMPs that are significantly associated with aortic diameters in patients with MFS. Seven of these DMPs (25%) could be allocated to a gene that was previously associated with cardiovascular diseases (HDAC4, IGF2BP3, CASZ1, SDK1, PCDHGA1, DIO3, PTPRN2). Moreover, we identified seven DMPs that were significantly associated with aortic diameter change and five DMP's that associated with clinical events. No significant associations at p < 10-8 or p < 10-6 were found with any of the non-cardiovascular phenotypic MFS features. Investigating DMRs, clusters were seen mostly on X- and Y, and chromosome 18-22. The remaining DMRs indicated involvement of a large family of protocadherins on chromosome 5, which were not reported in MFS before. CONCLUSION: This EWAS in patients with MFS has identified a number of methylation loci significantly associated with aortic diameters, aortic dilatation rate and aortic events. Our findings add to the slowly growing literature on the regulation of gene expression in MFS patients.

A Novel Hypothesis: A Role for Follicle Stimulating Hormone in Abdominal Aortic Aneurysm Development in Postmenopausal Women.

An abdominal aortic aneurysm (AAA) is a dilatation of the abdominal aorta, which can potentially be fatal due to exsanguination following rupture. Although AAA is less prevalent in women, women with AAA have a more severe AAA progression compared to men as reflected by enhanced aneurysm growth rates and a higher rupture risk. Women are diagnosed with AAA at an older age than men, and in line with increased osteoporosis and cardiovascular events, the delayed AAA onset has been attributed to the reduction of the protective effect of oestrogens during the menopausal transition. However, new insights have shown that a high follicle stimulating hormone (FSH) level during menopause may also play a key role in those diseases. In this report we hypothesize that FSH may aggravate AAA development and progression in postmenopausal women via a direct and/or indirect role, promoting aorta pathology. Since FSH receptors (FSHR) are reported on many other cell types than granulosa cells in the ovaries, it is feasible that FSH stimulation of FSHR-bearing cells such as aortic endothelial cells or inflammatory cells, could promote AAA formation directly. Indirectly, AAA progression may be influenced by an FSH-mediated increase in osteoporosis, which is associated with aortic calcification. Also, an FSH-mediated decrease in cholesterol uptake by the liver and an increase in cholesterol biosynthesis will increase the cholesterol level in the circulation, and subsequently promote aortic atherosclerosis and inflammation. Lastly, FSH-induced adipogenesis may lead to obesity-mediated dysfunction of the microvasculature of the aorta and/or modulation of the periaortic adipose tissue. Thus the long term increased plasma FSH levels during the menopausal transition may contribute to enhanced AAA disease in menopausal women and could be a potential novel target for treatment to lower AAA-related events in women.

Aortic distensibility in Marfan syndrome: a potential predictor of aortic events?

OBJECTIVES: Patients with Marfan syndrome (MFS) are prone to develop aortic aneurysms due to fragmentation of elastic fibres, resulting in reduced distensibility of the aorta. Reduced distensibility was previously shown to predict progressive descending aorta dilatation. Here, we investigated longitudinal changes in distensibility, as a potential predictor of aortic events. METHODS: This retrospective study included all patients with MFS with at least four cardiac magnetic resonance examinations performed between 1996 and 2012. Aortic distensibility was assessed, in the ascending (level 1), proximal descending (level 2) and distal descending (level 3) aorta. Changes in distensibility were studied using linear mixed-effects regression models. RESULTS: In total, 35 patients with MFS (age at inclusion 28 (IQR 23-32) years, 54% men) were included. Mean aortic distensibility was already low (between 2.9×10-3/mm Hg/year and 6.4×10-3/mm Hg/year) at all levels at baseline, and significantly decreased over time at levels 2 and 3 (respectively, p=0.012 and p=0.002). The rate of distensibility loss per year (×10-3/mm Hg/year) was 0.01, 0.03 and 0.06×10-3/mm Hg at levels 1, 2 and 3, respectively. At inclusion, men exhibited very low distensibility, whereas women showed moderately reduced distensibility, gradually decreasing with age.Aortic dilatation rate at level 2 was associated with reduced aortic distensibility. However, we could not demonstrate a direct correlation between distensibility and clinical events during a follow-up of 22 years. CONCLUSION: Patients with MFS display reduced aortic distensibility already at an early age, inversely relating to aortic dilatation rate. However, in this selected patient group, distensibility seems less suitable as an individual predictor of aortic events.

Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4).

Multiple layers of vascular smooth muscle cells (vSMCs) are present in blood vessels forming the media of the vessel wall. vSMCs provide a vessel wall structure, enabling it to contract and relax, thus modulating blood flow. They also play a crucial role in the development of vascular diseases, such as atherosclerosis and aortic aneurysm formation. vSMCs display a remarkable high degree of plasticity. At present, the number of different vSMC phenotypes has only partially been characterized. By mapping vSMC phenotypes in detail and identifying triggers for phenotype switching, the relevance of the different phenotypes in vascular disease may be identified. Up until recently, vSMCs were classified as either contractile or dedifferentiated (ie, synthetic). However, single-cell RNA sequencing studies revealed such dedifferentiated arterial vSMCs to be highly diverse. Currently, no consensus exist about the number of vSMC phenotypes. Therefore, we reviewed the data from relevant single-cell RNA sequencing studies, and classified a total of 6 vSMC phenotypes. The central dedifferentiated vSMC type that we classified is the mesenchymal-like phenotype. Mesenchymal-like vSMCs subsequently seem to differentiate into fibroblast-like, macrophage-like, osteogenic-like, and adipocyte-like vSMCs, which contribute differentially to vascular disease. This phenotype switching between vSMCs requires the transcription factor KLF4 (Kruppel-like factor 4). Here, we performed an integrated analysis of the data about the recently identified vSMC phenotypes, their associated gene expression profiles, and previous vSMC knowledge to better understand the role of vSMC phenotype transitions in vascular pathology.

Parallel Murine and Human Aortic Wall Genomics Reveals Metabolic Reprogramming as Key Driver of Abdominal Aortic Aneurysm Progression.

Background While numerous interventions effectively interfered with abdominal aortic aneurysm (AAA) formation/progression in preclinical models, none of the successes translated into clinical success. Hence, a systematic exploration of parallel and divergent processes in clinical AAA disease and its 2 primary models (the porcine pancreatic elastase and angiotensin-II infusion [AngII] murine model) was performed to identify mechanisms relevant for aneurysm disease. Methods and Results This study combines Movat staining and pathway analysis for histological and genomic comparisons between clinical disease and its models. The impact of a notable genomic signal for metabolic reprogramming was tested in a rescue trial (AngII model) evaluating the impact of 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15)-mediated interference with main glycolytic switch PFKFB3. Histological evaluation characterized the AngII model as a dissection model that is accompanied by adventitial fibrosis. The porcine pancreatic elastase model showed a transient inflammatory response and aortic dilatation, followed by stabilization and fibrosis. Normalization of the genomic responses at day 14 confirmed the self-limiting nature of the porcine pancreatic elastase model. Clear parallel genomic responses with activated adaptive immune responses, and particularly strong signals for metabolic switching were observed in human AAA and the AngII model. Rescue intervention with the glycolysis inhibitor PFK15 in the AngII model showed that interference with the glycolytic switching quenches aneurysm formation. Conclusions Despite clear morphological contrasts, remarkable genomic parallels exist for clinical AAA disease and the AngII model. The metabolic response appears causatively involved in AAA progression and provides a novel therapeutic target. The clear transient genomic response classifies the porcine pancreatic elastase model as a disease initiation model.

Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes.

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis; however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor-ß (TGF-ß). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-ß receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-ß-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.

Long-term clinical outcomes of losartan in patients with Marfan syndrome: follow-up of the multicentre randomized controlled COMPARE trial.

AIMS: The COMPARE trial showed a small but significant beneficial effect of 3-year losartan treatment on aortic root dilatation rate in adults with Marfan syndrome (MFS). However, no significant effect was found on clinical endpoints, possibly due to a short follow-up period. The aim of the current study was therefore to investigate the long-term clinical outcomes after losartan treatment. METHODS AND RESULTS: In the original COMPARE study (inclusion 2008-2009), adult patients with MFS (n = 233) were randomly allocated to either the angiotensin-II receptor blocker losartan® on top of regular treatment (ß-blockers in 71% of the patients) or no additional medication. After the COMPARE trial period of 3 years, study subjects chose to continue their losartan medication or not. In a median follow-up period of 8 years, 75 patients continued losartan medication, whereas 78 patients, originally allocated to the control group, never used losartan after inclusion. No differences existed between baseline characteristics of the two groups except for age at inclusion [losartan 34 (interquartile range, IQR 26-43) years, control 41 (IQR 30-52) years; P = 0.031], and ß-blocker use (losartan 81%, control 64%; P = 0.022). A pathological FBN1 mutation was present in 76% of patients and 58% of the patients were male. Clinical endpoints, defined as all-cause mortality, aortic dissection/rupture, elective aortic root replacement, reoperation, and vascular graft implantation beyond the aortic root, were compared between the two groups. A per-patient composite endpoint was also analysed. Five deaths, 14 aortic dissections, 23 aortic root replacements, 3 reoperations, and 3 vascular graft implantations beyond the aortic root occurred during follow-up. Except for aortic root replacement, all endpoints occurred in patients with an operated aortic root. Patients who used losartan during the entire follow-up period showed a reduced number of events compared to the control group (death: 0 vs. 5, P = 0.014; aortic dissection: 3 vs. 11, P = 0.013; elective aortic root replacement: 10 vs. 13, P = 0.264; reoperation: 1 vs. 2, P = 0.463; vascular graft implantations beyond the aortic root 0 vs. 3, P = 0.071; and composite endpoint: 14 vs. 26, P = 0.019). These results remained similar when corrected for age and ß-blocker use in a multivariate analysis. CONCLUSION: These results suggest a clinical benefit of combined losartan and ß-blocker treatment in patients with MFS.

S100A9 Links Inflammation and Repair in Myocardial Infarction.

RATIONALE: The alarmin S100A9 has been identified as a potential therapeutic target in myocardial infarction. Short-term S100A9 blockade during the inflammatory phase post-myocardial infarction inhibits systemic and cardiac inflammation and improves cardiac function long term. OBJECTIVE: To evaluate the impact of S100A9 blockade on postischemic cardiac repair. METHODS AND RESULTS: We assessed cardiac function, hematopoietic response, and myeloid phagocyte dynamics in WT (wild type) C57BL/6 mice with permanent coronary artery ligation, treated with the specific S100A9 blocker ABR-238901 for 7 or 21 days. In contrast to the beneficial effects of short-term therapy, extended S100A9 blockade led to progressive deterioration of cardiac function and left ventricle dilation. The treatment reduced the proliferation of Lin-Sca-1+c-Kit+ hematopoietic stem and progenitor cells in the bone marrow and the production of proreparatory CD150+CD48-CCR2+ hematopoietic stem cells. Monocyte trafficking from the spleen to the myocardium and subsequent phenotype switching to reparatory Ly6CloMerTKhi macrophages was also impaired, leading to inefficient efferocytosis, accumulation of apoptotic cardiomyocytes, and a larger myocardial scar. The transcription factor Nur77 (Nr4a1 [nuclear receptor subfamily 4 group A member 1]) mediates the transition from inflammatory Ly6Chi monocytes to reparatory Ly6Clo macrophages. S100A9 upregulated the levels and activity of Nur77 in monocytes and macrophages in vitro and in Ly6Chi/int monocytes in vivo, and S100A9 blockade antagonized these effects. Finally, the presence of reparatory macrophages in the myocardium was also impaired in S100A9-/- mice with permanent myocardial ischemia, leading to depressed cardiac function long term. CONCLUSIONS: We show that S100A9 plays an important role in both the inflammatory and the reparatory immune responses to myocardial infarction. Long-term S100A9 blockade negatively impacts cardiac recovery and counterbalances the beneficial effects of short-term therapy. These results define a therapeutic window targeting the inflammatory phase for optimal effects of S100A9 blockade as potential immunomodulatory treatment in acute myocardial infarction.

DLC1 is a direct target of activated YAP/TAZ that drives collective migration and sprouting angiogenesis.

Endothelial YAP/TAZ (YAP is also known as YAP1, and TAZ as WWTR1) signaling is crucial for sprouting angiogenesis and vascular homeostasis. However, the underlying molecular mechanisms that explain how YAP/TAZ control the vasculature remain unclear. This study reveals that the focal adhesion protein deleted-in-liver-cancer 1 (DLC1) is a direct transcriptional target of the activated YAP/TAZ-TEAD complex. We find that substrate stiffening and VEGF stimuli promote expression of DLC1 in endothelial cells. In turn, DLC1 expression levels are YAP and TAZ dependent, and constitutive activation of YAP is sufficient to drive DLC1 expression. DLC1 is needed to limit F-actin fiber formation, integrin-based focal adhesion lifetime and integrin-mediated traction forces. Depletion of endothelial DLC1 strongly perturbs cell polarization in directed collective migration and inhibits the formation of angiogenic sprouts. Importantly, ectopic expression of DLC1 is sufficient to restore migration and angiogenic sprouting in YAP-depleted cells. Together, these findings point towards a crucial and prominent role for DLC1 in YAP/TAZ-driven endothelial adhesion remodeling and collective migration during angiogenesis.This article has an associated First Person interview with the first author of the paper.