Role of the NO-GC/cGMP signaling pathway in platelet biomechanics.

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.

Increased platelet activation and development of thrombosis has been linked to a dysfunctional NO-GC/cGMP signaling pathway. How this pathway affects platelet stiffness, however, has not been studied yet. For the first time, we used novel microscopy techniques to investigate stiffness and shape of platelets in human and murine blood samples treated with cGMP modifying drugs. Stiffness contains information about biomechanical properties of the cytoskeleton, and shape quantifies the spreading behavior of platelets. We showed that the NO-GC/cGMP signaling pathway affects platelet stiffness, shape, and activation in human and murine blood. HIV-positive patients are often treated with medication that may disrupt the NO-GC/cGMP signaling pathway, leading to increased cardiovascular risk. We showed that treatment with cGMP-modifying drugs altered platelet shape and aggregation in blood from HIV-negative volunteers but not from HIV-positive patients treated with medication. Our study suggests that platelet stiffness and shape can be biomarkers for estimating cardiovascular risk.

Venous Thrombosis Assay in a Mouse Model of Cancer.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Compromised homeostasis in aged carotid arteries revealed by microstructural studies of elastic lamellae.

Healthy arteries are continuously subjected to diverse mechanical stimuli and adapt in order to maintain a mechanical homeostasis which is characterized by a uniform distribution of wall stresses. However, aging may compromise the homeostatic microenvironment within arteries. Structural heterogeneity has been suggested as a potential microstructural mechanism that could lead to homogeneous stress distribution across the arterial wall. Our previous study on the unfolding and stretching of the elastic lamellae revealed the underlying microstructural mechanism for equalizing the circumferential stresses through wall; inner elastic layers are wavier and unfold more than the outer layers which helps to evenly distribute lamellar stretching (Yu et al., 2018). In this study, we investigated the effect of aging on lamellar deformation and its implications for tissue homeostasis. Common carotid arteries from aged mice were imaged under a multi-photon microscope while subjected to biaxial extension and inflation at five different pressures ranging from 0 up to 120 mmHg. Lamellar unfolding during pressurization was then determined from the reconstructed cross-sectional images of elastic lamellae. Tissue-level circumferential stretch was combined with the lamellar unfolding to calculate lamellar stretching. Our results revealed that the straightness gradient of aged elastic lamellae is similar to the young ones. However, during pressurization, the inner elastic lamella of the aged mice unfolded significantly more than the inner layer in young arteries. An important finding of our study is the uneven increase in inter-lamellar space which contributed to a nonuniform stretching of the elastic lamellae of aged mice arteries, elevated stress gradient, and a shifting of the load-bearing component to adventitia. Our results shed light into the complex microstructural mechanisms that take place in aging and adversely affect arterial mechanical behavior and homeostasis.

Aging and Vascular Disease: A Multidisciplinary Overview.

Vascular aging, i.e., the deterioration of the structure and function of the arteries over the life course, predicts cardiovascular events and mortality. Vascular degeneration can be recognized before becoming clinically symptomatic; therefore, its assessment allows the early identification of individuals at risk. This opens the possibility of minimizing disease progression. To review these issues, a search was completed using PubMed, MEDLINE, and Google Scholar from 2000 to date. As a network of clinicians and scientists involved in vascular medicine, we here describe the structural and functional age-dependent alterations of the arteries, the clinical tools for an early diagnosis of vascular aging, and the cellular and molecular events implicated. It emerges that more studies are necessary to identify the best strategy to quantify vascular aging, and to design proper physical activity programs, nutritional and pharmacological strategies, as well as social interventions to prevent, delay, and eventually revert the disease.

Redox Dysregulation of Vascular Smooth Muscle Sirtuin-1 in Thoracic Aortic Aneurysm in Marfan Syndrome.

BACKGROUND: Thoracic aortic aneurysms (TAAs) are abnormal aortic dilatations and a major cardiovascular complication of Marfan syndrome. We previously demonstrated a critical role for vascular smooth muscle (VSM) SirT1 (sirtuin-1), a lysine deacetylase, against maladaptive aortic remodeling associated with chronic oxidative stress and aberrant activation of MMPs (matrix metalloproteinases). METHODS: In this study, we investigated whether redox dysregulation of SirT1 contributed to the pathogenesis of TAA using fibrillin-1 hypomorphic mice (Fbn1mgR/mgR), an established model of Marfan syndrome prone to aortic dissection/rupture. RESULTS: Oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal were significantly elevated in aortas of patients with Marfan syndrome. Moreover, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, particularly S-glutathionylation, were dramatically increased in aortas of Fbn1mgR/mgR mice, before induction of severe oxidative stress markers. Fbn1mgR/mgR aortas and VSM cells exhibited an increase in rOPTM of SirT1, coinciding with the upregulation of acetylated proteins, an index of decreased SirT1 activity, and increased MMP2/9 activity. Mechanistically, we demonstrated that TGFß (transforming growth factor beta), which was increased in Fbn1mgR/mgR aortas, stimulated rOPTM of SirT1, decreasing its deacetylase activity in VSM cells. VSM cell-specific deletion of SirT1 in Fbn1mgR/mgR mice (SMKO-Fbn1mgR/mgR) caused a dramatic increase in aortic MMP2 expression and worsened TAA progression, leading to aortic rupture in 50% of SMKO-Fbn1mgR/mgR mice, compared with 25% of Fbn1mgR/mgR mice. rOPTM of SirT1, rOPTM-mediated inhibition of SirT1 activity, and increased MMP2/9 activity were all exacerbated by the deletion of Glrx (glutaredoxin-1), a specific deglutathionylation enzyme, while being corrected by overexpression of Glrx or of an oxidation-resistant SirT1 mutant in VSM cells. CONCLUSIONS: Our novel findings strongly suggest a causal role of S-glutathionylation of SirT1 in the pathogenesis of TAA. Prevention or reversal of SirT1 rOPTM may be a novel therapeutic strategy to prevent TAA and TAA dissection/ruptures in individuals with Marfan syndrome, for which, thus far, no targeted therapy has been developed.

GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and often death. Here we report that deficiency of transcription factor GATA6 is a shared pathological feature of PA endothelial (PAEC) and smooth muscle cells (PASMC) in human PAH and experimental PH, which is responsible for maintenance of hyper-proliferative cellular phenotypes, pulmonary vascular remodeling and pulmonary hypertension. We further show that GATA6 acts as a transcription factor and direct positive regulator of anti-oxidant enzymes, and its deficiency in PAH/PH pulmonary vascular cells induces oxidative stress and mitochondrial dysfunction. We demonstrate that GATA6 is regulated by the BMP10/BMP receptors axis and its loss in PAECs and PASMC in PAH supports BMPR deficiency. In addition, we have established that GATA6-deficient PAEC, acting in a paracrine manner, increase proliferation and induce other pathological changes in PASMC, supporting the importance of GATA6 in pulmonary vascular cell communication. Treatment with dimethyl fumarate resolved oxidative stress and BMPR deficiency, reversed hemodynamic changes caused by endothelial Gata6 loss in mice, and inhibited proliferation and induced apoptosis in human PAH PASMC, strongly suggesting that targeting GATA6 deficiency may provide a therapeutic advance for patients with PAH.

Harmonic Distortion of Blood Pressure Waveform as a Measure of Arterial Stiffness.

Aging and disease alter the composition and elastic properties of the aortic wall resulting in shape changes in blood pressure waveform (BPW). Here, we propose a new index, harmonic distortion (HD), to characterize BPW and its relationship with other in vitro and in vivo measures. Using a Fourier transform of the BPW, HD is calculated as the ratio of energy above the fundamental frequency to that at the fundamental frequency. Male mice fed either a normal diet (ND) or a high fat, high sucrose (HFHS) diet for 2-10 months were used to study BPWs in diet-induced metabolic syndrome. BPWs were recorded for 20 s hourly for 24 h, using radiotelemetry. Pulse wave velocity (PWV), an in vivo measure of arterial stiffness, was measured in the abdominal aorta via ultrasound sonography. Common carotid arteries were excised from a subset of mice to determine the tangent modulus using biaxial tension-inflation test. Over a 24-h period, both HD and systolic blood pressure (SBP) show a large variability, however HD linearly decreases with increasing SBP. HD is also linearly related to tangent modulus and PWV with slopes significantly different between the two diet groups. Overall, our study suggests that HD is sensitive to changes in blood pressure and arterial stiffness and has a potential to be used as a noninvasive measure of arterial stiffness in aging and disease.

Biomechanical Properties of Mouse Carotid Arteries With Diet-Induced Metabolic Syndrome and Aging.

Metabolic syndrome increases the risk of cardiovascular diseases. Arteries gradually stiffen with aging; however, it can be worsened by the presence of conditions associated with metabolic syndrome. In this study, we investigated the combined effects of diet-induced metabolic syndrome and aging on the biomechanical properties of mouse common carotid arteries (CCA). Male mice at 2 months of age were fed a normal or a high fat and high sucrose (HFHS) diet for 2 (young group), 8 (adult group) and 18-20 (old group) months. CCAs were excised and subjected to in vitro biaxial inflation-extension tests and the Cauchy stress-stretch relationships were determined in both the circumferential and longitudinal directions. The elastic energy storage of CCAs was obtained using a four-fiber family constitutive model, while the material stiffness in the circumferential and longitudinal directions was computed. Our study showed that aging is a dominant factor affecting arterial remodeling in the adult and old mice, to a similar extent, with stiffening manifested with a significantly reduced capability of energy storage by ∼50% (p < 0.05) and decreases in material stiffness and stress (p < 0.05), regardless of diet. On the other hand, high fat high sucrose diet resulted in an accelerated arterial remodeling in the young group at pre-diabetic stage by affecting the circumferential material stiffness and stress (p < 0.05), which was eventually overshadowed by aging progression. These findings have important implications on the effects of metabolic syndrome on elastic arteries in the younger populations.

Aging and Hypercholesterolemia Differentially Affect the Unfolded Protein Response in the Vasculature of ApoE-/- Mice.

Background Persistent activation of endoplasmic reticulum stress and the unfolded protein response (UPR) induces vascular cell apoptosis, contributing to atherogenesis. Aging and hypercholesterolemia are 2 independent proatherogenic factors. How they affect vascular UPR signaling remains unclear. Methods and Results Transcriptome analysis of aortic tissues from high fat diet-fed and aged ApoE-/- mice revealed 50 overlapping genes enriched for endoplasmic reticulum stress- and UPR-related pathways. Aortae from control, Western diet (WD)-fed, and aged ApoE-/- mice were assayed for (1) 3 branches of UPR signaling (pancreatic ER eIF2-alpha kinase /alpha subunit of the eukaryotic translation initiation factor 1/activating transcription factor 4, inositol-requiring enzyme 1 alpha/XBP1s, activating transcription factor 6); (2) UPR-mediated protective adaptation (upregulation of immunoglobulin heavy chain-binding protein and protein disulfide isomerase); and (3) UPR-mediated apoptosis (induction of C/EBP homologous transcription factor, p-JNK, and cleaved caspase-3). Aortic UPR signaling was differentially regulated in the aged and WD-fed groups. Consumption of WD activated all 3 UPR branches; in the aged aorta, only the ATF6α arm was activated, but it was 10 times higher than that in the WD group. BiP and protein disulfide isomerase protein levels were significantly decreased only in the aged aorta despite a 5-fold increase in their mRNA levels. Importantly, the aortae of aged mice exhibited a substantially enhanced proapoptotic UPR compared with that of WD-fed mice. In lung tissues, UPR activation and the resultant adaptive/apoptotic responses were not significantly different between the 2 groups. Conclusions Using a mouse model of atherosclerosis, this study provides the first in vivo evidence that aging and an atherogenic diet activate differential aortic UPR pathways, leading to distinct vascular responses. Compared with dietary intervention, aging is associated with impaired endoplasmic reticulum protein folding and increased aortic apoptosis.

BCL11B Regulates Arterial Stiffness and Related Target Organ Damage.

[Figure: see text].

Aging-induced microbleeds of the mouse thalamus compared to sensorimotor and memory defects.

The aim of this study is to investigate the relationship between aging and brain vasculature health. Three groups of mice, 3, 17-18, and 24 months, comparable to young adult, middle age, and old human were studied. Prussian blue histology and fast imaging with steady precession T2∗-weighted magnetic resonance imaging were used to quantify structural changes in the brain across age groups. The novel object recognition test was used to assess behavioral changes associated with anatomical changes. This study is the first to show that the thalamus is the most vulnerable brain region in the mouse model for aging-induced vascular damage. Magnetic resonance imaging data document the timeline of accumulation of thalamic damage. Histological data reveal that the majority of vascular damage accumulates in the ventroposterior nucleus and mediodorsal thalamic nucleus. Functional studies indicate that aging-induced vascular damage in the thalamus is associated with memory and sensorimotor deficits. This study points to the possibility that aging-associated vascular disease is a factor in irreversible brain damage as early as middle age.

The Role of Sirtuin-1 in the Vasculature: Focus on Aortic Aneurysm.

Sirtuin-1 (SirT1) is a nicotinamide adenine dinucleotide-dependent deacetylase and the best characterized member of the sirtuins family in mammalians. Sirtuin-1 shuttles between the cytoplasm and the nucleus, where it deacetylates histones and non-histone proteins involved in a plethora of cellular processes, including survival, growth, metabolism, senescence, and stress resistance. In this brief review, we summarize the current knowledge on the anti-oxidant, anti-inflammatory, anti-apoptotic, and anti-senescence effects of SirT1 with an emphasis on vascular diseases. Specifically, we describe recent research advances on SirT1-mediated molecular mechanisms in aortic aneurysm (AA), and how these processes relate to oxidant stress and the heme-oxygenase (HO) system. HO-1 and HO-2 catalyze the rate-limiting step of cellular heme degradation and, similar to SirT1, HO-1 exerts beneficial effects in the vasculature through the activation of anti-oxidant, anti-inflammatory, anti-apoptotic, and anti-proliferative signaling pathways. SirT1 and HO-1 are part of an integrated system for cellular stress tolerance, and may positively interact to regulate vascular function. We further discuss sex differences in HO-1 and SirT1 activity or expression, and the potential interactions between the two proteins, in relation to the progression and severity of AA, as well as the ongoing efforts for translational applications of SirT1 activation and HO-1 induction in the treatment of cardiovascular diseases including AA.

Smooth muscle NADPH oxidase 4 promotes angiotensin II-induced aortic aneurysm and atherosclerosis by regulating osteopontin.

BACKGROUND AND AIMS: Angiotensin II (Ang II) is commonly used to induce aortic aneurysm and atherosclerosis in animal models. Ang II upregulates NADPH oxidase isoform Nox4 in aortic smooth muscle cells (SMCs) in mice. However, whether smooth muscle Nox4 is directly involved in Ang II-induced aortic aneurysm and atherosclerosis is unclear. METHODS & RESULTS: To address this, we used smooth muscle-specific Nox4 dominant-negative (SDN) transgenic mice, in which Nox4 activity is constitutively inhibited. In non-transgenic (NTg) mice, Ang II increased the expression of proteins known to contribute to both aortic aneurysm and atherosclerosis, namely osteopontin (OPN), collagen type I&III (Col I&III), matrix metalloproteinase 2 (MMP2), and vascular cell adhesion molecule 1 (VCAM1), which were all significantly downregulated in SDN mice. The number and size of Ang II-induced aorta collateral aneurysms and atherosclerotic lesions in the renal artery and aortic root of SDN mice were significantly decreased compared to NTg mice, and directly correlated with a decrease in OPN expression. Replenishing OPN in SDN SMCs, increased the expression of Col I&III, MMP2, and VCAM1, and promoted SMC proliferation, migration, and inflammation. CONCLUSIONS: Our data demonstrate that smooth muscle Nox4 directly promotes the development of Ang II-induced aortic aneurysm and atherosclerosis, at least in part, through regulating OPN expression.

Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators.

Sirtuin-1 (SirT1) catalyzes NAD+-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regulate SirT1 activity. Technical barriers against measuring endogenous SirT1 activity have limited characterization of SirT1 in disease and its activation by small molecules. Herein, we developed a relative quantitative mass spectrometry-based technique for measuring endogenous SirT1 activity (RAMSSAY/RelAtive Mass Spectrometry Sirt1 Activity assaY) in cell and tissue homogenates using a biotin-labeled, acetylated p53-derived peptide as a substrate. We demonstrate that oxidative and metabolic stress diminish SirT1 activity in the hepatic cell line HepG2. Moreover, pharmacologic molecules including nicotinamide and EX-527 attenuate SirT1 activity; purported activators of SirT1, the polyphenol S17834, the polyphenol resveratrol, or the non-polyphenolic Sirtris compound SRT1720, failed to activate endogenous SirT1 significantly. Furthermore, we provide evidence that feeding a high fat high sucrose diet (HFHS) to mice inhibits endogenous SirT1 activity in mouse liver. In summary, we introduce a robust, specific and sensitive mass spectrometry-based assay for detecting and quantifying endogenous SirT1 activity using a biotin-labeled peptide in cell and tissue lysates. With this assay, we determine how pharmacologic molecules and metabolic and oxidative stress regulate endogenous SirT1 activity. The assay may also be adapted for other sirtuin isoforms.

Micromechanics of elastic lamellae: unravelling the role of structural inhomogeneity in multi-scale arterial mechanics.

Microstructural deformation of elastic lamellae plays important roles in maintaining arterial tissue homeostasis and regulating vascular smooth muscle cell fate. Our study unravels the underlying microstructural origin that enables elastic lamellar layers to evenly distribute the stresses through the arterial wall caused by intraluminal distending pressure, a fundamental requirement for tissue and cellular function. A new experimental approach was developed to quantify the spatial organization and unfolding of elastic lamellar layers under pressurization in mouse carotid arteries by coupling physiological extension-inflation and multiphoton imaging. Tissue-level circumferential stretch was obtained from analysis of the deformation of a thick-walled cylinder. Our results show that the unfolding and extension of lamellar layers contribute simultaneously to tissue-level deformation. The inner lamellar layers are wavier and unfold more than the outer layers. This waviness gradient compensates the larger tissue circumferential stretch experienced at the inner surface, thus equalizing lamellar layer extension through the arterial wall. Discoveries from this study reveal the importance of structural inhomogeneity in maintaining tissue homeostasis through the arterial wall, and may have profound implications on vascular remodelling in aging and diseases, as well as in tissue engineering of functional blood vessels.

High-Fat, High-Sugar Diet-Induced Subendothelial Matrix Stiffening is Mitigated by Exercise.

Consumption of a high-fat, high-sugar diet and sedentary lifestyle are correlated with bulk arterial stiffening. While measurements of bulk arterial stiffening are used to assess cardiovascular health clinically, they cannot account for changes to the tissue occurring on the cellular scale. The compliance of the subendothelial matrix in the intima mediates vascular permeability, an initiating step in atherosclerosis. High-fat, high-sugar diet consumption and a sedentary lifestyle both cause micro-scale subendothelial matrix stiffening, but the impact of these factors in concert remains unknown. In this study, mice on a high-fat, high-sugar diet were treated with aerobic exercise or returned to a normal diet. We measured bulk arterial stiffness through pulse wave velocity and subendothelial matrix stiffness ex vivo through atomic force microscopy. Our data indicate that while diet reversal mitigates high-fat, high-sugar diet-induced macro- and micro-scale stiffening, exercise only significantly decreases micro-scale stiffness and not macro-scale stiffness, during the time-scale studied. These data underscore the need for both healthy diet and exercise to maintain vascular health. These data also indicate that exercise may serve as a key lifestyle modification to partially reverse the deleterious impacts of high-fat, high-sugar diet consumption, even while macro-scale stiffness indicators do not change.

Catalyzing Interdisciplinary Research and Training: Initial Outcomes and Evolution of the Affinity Research Collaboratives Model.

Team science has been recognized as critical to solving increasingly complex biomedical problems and advancing discoveries in the prevention, diagnosis, and treatment of human disease. In 2009, the Evans Center for Interdisciplinary Biomedical Research (ECIBR) was established in the Department of Medicine at Boston University School of Medicine as a new organizational paradigm to promote interdisciplinary team science. The ECIBR is made up of affinity research collaboratives (ARCs), consisting of investigators from different departments and disciplines who come together to study biomedical problems that are relevant to human disease and not under interdisciplinary investigation at the university. Importantly, research areas are identified by investigators according to their shared interests. ARC proposals are evaluated by a peer review process, and collaboratives are funded annually for up to three years.Initial outcomes of the first 12 ARCs show the value of this model in fostering successful biomedical collaborations that lead to publications, extramural grants, research networking, and training. The most successful ARCs have been developed into more sustainable organizational entities, including centers, research cores, translational research projects, and training programs.To further expand team science at Boston University, the Interdisciplinary Biomedical Research Office was established in 2015 to more fully engage the entire university, not just the medical campus, in interdisciplinary research using the ARC mechanism. This approach to promoting team science may be useful to other academic organizations seeking to expand interdisciplinary research at their institutions.

Dimethyl Fumarate ameliorates pulmonary arterial hypertension and lung fibrosis by targeting multiple pathways.

Pulmonary arterial hypertension (PAH) is a fatal condition for which there is no cure. Dimethyl Fumarate (DMF) is an FDA approved anti-oxidative and anti-inflammatory agent with a favorable safety record. The goal of this study was to assess the effectiveness of DMF as a therapy for PAH using patient-derived cells and murine models. We show that DMF treatment is effective in reversing hemodynamic changes, reducing inflammation, oxidative damage, and fibrosis in the experimental models of PAH and lung fibrosis. Our findings indicate that effects of DMF are facilitated by inhibiting pro-inflammatory NFκB, STAT3 and cJUN signaling, as well as ßTRCP-dependent degradation of the pro-fibrogenic mediators Sp1, TAZ and ß-catenin. These results provide a novel insight into the mechanism of its action. Collectively, preclinical results demonstrate beneficial effects of DMF on key molecular pathways contributing to PAH, and support its testing in PAH treatment in patients.