Pilot study to evaluate the safety and effectiveness of etidronate treatment for arterial calcification due to deficiency of CD73 (ACDC).

BACKGROUND: Arterial calcification due to deficiency of CD73 (ACDC; OMIM 211800) is a rare genetic disease resulting in calcium deposits in arteries and small joints causing claudication, resting pain, severe joint pain, and deformities. Currently, there are no standard treatments for ACDC. Our previous work identified etidronate as a potential targeted ACDC treatment, using in vitro and in vivo disease models with patient-derived cells. In this study, we test the safety and effectiveness of etidronate in attenuating the progression of lower-extremity arterial calcification and vascular blood flow based on the computed tomography (CT) calcium score and ankle-brachial index (ABI). METHODS: Seven adult patients with a confirmed genetic diagnosis of ACDC were enrolled in an open-label, nonrandomized, single-arm pilot study for etidronate treatment. They took etidronate daily for 14 days every 3 months and were examined at the NIH Clinical Center bi-annually for 3 years. They received a baseline evaluation as well as yearly follow up after treatment. Study visits included imaging studies, exercise tolerance tests with ABIs, clinical blood and urine testing, and full dental exams. RESULTS: Etidronate treatment appeared to have slowed the progression of further vascular calcification in lower extremities as measured by CT but did not have an effect in reversing vascular and/or periarticular joint calcifications in our small ACDC cohort. CONCLUSIONS: Etidronate was found to be safe and well tolerated by our patients and, despite the small sample size, appeared to show an effect in slowing the progression of calcification in our ACDC patient cohort.(ClinicalTrials.gov Identifier NCT01585402).

Mast cell activation and degranulation in acute artery injury: A target for post-operative therapy.

The increasing incidence of cardiovascular disease (CVD) has led to a significant ongoing need to address this surgically through coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI). From this, there continues to be a substantial burden of mortality and morbidity due to complications arising from endothelial damage, resulting in restenosis. Whilst mast cells (MC) have been shown to have a causative role in atherosclerosis and other vascular diseases, including restenosis due to vein engraftment; here, we demonstrate their rapid response to arterial wire injury, recapitulating the endothelial damage seen in PCI procedures. Using wild-type mice, we demonstrate accumulation of MC in the femoral artery post-acute wire injury, with rapid activation and degranulation, resulting in neointimal hyperplasia, which was not observed in MC-deficient KitW-sh/W-sh mice. Furthermore, neutrophils, macrophages, and T cells were abundant in the wild-type mice area of injury but reduced in the KitW-sh/W-sh mice. Following bone-marrow-derived MC (BMMC) transplantation into KitW-sh/W-sh mice, not only was the neointimal hyperplasia induced, but the neutrophil, macrophage, and T-cell populations were also present in these transplanted mice. To demonstrate the utility of MC as a target for therapy, we administered the MC stabilizing drug, disodium cromoglycate (DSCG) immediately following arterial injury and were able to show a reduction in neointimal hyperplasia in wild-type mice. These studies suggest a critical role for MC in inducing the conditions and coordinating the detrimental inflammatory response seen post-endothelial injury in arteries undergoing revascularization procedures, and by targeting the rapid MC degranulation immediately post-surgery with DSCG, this restenosis may become a preventable clinical complication.

Circulating Ectonucleotidases Signal Impaired Myocardial Perfusion at Rest and Stress.

Background Ectonucleotidases maintain vascular homeostasis by metabolizing extracellular nucleotides, modulating inflammation and thrombosis, and potentially, myocardial flow through adenosine generation. Evidence implicates dysfunction or deficiency of ectonucleotidases CD39 or CD73 in human disease; the utility of measuring levels of circulating ectonucleotidases as plasma biomarkers of coronary artery dysfunction or disease has not been previously reported. Methods and Results A total of 529 individuals undergoing clinically indicated positron emission tomography stress testing between 2015 and 2019 were enrolled in this single-center retrospective analysis. Baseline demographics, clinical data, nuclear stress test, and coronary artery calcium score variables were collected, as well as a blood sample. CD39 and CD73 levels were assessed as binary (detectable, undetectable) or continuous variables using ELISAs. Plasma CD39 was detectable in 24% of White and 8% of Black study participants (P=0.02). Of the clinical history variables examined, ectonucleotidase levels were most strongly associated with underlying liver disease and not other traditional coronary artery disease risk factors. Intriguingly, detection of circulating ectonucleotidase was inversely associated with stress myocardial blood flow (2.3±0.8 mL/min per g versus 2.7 mL/min per g±1.1 for detectable versus undetectable CD39 levels, P<0.001) and global myocardial flow reserve (Pearson correlation between myocardial flow reserve and log(CD73) -0.19, P<0.001). A subanalysis showed these differences held true independent of liver disease. Conclusions Vasodilatory adenosine is the expected product of local ectonucleotidase activity, yet these data support an inverse relationship between plasma ectonucleotidases, stress myocardial blood flow (CD39), and myocardial flow reserve (CD73). These findings support the conclusion that plasma levels of ectonucleotidases, which may be shed from the endothelial surface, contribute to reduced stress myocardial blood flow and myocardial flow reserve.

Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFß-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

Molecular Mechanisms of Vascular Health: Insights From Vascular Aging and Calcification.

Cardiovascular disease is the most common cause of death worldwide, especially beyond the age of 65 years, with the vast majority of morbidity and mortality due to myocardial infarction and stroke. Vascular pathology stems from a combination of genetic risk, environmental factors, and the biologic changes associated with aging. The pathogenesis underlying the development of vascular aging, and vascular calcification with aging, in particular, is still not fully understood. Accumulating data suggests that genetic risk, likely compounded by epigenetic modifications, environmental factors, including diabetes and chronic kidney disease, and the plasticity of vascular smooth muscle cells to acquire an osteogenic phenotype are major determinants of age-associated vascular calcification. Understanding the molecular mechanisms underlying genetic and modifiable risk factors in regulating age-associated vascular pathology may inspire strategies to promote healthy vascular aging. This article summarizes current knowledge of concepts and mechanisms of age-associated vascular disease, with an emphasis on vascular calcification.

Medial Arterial Calcification: A Significant and Independent Contributor of Peripheral Artery Disease.

Over 200 million individuals worldwide are estimated to have peripheral artery disease (PAD). Although the term peripheral can refer to any outer branch of the vasculature, the focus of this review is on lower-extremity arteries. The initial sequelae of PAD often include movement-induced cramping pain in the hips and legs or loss of hair and thinning of the skin on the lower limbs. PAD progresses, sometimes rapidly, to cause nonhealing ulcers and critical limb ischemia which adversely affects mobility and muscle tone; acute limb ischemia is a medical emergency. PAD causes great pain and a high risk of amputation and ultimately puts patients at significant risk for major adverse cardiovascular events. The negative impact on patients' quality of life, as well as the medical costs incurred, are huge. Atherosclerotic plaques are one cause of PAD; however, emerging clinical data now shows that nonatherosclerotic medial arterial calcification (MAC) is an equal and distinct contributor. This ATVB In Focus article will present the recent clinical findings on the prevalence and impact of MAC in PAD, discuss the known pathways that contribute specifically to MAC in the lower extremity, and highlight gaps in knowledge and tools that limit our understanding of MAC pathogenesis.

PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1.

OBJECTIVE: Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow, which is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. In this study, we determine the role of PGC1α (peroxisome proliferator gamma coactivator-1α)-TERT (telomerase reverse transcriptase)-HMOX1 (heme oxygenase-1) during shear stress in vitro and in vivo. Approach and Results: Here, we have identified PGC1α as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared with oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) showed increased PGC1α expression and its transcriptional coactivation. PGC1α was required for laminar FSS-induced expression of TERT in vitro and in vivo via its association with ERRα(estrogen-related receptor alpha) and KLF (Kruppel-like factor)-4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status, HMOX1 was required for endothelial alignment to laminar FSS. CONCLUSIONS: These data suggest an important role for a PGC1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

Forestalling age-impaired angiogenesis and blood flow by targeting NOX: Interplay of NOX1, IL-6, and SASP in propagating cell senescence.

In an aging population, intense interest has shifted toward prolonging health span. Mounting evidence suggests that cellular reactive species are propagators of cell damage, inflammation, and cellular senescence. Thus, such species have emerged as putative provocateurs and targets for senolysis, and a clearer understanding of their molecular origin and regulation is of paramount importance. In an inquiry into signaling triggered by aging and proxy instigator, hyperglycemia, we show that NADPH Oxidase (NOX) drives cell DNA damage and alters nuclear envelope integrity, inflammation, tissue dysfunction, and cellular senescence in mice and humans with similar causality. Most notably, selective NOX1 inhibition rescues age-impaired blood flow and angiogenesis, vasodilation, and the endothelial cell wound response. Indeed, NOX1i delivery in vivo completely reversed age-impaired hind-limb blood flow and angiogenesis while disrupting a NOX1-IL-6 senescence-associated secretory phenotype (SASP) proinflammatory signaling loop. Relevant to its comorbidity with age, clinical samples from diabetic versus nondiabetic subjects reveal as operant this NOX1-mediated vascular senescence and inflammation in humans. On a mechanistic level, our findings support a previously unidentified role for IL-6 in this feedforward inflammatory loop and peroxisome proliferator-activated receptor gamma (PPARγ) down-regulation as inversely modulating p65-mediated NOX1 transcription. Targeting this previously unidentified NOX1-SASP signaling axis in aging is predicted to be an effective strategy for mitigating senescence in the vasculature and other organ systems.

Medial Arterial Calcification: JACC State-of-the-Art Review.

Medial arterial calcification (MAC) is a chronic systemic vascular disorder distinct from atherosclerosis that is frequently but not always associated with diabetes mellitus, chronic kidney disease, and aging. MAC is also a part of more complex phenotypes in numerous less common diseases. The hallmarks of MAC include disseminated and progressive precipitation of calcium phosphate within the medial layer, a prolonged and clinically silent course, and compromise of hemodynamics associated with chronic limb-threatening ischemia. MAC increases the risk of complications during vascular interventions and mitigates their outcomes. With the exception of rare monogenetic defects affecting adenosine triphosphate metabolism, MAC pathogenesis remains unknown, and causal therapy is not available. Implementation of genetics and omics-based approaches in research recognizing the critical importance of calcium phosphate thermodynamics holds promise to unravel MAC molecular pathogenesis and to provide guidance for therapy. The current state of knowledge concerning MAC is reviewed, and future perspectives are outlined.

Building Your Mentoring Network in Your Early Career.

The pursuit of knowledge, curiosity about the natural world, and a drive to better the human condition are several of the many motivations that encourage someone to further their education in the biological sciences. However noble the intentions, success in an academic graduate program, and perhaps more importantly, in the career options that follow, is not guaranteed. While it is often said that a trainee needs support from their mentors and network to succeed, the Arteriosclerosis, Thrombosis and Vascular Biology Early Career Committee has observed, through our many interactions, both face to face and in the virtual space, that many trainees do not appreciate that building their mentoring network is an active process, and the trainee has more agency in the relationship than perhaps they perceive. In the article below, we discuss our views on building relationships and identifying mentors at different levels and for different purposes. We also highlight events hosted by the Arteriosclerosis, Thrombosis and Vascular Biology Early Career Committee at Vascular Discoveries, Scientific Sessions, and in the virtual space that can help you at the critical career stage.

Isolation of Human Primary Valve Cells for In vitro Disease Modeling.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

Multifocal calcific periarthritis with distinctive clinical and radiological features in patients with CD73 deficiency.

OBJECTIVES: Arterial calcification due to deficiency of CD73 (ACDC) is a hereditary autosomal recessive ectopic mineralization syndrome caused by loss-of-function mutations in the ecto-5'-nucleotidase gene. Periarticular calcification has been reported but the clinical characterization of arthritis as well as the microstructure and chemical composition of periarticular calcifications and SF crystals has not been systematically investigated. METHODS: Eight ACDC patients underwent extensive rheumatological and radiological evaluation over a period of 11 years. Periarticular and synovial biopsies were obtained from four patients. Characterization of crystal composition was evaluated by compensated polarized light microscopy, Alizarin Red staining for synovial fluid along with X-ray diffraction and X-ray micro tomosynthesis scanner for periarticular calcification. RESULTS: Arthritis in ACDC patients has a clinical presentation of mixed erosive-degenerative joint changes with a median onset of articular symptoms at 17 years of age and progresses over time to the development of fixed deformities and functional limitations of small peripheral joints with, eventually, larger joint and distinct axial involvement later in life. We have identified calcium pyrophosphate and calcium hydroxyapatite (CHA) crystals in SF specimens and determined that CHA crystals are the principal component of periarticular calcifications. CONCLUSION: This is the largest study in ACDC patients to describe erosive peripheral arthropathy and axial enthesopathic calcifications over a period of 11 years and the first to identify the composition of periarticular calcifications and SF crystals. ACDC should be considered among the genetic causes of early-onset OA, as musculoskeletal disease signs may often precede vascular symptoms.

Dysregulation of FOXO1 (Forkhead Box O1 Protein) Drives Calcification in Arterial Calcification due to Deficiency of CD73 and Is Present in Peripheral Artery Disease.

OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.