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).

Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

Long-term health outcomes associated with hydration status.

Body water balance is determined by fundamental homeostatic mechanisms that maintain stable volume, osmolality and the composition of extracellular and intracellular fluids. Water balance is maintained by multiple mechanisms that continuously match water losses through urine, the skin, the gastrointestinal tract and respiration with water gains achieved through drinking, eating and metabolic water production. Hydration status is determined by the state of the water balance. Underhydration occurs when a decrease in body water availability, due to high losses or low gains, stimulates adaptive responses within the water balance network that are aimed at decreasing losses and increasing gains. This stimulation is also accompanied by cardiovascular adjustments. Epidemiological and experimental studies have linked markers of low fluid intake and underhydration - such as increased plasma concentration of vasopressin and sodium, as well as elevated urine osmolality - with an increased risk of new-onset chronic diseases, accelerated aging and premature mortality, suggesting that persistent activation of adaptive responses may be detrimental to long-term health outcomes. The causative nature of these associations is currently being tested in interventional trials. Understanding of the physiological responses to underhydration may help to identify possible mechanisms that underlie potential adverse, long-term effects of underhydration and inform future research to develop preventative and treatment approaches to the optimization of hydration status.

Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Derived myeloid lineage induced pluripotent stem as a platform to study human C-C chemokine receptor type 5Δ32 homozygotes.

The C-C chemokine receptor type 5 (CCR5) expressed on immune cells supports inflammatory responses by directing cells to the inflammation site. CCR5 is also a major coreceptor for macrophage tropic human immunodeficiency viruses (R5-HIV-1) and its variants can confer protection from HIV infection, making it an ideal candidate to target for therapy. We developed a stepwise protocol that differentiates induced pluripotent stem cells (iPSCs) from individuals homozygous for the CCR5Δ32 variant and healthy volunteers into myeloid lineage induced monocytes (iMono) and macrophages (iMac). By characterizing iMono and iMac against their primary counterparts, we demonstrated that CCR5Δ32 homozygous cells are endowed with similar pluripotent potential for self-renewal and differentiation as iPSC lines generated from non-variant individuals while also showing resistance to HIV infection. In conclusion, these cells are a platform to investigate CCR5 pathophysiology in HIV-positive and negative individuals and to help develop novel therapies.

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.

Middle-age high normal serum sodium as a risk factor for accelerated biological aging, chronic diseases, and premature mortality.

BACKGROUND: It is known that some people age faster than others, some people live into old age disease-free, while others develop age-related chronic diseases. With a rapidly aging population and an emerging chronic diseases epidemic, finding mechanisms and implementing preventive measures that could slow down the aging process has become a new challenge for biomedical research and public health. In mice, lifelong water restriction shortens the lifespan and promotes degenerative changes. Here, we test the hypothesis that optimal hydration may slow down the aging process in humans. METHODS: We performed a cohort analysis of data from the Atherosclerosis Risk in Communities study with middle-age enrollment (45-66 years, n = 15,752) and 25 years follow-up. We used serum sodium, as a proxy for hydration habits. To estimate the relative speed of aging, we calculated the biological age (BA) from age-dependent biomarkers and assessed risks of chronic diseases and premature mortality. FINDINGS: The analysis showed that middle age serum sodium >142 mmol/l is associated with a 39% increased risk to develop chronic diseases (hazard ratio [HR] = 1.39, 95% confidence interval [CI]:1.18-1.63) and >144 mmol/l with 21% elevated risk of premature mortality (HR = 1.21, 95% CI:1.02-1.45). People with serum sodium >142 mmol/l had up to 50% higher odds to be older than their chronological age (OR = 1.50, 95% CI:1.14-1.96). A higher BA was associated with an increased risk of chronic diseases (HR = 1.70, 95% CI:1.50-1.93) and premature mortality (HR = 1.59, 95% CI 1.39-1.83). INTERPRETATION: People whose middle-age serum sodium exceeds 142 mmol/l have increased risk to be biologically older, develop chronic diseases and die at younger age. Intervention studies are needed to confirm the link between hydration and aging. FUNDING: This work was funded by Intramural Research program of the National Heart, Lung, and Blood Institute (NHLBI). The ARIC study has been funded in whole or in part with federal funds from the NHLBI; the National Institutes of Health (NIH); and the Department of Health and Human Services.

Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration.

Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch's-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.

3D printed biomimetic flexible blood vessels with iPS cell-laden hierarchical multilayers.

Successful recovery from vascular diseases has typically relied on the surgical repair of damaged blood vessels (BVs), with the majority of current approaches involving the implantation of autologous BVs, which is plagued by donor site tissue damage. Researchers have attempted to develop artificial vessels as an alternative solution to traditional approaches to BV repair. However, the manufacturing of small-diameter (< 6 mm) BVs is still considered one of the biggest challenges due to its difficulty in the precise fabrication and the replication of biomimetic architectures. In this study, we successfully developed 3D printed flexible small-diameter BVs that consist of smooth muscle cells and a vascularized endothelium. In the developed artificial BV, a rubber-like elastomer was printed as the outermost layer of the vessel, which demonstrated enhanced mechanical properties, while and human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iSMCs) and endothelial cells (iECs) embedded fibrinogen solutions were coaxially extruded with thrombin solution to form cell-laden fibrin gel inner layers. Our results showed that the 3D BVs possessed proper mechanical properties, and the cells in the fibrin layers substantially proliferated over time to form a stable BV construct. Our study demonstrated that the 3D printed flexible small-diameter BV using iPSCs could be a promising platform for the treatment of vascular diseases.

Human induced pluripotent stem cells generated from STING-associated vasculopathy with onset in infancy (SAVI) patients with a heterozygous mutation in the STING gene.

We have successfully created induced pluripotent stem cells (iPSC) from patients carrying a heterozygous mutation in the gene encoding STING. The gain-of-function mutation leads to constitutive activation of STING which leads to the development of the disease STING-associated vasculopathy with onset in infancy (SAVI). The iPSC lines derived from the SAVI patitents are shown to be morphologically and phenotypically normal and have the potential to self renew and differentiate into the three germ layers. These iPSC provide a powerful tools to investigate the role of STING in the regulation of immune responses and vascular renegeration.

Human induced pluripotent stem cells generated from a patient with a homozygous mutation in the Lyn kinase gene.

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of the patient with a germline mutation in the coding region of the LYN kinase gene. This gain of function (GOF) mutation eliminates the inhibitory tyrosine (Y) at the position p.Y508, with an unknown established disease etiology. The iPSC carrying germline mutation in LYN are phenotypically normal, and they have capacity to differentiate toward the three germ layers. These iPSCs are critical for studying this unknown disease etiology and to the further understand the role of Lyn kinases in autoimmune disease.

Human induced pluripotent stem cells generated from Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome patients with a homozygous mutation in the PSMB8 gene (NIHTVBi016-A, NIHTVBi017-A, NIHTVBi018-A).

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts and peripheral blood mononuclear cells from patients with a homozygous missense mutation in the gene encoding PSMB8. Biallelic loss of function mutations in this gene are responsible for the PSMB8 deficiency termed Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). The iPSC carrying the homozygous PSMB8 gene mutation (c.224C > T, T75M) are phenotypically normal and have the capacity to differentiate toward the three germ layers. These iPSC have great potential to study the role of PMSB8 in the regulation of immune responses and other cellular pathways.

Diagnosis and discovery: Insights from the NIH Undiagnosed Diseases Program.

Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses, and improve patient care for patients with rare conditions.

Perspectives on Cognitive Phenotypes and Models of Vascular Disease.

Clinical investigations have established that vascular-associated medical conditions are significant risk factors for various kinds of dementia. And yet, we are unable to associate certain types of vascular deficiencies with specific cognitive impairments. The reasons for this are many, not the least of which are that most vascular disorders are multi-factorial and the development of vascular dementia in humans is often a multi-year or multi-decade progression. To better study vascular disease and its underlying causes, the National Heart, Lung, and Blood Institute of the National Institutes of Health has invested considerable resources in the development of animal models that recapitulate various aspects of human vascular disease. Many of these models, mainly in the mouse, are based on genetic mutations, frequently using single-gene mutations to examine the role of specific proteins in vascular function. These models could serve as useful tools for understanding the association of specific vascular signaling pathways with specific neurological and cognitive impairments related to dementia. To advance the state of the vascular dementia field and improve the information sharing between the vascular biology and neurobehavioral research communities, National Heart, Lung, and Blood Institute convened a workshop to bring in scientists from these knowledge domains to discuss the potential utility of establishing a comprehensive phenotypic cognitive assessment of a selected set of existing mouse models, representative of the spectrum of vascular disorders, with particular attention focused on age, sex, and rigor and reproducibility. The workshop highlighted the potential of associating well-characterized vascular disease models, with validated cognitive outcomes, that can be used to link specific vascular signaling pathways with specific cognitive and neurobehavioral deficits.

Middle age serum sodium levels in the upper part of normal range and risk of heart failure.

AIMS: With increasing prevalence of heart failure (HF) owing to the ageing population, identification of modifiable risk factors is important. In a mouse model, chronic hypohydration induced by lifelong water restriction promotes cardiac fibrosis. Hypohydration elevates serum sodium. Here, we evaluate the association of serum sodium at middle age as a measure of hydration habits with risk to develop HF. METHODS AND RESULTS: We analysed data from Atherosclerosis Risk in Communities study with middle age enrolment (45-66 years) and 25 years of follow-up. Participants without water balance dysregulation were selected: serum sodium within normal range (135-146 mmol/L), not diabetic, not obese and free of HF at baseline (N = 11 814). In time-to-event analysis, HF risk was increased by 39% if middle age serum sodium exceeded 143 mmol/L corresponding to 1% body weight water deficit [hazard ratio 1.39, 95% confidence interval (CI) 1.14-1.70]. In a retrospective case-control analysis performed on 70- to 90-year-old attendees of Visit 5 (N = 4961), serum sodium of 142.5-143 mmol/L was associated with 62% increase in odds of left ventricular hypertrophy (LVH) diagnosis [odds ratio (OR) 1.62, 95% CI 1.03-2.55]. Serum sodium above 143 mmol/L was associated with 107% increase in odds of LVH (OR 2.07, 95% CI 1.30-3.28) and 54% increase in odds of HF (OR 1.54, 95% CI 1.06-2.23). As a result, prevalence of HF and LVH was increased among 70- to 90-year-old participants with higher middle age serum sodium. CONCLUSION: Middle age serum sodium above 142 mmol is a risk factor for LVH and HF. Maintaining good hydration throughout life may slow down decline in cardiac function and decrease prevalence of HF.