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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.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL1 and SHARPIN, and is essential for proper immune responses. Patients with HOIP and HOIL1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage. In mice, the loss ofSharpinleads to severe dermatitis due to excessive cell death in keratinocytes. Here we report the first patient with SHARPIN deficiency, manifesting fever, arthritis, colitis, chronic otitis media and hepatic glycogenosis but unexpectedly, not associated with dermatologic manifestations. Mechanistically, fibroblasts and B cells from patients with all three LUBAC deficiencies showed attenuated canonical NF-B response and propensity to apoptosis mediated by TNF superfamily members. Furthermore, the SHARPIN deficient patient showed substantial reduction of adenoidal germinal center B cell development. Treatment of the SHARPIN deficient patient with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical role of LUBAC as a gatekeeper for apoptosis-mediated immune dysregulation in humans.

While most cases of vascular dementia represent complex interactions between host genetics and environmental factors, mendelian forms of vascular dementia also exist. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian disease characterized by progressive vascular deterioration, cognitive deficits, and strokes. Mutations in the NOTCH3 receptor underlies the pathologies in CADASIL. NOTCH3 is primarily expressed in vascular smooth muscle cells (vSMCs) and its’ expression is critical for differentiation and functional integrity of arterial vSMCs, albeit through unclear mechanism(s). To elucidate the contribution of NOTCH3 in the maintenance of cerebral vascular architecture and function, we performed micro-computed tomography (micro-CT) on the brains of aged Notch3 -deficient animals. Micro-CT assessment of the cerebral vasculature architecture showed significant abnormalities including severe vessel dilation and tortuosity (dolicoectasia) of the middle cerebral artery and its branches in the Notch3 -/- compared to aged-match controls. To identify the molecular pathway from NOTCH3 dysregulation to the observed cerebral vascular dysfunction, we performed single-cell RNASeq on cerebral arteries isolated from young (4w) and old (104w) Notch3 -/- animals. Evaluation of the vSMC-specific transcriptomes indicated significant loss of proteins associated with muscle contraction and increased extracellular matrix production in animals that lack NOTCH3. Using a combination of immunofluorescence microscopy and in vitro functional assays, we confirmed that continued expression of Notch3 is a critical requirement for maintenance of vSMC contractile function. Impaired contractility also affected flow of cerebrospinal fluid in the parenchyma of Notch3 -/- . MRI and behavioral assessments were performed in the Notch3 -/- animals to elucidate the relationship between impaired vascular contractility to cognitive function. Taken together these findings link the molecular dysfunction of NOTCH3 through its regulation of vascular contractility and cerebral vessel architecture to altered neurological function and clarify the molecular pathways to cellular pathology of Notch3 driven dementias.

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.

As an innovative additive manufacturing process, 4D printing can be utilized to generate predesigned, self-assembly structures which can actuate time-dependent, and dynamic shape-changes. Compared to other manufacturing techniques used for tissue engineering purposes, 4D printing has the advantage of being able to fabricate reprogrammable dynamic tissue constructs that can promote uniform cellular growth and distribution. For this study, a digital light processing (DLP)-based printing technique was developed to fabricate 4D near-infrared (NIR) light-sensitive cardiac constructs with highly aligned microstructure and adjustable curvature. As the curvature of the heart is varied across its surface, the 4D cardiac constructs can change their shape on-demand to mimic and recreate the curved topology of myocardial tissue for seamless integration. To mimic the aligned structure of the human myocardium and to achieve the 4D shape change, a NIR light-sensitive 4D ink material, consisting of a shape memory polymer and graphene, was created to fabricate microgroove arrays with different widths. The results of our study illustrate that our innovative NIR-responsive 4D constructs exhibit the capacity to actuate a dynamic and remotely controllable spatiotemporal transformation. Furthermore, the optimal microgroove width was discovered via culturing human induced pluripotent stem cell-derived cardiomyocytes and mesenchymal stem cells onto the constructs' surface and analyzing both their cellular morphology and alignment. The cell proliferation profiles and differentiation of tricultured human-induced pluripotent stem cell-derived cardiomyocytes, mesenchymal stem cells, and endothelial cells, on the printed constructs, were also studied using a Cell Counting Kit-8 and immunostaining. Our results demonstrate a uniform distribution of aligned cells and excellent myocardial maturation on our 4D curved cardiac constructs. This study not only provides an efficient method for manufacturing curved tissue architectures with uniform cell distributions, but also extends the potential applications of 4D printing for tissue regeneration.

Background/Purpose: CD73 is a ecto-5′-nucleotidase located on the plasma membrane that generates adenosine from AMP in the extracellular space, and functions as a checkpoint against immune and vascular activation. CD73 inhibitors that eliminate this immune checkpoint blockade are being studied in phase 2/3 cancer clinical trials. We recently identified that lack of CD73 promotes vascular thromboinflammation and neutrophil extracellular trap (NET) formation in mice. The role of CD73 in human NETosis, however, remains to be studied. Arterial calcification due to deficiency of CD73 (ACDC) is a rare, autosomal recessive condition (~20 patients globally) that results in occlusive peripheral vascular disease. We hypothesized that the congenital absence of CD73 and its function as an immune checkpoint would heighten innate immune activation in patients with ACDC. Methods: To test this hypothesis, six patients with molecularly confirmed ACDC were recruited to the NIH for mechanistic studies. Using purified neutrophils from ACDC patients and healthy controls, spontaneous and LPS-induced NET formation was quantified by both Sytox extracellular DNA (eDNA) labeling and NET-associated MPO activity. Results: Absence of surface CD73 was confirmed by flow cytometry. Relative to healthy controls, four of six (4/6) patients with ACDC demonstrated increased ex vivo spontaneous NET formation, but not LPS-triggered NETosis. The four ACDC patients with increased spontaneous NETosis had a mean 1.7-fold increase in Sytox-stained eDNA and 2-fold increase in MPO activity compared with contemporary healthy controls. Conclusion: These data reveal marked, spontaneous NETosis in patients deficient in the ectoenzyme CD73. Comparison with LPS-stimulated neutrophils demonstrates near maximal NETosis at baseline in patients with ACDC, consistent with a model where CD73 tonically suppresses neutrophil activation to maintain vascular and immune homeostasis. Ongoing studies are exploring the mechanism of spontaneous NETosis, thrombin formation, and fibrinolysis in patients with ACDC. Taken together, these data will have relevance to patients with ACDC, and inform the thromboinflammatory risk of cancer patients receiving CD73 inhibitors.

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.

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.

A 54-year old female patient with the genetic disease of arterial calcification due to deficiency of CD73 was studied under the Undiagnosed Disease Program of the National Institutes of Health. She presented with symptoms of claudication in her 40s and later developed arthritic symptoms, ectopic calcification in her left hand and severe arterial calcifications of the lower extremities. Since little was known about the composition of the calcifications in arterial calcification due to deficiency of CD73, we investigated their chemical identity and microscopic morphology in this patient with imaging and x-ray diffraction analysis. We found that, microscopically, the bulk calcifications consisted of fragments of either solid or porous internal structure. Both periarticular and arterial calcifications were primarily hydroxyapatite crystals of the same crystalline anisotropy, but different crystalline grain sizes. This was consistent with the presence of hydroxyapatite crystals along with birefringent calcium pyrophosphate dihydrate crystals in the synovial fluid of the patients by polarized light microscopy. The result suggests that tissue calcification in both locations follow a similar biochemical mechanism caused by an increase in extracellular tissue-nonspecific alkaline phosphatase activity.

To clarify how smoking leads to heart attacks and stroke, we developed an endothelial cell model (iECs) generated from human induced Pluripotent Stem Cells (iPSC) and evaluated its responses to tobacco smoke. These iECs exhibited a uniform endothelial morphology, expressed markers PECAM1/CD31, VWF/von Willebrand Factor, and CDH5/VE-Cadherin, and exhibited tube formation and Acetyl-LDL uptake comparable to primary endothelial cells (EC). RNA sequencing (RNAseq) revealed a robust correlation coefficient between iECs and EC (R = 0.76), whereas gene responses to smoke were qualitatively nearly identical between iECs and primary ECs (R=0.86). Further analysis of transcriptional responses implicated eighteen transcription factors in regulating responses to smoke treatment, and identified gene sets regulated by each transcription factor, including oxidative stress, DNA damage/repair, ER stress, apoptosis, and cell cycle arrest. Assays for 42 cytokines in HUVEC cells and iECs identified 23 cytokines that responded dynamically to cigarette smoke. These cytokines and cellular stress response pathways describe endothelial responses for lymphocyte attachment, activation of coagulation and complement, lymphocyte growth factors, and inflammation and fibrosis; EC-initiated events that collectively lead to atherosclerosis. Thus, these studies validate the iEC model and identify transcriptional response networks by which ECs respond to tobacco smoke. Our results systematically trace how ECs use these response networks to regulate genes and pathways, and finally cytokine signals to other cells, to initiate the diverse processes that lead to atherosclerosis and cardiovascular disease.

As the field of medicine is striving forward heralded by a new era of next-generation sequencing (NGS) and integrated technologies such as bioprinting and biological material development, the utility of rare monogenetic vascular disease modeling in this landscape is starting to emerge. With their genetic simplicity and broader applicability, these patient-specific models are at the forefront of modern personalized medicine. As a collective, rare diseases are a significant burden on global healthcare systems, and rare vascular diseases make up a significant proportion of this. High costs are due to a lengthy diagnostic process, affecting all ages from infants to adults, as well as the severity and chronic nature of the disease. Their complex nature requires sophisticated disease models and integrated approaches involving multidisciplinary teams. Here, we review these emerging vascular disease models, how they contribute to our understanding of the pathomechanisms in rare vascular diseases and provide useful platforms for therapeutic discovery.

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.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.The analysis showed that middle age serum sodium142 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) and144 mmol/l with 21% elevated risk of premature mortality (HR = 1.21, 95% CI:1.02-1.45). People with serum sodium142 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).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.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.

Receptor Interacting Protein Kinase 1 (RIPK1) is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is post-translationally regulated by well characterised ubiquitylation and phosphorylation events, as well as caspase-8 mediated cleavage1–7. The physiological relevance of this cleavage remains unclear, though it is believed to inhibit activation of RIPK3 and necroptosis8. Here we show that heterozygous missense mutations p.D324N, p.D324H and p.D324Y prevent caspase cleavage of RIPK1 in humans and result in early-onset periodic fever episodes and severe intermittent lymphadenopathy, a condition we designate ‘Cleavage-resistant RIPK1-Induced Autoinflammatory’ (CRIA) syndrome. To define the mechanism for this disease we generated a cleavage-resistant Ripk1D325A mutant mouse strain. While Ripk1-/- mice die postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life.

Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class IIa HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class IIa HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease.

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.

Background Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations. Objective To explore the pathophysiology and the underlying mechanisms of TNF inhibitor response in these patients. Methods We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in DADA2 patients and studied their response to TNF inhibitor treatment. Results We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and NF-κB pathways in patients’ primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation towards the pro-inflammatory M1 macrophage subset and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2. Conclusion Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells and the endothelial cell damage is rescued with anti-TNF treatment.

There are more than 7000 described rare diseases, most lacking specific treatment. Autosomal-dominant hyper-IgE syndrome (AD-HIES, also known as Job's syndrome) is caused by mutations in STAT3. These patients present with immunodeficiency accompanied by severe nonimmunological features, including skeletal, connective tissue, and vascular abnormalities, poor postinfection lung healing, and subsequent pulmonary failure. No specific therapies are available for these abnormalities. Here, we investigated underlying mechanisms in order to identify therapeutic targets. Histological analysis of skin wounds demonstrated delayed granulation tissue formation and vascularization during skin-wound healing in AD-HIES patients. Global gene expression analysis in AD-HIES patient skin fibroblasts identified deficiencies in a STAT3-controlled transcriptional network regulating extracellular matrix (ECM) remodeling and angiogenesis, with hypoxia-inducible factor 1α (HIF-1α) being a major contributor. Consistent with this, histological analysis of skin wounds and coronary arteries from AD-HIES patients showed decreased HIF-1α expression and revealed abnormal organization of the ECM and altered formation of the coronary vasa vasorum. Disease modeling using cell culture and mouse models of angiogenesis and wound healing confirmed these predicted deficiencies and demonstrated therapeutic benefit of HIF-1α-stabilizing drugs. The study provides mechanistic insights into AD-HIES pathophysiology and suggests potential treatment options for this rare disease.

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of a patient with a homozygous p.Leu272Pro mutation in the gene encoding the linear deubiquitinase OTULIN. Biallelic loss of function mutations in this gene are responsible for the OTULIN deficiency termed Otulipenia or OTULIN-related autoinflammatory syndrome (ORAS). The iPSC carrying homozygous L272P OTULIN gene mutations are phenotypically normal and they have capacity to differentiate toward the three germ layers. These iPSC have great potential to study the role of linear ubiquitination in the regulation of immune responses and other cellular pathways.

Supplemental Digital Content is available in the text., 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.

A 4D cardiac patch with physiological adaptability may improve iPSC cell therapy for the treatment of myocardial infarction., There has been considerable progress in engineering cardiac scaffolds for the treatment of myocardial infarction (MI). However, it is still challenging to replicate the structural specificity and variability of cardiac tissues using traditional bioengineering approaches. In this study, a four-dimensional (4D) cardiac patch with physiological adaptability has been printed by beam-scanning stereolithography. By combining a unique 4D self-morphing capacity with expandable microstructure, the specific design has been shown to improve both the biomechanical properties of the patches themselves and the dynamic integration of the patch with the beating heart. Our results demonstrate improved vascularization and cardiomyocyte maturation in vitro under physiologically relevant mechanical stimulation, as well as increased cell engraftment and vascular supply in a murine chronic MI model. This work not only potentially provides an effective treatment method for MI but also contributes a cutting-edge methodology to enhance the structural design of complex tissues for organ regeneration.

We have successfully generated induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells of five patients with Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These cells carry the genetic NOTCH3 mutation present in their parental cells. These iPSC cells exhibited normal karyotype and phenotype, which were sustained through propagation. Furthermore, these iPSCs displayed the capacity of differentiating toward the three germ layers in vitro.

Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2′-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs.

Histopathology protocols often require sectioning and processing of numerous microscopy slides to survey a sample. Trade-offs between workload and sampling density means that small features can be missed. Aiming to reduce the workload of routine histology protocols and the concern over missed pathology in skipped sections, we developed a prototype x-ray tomographic scanner dedicated to rapid scouting and identification of regions of interest in pathology specimens, thereby allowing targeted histopathology analysis to replace blanket searches. In coronary artery samples of a deceased HIV patient, the scanner, called Tomopath, obtained depth-resolved cross-sectional images at 15 µm resolution in a 15-minute scan, which guided the subsequent histological sectioning and microscopy. When compared to a commercial tabletop micro-CT scanner, the prototype provided several-fold contrast-to-noise ratio in 1/11th the scan time. Correlated tomographic and histological images revealed two types of micro calcifications: scattered loose calcifications typically found in atherosclerotic lesions; isolated focal calcifications in one or several cells in the internal elastic lamina and occasionally in the tunica media, which we speculate were the initiation of medial calcification linked to kidney disease, but rarely detected at this early stage due to their similarity to particle contaminants introduced during histological processing, if not for the evidence from the tomography scan prior to sectioning. Thus, in addition to its utility as a scouting tool, in this study it provided complementary information to histological microscopy. Overall, the prototype scanner represents a step toward a dedicated scouting and complementary imaging tool for routine use in pathology labs.