CHOP-mediated IL-23 overexpression does not drive colitis in experimental spondyloarthritis.

HLA-B27 is a major risk factor for spondyloarthritis (SpA), yet the underlying mechanisms remain unclear. HLA-B27 misfolding-induced IL-23, which is mediated by endoplasmic reticulum (ER) stress has been hypothesized to drive SpA pathogenesis. Expression of HLA-B27 and human ß2m (hß2m) in rats (HLA-B27-Tg) recapitulates key SpA features including gut inflammation. Here we determined whether deleting the transcription factor CHOP (Ddit3-/-), which mediates ER-stress induced IL-23, affects gut inflammation in HLA-B27-Tg animals. ER stress-mediated Il23a overexpression was abolished in CHOP-deficient macrophages. Although CHOP-deficiency also reduced Il23a expression in immune cells isolated from the colon of B27+ rats, Il17a levels were not affected, and gut inflammation was not reduced. Rather, transcriptome analysis revealed increased expression of pro-inflammatory genes, including Il1a, Ifng and Tnf in HLA-B27-Tg colon tissue in the absence of CHOP, which was accompanied by higher histological Z-scores. RNAScope localized Il17a mRNA to the lamina propria of the HLA-B27-Tg rats and revealed similar co-localization with Cd3e (CD3) in the presence and absence of CHOP. This demonstrates that CHOP-deficiency does not improve, but rather exacerbates gut inflammation in HLA-B27-Tg rats, indicating that HLA-B27 is not promoting gut disease through ER stress-induced IL-23. Hence, CHOP may protect rats from more severe HLA-B27-induced gut inflammation.

Endothelial cell dysfunction in cardiac disease: driver or consequence?

The vascular endothelium is a multifunctional cellular system which directly influences blood components and cells within the vessel wall in a given tissue. Importantly, this cellular interface undergoes critical phenotypic changes in response to various biochemical and hemodynamic stimuli, driving several developmental and pathophysiological processes. Multiple studies have indicated a central role of the endothelium in the initiation, progression, and clinical outcomes of cardiac disease. In this review we synthesize the current understanding of endothelial function and dysfunction as mediators of the cardiomyocyte phenotype in the setting of distinct cardiac pathologies; outline existing in vivo and in vitro models where key features of endothelial cell dysfunction can be recapitulated; and discuss future directions for development of endothelium-targeted therapeutics for cardiac diseases with limited existing treatment options.

Bone health is improving over time: data from Framingham cohorts.

Hip fractures have steadily declined in the USA. We found that bone health, as measured by bone mineral density, has significantly improved over the past 30 years. Our findings contradict previous studies and offer one explanation for the decline in hip fractures. PURPOSE: Despite the widespread undertreatment of osteoporosis, hip fractures have been declining in the USA. The reasons for this decline are unclear; however, one possible explanation could be that the bone health has improved over time. METHODS: To determine the trends in bone density in the USA, we analyzed the bone mineral density scans of 7216 subjects across three generations in the Framingham Heart Study. We compared the mean femoral bone mineral density (BMD) between cohorts then constructed a linear regression model controlling for age, sex, BMI, and smoking rates. RESULTS: We observed that the mean BMD of each successive Framingham cohort increased (p < 0.001). After controlling for age, subjects born later had higher BMD. The results from the linear-regression model developed on the original cohort indicated that the BMD of the women from the offspring and third generation were higher than what would be predicted. Younger generations demonstrated higher activity scores (p < 0.001), and lower smoking rates (p = 0.045). CONCLUSION: These data suggest that bone health, measured by bone mineral density scans, is improving in later generations, in part due to decreased smoking rates and higher rates of activity.

VEGF Secretion Drives Bone Formation in Classical MAP2K1+ Melorheostosis.

Patients with classical melorheostosis exhibit exuberant bone overgrowth in the appendicular skeleton, resulting in pain and deformity with no known treatment. Most patients have somatic, mosaic mutations in MAP2K1 (encoding the MEK1 protein) in osteoblasts and overlying skin. As with most rare bone diseases, lack of affected tissue has limited the opportunity to understand how the mutation results in excess bone formation. The aim of this study was to create a cellular model to study melorheostosis. We obtained patient skin cells bearing the MAP2K1 mutation (affected cells), and along with isogenic control normal fibroblasts reprogrammed them using the Sendai virus method into induced pluripotent stem cells (iPSCs). Pluripotency was validated by marker staining and embryoid body formation. iPSCs were then differentiated to mesenchymal stem cells (iMSCs) and validated by flow cytometry. We confirmed retention of the MAP2K1 mutation in iMSCs with polymerase chain reaction (PCR) and confirmed elevated MEK1 activity by immunofluorescence staining. Mutation-bearing iMSCs showed significantly elevated vascular endothelial growth factor (VEGF) secretion, proliferation and collagen I and IV secretion. iMSCs were then differentiated into osteoblasts, which showed increased mineralization at 21 days and increased VEGF secretion at 14 and 21 days of differentiation. Administration of VEGF to unaffected iMSCs during osteogenic differentiation was sufficient to increase mineralization. Blockade of VEGF by bevacizumab reduced mineralization in iMSC-derived affected osteoblasts and in affected primary patient-derived osteoblasts. These data indicate that patient-derived induced pluripotent stem cells recreate the elevated MEK1 activity, increased mineralization, and increased proliferation seen in melorheostosis patients. The increased bone formation is driven, in part, by abundant VEGF secretion. Modifying the activity of VEGF (a known stimulator of osteoblastogenesis) represents a promising treatment pathway to explore. iPSCs may have wide applications to other rare bone diseases. © 2023 American Society for Bone and Mineral Research (ASBMR).

Reverse engineering the FRAX algorithm: Clinical insights and systematic analysis of fracture risk.

The Fracture Risk Assessment Tool (FRAX) is a computational tool developed to predict the 10-year probability of hip fracture and major osteoporotic fracture based on inputs of patient characteristics, bone mineral density (BMD), and a set of seven clinical risk factors. While the FRAX tool is widely available and clinically validated, its underlying algorithm is not public. The relative contribution and necessity of each input parameter to the final FRAX score is unknown. We systematically collected hip fracture risk scores from the online FRAX calculator for osteopenic Caucasian women across 473,088 unique inputs. This dataset was used to dissect the FRAX algorithm and construct a reverse-engineered fracture risk model to assess the relative contribution of each input variable. Within the reverse-engineered model, age and T-Score were the strongest contributors to hip fracture risk, while BMI had marginal contribution. Of the clinical risk factors, parent history of fracture and ongoing glucocorticoid treatment had the largest additive effect on risk score. A generalized linear model largely recapitulated the FRAX tool with an R2 of 0.91. Observed effect sizes were then compared to a true patient population by creating a logistic regression model of the Study of Osteoporotic Fractures (SOF) cohort, which closely paralleled the effect sizes seen in the reverse-engineered fracture risk model. Analysis identified several clinically relevant observations of interest to FRAX users. The role of major osteoporotic fracture risk prediction in contributing to an indication of treatment need is very narrow, as the hip fracture risk prediction accounted for 98% of treatment indications for the SOF cohort. Removing any risk factor from the model substantially decreased its accuracy and confirmed that more parsimonious models are not ideal for fracture prediction. For women 65 years and older with a previous fracture, 98% of FRAX combinations exceeded the treatment threshold, regardless of T-score or other factors. For women age 70+ with a parent history of fracture, 99% of FRAX combinations exceed the treatment threshold. Based on these analyses, we re-affirm the efficacy of the FRAX as the best tool for fracture risk assessment and provide deep insight into the interplay between risk factors.

Fibroblasts from Patients with Melorheostosis Promote Angiogenesis in Healthy Endothelial Cells through Secreted Factors.

Melorheostosis is a rare sclerosing bone disease with associated vascular abnormalities in skin and bone, which is caused by somatic mosaic single nucleotide variations in the MAP2K1 gene, which encodes MAPK/extracellular signal‒regulated kinase (ERK) kinase 1. However, disease pathogenesis is poorly understood. Using patient-derived cells, we found that affected skin fibroblasts carrying the single nucleotide variations have increased activation of ERK1/2, which results in increased expression and secretion of proangiogenic factors, including VEGF. VEGF secretion was strongly reduced in affected cells after treatment with MAPK/ERK kinase 1 inhibitor trametinib. Treatment of healthy endothelial cells on matrigel with conditioned medium from affected fibroblasts induces the adoption of a proangiogenic phenotype. Direct coculture of fibroblasts and endothelial cells further shows that both secreted factors and extracellular matrix are capable of inducing a proangiogenic phenotype in healthy endothelial cells. Blocking VEGF with bevacizumab reduces the proangiogenic effect of affected fibroblasts in both the matrigel and direct coculture angiogenesis models, indicating that elevated VEGF secretion is a key mediator of increased angiogenesis in melorheostosis tissue. In conclusion, this work identifies the role of several important molecular mediators in the pathogenesis of melorheostosis, including MAPK/ERK kinase 1, phosphorylated ERK1/2, and VEGF, all of which have clinically available pharmacologic inhibitors, which could be further explored as therapeutic targets.

Incorporating a structural extracellular matrix gradient into a porcine urinary bladder matrix-based hydrogel dermal scaffold.

The increasing prevalence of chronic, nonhealing wounds necessitates the investigation of full-thickness skin substitutes conducive to host integration and wound closure. Extracellular matrix (ECM)-based hydrogel scaffolds mimic the physiological matrix environment of dermal cells, thereby conferring favorable cellular adhesion, infiltration, and proliferation. However, low-concentration ECM hydrogels rapidly lose mechanical strength as they degrade, leaving them susceptible to shrinkage from fibroblast-mediated contraction. Conversely, high-concentration ECM hydrogels are typically too dense to permit nutrient diffusion and cellular migration. This study investigates the design and fabrication of a graded-concentration hydrogel composed of porcine urinary bladder matrix (UBM) as a dermal scaffold for potential use in chronic wound treatment. Our method of UBM isolation and decellularization effectively removed native DNA while preserving matrix proteins. Hydrogels composed of a range of decellularized UBM (dUBM) concentrations were characterized and used to design a three-tiered gradient hydrogel that promoted cellular activity and maintained structural integrity. The gradient dUBM hydrogel showed stability of cross-sectional area during collagenase degradation, despite considerable loss of mass. The gradient dUBM hydrogel also resisted fibroblast-mediated contraction while supporting high surface cell viability, demonstrating the mechanical support provided by denser layers of dUBM. Overall, incorporation of an ECM concentration gradient into a porcine UBM-based hydrogel scaffold capitalizes on the unique advantages of both high and low-concentration ECM hydrogels, and mitigates the structural weaknesses that have limited the efficacy of hydrogel dermal scaffolds for chronic wounds. Our gradient design shows promise for future development of stable, pro-regenerative wound scaffolds with customized architectures using 3D printing.

Assessment of decellularized pericardial extracellular matrix and poly(propylene fumarate) biohybrid for small-diameter vascular graft applications.

Autologous grafts are the current gold standard of care for coronary artery bypass graft surgeries, but are limited by availability and plagued by high failure rates. Similarly, tissue engineering approaches to small diameter vascular grafts using naturally derived and synthetic materials fall short, largely due to inappropriate mechanical properties. Alternatively, decellularized extracellular matrix from tissue is biocompatible and has comparable strength to vessels, while poly(propylene fumarate) (PPF) has shown promising results for vascular grafts. This study investigates the integration of decellularized pericardial extracellular matrix (dECM) and PPF to create a biohybrid scaffold (dECM+PPF) suitable for use as a small diameter vascular graft. Our method to decellularize the ECM was efficient at removing DNA content and donor variability, while preserving protein composition. PPF was characterized and added to dECM, where it acted to preserve dECM against degradative effects of collagenase without disturbing the material's overall mechanics. A transport study showed that diffusion occurs across dECM+PPF without any effect from collagenase. The modulus of dECM+PPF matched that of human coronary arteries and saphenous veins. dECM+PPF demonstrated ample circumferential stress, burst pressure, and suture retention strength to survive in vivo. An in vivo study showed re-endothelialization and tissue growth. Overall, the dECM+PPF biohybrid presents a robust solution to overcome the limitations of the current methods of treatment for small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: In creating a dECM+PPF biohybrid graft, we have observed phenomena that will have a lasting impact within the scientific community. First, we found that we can reduce donor variability through decellularization, a unique use of the decellularization process. Additionally, we coupled a natural material with a synthetic polymer to capitalize on the benefits of each: the cues provided to cells and the ability to easily tune material properties, respectively. This principle can be applied to other materials in a variety of applications. Finally, we created an off-the-shelf alternative to autologous grafts with a newly developed material that has yet to be utilized in any scaffolds. Furthermore, bovine pericardium has not been investigated as a small diameter vascular graft.

Microraft array-based platform for sorting of viable microcolonies based on cell-lethal immunoassay of intracellular proteins in microcolony biopsies.

The majority of bioassays are cell-lethal and thus cannot be used for cell assay and selection prior to live-cell sorting. A quad microraft array-based platform was developed to perform semi-automated cell sampling, bioassay, and banking on ultra-small sample sizes. The system biopsies and collects colony fragments, quantifies intracellular protein levels via immunostaining, and then retrieves the living mother colonies based on the fragments' immunoassay outcome. To accomplish this, a magnetic, microwell-based plate was developed to mate directly above the microraft array and capture colony fragments with a one-to-one spatial correspondence to their mother colonies. Using the Signal Transducer and Activator of Transcription 3 (STAT3) model pathway in basophilic leukemia cells, the system was used to sort cells based on the amount of intracellular STAT3 protein phosphorylation (pSTAT3). Colonies were detected on quad arrays using bright field microscopy with 96 ± 20% accuracy (true-positive rate), 49 ± 3% of the colonies were identified as originating from a single cell, and the majority (95 ± 3%) of biopsied clonal fragments were successfully collected into the microwell plate for immunostaining. After assay, biopsied fragments were matched back to their mother colonies and mother colonies with fragments possessing the greatest and least pSTAT3/STAT3 were resampled for expansion and downstream biological assays for pSTAT3/STAT3 and immune granule exocytosis. This approach has the potential to enable colony screening and sorting based on assays not compatible with cell viability, greatly expanding the cell selection criteria available to identify cells with unique phenotypes for subsequent biomedical research.

Characterization of Tensioned PDMS Membranes for Imaging Cytometry on Microraft Arrays.

Polydimethylsiloxane (PDMS) membranes can act as sensing elements, barriers, and substrates, yet the low rigidity of the elastomeric membranes can limit their practical use in devices. Microraft arrays rely on a freestanding PDMS membrane as a substrate for cell arrays used in imaging cytometry and cellular isolation. However, the underlying PDMS membrane deforms under the weight of the cell media, making automated analytical microscopy (and thus cytometry and cell isolation) challenging. Here we report the development of microfabrication strategies and physically motivated mathematical modeling of membrane deformation of PDMS microarrays. Microraft arrays were fabricated with mechanical tension stored within the PDMS substrate. These membranes deformed 20× less than that of arrays fabricated using prior methods. Modeling of the deformation of pretensioned arrays using linear membrane theory yielded ≤15% error in predicting the array deflection and predicted the impact of cure temperatures up to 120 °C. A mathematical approach was developed to fit models of microraft shape to sparse real-world shape measurements. Automated imaging of cells on pretensioned microarrays using the focal planes predicted by the model produced high quality fluorescence images of cells, enabling accurate cell area quantification (<4% error) at increased speed (13×) relative to conventional methods. Our microfabrication method and simplified, linear modeling approach is readily applicable to control the deformation of similar membranes in MEMs devices, sensors, and microfluidics.