Angiopoietin-2 reverses endothelial cell dysfunction in progeria vasculature.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder in children caused by a point mutation in the lamin A gene, resulting in a toxic form of lamin A called progerin. Accelerated atherosclerosis leading to heart attack and stroke are the major causes of death in these patients. Endothelial cell (EC) dysfunction contributes to the pathogenesis of HGPS related cardiovascular diseases (CVD). Endothelial cell-cell communications are important in the development of the vasculature, and their disruptions contribute to cardiovascular pathology. However, it is unclear how progerin interferes with such communications that lead to vascular dysfunction. An antibody array screening of healthy and HGPS patient EC secretomes identified Angiopoietin-2 (Ang2) as a down-regulated signaling molecule in HGPS ECs. A similar down-regulation of Ang2 mRNA and protein was detected in the aortas from an HGPS mouse model. Addition of Ang2 to HGPS ECs rescues vasculogenesis, normalizes endothelial cell migration and gene expression, and restores nitric oxide bioavailability through eNOS activation. Furthermore, Ang2 addition reverses unfavorable paracrine effects of HGPS ECs on vascular smooth muscle cells. Lastly, by utilizing adenine base editor (ABE)-corrected HGPS ECs and progerin-expressing HUVECs, we demonstrated a negative correlation between progerin and Ang2 expression. Lastly, our results indicated that Ang2 exerts its beneficial effect in ECs through Tie2 receptor binding, activating an Akt-mediated pathway. Together, these results provide molecular insights into EC dysfunction in HGPS and suggest that Ang2 treatment has potential therapeutic effects in HGPS-related CVD.

Mechanotransduction of the vasculature in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder that causes severe cardiovascular disease, resulting in the death of patients in their teenage years. The disease pathology is caused by the accumulation of progerin, a mutated form of the nuclear lamina protein, lamin A. Progerin binds to the inner nuclear membrane, disrupting nuclear integrity, and causes severe nuclear abnormalities and changes in gene expression. This results in increased cellular inflammation, senescence, and overall dysfunction. The molecular mechanisms by which progerin induces the disease pathology are not fully understood. Progerin's detrimental impact on nuclear mechanics and the role of the nucleus as a mechanosensor suggests dysfunctional mechanotransduction could play a role in HGPS. This is especially relevant in cells exposed to dynamic, continuous mechanical stimuli, like those of the vasculature. The endothelial (ECs) and smooth muscle cells (SMCs) within arteries rely on physical forces produced by blood flow to maintain function and homeostasis. Certain regions within arteries produce disturbed flow, leading to an impaired transduction of mechanical signals, and a reduction in cellular function, which also occurs in HGPS. In this review, we discuss the mechanics of nuclear mechanotransduction, how this is disrupted in HGPS, and what effect this has on cell health and function. We also address healthy responses of ECs and SMCs to physiological mechanical stimuli and how these responses are impaired by progerin accumulation.

Ammonia transporter RhBG initiates downstream signaling and functional responses by activating NFκB.

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG-MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG-MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors.

Bioengineered Model of Human LGMD2B Skeletal Muscle Reveals Roles of Intracellular Calcium Overload in Contractile and Metabolic Dysfunction in Dysferlinopathy.

Dysferlin is a multi-functional protein that regulates membrane resealing, calcium homeostasis, and lipid metabolism in skeletal muscle. Genetic loss of dysferlin results in limb girdle muscular dystrophy 2B/2R (LGMD2B/2R) and other dysferlinopathies - rare untreatable muscle diseases that lead to permanent loss of ambulation in humans. The mild disease severity in dysferlin-deficient mice and diverse genotype-phenotype relationships in LGMD2B patients have prompted the development of new in vitro models for personalized studies of dysferlinopathy. Here the first 3-D tissue-engineered hiPSC-derived skeletal muscle ("myobundle") model of LGMD2B is described that exhibits compromised contractile function, calcium-handling, and membrane repair, and transcriptomic changes indicative of impaired oxidative metabolism and mitochondrial dysfunction. In response to the fatty acid (FA) challenge, LGMD2B myobundles display mitochondrial deficits and intracellular lipid droplet (LD) accumulation. Treatment with the ryanodine receptor (RyR) inhibitor dantrolene or the dissociative glucocorticoid vamorolone restores LGMD2B contractility, improves membrane repair, and reduces LD accumulation. Lastly, it is demonstrated that chemically induced chronic RyR leak in healthy myobundles phenocopies LGMD2B contractile and metabolic deficit, but not the loss of membrane repair capacity. Together, these results implicate intramyocellular Ca2+ leak as a critical driver of dysferlinopathic phenotype and validate the myobundle system as a platform to study LGMD2B pathogenesis.

Multi-cellular engineered living systems to assess reproductive toxicology.

Toxicants and some drugs can negatively impact reproductive health. Many toxicants haven't been tested due to lack of available models. The impact of many drugs taken during pregnancy to address maternal health may adversely affect fetal development with life-long effects and clinical trials do not examine toxicity effects on the maternal-fetal interface, requiring indirect assessment of safety and efficacy. Due to current gaps in reproductive toxicological knowledge and limitations of animal models, multi-cellular engineered living systems may provide solutions for modeling reproductive physiology and pathology for chemical and xenobiotic toxicity studies. Multi-cellular engineered living systems, such as microphysiological systems (MPS) and organoids, model of functional units of tissues. In this review, we highlight the key functions and structures of human reproductive organs and well-known representative toxicants afflicting these systems. We then discuss current approaches and specific studies where scientists have used MPS or organoids to recreate in vivo markers and cellular responses of the female and male reproductive system, as well as pregnancy-associated placenta formation and embryo development. We provide specific examples of organoids and organ-on-chip that have been used for toxicological purposes with varied success. Finally, we address current issues related to usage of MPS, emerging techniques for improving upon these complications, and improvements needed to make MPS more capable in assessing reproductive toxicology. Overall, multi-cellular engineered living systems have considerable promise to serve as a suitable, alternative reproductive biological model compared to animal studies and 2D culture.

Genetic changes from type I interferons and JAK inhibitors: clues to drivers of juvenile dermatomyositis.

OBJECTIVE: To better understand the pathogenesis of juvenile dermatomyositis (JDM), we examined the effect of the cytokines type I interferons (IFN I) and JAK inhibitor drugs (JAKi) on gene expression in bioengineered pediatric skeletal muscle. METHODS: Myoblasts from three healthy pediatric donors were used to create three-dimensional skeletal muscle units termed myobundles. Myobundles were treated with IFN I, either IFNα or IFNß. A subset of IFNß-exposed myobundles was treated with JAKi tofacitinib or baricitinib. RNA sequencing analysis was performed on all myobundles. RESULTS: Seventy-six myobundles were analysed. Principal component analysis showed donor-specific clusters of gene expression across IFNα and IFNß-exposed myobundles in a dose-dependent manner. Both cytokines upregulated interferon response and proinflammatory genes; however, IFNß led to more significant upregulation. Key downregulated pathways involved oxidative phosphorylation, fatty acid metabolism and myogenesis genes. Addition of tofacitinib or baricitinib moderated the gene expression induced by IFNß, with partial reversal of upregulated inflammatory and downregulated myogenesis pathways. Baricitinib altered genetic profiles more than tofacitinib. CONCLUSION: IFNß leads to more pro-inflammatory gene upregulation than IFNα, correlating to greater decrease in contractile protein gene expression and reduced contractile force. JAK inhibitors, baricitinib more so than tofacitinib, partially reverse IFN I-induced genetic changes. Increased IFN I exposure in healthy bioengineered skeletal muscle leads to IFN-inducible gene expression, inflammatory pathway enrichment, and myogenesis gene downregulation, consistent with what is observed in JDM.

Effect of type I interferon on engineered pediatric skeletal muscle: a promising model for juvenile dermatomyositis.

OBJECTIVE: To investigate pathogenic mechanisms underlying JDM, we defined the effect of type I IFN, IFN-α and IFN-ß, on pediatric skeletal muscle function and expression of myositis-related proteins using an in vitro engineered human skeletal muscle model (myobundle). METHODS: Primary myoblasts were isolated from three healthy pediatric donors and used to create myobundles that mimic functioning skeletal muscle in structural architecture and physiologic function. Myobundles were exposed to 0, 5, 10 or 20 ng/ml IFN-α or IFN-ß for 7 days and then functionally tested under electrical stimulation and analyzed immunohistochemically for structural and myositis-related proteins. Additionally, IFN-ß-exposed myobundles were treated with Janus kinase inhibitors (JAKis) tofacitinib and baricitinib. These myobundles were also analyzed for contractile force and immunohistochemistry. RESULTS: IFN-ß, but not IFN-α, was associated with decreased contractile tetanus force and slowed twitch kinetics. These effects were reversed by tofacitinib and baricitinib. Type I IFN paradoxically reduced myobundle fatigue, which did not reverse after JAKi. Additionally, type I IFN correlated with MHC I upregulation, which normalized after JAKi treatment, but expression of myositis-specific autoantigens Mi-2, melanocyte differentiation-associated protein 5 and the endoplasmic reticulum stress marker GRP78 were variable and donor specific after type I IFN exposure. CONCLUSION: IFN-α and IFN-ß have distinct effects on pediatric skeletal muscle and these effects can partially be reversed by JAKi treatment. This is the first study illustrating effective use of a three-dimensional human skeletal muscle model to investigate JDM pathogenesis and test novel therapeutics.

PCSK9 activation promotes early atherosclerosis in a vascular microphysiological system.

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P-) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P- equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

The Potential of Deep Learning to Advance Clinical Applications of Computational Biomechanics.

When combined with patient information provided by advanced imaging techniques, computational biomechanics can provide detailed patient-specific information about stresses and strains acting on tissues that can be useful in diagnosing and assessing treatments for diseases and injuries. This approach is most advanced in cardiovascular applications but can be applied to other tissues. The challenges for advancing computational biomechanics for real-time patient diagnostics and treatment include errors and missing information in the patient data, the large computational requirements for the numerical solutions to multiscale biomechanical equations, and the uncertainty over boundary conditions and constitutive relations. This review summarizes current efforts to use deep learning to address these challenges and integrate large data sets and computational methods to enable real-time clinical information. Examples are drawn from cardiovascular fluid mechanics, soft-tissue mechanics, and bone biomechanics. The application of deep-learning convolutional neural networks can reduce the time taken to complete image segmentation, and meshing and solution of finite element models, as well as improving the accuracy of inlet and outlet conditions. Such advances are likely to facilitate the adoption of these models to aid in the assessment of the severity of cardiovascular disease and the development of new surgical treatments.

Lonafarnib and everolimus reduce pathology in iPSC-derived tissue engineered blood vessel model of Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal genetic disease that accelerates atherosclerosis. With a limited pool of HGPS patients, clinical trials face unique challenges and require reliable preclinical testing. We previously reported a 3D tissue engineered blood vessel (TEBV) microphysiological system fabricated with iPSC-derived vascular cells from HGPS patients. HGPS TEBVs exhibit features of HGPS atherosclerosis including loss of smooth muscle cells, reduced vasoactivity, excess extracellular matrix (ECM) deposition, inflammatory marker expression, and calcification. We tested the effects of HGPS therapeutics Lonafarnib and Everolimus separately and together, currently in Phase I/II clinical trial, on HGPS TEBVs. Everolimus decreased reactive oxygen species levels, increased proliferation, reduced DNA damage in HGPS vascular cells, and improved vasoconstriction in HGPS TEBVs. Lonafarnib improved shear stress response of HGPS iPSC-derived endothelial cells (viECs) and reduced ECM deposition, inflammation, and calcification in HGPS TEBVs. Combination treatment with Lonafarnib and Everolimus produced additional benefits such as improved endothelial and smooth muscle marker expression and reduced apoptosis, as well as increased TEBV vasoconstriction and vasodilation. These results suggest that a combined trial of both drugs may provide cardiovascular benefits beyond Lonafarnib, if the Everolimus dose can be tolerated.

Chemotherapeutic drug screening in 3D-Bioengineered human myobundles provides insight into taxane-induced myotoxicities.

Two prominent frontline breast cancer (BC) chemotherapies commonly used in combination, doxorubicin (DOX) and docetaxel (TAX), are associated with long-lasting cardiometabolic and musculoskeletal side effects. Whereas DOX has been linked to mitochondrial dysfunction, mechanisms underlying TAX-induced myotoxicities remain uncertain. Here, the metabolic and functional consequences of TAX ± DOX were investigated using a 3D-bioengineered model of adult human muscle and a drug dosing regimen designed to resemble in vivo pharmacokinetics. DOX potently reduced mitochondrial respiratory capacity, 3D-myobundle size, and contractile force, whereas TAX-induced acetylation and remodeling of the microtubule network led to perturbations in glucose uptake, mitochondrial respiratory sensitivity, and kinetics of fatigue, without compromising tetanic force generation. These findings suggest TAX-induced remodeling of the microtubule network disrupts glucose transport and respiratory control in skeletal muscle and thereby have important clinical implications related to the cardiometabolic health and quality of life of BC patients and survivors.

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle.

Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle ("myobundles"). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p < 0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo.

Late onset cardiovascular dysfunction in adult mice resulting from galactic cosmic ray exposure.

The complex and inaccessible space radiation environment poses an unresolved risk to astronaut cardiovascular health during long-term space exploration missions. To model this risk, healthy male c57BL/6 mice aged six months (corresponding to an astronaut of 34 years) were exposed to simplified galactic cosmic ray (GCR5-ion; 5-ion sim) irradiation at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratories (BNL). Multi-modal cardiovascular functional assessments performed longitudinally and terminally revealed significant impairment in cardiac function in mice exposed to GCR5-ion compared to unirradiated controls, gamma irradiation, or single mono-energetic ions (56Fe or 16O). GCR5-ion-treated mice exhibited increased arterial elastance likely mediated by disruption of elastin fibers. This study suggests that a single exposure to GCR5-ion is associated with deterioration in cardiac structure and function that becomes apparent long after exposure, likely associated with increased morbidity and mortality. These findings represent important health considerations when preparing for successful space exploration.

Principles for the design of multicellular engineered living systems.

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell-cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the "black box" of living cells.

In Situ Fabrication and Perfusion of Tissue-Engineered Blood Vessel Microphysiological System.

Human tissue-engineered blood vessels (TEBVs) that exhibit vasoactivity can be used to test drug toxicity, modulate pro-inflammatory cytokines, and model disease states in vitro. We developed a novel device to fabricate arteriole-scale human endothelialized TEBVs in situ with smaller volumes and higher throughput than previously reported. Both primary and induced pluripotent stem cell (iPSC)-derived cells can be used. Four collagen TEBVs with 600µm inner diameter and 2.9 mm outer diameter are fabricated by pipetting a solution of collagen and medial cells into a three-layer acrylic mold. After gelation, the TEBVs are released from the mold and dehydrated. After suturing the TEBVs in place and changing the mold parts to form a perfusion chamber, the TEBVs are endothelialized in situ, and then media is perfused through the lumen. By removing 90% of the water after gelation, the TEBVs become mechanically strong enough for perfusion at the physiological shear stress of 0.4 Pa within 24 h of fabrication and maintain function for at least 5 weeks.

Tissue engineered skeletal muscle model of rheumatoid arthritis using human primary skeletal muscle cells.

Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily targeting the joints. Autoreactive immune cells involved in RA affect other tissues, including skeletal muscle. Patients with RA experience diminished physical function, limited mobility, reduced muscle function, chronic pain, and increased mortality. To explore the impact of RA on skeletal muscle, we engineered electrically responsive, contractile human skeletal muscle constructs (myobundles) using primary skeletal muscle cells isolated from the vastus lateralis muscle of 11 RA patients (aged 57-74) and 10 aged healthy donors (aged 55-76), as well as from the hamstring muscle of six young healthy donors (less than 18 years of age) as a benchmark. Since all patients were receiving treatment for the disease, RA disease activity was mild. In 2D culture, RA myoblast purity, growth rate, and senescence were not statistically different than aged controls; however, RA myoblast purity showed greater variance compared to controls. Surprisingly, in 3D culture, contractile force production by RA myobundles was greater compared to aged controls. In support of this finding, assessment of RA myofiber maturation showed increased area of sarcomeric α-actinin (SAA) expression over time compared to aged controls. Furthermore, a linear regression test indicated a positive correlation between SAA protein levels and tetanus force production in RA and controls. Our findings suggest that medications prescribed to RA patients may maintain-or even enhance-muscle function, and this effect is retained and observed in in vitro culture. Future studies regarding the effects of RA therapeutics on RA skeletal muscle, in vivo and in vitro, are warranted.

Biofabrication of tissue engineering vascular systems.

Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the United States and many other developed countries. Tissue engineered vascular systems (TEVS) can serve as grafts for CVD treatment and be used as in vitro model systems to examine the role of various genetic factors during the CVD progressions. Current focus in the field is to fabricate TEVS that more closely resembles the mechanical properties and extracellular matrix environment of native vessels, which depends heavily on the advance in biofabrication techniques and discovery of novel biomaterials. In this review, we outline the mechanical and biological design requirements of TEVS and explore the history and recent advances in biofabrication methods and biomaterials for tissue engineered blood vessels and microvascular systems with special focus on in vitro applications. In vitro applications of TEVS for disease modeling are discussed.

Differentiation and characterization of human iPSC-derived vascular endothelial cells under physiological shear stress.

Induced pluripotent stem cells (iPSCs) offer a potentially unlimited source to generate endothelial cells (ECs) for numerous applications. Here, we describe a 7-day protocol to differentiate up to 55 million vascular endothelial cells (viECs) from 3.5 million human iPSCs using small molecules to regulate specific transcription factors. We also describe a parallel-plate flow chamber system to study EC behavior under physiological shear stress. For complete details on the use and execution of this protocol, please refer to Atchison et al. (2020).

Development and Application of Endothelial Cells Derived From Pluripotent Stem Cells in Microphysiological Systems Models.

The vascular endothelium is present in all organs and blood vessels, facilitates the exchange of nutrients and waste throughout different organ systems in the body, and sets the tone for healthy vessel function. Mechanosensitive in nature, the endothelium responds to the magnitude and temporal waveform of shear stress in the vessels. Endothelial dysfunction can lead to atherosclerosis and other diseases. Modeling endothelial function and dysfunction in organ systems in vitro, such as the blood-brain barrier and tissue-engineered blood vessels, requires sourcing endothelial cells (ECs) for these biomedical engineering applications. It can be difficult to source primary, easily renewable ECs that possess the function or dysfunction in question. In contrast, human pluripotent stem cells (hPSCs) can be sourced from donors of interest and renewed almost indefinitely. In this review, we highlight how knowledge of vascular EC development in vivo is used to differentiate induced pluripotent stem cells (iPSC) into ECs. We then describe how iPSC-derived ECs are being used currently in in vitro models of organ function and disease and in vivo applications.