A Systematic Comparison of Normal Structure and Function of the Greater Thoracic Vessels.

The greater thoracic vessels are central to a well-functioning circulatory system and are often targeted in congenital heart surgeries, yet the structure and function of these vessels have not been well studied. Here we use consistent methods to quantify and compare microstructural features and biaxial biomechanical properties of the following six greater thoracic vessels in wild-type mice: ascending thoracic aorta, descending thoracic aorta, right subclavian artery, right pulmonary artery, thoracic inferior vena cava, and superior vena cava. Specifically, we determine volume fractions and orientations of the structurally significant wall constituents (i.e., collagen, elastin, and cell nuclei) using multiphoton imaging, and we quantify vasoactive responses and mechanobiologically relevant mechanical quantities (e.g., stress, stiffness) using computer-controlled biaxial mechanical testing. Similarities and differences across systemic, pulmonary, and venous circulations highlight underlying design principles of the vascular system. Results from this study represent another step towards understanding growth and remodeling of greater thoracic vessels in health, disease, and surgical interventions by providing baseline information essential for developing and validating predictive computational models.

Tempol improves aortic mechanics in a mouse model of hypertension.

Hypertension-induced arterial remodeling is thought to be a response to increases in both mechanical stress and oxidative stress. The superoxide dismutase mimetic Tempol has been shown to reduce adverse aortic remodeling in multiple murine models of hypertension but in the absence of a detailed assessment of the biaxial biomechanics. We show that concurrent treatment with Tempol in a common mouse model of systemic hypertension results in modest reductions in both wall thickening and circumferential material stiffness that yet work together to achieve a significant reduction in calculated aortic pulse wave velocity. Reducing elevated values of pulse wave velocity engenders multiple benefits to cardiovascular function.

Effects of Age, Sex, and Extracellular Matrix Integrity on Aortic Dilatation and Rupture in a Mouse Model of Marfan Syndrome.

BACKGROUND: Transmural failure of the aorta is responsible for substantial morbidity and mortality; it occurs when mechanical stress exceeds strength. The aortic root and ascending aorta are susceptible to dissection and rupture in Marfan syndrome, a connective tissue disorder characterized by a progressive reduction in elastic fiber integrity. Whereas competent elastic fibers endow the aorta with compliance and resilience, cross-linked collagen fibers confer stiffness and strength. We hypothesized that postnatal reductions in matrix cross-linking increase aortopathy when turnover rates are high. METHODS: We combined ex vivo biaxial mechanical testing with multimodality histological examinations to quantify expected age- and sex-dependent structural vulnerability of the ascending aorta in Fbn1C1041G/+ Marfan versus wild-type mice without and with 4-week exposures to ß-aminopropionitrile, an inhibitor of lysyl oxidase-mediated cross-linking of newly synthesized elastic and collagen fibers. RESULTS: We found a strong ß-aminopropionitrile-associated sexual dimorphism in aortic dilatation in Marfan mice and aortic rupture in wild-type mice, with dilatation correlating with compromised elastic fiber integrity and rupture correlating with compromised collagen fibril organization. A lower incidence of rupture of ß-aminopropionitrile-exposed Marfan aortas associated with increased lysyl oxidase, suggesting a compensatory remodeling of collagen that slows disease progression in the otherwise compromised Fbn1C1041G/+ aorta. CONCLUSIONS: Collagen fiber structure and function in the Marfan aorta are augmented, in part, by increased lysyl oxidase in female and especially male mice, which improves structural integrity, particularly via fibrils in the adventitia. Preserving or promoting collagen cross-linking may represent a therapeutic target for an otherwise vulnerable aorta.

Persistent non-homeostatic remodeling of aortic collagen following a brief episode of hypertension: A computational study.

The healthy adult aorta exhibits a remarkable homeostatic ability to respond to sustained changes in hemodynamic loads under many circumstances, but this mechanical homeostasis can be compromised or lost in natural aging and diverse pathological processes. Herein, we investigate persistent non-homeostatic changes in the composition and mechanical properties of the thoracic aorta in adult wild-type mice following 14 days of angiotensin II-induced hypertension. We employ a multiscale computational model of arterial growth and remodeling driven by mechanosensitive and angiotensin II-related cell signaling pathways. We find that experimentally observed findings can only be recapitulated computationally if the collagen deposited during the transient period of hypertension has altered properties (deposition stretch, fiber angle, crosslinking) compared with the collagen produced in the original homeostatic state. Some of these changes are predicted to persist for at least six months after blood pressure is restored to normal levels, consistent with the experimental findings.

Biomechanical and transcriptional evidence that smooth muscle cell death drives an osteochondrogenic phenotype and severe proximal vascular disease in progeria.

Hutchinson-Gilford Progeria Syndrome results in rapid aging and severe cardiovascular sequelae that accelerate near end-of-life. We found a progressive disease process in proximal elastic arteries that was less evident in distal muscular arteries. Changes in aortic structure and function were then associated with changes in transcriptomics assessed via both bulk and single cell RNA sequencing, which suggested a novel sequence of progressive aortic disease: adverse extracellular matrix remodeling followed by mechanical stress-induced smooth muscle cell death, leading a subset of remnant smooth muscle cells to an osteochondrogenic phenotype that results in an accumulation of proteoglycans that thickens the aortic wall and increases pulse wave velocity, with late calcification exacerbating these effects. Increased central artery pulse wave velocity is known to drive left ventricular diastolic dysfunction, the primary diagnosis in progeria children. It appears that mechanical stresses above ~ 80 kPa initiate this progressive aortic disease process, explaining why elastic lamellar structures that are organized early in development under low wall stresses appear to be nearly normal whereas other medial constituents worsen progressively in adulthood. Mitigating early mechanical stress-driven smooth muscle cell loss/phenotypic modulation promises to have important cardiovascular implications in progeria patients.

FN (Fibronectin)-Integrin α5 Signaling Promotes Thoracic Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

BACKGROUND: Marfan syndrome, caused by mutations in the gene for fibrillin-1, leads to thoracic aortic aneurysms (TAAs). Phenotypic modulation of vascular smooth muscle cells (SMCs) and ECM (extracellular matrix) remodeling are characteristic of both nonsyndromic and Marfan aneurysms. The ECM protein FN (fibronectin) is elevated in the tunica media of TAAs and amplifies inflammatory signaling in endothelial and SMCs through its main receptor, integrin α5ß1. We investigated the role of integrin α5-specific signals in Marfan mice in which the cytoplasmic domain of integrin α5 was replaced with that of integrin α2 (denoted α5/2 chimera). METHODS: We crossed α5/2 chimeric mice with Fbn1mgR/mgR mice (mgR model of Marfan syndrome) to evaluate the survival rate and pathogenesis of TAAs among wild-type, α5/2, mgR, and α5/2 mgR mice. Further biochemical and microscopic analysis of porcine and mouse aortic SMCs investigated molecular mechanisms by which FN affects SMCs and subsequent development of TAAs. RESULTS: FN was elevated in the thoracic aortas from Marfan patients, in nonsyndromic aneurysms, and in mgR mice. The α5/2 mutation greatly prolonged survival of Marfan mice, with improved elastic fiber integrity, mechanical properties, SMC density, and SMC contractile gene expression. Furthermore, plating of wild-type SMCs on FN decreased contractile gene expression and activated inflammatory pathways whereas α5/2 SMCs were resistant. These effects correlated with increased NF-kB activation in cultured SMCs and mgR aortas, which was alleviated by the α5/2 mutation or NF-kB inhibition. CONCLUSIONS: FN-integrin α5 signaling is a significant driver of TAA in the mgR mouse model. This pathway thus warrants further investigation as a therapeutic target.

Smooth Muscle Cell Death Drives an Osteochondrogenic Phenotype and Severe Proximal Vascular Disease in Progeria.

Hutchinson-Gilford Progeria Syndrome results in rapid aging and severe cardiovascular sequelae that accelerate near end of life. We associate progressive deterioration of arterial structure and function with single cell transcriptional changes, which reveals a rapid disease process in proximal elastic arteries that largely spares distal muscular arteries. These data suggest a novel sequence of progressive vascular disease in progeria: initial extracellular matrix remodeling followed by mechanical stress-induced smooth muscle cell death in proximal arteries, leading a subset of remnant smooth muscle cells to an osteochondrogenic phenotypic modulation that results in an accumulation of proteoglycans that thickens the wall and increases pulse wave velocity, with late calcification exacerbating these effects. Increased pulse wave velocity drives left ventricular diastolic dysfunction, the primary diagnosis in progeria children. Mitigating smooth muscle cell loss / phenotypic modulation promises to have important cardiovascular implications in progeria patients.

Role of Axl in target organ inflammation and damage due to hypertensive aortic remodeling.

We have shown that excessive endothelial cell stretch causes release of growth arrest-specific 6 (GAS6), which activates the tyrosine kinase receptor Axl on monocytes and promotes immune activation and inflammation. We hypothesized that GAS6/Axl blockade would reduce renal and vascular inflammation and lessen renal dysfunction in the setting of chronic aortic remodeling. We characterized a model of aortic remodeling in mice following a 2-wk infusion of angiotensin II (ANG II). These mice had chronically increased pulse wave velocity, and their aortas demonstrated increased mural collagen. Mechanical testing revealed a marked loss of Windkessel function that persisted for 6 mo following ANG II infusion. Renal function studies showed a reduced ability to excrete a volume load, a progressive increase in albuminuria, and tubular damage as estimated by periodic acid Schiff staining. Treatment with the Axl inhibitor R428 beginning 2 mo after ANG II infusion had a minimal effect on aortic remodeling 2 mo later but reduced the infiltration of T cells, γ/δ T cells, and macrophages into the aorta and kidney and improved renal excretory capacity, reduced albuminuria, and reduced evidence of renal tubular damage. In humans, circulating Axl+/Siglec6+ dendritic cells and phospho-Axl+ cells correlated with pulse wave velocity and aortic compliance measured by transesophageal echo, confirming chronic activation of the GAS6/Axl pathway. We conclude that brief episodes of hypertension induce chronic aortic remodeling, which is associated with persistent low-grade inflammation of the aorta and kidneys and evidence of renal dysfunction. These events are mediated at least in part by GAS6/Axl signaling and are improved with Axl blockade.NEW & NOTEWORTHY In this study, a brief, 2-wk period of hypertension in mice led to progressive aortic remodeling, an increase in pulse wave velocity, and evidence of renal injury, dysfunction, and albuminuria. This end-organ damage was associated with persistent renal and aortic infiltration of CD8+ and γ/δ T cells. We show that this inflammatory response is likely due to GAS6/Axl signaling and can be ameliorated by blocking this pathway. We propose that the altered microvascular mechanical forces caused by increased pulse wave velocity enhance GAS6 release from the endothelium, which in turn activates Axl on myeloid cells, promoting the end-organ damage associated with aortic stiffening.

Predicting and understanding arterial elasticity from key microstructural features by bidirectional deep learning.

Microstructural features and mechanical properties are closely related in all soft biological tissues. Both yet exhibit considerable inter-individual differences and are affected by factors such as aging and disease and its progression. Histological analysis, modern in situ imaging, and biomechanical testing have deepened our understanding of these complex interrelations, yet two key questions remain: (1) Given the specific microstructure, can one predict the macroscopic mechanical properties without mechanical testing? (2) Can one quantify individual contributions of the different microstructural features to the macroscopic mechanical properties in an automated, systematic and largely unbiased way? Here we propose a bidirectional deep learning architecture to address these two questions. Our architecture uses data from standard histological analyses, two-photon microscopy and biaxial biomechanical testing. Its capabilities are demonstrated by predicting with high accuracy (R2=0.92) the evolving mechanical properties of the murine aorta during maturation and aging. Moreover, our architecture reveals that the extracellular matrix composition and organization are the most prominent factors governing the macroscopic mechanical properties of the tissues studied herein. STATEMENT OF SIGNIFICANCE: We present a physics-informed machine learning architecture that can predict macroscopic mechanical properties of arterial tissue with high accuracy (R2=0.92) from the tissue microstructure (characterized by imaging data). For the first time, this architecture enables also a fully automatic and largely unbiased quantification of the relevance of different microstructural features (such as collagen volume fraction and fiber straightness) for the macroscopic mechanical properties. This approach opens up unprecedented ways to predictive mechanical modeling of soft biological tissues. Moreover, it provides quantitative insights into the relation between tissue microstructure and its macroscopic properties that promise to play an important role in future tissue engineering.

Fibronectin-Integrin α5 Signaling in Vascular Complications of Type 1 Diabetes.

Vascular complications are a major cause of illness and death in patients with type 1 diabetes (T1D). Diabetic vascular basement membranes are enriched in fibronectin (FN), an extracellular matrix protein that amplifies inflammatory signaling in endothelial cells through its main receptor, integrin α5ß1. Binding of the integrin α5 cytoplasmic domain to phosphodiesterase 4D5 (PDE4D5), which increases phosphodiesterase catalytic activity and inhibits antiinflammatory cAMP signaling, was found to mediate these effects. Here, we examined mice in which the integrin α5 cytoplasmic domain is replaced by that of α2 (integrin α5/2) or the integrin α5 binding site in PDE4D is mutated (PDE4Dmut). T1D was induced via injection of streptozotocin and hyperlipidemia induced via injection of PCSK9 virus and provision of a high-fat diet. We found that in T1D and hyperlipidemia, the integrin α5/2 mutation reduced atherosclerosis plaque size by ∼50%, with reduced inflammatory cell invasion and metalloproteinase expression. Integrin α5/2 T1D mice also had improved blood-flow recovery from hindlimb ischemia and improved biomechanical properties of the carotid artery. By contrast, the PDE4Dmut had no beneficial effects in T1D. FN signaling through integrin α5 is thus a major contributor to diabetic vascular disease but not through its interaction with PDE4D.

Simulating progressive intramural damage leading to aortic dissection using DeepONet: an operator-regression neural network.

Aortic dissection progresses mainly via delamination of the medial layer of the wall. Notwithstanding the complexity of this process, insight has been gleaned by studying in vitro and in silico the progression of dissection driven by quasi-static pressurization of the intramural space by fluid injection, which demonstrates that the differential propensity of dissection along the aorta can be affected by spatial distributions of structurally significant interlamellar struts that connect adjacent elastic lamellae. In particular, diverse histological microstructures may lead to differential mechanical behaviour during dissection, including the pressure-volume relationship of the injected fluid and the displacement field between adjacent lamellae. In this study, we develop a data-driven surrogate model of the delamination process for differential strut distributions using DeepONet, a new operator-regression neural network. This surrogate model is trained to predict the pressure-volume curve of the injected fluid and the damage progression within the wall given a spatial distribution of struts, with in silico data generated using a phase-field finite-element model. The results show that DeepONet can provide accurate predictions for diverse strut distributions, indicating that this composite branch-trunk neural network can effectively extract the underlying functional relationship between distinctive microstructures and their mechanical properties. More broadly, DeepONet can facilitate surrogate model-based analyses to quantify biological variability, improve inverse design and predict mechanical properties based on multi-modality experimental data.

Critical Pressure of Intramural Delamination in Aortic Dissection.

Computational models of aortic dissection can examine mechanisms by which this potentially lethal condition develops and propagates. We present results from phase-field finite element simulations that are motivated by a classical but seldom repeated experiment. Initial simulations agreed qualitatively and quantitatively with data, yet because of the complexity of the problem it was difficult to discern trends. Simplified analytical models were used to gain further insight. Together, simplified and phase-field models reveal power-law-based relationships between the pressure that initiates an intramural tear and key geometric and mechanical factors-insult surface area, wall stiffness, and tearing energy. The degree of axial stretch and luminal pressure similarly influence the pressure of tearing, which was ~88 kPa for healthy and diseased human aortas having sub-millimeter-sized initial insults, but lower for larger tear sizes. Finally, simulations show that the direction a tear propagates is influenced by focal regions of weakening or strengthening, which can drive the tear towards the lumen (dissection) or adventitia (rupture). Additional data on human aortas having different predisposing disease conditions will be needed to extend these results further, but the present findings show that physiologic pressures can propagate initial medial defects into delaminations that can serve as precursors to dissection.

Mechanisms of Hypoxia-Induced Pulmonary Arterial Stiffening in Mice Revealed by a Functional Genetics Assay of Structural, Functional, and Transcriptomic Data.

Hypoxia adversely affects the pulmonary circulation of mammals, including vasoconstriction leading to elevated pulmonary arterial pressures. The clinical importance of changes in the structure and function of the large, elastic pulmonary arteries is gaining increased attention, particularly regarding impact in multiple chronic cardiopulmonary conditions. We establish a multi-disciplinary workflow to understand better transcriptional, microstructural, and functional changes of the pulmonary artery in response to sustained hypoxia and how these changes inter-relate. We exposed adult male C57BL/6J mice to normoxic or hypoxic (FiO2 10%) conditions. Excised pulmonary arteries were profiled transcriptionally using single cell RNA sequencing, imaged with multiphoton microscopy to determine microstructural features under in vivo relevant multiaxial loading, and phenotyped biomechanically to quantify associated changes in material stiffness and vasoactive capacity. Pulmonary arteries of hypoxic mice exhibited an increased material stiffness that was likely due to collagen remodeling rather than excessive deposition (fibrosis), a change in smooth muscle cell phenotype reflected by decreased contractility and altered orientation aligning these cells in the same direction as the remodeled collagen fibers, endothelial proliferation likely representing endothelial-to-mesenchymal transitioning, and a network of cell-type specific transcriptomic changes that drove these changes. These many changes resulted in a system-level increase in pulmonary arterial pulse wave velocity, which may drive a positive feedback loop exacerbating all changes. These findings demonstrate the power of a multi-scale genetic-functional assay. They also highlight the need for systems-level analyses to determine which of the many changes are clinically significant and may be potential therapeutic targets.

Evolving structure-function relations during aortic maturation and aging revealed by multiphoton microscopy.

The evolving microstructure and mechanical properties that promote homeostasis in the aorta are fundamental to age-specific adaptations and disease progression. We combine ex vivo multiphoton microscopy and biaxial biomechanical phenotyping to quantify and correlate layer-specific microstructural parameters, for the primary extracellular matrix components (fibrillar collagen and elastic lamellae) and cells (endothelial, smooth muscle, and adventitial), with mechanical properties of the mouse aorta from weaning through natural aging up to one year. The aging endothelium was characterized by progressive reductions in cell density and altered cellular orientation. The media similarly showed a progressive decrease in smooth muscle cell density and alignment though with inter-lamellar widening from intermediate to older ages, suggesting cell hypertrophy, matrix accumulation, or both. Despite not changing in tissue thickness, the aging adventitia exhibited a marked thickening and straightening of collagen fiber bundles and reduction in cell density, suggestive of age-related remodeling not growth. Multiple microstructural changes correlated with age-related increases in circumferential and axial material stiffness, among other mechanical metrics. Because of the importance of aging as a risk factor for cardiovascular diseases, understanding the normal progression of structural and functional changes is essential when evaluating superimposed disease-related changes as a function of the age of onset.

Differential propensity of dissection along the aorta.

Aortic dissections progress, in part, by delamination of the wall. Previous experiments on cut-open segments of aorta demonstrated that fluid injected within the wall delaminates the aorta in two distinct modes: stepwise progressive tearing in the abdominal aorta and a more prevalent sudden mode of tearing in the thoracic aorta that can also manifest in other regions. A microstructural understanding that delineates these two modes of tearing has remained wanting. We implemented a phase-field finite-element model of the aortic wall, motivated in part by two-photon imaging, and found correlative relations for the maximum pressure prior to tearing as a function of local geometry and material properties. Specifically, the square of the pressure of tearing relates directly to both tissue stiffness and the critical energy of tearing and inversely to the square root of the torn area; this correlation explains the sudden mode of tearing and, with the microscopy, suggests a mechanism for progressive tearing. Microscopy also confirmed that thick interlamellar radial struts are more abundant in the abdominal region of the aorta, where progressive tearing was observed previously. The computational results suggest that structurally significant radial struts increase tearing pressure by two mechanisms: confining the fluid by acting as barriers to flow and increasing tissue stiffness by holding the adjacent lamellae together. Collectively, these two phase-field models provide new insights into the mechanical factors that can influence intramural delaminations that promote aortic dissection.

Progressive Microstructural Deterioration Dictates Evolving Biomechanical Dysfunction in the Marfan Aorta.

Medial deterioration leading to thoracic aortic aneurysms arises from multiple causes, chief among them mutations to the gene that encodes fibrillin-1 and leads to Marfan syndrome. Fibrillin-1 microfibrils associate with elastin to form elastic fibers, which are essential structural, functional, and instructional components of the normal aortic wall. Compromised elastic fibers adversely impact overall structural integrity and alter smooth muscle cell phenotype. Despite significant progress in characterizing clinical, histopathological, and mechanical aspects of fibrillin-1 related aortopathies, a direct correlation between the progression of microstructural defects and the associated mechanical properties that dictate aortic functionality remains wanting. In this paper, age-matched wild-type, Fbn1 C1041G/+, and Fbn1 mgR/mgR mouse models were selected to represent three stages of increasing severity of the Marfan aortic phenotype. Ex vivo multiphoton imaging and biaxial mechanical testing of the ascending and descending thoracic aorta under physiological loading conditions demonstrated that elastic fiber defects, collagen fiber remodeling, and cell reorganization increase with increasing dilatation. Three-dimensional microstructural characterization further revealed radial patterns of medial degeneration that become more uniform with increasing dilatation while correlating strongly with increased circumferential material stiffness and decreased elastic energy storage, both of which comprise aortic functionality.

Mechanics-driven mechanobiological mechanisms of arterial tortuosity.

Arterial tortuosity manifests in many conditions, including hypertension, genetic mutations predisposing to thoracic aortopathy, and vascular aging. Despite evidence that tortuosity disrupts efficient blood flow and that it may be an important clinical biomarker, underlying mechanisms remain poorly understood but are widely appreciated to be largely biomechanical. Many previous studies suggested that tortuosity may arise via an elastic structural buckling instability, but the novel experimental-computational approach used here suggests that tortuosity arises from mechanosensitive, cell-mediated responses to local aberrations in the microstructural integrity of the arterial wall. In particular, computations informed by multimodality imaging show that aberrations in elastic fiber integrity, collagen alignment, and collagen turnover can lead to a progressive loss of structural stability that entrenches during the development of tortuosity. Interpreted in this way, microstructural defects or irregularities of the arterial wall initiate the condition and hypertension is a confounding factor.

Smart Approaches to Food Waste Final Disposal.

Food waste, among the organic wastes, is one of the most promising substrates to be used as a renewable resource. Wide availability of food waste and the high greenhouse gas impacts derived from its inappropriate disposal, boost research through food waste valorization. Several innovative technologies are applied nowadays, mainly focused on bioenergy and bioresource recovery, within a circular economy approach. Nevertheless, food waste treatment should be evaluated in terms of sustainability and considering the availability of an optimized separate collection and a suitable treatment facility. Anaerobic codigestion of waste-activated sludge with food waste is a way to fully utilize available anaerobic digestion plants, increasing biogas production, energy, and nutrient recovery and reducing greenhouse gas (GHG) emissions. Codigestion implementation in Europe is explored and discussed in this paper, taking into account different food waste collection approaches in relation to anaerobic digestion treatment and confirming the sustainability of the anaerobic process based on case studies. Household food waste disposal implementation is also analyzed, and the results show that such a waste management system is able to reduce GHG emissions due to transport reduction and increase wastewater treatment performance.

Does the Knowledge of the Local Thickness of Human Ascending Thoracic Aneurysm Walls Improve Their Mechanical Analysis?

Ascending thoracic aortic aneurysm (ATAA) ruptures are life threatening phenomena which occur in local weaker regions of the diseased aortic wall. As ATAAs are evolving pathologies, their growth represents a significant local remodeling and degradation of the microstructural architecture and thus their mechanical properties. To address the need for deeper study of ATAAs and their failure, it is required to analyze the mechanical behavior at the sub-millimeter scale by making use of accurate geometrical and kinematical measurements during their deformation. For this purpose, we propose a novel methodology that combined an accurate tool for thickness distribution measurement of the arterial wall, digital image correlation to assess local strain fields and bulge inflation to characterize the physiological and failure response of flat unruptured human ATAA specimens. The analysis of the heterogeneity of the local thickness and local physiological stress and strain was carried out for each investigated subject. At the subject level, our results state the presence of a non-consistent relationship between the local wall thickness and the local physiological strain field and high heterogeneity of the variables. At the inter-subject level, thicknesses were studied in relation to physiological strain and stress and load at rupture. The rupture pressure was correlated with neither the average thickness nor the lowest thickness of the specimens. Our results confirm that intrinsic material strength (hence structure) differs a lot from a subject to another and even within the same subject.

Recent developments in biohythane production from household food wastes: A review.

Biohythane is a hydrogen-methane blend with hydrogen concentration between 10 and 30% v/v. It can be produced from different organic substrates by two sequential anaerobic stages: a dark fermentation step followed by a second an anaerobic digestion step, for hydrogen and methane production, respectively. The advantages of this blend compared to either hydrogen or methane, as separate biofuels, are first presented in this work. The two-stage anaerobic process and the main operative parameters are then discussed. Attention is focused on the production of biohythane from household food wastes, one of the most abundant organic substrate available for anaerobic digestion: the main milestones and the future trends are exposed. In particular, the possibility to co-digest food wastes and sewage sludge to improve the process yield is discussed. Finally, the paper illustrates the developments of biohythane application in the automotive sector as well as its reduced environmental burden.