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Background: Thoracic aortopathy associates with extracellular matrix remodeling and altered biomechanical properties. We sought to quantify the natural history of thoracic aortopathy in a common mouse model and to correlate measures of wall remodeling such as aortic dilatation or localized mural defects with evolving microstructural composition and biomechanical properties of the wall. Methods: We combined a high-resolution multimodality imaging approach (panoramic digital image correlation and optical coherence tomography) with histopathologic examinations and biaxial mechanical testing to correlate spatially, for the first time, macroscopic mural defects and medial degeneration within the ascending aorta with local changes in aortic wall composition and mechanical properties. Results: Findings revealed strong correlations between local decreases in elastic energy storage and increases in circumferential material stiffness with increasing proximal aortic diameter and especially mural defect size. Mural defects tended to exhibit a pronounced biomechanical dysfunction that is driven by an altered organization of collagen and elastic fibers. Conclusions: While aneurysmal dilatation is often observed within particular segments of the aorta, dissection and rupture initiate as highly localized mechanical failures. We show that wall composition and material properties are compromised in regions of local mural defects, which further increases the dilatation and overall structural vulnerability of the wall. Identification of therapies focused on promoting robust collagen accumulation may protect the wall from these vulnerabilities and limit the incidence of dissection and rupture.

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 similar to 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.

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 α5β1. 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 Fbn1 mgR/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.

Background: Arteriovenous fistulae (AVF) are the gold standard for vascular access for hemodialysis. Although the vein must thicken and dilate for successful hemodialysis, excessive wall thickness leads to stenosis causing AVF failure. Since TGF-β (transforming growth factor-beta) regulates ECM (extracellular matrix) deposition and smooth muscle cell (SMC) proliferation—critical components of wall thickness—we hypothesized that disruption of TGF-β signaling prevents excessive wall thickening during venous remodeling. Methods: A mouse aortocaval fistula model was used. SB431542—an inhibitor of TGF-β receptor I—was encapsulated in nanoparticles and applied to the AVF adventitia in C57BL/6J mice. Alternatively, AVFs were created in mice with conditional disruption of TGF-β receptors in either SMCs or endothelial cells. Doppler ultrasound was performed serially to confirm patency and to measure vessel diameters. AVFs were harvested at predetermined time points for histological and immunofluorescence analyses. Results: Inhibition of TGF-β signaling with SB431542-containing nanoparticles significantly reduced p-Smad2–positive cells in the AVF wall during the early maturation phase (days 7–21) and was associated with decreased AVF wall thickness that showed both decreased collagen density and decreased SMC proliferation. SMC-specific TGF-β signaling disruption decreased collagen density but not SMC proliferation or wall thickness. Endothelial cell–specific TGF-β signaling disruption decreased both collagen density and SMC proliferation in the AVF wall and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Conclusions: Endothelial cell–targeted TGF-β inhibition may be a translational strategy to improve AVF patency.

Background: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Methods: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell–derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. Results: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain ( MYH7 ) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin–nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein–W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Conclusions: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Expression of a neuropilin-like protein, DCBLD2, is reduced in human calcific aortic valve disease (CAVD). DCBLD2-deficient mice develop bicuspid aortic valve (BAV) and CAVD, which is more severe in BAV mice compared with tricuspid littermates. In vivo and in vitro studies link this observation to up-regulated bone morphogenic protein (BMP)2 expression in the presence of DCBLD2 down-regulation, and enhanced BMP2 signaling in BAV, indicating that a combination of genetics and BAV promotes aortic valve calcification and stenosis. This pathway may be a therapeutic target to prevent CAVD progression in BAV.

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.

Background: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of abdominal aortic aneurysm (AAA). Imaging aortic MMP activity, especially using positron emission tomography to access high sensitivity, quantitative data, could potentially improve AAA risk stratification. Here, we describe the design, synthesis, characterization, and evaluation in murine AAA and human aortic tissue of a first-in-class MMP-targeted positron emission tomography radioligand, 64 Cu-RYM2. Methods: The broad spectrum MMP inhibitor, RYM2 was synthetized, and its potency as an MMP inhibitor was evaluated by a competitive inhibition assay. Toxicology studies were performed. Tracer biodistribution was evaluated in a murine model of AAA induced by angiotensin II infusion in Apolipoprotein E-deficient mice. 64 Cu-RYM2 binding to normal and aneurysmal human aortic tissues was assessed by autoradiography. Results: RYM2 functioned as an MMP inhibitor with nanomolar affinities. Toxicology studies showed no adverse reaction in mice. Upon radiolabeling with Cu-64, the resulting tracer was stable in murine and human blood in vitro. Biodistribution and metabolite analysis in mice showed rapid renal clearance and acceptable in vivo stability. In vivo positron emission tomography/computed tomography in a murine model of AAA showed a specific aortic signal, which correlated with ex vivo measured MMP activity and Cd68 gene expression. 64 Cu-RYM2 specifically bound to normal and aneurysmal human aortic tissues in correlation with MMP activity. Conclusions: 64 Cu-RYM2 is a first-in-class MMP-targeted positron emission tomography tracer with favorable stability, biodistribution, performance in preclinical AAA, and importantly, specific binding to human tissues. These data set the stage for 64 Cu-RYM2-based translational imaging studies of vessel wall MMP activity, and indirectly, inflammation, in AAA.

Background: Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. Methods: The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h –/– mice to lethally irradiated Ldlr –/– mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr –/– or Ch25h–/–;Ldlr–/– mice . The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage–tracing mouse model, Myh11 ERT2CRE mT/mG;Ldlr –/– , adoptively transferred with wild-type or Ch25h –/– mice bone marrow followed by 12 weeks of Western diet feeding. Results: We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB–mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC–mediated modulation of liver X receptor or SREBP (sterol regulatory element–binding protein) transcriptional activity. Conclusions: Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

BackgroundMarfan syndrome, caused by mutations in the gene for the extracellular matrix (ECM) glycoprotein fibrillin-1, leads to thoracic aortic aneurysms (TAAs). Phenotypic modulation of vascular smooth muscle cells (SMCs) and ECM remodeling are characteristics of both non-syndromic and Marfan aneurysms. The ECM protein fibronectin (FN) is elevated in the tunica media of TAAs and amplifies inflammatory signaling in endothelial and SMCs through its main receptor, integrin α5β1. 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).MethodsWe used α5/2 chimera mouse crossed with Fbn1mgR/mgR genetic background (mgR, a mouse model of Marfan syndrome) to compare 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 allowed us to identify the molecular mechanisms by which FN affects SMCs and subsequent development of TAAs.ResultsFN was elevated in the thoracic aortas from Marfan patients, in non-syndromic aneurysms and in the mgR mouse model of Marfan syndrome. 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, in vitro, plating of wild-type, but not α5/2, SMCs on FN decreased contractile gene expression and activated inflammatory pathways. These effects correlated with increased NF-kB activation and immune cell infiltration in the mgR aortas, which was rescued in the α5/2 mgR aortas.ConclusionsFN-integrin α5 signaling is a significant driver of TAA in the mgR mouse model. This pathway warrants further investigation as a therapeutic target.

A significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

Background Mechanical homeostasis promotes proper aortic structure and function. Pathological conditions may arise, in part, from compromised or lost homeostasis. There is thus a need to quantify the homeostatic state and when it emerges. Here we quantify changes in mechanical loading, geometry, structure, and function of the murine aorta from the late prenatal period into maturity. Results Our data suggest that a homeostatic set-point is established by postnatal day P2 for the flow-induced shear stress experienced by endothelial cells; this value deviates from its set-point from P10 to P21 due to asynchronous changes in mechanical loading (flow, pressure) and geometry (radius, wall thickness), but is restored thereafter consistent with homeostasis. Smooth muscle contractility also decreases during this period of heightened matrix deposition but is also restored in maturity. The pressure-induced mechanical stress experienced by intramural cells initially remains low despite increasing blood pressure, and then increases while extracellular matrix accumulates. Conclusions These findings suggest that cell-level mechanical homeostasis emerges soon after birth to allow mechanosensitive cells to guide aortic development, with deposition of matrix after P2 increasingly stress shielding intramural cells. The associated tissue-level set-points that emerge for intramural stress can be used to assess and model the aorta that matures biomechanically by P56.

Alloantibodies in presensitized transplant candidates deposit complement membrane attack complexes (MACs) on graft endothelial cells (ECs), increasing risk of CD8(+) T cell–mediated acute rejection. We recently showed that human ECs endocytose MACs into Rab5(+) endosomes, creating a signaling platform that stabilizes NF-κB–inducing kinase (NIK) protein. Endosomal NIK activates both noncanonical NF-κB signaling to synthesize pro–IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β. IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4(+) T (Tem) cells. Here, we report that IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB–dependent process in which IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8(+) Tem cells. Blocking NLRP3 inflammasome assembly, IL-1 receptor, or IL-15 on ECs inhibited the augmented CD8(+) Tem cell responses, indicating that this pathway is not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice, and enhanced intimal CD8(+) T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Background: Ischemia reperfusion injury (IRI) predisposes to the formation of donor-specific antibodies, a factor contributing to chronic rejection and late allograft loss. Methods: We describe a mechanism underlying the correlative association between IRI and donor-specific antibodies by using humanized models and patient specimens. Results: IRI induces immunoglobulin M–dependent complement activation on endothelial cells that assembles an NLRP3 (NOD-like receptor pyrin domain-containing protein 3) inflammasome via a Rab5-ZFYVE21-NIK axis and upregulates ICOS-L (inducible costimulator ligand) and PD-L2 (programmed death ligand 2). Endothelial cell–derived interleukin-18 (IL-18) selectively expands a T-cell population (CD4+CD45RO+PD-1 hi ICOS+CCR2+CXCR5–) displaying features of recently described T peripheral helper cells. This population highly expressed IL-18R1 and promoted donor-specific antibodies in response to IL-18 in vivo. In patients with delayed graft function, a clinical manifestation of IRI, these cells were Ki-67+IL-18R1+ and could be expanded ex vivo in response to IL-18. Conclusions: IRI promotes elaboration of IL-18 from endothelial cells to selectively expand alloreactive IL-18R1+ T peripheral helper cells in allograft tissues to promote donor-specific antibody formation.

Smooth muscle cell (SMC) proliferation has been thought to limit the progression of thoracic aortic aneurysm and dissection (TAAD) because loss of medial cells associates with advanced disease. We investigated effects of SMC proliferation in the aortic media by conditional disruption of Tsc1, which hyperactivates mTOR complex 1. Consequent SMC hyperplasia led to progressive medial degeneration and TAAD. In addition to diminished contractile and synthetic functions, fate-mapped SMCs displayed increased proteolysis, endocytosis, phagocytosis, and lysosomal clearance of extracellular matrix and apoptotic cells. SMCs acquired a limited repertoire of macrophage markers and functions via biogenesis of degradative organelles through an mTOR/β-catenin/MITF-dependent pathway, but were distinguishable from conventional macrophages by an absence of hematopoietic lineage markers and certain immune effectors even in the context of hyperlipidemia. Similar mTOR activation and induction of a degradative SMC phenotype in a model of mild TAAD due to Fbn1 mutation greatly worsened disease with near-uniform lethality. The finding of increased lysosomal markers in medial SMCs from clinical TAAD specimens with hyperplasia and matrix degradation further supports the concept that proliferation of degradative SMCs within the media causes aortic disease, thus identifying mTOR-dependent phenotypic modulation as a therapeutic target for combating TAAD.

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3β-hydroxysterol Δ(24)-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45(+)CD11b(+) cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)–dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.

Aortic dissection and rupture are triggered by decreased vascular wall strength and/or increased mechanical loads. We investigated the role of mTOR signaling in aortopathy using a well-described model of angiotensin II-induced dissection, aneurysm, or rupture of the suprarenal abdominal aorta in Apoe-deficient mice. Although not widely appreciated, nonlethal hemorrhagic lesions present as pseudoaneurysms without significant dissection in this model. Angiotensin II-induced aortic tears result in free rupture, contained rupture with subadventitial hematoma (forming pseudoaneurysms), dilatation, or healing, while the media invariably thickens regardless of mural tears. Medial thickening results from smooth muscle cell hypertrophy and extracellular matrix accumulation, including matricellular proteins. Angiotensin II activates mTOR signaling in vascular wall cells, and inhibition of mTOR signaling by rapamycin prevents aortic rupture but promotes dissection. Decreased aortic rupture correlates with decreased inflammation and metalloproteinase expression, whereas extensive dissection correlates with induction of matricellular proteins that modulate adhesion of vascular cells. Thus, mTOR activation in vascular wall cells determines whether aortic tears progress to dissection or rupture. Previous mechanistic studies of aortic aneurysm and dissection by angiotensin II in Apoe-deficient mice should be reinterpreted as clinically relevant to pseudoaneurysms, and mTOR inhibition for aortic disease should be explored with caution.

Background Dysfunction and inflammation of hearts subjected to cold ischemic preservation may differ between left and right ventricles, suggesting distinct strategies for amelioration. Methods and Results Explanted murine hearts subjected to cold ischemia for 0, 4, or 8 h in preservation solution were assessed for function during 60 min of warm perfusion and then analyzed for cell death and inflammation by immunohistochemistry and western blotting and total RNA sequencing. Increased cold ischemic times led to greater left ventricle (LV) dysfunction compared to right ventricle (RV). The LV experienced greater cell death assessed by TUNEL+ cells and cleaved caspase‐3 expression (n = 4). While IL‐6 protein levels were upregulated in both LV and RV, IL‐1β, TNFα, IL‐10, and MyD88 were disproportionately increased in the LV. Inflammasome components (NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), adaptor molecule apoptosis‐associated speck‐like protein containing a CARD (ASC), cleaved caspase‐1) and products (cleaved IL‐1β and gasdermin D) were also more upregulated in the LV. Pathway analysis of RNA sequencing showed increased signaling related to tumor necrosis factor, interferon, and innate immunity with ex‐vivo ischemia, but no significant differences were found between the LV and RV. Human donor hearts showed comparable inflammatory responses to cold ischemia with greater LV increases of TNFα, IL‐10, and inflammasomes (n = 3). Conclusions Mouse hearts subjected to cold ischemia showed time‐dependent contractile dysfunction and increased cell death, inflammatory cytokine expression and inflammasome expression that are greater in the LV than RV. However, IL‐6 protein elevations and altered transcriptional profiles were similar in both ventricles. Similar changes are observed in human hearts., Increasing duration of murine donor heart storage with subsequent ex‐vivo perfusion using clinically utilized histidine‐tryptophan‐ketoglutarate preservation solution induced progressively higher expression of IL‐1β, MyD88, TNFα, IL‐6, MCP1, and inflammasome components. This inflammatory response was disproportionately greater in the left ventricle compared to the right ventricle. We confirm that LV contractility and relaxation were also degraded to a greater degree in the left ventricle compared to the right ventricle following progressively longer periods of preservation. Murine findings corroborated with results from human preserved donor hearts where we also observed selective upregulation of TNFα, MCP1, and inflammasome components (Nlrp3, cleaved IL‐1β‐p17) in the left ventricle compared to the right ventricle during cold preservation.

[EN] Hypertension induces significant aortic remodelling, often adaptive but sometimes not. To identify immuno-mechanical mechanisms responsible for differential remodelling, we studied thoracic aortas from 129S6/SvEvTac and C57BL/6 J mice before and after continuous 14-day angiotensin II infusion, which elevated blood pressure similarly in both strains. Histological and biomechanical assessments of excised vessels were similar at baseline, suggesting a common homeostatic set-point for mean wall stress. Histology further revealed near mechano-adaptive remodelling of the hypertensive 129S6/SvEvTac aortas, but a grossly maladaptive remodelling of C57BL/6 J aortas. Bulk RNA sequencing suggested that increased smooth muscle contractile processes promoted mechano-adaptation of 129S6/SvEvTac aortas while immune processes prevented adaptation of C57BL/6 J aortas. Functional studies confirmed an increased vasoconstrictive capacity of the former while immunohistochemistry demonstrated marked increases in inflammatory cells in the latter. We then used multiple computational biomechanical models to test the hypothesis that excessive adventitial wall stress correlates with inflammatory cell infiltration. These models consistently predicted that increased vasoconstriction against an increased pressure coupled with modest deposition of new matrix thickens the wall appropriately, restoring wall stress towards homeostatic consistent with adaptive remodelling. By contrast, insufficient vasoconstriction permits high wall stresses and exuberant inflammation-driven matrix deposition, especially in the adventitia, reflecting compromised homeostasis and gross maladaptation., This work was supported by grants from NIH (grant nos. R01HL105297, P01 HL134605, U01 HL142518, R01 HL146723), Netherlands Organisation for Scientific Research (grant no. Rubicon 452172006) and the European Union¿s Horizon 2020 research and innovation program (grant no. 793805)

Medial degeneration is a common histopathological finding in aortopathy and is considered a mechanism for dilatation. We investigated if medial degeneration is specific for sporadic thoracic aortic aneurysms versus nondilated aortas. Specimens were graded by pathologists, blinded to the clinical diagnosis, according to consensus histopathological criteria. The extent of medial degeneration by qualitative (semi-quantitative) assessment was not specific for aneurysmal compared to nondilated aortas. In contrast, blinded quantitative assessment of elastin amount and medial cell number distinguished aortic aneurysms and referent specimens, albeit with marked overlap in results. Specifically, the medial fraction of elastin decreased from dilution rather than loss of protein as cross-sectional amount was maintained while the cross-sectional number, though not density, of smooth muscle cells increased in proportion to expansion of the media. Furthermore, elastic lamellae did not thin and interlamellar distance did not diminish as expected for lumen dilatation, implying a net gain of lamellar elastin and intralamellar cells or extracellular matrix during aneurysmal wall remodeling. These findings support the concepts that: (1) medial degeneration need not induce aortic aneurysms, (2) adaptive responses to altered mechanical stresses increase medial tissue, and (3) greater turnover, not loss, of mural cells and extracellular matrix associates with aortic dilatation.

Epigenetic modifications of the genome, including DNA methylation, histone methylation/acetylation and noncoding RNAs, have been reported to play a fundamental role in regulating immune response during the progression of atherosclerosis. SETDB2 is a member of the KMT1 family of lysine methyltransferases and members of this family typically methylate histone H3 Lys9 (H3K9), an epigenetic mark associated with gene silencing and previous studies have shown SETDB2 is involved in innate and adaptive immunity, the pro-inflammatory response and hepatic lipid metabolism. Here we report that the expression of SETDB2 is markedly upregulated in human and murine atherosclerotic lesions. The upregulation of SETDB2 is observed in pro-inflammatory M1, but not anti-inflammatory M2 macrophages (MΦ). Notably, we found that genetic deletion of SETDB2 in hematopoietic cells promotes vascular inflammation and enhances the progression of atherosclerosis in bone marrow transfer studies in LDLR knockout mice. Single cell RNA-Seq analysis in isolated CD45+ cells from atherosclerotic plaques from mice with SETDB2 deficient bone marrow revealed a significant increase in inflammatory macrophage population and enhanced expression of genes involved in inflammation, myeloid cell recruitment and lipid metabolism. Additionally, we found that loss of SETDB2 in hematopoietic cells is associated with macrophage accumulation in atherosclerotic lesions, macrophage proliferation and attenuated efferocytosis. Overall, these studies identify SETDB2 as an important inflammatory cell regulator that controls macrophage activation in atherosclerotic plaques.

Transplantation of pancreatic islets has been shown to be effective, in some patients, for the long-term treatment of type 1 diabetes. However, transplantation of islets into either the portal vein or the subcutaneous space can be limited by insufficient oxygen transfer, leading to islet loss. Furthermore, oxygen diffusion limitations can be magnified when islet numbers are increased dramatically, as in translating from rodent studies to human-scale treatments. To address these limitations, an islet transplantation approach using an acellular vascular graft as a vascular scaffold has been developed, termed the BioVascular Pancreas (BVP). To create the BVP, islets are seeded as an outer coating on the surface of an acellular vascular graft, using fibrin as a hydrogel carrier. The BVP can then be anastomosed as an arterial (or arteriovenous) graft, which allows fully oxygenated arterial blood with a pO2 of roughly 100 mmHg to flow through the graft lumen and thereby supply oxygen to the islets. In silico simulations and in vitro bioreactor experiments show that the BVP design provides adequate survivability for islets and helps avoid islet hypoxia. When implanted as end-to-end abdominal aorta grafts in nude rats, BVPs were able to restore near-normoglycemia durably for 90 days and developed robust microvascular infiltration from the host. Furthermore, pilot implantations in pigs were performed, which demonstrated the scalability of the technology. Given the potential benefits provided by the BVP, this tissue design may eventually serve as a solution for transplantation of pancreatic islets to treat or cure type 1 diabetes.

Conventional in vitro methods for biological evaluation of intra-arterial devices such as stents fail to accurately predict cytotoxicity and remodeling events. An ex vivo flow-tunable vascular bioreactor system (VesselBRx), comprising intra- and extra-luminal monitoring capabilities, addresses these limitations. VesselBRx mimics the in vivo physiological, hyperplastic, and cytocompatibility events of absorbable magnesium (Mg)-based stents in ex vivo stent-treated porcine and human coronary arteries, with in-situ and real-time monitoring of local stent degradation effects. Unlike conventional, static cell culture, the VesselBRx perfusion system eliminates unphysiologically high intracellular Mg(2+) concentrations and localized O(2) consumption resulting from stent degradation. Whereas static stented arteries exhibited only 20.1% cell viability and upregulated apoptosis, necrosis, metallic ion, and hypoxia-related gene signatures, stented arteries in VesselBRx showed almost identical cell viability to in vivo rabbit models (~94.0%). Hyperplastic intimal remodeling developed in unstented arteries subjected to low shear stress, but was inhibited by Mg-based stents in VesselBRx, similarly to in vivo. VesselBRx represents a critical advance from the current static culture standard of testing absorbable vascular implants.

Cardiac allograft vasculopathy (CAV) is a pathologic immune-mediated remodelling of the vasculature in transplanted hearts and, by impairing perfusion, is the major cause of late graft loss. Although best understood following cardiac transplantation, similar forms of allograft vasculopathy occur in other vascularized organ grafts and some features of CAV may be shared with other immune-mediated vasculopathies. Here, we describe the incidence and diagnosis, the nature of the vascular remodelling, immune and non-immune contributions to pathogenesis, current therapies, and future areas of research in CAV.

Atherosclerosis is a progressive vascular disease triggered by interplay between abnormal shear stress and endothelial lipid retention. A combination of these and, potentially, other factors leads to a chronic inflammatory response in the vessel wall, which is thought to be responsible for disease progression characterized by a buildup of atherosclerotic plaques. Yet molecular events responsible for maintenance of plaque inflammation and plaque growth have not been fully defined. Here we show that endothelial transforming growh factor β (TGF-β) signalling is one of the primary drivers of atherosclerosis-associated vascular inflammation. Inhibition of endothelial TGF-β signalling in hyperlipidemic mice reduces vessel wall inflammation and vascular permeability and leads to arrest of disease progression and regression of established lesions. These proinflammatory effects of endothelial TGF-β signalling are in stark contrast with its effects in other cell types and identify it as an important driver of atherosclerotic plaque growth and show the potential of cell-type-specific therapeutic intervention aimed at control of this disease.