Tumor endothelial cell autophagy is a key vascular-immune checkpoint in melanoma.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

Methods for Isolation of Tumor-Associated Endothelial Cells for Surface Protein Analysis and Sorting by Flowcytometry.

Polychromatic flowcytometry is increasingly used for simultaneously analyzing multiple intracellular and cell-surface proteins on a given cell population. Here we describe a flowcytometry-based method to analyze various proteins on the surface of endothelial cells (which comprise of less than 0.5% of the tumor microenvironment) and concurrently sort the live endothelial cells for the downstream applications such as gene expression by conventional quantitative PCR or by single-cell RNA sequencing.

Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis.

Endothelial cell autophagy in homeostasis and cancer.

Autophagy, the major lysosomal pathway for the degradation and recycling of cytoplasmic materials, is increasingly recognized as a major player in endothelial cell (EC) biology and vascular pathology. Particularly in solid tumors, tumor microenvironmental stress such as hypoxia, nutrient deprivation, inflammatory mediators, and metabolic aberrations stimulates autophagy in tumor-associated blood vessels. Increased autophagy in ECs may serve as a mechanism to alleviate stress and restrict exacerbated inflammatory responses. However, increased autophagy in tumor-associated ECs can re-model metabolic pathways and affect the trafficking and surface availability of key mediators and regulators of the interplay between EC and immune cells. In line with this, heightened EC autophagy is involved in pathological angiogenesis, inflammatory, and immune responses. Here, we review major cellular and molecular mechanisms regulated by autophagy in ECs under physiological conditions and discuss recent evidence implicating EC autophagy in tumor angiogenesis and immunosurveillance.

Creation of Abdominal Aortic Aneurysms in Sheep by Extrapolation of Rodent Models: Is It Feasible?

BACKGROUND: Abdominal aortic aneurysms (AAAs) are a potentially deathly disease, needing surgical or endovascular treatment. To evaluate potentially new diagnostic tools and treatments, a large animal model, which resembles not only the morphological characteristics but also the pathophysiological background, would be useful. METHODS: Rodent animal aneurysm models were extrapolated to sheep. Four groups were created: intraluminal infusion with an elastase-collagenase solution (n = 4), infusion with elastase-collagenase solution combined with proximal stenosis (n = 7), aortic xenograft (n = 3), and elastase-collagenase-treated xenograft (n = 4). At fixed time intervals (6, 12, and 24 weeks), computer tomography and autopsy with histological evaluation were performed. RESULTS: The described models had a high perioperative mortality (45%), due to acute aortic thrombosis or fatale hemorrhage. A maximum aortic diameter increase of 30% was obtained in the protease-stenosis group. In the protease-treated groups, some histological features of human AAAs, such as inflammation, thinning of the media, and loss of elastin could be reproduced. In the xenotransplant groups, a pronounced inflammatory reaction was visible at the start. In all models, inflammation decreased and fibrosis occurred at long follow-up, 24 weeks postoperatively. CONCLUSIONS: None of the extrapolated small animal aneurysm models could produce an AAA in sheep with similar morphological features as the human disease. Some histological findings of human surgical specimens could be reproduced in the elastase-collagenase-treated groups. Long-term histological evaluation indicated stabilization and healing of the aortic wall months after the initial stimulus.

The Effect of a Nonpeptide Angiotensin II Type 2 Receptor Agonist, Compound 21, on Aortic Aneurysm Growth in a Mouse Model of Marfan Syndrome.

INTRODUCTION: Available evidence suggests that the renin-angiotensin-aldosterone (RAA) system is a good target for medical intervention on aortic root dilatation in Marfan syndrome (MFS). The effect of Compound 21 (C21), a nonpeptide angiotensin II type 2 receptor agonist, on aneurysm progression was tested. METHODS: Mice with a mutation in fibrillin-1 (Fbn1) and wild-type mice were treated with vehicle, losartan, C21, enalapril, or a combination. Blood pressure, aortic root diameter, and histological slides were evaluated. RESULTS: All groups had a comparable blood pressure. Echographic evaluation of the aortic root diameter revealed a protective effect of angiotensin II type 1 receptor antagonist (losartan) and no effect of C21 treatment. None of the treatments had a beneficial effect on the histological changes in MFS. DISCUSSION: This study confirms that angiotensin II type 1 receptor antagonism (losartan) decreases aortic aneurysm growth in a mouse model of MFS. A nonpeptide angiotensin II type 2 receptor agonist (C21), at the doses studied, was ineffective. Future studies are warranted to further elucidate the exact role of the RAA system in aneurysm formation in MFS and identify alternative targets for intervention.

Remnant Epitope Autoimmunity in Human Abdominal Aortic Aneurysm: A Pilot Study with Elastin Peptides.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a prevalent disease affecting around 5% of the population aged more than 65 years. The exact etiology and physiopathology of AAA still raises questions, and elective surgery is currently the only treatment option for this often progressive disease. In this study, we hypothesized and tested a pathophysiological model that depicts AAA as an inflammation-triggered autoimmune disease with remnant vessel wall peptide fragments as the antigen. METHODS: A pilot study with male AAA patients (n = 14) and male controls (n = 8) was conducted. In both study groups, peripheral blood monocytes and plasma were separated from whole blood by centrifugation. An ELISpot test was performed on cultured white blood cells for the presence of elastin-specific T-lymphocytes. An Enzyme-linked immuno sorbent assay (ELISA) was performed on plasma for the presence of elastin-specific IgG molecules. RESULTS: ELISpot interferon-gamma secretion in AAA (7.7 ± 9.5%) and control (4.6 ± 3.5%) and ELISA anti-elastin IgG titer in AAA (77.5 ± 17.8%) and control (78.2 ± 31.5%) were not significantly different (P = 0.94 and P = 0.55, respectively). Both results are expressed as a percentage relative to the respective positive and negative control. CONCLUSIONS: The results of our pilot study did not indicate a clear and invariable autoimmune process directed against remnant elastin peptide fragments. Further research into the model mechanics and a possible antigen is still necessary. In the mean time, the model as presented here already offers a pathophysiological framework to further research into the possible remnant epitope-driven AAA etiology.

An electro-responsive hydrogel for intravascular applications: an in vitro and in vivo evaluation.

There is a growing interest in using hydrogels for biomedical applications, because of more favourable characteristics. Some of these hydrogels can be activated by using particular stimuli, for example electrical fields. These stimuli can change the hydrogel shape in a predefined way. It could make them capable of adaptation to patient-specific anatomy even post-implantation. This is the first paper aiming to describe in vivo studies of an electro-responsive, Pluronic F127 based hydrogel, for intravascular applications. Pluronic methacrylic acid hydrogel (PF127/MANa) was in vitro tested for its haemolytic and cytotoxic effects. Minimal invasive implantation in the carotid artery of sheep was used to evaluate its medium-term biological effects, through biochemical, macroscopic, radiographic, and microscopic evaluation. Indirect and direct testing of the material gave no indication of the haemolytic effects of the material. Determination of fibroblast viability after 24 h of incubation in an extract of the hydrogel showed no cytotoxic effects. Occlusion was obtained within 1 h following in vivo implantation. Evaluation at time of autopsy showed a persistent occlusion with no systemic effects, no signs of embolization and mild effects on the arterial wall. An important proof-of-concept was obtained showing biocompatibility and effectiveness of a pluronic based electro-responsive hydrogel for obtaining an arterial occlusion with limited biological impact. So the selected pluronic-methacrylic acid based hydrogel can be used as an endovascular occlusion device. More importantly it is the first step in further development of electro-active hydrogels for a broad range of intra-vascular applications (e.g. system to prevent endoleakage in aortic aneurysm treatment, intra-vascular drug delivery).

Mechanical support of the pressure overloaded right ventricle: an acute feasibility study comparing low and high flow support.

The objectives of this study were to assess the feasibility of low flow right ventricular support and to describe the hemodynamic effects of low versus high flow support in an animal model of acute right ventricular pressure overload. A Synergy Pocket Micro-pump (HeartWare International, Framingham, MA) was implanted in seven sheep. Blood was withdrawn from the right atrium to the pulmonary artery. Hemodynamics and pressure-volume loops were recorded in baseline conditions, after banding the pulmonary artery, and after ligating the right coronary artery in these banded sheep. End-organ perfusion (reflected by total cardiac output and arterial blood pressure) improved in all conditions. Intrinsic right ventricular contractility was not significantly impacted by support. Diastolic unloading of the pressure overloaded right ventricle (reflected by decreases in central venous pressure, end-diastolic pressure and volume, and ventricular capacitance) was successful, but with a concomitant and flow-dependent increase of the systolic afterload. This unloading diminished with right ventricular ischemia. Right ventricular mechanical support improves arterial blood pressure and cardiac output. It provides diastolic unloading of the right ventricle, but with a concomitant and right ventricular assist device flow-dependent increase of systolic afterload. These effects are most distinct in the pressure overloaded right ventricle without profound ischemic damage. We advocate the low flow strategy, which is potentially beneficial for the afterload sensitive right ventricle and has the advantage of avoiding excessive increases in pulmonary artery pressure when pulmonary hypertension exists. This might protect against the development of pulmonary edema and hemorrhage.

Strain assessment in the carotid artery wall using ultrasound speckle tracking: validation in a sheep model.

The aim of this study was to validate carotid artery strain assessment in-vivo using ultrasound speckle tracking. The left carotid artery of five sheep was exposed and sonomicrometry crystals were sutured onto the artery wall to obtain reference strain. Ultrasound imaging was performed at baseline and stress, followed by strain estimation using an in-house speckle tracking algorithm tuned for vascular applications. The correlation between estimated and reference strain was r = 0.95 (p < 0.001) and r = 0.87 (p < 0.01) for longitudinal and circumferential strain, respectively. Moreover, acceptable limits of agreement were found in Bland-Altman analysis (longitudinally: -0.15 to 0.42%, circumferentially: -0.54 to 0.50%), which demonstrates the feasibility of estimating carotid artery strain using ultrasound speckle tracking. However, further studies are needed to test the algorithm on human in-vivo data and to investigate its potential to detect subclinical cardiovascular disease and characterize atherosclerotic plaques.

In situ evolution of the mechanical properties of stretchable and non-stretchable ePTFE vascular grafts and adjacent native vessels.

INTRODUCTION: The purpose of this study was to evaluate the evolution of the mechanical properties of stretchable and non-stretchable ePTFE vascular grafts over time following implantation, as well as those of the adjacent native vessels. METHODS: One stretchable and one non-stretchable graft were implanted in either carotid position of six sheep. After twelve weeks, the samples, as well as the distal adjacent native vessel, were explanted and evaluated mechanically by means of uniaxial tensile tests. These tests were performed in the axial as well as in the circumferential direction. Also, patches of these grafts were implanted subcutaneously in the abdominal wall of the same animals. At 2, 4, 6, and 8 weeks, patches were explanted and evaluated mechanically. Baseline mechanical tensile tests were also performed on non-implanted grafts and on native sheep carotid arteries. RESULTS: Statistical analysis shows a significant effect of implantation time on the tensile mechanical properties of ePTFE vascular grafts. This effect is present for stretchable as well as non-stretchable grafts, in axial as well as in circumferential direction of the material. Also the adjacent native vessels have a tendency to lose mechanical stiffness and strength in the circumferential direction, an effect which is most pronounced for stretchable grafts. CONCLUSIONS: The mechanical properties of ePTFE vascular grafts and the adjacent native vessels significantly change as a function of implantation time. Compared to the native vessels, the grafts are still significantly stronger and stiffer, though longer term experiments are needed to evaluate whether and how much the decline would continue in time.