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Tissue maintenance and repair depend on the integrated activity of multiple cell types 1 . Whereas the contributions of epithelial 2 , 3 , immune 4 , 5 and stromal cells 6 , 7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here we uncover important roles of enteric glial cells (EGCs) in intestinal homeostasis, immunity and tissue repair. We demonstrate that infection of mice with Heligmosomoides polygyrus leads to enteric gliosis and the upregulation of an interferon gamma (IFNγ) gene signature. IFNγ-dependent gene modules were also induced in EGCs from patients with inflammatory bowel disease 8 . Single-cell transcriptomics analysis of the tunica muscularis showed that glia-specific abrogation of IFNγ signalling leads to tissue-wide activation of pro-inflammatory transcriptional programs. Furthermore, disruption of the IFNγ–EGC signalling axis enhanced the inflammatory and granulomatous response of the tunica muscularis to helminths. Mechanistically, we show that the upregulation of Cxcl10 is an early immediate response of EGCs to IFNγ signalling and provide evidence that this chemokine and the downstream amplification of IFNγ signalling in the tunica muscularis are required for a measured inflammatory response to helminths and resolution of the granulomatous pathology. Our study demonstrates that IFNγ signalling in enteric glia is central to intestinal homeostasis and reveals critical roles of the IFNγ–EGC–CXCL10 axis in immune response and tissue repair after infectious challenge. Enteric glial cells have tissue-wide immunoregulatory roles through the upregulation of IFNγ-dependent genes both at steady state and after parasite infection, promoting immune homeostasis and CXCL10-mediated tissue repair after pathogen-induced intestinal damage in mice.

Aging is the most important risk factor for the development of cardiovascular diseases. Senescent cells release plethora of factors commonly known as the senescence-associated secretory phenotype, which can modulate the normal function of the vascular wall. It is currently not well understood if and how endothelial cell senescence can affect adventitial niche. The aim of this study was to characterize oxidative stress-induced endothelial cells senescence and identify their paracrine effects on the primary cell type of the adventitia, the fibroblasts. Human aortic endothelial cells (HAEC) were treated with hydrogen peroxide to induce premature senescence. Mass spectrometry analysis identified several proteomic changes in senescent HAEC with top upregulated secretory protein growth differentiation factor 15 (GDF-15). Treatment of the human adventitial fibroblast cell line (hAdv cells) with conditioned medium (CM) from senescent HAEC resulted in alterations in the proteome of hAdv cells identified in mass spectrometry analysis. Majority of differentially expressed proteins in hAdv cells treated with CM from senescent HAEC were involved in the uptake and metabolism of lipoproteins, mitophagy and ferroptosis. We next analyzed if some of these changes and pathways might be regulated by GDF-15. We found that recombinant GDF-15 affected some ferroptosis-related factors (e.g. ferritin) and decreased oxidative stress in the analyzed adventitial fibroblast cell line, but it had no effect on erastin-induced cell death. Contrary, silencing of GDF-15 in hAdv cells was protective against this ferroptotic stimuli. Our findings can be of importance for potential therapeutic strategies targeting cell senescence or ferroptosis to alleviate vascular diseases.

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.

Background: Arteriovenous fistula (AVF) stenosis remains an important cause of AVF maturation failure, for which there are currently no effective therapies. We examined the pattern and phenotype of cellular proliferation at different timepoints in a mouse model characterized by a peri-anastomotic AVF stenosis. Methods: Standard immunohistochemical analyses for cellular proliferation and macrophage infiltration were performed at 2, 7 and 14 d on our validated mouse model of AVF stenosis to study the temporal profile, geographical location and cellular phenotype of proliferating and infiltrating cells in this model. Results: Adventitial proliferation and macrophage infiltration (into the adventitia) began at 2 d, peaked at 7 d and then declined over time. Surprisingly, there was minimal macrophage infiltration or proliferation in the neointimal region at either 7 or 14 d, although endothelial cell proliferation increased rapidly between 2 d and 7 d, and peaked at 14 d. Conclusions: Early and rapid macrophage infiltration and cellular proliferation within the adventitia could play an important role in the downstream pathways of both neointimal hyperplasia and inward or outward remodelling.

Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries characterized by leukocyte accumulation in the vessel wall. Both innate and adaptive immune responses contribute to atherogenesis, but the identity of atherosclerosis-relevant antigens and the role of antigen presentation in this disease remain poorly characterized. We developed live-cell imaging of explanted aortas to compare the behavior and role of APCs in normal and atherosclerotic mice. We found that CD4+ T cells were capable of interacting with fluorescently labeled (CD11c-YFP+) APCs in the aortic wall in the presence, but not the absence, of cognate antigen. In atherosclerosis-prone Apoe-/-CD11c-YFP+ mice, APCs extensively interacted with CD4+ T cells in the aorta, leading to cell activation and proliferation as well as secretion of IFN-γ and TNF-α. These cytokines enhanced uptake of oxidized and minimally modified LDL by macrophages. We conclude that antigen presentation by APCs to CD4+ T cells in the arterial wall causes local T cell activation and production of proinflammatory cytokines, which promote atherosclerosis by maintaining chronic inflammation and inducing foam cell formation.

Vulnerable plaque is closely related to the occurrence of ischemic stroke. Therefore, early accurate identification of plaque vulnerability is crucial in risk stratification. In the development of vulnerable plaques, the change of the adventitia is earlier than that of the intima. Currently, researchers focused on the ultrasound detection of intraplaque and intima, but adventitia was often ignored in the examination. Real-time elastography technology (RTE) provides an estimation of adventitia stiffness, and contrast-enhanced ultrasound (CEUS) provides the quantification of adventitial VV. Therefore, we aimed to evaluate the value of adventitia in the early diagnosis of plaque vulnerability by combining CEUS and RTE based on histopathology. Rabbit carotid atherosclerosis models were established, and CEUS and RTE were performed. Normalized maximal video-intensity enhancement (MVE) was calculated to quantify adventitial VV density, and strain values were acquired to evaluate the adventitial elasticity. After removal of the lesion lumen, histological analysis of each excised plaque and adventitia was performed, and vulnerable plaques ( n = 32) and stable group ( n = 13) were distinguished. Normalized MVE of the adventitial VV and adventitial strain values in the vulnerable group was significantly higher than those in the stable group. Normalized MVE and strain values had a positive linear correlation with histological findings. Normalized MVE of the adventitial VV combined with adventitial strain values could identify plaque vulnerability with the area under the curve of 0.913 (sensitivity 90% and specificity 97%). Accordingly, the multimodal ultrasound detection strategy of adventitia has a high diagnostic value for early plaque vulnerability.

Through mechanobiological control of the extracellular matrix, and hence local stiffness, smooth muscle cells of the media and fibroblasts of the adventitia play important roles in arterial homeostasis, including adaptations to altered hemodynamics, injury, and disease. We present a new approach to model arterial wall mechanics that seeks to define better the mechanical environments of the media and adventitia while avoiding the common prescription of a traction-free reference configuration. Specifically, we employ the concept of constituent-specific deposition stretches from the growth and remodeling literature and define a homeostatic state at physiologic pressure and axial stretch that serves as a convenient biologically and clinically relevant reference configuration. Information from histology and multiphoton imaging is then used to prescribe structurally motivated constitutive relations for a bi-layered model of the wall. The utility of this approach is demonstrated by describing in vitro measured biaxial pressure–diameter and axial force–length responses of murine carotid arteries and predicting the associated intact and radially cut traction-free configurations. The latter provides a unique validation while confirming that this constrained mixture approach naturally recovers estimates of residual stresses, which are fundamental to wall mechanics, without the usual need to prescribe an opening angle that is only defined conveniently on cylindrical geometries and cannot be measured in vivo . Among other findings, the model suggests that medial and adventitial stresses can be nearly uniform at physiologic loads, albeit at separate levels, and that the adventitia bears increasingly more load at supra-physiologic pressures while protecting the media from excessive stresses.

Weight loss after bariatric surgery decreases the earlier expansion of the adventitial vasa vasorum (VV), a biomarker of early atheromatous disease. However, no data are available regarding weight loss achieved by very low calorie ketogenic diets (VLCKD) on VV and lipid-based atherogenic indices. A randomized clinical trial was performed to examine changes in adventitial VV density in 20 patients with moderate obesity who underwent a 6-month very low calorie ketogenic diet (VLCKD, 600–800 kcal/day), and 10 participants with hypocaloric diet based on the Mediterranean Diet (MedDiet, estimated reduction of 500 kcal on the usual intake). Contrast-enhanced carotid ultrasound was used to assess the VV. Body composition analysis was also used. The atherogenic index of plasma (log (triglycerides to high-density lipoprotein cholesterol ratio)) and the triglyceride-glucose index were calculated. Serum concentrations of soluble intercellular adhesion molecule 1 (sICAM-1), and soluble vascular cell adhesion molecule 1 (sVCAM-1) were measured. The impact of weight on quality of life-lite (IWQOL-Lite) questionnaire was administered. Participants of intervention groups displayed a similar VV values. Significant improvements of BMI (−5.3 [−6.9 to −3.6] kg/m2, p < 0.001), total body fat (−7.0 [−10.7 to −3.3] %, p = 0.003), and IWQOL-Lite score (−41.4 [−75.2 to −7.6], p = 0.027) were observed in VLCKD group in comparison with MedDiet group. Although after a 6-months follow-up period VV density (mean, right and left sides) did not change significantly in any group, participants in the VLCKD exhibited a significantly decrease both in their atherogenic index of plasma and serum concentration of sICAM-1. A 6-month intervention with VLCKD do not impact in the density of the adventitial VV in subjects with moderate obesity, but induces significant changes in markers of endothelial dysfunction and CV risk.