The role of carcinoembryonic antigen-related cell adhesion molecule 1 in cancer

The Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a, is a member of the immunoglobulin superfamily. CEACAM1 was shown to be a prognostic marker in patients suffering from cancer. In this review, we summarize pre-clinical and clinical evidence linking CEACAM1 to tumorigenicity and cancer progression. Furthermore, we discuss potential CEACAM1-based mechanisms that may affect cancer biology

Influence of CEACAM1 on endothelial function

Dem Endothel, welches die luminale Oberfläche aller Blutgefäße auskleidet, kommt eine wichtige Barrierefunktion zwischen Blut und Gewebe zu. Nur durch eine bedarfsgerechte Justierung dieser Barriere, die den Durchtritt von Molekülen und Zellen reguliert, kann die Gewebehomöostase aufrechterhalten werden. Dabei ist das Endothel nicht nur passive Barriere, sondern auch an dieser dynamischen Regulation aktiv beteiligt. Störungen oder Fehlregulationen dieser Prozesse führen zu Pathologien, z.B. Arteriosklerose. Es ist seit längerem bekannt, dass Carcinoembryonic antigen–related cell adhesion molecule-1 (CEACAM1), ein Mitglied der Immunglobulin-Superfamilie, die Bildung und Morphogenese neuer Blutgefäße beeinflusst. Die spontane Entwicklung kleiner Arteriosklerose-ähnlicher Läsionen in CEACAM1 knockout (Cc1-/-) Mäusen zeigt, dass CEACAM1 auch für die Homöostase ausgereifter Blutgefäße von Bedeutung ist. Ziel dieser Dissertationsarbeit war daher, den Einfluss von CEACAM1 auf wesentliche Aspekte der Endothelfunktion in Aorten in situ bzw. in Endothelzellkulturen in vitro zu analysieren. Es konnte zunächst gezeigt werden, dass CEACAM1-defiziente Endothelzellen im Vergleich zu Wildtyp (WT) Endothelzellen eine rundlichere Zellmorphologie mit meanderförmigen Zellgrenzen und interzellulären Lücken aufweisen. Diese morphologischen Unterschiede stimmen mit Befunden in situ an Aorten von WT und Cc1-/- Mäusen überein. Weiterhin wurde eine Translokation der endothelialen NO-Synthase (eNOS) von der Zellmembran in den peri-nukleären Bereich bei CEACAM1-Defizienz festgestellt. Die erhobenen Daten bieten zwei mögliche Erklärungen dafür. Einerseits könnte CEACAM1 durch Interaktion mit eNOS als Membrananker fungieren. Daneben wiesen CEACAM1-defiziente Endothelzellen eine erhöhte Expression des Enzyms APT1 auf, welches eNOS depalmitoyliert. Die daraus resultierende, ebenfalls nachgewiesene geringere Palmitoylierung könnte auch zur verminderten Membran-lokalisation von eNOS beitragen. Zur endothelialen Funktion gehört, die Adhäsion von Blutzellen an die Gefäßwand weitestgehend zu beschränken. CEACAM1-defiziente Endothelzellen zeigten im Vergleich zu WT Endothelzellen eine verstärkte Adhäsivität gegenüber murinen und humanen Monozyten. Ähnliche Unterschiede wurden für Aortenexplantate aus WT und Cc1-/- Mäusen festgestellt. Dies ist einerseits mit einer verstärkten Expression des Zelladhäsionsmoleküls ICAM-1 bei CEACAM1-Defizienz erklärbar. Darüber hinaus vermittelt die Glykokalyx anti-adhäsive Eigenschaften. Aus Vorbefunden war bekannt, dass die endotheliale Glykokalyx in der Aorta von Cc1-/- Mäuse reduziert ist. Im Rahmen dieser Arbeit konnte dies auf eine verstärkte Expression der Glykokalyx-degradierenden Enzyme MMP9, Chondroitinase sowie Hyaluronidase-2 in Cc1-/- Endothelzellen zurückgeführt werden. Eine erhöhte Permeabilität stellt einen Indikator für ein dysfunktionales Endothel, eines der initialen Schritte in der Pathogenese der Arteriosklerose, dar. Zur Analyse der aortalen Permeabilität wurde ein modifizierter Miles-Assay etabliert. Unter Verwendung etablierter muriner Arteriosklerosemodelle konnte gezeigt werden, dass dieser Assay eine Störung der vaskulären Permeabilität bereits vor Auftreten makroskopischer Veränderungen zuverlässig detektiert. Im Rahmen der folgenden Analysen an WT und Cc1-/- Mäusen zeigte sich ein altersabhängiger Effekt von CEACAM1 auf die Gefäßpermeabilität: Aorten von 3 Monate alten Cc1-/- Mäuse wiesen eine im Vergleich zum WT erhöhte Gefäßpermeabilität auf, welche wahrscheinlich Folge einer verzögerten Gefäßreifung ist. Im Alter von 9 Monaten zeigte sich dagegen ein entgegengesetztes Bild. Dies wurde auf eine verstärkte Expression des die Barriere schädigenden Inflammationsmediators TNF-α in 9 Monate alten WT Mäusen zurückgeführt. Außerdem modulierte CEACAM1 die TNF-α-vermittelte Lockerung der endothelialen Barriere, indem es die Phosphorylierung von Adherens Junction Proteinen beeinflusste. Basal stabilisierte CEACAM1 die endotheliale Barriere durch Hemmung der Phosphorylierung von Caveolin-1, welches Adherens Junctions destabilisiert. Unter Einfluss von TNF-α war CEACAM1 verstärkt im Bereich von Adherens Junctions lokalisiert und rekrutierte dort Src-Kinase. Src-Kinase wiederum destabilisierte Adherens Junctions durch Phosphorylierung von β-Catenin, was in verstärkter Gefäßpermeabilität resultierte. Dagegen führte TNF-α in CEACAM1-defizienten Endothelzellen zu einer Dephosphorylierung von Caveolin-1 und β-Catenin, wodurch Adherens Junctions und damit die endotheliale Barriere stabilisiert wurden. Diese CEACAM1-abhängige differenzielle Regulation der Stabilität von Adherens Junctions unter TNF-α trägt wahrscheinlich maßgeblich zu den Unterschieden der vaskulären Permeabilität in 3 bzw. 9 Monate alten WT und Cc1-/- Mäusen bei. Zusammenfassend konnte im Rahmen dieser Arbeit nachgewiesen werden, dass CEACAM1 zentrale Funktionen des Endothels und hierüber die Homöostase reifer Gefäße beeinflusst. Da eine Expression von CEACAM1 auch in arteriosklerotischen Plaques nachgewiesen werden konnte, soll in weiteren Untersuchungen auch der Beitrag von CEACAM1 zur arteriosklerotischen Plaquebildung analysiert werden.The endothelium forms the inner surface of blood vessels and therefore is critically involved in forming a barrier between the intraluminal blood and the surrounding tissue. Adequate regulation of this barrier regarding extravasation of molecules and cells is mandatory to maintain tissue homeostasis. Thereby the endothelium is not only a passive barrier but regulates barrier function in an active and dynamic manner. Malfunction and dysregulation of these processes promote pathologies, i.e. atherosclerosis. It is known, that Carcinoembryonic antigen–related cell adhesion molecule-1 (CEACAM1), a member of the immunoglobulin superfamily, modulates the formation and morphogenesis of new blood vessels. In addition, spontaneous development of small atherosclerosis-like lesions within the aorta of CEACAM1-deficient (Cc1-/-) mice indicates the involvement of CEACAM1 in homeostasis of mature blood vessels. Therefore, the aim of this study was to analyze the effect of CEACAM1 on central aspects of endothelial function in situ (aortic tissue) and in vitro (endothelial cell cultures). First it was shown, that CEACAM1-deficient endothelial cells display a rather round cellular morphology with meandering cell boundaries and inter-cellular gaps compared to CEACAM1-expressing cells. These morphologic differences are in agreement with in situ observations in WT and Cc1-/- mice. Furthermore, CEACAM1 deficiency resulted in translocation of endothelial NO-Synthase (eNOS) from the cell membrane towards the peri-nuclear compartment within endothelial cells. Analysis of the underlying mechanisms suggests two scenarios. On the one hand, CEACAM1 might serve as a membrane anchor due to physical interaction with eNOS. On the other hand, expression of the de-palmitoylating enzyme APT1 is upregulated and eNOS palmitoylation is reduced in CEACAM1-deficient endothelial cells. This might also contribute to its reduced location at the cell membrane. Under physiological conditions the endothelium limits adhesion of blood cells to the vascular wall. However, adhesion of murine and human monocytes to cultured endothelial cells was increased when endothelial CEACAM1 was absent. Similar results were obtained using aortic explants of WT and Cc1-/- mice. This was attributed to the increased expression of cell adhesion molecule ICAM-1 in Cc1-/- endothelial cells. Furthermore, the glycocalyx of endothelial cells greatly contributes to anti- adhesive properties. Based on preliminary results which showed a reduced endothelial glycocalyx in aortae from Cc1-/- mice compared to WT mice, we found a higher expression of the glycocalyx-degrading enzymes MMP-9, chondroitinase and hyaluronidase-2 in Cc1-/- compared to WT endothelial cells. Enhanced vascular permeability indicates a dysfunctional endothelium, which is the initial step in the pathogenesis of atherosclerosis. To analyze aortic permeability a modified Miles-assay was established. Using well-characterized murine atherosclerosis models, it was shown that this assay reliably detects alterations in vascular permeability prior to macroscopically visible morphological alterations. CEACAM1 had an age-dependent effect on vascular permeability: aortae from 3 months old Cc1-/- mice showed increased vascular permeability for Evans blue compared to aortae from age-matched WT mice most likely due to delayed vascular maturation. Interestingly, this situation was completely reversed at the age of 9 months. This was attributed to enhanced aortic expression of the permeability promoting inflammatory mediator TNF-α in 9 month old WT mice compared to age-matched Cc1-/- mice. Moreover, CEACAM1 modulated the TNF-α-dependent increase in vascular permeability by affecting adherens junction phosphorylation. Under basal conditions CEACAM1 stabilizes endothelial barrier by inhibition of caveolin-1 phosphorylation known to destabilize adherens junctions. In the presence of TNF-α, CEACAM1 translocate to endothelial adherens junctions and recruits Src kinase which destabilizes adherens junctions by phosphorylation of β-catenin resulting in enhanced vascular permeability. In contrast, in CEACAM1-deficient endothelial cells TNF-α promotes dephosphorylation of caveolin-1 and β-catenin thus stabilizing adherens junction complexes and endothelial barrier. This CEACAM1-dependent differential regulation of adherens junction complex stability presumably contributes substantially to the differences in vascular permeability observed in WT and Cc1-/- mice at the age of 3 and 9 months, respectively. In summary, this study shows that CEACAM1 influences central aspects of endothelial functions and thereby affects homeostasis of mature blood vessels. Since expression of CEACAM1 was observed in endothelium covering atherosclerotic plaques, further studies will investigate the contribution of CEACAM1 to atherosclerotic plaque formation

Pro-angiogenic effects of pregnancy-specific glycoproteins in endothelial and extravillous trophoblast cells

We previously reported that binding to heparan sulfate (HS) is required for the ability of the placentally secreted pregnancy-specific glycoprotein 1 (PSG1) to induce endothelial tubulogenesis. PSG1 is composed of four immunoglobulin-like domains but which domains of the protein bind to HS remains unknown. To analyze the interaction of PSG1 with HS, we generated several recombinant proteins, including the individual domains, chimeric proteins between two PSG1 domains, and mutants. Using flow cytometric and surface plasmon resonance studies, we determined that the B2 domain of PSG1 binds to HS and that the positively charged amino acids encompassed between amino acids 43-59 are required for this interaction. Furthermore, we showed that the B2 domain of PSG1 is required for the increase in the formation of tubes by endothelial cells (EC) including a human endometrial EC line and two extravillous trophoblast (EVT) cell lines and for the pro-angiogenic activity of PSG1 observed in an aortic ring assay. PSG1 enhanced the migration of ECs while it increased the expression of matrix metalloproteinase-2 in EVTs, indicating that the pro-angiogenic effect of PSG1 on these two cell types may be mediated by different mechanisms. Despite differences in amino acid sequence, we observed that all human PSGs bound to HS proteoglycans and confirmed that at least two other members of the family, PSG6 and PSG9, induce tube formation. These findings contribute to a better understanding of the pro-angiogenic activity of human PSGs and strongly suggest conservation of this function among all PSG family members.Fil: Rattila, Shemona. Western University Of Health Sciences.; Estados UnidosFil: Kleefeldt, Florian. Julius Maximilians Universitat de Wurzburgo; AlemaniaFil: Ballesteros Gomez, Angela Patricia. National Institutes of Health; Estados UnidosFil: Beltrame, Jimena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Ribeiro, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Ergün, Süleyman. Julius Maximilians Universitat de Wurzburgo; AlemaniaFil: Dveksler, Gabriela. Western University Of Health Sciences.; Estados Unido

Contribution of adventitia-derived stem and progenitor cells to new vessel formation in tumors

Blocking tumor vascularization has not yet come to fruition to the extent it was hoped for, as angiogenesis inhibitors have shown only partial success in the clinic. We hypothesized that under- appreciated vascular wall-resident stem and progenitor cells (VW-SPCs) might be involved in tumor vascularization and influence effectiveness of anti-angiogenic therapy. Indeed, in patient samples, we observed that vascular adventitia-resident CD34$^+$ VW-SPCs are recruited to tumors in situ from co-opted vessels. To elucidate this in detail, we established an ex vivo model using concomitant embedding of multi-cellular tumor spheroids (MCTS) and mouse aortic rings (ARs) into collagen gels, similar to the so-called aortic ring assay (ARA). Moreover, ARA was modified by removing the ARs’ adventitia that harbors VW-SPCs. Thus, this model enabled distinguishing the contribution of VW-SPCs from that of mature endothelial cells (ECs) to new vessel formation. Our results show that the formation of capillary-like sprouts is considerably delayed, and their number and network formation were significantly reduced by removing the adventitia. Substituting iPSC-derived neural spheroids for MCTS resulted in distinct sprouting patterns that were also strongly influenced by the presence or absence of VW-SPCs, also underlying the involvement of these cells in non-pathological vascularization. Our data suggest that more comprehensive approaches are needed in order to block all of the mechanisms contributing to tumor vascularization

Bone marrow-independent adventitial macrophage progenitor cells contribute to angiogenesis

Pathological angiogenesis promotes tumor growth, metastasis, and atherosclerotic plaque rupture. Macrophages are key players in these processes. However, whether these macrophages differentiate from bone marrow-derived monocytes or from local vascular wall-resident stem and progenitor cells (VW-SCs) is an unresolved issue of angiogenesis. To answer this question, we analyzed vascular sprouting and alterations in aortic cell populations in mouse aortic ring assays (ARA). ARA culture leads to the generation of large numbers of macrophages, especially within the aortic adventitia. Using immunohistochemical fate-mapping and genetic in vivo-labeling approaches we show that 60% of these macrophages differentiate from bone marrow-independent Ly6c$^{+}$/Sca-1$^{+}$ adventitial progenitor cells. Analysis of the NCX$^{−/-}$ mouse model that genetically lacks embryonic circulation and yolk sac perfusion indicates that at least some of those progenitor cells arise yolk sac-independent. Macrophages represent the main source of VEGF in ARA that vice versa promotes the generation of additional macrophages thereby creating a pro-angiogenetic feedforward loop. Additionally, macrophage-derived VEGF activates CD34$^{+}$ progenitor cells within the adventitial vasculogenic zone to differentiate into CD31$^{+}$ endothelial cells. Consequently, depletion of macrophages and VEGFR2 antagonism drastically reduce vascular sprouting activity in ARA. In summary, we show that angiogenic activation induces differentiation of macrophages from bone marrow-derived as well as from bone marrow-independent VW-SCs. The latter ones are at least partially yolk sac-independent, too. Those VW-SC-derived macrophages critically contribute to angiogenesis, making them an attractive target to interfere with pathological angiogenesis in cancer and atherosclerosis as well as with regenerative angiogenesis in ischemic cardiovascular disorders

Chronic Obstructive Pulmonary Disease and the Cardiovascular System: Vascular Repair and Regeneration as a Therapeutic Target

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and encompasses chronic bronchitis and emphysema. It has been shown that vascular wall remodeling and pulmonary hypertension (PH) can occur not only in patients with COPD but also in smokers with normal lung function, suggesting a causal role for vascular alterations in the development of emphysema. Mechanistically, abnormalities in the vasculature, such as inflammation, endothelial dysfunction, imbalances in cellular apoptosis/proliferation, and increased oxidative/nitrosative stress promote development of PH, cor pulmonale, and most probably pulmonary emphysema. Hypoxemia in the pulmonary chamber modulates the activation of key transcription factors and signaling cascades, which propagates inflammation and infiltration of neutrophils, resulting in vascular remodeling. Endothelial progenitor cells have angiogenesis capabilities, resulting in transdifferentiation of the smooth muscle cells via aberrant activation of several cytokines, growth factors, and chemokines. The vascular endothelium influences the balance between vaso-constriction and -dilation in the heart. Targeting key players affecting the vasculature might help in the development of new treatment strategies for both PH and COPD. The present review aims to summarize current knowledge about vascular alterations and production of reactive oxygen species in COPD. The present review emphasizes on the importance of the vasculature for the usually parenchyma-focused view of the pathobiology of COPD

Peroxisomes in the mouse parotid glands: An in-depth morphological and molecular analysis

Background: The parotid gland is a major salivary gland that has important roles in the digestive and immune system. Peroxisomes are ubiquitous, single-membrane-bound organelles that are present in all eukaryotic cells. Peroxisomes help mediate lipid and reactive oxygen species metabolism, as well as polyunsaturated fatty acid, cholesterol and plasmalogen synthesis. Much of the knowledge on peroxisomes has derived from metabolic organs, however no detailed knowledge is available on peroxisomes in the parotid glands. We thus aimed to comprehensively delineate the localization and characterization of peroxisomal proteins in the murine parotid gland. Methods: We characterized peroxisomes in the acinar and striated duct cells of the murine parotid gland by fluorescence and electron microscopy, as well as protein and mRNA expression analyses for important peroxisomal genes and proteins. Results: We found that peroxisomes are present in all cell types of the mouse parotid gland, however, exhibit notable cell-specific differences in their abundance and enzyme content. We also observed that mouse parotid glands contain high levels of peroxisomal beta-oxidation enzymes (including Acox1, Mfp2 and Acaa1), catalase and other peroxisomal anti-oxidative enzymes. Conclusions: This data suggests that peroxisomes are highly abundant in the murine parotid gland and might help to protect against oxidative stress. This comprehensive description of peroxisomes in the parotid gland lays the groundwork for further research concerning their role in the pathogenesis of parotid gland diseases and tumors. (C) 2021 Elsevier GmbH. All rights reserved

Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

Aging is an independent risk factor for cardiovascular diseases and therefore of particular interest for the prevention of cardiovascular events. However, the mechanisms underlying vascular aging are not well understood. Since carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is crucially involved in vascular homeostasis, we sought to identify the role of CEACAM1 in vascular aging. Using human internal thoracic artery and murine aorta, we show that CEACAM1 is upregulated in the course of vascular aging. Further analyses demonstrated that TNF‐α is CEACAM1‐dependently upregulated in the aging vasculature. Vice versa, TNF‐α induces CEACAM1 expression. This results in a feed‐forward loop in the aging vasculature that maintains a chronic pro‐inflammatory milieu. Furthermore, we demonstrate that age‐associated vascular alterations, that is, increased oxidative stress and vascular fibrosis, due to increased medial collagen deposition crucially depend on the presence of CEACAM1. Additionally, age‐dependent upregulation of vascular CEACAM1 expression contributes to endothelial barrier impairment, putatively via increased VEGF/VEGFR‐2 signaling. Consequently, aging‐related upregulation of vascular CEACAM1 expression results in endothelial dysfunction that may promote atherosclerotic plaque formation in the presence of additional risk factors. Our data suggest that CEACAM1 might represent an attractive target in order to delay physiological aging and therefore the transition to vascular disorders such as atherosclerosis

Continuous extracorporeal femoral perfusion model for intravascular ultrasound, computed tomography and digital subtraction angiography.

ObjectivesWe developed a novel human cadaveric perfusion model with continuous extracorporeal femoral perfusion suitable for performing intra-individual comparison studies, training of interventional procedures and preclinical testing of endovascular devices. Objective of this study was to introduce the techniques and evaluate the feasibility for realistic computed tomography angiography (CTA), digital subtraction angiography (DSA) including vascular interventions, and intravascular ultrasound (IVUS).MethodsThe establishment of the extracorporeal perfusion was attempted using one formalin-fixed and five fresh-frozen human cadavers. In all specimens, the common femoral and popliteal arteries were prepared, introducer sheaths inserted, and perfusion established by a peristaltic pump. Subsequently, we performed CTA and bilateral DSA in five cadavers and IVUS on both legs of four donors. Examination time without unintentional interruption was measured both with and without non-contrast planning CT. Percutaneous transluminal angioplasty and stenting was performed by two interventional radiologists on nine extremities (five donors) using a broad spectrum of different intravascular devices.ResultsThe perfusion of the upper leg arteries was successfully established in all fresh-frozen but not in the formalin-fixed cadaver. The experimental setup generated a stable circulation in each procedure (ten upper legs) for a period of more than six hours. Images acquired with CT, DSA and IVUS offered a realistic impression and enabled the sufficient visualization of all examined vessel segments. Arterial cannulating, percutaneous transluminal angioplasty as well as stent deployment were feasible in a way that is comparable to a vascular intervention in vivo. The perfusion model allowed for introduction and testing of previously not used devices.ConclusionsThe continuous femoral perfusion model can be established with moderate effort, works stable, and is utilizable for medical imaging of the peripheral arterial system using CTA, DSA and IVUS. Therefore, it appears suitable for research studies, developing skills in interventional procedures and testing of new or unfamiliar vascular devices