Blood Vessel Remodeling Research Articles

Chronic inflammation, lymphangiogenesis, and effect of an anti-VEGFR therapy in a mouse model and in human patients with aspiration pneumonia.

Chronic inflammation induces lymphangiogenesis and blood vessel remodelling. Since aged pneumonia patients often have repeated episodes of aspiration pneumonia, the pathogenesis may involve chronic inflammation. For lymphangiogenesis, VEGFR-3 and its ligand VEGF-C are key factors. No previous studies have examined chronic inflammation or vascular changes in aspiration pneumonia or its mouse models. In lung inflammation, little is known about the effect of blocking VEGFR-3 on lung lymphangiogenesis and, moreover, its effect on the disease condition. This study aimed to establish a mouse model of aspiration pneumonia, examine the presence of chronic inflammation and vascular changes in the model and in patients, and evaluate the effect of inhibiting VEGFR-3 on the lymphangiogenesis and disease condition in this model. To induce aspiration pneumonia, we repeated inoculation of pepsin at low pH and LPS into mice for 21-28 days, durations in which bronchioalveolar lavage and plasma leakage in the lung suggested the presence of exaggerated inflammation. Conventional and immunohistochemical analysis of tracheal whole mounts suggested the presence of chronic inflammation, lymphangiogenesis, and blood vessel remodelling in the model. Quantitative RT-PCR of the trachea and lung suggested the involvement of lymphangiogenic factor VEGF-C, VEGFR-3, and pro-inflammatory cytokines. In the lung, the aspiration model showed the presence of chronic inflammation and exaggerated lymphangiogenesis. Treatment with the VEGFR inhibitor axitinib or the VEGFR-3 specific inhibitor SAR131675 impaired lymphangiogenesis in the lung and improved oxygen saturation in the aspiration model. Since the lung is the main site of aspiration pneumonia, the changes were intensive in the lung and mild in the trachea. Human lung samples also showed the presence of chronic inflammation and exaggerated lymphangiogenesis, suggesting the relevance of the model to the disease. These results suggest lymphatics in the lung as a new target of analysis and therapy in aspiration pneumonia.

Arteriogenic expansion of extratumoral macrovessels - impact of vascular ageing.

The critical role of the vasculature in cancer progression is predominantly studied at the capillary level, and often equated with angiogenesis. However, the mechanisms that ensure the supply of increasing blood volume to the expanding tumor microcirculation remain presently unclear. Here we used established mouse tumor models to document the enlargement (arteriogenesis), of macroscopic feeding vessels at considerable distances upstream from the malignant lesion, but not contralaterally. These changes are not affected by the procoagulant host tissue factor (TF), but are modulated by vascular ageing and atherosclerosis in ApoE-/- mice. Moreover, arteriogenic growth involves infiltration of bone marrow derived (YFP-labeled) cells and changes the gene expression profiles in the vessel wall. Thus, our observations suggest that in addition to local angiogenesis tumors influence distant remodeling of regional macroscopic blood vessels in amanner that is modulated by certain vascular comorbidities.

An integrated assessment of histopathological changes of the enteric neuromuscular compartment in experimental colitis.

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.

107 - Expression and Redox Contribution of Cytoglobin to the Smooth Muscle Fibroproliferative Phenotype during Vascular Remodeling in Human Blood Vessels

107 - Expression and Redox Contribution of Cytoglobin to the Smooth Muscle Fibroproliferative Phenotype during Vascular Remodeling in Human Blood Vessels

Abstract 8: The protein tyrosine phosphatase DEP-1/PTPRJ is an essential promoter of vascular permeability, angiogenesis and tumor progression

Abstract Angiogenesis and increased vessel permeability play key roles during tumor growth and cancer progression. Therefore, much current effort in the field aims at understanding the molecular mechanisms underlying the formation and remodeling of blood vessels as a way to identify new approaches to harness these processes. In this context, we have been focusing our efforts on the protein tyrosine phosphatase DEP-1 (also named PTPRJ or CD148), which was initially identified as a negative regulator of cell proliferation and VEGFR2 phosphorylation when highly expressed in quiescent endothelial cells. However, using an RNAi knockdown approach, we demonstrated for the first time that through its ability to dephosphorylate the Src inhibitory Y529, DEP-1 was also a positive regulator of Src activation in VEGF-stimulated endothelial cells. As Src plays key roles in the promotion of angiogenesis and vascular permeability, we further demonstrated that DEP-1 was indeed a promoter of these vascular functions in vitro, and that the tyrosine phosphorylation of its C-terminal tail, allowing Src interaction and activation, was required for this. Interestingly, the catalytic inactivation of DEP-1 in mice led to increased endothelial cell proliferation, but also disorganized vascular structures, in contrast with the apparent absence of phenotype in DEP-1 knock-out (KO) mice models. In this study, we evaluated the biological consequences of the loss of DEP-1 expression on normal and pathological angiogenesis using a complete KO mouse model. We first confirmed that DEP-1 was required for Src activation and the phosphorylation of its substrates VE-cadherin and FAK in mice lung lysates upon systemic VEGF stimulation. Consistent with these observations, VEGF-induced vascular leakage was abrogated in the DEP-1 KO mice, as determined using a modified Miles assay. Capillary formation from mouse aortic rings ex vivo and in Matrigel plugs was also impaired, demonstrating the key positive role of DEP-1 in permeability and angiogenesis. Ischemia-induced angiogenesis was also deficient in KO mice, as demonstrated by reduced limb blood flow and capillary formation in a hind limb ischemia model. To assess tumor-associated angiogenesis, the highly metastatic Mvt-1 mouse mammary tumor cells were orthotopically injected in WT and DEP-1 KO mice. The growth of tumors in DEP-1 KO mice was reduced by about 50% compared to those grown in WT animals, due to inhibition of tumor angiogenesis. As tumor-associated and leaky blood vessels contribute to dissemination of cancer cells, a 50-60% decrease in the number and size of spontaneous and experimental metastases were also observed in the lungs of DEP-1 KO mice. Altogether, these results demonstrate for the first time that DEP-1 is an essential driver of VEGF-dependent Src activation, permeability, and angiogenesis in vivo, therefore acting as a promoter of tumor cell growth and dissemination. Citation Format: Patrick Fournier, Sylvie Dussault, Alfredo Fusco, Alain Rivard, Isabelle Royal. The protein tyrosine phosphatase DEP-1/PTPRJ is an essential promoter of vascular permeability, angiogenesis and tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 8. doi:10.1158/1538-7445.AM2014-8

Effects of exercise training on pulmonary vessel muscularization and right ventricular function in an animal model of COPD.

BackgroundRight ventricular dysfunction in COPD is common, even in the absence of pulmonary hypertension. The aim of the present study was to examine the effects of high intensity interval training (HIIT) on right ventricular (RV) function, as well as pulmonary blood vessel remodeling in a mouse model of COPD.Methods42 female A/JOlaHsd mice were randomized to exposure to either cigarette smoke or air for 6 hours/day, 5 days/week for 14 weeks. Mice from both groups were further randomized to sedentariness or HIIT for 4 weeks. Cardiac function was evaluated by echocardiography and muscularization of pulmonary vessel walls by immunohistochemistry.ResultsSmoke exposure induced RV systolic dysfunction demonstrated by reduced tricuspid annular plane systolic excursion. HIIT in smoke-exposed mice reversed RV dysfunction. There were no significant effects on the left ventricle of neither smoke exposure nor HIIT. Muscularization of the pulmonary vessels was reduced after exercise intervention, but no significant effects on muscularization were observed from smoke exposure.ConclusionsRV function was reduced in mice exposed to cigarette smoke. No Increase in pulmonary vessel muscularization was observed in these mice, implying that other mechanisms caused the RV dysfunction. HIIT attenuated the RV dysfunction in the smoke exposed mice. Reduced muscularization of the pulmonary vessels due to HIIT suggests that exercise training not only affects the heart muscle, but also has important effects on the pulmonary vasculature.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-014-0117-y) contains supplementary material, which is available to authorized users.

Abstract 166: Matrix Metalloproteinase-9 Mediates Post-hypoxic Vascular Pruning Of Cerebral Blood Vessels By Degrading Laminin And Claudin-5

Objective: Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. The goal of this study was to examine the role of matrix metalloproteinase-9 (MMP-9) in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia. Approach and Results: In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both in the hypoxic angiogenic response and in the post-hypoxic pruning response. Compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells (BECs) but not pericytes. While immunofluorescent and flow cytometry analyses showed that hypoxia led to reduced expression of laminin and claudin-5 in wild-type BECs, this decrease was absent in MMP-9 KO BECs. Conclusions: These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5. Our data support the concept that MMP-9 inhibition might provide therapeutic benefit in the treatment of ischemic stroke, by preventing post-hypoxic vascular pruning, thereby optimizing vascular density and integrity.

Localized micro- and nano-scale remodelling in the diabetic aorta

Diabetes is strongly associated with cardiovascular disease, but the mechanisms, structural and biomechanical consequences of aberrant blood vessel remodelling remain poorly defined. Using an experimental (streptozotocin, STZ) rat model of diabetes, we hypothesized that diabetes enhances extracellular protease activity in the aorta and induces morphological, compositional and localized micromechanical tissue remodelling. We found that the medial aortic layer underwent significant thickening in diabetic animals but without significant changes in collagen or elastin (abundance). Scanning acoustic microscopy demonstrated that such tissue remodelling was associated with a significant decrease in acoustic wave speed (an indicator of reduced material stiffness) in the inter-lamellar spaces of the vessel wall. This index of decreased stiffness was also linked to increased extracellular protease activity (assessed by semi-quantitative in situ gelatin zymography). Such a proteolytically active environment may affect the macromolecular structure of long-lived extracellular matrix molecules. To test this hypothesis, we also characterized the effects of diabetes on the ultrastructure of an important elastic fibre component: the fibrillin microfibril. Using size exclusion chromatography and atomic force microscopy, we isolated and imaged microfibrils from both healthy and diabetic aortas. Microfibrils derived from diabetic tissues were fragmented, morphologically disrupted and weakened (as assessed following molecular combing). These structural and functional abnormalities were not replicated by in vitro glycation. Our data suggest that proteolysis may be a key driver of localized mechanical change in the inter-lamellar space of diabetic rat aortas and that structural proteins (such as fibrillin microfbrils) may be biomarkers of diabetes induced damage.

Trophic action of sympathetic nerves reduces arterial smooth muscle Ca(2+) sensitivity during early post-natal development in rats.

A decrease in the Ca(2+) sensitivity of smooth muscle contraction is a hallmark of functional remodelling of blood vessels during development. However, the responsible factors are largely unknown. Here, we tested the hypothesis that the post-natal decline of arterial Ca(2+) sensitivity is the result of trophic effects of sympathetic nerves. Contractile responses, intracellular Ca(2+) levels and protein expression profiles were compared in saphenous arteries from young (1- and 2-week-old) and adult rats using wire myography, Ca(2+) fluorimetry and Western blotting respectively. We observed a lower Ca(2+) sensitivity of contractions induced by methoxamine, an agonist of α1 -adrenoceptors, and U46619, an agonist of thromboxane A2 receptors, in arteries from adult as compared to young animals. Post-natal maturation was associated with stronger expression of regulatory proteins mediating Ca(2+) -dependent contraction (myosin light chain kinase (MLCK), myosin targeting subunit (MYPT1) and h-caldesmon) and weaker expression of proteins regulating Ca(2+) -independent contraction (Rho kinase, extracellular-regulated kinases (ERK1/2) and mitogen-activated protein kinases p38 MAPK) in vessels from adult rats. To eliminate the trophic action of sympathetic nerves, we performed lumbar sympathectomy in adult rats. This resulted in higher Ca(2+) sensitivity of agonist-induced contractions in denervated as compared to control arteries. Furthermore, denervated arteries contained less MLCK, MYPT1 and h-caldesmon and more ERK1/2 and p38 MAPK. Sympathetic denervation reverses developmental changes both in Ca(2+) sensitivity and in the expression of regulatory proteins back to the early post-natal phenotype in the rat saphenous artery. We conclude that trophic effects of sympathetic nerves govern functional remodelling of arteries during early post-natal development.

Nestin(+) tissue-resident multipotent stem cells contribute to tumor progression by differentiating into pericytes and smooth muscle cells resulting in blood vessel remodeling.

Tumor vessels with resistance to anti-angiogenic therapy are characterized by the normalization of the vascular structures through integration of mature pericytes and smooth muscle cells (SMC) into the vessel wall, a process termed vessel stabilization. Unfortunately, stabilization-associated vascular remodeling can result in reduced sensitivity to subsequent anti-angiogenic therapy. We show here that blockade of VEGF by bevacizumab induces stabilization of angiogenic tumor blood vessels in human tumor specimen by recruiting Nestin-positive cells, whereas mature vessels down-regulated Nestin-expression. Using xenograft tumors growing on bone-marrow (BM) chimera of C57Bl/6 wildtype and Nestin-GFP transgenic mice, we show for first time that Nestin(+) cells inducing the maturation of tumor vessels do not originate from the BM but presumably reside within the adventitia of adult blood vessels. Complementary ex vivo experiments using explants of murine aortas revealed that Nestin(+) multipotent stem cells (MPSCs) are mobilized from their niche and differentiated into pericytes and SMC through the influence of tumor-cell-secreted factors. We conclude that tissue-resident Nestin(+) cells are more relevant than BM-derived cells for vessel stabilization and therefore have to be considered in future strategies for anti-angiogenic therapy. The identification of proteins mediating recruitment or differentiation of local Nestin(+) cells with potential stem cell character to angiogenic blood vessels may allow the definition of new therapeutic targets to reduce tumor resistance against anti-angiogenic drugs.

THU0517 Tie2 Signaling Induces A Pro-Inflammatory Phenotype in Rheumatoid Arthritis and Psoriatic Arthritis Macrophages

BackgroundTie2 is a tyrosine kinase receptor that has an essential role in blood vessel remodeling and angiogenesis, through its activation by its ligands angiopoietin (Ang)-1 and Ang-2. Tie2 and its...

Trophoblast Invasion and Blood Vessel Remodeling Are Altered in a Rat Model of Lifelong Maternal Obesity

Maternal obesity is associated with an increased risk of a number of pregnancy complications, including fetal demise, which may be linked to impaired placental development as a result of altered trophoblast invasion and vessel remodeling. Therefore, we examined these parameters in pregnant rats fed a control (normal weight) or high fat (HF) diet (obese) at 2 critical times of rat placental development. Early trophoblast invasion was increased by approximately 2-fold in HF-fed dams with a concomitant increase in the expression of matrix metalloproteinase 9 protein, a mediator of tissue remodeling and invasion. Furthermore, we observed significantly higher levels of smooth muscle actin surrounding the placental spiral arteries of HF-fed dams, suggesting impaired spiral artery remodeling. Taken together, the results of this study suggest that altered placental development is an important contributor to the poor pregnancy outcomes and increased fetal demise in our model of lifelong maternal obesity.

Abstract 563: Mechanisms of VEGFR3-Dependent Pathological Lymphangiogenesis

Vascular remodeling, i.e., the growth and/or enlargement of both blood and lymphatic vessels, is tightly associated with inflammatory diseases, such as chronic airway inflammation, rheumatoid arthritis, inflammatory bowl disease, and chronic inflammatory skin psoriasis. The lymphatic system collects extravasated fluid, macromolecules, and immune cells from tissues and returns them to the blood circulation. While VEGFR2 and its ligand VEGF-A functions as master regulators of vasculogenesis and angiogenesis, VEGFR3 is critical for lymphangiogenesis. These conclusions are based on observations in genetically modified mice for VEGFR3 and its ligands. VEGFR3 is expressed on the blood and lymphatic endothelium during development, and is restricted to the lymphatics in the adult. Extensive studies have thus far focused on the role of VEGFR3 in lymphangiogenesis during development, however the function, regulation and intracellular mediators of the VEGFR3-dependent pathways in pathologic formation of lymphatic vessels during diseases remain poorly characterized. Our recent studies have identified two critical intracellular mediators, AIP1 and Bmx, in regulating VEGFR3 expression and activity. Genetic deletion of AIP1, a Ras-GAP protein, delays developmental lymphangiogenesis in neonatal skin and mesentery, and mounts weaker VEGF-C-induced cornea lymphangiogenesis. Deletion of Bmx, a non-receptor tyrosine kinase, has no effect on developmental lymphangiogenesis but blunts pathological lymphangiogenesis in mouse models. Mechanistically, AIP1 and Bmx sequentially regulate different steps during the VEGFR3 activation. AIP1 constitutively binds to VEGFR3 and enhances VEGFR3 endocytosis and stability. Bmx is not expressed in developmental lymphatics but is induced in response to pathological stimuli such as ischemia, and associates with an active form of VEGFR3 to regulate its downstream signaling without an effect on receptor phosphorylation. I will report the most recent progress as to how AIP1 and Bmx coordinately regulate VEGFR3-dependent lymphangiogenesis.

Fusing VE-Cadherin to α-Catenin Impairs Fetal Liver Hematopoiesis and Lymph but Not Blood Vessel Formation

We have recently shown that genetic replacement of VE-cadherin by a VE-cadherin-α-catenin fusion construct strongly impairs opening of endothelial cell contacts during leukocyte extravasation and induction of vascular permeability in adult mice. Here we show that this mutation leads to lethality at midgestation on a clean C57BL/6 background. Investigating the reasons for embryonic lethality, we observed a lack of fetal liver hematopoiesis and severe lymphedema but no detectable defects in blood vessel formation and remodeling. As for the hematopoiesis defect, VE-cadherin-α-catenin affected neither the generation of hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium nor their differentiation into multiple hematopoietic lineages. Instead, HSPCs accumulated in the fetal circulation, suggesting that their entry into the fetal liver was blocked. Edema formation was caused by disturbed lymphatic vessel development. Lymphatic progenitor cells of VE-cadherin-α-catenin-expressing embryos were able to leave the cardinal vein and migrate to the site of the first lymphatic vessel formation, yet subsequently, these cells failed to form large lumenized lymphatic vessels. Thus, stabilizing endothelial cell contacts by a covalent link between VE-cadherin and α-catenin affects recruitment of hematopoietic progenitors into the fetal liver and the development of lymph but not blood vessels.

Effect of antiprogesterone RU486 on VEGF expression and blood vessel remodeling on ovarian follicles before ovulation.

BackgroundThe success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P4) remains to be clarified, in particular when its concentration rapidly increases before ovulation.AimThis in vivo study was designed to clarify the effect promoted by a P4 receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration.Material and MethodsPreovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture.Results and ConclusionsVEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P4 antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may affect the functionality of the corpus luteum (CL) and the success of pregnancy.

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

Long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The impact of the immune system on tumor: angiogenesis and vascular remodeling.

Angiogenesis, the formation of new blood vessels, as well as inflammation with massive infiltration of leukocytes are hallmarks of various tumor entities. Various epidemiological, clinical, and experimental studies have not only demonstrated a link between chronic inflammation and cancer onset but also shown that immune cells from the bone marrow such as tumor-infiltrating macrophages significantly influence tumor progression. Tumor angiogenesis is critical for tumor development as tumors have to establish a blood supply in order to progress. Although tumor cells were first believed to fuel tumor angiogenesis, numerous studies have shown that the tumor microenvironment and infiltrating immune cell subsets are important for regulating the process of tumor angiogenesis. These infiltrates involve the adaptive immune system including several types of lymphocytes as well as cells of the innate immunity such as macrophages, neutrophils, eosinophils, mast cells, dendritic cells, and natural killer cells. Besides their known immune function, these cells are now recognized for their crucial role in regulating the formation and the remodeling of blood vessels in the tumor. In this review, we will discuss for each cell type the mechanisms that regulate the vascular phenotype and its impact on tumor growth and metastasis.

Sénescence cellulaire et physiopathologie des maladies respiratoires chroniques : rôle dans la bronchopneumopathie chronique obstructive

Knowledge of the biology of cellular senescence has improved markedly in recent years, helping us to understand the aging process. It is now clear that cellular senescence is involved in the pathogenesis of many age-related diseases, including respiratory diseases such as chronic obstructive pulmonary disease (COPD). COPD occupies a special place among chronic respiratory diseases because of its frequency and socio-economic impact. The high morbidity and mortality associated with COPD are related to multiple systemic manifestations independent of the severity of airway obstruction. COPD, although most often due to smoking, is also an aging-related respiratory disease. According to a newly developed concept, lung-cell senescence could play a key role in the pathophysiology of COPD, including remodeling of blood vessels and lung parenchyma, as well as the characteristic inflammatory process. Systemic manifestations of COPD, including cardiovascular disease, weight loss, bone demineralization and muscle dysfunction, may reflect a general process of premature aging secondary to the pulmonary changes.

Analysis of TNF-alpha-mediated cerebral pericyte remodeling.

As well as being a central regulator of inflammatory and immune-mediated events, TNF-α also influences vascular remodeling, resulting in alterations in the structure and function of blood vessels. In addition to endothelial cells, pericytes are another type of vascular cell that significantly contribute to the development, maturation, stabilization, and remodeling of blood vessels. To investigate the regulatory influence of different factors on pericyte behavior, we recently described a novel yet simple approach of isolating and culturing highly pure, high density cultures of mouse brain pericytes. In this chapter, we briefly describe this culture system, as well as methods for examining different aspects of pericyte behavior, including cell adhesion, cell migration, and cell proliferation. These assays can be used to examine the influence of TNF-α or any other factor on pericyte behavior.