Heterogeneity of endothelial VE-PTP downstream polarization, Tie2 activation, junctional claudin-5, and permeability in the aorta and vena cava.

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymphatic Expansion.

BACKGROUND: Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. METHODS: Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. RESULTS: Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. CONCLUSIONS: Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.

Lymphatic Proliferation Ameliorates Pulmonary Fibrosis after Lung Injury.

Despite many reports about pulmonary blood vessels in lung fibrosis, the contribution of lymphatics to fibrosis is unknown. We examined the mechanism and consequences of lymphatic remodeling in mice with lung fibrosis after bleomycin injury or telomere dysfunction. Widespread lymphangiogenesis was observed after bleomycin treatment and in fibrotic lungs of prospero homeobox 1-enhanced green fluorescent protein (Prox1-EGFP) transgenic mice with telomere dysfunction. In loss-of-function studies, blocking antibodies revealed that lymphangiogenesis 14 days after bleomycin treatment was dependent on vascular endothelial growth factor (Vegf) receptor 3 signaling, but not on Vegf receptor 2. Vegfc gene and protein expression increased specifically. Extensive extravasated plasma, platelets, and macrophages at sites of lymphatic growth were potential sources of Vegfc. Lymphangiogenesis peaked at 14 to 28 days after bleomycin challenge, was accompanied by doubling of chemokine (C-C motif) ligand 21 in lung lymphatics and tertiary lymphoid organ formation, and then decreased as lung injury resolved by 56 days. In gain-of-function studies, expansion of the lung lymphatic network by transgenic overexpression of Vegfc in club cell secretory protein (CCSP)/VEGF-C mice reduced macrophage accumulation and fibrosis and accelerated recovery after bleomycin treatment. These findings suggest that lymphatics have an overall protective effect in lung injury and fibrosis and fit with a mechanism whereby lung lymphatic network expansion reduces lymph stasis and increases clearance of fluid and cells, including profibrotic macrophages.

Vascular Endothelial Growth Factor C for Polycystic Kidney Diseases.

Polycystic kidney diseases (PKD) are genetic disorders characterized by progressive epithelial cyst growth leading to destruction of normally functioning renal tissue. Current therapies have focused on the cyst epithelium, and little is known about how the blood and lymphatic microvasculature modulates cystogenesis. Hypomorphic Pkd1(nl/nl) mice were examined, showing that cystogenesis was associated with a disorganized pericystic network of vessels expressing platelet/endothelial cell adhesion molecule 1 and vascular endothelial growth factor receptor 3 (VEGFR3). The major ligand for VEGFR3 is VEGFC, and there were lower levels of Vegfc mRNA within the kidneys during the early stages of cystogenesis in 7-day-old Pkd1(nl/nl) mice. Seven-day-old mice were treated with exogenous VEGFC for 2 weeks on the premise that this would remodel both the VEGFR3(+) pericystic vascular network and larger renal lymphatics that may also affect the severity of PKD. Treatment with VEGFC enhanced VEGFR3 phosphorylation in the kidney, normalized the pattern of the pericystic network of vessels, and widened the large lymphatics in Pkd1(nl/nl) mice. These effects were associated with significant reductions in cystic disease, BUN and serum creatinine levels. Furthermore, VEGFC administration reduced M2 macrophage pericystic infiltrate, which has been implicated in the progression of PKD. VEGFC administration also improved cystic disease in Cys1(cpk/cpk) mice, a model of autosomal recessive PKD, leading to a modest but significant increase in lifespan. Overall, this study highlights VEGFC as a potential new treatment for some aspects of PKD, with the possibility for synergy with current epithelially targeted approaches.

Pulmonary lymphangiectasia resulting from vascular endothelial growth factor-C overexpression during a critical period.

RATIONALE: Lymphatic vessels in the respiratory tract normally mature into a functional network during the neonatal period, but under some pathological conditions they can grow as enlarged, dilated sacs that result in the potentially lethal condition of pulmonary lymphangiectasia. OBJECTIVE: We sought to determine whether overexpression of the lymphangiogenic growth factor (vascular endothelial growth factor-C [VEGF-C]) can promote lymphatic growth and maturation in the respiratory tract. Unexpectedly, perinatal overexpression of VEGF-C in the respiratory epithelium led to a condition resembling human pulmonary lymphangiectasia, a life-threatening disorder of the newborn characterized by respiratory distress and the presence of widely dilated lymphatics. METHODS AND RESULTS: Administration of doxycycline to Clara cell secretory protein-reverse tetracycline-controlled transactivator/tetracycline operator-VEGF-C double-transgenic mice during a critical period from embryonic day 15.5 to postnatal day 14 was accompanied by respiratory distress, chylothorax, pulmonary lymphangiectasia, and high mortality. Enlarged sac-like lymphatics were abundant near major airways, pulmonary vessels, and visceral pleura. Side-by-side comparison revealed morphological features similar to pulmonary lymphangiectasia in humans. The condition was milder in mice given doxycycline after age postnatal day 14 and did not develop after postnatal day 35. Mechanistic studies revealed that VEGF recptor (VEGFR)-3 alone drove lymphatic growth in adult mice, but both VEGFR-2 and VEGFR-3 were required for the development of lymphangiectasia in neonates. VEGFR-2/VEGFR-3 heterodimers were more abundant in the dilated lymphatics, consistent with the involvement of both receptors. Despite the dependence of lymphangiectasia on VEGFR-2 and VEGFR-3, the condition was not reversed by blocking both receptors together or by withdrawing VEGF-C. CONCLUSIONS: The findings indicate that VEGF-C overexpression can induce pulmonary lymphangiectasia during a critical period in perinatal development.

Neutrophil dependence of vascular remodeling after Mycoplasma infection of mouse airways.

Vascular remodeling is a feature of sustained inflammation in which capillaries enlarge and acquire the phenotype of venules specialized for plasma leakage and leukocyte recruitment. We sought to determine whether neutrophils are required for vascular remodeling in the respiratory tract by using Mycoplasma pulmonis infection as a model of sustained inflammation in mice. The time course of vascular remodeling coincided with the influx of neutrophils during the first few days after infection and peaked at day 5. Depletion of neutrophils with antibody RB6-8C5 or 1A8 reduced neutrophil influx and vascular remodeling after infection by about 90%. Similarly, vascular remodeling after infection was suppressed in Cxcr2(-/-) mice, in which neutrophils adhered to the endothelium of venules but did not extravasate into the tissue. Expression of the venular adhesion molecule P-selectin increased in endothelial cells from day 1 to day 3 after infection, as did expression of the Cxcr2-receptor ligands Cxcl1 and Cxcl2. Tumor necrosis factor α (TNFα) expression increased more than sixfold in the trachea of wild-type and Cxcr2(-/-) mice, but intratracheal administration of TNFα did not induce vascular remodeling similar to that seen in infection. We conclude that neutrophil influx is required for remodeling of capillaries into venules in the airways of mice with Mycoplasma infection and that TNFα signaling is necessary but not sufficient for vascular remodeling.

Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.

Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1-enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C-producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.

Transgenic overexpression of interleukin-1ß induces persistent lymphangiogenesis but not angiogenesis in mouse airways.

These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1ß mice that conditionally overexpress IL-1ß in Clara cells to determine whether IL-1ß can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1ß production, influx of neutrophils and macrophages, and conspicuous and persistent lymphangiogenesis, but surprisingly no angiogenesis. Gene profiling showed many up-regulated genes, including chemokines (Cxcl1, Ccl7), cytokines (tumor necrosis factor α, IL-1ß, and lymphotoxin-ß), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1ß overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1ß induced lymphangiogenesis. Inactivation of vascular endothelial growth factor (VEGF)-C and VEGF-D by adeno-associated viral vector-mediated soluble VEGFR-3 (VEGF-C/D Trap) completely blocked lymphangiogenesis, showing its dependence on VEGFR-3 ligands. Consistent with this mechanism, VEGF-C immunoreactivity was present in some Aif1/Iba1-immunoreactive macrophages. Because neutrophils contribute to IL-1ß-induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1ß/CXCR2(-/-) mice had the usual IL-1ß-mediated lymphangiogenesis but no neutrophil recruitment, suggesting that neutrophils are not essential. IL1R1 immunoreactivity was found on some epithelial basal cells and neuroendocrine cells, suggesting that these cells are targets of IL-1ß, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1ß overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.

Cathepsin L protects mice from mycoplasmal infection and is essential for airway lymphangiogenesis.

Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.

Functionally specialized junctions between endothelial cells of lymphatic vessels.

Recirculation of fluid and cells through lymphatic vessels plays a key role in normal tissue homeostasis, inflammatory diseases, and cancer. Despite recent advances in understanding lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature. 438:946-953), the cellular features responsible for entry of fluid and cells into lymphatics are incompletely understood. We report the presence of novel junctions between endothelial cells of initial lymphatics at likely sites of fluid entry. Overlapping flaps at borders of oak leaf-shaped endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the sides by discontinuous button-like junctions (buttons) that differed from conventional, continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. However, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin) and tight junction-associated proteins, including occludin, claudin-5, zonula occludens-1, junctional adhesion molecule-A, and endothelial cell-selective adhesion molecule. In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity, but platelet/endothelial cell adhesion molecule-1 was not. Growing tips of lymphatic sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather than immature ones. Our findings suggest that fluid enters throughout initial lymphatics via openings between buttons, which open and close without disrupting junctional integrity, but most leukocytes enter the proximal half of initial lymphatics.

Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.

Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells.

Shear stress control of vascular leaks and atheromas through Tie2 activation by VE-PTP sequestration.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) influences endothelial barrier function by regulating the activation of tyrosine kinase receptor Tie2. We determined whether this action is linked to the development of atherosclerosis by examining the influence of arterial shear stress on VE-PTP, Tie2 activation, plasma leakage, and atherogenesis. We found that exposure to high average shear stress led to downstream polarization and endocytosis of VE-PTP accompanied by Tie2 activation at cell junctions. In aortic regions with disturbed flow, VE-PTP was not redistributed away from Tie2. Endothelial cells exposed to high shear stress had greater Tie2 activation and less macromolecular permeability than regions with disturbed flow. Deleting endothelial VE-PTP in VE-PTPiECKO mice increased Tie2 activation and reduced plasma leakage in atheroprone regions. ApoE-/- mice bred with VE-PTPiECKO mice had less plasma leakage and fewer atheromas on a high-fat diet. Pharmacologic inhibition of VE-PTP by AKB-9785 had similar anti-atherogenic effects. Together, the findings identify VE-PTP as a novel target for suppression of atherosclerosis.

Pericyte requirement for anti-leak action of angiopoietin-1 and vascular remodeling in sustained inflammation.

Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis, or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonis infection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.

Lymphotoxin-alpha contributes to lymphangiogenesis.

Lymphotoxin-α (LTα), lymphotoxin-ß (LTß), and tumor necrosis factor-α (TNFα) are inflammatory mediators that play crucial roles in lymphoid organ development. We demonstrate here that LTα also contributes to the function of lymphatic vessels and to lymphangiogenesis during inflammation. LTα(-/-) mice exhibited reduced lymph flow velocities and increased interstitial fluid pressure. Airways of LTß(-/-) mice infected with Mycoplasma pulmonis had significantly more lymphangiogenesis than wild type (WT) or LTα(-/-) mice, as did the skin draining immunization sites of LTß(-/-) mice. Macrophages, B cells, and T cells, known sources of LT and TNFα, were apparent in the skin surrounding the immunization sites as were LTα, LTß, and TNFα mRNAs. Ectopic expression of LTα led to the development of LYVE-1 and Prox1-positive lymphatic vessels within tertiary lymphoid organs (TLOs). Quantification of pancreatic lymphatic vessel density in RIPLTαLTß(-/-) and WT mice revealed that LTα was sufficient for inducing lymphangiogenesis and that LTß was not required for this process. Kidneys of inducible LTα transgenic mice developed lymphatic vessels before the appearance of obvious TLOs. These data indicate that LTα plays a significant role in lymphatic vessel function and in inflammation-associated lymphangiogenesis.

Buttons and Zippers: Endothelial Junctions in Lymphatic Vessels.

Button-like junctions are discontinuous contacts at the border of oak-leaf-shaped endothelial cells of initial lymphatic vessels. These junctions are distinctively different from continuous zipper-like junctions that create the endothelial barrier in collecting lymphatics and blood vessels. Button junctions are point contacts, spaced about 3 µm apart, that border valve-like openings where fluid and immune cells enter lymphatics. In intestinal villi, openings between button junctions in lacteals also serve as entry routes for chylomicrons. Like zipper junctions that join endothelial cells, buttons consist of adherens junction proteins (VE-cadherin) and tight junction proteins (claudin-5, occludin, and others). Buttons in lymphatics form from zipper junctions during embryonic development, can convert into zippers in disease or after experimental genetic or pharmacological manipulation, and can revert back to buttons with treatment. Multiple signaling pathways and local microenvironmental factors have been found to contribute to button junction plasticity and could serve as therapeutic targets in pathological conditions ranging from pulmonary edema to obesity.

Imaging Blood Vessels and Lymphatics in Mouse Trachea Wholemounts.

Changes in blood vessels and lymphatics in health and disease are easier to understand and interpret when studied microscopically in three dimensions. The mouse trachea is a simple, yet powerful, and versatile model system in which to achieve this. We describe practical immunohistochemical methods for fluorescence and confocal microscopy of wholemounts of the mouse trachea to achieve this purpose in which the entire vasculature can be visualized from the organ level to the cellular and subcellular level. Blood vessels and lymphatics have highly stereotyped vascular architectures that repeat in arcades between the tracheal cartilages. Arterioles, capillaries, and venules can be easily identified for the blood vessels, while the lymphatics consist of initial lymphatics and collecting lymphatics. Even small abnormalities in either blood vessels or lymphatics can be noticed and evaluated in three dimensions. We and others have used the mouse trachea for examining in situ angiogenesis and lymphangiogenesis, vascular development and regression, vessel patency, differences in transgenic mice, and pathological changes, such as increased vascular permeability induced by inflammatory mediators.

Steroid-resistant lymphatic remodeling in chronically inflamed mouse airways.

Angiogenesis and lymphangiogenesis participate in many inflammatory diseases, and their reversal is thought to be beneficial. However, the extent of reversibility of vessel remodeling is poorly understood. We exploited the potent anti-inflammatory effects of the corticosteroid dexamethasone to test the preventability and reversibility of vessel remodeling in Mycoplasma pulmonis-infected mice using immunohistochemistry and quantitative RT-PCR. In this model robust immune responses drive rapid and sustained changes in blood vessels and lymphatics. In infected mice not treated with dexamethasone, capillaries enlarged into venules expressing leukocyte adhesion molecules, sprouting angiogenesis and lymphangiogenesis occurred, and the inflammatory cytokines tumor necrosis factor and interleukin-1 increased. Concurrent dexamethasone treatment largely prevented the remodeling of blood vessels and lymphatics. Dexamethasone also significantly reduced cytokine expression, bacterial burden, and leukocyte influx into airways and lungs over 4 weeks of infection. In contrast, when infection was allowed to proceed untreated for 2 weeks and then was treated with dexamethasone for 4 weeks, most blood vessel changes reversed but lymphangiogenesis did not, suggesting that different survival mechanisms apply. Furthermore, dexamethasone significantly reduced the bacterial burden and influx of lymphocytes but not of neutrophils or macrophages or cytokine expression. These findings show that lymphatic remodeling is more resistant than blood vessel remodeling to corticosteroid-induced reversal. We suggest that lymphatic remodeling that persists after the initial inflammatory response has resolved may influence subsequent inflammatory episodes in clinical situations.

Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor in tumors, but less is known about what drives vascular remodeling in inflammation, where plasma leakage and leukocyte influx are prominent features. In chronic airway inflammation in mice infected by the bacterium Mycoplasma pulmonis (M. pulmonis), the segment of the microvasculature that supports leukocyte adhesion and migration expands through remodeling of capillaries into vessels with features of venules. Here, we report that the angiopoietin/Tie2 pathway is an essential driving force for capillary remodeling into venules in M. pulmonis-infected mouse airways. Similar to M. pulmonis infection, systemic overexpression of angiopoietin-1 (Ang1) resulted in remodeling of airway capillaries into venular-like vessels that expressed venous markers like P-selectin, ICAM-1, and EphB4 and were sites of leukocyte adhesion during lipopolysaccharide-induced acute inflammation. Ang1 and Ang2 protein increased in M. pulmonis-infected mouse airways but came from different cellular sources: Ang1 was expressed in infiltrating neutrophils and Ang2 in endothelial cells. Indeed, systemic administration of soluble Tie2 inhibited capillary remodeling, induction of venous markers, and leukocyte influx in M. pulmonis-infected mouse airways. Together, these findings suggest that blockade of the Ang/Tie2 pathway may represent a therapeutic approach in airway inflammation.

Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung.

Exaggerated levels of VEGF (vascular endothelial growth factor) are present in persons with asthma, but the role(s) of VEGF in normal and asthmatic lungs has not been defined. We generated lung-targeted VEGF(165) transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (T(H)2)-mediated inflammation. In these mice, VEGF induced, through IL-13-dependent and -independent pathways, an asthma-like phenotype with inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF also enhanced respiratory antigen sensitization and T(H)2 inflammation and increased the number of activated DC2 dendritic cells. In antigen-induced inflammation, VEGF was produced by epithelial cells and preferentially by T(H)2 versus T(H)1 cells. In this setting, it had a critical role in T(H)2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization and is crucial in adaptive T(H)2 inflammation. VEGF regulation may be therapeutic in asthma and other T(H)2 disorders.

Rapid remodeling of airway vascular architecture at birth.

Recent advances have documented the development of lung vasculature before and after birth, but less is known of the growth and maturation of airway vasculature. We sought to determine whether airway vasculature changes during the perinatal period and when the typical adult pattern develops. On embryonic day 16.5 mouse tracheas had a primitive vascular plexus unlike the adult airway vasculature, but instead resembling the yolk sac vasculature. Soon after birth (P0), the primitive vascular plexus underwent abrupt and extensive remodeling. Blood vessels overlying tracheal cartilage rings regressed from P1 to P3 but regrew from P4 to P7 to form the hierarchical, segmented, ladder-like adult pattern. Hypoxia and HIF-1α were present in tracheal epithelium over vessels that survived but not where they regressed. These findings reveal the plasticity of airway vasculature after birth and show that these vessels can be used to elucidate factors that promote postnatal vascular remodeling and maturation.