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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. Vegfcgene 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 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 pulmonisinfection 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 Mycoplasmainfection and that TNFα signaling is necessary but not sufficient for vascular remodeling.

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 pulmonisinfection 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.

Inhibition of platelet-derived growth factor-B (PDGF-B) has multiple effects on tumors, including loss of pericytes, regression of some vessels, normalization of other vessels, and reduction of interstitial pressure. PDGF-B inhibition also increases the efficacy of cancer therapeutics, but the role on tumor vessel efficiency and drug delivery is unclear. We sought to determine whether inhibition of PDGF-B signaling can increase delivery and efficacy of cyclophosphamide in Lewis lung carcinomas or RIP-Tag2 tumors. PDGF-B blockade in Lewis lung carcinoma tumors by the DNA aptamer AX102 for 14 days increased the number of perfused tumor vessels marked by lectin in the bloodstream by 50%. AX102 also increased the width of sleeves of viable tumor cells around blood vessels by 66%, increased tumor cell proliferation by 90%, and increased intratumoral delivery of Hoechst 33342 by 78%. A low dose of cyclophosphamide (20 mg/kg) reduced tumor cell proliferation by 31% when combined with AX102 but not when given alone. Synergy of cyclophosphamide and AX102 on tumor cell proliferation also was found in RIP-Tag2 tumors. Similarly, the PDGF receptor signaling inhibitor imatinib increased delivery of cyclophosphamide and reduced tumor burden in RIP-Tag2 mice, without evidence of tumor cell sensitization to chemotherapy. Together, these findings indicate that inhibition of PDGF-B signaling promotes the delivery and efficacy of chemotherapeutic agents by increasing the efficiency of tumor blood vessels.

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. pulmonisinfection 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.

Vascular remodeling is a feature of chronic inflammation during which capillaries transform into venules that expand the region of the vasculature in which leakage and leukocyte emigration both occur. Recently, we found that angiopoietin/Tie2 receptor signaling drives the transformation of capillaries into venules at an early stage of the sustained inflammatory response in the airways of mice infected with Mycoplasma pulmonis. However, the precise contributions of both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are not clear. In this study, we sought to determine the contribution of Ang2 to this vascular remodeling. Ang2 mRNA expression levels increased and phosphorylated Tie2 immunoreactivity in mucosal blood vessels decreased, indicative of diminished receptor signaling after infection. Selective inhibition of Ang2 throughout the infection by administration of either of two distinct function-blocking antibodies reduced the suppression of Tie2 phosphorylation and decreased the remodeling of mucosal capillaries into venules, the amount of leukocyte influx, and disease severity. These findings are consistent with Ang2 acting as an antagonist of Tie2 receptors and the reduction of Tie2 phosphorylation in endothelial cells rendering the vasculature more responsive to cytokines that promote both vascular remodeling and the consequences of inflammation after M. pulmonisinfection. By blocking such changes, Ang2 inhibitors may prove beneficial in the treatment of sustained inflammation in which vascular remodeling, leakage, and leukocyte influx contribute to its pathophysiology.

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. pulmonisinfection, 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.

Ricinus communisagglutinin I (RCA I), a galactose-binding lectin from castor beans, binds to endothelial cells at sites of plasma leakage, but little is known about the amount and functional consequences of binding to tumor endothelial cells. We addressed this issue by examining the effects of RCA I on blood vessels of spontaneous pancreatic islet-cell tumors in RIP-Tag2 transgenic mice. After intravenous injection, RCA I bound strongly to tumor vessels but not to normal blood vessels. At 6 minutes, RCA I fluorescence of tumor vessels was largely diffuse, but over the next hour, brightly fluorescent dots appeared as the lectin was internalized by endothelial cells. RCA I injection led to a dose- and time-dependent decrease in vascular endothelial growth factor receptor-2 (VEGFR-2) immunoreactivity in tumor endothelial cells, with 95% loss over 6 hours. By comparison, VEGFR-3, CD31, and CD105 had decreases in the range of 21% to 33%. Loss of VEGFR-2 was followed by increased activated caspase-3 in tumor vessels. Prior inhibition of VEGF signaling by AG-028262 decreased RCA I binding and internalization into tumor vessels. These findings indicate RCA I preferentially binds to and is internalized by tumor endothelial cells, which leads to VEGFR-2 down-regulation, endothelial cell apoptosis, and tumor vessel regression. Together, the results illustrate the selective impact of RCA I on VEGF signaling in tumor blood vessels.

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-1 (Ang1) and angiopoietin-2 (Ang2) have complex actions in angiogenesis and vascular remodeling due to their effects on Tie2 receptor signaling. Ang2 blocks Ang1-mediated activation of Tie2 in endothelial cells under certain conditions but is a Tie2 receptor agonist in others. We examined the effects of selective inhibitors of Ang1 (mL4-3) or Ang2 (L1-7[N]), alone or in combination, on the vasculature of human Colo205 tumors in mice. The Ang2 inhibitor decreased the overall abundance of tumor blood vessels by reducing tumor growth and keeping vascular density constant. After inhibition of Ang2, tumor vessels had many features of normal blood vessels (normalization), as evidenced by junctional accumulation of vascular endothelial-cadherin, junctional adhesion molecule-A, and platelet/endothelial cell adhesion molecule-1 in endothelial cells, increased pericyte coverage, reduced endothelial sprouting, and remodeling into smaller, more uniform vessels. The Ang1 inhibitor by itself had little noticeable effect on the tumor vasculature. However, when administered with the Ang2 inhibitor, the Ang1 inhibitor prevented tumor vessel normalization, but not the reduction in tumor vascularity produced by the Ang2 inhibitor. These findings are consistent with a model whereby inhibition of Ang2 leads to normalization of tumor blood vessels by permitting the unopposed action of Ang1, but decreases tumor vascularity primarily by blocking Ang2 actions.

Both Eph receptors and ephrin ligands have been implicated in blood vessel and neuronal development. Recent studies suggested that EphA2 inhibition reduces tumor angiogenesis, but its role in blood vessel development and inflammation is unclear. We examined these issues using either airways of pathogen-free, EphA2-deficient mice at various ages or EphA2-deficient mice whose airways were inflamed by either Mycoplasma pulmonisinfection or ovalbumin sensitization and challenge. EphA2-deficient mice had fewer capillaries, a greater number of endothelial sprouts, and greater capillary diameters than age-matched, wild-type control mice. Moreover, capillaries in EphA2-deficient mice had significantly less pericyte coverage, suggesting abnormal interactions between endothelial cells and pericytes. These differences were apparent in early postnatal life but decreased during progression into adulthood. In inflamed airways, significantly more angiogenesis and lymphangiogenesis, a greater number of infiltrating leukocytes, and higher expression levels of inflammatory cytokine mRNA were present in EphA2-deficient mice after M. pulmonisinfection. Additionally, in allergic airway inflammation with ovalbumin sensitization and challenge, a greater number of lymphatic sprouts and infiltrating leukocytes, higher mRNA expression levels of TH2 cytokines and chemokines related to allergic airway inflammation, and enhanced airway hyper-responsiveness were present in EphA2-deficient mice. We conclude that defective pericyte coverage causes capillary defects, abundant endothelial sprouts, and thick capillary diameters in EphA2-deficient mice, indicating that these animals have exaggerated responses to airway inflammation.

The integrin α5β1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of α5β1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonisinfection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, α5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have α5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the α5β1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonisinfection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.

Integrin α5β1is among the proteins overexpressed on tumor vessels and is a potential target for diagnostics and therapeutics. Here, we mapped the distribution of α5β1integrin in three murine tumor models and identified sites of expression that are rapidly accessible to intravascular antibodies. When examined by conventional immunohistochemistry, α5β1integrin expression was strong on most blood vessels in RIP-Tag2 transgenic mouse tumors, adenomatous polyposis coli (apc) mouse adenomas, and implanted MCa-IV mammary carcinomas. Expression increased during malignant progression in RIP-Tag2 mice. However, immunoreactivity was also strong in normal pancreatic ducts, intestinal smooth muscle, and several other sites. To determine which sites of expression were rapidly accessible from the bloodstream, we intravenously injected anti-α5β1integrin antibody and 10 minutes to 24 hours later examined the amount and distribution of labeling. The injected antibody strongly labeled tumor vessels at all time points but did not label most normal blood vessels or gain access to pancreatic ducts or intestinal smooth muscle. Intense vascular labeling by anti-α5β1integrin antibody co-localized with the uniform CD31 immunoreactivity of tumor vessels and contrasted sharply with the patchy accumulation of nonspecific IgG at sites of leakage. This strategy of injecting antibodies revealed the uniform overexpression and rapid accessibility of α5β1integrin on tumor vessels and may prove useful in assessing other potential therapeutic targets in cancer.

Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivophage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivophage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivophage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

Angiogenesis and vascular remodeling occurs in many inflammatory diseases, including asthma. In this study, we determined the time course and reversibility of the angiogenesis and vascular remodeling produced by vascular endothelial growth factor (VEGF) in a tet-on inducible transgenic system driven by the CC10 promoter in airway epithelium. One day after switching on VEGF expression, endothelial sprouts arose from venules, grew toward the epithelium, and were abundant by 3 to 5 days. Vessel density reached twice baseline by 7 days. Many new vessels were significantly larger than normal, were fenestrated, and penetrated the epithelium. Despite their mature appearance at 7 days suggested by their pericyte coat and basement membrane, the new vessels started to regress within 3 days when VEGF was switched off, showing stasis and luminal occlusion, influx of inflammatory cells, and retraction and apoptosis of endothelial cells and pericytes. Vessel density returned to normal within 28 days after VEGF withdrawal. Our study showed the dynamic nature of airway angiogenesis and regression. Blood vessels can respond to VEGF by sprouting angiogenesis within a few days, but regress more slowly after VEGF withdrawal, and leave a historical record of their previous extent in the form of empty basement membrane sleeves.

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.

Often described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered 99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1-μm confocal optical sections, were <2.5 μm in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors.

Endothelial cells of tumor vessels have well-documented alterations, but it is less clear whether pericytes on these vessels are abnormal or even absent. Here we report that α-smooth muscle actin (α-SMA) and desmin-immunoreactive pericytes were present on 97% of blood vessels viewed by confocal microscopy in 100-μm-thick sections of three different spontaneous or implanted tumors in mice. However, the cells had multiple abnormalities. Unlike pericytes on capillaries in normal pancreatic islets, which had desmin but not α-SMA immunoreactivity, pericytes on capillary-size vessels in insulinomas in RIP-Tag2 transgenic mice expressed both desmin and α-SMA. Furthermore, pericytes in RIP-Tag2 tumors, as well as those in MCa-IV breast carcinomas and Lewis lung carcinomas, had an abnormally loose association with endothelial cells and extended cytoplasmic processes deep into the tumor tissue. α-SMA-positive pericytes also covered 73% of endothelial sprouts in RIP-Tag2 tumors and 92% of sprouts in the other tumors. Indeed, pericyte sleeves were significantly longer than the CD31-immunoreactive endothelial cell sprouts themselves in all three types of tumors. All three tumors also contained α-SMA-positive myofibroblasts that resembled pericytes but were not associated with blood vessels. We conclude that pericytes are present on most tumor vessels but have multiple abnormalities, including altered expression of marker proteins. In contrast to some previous studies, the almost ubiquitous presence of pericytes on tumor vessels found in the present study may be attributed to our use of both desmin and α-SMA as markers and 100-μm-thick tissue sections. The association of pericytes with endothelial sprouts raises the possibility of an involvement in sprout growth or retraction in tumors.

Angiogenesis and vascular remodeling are features of many chronic inflammatory diseases. When diseases evolve slowly, the accompanying changes in the microvasculature would seem to be similarly gradual. Here we report that the rate of endothelial cell proliferation and the size of blood vessels increases rapidly after the onset of an infection that leads to chronic inflammatory airway disease. In C3H mice inoculated with Mycoplasma pulmonis, the tracheal microvasculature, made visible by perfusion of Lycopersicon esculentumlectin, rapidly enlarged from 4 to 7 days after infection and then plateaued. Diameters of arterioles, capillaries, and venules increased on average 148, 214, and 74%, respectively. Endothelial cell proliferation, measured by bromodeoxyuridine (BrdU) labeling, peaked at 5 days (18 times the pathogen-free value), declined sharply until day 9, but remained at ∼3 times the pathogen-free value for at least 28 days. Remodeled capillaries and venules were sites of focal plasma leakage and extensive leukocyte adherence. Most systemic manifestations of the infection occurred well after the peak of endothelial proliferation, and the humoral immune response to M. pulmoniswas among the latest, increasing after 14 days. These data show that endothelial cell proliferation and microvascular remodeling occur at an early stage of chronic airway disease and suggest that the vascular changes precede widespread tissue remodeling.

Leakiness of blood vessels in tumors may contribute to disease progression and is key to certain forms of cancer therapy, but the structural basis of the leakiness is unclear. We sought to determine whether endothelial gaps or transcellular holes, similar to those found in leaky vessels in inflammation, could explain the leakiness of tumor vessels. Blood vessels in MCa-IV mouse mammary carcinomas, which are known to be unusually leaky (functional pore size 1.2–2 μm), were compared to vessels in three less leaky tumors and normal mammary glands. Vessels were identified by their binding of intravascularly injected fluorescent cationic liposomes and Lycopersicon esculentumlectin and by CD31 (PECAM) immunoreactivity. The luminal surface of vessels in all four tumors had a defective endothelial monolayer as revealed by scanning electron microscopy. In MCa-IV tumors, 14% of the vessel surface was lined by poorly connected, overlapping cells. The most superficial lining cells, like endothelial cells, had CD31 immunoreactivity and fenestrae with diaphragms, but they had a branched phenotype with cytoplasmic projections as long as 50 μm. Some branched cells were separated by intercellular openings (mean diameter 1.7 μm; range, 0.3–4.7 μm). Transcellular holes (mean diameter 0.6 μm) were also present but were only 8% as numerous as intercellular openings. Some CD31-positive cells protruded into the vessel lumen; others sprouted into perivascular tumor tissue. Tumors in RIP-Tag2 mice had, in addition, tumor cell-lined lakes of extravasated erythrocytes. We conclude that some tumor vessels have a defective cellular lining composed of disorganized, loosely connected, branched, overlapping or sprouting endothelial cells. Openings between these cells contribute to tumor vessel leakiness and may permit access of macromolecular therapeutic agents to tumor cells.

We sought to determine whether the changes in microvascular endothelial cells (EC) caused by a polarized chronic inflammatory stimulus depend on proximity to the stimulus. C3H mice were infected with Mycoplasma pulmonis, which attaches to the airway epithelium and creates a polarized inflammatory stimulus across the airway wall. At 1, 2, or 4 weeks, the tracheal vasculature was stained by perfusion of silver nitrate to mark EC borders or biotinylated Lycopersicon esculentum. lectin to label the EC surface and adherent leukocytes. E-selectin immunoreactivity and EC proliferation were also localized. We found that the size, shape, and immunoreactivity for adhesion molecules on EC nearest the airway lumen (subepithelial EC) were different from those on the opposite surface of the same vessels. Subepithelial EC were smaller, more irregular in shape, had greater E-selectin immunoreactivity, and had twice as many adherent leukocytes. In contrast, proliferating EC were uniformly distributed around the vessel circumference. We conclude that the polarized stimulus created by M. pulmonisinfection differentially changes the size, shape, and function of EC nearest the airway epithelium. This heterogeneity may result from a gradient of inflammatory mediators that triggers the influx of leukocytes into the airway lumen.

Chronic inflammation is associated with blood vessel proliferation and enlargement and changes in vessel phenotype. We sought to determine whether these changes represent different types of angiogenesis and whether they are stimulus dependent. Chronic airway inflammation, produced by infection with Mycoplasma pulmonis, was compared in strains of mice known to be resistant (C57BL/6) or susceptible (C3H). Tracheal vascularity, assessed in whole mounts after Lycopersicon esculentumlectin staining, increased in both strains at 1, 2, 4, and 8 weeks after infection, but the type of vascular remodeling was different. The number of vessels doubled in tracheas of C57BL/6 mice, with corresponding increases of capillaries and venules. In contrast, neither the number nor the length of vessels changed in C3H mice. Instead, vessel diameter and endothelial cell number doubled, and the proportion of venules doubled with a corresponding decrease of capillaries. Although the infection had no effect on baseline plasma leakage, in both strains it potentiated the leakage produced by substance P. We conclude that the same stimulus can result in blood vessel proliferation or enlargement, depending on the host response. Endothelial cells proliferate in both cases, but in one case new capillaries form whereas in the other capillaries convert to venules.