Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.     

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.Lymphatic vessels undergo changes in many inflammatory lung diseases, where lymphatic proliferation, enlargement, and regression have been described.^1,2 Examples include asthma, where lymphatics regress,³ chronic obstructive pulmonary disease (COPD) and pneumonia, where they proliferate,^4–6 and idiopathic pulmonary fibrosis, where they undergo abnormal growth and remodeling in the lung parenchyma^7,8 but regress in subpleural and interlobular compartments.⁹Although lymphatics are well known to drain interstitial fluid and serve as conduits for antigen-presenting cells and lymphocytes from the lung,^10–12 little has been learned about the mechanism and functional implications of lymphatic changes in pulmonary inflammation. Regardless of the impact of lymphangiogenesis on disease pathophysiological characteristics, the presence of edema in inflammatory lung disease indicates that the amount of plasma leakage exceeds the fluid drainage capacity through lymphatics and other routes.Lymphatics proliferate in many settings of sustained inflammation, including psoriasis,¹³ rheumatoid arthritis,¹⁴ and inflammatory bowel disease,¹⁵ but it is still unclear whether proliferation of lymphatics worsens or ameliorates disease severity. Promotion of lymphatic growth by transgenic overexpression of vascular endothelial growth factor (VEGF)-C reduces the severity of skin inflammation.¹⁶ This effect has not been examined in the lung, and it is unknown whether it is typical of inflammatory conditions in other organs. It is also unclear whether lung lymphatics exhibit the same plasticity in inflammation as those in other organs.Previous studies had shown that tracheal lymphatics undergo widespread growth and remodeling after infection. During the first 4 weeks after infection, tracheal lymphatics undergo even more extensive changes than blood vessels.^17,18 However, sensitization and challenge of lungs to house dust mite allergen for 2 weeks has no detectable effect on the number of lung lymphatics.¹⁹ Little is known about the effects on lung lymphatics of other conditions of sustained inflammation.We, therefore, used a mouse model of sustained lung inflammation produced by respiratory tract infection by Mycoplasma pulmonis bacteria to determine the response of lung lymphatics to sustained inflammation and to compare changes in the lung with those in the trachea. With the presumption that lymphangiogenesis does occur in the lung, we sought to determine exactly when and where. During the period of 1 to 4 weeks after infection, we closely observed the distribution of the changes in the lung to address the possibility that lymphatic growth or remodeling was regionally specific.We also investigated the driving mechanism for lymphatic growth in lungs in this model. Because of compelling evidence that lymphatic growth in the trachea and other settings is driven by VEGF-C activation of VEGF receptor (VEGFR)-3 signaling,²⁰ we compared the effects in the lung and trachea of blocking VEGFR-2 and VEGFR-3 administered individually or together.Consistent with this reasoning, previous studies revealed that lymphangiogenesis in the trachea after M. pulmonis infection was completely inhibited by a function-blocking antibody to VEGFR-3.¹⁷ Similar results have been obtained in skin²¹ and cornea.²² However, lymphangiogenesis under some conditions is also partially reduced by selective inhibition of VEGFR-2, examples being skin,²³ cornea,²⁴ lymph nodes,²⁵ arthritic joints,²⁴ and tumors.²⁶ The latter mechanism could reflect effects of VEGFR-2 blockade directly on lymphatics or indirectly through changes in leukocytes or other cells that produce lymphangiogenic factors.The present study of lymphatic remodeling in sustained bronchopneumonia produced by M. pulmonis infection addressed the question of whether lymphatics grow, undergo remodeling, or regress during the development of bronchopneumonia. The study also examined the time course of changes in lymphatics, whether the distribution of lymphangiogenesis coincides with the widespread inflammatory changes in the lung, and whether lymphatic growth and remodeling in the lung is driven by changes in signaling of VEGFR-3, VEGFR-2, or both.The experiments revealed that some lymphatics in the lung underwent profound changes after M. pulmonis infection. Sprouting lymphangiogenesis was evident at 1 week and was more pronounced at 2 and 4 weeks. Strikingly, expansion of the lymphatic network was restricted to regions of bronchus-associated lymphoid tissue (BALT) that formed in the lung around bronchi and major pulmonary vessels. Lymphatics in more peripheral regions of the lung did not exhibit these changes, despite the presence of inflammatory cells. Growth of lymphatics in BALT was blocked 99% by inhibition of VEGFR-2 and VEGFR-3 together. Inhibition of VEGFR-3 alone resulted in 83% reduction, whereas inhibition of VEGFR-2 alone resulted in 68% reduction. Inhibition of lymphangiogenesis in BALT by blocking VEGFR-2 and VEGFR-3 did not prevent the formation of BALT.     

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.Neutrophils are key effector cells of innate immunity that rapidly arrive at sites of tissue injury to kill bacteria and interact with macrophages and other cells to orchestrate a coordinated immune cell and cytokine response to injury.^1–4 Neutrophils are involved in many inflammatory diseases of the airways and lung, including pneumonia, acute lung injury, sepsis, asthma, cystic fibrosis, bronchitis, and chronic obstructive lung disease,⁵ also contribute to tissue damage in inflammatory conditions of other organs, and play a role in arterial remodeling in atherosclerosis.⁴The signals and events that bring neutrophils to sites of inflammation are well characterized.^6–8 These include expression of endothelial cell adhesion molecules to induce rolling and firm attachment, followed by extravasation into tissues where they release cytokines and other products that can kill bacteria and promote tissue remodeling. The dominant mechanism driving neutrophil influx may be organ-specific.^9,10 Blood vessels of the microcirculation undergo numerous changes in sustained inflammation, and these include structural and functional remodeling of endothelial cells and pericytes.^11–14 Among these changes, capillaries transform into venules that support plasma leakage and leukocyte influx. The contribution of neutrophils to this remodeling is not well understood. Circumferential vessel enlargement is a prominent feature of vascular remodeling–sustained airway inflammation^15–23 and is distinct from more familiar and better-documented types of sprouting angiogenesis.²⁴We asked whether incoming neutrophils contribute to the vascular remodeling, with the thought that the initial wave of leukocyte influx could render blood vessels more efficient for leukocyte adhesion and transmigration. Although leukocyte influx is known to accompany blood vessel remodeling,^15,18,22 it is unknown whether there is a causal relationship and, if so, what is the underlying mechanism? Neutrophils are attractive candidates for contributing to vascular remodeling because they are among the first leukocytes to enter inflamed tissues^4,6,25 and can produce cytokines, growth factors, proteases, and reactive oxygen species that have profound vascular effects.^2–4,26With this background, we sought to determine whether neutrophils are essential for the vascular remodeling that occurs soon after Mycoplasma pulmonis infection, when capillaries transform into venules. In particular, we asked whether neutrophil influx coincides spatially and temporally with vascular remodeling, can vascular remodeling be prevented by neutrophil depletion, and if Cxcr2 signaling is required for the neutrophil influx that accompanies vascular remodeling?To address these questions we examined the relationship between neutrophil influx and vascular remodeling during the first week after M. pulmonis infection of the respiratory tract of mice. The approach was to compare the time course of neutrophil influx and vascular remodeling in the trachea and then determine whether the remodeling was blocked by neutrophil depletion by either of two different antineutrophil antibodies: RB6-8C5 or 1A8. We also tested whether vascular remodeling was prevented by genetic deletion of Cxcr2, which mediates the actions of the chemotactic chemokines Cxcl1 and Cxcl2, which bring neutrophils into inflamed tissues. Because previous studies have shown that vascular remodeling was inhibited by blocking tumor necrosis factor α (TNFα) signaling,¹⁹ we asked whether TNFα expression was increased in wild-type and Cxcr2^−/− mice and whether intratracheal administration of TNFα was sufficient to induce vascular remodeling similar to that seen after infection. Other studies examined the expression of the Cxcr2 ligands, Cxcl1 and Cxcl2. Together, the experiments showed that neutrophil influx was required for vascular remodeling after M. pulmonis infection, and that TNF signaling was necessary but not sufficient for vascular remodeling.     

Lymphangiogenesis Is Induced by Mycobacterial Granulomas via Vascular Endothelial Growth Factor Receptor-3 and Supports Systemic T-Cell Responses against Mycobacterial Antigen

Granulomatous inflammation is characteristic of many autoimmune and infectious diseases. The lymphatic drainage of these inflammatory sites remains poorly understood, despite an expanding understanding of lymphatic role in inflammation and disease. Here, we show that the lymph vessel growth factor Vegf-c is up-regulated in Bacillus Calmette-Guerin– and Mycobacterium tuberculosis–induced granulomas, and that infection results in lymph vessel sprouting and increased lymphatic area in granulomatous tissue. The observed lymphangiogenesis during infection was reduced by inhibition of vascular endothelial growth factor receptor 3. By using a model of chronic granulomatous infection, we also show that lymphatic remodeling of tissue persists despite resolution of acute infection and a 10- to 100-fold reduction in the number of bacteria and tissue-infiltrating leukocytes. Inhibition of vascular endothelial growth factor receptor 3 decreased the growth of new vessels, but also reduced the proliferation of antigen-specific T cells. Together, our data show that granuloma–up-regulated factors increase granuloma access to secondary lymph organs by lymphangiogenesis, and that this process facilitates the generation of systemic T-cell responses to granuloma-contained antigens.The lymphatic system is made of a network of tissue-resident lymphatic endothelial vessels that drain extracellular fluid to the lymph nodes and back into blood circulation, a process that is critical in maintaining body fluid balance. Lymphatics also play a critical role in transporting dendritic cells (DCs) of the immune system, which may contain bacterial, viral, or fungal peptides, to T- and B-cell areas in the lymph nodes. Afferent lymph vessels express high levels of chemokines CCL19/21, which bind to CCR7 on activated DCs and induce their migration across lymphatic endothelial cells toward lymph nodes.^{1, 2, 3} Soluble antigen alone can also flow through the lymph to the lymph nodes, where it can be acquired by lymph node–resident DCs and presented to T and B cells.^{4, 5} Through these processes, adaptive immunity and clonal expansion of lymphocytes are initiated during infection.Although the role and requirement of lymphatics during steady-state conditions are well studied, the mechanisms and consequences of lymphangiogenesis during inflammation are far less so by comparison. Lymphangiogenesis is induced during neonatal development, as well as postdevelopment (inflammation, infection, and tumor growth) by vascular endothelial growth factor (VEGF)-C and VEGF-D binding to vessel-expressed VEGF receptor 3 (VEGFR3).^{6, 7, 8, 9} CD11b⁺ monocytes have been identified as an important initiators of lymphangiogenesis because they produce VEGF-C and VEGF-D after proinflammatory stimuli^{10, 11, 12} and can integrate into pre-existing lymph vessels and transdifferentiate into lymphatic endothelial-like cells.¹³ Recent evidence shows an unappreciated role for lymphatics and lymphangiogenesis beyond transportation of antigen-presenting cells and peptides to the lymph nodes. These functions include direct modulation of DC and T-cell activation or tolerance,^{14, 15, 16, 17} the presentation of antigens,^{18, 19} and egress of T cells from lymph nodes.^{20, 21} The growing appreciation of diversity in lymphatic function ensures the importance of understanding lymphangiogenesis during infection and inflammation.Granulomatous immune responses are associated with many infectious and autoimmune diseases. The granuloma itself is a macrophage-dominated collection of leukocytes that forms with defined spatial and organizational arrangement, and these sites are important in the protection and pathology during granulomatous diseases.^{22, 23, 24, 25} During infectious disease, granulomas contain the immune response-inducing antigens, and so engagement between the peripheral immune organs and these antigens is required. Lymphatic vessels are important because they are routes that soluble and DC-carried antigens use to reach the lymph nodes from granulomatous tissue. The relationship between the granulomas and lymphoid vessels, especially in the context of lymphangiogenesis, is not yet understood. Here, we used two different mycobacterial models of granulomatous inflammation to investigate this relationship. This first involves high-dose infection with the Bacillus Calmette-Guerin (BCG) strain of mycobacterium, which induces acute granulomatous inflammation in the liver 3 weeks after infection. Resolution of inflammation after 3 weeks results in reduced, but persistent, BCG-containing granulomas in the chronic stages of infection. Granulomatous inflammation of the liver is a characteristic pathology of diseases including histoplasmosis^{26, 27, 28} and schistosomiasis,^{29, 30, 31} and many tuberculosis patients also have tubercle granulomas in their livers.^{32, 33, 34} We also used a mouse model involving aerosol infection in the lung with Mycobacterium tuberculosis (MTB). This model is distinct from systemic BCG infection because acute granulomatous inflammation does not resolve, and mice eventually succumb to disease resulting from increasing granuloma and bacterial burden. Understanding the relationship between granulomatous inflammation and lymphangiogenesis will undoubtedly involve an understanding of the infectious context given that granulomas can occur in different organs and the fact that lymphatic form and function are adapted to the anatomy of the tissue.Here, using both models, we show that granulomatous inflammation induces lymphangiogenesis and that the biology of this process has a regulatory role in the proliferation of mycobacterial-specific T cells.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

Trophoblast- and Vascular Smooth Muscle Cell-Derived MMP-12 Mediates Elastolysis during Uterine Spiral Artery Remodeling

During the first trimester of pregnancy, the uterine spiral arteries are remodeled, creating heavily dilated conduits that lack maternal vasomotor control but allow the placenta to meet an increasing requirement for nutrients and oxygen. To effect permanent vasodilatation, the internal elastic lamina and medial elastin fibers must be degraded. In this study, we sought to identify the elastolytic proteases involved in this process. Primary first-trimester cytotrophoblasts (CTBs) derived from the placenta exhibited intracellular and membrane-associated elastase activity; membrane-associated activity was primarily attributable to matrix metalloproteinases (MMP). Indeed, Affymetrix microarray analysis and immunocytochemistry implicated MMP-12 (macrophage metalloelastase) as a key mediator of elastolysis. Cultured human aortic smooth muscle cells (HASMCs) exhibited constitutive membrane-associated elastase activity and inducible intracellular elastase activity; these cells also expressed MMP-12 protein. Moreover, a specific inhibitor of MMP-12 significantly reduced CTB- and HASMC-mediated elastolysis in vitro, to 31.7 ± 10.9% and 23.3 ± 8.7% of control levels, respectively. MMP-12 is expressed by both interstitial and endovascular trophoblasts in the first-trimester placental bed and by vascular SMCs (VSMCs) in remodeling spiral arteries. Perfusion of isolated spiral artery segments with CTB-conditioned medium stimulated MMP-12 expression in medial VSMCs. Our data support a model in which trophoblasts and VSMCs use MMP-12 cooperatively to degrade elastin during vascular remodeling in pregnancy, with the localized release of elastin peptides and CTB-derived factors amplifying elastin catabolism.Transformation of the uterine spiral arteries during the first 20 weeks of gestation ensures that a constant supply of blood is delivered to the developing placenta, at an optimal rate of flow.^1–3 This allows the placenta to meet an increasing requirement for nutrients and oxygen and enables the developing fetus to attain its growth potential. The remodeling process leads to vessel dilatation, loss of spirality, and decreased vasoactivity, allowing a nonpulsatile low-pressure supply of blood to be delivered to placental villi at the maternofetal interface. Early alterations in arterial structure include endothelial vacuolation, hypertrophy of vascular smooth muscle cells (VSMCs), and disruption of medial smooth muscle layers, which occur in the absence of fetal-derived trophoblast and correlate with perivascular accumulation of macrophages and uterine natural killer (uNK) cells.^4,5 After colonization of the uterine decidua and myometrium by extravillous cytotrophoblast (EVT), endothelial cells and VSMCs are lost from the arterial wall and replaced by trophoblast embedded in a fibrinoid matrix. Remodeling is regulated in a spatial and temporal manner, such that the successive steps of trophoblast adherence, intravasation, fibrinoid deposition, and mural incorporation are effected without any loss in vessel integrity. A complex and highly orchestrated combination of vascular cell apoptosis, dedifferentiation, and matrix breakdown is probably required to achieve this alteration in vessel wall structure.^5–9Two distinct populations of EVT originate from anchoring placental villi and contribute to vessel transformation.^10,11 Interstitial EVT invade the uterine wall, migrating through the decidua and myometrium to adopt a perivascular position. Endovascular EVT enter the lumen of the spiral arteries and migrate as far as the first third of the myometrium, colonizing the arterial wall from within. Impaired arterial remodeling is distinguished by shallow EVT invasion, decreased numbers of EVT, and the persistence of muscular, narrow-bore arteries, and is associated with second trimester miscarriage,¹² preterm labor,¹³ pre-eclampsia,¹⁴ and fetal growth restriction.¹⁵To effect a permanent increase in vessel diameter it is crucial that elastin fibers within each artery are catabolized, eliminating their capacity for stretch and recoil. Myometrial segments of the spiral arteries possess an internal elastic lamina (IEL), and the musculo-elastic media of both decidual and myometrial arteries is rich in elastic fibers.^16,17 During pregnancy, EVT traverse the IEL during mural incorporation,¹⁸ thus it is highly likely that they possess elastase activity: indeed, first-trimester EVT synthesize and secrete the elastolytic proteases matrix metalloproteinase-2 (MMP-2), MMP-7, MMP-9, cathepsin B, and cathepsin L.^19,20 Although both uNK cells and macrophages produce enzymes capable of elastolysis,⁵ uNK cells are not abundant in myometrium,²¹ and elastin breakdown is associated with the presence of endovascular EVT¹⁷ rather than macrophages.²² Previous studies have demonstrated that the availability of nitric oxide (NO) can influence protease expression and activity,^23–26 and we have shown NO to be an important regulator of trophoblast function.^27–29 As dysregulation of NO production has been implicated in the pathogenesis of pre-eclampsia and intrauterine growth restriction (IUGR),^30–32 NO availability may regulate the process of arterial remodeling by controlling trophoblast elastolysis.Rodent models of atherosclerosis have highlighted a role for VSMC-derived cathepsins as mediators of IEL breakdown during lesion formation,³³ demonstrating that the arterial wall may be a potential source of elastases. Similarly, caspase-2, −3, and −7 derived from apoptotic VSMCs have been implicated as mediators of elastin breakdown.³⁴ Thus, during the process of spiral artery transformation, resident VSMCs may also be stimulated to produce elastase(s) in response to pregnancy hormones, trophoblast invasion, or soluble factors released by cells within the placental bed. In this study we have investigated the origin and identity of the proteases involved in mediating elastin breakdown during spiral artery remodeling.     

Dermal Collagen and Lipid Deposition Correlate with Tissue Swelling and Hydraulic Conductivity in Murine Primary Lymphedema

Primary lymphedema is a congenital pathology of dysfunctional lymphatic drainage characterized by swelling of the limbs, thickening of the dermis, and fluid and lipid accumulation in the underlying tissue. Two mouse models of primary lymphedema, the Chy mouse and the K14-VEGFR-3-Ig mouse, both lack dermal lymphatic capillaries and exhibit a lymphedematous phenotype attributable to disrupted VEGFR-3 signaling. Here we show that the differences in edematous tissue composition between these two models correlated with drastic differences in hydraulic conductivity. The skin of Chy mice possessed significantly higher levels of collagen and fat, whereas K14-VEGFR-3-Ig mouse skin composition was relatively normal, as compared with their respective wild-type controls. Functionally, this resulted in a greatly increased dermal hydraulic conductivity in K14-VEGFR3-Ig, but not Chy, mice. Our data suggest that lymphedema associated with increased collagen and lipid accumulation counteracts an increased hydraulic conductivity associated with dermal swelling, which in turn further limits interstitial transport and swelling. Without lipid and collagen accumulation, hydraulic conductivity is increased and overall swelling is minimized. These opposing tissue responses to primary lymphedema imply that tissue remodeling—predominantly collagen and fat deposition—may dictate tissue swelling and govern interstitial transport in lymphedema.Primary or congenital lymphedema is a pathological condition in which excess fluid accumulates in the limb because of dysfunctional lymphatic drainage.^1,2 In humans, primary lymphedema has been linked to mutations in lymphatic endothelial cell genes that result in malformations in lymphatic valve and mural structure or insufficient organization of lymphatic capillaries.^3–8 As a chronic pathology, lymphedema results in characteristic morphological changes including remodeling of the skin and subcutaneous extracellular matrix (ECM) and accumulation of lipids.^9–12 Lymphatic function is tightly controlled by the mechanical properties of the tissue via anchoring filaments that attach lymphatic endothelium to the surrounding ECM,^13,14 such that structural changes can further retard interstitial fluid clearance.^11,15 No treatment to date can truly restore tissue fluid balance or improve lymphatic function, but there has been success using compression sleeves, massage, and surgical removal of tissue in limiting the pathology.¹⁶ These successes further underscore lymphedema as not simply a disease of lymphatic transport, but a pathology governed by the ECM.To recreate the pathology of primary lymphedema in mouse models, lymphatic genes have been targeted to disrupt proper formation of lymphatic vessels during development, but many of these are lethal, including the deletion of Foxc2,^3,7 VEGFR-3,^3,7 VEGF-C,¹⁷ or Prox-1.¹⁸ Heterozygote mutations or deletions of these genes, however, are sometimes viable and may present poorly formed lymphatic vessels, an edematous phenotype in adulthood, or failed responses to interstitial challenge.^3,7,17–19 Although the lymphedema exhibited in such models never entirely recapitulates the extent of swelling of whole limbs or pathological asymmetry found in humans, such models provide an excellent platform for studying the consequential dermal pathology of lymphedema and potential treatments.The Chy mouse and the K14-VEGFR-3-Ig mouse are two such models previously developed targeting VEGFR-3 signaling.^20,21 The Chy mouse possesses a heterozygous VEGFR-3 mutation in the tyrosine kinase domain, preventing phosphorylation and resulting in early developmental deficiencies in some lymphatic vessels and chylous ascites as newborns.²⁰ Adult Chy mice lack dermal lymphatics.^20,22 In contrast, the K14-VEGFR-3-Ig mouse secretes a soluble variant of VEGFR-3, formed by the fusion of the extracellular ligand-binding domain of VEGFR-3 and an IgG Fc domain, in the epidermis under the keratin-14 (K14) promoter.²¹ The secreted VEGFR-3 appropriates VEGF-C, preventing lymphatic capillary development in the skin.²¹ No abnormal blood vascular phenotypes have been reported in these mice resulting from these mutations. Both mouse models exhibit lymphedema, particularly in the lower limbs, tail, and snout, and tissue histology shows dermal remodeling and fluid accumulation in the hypodermis.^20,21 Symptomatically, these models represent features of the human disease arising from VEGFR-3 and VEGF-C mutations⁸ and provide a platform for dermal transport consequences in lymphedema.Interstitial fluid pressure (IFP) provides the driving force for flow through tissues while the hydraulic conductivity (K) of the tissue determines its resistance to flow. Fluid moves more freely through tissues with a higher K, potentially limiting the swelling load on the ECM. Factors influencing tissue hydraulic conductivity include tissue hydration,^23,24 matrix composition,^25,26 and IFP.²⁷ Small changes in matrix composition or IFP can result in large changes to hydraulic conductivity.²⁸ We therefore hypothesized that tissue composition changes associated with dysfunctional local lymphatic drainage likely alter tissue hydraulic conductivity and interstitial fluid transport that would dictate the functional manifestation of lymphedema. Tissue collagen, lipid, and water content were measured to determine tissue compositional changes in these mice, and interstitial transport was measured by applying a quantitative in situ model of tissue hydraulic conductivity. Despite both models lacking dermal lymphatics, we found that the tissue compositional changes were quite different between the two models, resulting in large differences in interstitial transport properties. This demonstrates that lymphatic transport deficiencies alone do not determine the extent of lymphedema, but rather that tissue composition plays a critical and potentially compounding influence.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Regression of Allograft Airway Fibrosis: The Role of MMP-Dependent Tissue Remodeling in Obliterative Bronchiolitis after Lung Transplantation

Obliterative bronchiolitis after lung transplantation is a chronic inflammatory and fibrotic condition of small airways. The fibrosis associated with obliterative bronchiolitis might be reversible. Matrix metalloproteinases (MMPs) participate in inflammation and tissue remodeling. MMP-2 localized to myofibroblasts in post-transplant human obliterative bronchiolitis lesions and to allograft fibrosis in a rat intrapulmonary tracheal transplant model. Small numbers of infiltrating T cells were also observed within the fibrosis. To modulate inflammation and tissue remodeling, the broad-spectrum MMP inhibitor SC080 was administered after the allograft was obliterated, starting at post-transplant day 21. The allograft lumen remained obliterated after treatment. Only low-dose (2.5 mg/kg per day) SC080 significantly reduced collagen deposition, reduced the number of myofibroblasts and the infiltration of T cells in association with increased collagenolytic activity, increased MMP-2 gene expression, and decreased MMP-8, MMP-9, and MMP-13 gene expression. In in vitro experiments using cultured myofibroblasts, a relatively low concentration of SC080 increased MMP-2 activity and degradation of type I collagen. Moreover, coculture with T cells facilitated persistence of myofibroblasts, suggesting a role for T-cell infiltration in myofibroblast persistence in fibrosis. By combining low-dose SC080 with cyclosporine in vivo at post-transplant day 28, partial reversal of obliterative fibrosis was observed at day 42. Thus, modulating MMP activity might reverse established allograft airway fibrosis by regulating inflammation and tissue remodeling.Chronic allograft dysfunction after lung transplantation is manifested by obliterative bronchiolitis (OB), a fibroproliferative obstructive lesion in small airways, and its clinical correlate, bronchiolitis obliterans syndrome (BOS).^1,2 Once the fibrotic process of OB is initiated, conventional immunosuppression is usually ineffective.³ The traditional pathological perspective is that fibrosis is the end result of damage: scar tissue, with no possibility of return to the pre-existing structure.⁴ However, increasing evidence suggests that fibrosis still undergoes dynamic remodeling and is potentially a reversible process. For example, the resolution of liver fibrosis is well documented both clinically and experimentally. In animal experiments, up-regulation or overexpression of matrix metalloproteinases (MMPs) capable of degrading interstitial type I and type III collagen (including MMP-1,⁵ MMP-8,⁶ MMP-13,⁷and MMP-2 and MMP-14^8,9) is associated with the regression of liver fibrosis. Pulmonary fibrosis has also been shown to be conditionally reversible.¹⁰One possible mechanism rendering fibrosis unlikely to resolve is the aberrant persistence of myofibroblasts, an active form of fibroblasts positive for α-smooth muscle actin (α-SMA), which leads to production of extracellular matrix (ECM) in excess of MMP-dependent ECM degradation.¹¹ Unresolved inflammation can be an important contributor to this mechanism.¹⁰ Accumulating evidence suggests that chronic fibrotic conditions are mediated by complex interactions between immune and nonimmune cells, in which the persistence of a relatively low grade of inflammation continuously stimulates resident stromal cells^12,13 and provides survival signals to myofibroblasts.¹⁴ For instance, the resolution of liver fibrosis encountered in alcohol-induced and virus-related fibrosis occurs only after remedy of the underlying cause.^15,16 Moreover, in experimental models of fibrosis, reversal of fibrosis has occurred in one-hit injury models such as bleomycin-induced pulmonary fibrosis,¹⁷ in which the initial tissue injury leads to fibrosis but the tissue injury or inflammation is not continuous.^8,9Along those lines, OB after lung transplantation is a fibrotic and chronic inflammatory condition¹⁸ in which myofibroblasts persist.¹⁹ The intrapulmonary tracheal transplant model of OB is a unique animal model in which persistent alloantigen from the donor trachea within the pulmonary milieu causes continuous alloantigen-induced inflammation and results in robust fibrosis in the allograft lumen.²⁰ We have previously demonstrated that myofibroblasts expressing high levels of collagen and MMP-2 and MMP-14 play a central role in the remodeling of established allograft airway fibrosis.²⁰ Given that MMPs also play important but complex roles in the trafficking of immune responsive cells,²⁰ MMPs involved in both tissue remodeling and inflammation may play key roles in the reversal of fibrosis.We therefore hypothesized that allograft airway fibrosis is a potentially reversible process involving MMPs. Here, we demonstrate expression patterns of MMPs in established human OB lesions and describe the roles of MMPs in the remodeling of collagen matrix, myofibroblasts, and immune responsive cells using in vivo and in vitro models with SC080, a general MMP inhibitor. Finally, we demonstrate for the first time reversibility of allograft airway fibrosis by combining immunosuppression with a low dose of SC080.     

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

The Calreticulin-Binding Sequence of Thrombospondin 1 Regulates Collagen Expression and Organization During Tissue Remodeling

Amino acids 17-35 of the thrombospondin1 (TSP1) N-terminal domain (NTD) bind cell surface calreticulin to signal focal adhesion disassembly, cell migration, and anoikis resistance in vitro. However, the in vivo relevance of this signaling pathway has not been previously determined. We engineered local in vivo expression of the TSP1 calreticulin-binding sequence to determine the role of TSP1 in tissue remodeling. Surgical sponges impregnated with a plasmid encoding the secreted calreticulin-binding sequence [NTD (1-35)-EGFP] or a control sequence [mod NTD (1-35)-EGFP] tagged with enhanced green fluorescent protein were implanted subcutaneously in mice. Sponges expressing NTD (1-35)-EFGP formed a highly organized capsule despite no differences in cellular composition, suggesting stimulation of collagen deposition by the calreticulin-binding sequence of TSP1. TSP1, recombinant NTD, or a peptide of the TSP1 calreticulin-binding sequence (hep I) increased both collagen expression and matrix deposition by fibroblasts in vitro. TSP1 stimulation of collagen was inhibited by a peptide that blocks TSP1 binding to calreticulin, demonstrating the requirement for cell surface calreticulin. Collagen stimulation was independent of TGF-β activity and Smad phosphorylation but was blocked by an Akt inhibitor, suggesting that signaling through the Akt pathway is important for regulation of collagen through TSP1 binding to calreticulin. These studies identify a novel function for the NTD of TSP1 as a mediator of collagen expression and deposition during tissue remodeling.Tissue remodeling is a highly orchestrated process that requires coordinated regulation of cell migration, proliferation, extracellular matrix deposition and remodeling, and eventual cell regression. The extracellular matrix provides both biochemical and mechanical cues to regulate these complex cellular responses to injury and repair. A family of extracellular matrix proteins, the matricellular proteins, has been shown to regulate cell behavior and extracellular matrix deposition during tissue remodeling and wound repair.^1–3Thrombospondin 1 (TSP1) is a multifunctional, matricellular protein that constitutes 25% of the protein released from the α-granules of activated platelets.^4,5 TSP1 is present in wounds and expressed by cells involved in wound healing, including macrophages, fibroblasts, endothelial cells, and vascular smooth muscle cells.^6–9 TSP1 knockout mice display compromised wound healing, characterized by reduced macrophage infiltration and a delay in capillary angiogenesis, but persistence of granulation tissue.⁹ It induces focal adhesion disassembly, stimulates cell motility, activates latent transforming growth factor-β (TGF-β), and inhibits nitric oxide signaling.^10–12 Depending on whether the N- or C-terminal domain of TSP1 is engaged, it is either anti- or proangiogenic.^12–14 TSP1 can be proapoptotic to endothelial cells, but it also stimulates cell survival by signaling resistance to anoikis.¹⁵ These diverse and sometimes paradoxical activities can be ascribed to its interactions with multiple receptors, including integrins, syndecans,^13,16 CD47,¹⁷ CD36,¹⁸ low-density lipoprotein receptor-related protein 1 (LRP1),¹⁹ and calreticulin (CRT).²⁰To date, the role of TSP1 in tissue remodeling has largely been studied through injury models in TSP1-null mice.^9,21,22 These models have been useful for identifying many functions of TSP1 but are limited in their ability to mimic tissue remodeling in a normal organism, in which TSP1 expression, proteolysis, and interactions with multiple receptors will be modulated both temporally and spatially. The susceptibility of TSP1 to proteolytic cleavage by a wide spectrum of proteases suggests that cells are likely to be exposed to fragments of TSP1 during tissue remodeling.²³ Both the N- and C-terminal domains can be detected separately from the full-length TSP1 molecule in vivo.^23,24 Therefore, ongoing questions include whether TSP1 can signal simultaneously through multiple receptors and whether isolated domains elicit responses distinct from the intact molecule. For these reasons, in vivo models expressing isolated TSP1 domains on a wild-type genetic background are relevant to the physiological conditions of TSP1 in wound healing.Previously, we showed that amino acids 17-35 of the N-terminal domain (NTD) of TSP1 signal focal adhesion disassembly and increased cell migration in vitro.²⁵ Furthermore, signaling through this sequence prevents anoikis.¹⁹ This sequence in the NTD binds to a cell surface cocomplex of CRT and LRP1 and stimulates signaling through focal adhesion kinase (FAK), extracellular signal related kinase (ERK), and phosphoinositide 3-kinase (PI3 kinase), which results in transient phosphorylation of Akt and down-regulation of Rho kinase.^26,27 Signaling downstream from TSP1 engagement of the CRT/LRP1 cocomplex induces an intermediate state of adhesion in endothelial cells, fibroblasts, and vascular smooth muscle cells.^25,26 Intermediate adhesion is characterized by a reduced number of focal adhesions and actin stress fibers without the loss of cell attachment or spreading.²⁸ This intermediate adhesive state precedes migration in response to the TSP1 CRT-binding sequence.^28,29 Induction of intermediate adhesion, cell migration, and anoikis resistance are similarly regulated by TSP1, a recombinant trimeric form of the NTD (NoC1), and by a synthetic peptide comprising the CRT-binding sequence (aa 17-35, hep I peptide). Furthermore, TSP1 binding to aa19-36 in the NTD of CRT is necessary for TSP-CRT binding and induction of signaling, and cells lacking this site in CRT do not respond to TSP1.^15,30,31The role of TSP1 binding to the CRT-LRP1 complex in vivo is unknown. Based on previous studies, we hypothesized that local expression of the secreted CRT-binding sequence of TSP1 at sites of injury in vivo would signal intermediate cell adhesion and migration of CRT-expressing cells to increase cellularity of wounds. To test this hypothesis, we used an in vivo mouse model of the foreign body response to drive local expression of a secreted enhanced green fluorescent protein (EGFP)-tagged fusion protein of the TSP1 CRT-binding sequence. Unexpectedly, our results showed that the CRT-binding sequence of TSP1 stimulates the formation of a highly organized collagen capsule, which reduced cellular infiltration into the sponges. In vitro studies confirmed that TSP1 stimulates fibrillar collagen expression by fibroblasts and increased incorporation of collagen into the extracellular matrix in a CRT-dependent manner. Although TSP1 can activate latent TGF-β, the recombinant TSP1 NTD protein NoC1 stimulated collagen independently of both TGF-β activity and Smad2 phosphorylation. Rather, the CRT-binding sequence requires Akt activity to stimulate collagen. These studies identify a previously unknown role for the NTD of TSP1 in tissue remodeling through a CRT-dependent TGF-β–independent stimulation of collagen matrix formation.     

Decidual Natural Killer Cell Interactions with Trophoblasts Are Impaired in Pregnancies at Increased Risk of Preeclampsia

Transformation of the uterine spiral arteries (SAs) during pregnancy is critical to support the developing fetus, and is impaired in some pregnancy disorders, including preeclampsia. Decidual natural killer (dNK) cells play a role in SA remodeling, although their interactions with fetal trophoblast remain unclear. A uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of the extent of SA remodeling; we have used this technique to characterize dNK cells from pregnancies with normal (normal RI) and impaired (high RI) SA remodeling, which display least and highest risk of developing preeclampsia, respectively. We examined the impact of dNK cell secreted factors on trophoblast motility, chemoattraction, and signaling pathways to determine the contribution of dNK cells to SA transformation. We demonstrated that the chemoattraction of the trophoblast by dNK cells is impaired in pregnancies with high RI, as is the ability to induce trophoblast outgrowth from placental villous explants. These processes are dependent on activation of the extracellular signal–regulated kinase 1/2 and phosphatidylinositol 3-kinase–Akt signaling pathways, which were altered in trophoblasts incubated with secreted factors from dNK cells from high RI pregnancies. Therefore, by characterizing pregnancies using uterine artery Doppler RI before dNK cell isolation, we have identified that impaired dNK-trophoblast interactions may lead to poor placentation. These findings have implications for pregnancy pathological conditions, such as preeclampsia.After implantation of the blastocyst, the pregnant uterus (decidua) undergoes physiological changes to ensure a successful pregnancy. Key to this is the transformation of the uterine spiral arteries (SAs) from high-resistance, low-flow vessels to low-resistance, high-flow vessels. This establishes an increased blood flow to the intervillous space, allowing nutrient and gas exchange between the maternal blood flow and fetal placenta.¹ SA transformation is under the control of extravillous trophoblast (EVT), specialized fetal cells derived from the placenta that invade into the decidua and remodel the SA by inducing apoptosis of endothelial cells and vascular smooth muscle cells (VSMCs).^2,3 The VSMCs undergo induced hypertrophy and disruption of VSMC layers, leading to motility and dedifferentiation of vascular smooth muscle cells.^4,5 Trophoblasts then line the SA in place of the absent vascular cells. The essential presence of trophoblasts in SA transformation is inferred from pregnancy disorders, such as preeclampsia and intrauterine growth restriction, that display reduced trophoblast invasion and reduced remodeling of the arteries in the first trimester of pregnancy.^6,7 The reason for decreased EVT invasion in these pathological conditions is unknown.It is increasingly recognized that transformation of SA begins in a trophoblast-independent manner, when only maternal cells are present surrounding the spiral arteries.⁸ Several different maternal cells exist in the decidua in the first trimester of pregnancy, including leukocytes, of which 70% are decidual natural killer (dNK) cells. These differ from peripheral blood natural killer (pbNK) cells in their surface receptor expression; notably, dNK cells are predominantly CD56^brightCD16⁻, whereas pbNK cells are predominantly CD56^dimCD16⁺. Decidual NK cells are thought to have a cytokine-secreting role as opposed to the cytotoxic role of pbNK cells,⁹ because dNK cells do not kill trophoblasts in a normal pregnancy,¹⁰ despite possessing the same cytotoxic capacity as pbNK cells in terms of expression of the cytolytic proteins, perforin and granzyme.¹¹ Disruption of the vasculature when the SAs are surrounded by dNK cells, but the trophoblasts are absent, has been identified⁸ and has implicated dNK cells in SA remodeling via both direct interaction with the vascular cells and indirect interaction with trophoblasts.Decidual NK cells are proposed to contribute directly to SA remodeling by expression of secreted factors that disrupt vascular cell interactions,¹² including matrix metalloproteinases that disrupt vascular extracellular matrix connections, therefore enabling migration of VSMCs from the vessel.^8,13 Some apoptosis of VSMCs and endothelial cells during remodeling has also been attributed to dNK cells via an Fas-ligand pathway.¹⁴ Decidual NK cells may also have indirect effects on remodeling by controlling EVT invasion, by both promotion and inhibition. EVT invasion is dependent on motility and chemotaxis. dNK cells have been demonstrated to increase EVT motility via hepatocyte growth factor secretion,¹⁴ and to play a role in chemoattraction of the EVT to the sites of remodeling, in particular through expression of the chemokines IL-8 and CXCL10.¹⁵ They have also been reported to decrease trophoblast invasion via an interferon-γ–secreted mechanism.¹⁶ However, many chemokines have been identified as secreted by dNK cells, and these may also play a role in the promotion of invasion of EVT through the decidua.^17–19Although there is strong evidence for the role of dNK cells in mouse SA remodeling,²⁰ and human dNK cells have been linked to pregnancy disorders associated with poor SA remodeling,^21,22 the key role of dNK cells in human pregnancy is proposed to occur during the first trimester of pregnancy, hampering studies because of a lack of access to tissue. When first-trimester termination of pregnancy samples is used for isolation of dNK cells, the outcome of that pregnancy, if brought to term, and the extent of remodeling are unknown. Uterine artery Doppler resistance index (RI) in the first trimester can be used as a proxy measure of the extent to which remodeling of the spiral arteries has occurred,^23,24 and can, therefore, be used as a technique to separate pregnancies of a normal and high RI, which are at least risk (<1%) and most risk (21%) of developing preeclampsia, respectively.^14,25 We can, therefore, examine the role of dNK cells isolated from these pregnancies. We have previously demonstrated that dNK cells isolated from high RI pregnancies are less able to induce vascular cell apoptosis.¹⁴ In this study, we examined the interactions of dNK cells from normal and high RI pregnancies with trophoblasts, to determine the contribution of dNK to trophoblast-induced SA remodeling.     

Myoglobin Overexpression Inhibits Reperfusion in the Ischemic Mouse Hindlimb through Impaired Angiogenesis but Not Arteriogenesis

Adaptive vascular remodeling in response to arterial occlusion takes the form of capillary growth (angiogenesis) and outward remodeling of pre-existing collateral arteries (arteriogenesis). However, the relative contributions of angiogenesis and arteriogenesis toward the overall reperfusion response are both highly debated and poorly understood. Here, we tested the hypothesis that myoglobin overexpressing transgenic mice (MbTg⁺) exhibit impaired angiogenesis in the setting of normal arteriogenesis in response to femoral artery ligation, and thereby serve as a model for disconnecting these two vascular growth processes. After femoral artery ligation, MbTg⁺ mice were characterized by delayed distal limb reperfusion (by laser Doppler perfusion imaging), decreased foot use, and impaired distal limb muscle angiogenesis in both glycolytic and oxidative muscle fiber regions at day 7. Substantial arteriogenesis occurred in the primary collaterals supplying the ischemic limb in both wild-type and MbTg⁺ mice; however, there were no significant differences between groups, indicating that myoglobin overexpression does not affect arteriogenesis. Together, these results uniquely demonstrate that functional collateral arteriogenesis alone is not necessarily sufficient for adequate reperfusion after arterial occlusion. Angiogenesis is a key component of an effective reperfusion response, and clinical strategies that target both angiogenesis and arteriogenesis could yield the most efficacious treatments for peripheral arterial disease.Peripheral arterial disease (PAD) is caused by atherosclerosis and is characterized by the progressive and often complete occlusion of large- and medium-size arteries at sites other than the heart. PAD most often occurs in the lower limbs, with progressive PAD leading to the debilitating consequences of intermittent claudication and critical limb ischemia. Given the high prevalence (>20% of those >65 years of age¹) and economic impact ($4.4 billion estimated treatment costs²) of PAD, along with few therapeutic options, there is a critical need for developing new therapeutic modalities. One promising approach entails stimulating adaptive vascular remodeling to enhance perfusion around occlusions. To date, however, trials using this approach have largely failed. An improper understanding of the balance of angiogenesis versus arteriogenesis has been cited as a reason for many of these failures.^{3, 4, 5}Adaptive vascular remodeling to arterial occlusion(s) can be broken down into two aspects. First, in ischemic tissues downstream of an arterial occlusion, capillaries grow from existing vessels via angiogenesis, expanding blood flow distribution throughout the ischemic tissue. In contrast, collateral arteries around the occlusion are stimulated to undergo structural lumenal expansion (ie, arteriogenesis) that allows for greater in-flow into the distal, ischemic tissue. Therapeutic clinical trials have largely focused on only one process (either angiogenesis or arteriogenesis).^{3, 4, 5} The most direct examples of an unbalanced approach come from the early and prominent failures of many large clinical trials using predominantly angiogenic factors (eg, vascular endothelial growth factor and hypoxia inducible factor 1-α) to induce angiogenesis.^{6, 7, 8} However, trials targeting factors chosen specifically for their arteriogenic potential (eg, fibroblast growth factor 2 or granulocyte macrophage-colony stimulating factor) have also reported only marginal success.^{9, 10, 11, 12} A more fruitful strategy was recently hinted at in a study by West et al,¹³ which suggested that even in the presence of increased perfusion pressure to the distal tissue after a percutaneous intervention, revascularization is unable to restore microvascular perfusion in PAD patients. This suggests that microvascular perfusion impairments must be addressed for full functional recovery. Moreover, strategies that do not change large vessel occlusion but alter angiogenesis can be clinically beneficial. In summary, these findings suggest the need to better understand how angiogenesis and arteriogenesis work together to improve reperfusion after arterial occlusion.Data demonstrating how angiogenesis and arteriogenesis separately contribute to reperfusion after arterial occlusion could outline how targeting both aspects of neovascularization could improve therapy. However, being able to identify how angiogenesis and arteriogenesis each contribute to reperfusion after ischemic injury requires both a condition that does not simultaneously impact arteriogenesis and angiogenesis, and methods for separately quantifying angiogenesis and arteriogenesis. Myoglobin overexpression in skeletal muscle may serve as a unique stimulus that has an effect on tissue reperfusion during ischemia, but has uncoupled effects on arteriogenesis and angiogenesis. Skeletal muscle myoglobin overexpression has been previously documented to impair angiogenesis and reperfusion in a severe hindlimb ischemia model.¹⁴ The impaired angiogenesis was proposed to arise from the excess myoglobin acting as a sink for nitric oxide,^{14, 15} which resulted in the loss of key trophic and angiogenic factors during ischemia.^{16, 17} The close physical association of the endothelium with muscle fibers required for this mechanism occurs at the capillary level, but the greater separation of collateral arteries from the parenchymal muscle tissue reduces the potential for this mechanism on arteriogenesis.^{18, 19} The potential angiogenesis-specific impairment is further supported by the delayed time course of the perfusion deficit.^{14, 20} However, because the more severe ischemia model was chosen, the ability to assess arteriogenesis was limited.²¹ Alternatively, a milder hindlimb ischemia model [ie, femoral artery ligation (FAL) without excision] can be used to produce consistent collateral artery remodeling needed for the precise quantification of arteriogenesis.^{21, 22, 23} Therefore, we hypothesized that coupling the MbTg⁺ transgenic model with the milder FAL ischemic stimulus would permit quantification of the degree to which impairment in angiogenesis alone can contribute to the reperfusion response after arterial occlusion. Here, we present a unique dataset demonstrating that angiogenesis is required along with normal arteriogenesis for a more fully effective revascularization response.     

Transgenic Overexpression of Interleukin-1β Induces Persistent Lymphangiogenesis But Not Angiogenesis in Mouse Airways

These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1β mice that conditionally overexpress IL-1β in Clara cells to determine whether IL-1β can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1β 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-1β, and lymphotoxin-β), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1β overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1β 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-1β–induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1β/CXCR2^−/− mice had the usual IL-1β-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-1β, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1β overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.IL-1β is a key inflammatory cytokine found in many pathologic conditions and is responsible for triggering multiple downstream inflammatory pathways.¹ Inhibiting IL-1 signaling by neutralizing antibodies or by blocking IL1R1 receptors is effective in treating inflammation in numerous pathologic conditions.² However, IL-1β can be a two-edged sword. Depending on the context, IL-1β is responsible for deleterious effects by amplifying inflammation and also for protective effects, for example, by activating the immune system during infection.³IL-1β has a main role in the remodeling of many tissues, including the airways and lungs. Overexpression of IL-1β in adult mouse airways and lungs results in pulmonary inflammation and the recruitment of inflammatory cells, including neutrophils, enlargement of distal airspaces, and the induction of mucous metaplasia and airway fibrosis.⁴ In neonatal mice, overexpression of IL-1β results in the disruption of lung development characteristic of bronchopulmonary dysplasia,^5,6 and this effect is mediated in part by integrins.^7,8 Furthermore, in addition to its known effects on remodeling of many tissue types, IL-1β has been reported to induce angiogenesis in several experimental models and in human diseases, including the eye, arthritic joints, and tumors, mediated in part by recruitment of leukocytes that release other inflammatory mediators.^9–14Blood vessels and lymphatics of airways show a wide repertoire of responses to different inflammatory stimuli. Various patterns of blood vessel enlargement and angiogenic sprouting are found in mice with chronic airway inflammation.^15–17 For the most part, the cellular and molecular mediators that drive vascular changes are still poorly understood, but numerous cytokines and chemokines, including IL-1β, are up-regulated in Mycoplasma pulmonis infection.^17–20 M. pulmonis-infected mice also show profound lymphangiogenesis, mediated by vascular endothelial growth factor receptor (VEGFR)-3 signaling.²¹ Because IL-1β can activate NF-κB pathways to up-regulate vascular endothelial growth factor (VEGF)-C and -D, ligands for VEGFR-3,^22,23 IL-1β could also be a candidate for driving lymphangiogenesis. IL-1β is also known to up-regulate VEGF-C in vitro, a VEGFR-3 ligand that can drive lymphangiogenesis.²⁴ However, it has been difficult to dissect the effects of individual cytokines in bacterial infection, and the effects of IL-1β alone in airways have not been examined.With this background, we took advantage of bi-transgenic (CCSP/IL-1β) mice in which IL-1β is overexpressed in airways by the rat Clara cell secretory protein (CCSP) promoter in a doxycycline (Dox)-inducible fashion.⁴ This model permitted us to study the effects of overexpression of IL-1β alone on lymphangiogenesis and angiogenesis.The goal of this study was to determine whether selective overexpression of IL-1β in adult mouse airways would induce growth or remodeling of blood vessels or lymphatic vessels and to determine the involved cells and molecules. We also sought to learn if vessel remodeling persisted after IL-1β was turned off and if VEGFR-3 signaling drove the lymphangiogenesis. To approach these issues, we stained blood vessels and lymphatics immunohistochemically in whole mounts of tracheas from CCSP/IL-1β mice treated with Dox. We also used immunohistochemistry to identify airway cells that stained for IL1R1. Because IL-1β induced leukocyte influx, including abundant neutrophils, we tested whether neutrophils were essential for the effects of IL-1β on lymphatic vessels by examining lymphangiogenesis in CXCR2^−/− mice crossed to CCSP/IL-1β mice.We found that overexpression of IL-1β in mouse airways produced neutrophil and macrophage influx, expression of inflammatory cytokines and chemokines, and long-lasting lymphangiogenesis, but not angiogenesis. IL1R1 receptors were abundant on epithelial basal cells and neuroendocrine cells, but not on lymphatics. Inactivation of VEGFR-3 ligands by soluble VEGFR-3 (VEGF-C/D Trap) from an adeno-associated viral (AAV) vector completely blocked the lymphangiogenesis, indicative of the necessity of VEGFR-3 ligands, VEGF-C and/or VEGF-D. VEGF-C immunoreactivity was present in some recruited macrophages, but the lymphangiogenesis did not require the influx of neutrophils.