Angiogenesis in the lung.

Both systemic (tracheal and bronchial) and pulmonary circulations perfuse the lung. However, documentation of angiogenesis of either is complicated by the presence of the other. Well-documented angiogenesis of the systemic circulations have been identified in asthma, cystic fibrosis, chronic thromboembolism and primary carcinomas. Angiogenesis of the vasa vasorum, which are branches of bronchial arteries, is seen in the walls of large pulmonary vessels after a period of chronic hypoxia. Documentation of increased pulmonary capillaries has been shown in models of chronic hypoxia, after pneumonectomy and in some carcinomas. Although endothelial cell proliferation may occur as part of the repair process in several pulmonary diseases, it is separate from the unique establishment of new functional perfusing networks defined as angiogenesis. Identification of the mechanisms driving the expansion of new vascular beds in the adult needs further investigation. Yet the growth factors and molecular mechanisms of lung angiogenesis remain difficult to separate from underlying disease sequelae.

Lymphangiogenesis in rat asthma model.

Although bronchial angiogenesis has been well documented in allergic asthma, lymphangiogenesis has not been widely studied. Therefore, we evaluated changes in lung lymphatics in a rat model of allergen-induced asthma using house dust mite (Der p 1; 100 µg/challenge). Additionally, properties of isolated lung lymphatic endothelial cells (CD45-, CD141+, LYVE-1+, Prox-1+) were studied in vitro. Three weeks after the onset of intranasal allergen exposure (twice-weekly), an increase in the number of lung lymphatic vessels was measured (34% increase) by lung morphometry. New lymphatic structures were seen predominantly in the peribronchial and periarterial interstitial space but also surrounding large airways. Isolated lymphatic endothelial cells from sensitized lungs showed enhanced proliferation (% Ki67+), chemotaxis, and tube formation (number and length) compared to lymphatic endothelial cells isolated from naive rat lungs. This hyper-proliferative lymphangiogenic phenotype was preserved through multiple cell passages (2-8). Lymphatic endothelial cells isolated from naive and HDM-sensitized rats produced similar in vitro levels of VEGF-C, VEGF-D, and VEGFR3 protein, each recognized as critical lymphangiogenic factors. Inhibition with anti-VEGFR (axitinib, 0.1 µM) blocked proliferation and chemotaxis. Results suggest that in vivo sensitization causes fundamental changes to lymphatic endothelium, which are retained in vitro, and may relate to VEGFR downstream signaling.

Effector T Cells and Ischemia-Induced Systemic Angiogenesis in the Lung.

Lymphocytes have been shown to modulate angiogenesis. Our previous work showed that T regulatory (Treg) cell depletion prevented angiogenesis. In the present study, we sought to examine T-cell populations during lung angiogenesis and subsequent angiostasis. In a mouse model of ischemia-induced systemic angiogenesis in the lung, we examined the time course (0-35 d) of neovascularization and T-cell phenotypes within the lung after left pulmonary artery ligation (LPAL). T cells increased and reached a maximum by 10 days after LPAL and then progressively decreased, suggestive of a modulatory role during the early phase of new vessel growth. Because others have shown IFN-γ to be angiostatic in tumor models, we focused on this effector T-cell cytokine to control the magnitude of angiogenesis. Results showed that IFN-γ protein is secreted at low levels after LPAL and that mice required Treg depletion to see the full effect of effector T cells. Using Foxp3(DTR) and diphtheria toxin to deplete T regulatory cells, increased numbers of effector T cells (CD8(+)) and/or increased capacity to secrete the prominent angiostatic cytokine IFN-γ (CD4(+)) were seen. In vitro culture of mouse systemic and pulmonary microvascular endothelial cells with IFN-γ showed increased endothelial cell apoptosis. CD8(-/-) mice and IFN-γR(-/-) mice showed enhanced angiogenesis compared with wild-type mice, confirming that, in this model, IFN-γ limits the extent of systemic neovascularization in the lung.

Lung Angiogenesis Requires CD4(+) Forkhead Homeobox Protein-3(+) Regulatory T Cells.

Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4(+) lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4(+) CD25(+) forkhead homeobox protein-3 (Foxp3)(+) cells was observed 3-5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3(+) Treg cells can be depleted with diphtheria toxin (DT; Foxp3(DTR)), we unexpectedly found that Foxp3(+) Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3(gfp) controls). Adoptive transfer studies using CD4(+) CD25(+) splenocytes from congenic CD45.1 mice into Foxp3(+) Treg-depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3(gfp) controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-ß, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII](int), CD11C(+)) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX.

Angiogenesis and airway reactivity in asthmatic Brown Norway rats.

Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 µg/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5-3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, 19F/1H MR imaging at 3T following αvß3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in 19F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.

Characterization of early neovascular response to acute lung ischemia using simultaneous (19)F/ (1)H MR molecular imaging.

Angiogenesis is an important constituent of many inflammatory pulmonary diseases, which has been unappreciated until recently. Early neovascular expansion in the lungs in preclinical models and patients is very difficult to assess noninvasively, particularly quantitatively. The present study demonstrated that (19)F/(1)H MR molecular imaging with αvß3-targeted perfluorocarbon nanoparticles can be used to directly measure neovascularity in a rat left pulmonary artery ligation (LPAL) model, which was employed to create pulmonary ischemia and induce angiogenesis. In rats 3 days after LPAL, simultaneous (19)F/(1)H MR imaging at 3T revealed a marked (19)F signal in animals 2 h following αvß3-targeted perfluorocarbon nanoparticles [(19)F signal (normalized to background) = 0.80 ± 0.2] that was greater (p = 0.007) than the non-targeted (0.30 ± 0.04) and the sham-operated (0.07 ± 0.09) control groups. Almost no (19)F signal was found in control right lung with any treatment. Competitive blockade of the integrin-targeted particles greatly decreased the (19)F signal (p = 0.002) and was equivalent to the non-targeted control group. Fluorescent and light microscopy illustrated heavy decorating of vessel walls in and around large bronchi and large pulmonary vessels. Focal segmental regions of neovessel expansion were also noted in the lung periphery. Our results demonstrate that (19)F/(1)H MR molecular imaging with αvß3-targeted perfluorocarbon nanoparticles provides a means to assess the extent of systemic neovascularization in the lung.

Strain variation in response to lung ischemia: role of MMP-12.

BACKGROUND: Systemic neovascularization of the lung during chronic ischemia has been observed in all mammals studied. However, the proteins that orchestrate the complex interaction of new vessel growth and tunneling through lung tissue matrix have not been described. Although previous work has demonstrated the CXC chemokines are essential growth factors in the process of angiogenesis in mice and rats, key matrix proteins have not been identified. METHODS: Since the degradation of chemokines has been shown to be dependent on metalloproteinases (MMP), we first surveyed gene expression patterns (real time RT-PCR) of several lung matrix proteins in DBA/J (D2) mice and C57Bl/6 (B6) mice, strains known to have divergent parenchymal responses in other lung disease models. We studied changes in the time course of MMP-12 activity in D2 and B6 mice. Functional angiogenesis was determined 14 days after the onset of complete left lung ischemia induced by left pulmonary artery ligation (LPAL), using fluorescent microspheres. RESULTS: Our results confirmed higher levels of MMP-12 gene expression in D2 mice relative to B6, which corresponded to a phenotype of minimal systemic angiogenesis in D2 mice and more robust angiogenesis in B6 mice (p < 0.01). MMP-12 activity decreased over the course of 14 days in B6 mice whereas it increased in D2 mice (p < 0.05). MMP-12 was associated largely with cells expressing the macrophage marker F4/80. Genetic deficiency of MMP-12 resulted in significantly enhanced neovascularization (p < 0.01 from B6). CONCLUSION: Taken together, our results suggest macrophage-derived MMP-12 contributes to angiostasis in the ischemic lung.

Increased hyaluronan fragmentation during pulmonary ischemia.

Hyaluronan (HA), a glycosaminoglycan critical to the lung extracellular matrix, has been shown to dissociate into low-molecular-weight (LMW) HA fragments following exposure to injurious stimuli. In the present study we questioned whether lung HA changed during ischemia and whether changes had an effect on subsequent angiogenesis. After left pulmonary artery ligation (LPAL) in mice, we analyzed left lung homogenates immediately after the onset of ischemia (0 h) and intermittently for 14 days. The relative expression of HA synthase (HAS)1, HAS2, and HAS3 was determined by real-time RT-PCR, total HA in the lung was measured by an ELISA-like assay, gel electrophoresis was performed to determine changes in HA size distribution, and the activity of hyaluronidases was determined by zymography. A 50% increase in total HA was measured 16 h after the onset of ischemia and remained elevated for up to 7 days. Furthermore, a fourfold increase in LMW HA fragments (495-30 kDa) was observed by 4 h after LPAL. Both HAS1 and HAS2 showed increased expression 4-16 h after LPAL, yet no changes were seen in hyaluronidase activity. These results suggest that both HA fragmentation and activation of HA synthesis contribute to increased HA levels during lung ischemia. Delivery of LMW HA fragments in an in vitro tube formation assay or directly to the ischemic mouse lung in vivo both resulted in increased angiogenesis. We conclude that ischemic injury results in matrix fragmentation, which leads to stimulation of neovascularization.

Role of ROS in ischemia-induced lung angiogenesis.

Pulmonary artery obstruction and subsequent lung ischemia have been shown to induce systemic angiogenesis despite preservation of normoxia. The underlying mechanisms, however, remain poorly understood. In a mouse model of lung ischemia induced by left pulmonary artery ligation (LPAL), we showed previously, the formation of a new systemic vasculature to the ischemic lung. We hypothesize that LPAL in the mouse increases reactive oxygen species (ROS) production, and these molecules play an initiating role in subsequent lung neovascularization. We used oxidant-sensitive dyes (DHE and H(2)DCF-DA) to quantify ROS and measured the antioxidant-reduced glutathione (GSH) and its oxidized form (GSSG) as indicators of ROS levels after LPAL. The magnitude of systemic neovascularization was determined by measuring systemic blood flow to the left lung with radiolabeled microspheres 14 days after LPAL. An increase in ROS was observed early (30 min: 55% increase in H(2)DCF-DA) after LPAL, with a return to baseline by 24 h. GSH/GSSG was decreased (∼50%) 4 h after LPAL, suggesting earlier ROS upregulation. Mice treated with the antioxidant N-acetylcysteine showed attenuated angiogenesis (62% of wild-type LPAL), and mice lacking Nrf2, a transcription factor important for antioxidant synthesis, resulted in increased neovascularization (207% of wild-type LPAL). Overall, GSH/GSSG was inversely associated with the magnitude of neovascularization. These results demonstrate that LPAL induces an early and transient ROS upregulation, and ROS appear to play a role in promoting ischemia-induced angiogenesis.

Differential activity of pro-angiogenic CXC chemokines.

We showed previously in a mouse model of lung ischemia-induced angiogenesis, enhanced expression of the three ELR+ CXC chemokines (KC, LIX, and MIP-2) and that blockade of the ligand receptor CXCR(2) limited neovascularization. The present study was undertaken to determine the relative abundance and angiogenic potential of the three CXC chemokines and whether RhoA activation explained the measured differences in potencies. We found that LIX showed the greatest absolute amount in the in vivo model 4 h after left pulmonary artery obstruction (LIX>KC>MIP-2; p<0.05). In vitro, LIX induced the greatest degree of arterial endothelial cell chemotaxis and KC was without effect. A significant increase (approximately 40%) in active RhoA was observed with both LIX and MIP-2 compared with vehicle control (p<0.05). On average, LIX induced the greatest amount of tube formation within pleural tissue in culture. Thus, the results of the present study suggest that among the three ELR+ CXC chemokines, LIX predominates in eliciting a pro-angiogenic phenotype.

Inhibition of CXCR2 attenuates bronchial angiogenesis in the ischemic rat lung.

Under conditions of chronic pulmonary ischemia, the bronchial circulation undergoes massive proliferation. However, little is known regarding the mechanisms that promote neovascularization. An expanding body of literature implicates the glutamic acid-leucine-arginine (ELR+) CXC chemokines and their G protein-coupled receptor, CXCR(2), as key proangiogenic components in the lung. We used a rat model of chronic pulmonary ischemia induced by left pulmonary artery ligation (LPAL) to study bronchial angiogenesis. Using a methacrylate mixture, we cast the systemic vasculature of the rat lung at weekly intervals after LPAL. Twenty-one days after LPAL, numerous large, tortuous bronchial arteries were observed surrounding the left main bronchus that penetrated the left lung parenchyma. In stark contrast, the right lung was essentially devoid of vessels. We quantified bronchial neovascularization using 15-microm radiolabeled microspheres to measure systemic blood flow to the left lung (n = 12 rats). Results showed that by 21 days after LPAL, bronchial blood flow to the ischemic left lung had increased >10-fold compared with controls 2 days after LPAL (P < 0.01). Focusing on the predominant rat CXC chemokine that signals through CXCR(2), we measured increased levels of cytokine-induced neutrophil chemoattractant-3 protein expression in left lung homogenates early (4 and 24 h; n = 10 rats) after LPAL relative to paired right lung controls (P < 0.01). Treatment with a neutralizing antibody to CXCR(2) resulted in a significant decrease in neovascularization 21 days after LPAL (n = 9 rats; P < 0.01). Our results confirm the time course of bronchial angiogenesis in the rat and suggest the importance of CXC chemokines in promoting systemic neovascularization in the lung.

CD11b+ interstitial macrophages are required for ischemia-induced lung angiogenesis.

The importance of myeloid cells in promoting neovascularization has been shown in a number of pathological settings in several organs. However, the specific role of macrophages in promoting systemic angiogenesis during pulmonary ischemia is not fully determined. Our past work suggested that cells of monocytic lineage contributed to systemic angiogenesis in the lung since clodronate-induced depletion of all macrophages resulted in attenuated neovascularization. Our current goals were to define the population of macrophages important for systemic vessel growth into the lung after the onset of pulmonary ischemia in mice. Interstitial macrophages (CD64+ MerTK+ CD11b+ ) increased significantly as did the percent of CD45+ Ly6G+ neutrophils 1 day after the induction of left lung ischemia, despite the fact there was limited cell recruitment due to complete obstruction of the left pulmonary artery in this ischemia model. Since both interstitial macrophages and neutrophils express CD11b, we used CD11b+ DTR mice and showed the critical role for these cells since CD11b+ depleted mice showed no systemic angiogenesis 7 days after the onset of ischemia when compared to control mice. Coculture of mouse aortic endothelial cells with macrophages showed increased proliferation relative to endothelial cells in culture without inflammatory cells, or pulmonary artery endothelial cells. We conclude that CD11b+ leukocytes, trapped within the lung at the onset of ischemia, contribute to growth factor release, and are critical for new blood vessel proliferation.

Effects of airway distension on leukocyte recruitment in the mouse tracheal microvasculature.

We have shown previously that excessive distention of the rat trachea during mechanical ventilation results in enhanced leukocyte recruitment to the airway (Lim LH and Wagner EM. Am J Respir Crit Care Med 168:1068-1074, 2003). The objectives of this study were to develop a mouse model of positive end-expiratory pressure (PEEP)-induced leukocyte recruitment to the airway and begin to pursue molecular mechanisms that may contribute to the in vivo observation of increased leukocyte adhesion after PEEP exposure. We studied C57BL/6 wild-type mice and mice deficient in P-selectin or intercellular adhesion molecule-1 (ICAM-1) exposed to intermittent PEEP (8 cmH(2)O) applied five times for a 1-min duration, at 10-min intervals. After the imposed ventilatory stress, during normal ventilation (0.2 ml/breath, no PEEP), leukocyte adhesion in tracheal postcapillary venules was determined using intravital microscopy. PEEP induced a time-dependent increase in leukocyte adhesion that was significantly increased between 0 and 60 min (P < 0.01). Furthermore, PEEP-induced leukocyte adhesion at 60 min was ablated in P-selectin- and ICAM-1-deficient mice. These findings demonstrate the essential nature of both P-selectin and ICAM-1 within airway postcapillary venular endothelium for leukocyte recruitment after airway distension.

PEEP-induced changes in epithelial permeability in inbred mouse strains.

Inbred mouse strains have demonstrated a range of susceptibilities to inhaled environmental irritants. C57Bl/6J mice are highly susceptible while C3H/HeJ mice are resistant to ozone exposures, as assessed by lavaged protein. However, lavaged protein reflects a loss of both the endothelial and epithelial barrier. To determine whether basal differences exist in the epithelial barrier, we measured soluble tracer ((99m)technetium-diethylenetriamine pentaacetic acid, (99m)Tc-DTPA) clearance from the lung in spontaneously breathing, anesthetized mice and mice ventilated with increased lung volume with applied positive end-expiratory pressure (PEEP; 1, 6, or 10cmH(2)O). Both strains showed more rapid clearance during ventilation with 10cmH(2)O PEEP compared with other ventilation pressures (p<0.001). There was a substantial difference in clearance between the two strains during ventilation with 10cmH(2)O PEEP (mean half time for C57Bl/6J mice=19+/-4min versus 34+/-3min for C3H/HeJ mice; p<0.001). Thus, when lung volume is increased, the susceptible C57Bl/6J strain shows a greater change in epithelial barrier than the resistant C3H/HeJ strain. These results may reflect fundamental differences in lung architecture.

Anti-angiogenic Nanotherapy Inhibits Airway Remodeling and Hyper-responsiveness of Dust Mite Triggered Asthma in the Brown Norway Rat.

Although angiogenesis is a hallmark feature of asthmatic inflammatory responses, therapeutic anti-angiogenesis interventions have received little attention. Objective: Assess the effectiveness of anti-angiogenic Sn2 lipase-labile prodrugs delivered via αvß3-micellar nanotherapy to suppress microvascular expansion, bronchial remodeling, and airway hyper-responsiveness in Brown Norway rats exposed to serial house dust mite (HDM) inhalation challenges. Results: Anti-neovascular effectiveness of αvß3-mixed micelles incorporating docetaxel-prodrug (Dxtl-PD) or fumagillin-prodrug (Fum-PD) were shown to robustly suppress neovascular expansion (p<0.01) in the upper airways/bronchi of HDM rats using simultaneous 19F/1H MR neovascular imaging, which was corroborated by adjunctive fluorescent microscopy. Micelles without a drug payload (αvß3-No-Drug) served as a carrier-only control. Morphometric measurements of HDM rat airway size (perimeter) and vessel number at 21d revealed classic vascular expansion in control rats but less vascularity (p<0.001) after the anti-angiogenic nanotherapies. CD31 RNA expression independently corroborated the decrease in airway microvasculature. Methacholine (MCh) induced respiratory system resistance (Rrs) was high in the HDM rats receiving αvß3-No-Drug micelles while αvß3-Dxtl-PD or αvß3-Fum-PD micelles markedly and equivalently attenuated airway hyper-responsiveness and improved airway compliance. Total inflammatory BAL cells among HDM challenged rats did not differ with treatment, but αvß3+ macrophages/monocytes were significantly reduced by both nanotherapies (p<0.001), most notably by the αvß3-Dxtl-PD micelles. Additionally, αvß3-Dxtl-PD decreased BAL eosinophil and αvß3+ CD45+ leukocytes relative to αvß3-No-Drug micelles, whereas αvß3-Fum-PD micelles did not. Conclusion: These results demonstrate the potential of targeted anti-angiogenesis nanotherapy to ameliorate the inflammatory hallmarks of asthma in a clinically relevant rodent model.

Pulmonary ischemia induces lung remodeling and angiogenesis.

Cellular remodeling during angiogenesis in the lung is poorly described. Furthermore, it is the systemic vasculature of the lung and surrounding the lung that is proangiogenic when the pulmonary circulation becomes impaired. In a mouse model of chronic pulmonary thromboembolism, after left pulmonary artery ligation (LPAL), the intercostal vasculature, in proximity to the ischemic lung, proliferates and invades the lung (12). In the present study, we performed a detailed investigation of the kinetics of remodeling using histological sections of the left lung of C57Bl/6J mice after LPAL (4 h to 20 days) or after sham surgery. New vessels were seen within the thickened visceral pleura 4 days after LPAL predominantly in the upper portion of the left lung. Connections between new vessels within the pleura and pulmonary capillaries were clearly discerned by 7 days after LPAL. The visceral pleura and the lung parenchyma showed intense tissue remodeling, as evidenced by markedly elevated levels of both proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells. Rapidly dividing cells were predominantly macrophages and type II pneumocytes. The increased apoptotic activity was further quantified by caspase-3 activity, which showed a sixfold increase relative to naive lungs, by 24 h after LPAL. Because sham surgeries had little effect on measured parameters, we conclude that both thoracic wound healing and pulmonary ischemia are required for systemic neovascularization.

Changes in lung permeability after chronic pulmonary artery obstruction.

We have shown that left pulmonary artery ligation (LPAL) in mice causes a prompt angiogenic response, with new systemic vessels from intercostal arteries penetrating the pleura within 6 days. Because angiogenic vessels in other organs have been shown to exhibit increased permeability, we studied vascular permeability (Evans blue dye extravasation, lung wet weight-to-dry weight ratio, and lavaged protein) in naive C57BL/6 mice and 4 h, and 14 and 21 days after LPAL (4-6 mice/time point). We also measured radiolabel clearance as an index of functional perfusion after LPAL. Tracer clearance from the left lung was maximal by 6 days after LPAL and not different from right lungs. Thus a functional vasculature is established before 6 days of LPAL that results in normal tracer clearance. By 21 days after LPAL, Evans blue-albumin was significantly increased in the left lung relative to both 4 h (no vasculature) and 14 days after LPAL. Only after 21 days of LPAL was left lung wet weight-to-dry weight ratio significantly different from naive lungs. Additionally, lavaged protein was significantly increased both 4 h and 21 days after LPAL relative to control mice. Thus, using three different methods, results consistently demonstrated increased permeability to protein and water 21 days after LPAL. Although changes in surface area of perfusion might affect the interpretation of these results, blood flow measured with labeled microspheres indicated no change in left lung perfusion between 14 and 21 days of LPAL. Thus the lung vasculature, remodeled as a consequence of chronic pulmonary artery obstruction, demonstrates increased water and protein permeability.

Bronchial Artery Angiogenesis Drives Lung Tumor Growth.

Angiogenesis is vital for tumor growth but in well-vascularized organs such as the lung its importance is unclear. This situation is complicated by the fact that the lung has two separate circulations, the pulmonary and the systemic bronchial circulation. There are few relevant animal models of non-small cell lung cancer, which can be used to study the lung's complex circulations, and mice, lacking a systemic bronchial circulation cannot be used. We report here a novel orthotopic model of non-small cell lung cancer in rats, where we have studied the separate contributions of each of the two circulations for lung tumor growth. Results show that bronchial artery perfusion, quantified by fluorescent microspheres (206% increase in large tumors) or high-resolution computed tomography scans (276% increase in large tumors), parallels the growth in tumor volume, whereas pulmonary artery perfusion remained unchanged. Ablation of the bronchial artery after the initiation of tumor growth resulted in a decrease in tumor volume over a subsequent course of 4 weeks. These results demonstrate that although the existing pulmonary circulation can supply the metabolic needs for tumor initiation, further growth of the tumor requires angiogenesis from the highly proliferative bronchial circulation. This model may be useful to investigate new therapeutic approaches that target specifically the bronchial circulation. Cancer Res; 76(20); 5962-9. ©2016 AACR.

Role of IL-6 in systemic angiogenesis of the lung.

The multifunctional cytokine interleukin (IL)-6 has been shown to modulate inflammation and angiogenesis. In a mouse model of lung angiogenesis induced by chronic left pulmonary artery ligation (LPAL), we previously showed increased expression of IL-6 mRNA in lung homogenates 4 h after the onset of pulmonary ischemia. To determine whether IL-6 influences both new vessel growth and inflammatory cell influx, we studied wild-type (WT) and IL-6-deficient C57Bl/6J (KO) mice after LPAL (4 h and 1, 7, 14 days). We measured IL-6 protein of the lung by ELISA, the lavage cell profile of the left lung, and new systemic vessel growth with radiolabeled microspheres (14 days after LPAL) in WT and KO mice. We confirmed a 2.4-fold increase in IL-6 protein in the left lung of WT mice compared with right lung 4 h after LPAL. A significant increase in lavaged neutrophils (7.5% of total cells) was observed only in WT mice 4 h after LPAL. New vessel growth was significantly attenuated in KO relative to WT (0.7 vs. 1.9% cardiac output). In an additional series, treatment of WT mice with anti-neutrophil antibody demonstrated a reduction in lavaged neutrophils 4 h after LPAL; however, IL-6 protein remained elevated and neovascularization to the left lung (2.3% cardiac output) was not altered. These results demonstrate that IL-6 plays an important modulatory role in lung angiogenesis, but the changes are not dependent on trapped neutrophils.

Methods to track leukocyte and erythrocyte transit through the bronchial vasculature in sheep.

Study of the underlying mechanisms of leukocyte recruitment in the airway circulation is a crucial aspect of understanding the pathology of inflammatory airways disease. However, few in vivo studies have focused on leukocyte kinetics through the systemic airway vasculature. Because the bronchial vasculature of the sheep shows anatomical similarity to that of the human as well as being easily accessible for perfusion, we developed methods to study leukocyte transit through the sheep bronchial circulation. Leukocytes were isolated from whole blood after hypotonic lysis of red cells and labeled with 5-(and 6) carboxyfluorescein succinimidylester (CFSE; 0.1 microM), a green fluorescent dye. Red blood cells were labeled using PKH26-GL Red Fluorescent Cell Linker and served as a marker for blood flow and volume. Labeled leukocytes were tested for activation during the labeling process by monitoring surface levels of leukocyte adhesion molecules CD11b and L-selectin. When activated with phorbol myristate acetate (10 ng/ml), sheep leukocytes showed a marked increase in CD11b expression and a decrease in L-selectin. However, sheep leukocytes labeled with CFSE showed no significant increase of CD11b or shedding of L-selectin, suggesting that the labeling process did not significantly activate the adhesion properties of the cells. Aliquots containing both labeled erythrocytes and leukocytes were infused into the bronchial vasculature of the sheep through the cannulated bronchial artery at normal bronchial flow (0.6 ml/kg). Serial blood samples were withdrawn from the outflow of the bronchial vasculature (the left atrium) and analyzed using conventional flow cytometry. Retention of leukocytes and transit relative to red cell transit could then be evaluated with this technique. In addition, since cells are stably labeled, airway tissue samples can be removed and analyzed histologically to determine sites of extravasation. These methods offer an approach for examining leukocyte kinetics in situ in the airways of a relevant animal model.