(Chest. 2005;128:602S-608S.)
© 2005
American College of Chest Physicians
Imaging of Angiogenesis in Inflamed Airways and Tumors: Newly Formed Blood Vessels Are Not Alike and May Be Wildly Abnormal*
Parker B. Francis Lecture
Donald M. McDonald, MD, PhD and
Peter Baluk, MD, PhD
* From the Cardiovascular Research Institute, Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, CA.
Correspondence to: Donald M. McDonald, MD, Department of Anatomy, S1363, University of California, 513 Parnassus Ave, San Francisco, CA 94143-0452; e-mail: dmcd{at}itsa.ucsf.edu
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Heterogeneity of the Normal Microvasculature
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All blood vessels of the microvasculature have several features in common, including an endothelial cell lining, mural cells (smooth-muscle cells, pericytes) around the endothelial cells, and basement membrane enveloping both types of cells.1 Yet, the microvasculature has multiple levels of heterogeneity. The microvasculature of each organ is specialized in concert with the unique functions of each organ. Also, the microvasculature of every organ has a hierarchical organization of arterioles, capillaries, and venules, which have their own specialized structural and functional properties. Expression of distinct molecular markers is among these. Despite graded changes in BP and flow along the microcirculation, the transition in phenotype of endothelial cells and mural cells is abrupt from arteriole to capillary and capillary to venule.
In the lung, alveolar capillaries constitute by far the largest component of the microcirculation. Specialization of alveolar capillaries accommodates the large blood flow of the lung and provides the extensive surface area required for efficient gas exchange. By comparison, the microcirculation of the airways, including trachea, bronchi, and bronchioles, has markedly different specializations and only 1% of the blood flow of the pulmonary circulation.
One feature of the airway microcirculation is its simple architecture. Mucosal blood vessels of the mouse trachea are organized in segmental units aligned with the cartilaginous rings in the airway wall.23 Units consisting of arterioles, capillaries, and venules are arranged in a repetitive pattern along the airways (Fig. 1
, top left, A). Blood vessels in the airway mucosa are accompanied by abundant lymphatic vessels (Fig 1, top right, B). The importance of lymphatic vessels in the airway mucosa was underappreciated until methods were developed for reliable visualization of molecular markers such as lymphatic vessel endothelial receptor (LYVE)-1 on lymphatic endothelial cells.4 The simple, stereotyped architecture of blood vessels and lymphatic vessels of the airway mucosa can be exploited in determining the cellular and molecular features of normal vessels and changes occurring under pathologic conditions.
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Figure 1.. Microvasculature and lymphatic vessels in whole mounts of tracheal mucosa from pathogen-free (normal) and M pulmonis-infected mice. Top left, A: Simple architecture of mucosal blood vessels of normal mouse trachea made visible by perfusion of biotinylated Lycopersicon esculentum lectin, which binds to and highlights the luminal surface of endothelial cells. Top right, B: Delicate network of blood vessels (green, CD31 immunoreactivity) and larger blind-ended lymphatic vessels (red, LYVE-1 immunoreactivity) of normal mouse trachea stained by immunohistochemistry. Bottom left, C, and bottom right, D: Tracheas prepared by the same methods as shown in top left, A, and top right, B. Enlarged venules and proliferated lymphatic vessels in tracheas of mice infected with M pulmonis for 7 days (bottom left, C) or 28 days (bottom right, D). Abundant leukocytes (white arrows) are adherent to the luminal surface of the enlarged venules (bottom left, C) but are not present in the normal microvasculature (top left, A). Images in top left, A, and bottom left, C are from Thurston et al.3 Images in top right, B, and bottom right, D are from Baluk et al.4 Scale bar in bottom right, D; 50 µm in top left, A, and bottom left, C; 100 µm in top right, B, and bottom right, D.
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Microvascular Changes in Chronic Airway Inflammation
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We used the simple, well-organized microvasculature of the mouse trachea to address several questions relating to angiogenesis and lymphangiogenesis in chronic inflammation.45 In particular, we determined how rapidly blood vessels remodel, how the remodeled vessels differ from normal vessels, and whether the vascular modeling is reversible. The approach was to follow changes in the airways of pathogen-free animals after intranasal inoculation with the rodent respiratory pathogen Mycoplasma pulmonis, which leads to lifelong infection and inflammation of the airways.26 In comparison to the stereotyped normal vascular pattern (Fig 1, top left, A), the airways of mice infected with M pulmonis for 7 days have an impressively expanded vascular network consisting of capillaries transformed into enlarged venules with abundant adherent leukocytes (Fig 1, bottom left, C). The venular enlargement results from endothelial cell proliferation, which peaks 5 days after the infection and continues at an elevated level for at least several weeks.7
Prominent changes in the network of lymphatic vessels in the infected airway mucosa are even more conspicuous than the venular enlargement. After infection, proliferation of lymphatics exceeds blood vessel growth and remodeling (Fig 1, bottom right, D). The formation of new blood vessels and lymphatic vessels occurs sequentially, with blood vessel enlargement and proliferation dominating the first week and lymphatic vessel proliferation dominating the second and third weeks.4
To begin to learn more about growth factors that drive angiogenesis and lymphangiogenesis in the infected airways, we used selective antagonists for vascular endothelial growth factor (VEGF) receptors. Function-blocking antibodies that selectively bind VEGF receptor 1 or VEGF receptor 2 had no effect on microvascular remodeling or lymphangiogenesis after M pulmonis infection, thereby excluding VEGF (also known as VEGF-A), VEGF-B, and placental growth factor as essential in this condition.4 However, an antibody that selectively binds VEGF receptor 3 had no effect on blood vessel remodeling but abolished lymphangiogenesis after infection. Similarly, a soluble form of VEGF receptor 3 delivered by adenoviral vector blocked the lymphangiogenesis after M pulmonis infection without changing the angiogenesis.4 As soluble VEGF receptor 3 acts as a decoy receptor that selectively traps VEGF-C and VEGF-D, the inhibitory effects of soluble VEGF receptor 3 after infection implicates these growth factors in airway lymphangiogenesis.
Although VEGF seems not to be essential for vascular remodeling in airway inflammation after M pulmonis infection, we asked whether VEGF could stimulate angiogenesis in the airways under other circumstances. We used two approaches. First, we delivered into the airways an adenovirus coding VEGF, which transduced airway epithelial cells. These experiments showed that, indeed, overexpression of VEGF in the epithelium resulted in intense angiogenesis but no lymphangiogenesis in the airway mucosa.4 Second, we used a transgenic mouse model in which VEGF is overexpressed under regulation by a tet-on inducible system driven by the CC10 promoter in Clara cells of the airway epithelium.8 Experiments using this model verified that VEGF can rapidly drive angiogenesis in the airway mucosa. Interestingly, most of the newly formed blood vessels maintained their VEGF dependency and, thus, unlike most of the normal airway vasculature, regressed when the VEGF overexpression was turned off.8 Other experiments showed that under baseline conditions VEGF is a survival factor for approximately 20% of normal airway capillaries.9
The angiogenesis and vascular remodeling in the airway mucosa of mice with M. pulmonis infection is completely reversible. Treatment with dexamethasone or antibiotic can restore the segmental microvascular architecture and phenotype to normal in a few weeks.41011 By comparison, lymphangiogenesis that accompanies the infection appears to be largely irreversible. Continuous treatment for at least 12 weeks has little effect on the expanded network of lymphatics that develop after infection.4
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Abnormalities of Tumor Blood Vessels
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Newly formed blood vessels in growing tumors differ from those in chronic airway inflammation.12 By increasing blood flow, plasma leakage, and leukocyte influx into inflamed tissues, the proliferation and remodeling of blood vessels facilitate clearance of allergens, toxins, organisms, and debris. In cancer, angiogenesis supports tumor growth, but the increase in blood flow is generally not in scale with the increase in vascularity. One reason is that most blood vessels in tumors have multiple functional abnormalities, including high luminal resistance, high endothelial permeability, and marginal blood flow. In addition, interstitial pressure is unusually high in tumors.13
Tumor blood vessels are irregular in size, shape, and branching pattern, do not have the normal vascular hierarchy, and do not have recognizable features of arterioles, capillaries, or venules (Fig 2
, top left, A, and top right, B).15 Abnormalities are present in all components of the vessel wall (Fig 3
).121416 Endothelial cells, pericytes, and even the vascular basement membrane of tumor vessels have bizarre features not found in other conditions (Fig 3). Endothelial cells sprout and proliferate, consistent with their dynamic nature (Fig 2, bottom left, C, and bottom right, D). Defects in endothelial cell barrier function, due to abnormal cell-cell junctions and other changes, exaggerate vessel leakiness. This leakiness can be exploited in localizing tumors by imaging contrast media and in the delivery of macromolecular therapeutics.1517 Another unusual feature of tumor vessels is their dependency on growth factors. Blood vessels in some tumors depend on VEGF for survival; VEGF antagonists can therefore lead to tumor vessel regression and cessation of angiogenesis.16
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Figure 2.. Top left, A, and top right, B: Vascular casts contrasting the microvasculature in normal tissue and in tumor. Top left, A: Rat carotid sinus vasa vasorum have a simple organization and are partitioned into arterioles, capillaries, and venules. Top right, B: Human xenograft in nude mouse showing disorganized, highly branched, and sprouting tumor vessels than lack the hierarchy of arterioles, capillaries, and venules. Bottom left, C, and bottom right, D: Scanning electron micrographs showing the external surface of a blood vessel in a spontaneous islet cell tumor in a RIP-Tag2 transgenic mouse. The endothelial cells are extremely thin, have abundant tiny fenestrations, and have numerous sprouts (filopodia, white arrows). Region marked by the white box in bottom left, C is shown at higher magnification in bottom right, D. Images in top left, A, and top right, B are from McDonald and Choyke.15 Scale bar in shown in bottom right, D; 100 µm in top left, A, and top right, B; 3 µm in bottom left, C; 1 µm in bottom right, D.
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Figure 3.. Diagram illustrating three components of tumor blood vessels (endothelial cells, pericytes, and basement membrane) and the abnormalities associated with each component. Also listed are changes that occur in each component after treatment with an inhibitor of VEGF signaling. Data are from Baluk et al,12 McDonald and Foss,14 and Inai et al.16 VEGFR-2 = VEGF receptor 2.
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Endothelial cells of tumor blood vessels have multiple abnormalities in gene expression. Some proteins expressed in endothelial cells of tumor blood vessels have little or no expression in normal blood vessels. Integrin 
5ß1, the fibronectin receptor, is among the proteins overexpressed on endothelial cells of tumor vessels.181920 Although 5ß1 integrin is also expressed in smooth-muscle cells and some other normal cell types, it is not found in endothelial cells of most normal blood vessels and thus provides a potential therapeutic target on tumor blood vessels.
The key role of VEGF signaling in the survival of tumor blood vessels is evident from the actions of VEGF inhibitors. When VEGF signaling is blocked, a series of changes in endothelial cells leads to regression of tumor vessels. Early changes include loss of endothelial fenestrations, loss of the vascular lumen, and cessation of blood flow.16 Susceptible endothelial cells eventually undergo apoptosis, as evidenced by the expression of activated caspase-3. As many as 80% of tumor vessels undergo regression after inhibition of VEGF signaling.16 The roughly 20% of tumor vessels that survive treatment differ from those present before treatment because of the presence of new features reminiscent of normal blood vessels.16 Tumor vessel normalization appears to result from a balancing of the growth factor supply that determines vessel phenotype.1321
Pericytes in tumors also have multiple abnormalities (Fig 3). Unlike pericytes on normal blood vessels that cling tightly to endothelial cells and are enveloped by a tightly fitting basement membrane, most pericytes in tumors are loosely associated with endothelial cells and have extra layers of loosely fitting basement membrane (Fig 4
, top, A).22 In addition, abnormalities in gene expression may change molecular markers such as -smooth-muscle actin in pericytes. As a result, the identification of pericytes on tumor vessels can be problematic, and absence of markers can be misinterpreted as absence of pericytes.
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Figure 4.. Top, A: Diagram comparing the structure and close endothelial cell association of pericytes of a normal capillary with pericytes loosely associated with a tumor vessel. Bottom, B: Confocal microscopic image of a pancreatic islet cell tumor in a RIP-Tag2 transgenic mouse treated for 7 days (VEGF-Trap; Regeneron Pharmaceuticals; Tarrytown, NY), which blocks VEGF signaling by binding VEGF. Red strands (white arrows) are empty sleeves of basement membrane left behind by regressing tumor vessels. Image in top, A is modified from Morikawa et al22; and bottom, B from McDonald and Choyke.15 Scale bar is shown in bottom, B; 10 µm in top, A; 50 µm in bottom, B.
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The basement membrane that envelops endothelial cells and pericytes of tumor vessels may have extra layers that have no apparent association with the cells.23 These layers may be a product of previous generations of endothelial cells or pericytes at that location and reflect the dynamic nature of tumor vessels. Similarly, when tumor vessels regress in response to inhibition of VEGF signaling, the endothelial cells disappear but empty sleeves of vascular basement membrane remain (Fig 4, bottom, B).16 These basement membrane sleeves provide routes for rapid regrowth of tumor vessels when treatment with the anti-VEGF agent is stopped.
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Summary
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Blood vessels of the microvasculature have multiple levels of heterogeneity under normal conditions and become even more diverse under pathologic conditions. In chronic airway inflammation, new vessels form and some existing vessels undergo remodeling, whereby normal capillaries acquire the phenotype of venules that are sites of plasma leakage and leukocyte influx. In addition, airway lymphatics undergo extensive proliferation. Although the growth factors that promote vascular remodeling in chronic inflammation have not been identified, VEGF seems not to be essential. In contrast, lymphangiogenesis in inflamed airways is driven by the VEGF receptor 3 ligands, VEGF-C, and VEGF-D. Angiogenesis in tumors is different because new blood vessels that form differ from all types of normal vessels and have abnormalities that involve all components of the vessel wall. Blood vessels of tumors are dynamic, defective, and leaky. Also, tumor vessels may depend on VEGF for survival; therefore, inhibitors of VEGF signaling can stop angiogenesis and trigger tumor vessel regression. Dissimilarities in angiogenesis occurring under different pathologic conditions reflect the diversity of stimuli for new vessel growth and highlight the need for a broad range of inhibitors to eliminate the drive for blood vessel growth and remodeling that helps sustain disease processes.
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Acknowledgements
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The authors thank Dr. Hiroya Hashizume of University of California, San Francisco for the scanning electron micrographs, prepared in the Division of Microscopic Anatomy and Bio-imaging at Niigata University, Japan, showing surface views of tumor vessels. We also thank Dr. Moritz Konerding of University of Mainz, Germany, for the scanning electron micrograph of a vascular cast of tumor vessels; Dr. Tetsuichiro Inai of Kyushu University, Fukuoka, Japan, for the drawing of a blood vessel cross-section; Dr. Shunichi Morikawa of Tokyo Women’s Medical University, Japan, for the drawing of pericytes; and Dr. Norbert Voelkel of University of Colorado for many excellent suggestions regarding the article.
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Footnotes
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Abbreviations: LYVE = lymphatic vessel endothelial receptor; VEGF = vascular endothelial growth factor
Supported in part by National Institutes of Health grants P01 HL-24136 and R01 HL-59157 from the National Heart, Lung, and Blood Institute, and R01 CA082923 from the National Cancer Institute. Other funding was received from AngelWorks Foundation and the Vascular Mapping Project.
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References
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- Murphy, TJ, Thurston, G, Ezaki, T, et al Endothelial cell heterogeneity in venules of mouse airways induced by polarized inflammatory stimulus. Am J Pathol 1999;155,93-103[Abstract/Free Full Text]
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- Baluk, P, Tammela, T, Ator, E, et al Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J Clin Invest 2005;115,247-257[CrossRef][ISI][Medline]
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- Kim, S, Bell, K, Mousa, SA, et al Regulation of angiogenesis in vivo by ligation of integrin 5ß1 with the central cell-binding domain of fibronectin. Am J Pathol 2000;156,1345-1362[Abstract/Free Full Text]
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- Baluk, P, Morikawa, S, Haskell, A, et al Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. Am J Pathol 2003;163,1801-1815[Abstract/Free Full Text]
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Heterogeneity of the Normal...
Microvascular Changes in Chronic...
