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The Endothelium of the Pulmonary Microvessels

The Endothelium of the Pulmonary Microvessels

J Nippon Med Sch 1997; 64(6) (495)1

-Reviews-

The endothelium of the pulmonary microvessels

Oichi Kawanami

Department of Molecular Pathology, Institute of Gerontology, Nippon Medical School

Introduction

In the normal , the bronchial airways are lined by a pseudostratified arrangement of several types of epithelial cells such as ciliated cells, goblet cells, basal cells, Clara cells and undifferentiated bronchiolar cuboidal cells. They are adherent to the underlying which is com- posed of laminin, type IV and other Fig. 1 Type VII collagen, which is a main constitute of epidermal anchoring fibrils, is highly reactive proteoglycans. The basement membrane in the along the basement membrane zone (white tri- airways is subjacently supported by type VII col- angle) from trachea, brounchus to respiratory lagen which is a main composition of the electron bronchiole, suggesting that airway basal cells microscopic features of anchoring fibrils. Type VII retain a common characteristics with epidermal basal cells in producing type VII collagen. The collagen seems to precipitate as a distinct arc-form microvesels (arrow heads) being located sub- structure in the upper airways', but the fibrillar jacent to type VII collagen are originated exclu- structure becomes rather vague in the distal por- sively from bronchial system, but not from system. BL: bronchial tions of the bronchial airways despite a clear im- . ABC immunoperoxidase stain for type munohistological reaction to type VII collagen6 VII collagen (x75) (Fig. 1). The microvessels located subjacent to type VII collagen are originated exclusively from the bronchial artery system, and not from the pulmo- deemed to be in continuity with the lining endoth- nary artery system. The alveolar walls, which play elial cells of vessels distributed in the lung and any the most important role in gas-exchange function, other organs. never show a positive reactivity for type VII col- However, there is increasing evidence that the lagen. The epithelial lining in the alveolar walls in endothelial cells in the differ from the endoth- man is composed of only two types of epithelial elial cells of in visceral organs cells, type I and type II alveolar epithelial cells. The Furthermore microvascular endothelium, such as type I alveolar epithelial cells are exposed to air- that in the lung, apparently retains its own prop- space on one side, and the other side of the cells erties in morphology and function when compared firmly adheres to the single layer of basement with the endothelium in larger vessels in the same membrane, which in turn faces the flat cytoplasm of organ. Morphologically, endothelial the endothelial cells of the alveolar . The cells are classified into two types based on the capillary endothelial cells make up 30% of the lung ultrastructural features (Fig. 2): continuous type cells'. They cover the inner surface of vessels, and non-continuous type". Usually, capillary en- not only capillaries, but the aorta, and dothelial cells have flat cytoplasmic extensions with as well. They tightly attach to each other by jun- no defect (continuous type), and the cytoplasm of ctional structures which protect blood components other capillary endothelial cells contains either from leaking. Thus, any single endothelial cell is fenestrae or relatively large defects (non-continuous

* Professor 2(496)

ucts of which are central to endothelial repair and remodeling. Neovascularization implies that a pair of endothelial cells sprouts from parent vessels to induce new capillaries. Basic knowledge of this phenomenon has now gradually broadened in view of the complex interactions of growth factors, as well as proteoglycans and adhesion molecules".

1. Dual blood circulation in the lung The pulmonary vasculature includes dual arterial systems: to pulmonary artery and bronchial Fig. 2 Ultrastructural schema of two types of capil artery"-". They develop corresponding drainage -laries indicates. one is continuous and non- vessels for venous return to the . The pulmo- fenestrated type at the left and the other is discontinuous and fenestrated (diaphragm- nary artery originates from the right ventricle of forming; arrows) type at the right. The cyto the heart, and supplies venous blood to the vessels - plasms of both capillaries contain lots of which distribute along the bronchial trees. The pinocytotic vesicles. blood flow eventually reaches alveolar capillaries at areas of respiratory bronchioles. The alveolar capil- type) as in the hepatic sinusoid. The latter type of laries play an unequivocal role in the gas-exchange capillaries is localized in limited regions of some through the air-blood barrier of alveolar walls. It is organs. well understood that the alveolar blood flow returns A large number of studies describing the results to pulmonary veins through the drainage system of obtained from experiments on endothelial cells in the interlobular blood vessels which parallel fine vitro have accumulated In these studies, networks of vessels". The microvessels of endothelial cells were collected in relatively simple both pulmonary veins and bronchial veins are procedures" from vessels such as the human umbili- devoid of valves. In addition, extrapulmonary bron- cal cord ", saphenous vein" and pulmonary chial veins infrequently possess valves near the lung vessels". Since the natural characteristics of en- hilus26,29. And the pulmonary veins in rats have dothelial cell biology are rapidly modified in the regular thin bands of sphincters that cause pulmo- artificial environments of a culture dish20, and of nary by a physical stimulus to the brain". chicken chorioallantoic membranes21, it is not easy Using resin casting of the vasculature, Peao et all' to make any immediate inference as to in vivo also found vascular sphincters at the junction reactions from results based upon in vitro, experi- between alveolar capillaries and pulmonary ments. Thus, it is indispensable to categorize the in the mouse lung. They offer a structural setting vascular endothelial cells of the lung accord- for the existence of postcapillary control of blood ing to their in vivo, as against in vitro, biological flow in the . characteristics. Studies on the bronchial circulation system have Among all the organs of the body, the respiratory not been sufficiently carried out for a long period of microvessels seems to carry special properties in time although Leonard da Vinci in the 15th cen- terms of their dynamic dual blood circulations, and tury"," had depicted the bronchial arteries originat- their specific functions. The ultrastructual pheno- ing from the aorta. Cudkowicz and his group" have types of their endothelial cells are of interest as they made a great contribution by revealing the detailed may alter indifferent lung environments 14. distribution of the bronchial artery system using Particular interest exists in the repair processes arteriography. They showed histological changes in of endothelial cells following damage to thems22. In relation to various lung lesions. this condition, vascular endothelial cells, under (1) . Anatomy of the normal bronchial arteries profound influence of regulation", immedi- The origin of the bronchial arteries was shown to ately respond by expressing various genes" , prod- be located between the upper border of the fourth ( .197 ) 3

thoracic vertebra (T 4) and the lower border of T 6 in 90% of 40 fresh adult cadavers. The right bronchial arteries arise from the right intercostal bronchial artery trunk that originated from the thoracic aorta. And the one or two left bronchial arteries are directly connected to the thoracic aorta. Nagaishi et applied angiogram and resin casting techniques to examine the bronchial artery system. According to their results, the bronchial artery ultimately distributes in the mucosa of the bronchial wall, muscle and surrounding connective tissue layers, perichondrium, and large vessels. Extrapul- monary bronchial arteries supply their branches to Fig. 3 The pulmonary artery (PA) is visible situated par- alled to a bronchial airway which is branching at lymph nodes, and pleural and interlobular connec- the right lower corner (BR). Bronchial arteries are tive tissues. not obvious, but their microvessels (arrows) are Intrapulmonary bronchial arteries are located distributed along the connective tissue sheaths of the airways and large vessels. Those vessels are in a together with pulmonary arteries and lymph vessels close apposition to alveolar capillaries. Respiratory along the bronchial airways" (Fig. 3,4). Bronchial bronchiol (RB) and alveolar ducts (AD) are clearly seen in the middle of this picture. Scanning electron arteries appear much smaller in size when compar- micrograph of terminal bronchiole. ( x 1000 original) ed with associating pulmonary arteries in bron- chovascular connective tissue bands. It is almost impossible to differentiate the bronchial arteries The larger bronchi have a double venous net- from the pulmonary arteries at the periphery of work, one is located in the mucos a and the other in airways. The arterioles of bronchial circulation the connective tissue located external to the bron- have widely concentric muscle bundles bordered by chial cartilage. They are closely connected with . The vessels often appear each other. Approximately one-third of the normal totally occlusive in histologic Sections which have bronchial venous flow returns to azygos and hemi- given rise to the concept of "Sperrarterien" or azgos veins which terminate in the superior vena "blocking arteries" at th e sites of precapillary anas- cava or the coronary sinus. The majority of the tomoses with pulmonary arterioles". In the venous flow enters the left atrium via routes of precapillary regions, bronchial arterioles can regu- pulmonary veins. On the other hand, all the visible late their blood flow which drains into either bron- arteries in the pleura of the normal are exclu- chial veins or alveolar capillaries. Ultrastructural sively derived from the bronchial arteries. Inter- studies revealed that in contrast to smooth and thin lobular arterioles are also derived from the pleural cytoplasm in alveolar capillary endothelium", the arterial system, but not from arteries accompanying endothelial cells of microvessels from a bronchial the bronchi. The venous blood flow of the pleura artery are often characterized by the formation of exclusively returns to azygos or herniazygos fenestrae along their thin cytoplasmic segments",". vein -29. The presence of such fenestrations provides clues (3) Anastomoses of bronchial and pulmonary for identifying the bronchial artery origin of mi- circulations crovessels at an electron microscopic level (Fig. 2). The connections between the bronchial and pul- The fenestrated capillaries are present in other monary artery systems, bronchopulmonary anas- organs such as choriocapillaris36-36 pancreas", skin tomoses, are well known at the level of microvessels and muscle", ", nasal mucosa42 and others". along the airways, particularly at the terminally This structure of fenestrae differs from hepatic located bronchioles. In order to detect these commu- sinusoid",", but they heve been induced in the liver nications, we perfused the pulmonary and bronchial into which islet cells were transplanted". arteries of the rats with -containing warm (2) Anatomy of the normal bronchial veins saline". After the perfusion, carbon-containing (499)5

Fig. 5 Sprague Dawry (SD) rat for bronchial infusion by carbon-containing saline. In order to detect the broncho-pulmonary anastomoses, carbon-containing saline was infused through abdominal aorta after washing lungs by heparin-containing saline via vena cava inferior. The firm ligations at the ascending and abdominal aorta allowed pigmented fluid drain into the intercostal arteries and eventually bronchial arteries.

The former type may regulate blood flow and the latter may possibly be involved in angiogenesis50. It would be intriguing to evaluate interactions dis- played between of alveolar capillaries and myofibroblasts in alveolar interstitium in the lung.

2. Biological activities of bronchial artery in various lung diseases The complexity of dual blood circulation in the lung provides a very interesting basis for the path-

Fig. 6 Carbon particles initially filled intercostal arteries ogenesis of several lung diseases. arranged parallel to ribs. Via bronchial arteries, (1) Lung transplantation small vessels (arrows) which are located in the connective tissue sheaths of bronchial airways and In lung transplantation, it has been emphasized large vessels became filled by pigmented particles. that revascularization of the donor bronchial Pulmonary venules and adjacent alveolar capillaries arteries with the recipient internal thoracic artery occasionally contained them. HE stain (x 120). resulted in successful healing of airway anastomosis and lung implantation33.5. Without a systemic arte- tions. According to Nehls and Drenckhahn49, per- rial blood supply bronchial ischemia may cause icytes represent morphological and functional heter- early changes such as mucosal sloughing, granula- ogeneities: indicating alpha-smooth muscle actin- tion tissue formation, and bronchial dehiscence. As positive population in pre- and post-capillary area late complications, the airways often undergo con- and a population negative in the true-capillary zone. striction" which obstructs normal air flow in the (499)5

Fig. 5 Sprague Dawry (SD) rat for bronchial infusion by carbon-containing saline. In order to detect t he broncho-pulmonary anastomoses, carbon-containing saline was infused through abdominal aorta after washing lungs by heparin-containing saline via vena cava inferior. The firm ligations at the ascending and abdominal aorta allowed pigmented fluid drain into the intercostal arteries and eventually bronchial arteries.

The former type may regulate blood flow and the latter may possibly be involved in . It would be intriguing to evaluate interactions dis- played between pericytes of alveolar capillaries and myofibroblasts in alveolar interstitium in the lung.

2. Biological activities of bronchial artery in

various lung diseases

The complexity of dual blood circulation in the lung provides a very interesting basis for the path-

Fig. 6 Carbon particles initially filled intercostal arteries ogenesis of several lung diseases. arranged parallel to ribs. Via bronchial arteries, (1) Lung transplantation small vessels (arrows) which are located in the In lung transplantation, it has been emphasized connective tissue sheaths of bronchial airways and large vessels became filled by pigmented particles. that revascularization of the donor bronchial Pulmonary venules and adjacent alveolar capillaries arteries with the recipient internal thoracic artery occasionally contained them. HE stain (x120). resulted in successful healing of airway anastomosis and lung implantation. Without a systemic arte- tions. According to Nehls and Drenckhahn, per- rial blood supply bronchial ischemia may cause icytes represent morphological and functional heter- early changes such as mucosal sloughing, granula- ogeneities: indicating alpha-smooth muscle actin- tion tissue formation, and bronchial dehiscence. As positive population in pre- and post-capillary area late complications, the airways often undergo con- and a population negative in the true-capillary zone. striction which obstructs normal air flow in the 6(500)

were originating from adjacent bronchial arteries. Broncho-pulmonary anastomoses along the airways In the same samples they observed marked distor- tion of sizes and shapes in the alveolar capillaries involved. These results suggested that fibrotic transformation of the lung is associated with the local generation of angiogenic stimuli. It is consis- tent with a widely accepted concept that the bron- chial capillaries and venules can be hypertrophic, and that endothelial cells can regenerate rapidly to form new vessels. In contrast, the endothelium of the alveolar microvessels in pulmonary circulation does not appear able to vitally regenerate once these vessels are lost28,29. Infarction Causes of pulmonary infarction apparently differ from those in other organs. Experimental ligation of a pulmonary artery is said to fail to induce necrosis or infarction of the lung as long as the bronchial circulation is intact. In order to induce infarction, additional manupulations such as tying pulmonary

Fig. 7 This schema depicts the areas of anastomosis veins or physical compression of the lung are between bronchial arteries and alveolar capil- required besides pulmonary embolization. It is laries. This communication prevents tissue ne- known among clinicians that infarction rarely fol- crosis due to embolization of pulmonary lows pulmonary embolism except in the presence of arteries, and further plays an important role in the endothelial recruitment of alveolar capil- congestive heart failure, pulmonary , laries (see the details in the text). blood dyscrasis, neoplasm, the gravid or puerperal state. anastomoses of trachea and/or bronchus leading to (4) Sarcoidosis failure of transplantation. The bronchial arteries, In pulmonary sarcoidosis, Takemura et a1 demo- although they appear smale in size, provide vital nstrated that granuloma often involved large pulmo- functions for the survival of lung tissues. nary vessels indicating the evidence of sarcoid (2) Interstitial fibrosis angiitis. They also showed a relative decrease of In the studies of pulmonary fibrosis with intratra- pulmonary microvessels in the granulomatous cheal administration of bleomysin, Shraufnagel et lesions due to a loss of capillary beds. The relation al showed that intercapillary spaces became wider to bronchial blood circulation was not evaluated in and irregularly distorted in the alveolar sacs. This details in their studies, meanwhile, angiogenic fac- result suggests a consequential scarcity of alveolar tors were suggested to be released from epithelioid capillaries. It would imply loss of capillary beds due cells of the granulomata57. It remains to be evaluat- possibly to accumulation of collagenous matrices. ed whether sarcoidosis involves However, no angiogenetic activities are suggested. any neovascularization in eyes and bronchial In fact, histological changes of fibrosis was limited mucosa which are endoscopically evidenced. to patchy distribution in this experiment. Peao et (5)Tuberculosis al induced severe fibrosis in rat lungs by tracheal In pulmonary tuberculosis, the formation of small infusion of bleommysin and studied morphological pedunculated, pear-shaped "aneurysms of Rasmus- alterations of capillary networks using scanning sen" was found by Calmette in branches of pulmo- electron microscopy. They indicated that neovas- nary artery which traversed the walls of tubercu- cularization occurred after structural damage of lous cavities. He considered that this aneurysmal alveolar endothelium, and the new vessels possibly dilatation of the vessels could be bronchial artery (501)7

origin. Finding dilated bronchial arteries in the a pair of daughter cells, b) while these cells are in walls of tuberculous cavities , Wood and Millers the wall of parent capillary, a new lumen emerges, postulated that proliferation of pulmonary connec- c) the new lumen connects to a portion of the parent tive tissue was accompanied by the development of capillary lumen in the very early phase of the new vessels derived from the bronchial arteries. sprouting, d) endothelial sprout is further propelled, The bronchial arteries are most uniformly featured e) -endothelium interaction seems to have by the distortion, tortuosity and remarkable prolif- an important role in the sprout growth conducted by eration of endothelial cells in caseating tuberculous cytoplasmic caving on each other. The summary of lesions. The pulmonary arteries accompanying the angiogenesis in granulation tissue is contradictory involved bronchi may be thrombosed. The conten- to the observation reported by Folkman et a1 tion that hemoptysis comes from the bronchial They observed neovascularization in rabbit arteries is well supported in view of the large- and and showed that: a) mitosis follows migration of the thin-walled lacunae which can be seen connected to endothelial cells, b) tube structure is formed among enlarged bronchial arteries. Angiography of bron- the sprouted daughter cells, and c) pericytes dis- chial arteries often indicates higher densities in and appear during neovascularization and then reappear around a tuberculous lesion, which is consistent when sprouting ceases. These differences might be with an increase of microvessels derived from bron- related to stimuli derived from different environ- chial arteries. ments. In the granulation tissue formation, neovas- (6) Bronchiectasis, pneumothorax and granula- cularization is closely associated with fibroblast tion tissue proliferation. In contrast, the tumor implant on Liebow et al" found that the inflammatory cornea induces a rather arteficial condition as the responses and granulation tissue coincided with the environment originally lacks capillaries although it increase in bronchial arterial flow in the peribron- contains interstitial cells called keratocytes. chial tissues of bronchiectasis. They assumed that

neovascularization would be developed in granula- 3. Antigenic heterogeneity of microvascular tion tissue which took place as a part of a reaction endothelial cells in any other lung diseases as well. Vascular endothelial cells carry a variety of Following spontaneous pneumothorax, granula- antigens, genes of which are rapidly expressed in tion tissue formation apparently took place in areas response to injury" on their surface and/or in their subjacent to ruptured pleural tissue. Fourteen lung cytoplasm. The expression of such antigens varies samples obtained at a thoracotomy for the surgical according to the organs, stages of development treatment of subpleural bullae were histologically and maturation, and their sizes. Page et al demon- examined. These patients had episodes of sponta- strated several kinds of endothelial markers {EN 4, neous pneumothorax from a few days to 2 weeks (vWf), Pal-E and 44G4}, prior to the operation. The alveolar lumina located vascular adhesion molecules (ICAM-1, ELAM, subjacent to the rupture site showed invasion of VCAM and PECAM)25, the monocyte/endothelial hypetrophic myofibroblasts and accumulation of marker (OKM 5), and major histocompatibility com- collagenous matrices in various degrees. It was plex molecules Class I and II (MHC-I and -II). In clear that small capillaries were sprouted into their results capillary endothelial cells in the heart preexisting alveolar sacs in a close apposition to strongly expressed MHC-I and -II, ICAM, and adjacent small vessels in pleura. The endothelial OKM 5. In contrast, the large vessels distinctly cells initiating intraalveolar neovascularization expressed vWf and appeared to constitutively seemed to be derived from the bronchial arteries. express ELAM-1. These results suggest that the Angiogenesis occurs in the process of granulation capillary endothelial cells may be more efficient at tissue formation in any organ of the body. Wakui antigen presentation or more susceptible to immune demonstrated three-dimentional ultrastructures in attack in vivo. It is interesting to note that the the processes of angiogenesis and described that: a) capillary endothelium in myocardium showed only endothelial cell proliferation by mitosis gives rise to patchy reaction for vWf 8(502)

The lung alveolar capillary endothelial cells thrombomodulin on their cell surfaces. According to express HLA-DR antigens'. This is reminiscent of the electron microscopic observations, despite the coronary arteries and their capillaries of the adult lack of fenestrae in normal alveolar capillaries as heart. The experimental results of our experi- described previously", the fenestrated endothelial ments and Yamamoto et a1 showed that factor cells became apparent in the alveolar regions which VIII-related antigen is hardly revealed in the alveo- were invaded by adenocarcinoma cells and/or lar capillary endothelium in histologic sections. underwent interstitial fibrosis However, clearly positive for vWf were the endoth- elium of of large vessels, and the 4. Remodeling of the alveolar capillaries capillary endothelial cells located in peribronchial Such ultrstructural alteration of the alveolar connective tissues and pleural tissues. The blood capillary endothelial cells could be experimentally flow in these capillaries is apparently supplied by induced by the intratracheal infusion of silica in bronchial arteries. Besides, Reidy et al indicated rats". Following the transition processes of mitosis that endothelial injury induced incrase of vWf in and migration of the endothelial cells, cytoplasmic rats. Watababe briefly described how the mi- extension of the endothelial cells became adherent crovessels of bronchial blood circulation showed a to preexisting alveolar capillary tubes. The mi- quite distinct alkaline phosphatase reaction. On the crovessels in which endothelial cells mitosed were other hand, the cell surfaces of alveolar capillary closely situated to the vasculature of bronchial endothelial cells are intensively reactive for throm- artery blood circulation. In the study, we speculated bomodulin under normal condition as it was primar- that the endothelial cells could acquire their own ily detected in the Balb/c lung microvessels. phenotype of fenestrated cytoplasm after a latent This antigen expression becomes concealed when period of de novo maturation and differentiation they are embedded in collagenous matrices of alveo- (Fig. 10). The presence of fenestration may imply lar fibrosis. Furthermore, the alveolar capillaries endothelial metaplasia in the alveolar capillary". surrounded by fibrosis often became positive for Furthermore, vascular sprouts were evident in the vWf, such alterations of antigen expression fresh connective tissue or granulation tissue which might be regulated by transient expression of cyto- took place in peribronchiolar zones immediately kines induced in association with environmental adjacent to silicotic injury. On the other hand, changes such as . numerous capillary beds eventually disappeared in It is also interesting to show phenotypic altera- the peripheral alveolar structures as they became tions of alveolar capillary endothelial cells when fully embedded by fibrotic masses following tissue adenocarcinoma arises. Bronchioloalveolar car- necrosis by silica". From this result it became clear cinoma cells spread over and along the adjacent alveolar capillaries would be remodeled in three alveolar walls which retain normal interstitial different types: a) loss of capillary beds; b) endoth- matrix. We found that the alveolar capillary en- elial metaplasia possibly caused by endothelial cell dothelial cells in such cancerous areas immediately recruitment originated from bronchial artery side lost positive reactivity for thrombomodulin (Fig. 8). and c) neovascularization or vascular sprout in new Instead, they occasionally obtained a positive reac- granulation tissue in peripheral lung tissues. These tion for vWf in their cytoplasm. The positivity for changes might occur in common in various lung vWf in pulmonary microvessels becames conspicu- diseases, as also found in interstitial lung diseases of ous in the subpleural zone where alveolar structures man 14,35 were widely replaced by a large amount of col- lagenous tissues or scar tissues (Fig. 9). Although it 5. Angiogenetic mechanisms is uncertain that which vessels are newly sprouted The study of angiogenesis started with develop- among others and. which were preexisting alveolar ment of a transplant chamber that could be implant- capillaries, the cytoplasms of entire vessels in the ed in the rabbit ear skin. The chick embryo chor- lesion showed a positive reaction for vWf. In addi- ioallantoic membrane" became popular for study- tion, these vessels never showed an expression for ing tumor growth. The rabbit cornea has been (503)9

Fig. 8 The normal alveolar capillaries are highly reactive Fig. 10 Alveolar capillary endothelium, Which is originally for thrombomodulin (arrowheads), but the capill- non-fenestrated type, converts into typical fenes- aries of alveolar walls which are invaded by cancer trated (arrowhead) type after a latent period of cells of bronchioloalveolar carcinoma become differentiation in fibrotic environment (see th unreactive for it as shown in the lower third of this e text). CAP: allveolar capillary "Transmission elec- picture. ABC imnumoperoxidase reaction for throm- tron micrograph. ( x 11,500) bomodulin. (X75) Fig. 9 Factor VIII-related antigen (von Willebrand factor) (arrowheads) is located in most capillaries subjacent to bronchial mucosa, and the microvessels situated tion. On the second day, endothelial cells migrated in the alveolar walls with proliferating tumor cells often became reactive for factor VIII-related toward tumor implant through the fragmented base- antigen. Immunoperoxidase reaction for von Wille- ment membrane which may reflect local proteolysis brand factor. (X75) in response to the tumor stimulus. Migrating cells at the capillary tip did not mitose. Mitosis was evident utilized to demonstrate inflammatory neovascular- exclusively in the cells that were located in the ization. Angiogenesis factors as for as the cellular middle of the new capillary. He further described level is concerned involve lymphocytes, macro- how in all new capillary formation, whether embry- phages, corpus luteum cells, and adipocytes. Recent- onic or adult, capillary tubes were formed before ly, angiogenesis research has been further advanced flow was established. The pericytes disappeared in relation to the function of growth factors", extra- during capillary proliferation and reappeared when cellular molecules, adhesion molecules and the capillary matured and growth stopped. other antigens expressed on the endothelium. Neo- Neovascularization was also observed to occur vascularizatin is now presented under a complex around the implantation of "Sato lung cancer tis- background of stimuli derived from a large variety sue"" in the skin. The new capillaries appeared to of inflammatory cells and the constitutive tissue be originated from a terminal which made cells as well. numerous branchings. In this experiment, the (1) Tumor-related angiogenesis authors found that neovascularization rarely Wood et ally demonstrated an increase of bron- originated from veins and venules. chial arteries in primary malignant lung tumor. In the case of human lung cancer, the microvessel Cudkowicz supported this finding by providing counts in adenocarcinoma were significantly higher evidence for a higher blood flow of bronchial artery than in squamous cell carcinoma. The microves- in the patients with primary lung cancers, but not in sels in patients with stages I-II disease were signifi- control patients who had no tumor or patients with cantly less in number than that stage III disease. metastatic lung tumors. The mechanisms of tumor Patients with nodal metastasis had higher microves- angiogenesis was well reviewed by Folkman and his sel density than those without nodal metastasis. associates. According to the results obtained by These results suggested that microvessel density his group, resting endothelial cells in the preexisting correlates with histologic types, stages of disease, limbal vessels began to resemble regenerating en- and nodal status. dothelial cells within one day after tumor implanta- However, careful examinations are required 10(504) because neovascularization in the lung tissues Ausprunk et al described an absence of hepar- should be distinguished from the remnants of preex- an sulfate proteoglycans (ruthenium redlabeling) in isting alveolar capillaries and alveolar structures the region of the vessel sprouts. In contrast, Nicosia are vastly remodeled with an accumulation of and Madri showed the presence of both laminin fibrous components in alveolar sacs. and type IV collagen in very early vessel sprouts (2) Angiogenesis in acute lung injury developing from rat aortic tissue grown in plasma Angiogenesis bioactivity was found in the lower clot culture. Furthermore, Jerdan et aI75 applied respiratory tract in the patients who survived acute immunohistochernical techniques on unfixed frozen lung injury". The process of fibrosis is supposed to sctions and clearly detected positive reactions for represent an evolution of granulation tissue in alve- laminin and type IV collagen, and the presence of olar sacs because capillaries as well as myofibrob- proteoglycan and entactin was lasts were the principal cellular elements in both shown in sprouting vessels in cornea. Active cell structures. The lavage fluid from 72% of patients invasion, characteristic of capillary sprout growth with acute lung injury promoted endothelial cell as well as tumor metastasis, has been frequently migration, while lavage fluid from controls had no associated with a lack of basement membrane or its bioactivity. One of at least two moieties in lavage selective degradation"-".However, Jerdan et a1 fluid constituents appeared identical to basic fibrob- clearly showed that newly formed capillaries simul- last grwoth factor. The other was a 150-KD non- taneously developed basement membrane constitu- heparin binding protein that mediated endothelial ents, as was shown in the basement membrane cell migration in vitro, and it also promoted endoth- components in actively invading squamous cell car- elial attachment and the growth of new vessels in cinoma cells". Roles of compo- vivo in animal . Further evaluation should nents are to be further clarified in relation to be made concerning the histological: correlations neovascularization. whether they included a series of patients with acute interrstitial pneumonia or diffuse alveolar 6 and angiogenesis damage. Their was characterized by a (1) Fibroblast lack of vascular sprouts in each patients'. A growth factor for fibroblasts and endothelial (3) Extracellular matrix and angiogenesis cells was notified in endothelial cells and its deposit Extracellular martix components might modulate in subjacent extracellular matrix. Angiogenic the promotion of endothelial cell proliferation. activity was evidenced in vitro on the human am- Form et a1 observed in paraffin sections that the niotic membrane". Katz et a1 collected the wound proliferation of the microvascular endothelial cells fluid from the acute lesion of split-thickness skin was significantly greater on laminin matrix than on grafting. They concluded that a 25 kd molecular either plasma fibronectin, the interstitial collagen weight fragment was more effective as fibroblast- types I and III, or on the basement membrane and endothelial -growth factor than -derived collagen type IV matrices. Moreover, the growth of factor heterodimers or homodimers (28 to 35 kd). the endothelial cells on laminin could be modulated Meanwhile, both acidic and basic fibroblast growth by the presence of type IV collagen. And the appear- factors (aFGF, bFGF) are found to be potent sub- ance of laminin occurred through the newly formed stances for endothelial cell growth in postnatal vessels, as well as in individual cells at the migrat- stages as well". A group of FGFs are now named as ing, and proliferating tips in the in vivo murine FGF-1 (aFGF), FGF-2 (bFGF) and others up to FGF- cornea model. In contrast, the appearance of type 9 at this moment. IV collagen correlated with lumen formation and Basic FGF is a mitogen for endothelial cells, and was not detected at the vessel tips. The results is aso synthesized by endothelial cells possibly suggest that specific matrix components play a related to an autocrine function. In the normal significant role in orchestrating the growth and human tissues Cordon-Cardo et al" showed that differentiation of endothelial cells during an- expression of bFGF was ubiquitously detected in giogenesis. the basement membrane of blood vessels in a vari- (505)11

ety of tissues including lung. Large and intermedi- Vascular endothelial growth factor (VEGF) is a ate size blood vessels were homogeneously and secretory endothelial cell mitogen This factor intensely reactive for bFGF. In contrast, the expres- was secreted from tumor cells and initially thought sion of bFGF in capillaries was highly heterogene- to be responsible for the vascular hyperpermeability ous. The most intense reaction was observed in and was called vascular permeability factor branching capillaries. The epithelial cells lining the (VPF)106-108 Vascular permeability factor exerts a trachea and major bronchi stained strongly, revea- number of other effects on vascular endothelial ling a cytoplasmic pattern of reactivity. Staining of cells; e.g., VPF increases intracellular calcium, stim- minor bronchi was patchy or absent. Pneumocytes ulates inositol triphosphate formation, provokes in alveoli were not stained. Whereas the mechanism von Willebrand factor release, and stimulates tissue of bFGF secretion remains unknown, these data factor expression. VEGF-antibody stained im- suggest that bFGF is indeed secreted into vascular planted tumor cells (line 1 and line 10 bile basement membranes, and does in fact represent a carcinomas) in guinea pigs"'. In situ hybridization constitutive component of its structure under nor- demonstrated VPF mRNA in nearly all line 10 mal conditions. tumor cells but not in tumor vessels, indicating that In relation to fibroplasia and collagen accumula- immunohistochemical labeling of tumor vessels tion, a large number of mast cells appeared in lung with the antibodies to VPF peptides reflects uptake parenchyma as well as in the fluid of bron- of VPF, and not endogenous synthesis. In contrast, choalveolar lavage from patients with various inter- vessels more than 0.5 mm distant from tumors were stitial lung disorders. Inose et all' clearly demon- not hyperpermeable and did not exhibit immunohis- strated that mast cells in injured and fibrotic lungs tochemical staining for VPF. These studies indicate expressed bFGF mRNA and produced 17.8-kd that VPF is synthesized by tumor cells in vivo and bFGF protein. They also showed a release of bFGF accumulates in the nearby blood vessels, those are from mast cells when they underwent degranulation its target of action in vitro, suggesting a role of mast cell bFGF in the Breier et al 111showed intense expression of VEGF pulmonary fibrotic response. This result will recall in the ventricular neuroectoderm of embryonic and other interesting functions of mast cells related to postnatal brain when endothelial cells proliferate angiogenesis. It is widely open in future studies to rapidly but were reduced in the adult when endoth- clarify the real functions of mast cells in terms of elial cell proliferation has ceased. A persistent neovascularization and of appropriate expressions expression of VEGF was observed in epithelial cells of the FGF receptors adjacent to fenestrated endothelium, e.g. in choroid (2) Platelet-derived endothelial cell growth fac- plexus and in kidney glomeruli. The data are consis- tor tent with a role of VEGF as a multifunctional Platelet-derived endothelial cell growth factor regulator of endothelial cell growth and differentia- (Pd-ECGF) is an inducer of angiogenesis in vivo and tion. In this view, the fenestration formation has chemotactic activity for endothelial cells. The and vWf-reactivity of alveolar capillary endothelial presence of the factor in human has led to cells which appeared in the human fibrotic lungs as the suggestion that it might have a role in maintain- well as in experimentally induced pulmonary silico- ing the integrity of blood vessels. Amino acid and sis might involve regulations of VEGF functions. cDNA sequences of a vascular endothelial (4) VEGF mRNA expression in normal and cell mitogen was found to be homologous to platelet- pathologic tissues derived growth factor"'. However, Pd-ECGF lacks In order to understand physiological roles of a typical hydrophobic signal sequence of secretion VPF/VEGF in normal tissues, Monacci et al and the mechanism by which this factor is released examined normal rat tissues using Northern blot in vivo is not known. The receptors for platelet- analysis and in situ hybridization histochemistry. derived growth factor is described in human mi- VPF/VEGF mRNA was highest in the lung, espe- crovascular endothelial cells by Beitz et al. cially in pulmonary alveolar cell. However, the (3) Vascular endothelial growth factor capillary endothelial cells of alveolar walls did not 12(506)

significantly express VPE/VEGF mRNA, which 1 (VEGFR-1) gene, and they have analysed expres- strongly suggests that VPE/VEGF acts as a para- sion pattern during mouse embryogenesis. Based on

crine and not an autocrine factor in normal adult their results, VEGFR-1/flt-1 has a higher affinity to

tissues. The distinct cellular distribution of VPF/ VEGF than VEGF receptor-2 (flk-1) . VEGFR 1/flt-

VEGF mRNA in the lung suggests that the 1 mRNA was specifically localized in blood vessels

permeability-enhancing effects of this factor may be and capillaries of the developing organs"', closely involved in the regulation of capillary function in resembling the pattern of VEGFR 2/flk-1 transcript

this organ. Although expression of VPF/VEGF distribution. VEGF and its receptors have an impor-

mRNA does not correlate exclusively with either tant function both in the differentiation of the en-

angiogenesis or permeability, the widespread dothelial cell lineage and in the neovascularization

expression of VPF/VEGF in healthy adult tissues of developing organs, acting in a paracrine fashion.

lends continuing support to the hypothesis that this Senger et al demonstrated that VEGF induced factor plays an important, perhaps vital, role in the dermal microvascular endothelial cell expression of physiology of normal vasculature. mRNA coding the av and /3 3 heterodimers at the

Following recovery of acute lung injury with cell surface. VEGF also induced mRNA encoding oxygen, VEGF expression was present in alveolar (OPN), an avƒÀ3 ligand. Cooperative epithelial cells beginning at day 1 recovery"'. The mechanisms for VEGF regulation of endothelial cell

VEGF message was in alveolar epithelial cells migration involve the av/3 3 integrin, the avƒÀ 3 through out the lung by day 3 recovery. Compared ligand OPN, and cleavage of OPN. Alpha with alveolar epithelial cells, little or no expression v,8 3 integrins are expressed specifically on capil- was noted in large vessel endothelial cells, airway lary sprouts"'. Once granulation tissue filled the cells, or smooth muscle cells. Combined in situ wound and invasive angiogenesis terminated, the hybridization for VEGF and immunostaining for av/3 3 showed little or no expression in the granula- and other mesenchymal cells found no tion tissue microvasculature. Thus, av/3 3 plays a

VEGF message in those cell types. Double in situ fundamental role during invasive angiogenesis and hybridization for VEGF and surfactant protein-C granulation tissue formation in a healing wound. showed co-expression in a population of type II cells, but with an inverse relationship: cells with Conclusion abundant VEGF mRNA did not have abundant SP-C mRNA. They conclude that alveolar type II In the pulmonary microvessels, endothelial cells cells express increased VEGF mRNA during recov- express specific antigens on the cell surface and in ery from acute hyperoxia. These findings are con- the cytoplasm, which fairly represents their lineage sistent with a role for VEGF in regulating microvas- specificity of pulmonary or bronchial arteries. The cular endothelial repair after oxygen injury"' ultrastructure of these capillary endothelial cells In pathological synovial tissue of the patients also reflects their origin via non-fenestrated or with rheumatoid arthritis, VEGF polypeptide as fenestrated cytoplasms. The expression of antigens well as VEGF mRNA by in situ hybridization were and ultrastructural phenotypes easily fluctuate in strongly expressed in synovial lining cells, sub- accordance with the environmental changes . This synovial macrophages, fibroblasts surrounding mi- alteration might be regulated by inducing antigens crovessels, and cells , but which will be further regulated by a complex phe- not in vascular endothelial cells of synovial mem- nomenon of cytokine networks. In order to clarify brane. It is uncertain whether the microvasculature the sequential processes involved, the following endothelial cells take up the secreted VEGF, and the research studies are intriguing: functional mechanisms of VEGF for angiogenesis 1) Antigens on the cell surface or in the cyto- remains to be analysed . plasm should be identified in capillary endothelial (6) VEGF receptors and tissue constituents cells of each of the pulmonary and the bronchial Concerning the receptors of VEGF , Breier et al blood circulations. characterized the murine homolog of the human flt- 2) In vivo reactivities of microvessel endothelial (507)13 growth factors might differ under such different capillary . Physiol Rev 1973; 53: conditions as fetal development, maturation and 419-494. 8. Crapo JD, Barry BE, Gehr P, Bachofen M, Weibel aging in adults. ER: Cell number and cell characteristics of the 3) The endothelial growth factor functions normal human lung. Am Rev Respir Dis 1982; 125: should be characterized relevant to their replica- 332-337. tion, regeneration, proliferation, migration, matura- 9. Nonomura Y, Murota S, Sofue K, Nagai K: Blood Vessels-Endothelium and Smooth Muscle Cells tion, and differentiation. The processes of vascular "The Methods of New Biochemistry Experiments" sproutings or neovascularization would generate vol 10, (Japanese Society of Biochemistry ed.) 1993; particular interests for the control of inflammation Tokyo Kagaku Dojin. and neoplasia. 10. Pino RM: Cytochemical studies of the vascular 4) Inhibitory functions to the endothelial growth endothleium. "Cytochemical studies of the vascular activities should be defined, which might take place endothelium" (Pino RM, ed.) 1989; pp 1-38, Fisher, Stuttgart, New York. in vivo through the induction of antagonizing fac- 11. 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