Use of anti-basement membrane antibodies to distinguish blood vessel capillaries from lymphatic capillaries

A new method has been developed for identifying blood vessel capillaries and distinguishing them from lymphatic capillaries. Highly purified antibodies to two ubiquitous components of basement membrane--Type IV collagen and laminin--were applied to fresh-frozen and formalin-fixed tissue sections rich in blood vessel capillaries (granulation tissue), rich in lymphatic channels (small intestine and mesentery), and rich in both (skin, lung, and breast). Staining patterns were evaluated using standard immunofluorescence and immunoperoxidase techniques. Blood vessel capillaries contained intact basement membranes with linear staining for both Type IV collagen and laminin. Lymphatic capillaries, in contrast, uniformly lacked immunoreactivity with antibodies to these basement membrane components. These lymphatic capillaries, which were indistinguishable from blood vessel capillaries in both size (cross-sectional area) and shape (vessel eccentricity), were confirmed as lymphatics by dye injection studies. Our immunohistological technique detected 7-10 times more blood vessels than hematoxylin and eosin staining. Both benign and vessels than hematoxylin and eosin staining. Both benign and malignant tumors of blood vessel endothelium including two capillary hemangiomas and two angiosarcomas demonstrated extracellular linear staining for both Type IV collagen and laminin, whereas two lymphangiomas and two lymphangiectasias were negative. This method then provides a sensitive technique for detecting blood vessel capillaries in tissue sections and a reliable means of distinguishing blood vessel capillaries from lymphatic capillaries. This method may also aid in the identification of tumors whose origin is blood vessel endothelium.     

Accumulation of laminin and type IV collagen in the kidney in congenital nephrosis

The aim of this study was to evaluate qualitatively the occurrence of the basement membrane proteins laminin and type IV collagen in the kidneys of ten infants with congenital nephrotic syndrome of the Finnish type (CNF) aged from 3 to 23 months and to compare the results with those for age-matched controls. A slow accumulation of basement membrane (BM) material occurred in the glomerular mesangium, the peripheral capillaries, around atrophied tubules, and the renal vessels in the course of the disease. The staining pattern of accumulated material depended on the duration of the disease and subsequent renal parenchymal damage. Young CNF patients with slight morphological changes in the kidney had only focal and minimal increases in the amounts of mesangial matrix, but as the disease advanced, so the BMs of the glomerular capillaries, renal arteries, and atrophied tubules also became involved and were thicker than normal. The staining reaction was in all patients similar with antibodies against the fragment P1 of laminin and the 7-S domain of type IV collagen. The accumulation of BM material in CNF kidneys is regarded as a secondary phenomenon induced by an unknown pathogenetic defect in the metabolism of some BM component.     

Lymphatics in primary cutaneous melanoma.

Using Ulex europaeus I lectin (UEA1) to demonstrate endothelial cells, we have previously shown that frequency of capillary invasion correlates closely with maximum tumor thickness in primary cutaneous melanoma. UEA1 demonstrates both vascular and lymphatic capillaries; however, only vascular capillaries possess basement membranes. In order to ascertain whether these capillaries were lymphatic or vascular, we employed a double staining technique, using UEA1 in conjunction with a monoclonal anti-type IV collagen antibody. We studied 21 primary cutaneous melanomas. Seven of the 21 included lymphatic capillaries, while 14 did not. These lymphatic capillaries were very sparse and appeared to be residual dermal lymphatics rather than a result of lymphangiogenesis. Lymphatic permeation by melanoma was not seen in any of the tumors studied. There was no apparent association among tumor thickness, level of invasion, growth phase, necrosis, regression or mitotic index, and presence of lymphatics within the melanomas. Although scanty lymphatics are present in some primary cutaneous melanomas, this study does not suggest that lymphatic permeation plays a major role in the spread of melanoma to locoregional lymph nodes.     

Fibronectin, laminin, and collagen types I, III, IV and V in the healing rat colon anastomosis.

The purpose of this work was to study normal anastomotic healing in the rat colon. The unprepared sigmoid colon was divided and a colo-colostomy performed using a one-layer inverting technique. Frozen sections were taken and studied immunohistologically with specific antibodies to fibronectin, laminin and collagen types I, III, IV and V. From day one onwards a strong fibronectin reaction was observed in the anastomosis, reaching maximum staining intensity on postoperative day five. Type III collagen and pericellular type V collagen were at first detected in the anastomosis on day two. From day three onwards all collagens studied and laminin were present in the repair tissue (laminin and type IV and V collagen in the regenerating capillary walls). Maximum immunofluorescence was observed on day seven and it remained on a high level throughout the study, except for fibronectin, which weakened gradually after the fifth postoperative day. The results indicate that healing of the colon anastomosis occurs by rapid accumulation of connective tissue components between the inverted leaves of the colonic wall, as also new capillaries consisting of the basement membrane components, type IV and V collagen as well as laminin, are formed.     

Expression of extracellular matrix proteins and vimentin in testes of azoospermic man： an immunohistochemical and morphometric study

AIM: To investigate the changes in the extracellular matrix protein expression and the morphology of seminiferous tubules in the testis of 88 azoospermic men. METHODS: The patients were of the following categories: (1) 22 cases of Sertoli-cell-only syndrome, (2) 20 cases of spermatogenic arrest, and (3) 46 cases with hypospermatogenesis. Testicular sections were immunohistochemically stained for fibronectin, vimentin, laminin and collagen type IV. The seminiferous tubular diameter and the connective matrix zone (CMZ, the acellular zone between the basement membrane [BM] and the peritubular cells) thickness were measured. Seminiferous tubules were typed according to the thickness of the connective matrix in the lamina propria. The predominant tubule type and the Johnsen and Silber scores were determined. RESULTS: The mean tubular diameter were 119 +/- 27, 117 +/- 20, and 140 +/- 38 microm for Groups 1, 2, and 3, respectively. Both the laminin and the type IV collagen were localized to the epithelial BM and peritubular cells. In most of the tubules, BM and peritubular cells were separated by a homogenous acellular layer, the CMZ, in which laminin, type IV collagen, fibronectin and vimentin were not present. It is perceived that the worse the testicular histology, the higher the thickness of the CMZ. CONCLUSION: In testis with no or low sperm production, the diameter of the seminiferous tubules is decreased, the thickness of the seminiferous tubular wall is increased and a CMZ is formed between the peritubular cells and the BM. The thickness of CMZ is increasing with the advancement of testiclar deterioration. The most important morphologic predictive factor for spermiogenesis is the predominant     

Absence of α6(IV) collagen in kidney and skin of X-linked Alport syndrome patients

Ab stract. To identify the abnormalities of the type IV collagen α6 chain, α6(IV), in Alport syndrome, we examined renal and skin tissue using rat monoclonal antibodies against non-consensus amino acid sequences of α6(IV). Immunofluorescence of normal human kidney and skin tissue revealed linear α6(IV) staining in the basement membrane (BM) of Bowman’s capsule, in some tubules, and also in the epidermal BM. Renal specimens from five male patients of four families with X-linked Alport syndrome showed no reactivity for α6(IV) in Bowman’s capsules and tubules. In these patients, α1(IV) and α2(IV) were normal, whereas α3(IV), α4(IV), and α5(IV) were absent from the BMs of the kidney. In skin tissue of male patients, neither α5(IV) nor α6(IV) were detected. The epidermal BM of female heterozygotes with X-linked Alport syndrome showed a mosaic staining for α5(IV) and α6(IV). These findings indicate that, in addition to a disturbed α3(IV)-α4(IV)-α5(IV) network, patients with X-linked Alport syndrome have abnormalities in α6(IV) of the renal and epidermal BMs at the protein level. Received October 6, 1995; received in revised form April 25, 1996; accepted April 29, 1996     

Development of lymphatic vasculature and morphological characterization in rat kidney

Background

The mechanisms and morphological characteristics of lymphatic vascular development in embryonic kidneys remain uncertain.

Methods

We examined the distribution and characteristics of lymphatic vessels in developing rat kidneys using immunostaining for podoplanin, prox-1, Ki-67, type IV collagen (basement membrane: BM), and ??-smooth muscle actin (??SMA: pericytes or mural cells). We also examined the expression of VEGF-C.

Results

At embryonic day 17 (E17), podoplanin-positive lymphatic vessels were observed mainly in the kidney hilus. At E20, lymphatic vessels extended further into the developing kidneys along the interlobar vasculature. In 1-day-old pups (P1) to P20, lymphatic vessels appeared around the arcuate arteries and veins of the kidneys, with some reaching the developing cortex via interlobular vessels. In 8-week-old adult rats, lymphatic vessels were extensively distributed around the blood vasculature from the renal hilus to cortex. Only lymphatic capillaries lacking continuous BM and ??SMA-positive cells were present within adult kidneys, with none observed in renal medulla. VEGF-C was upregulated in the developing kidneys and expressed mainly in tubules. Importantly, the developing lymphatic vessels were characterized by endothelial cells immunopositive for podoplanin, prox-1, and Ki-67, with no surrounding BM or ??SMA-positive cells.

Conclusion

During nephrogenesis, lymphatic vessels extend from the renal hilus into the renal cortex along the renal blood vasculature. Podoplanin, prox-1, Ki-67, type IV collagen, and ??SMA immunostaining can detect lymphatic vessels during lymphangiogenesis.     

Immunohistologic distribution of basement membrane in oral squamous cell carcinoma

The distribution pattern of the basement membrane (BM) around tumor cells was determined in 72 oral squamous cell carcinomas by immunohistochemical staining with antibodies against human type IV collagen, laminin, and heparan sulfate proteoglycan. An intact continuous BM was found in 29 cases, whereas partial or widespread loss of the BM was detected in the other 43 cases (59.7%). Statistical analysis showed that the degree of BM loss was correlated with the degree of differentiation of tumor cells, but not with tumor size, and, most significantly, with the mode of cancer invasion and the incidence of lymph node metastasis. Carcinoma with a well-defined tumor-stromal boundary generally expressed an intact continuous BM. In contrast, the majority of diffusely invasive carcinomas lacked a continuous BM. Carcinomas with a widespread loss of BM structures showed a high frequency of regional lymph node metastasis (16 of 18 cases, 88.9%). © 1994 John Wiley & Sons, Inc.     

Effect of combined antisense oligonucleotides against high-glucose- and diabetes-induced overexpression of extracellular matrix components and increased vascular permeability

The effect of combined antisense oligonucleotides (AS-oligos) against overexpression of extracellular matrix (ECM) components, fibronectin, laminin, and collagen IV and on cell monolayer permeability was examined in rat microvascular endothelial cells (RMECs) grown in high glucose medium and on retinal vascular permeability in diabetic rats. RMECs grown in high glucose for 10 days and transfected with combined AS-oligos showed a significantly reduced fibronectin, laminin, and collagen IV protein level. In parallel studies, high-glucose-induced excess monolayer permeability was also reduced in RMECs transfected with the combined AS-oligos. Similarly, diabetic rats intravitreally injected with the combined AS-oligos and examined after 2 months of diabetes showed significant reduction in retinal fibronectin, laminin, and collagen IV expression. In addition, vascular permeability, as determined from extravasation of fluorescein isothiocyanate-BSA in the surrounding areas of the retinal capillaries, was partially reduced in the combined AS-oligos-treated diabetic retinas. Our results indicate that the combined AS-oligos strategy is effective in simultaneously reducing fibronectin, collagen IV, and laminin overexpression and reducing vascular leakage in the retinal capillaries of diabetic rat retinas. The findings suggest that abnormal synthesis of ECM components may contribute to vascular leakage in the diabetic retina.     

Ultrastructural pathological features of cerebrovascular malformations: a preliminary report

OBJECTIVE: Although cerebrovascular malformations have been characterized histologically, a systematic examination of such lesions by transmission electron microscopy has not been previously published. In this preliminary study, we describe the ultrastructural pathological features of cerebral arteriovenous malformations (AVMs) and cavernous malformations (CMs). METHODS: Using transmission electron microscopy, we examined three CMs and three AVMs microsurgically harvested from patients, for conventional indications. Normal control cerebral tissue was obtained from two patients undergoing surgery for epilepsy. Specific attention was directed at components of the vascular wall, endothelial cell (EC) morphology, intercellular tight junctions, and the subendothelial layer. RESULTS: In embolized AVM vessels, ECs were disrupted, with preservation of the underlying subendothelial vessel wall. Nidal vessel walls of AVMs showed disorganized collagen bundles. In CM specimens, ECs lined attenuated cavern walls that were composed of an amorphous material lacking organized collagen. Peripheral to the CMs, capillaries were often surrounded by rings of hemosiderin. Tight junctions between ECs were present in AVMs and control specimens, but substantial inter-EC gaps were found in CMs. Subendothelial smooth muscle cells were present in AVM and control specimens, but they were sparse or poorly characterized in CMs. CONCLUSION: Surgically resected vascular malformations demonstrate abnormal ultrastructural pathological features. The preoperative embolization of AVMs results in EC denudation, with preservation of vessel wall structural integrity. The thin walls of CMs, lacking significant subendothelial support, along with the rarity of intact tight junctions between ECs, may contribute to the known propensity of CMs for recurrent microhemorrhage.     

Effect of age and diabetes on type IV collagen and laminin in human kidney cortex

Specific radioimmunoassays for the 7-S domain of type IV collagen and the fragment P1 of laminin were used to quantify these basement membrane proteins in human kidney cortex at different ages and in some patients with diabetes mellitus. The antigens were solubilized by treating the tissue samples with the proteolytic enzymes collagenase, trypsin and pepsin. Total collagen content (as indicated by hydroxyproline concentration) increased with age, and the proportion of the collagen that could be solubilized by any enzyme treatment decreased. The type IV collagen concentration increased significantly with age, whereas the laminin concentration tended to decrease. In the one case of a type I diabetic the amounts of both antigens exceeded those in the age matched controls. In four type II diabetics the results were comparable with those for other aged cases. The distribution of the proteins was studied using the peroxidase-antiperoxidase method. The staining intensity and thickness of both antigens increased with age in the mesangium and Bowmans capsules, the change in type IV collagen staining being more evident. In diabetic patients these changes were more pronounced and other basement membranes appeared thicker in the stainings. These results indicate that basement membrane material accumulates in the kidney cortex during aging and that an alteration takes place in the composition of the basement membranes, the proportion of type IV collagen increasing and that of laminin decreasing.     

The origin of lymphatic capillaries in murine testes

It is known that the seminiferous tubules are bathed in a sea of lymph in mice, which are commonly used in reproductive and immunological studies. Although testicular lymphatic vessels arising from the tunica albuginea can be macroscopically observed in mice, the exact distribution of the lymphatic capillaries remains unclear. In the present study, we investigated the distribution of lymphatic capillaries in normal testes by immunohistochemical staining with monoclonal antibodies against lymph vessel endothelium HA-receptor 1 (LYVE-1) and a platelet endothelial cell adhesion molecule (CD31). Moreover, normal lymphocytes were locally injected into the testes of recipient mice, and their migration was investigated with the use of LYVE-1 and CD31. The results showed that lymphatic capillaries were in and just beneath the tunica albuginea but not in the interstitium between the seminiferous tubules. It was also noted that these were abundant in the thickened tunica albuginea adjacent to the epididymis, but they were scarce in the thin tunica albuginea opposite the epididymis. When normal lymphocytes were locally injected into testes, the injected lymphocytes migrated between the seminiferous tubules and then drained into the lymphatic vessels in the tunica albuginea. These results suggest that tissue fluid might drain from lymphatic capillaries that arise just beneath the tunica albuginea.     

Laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles

The newly developed laminin modified infection-preventing collagen membrane consists of a 3 component laminate, comprising 2 outer collagen layers and a central laminin layer. The 2 outer collagen layers (dense and porous layers) were fabricated by air-drying and freeze drying, respectively, and the laminin layer was formed by a straightforward liquid coating method. In addition, hyaluronan based microparticles containing silver sulfadiazine (AgSD) were incorporated into the 2 collagen layers (AgSD content 50 microg/cm2). Laminin coated collagen surfaces did not promote fibroblast attachment but showed a retarded fibroblast proliferation rate and an increased rate of collagen synthesis versus pure collagen surfaces. In an animal study, a laminin coating on a nonmedicated collagen membrane significantly increased both wound size reduction and vessel proliferation 7 days after application versus polyurethane film. Interestingly, the laminin coated AgSD medicated collagen membrane demonstrated higher wound size reduction and vessel proliferation and lower inflammation than the polyurethane control, suggesting that the laminin AgSD medicated collagen membrane substantially improves dermal wound healing.     

体外构建皮肤基底膜的组织学特征

目的观察制备的组织工程皮肤基底膜的组织学特征。方法取门诊正常儿童包皮环切术之包皮，采用胰蛋白酶胶原酶顺序消化得到角质形成细胞（KC）和成纤维细胞（Fb）悬液。制备复方壳多糖组织工程皮肤，浸没培养3d后，继续行气液界面培养。将培养7、10、15d的复方壳多糖组织工程皮肤用中性甲醛溶液固定后石蜡包埋、切片，行HE及高碘酸-雪夫（PAS）染色，并用免疫组织化学染色法观察基底膜的重要成分：Ⅳ型胶原、Ⅶ型胶原及层黏连蛋白（LN）的存在情况。结果HE染色可见培养的组织工程皮肤表皮结构分化良好，大致可分为基底层、棘层和角质层，各层均有数量不等的扁平梭形细胞。PAS染色显示真皮表皮间有一均匀红染的条带。免疫组织化学染色结果显示，Ⅳ型胶原、Ⅶ型胶原及LN呈阳性表达。结论复方壳多糖组织工程皮肤基底膜构建良好。     

Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41–59, BMI 26–38, cold ischemic time < 10 h). Collagen IV, pan‐laminin, perlecan and laminin‐α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin‐α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan‐laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan‐laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.     

Basement membrane alterations during development and regression of tubular cysts

Tubular cysts consisting of dilatation of the collecting ducts at the level of the subcapsular zone of the kidney were induced in newborn rabbits by a single injection of methylprednisolone acetate. We describe here the structural and compositional modifications of the tubular basement membrane (BM) during the formation, growth, and regression of the tubular cysts. During development of the tubular cysts the cystic BM appeared thickened and multilayered, with numerous matrix vesicles. Alcian blue- (AB) and ruthenium red- (RR) positive material distributed differently along the BM of control and cystic tubuli. While the amount of RR-positive material appeared increased in the cystic BM, no differences in the intensity of the AB staining could be discerned between normal and cystic tubuli. Immunofluorescent staining for laminin and type IV collagen appeared to be slightly decreased in the cystic tubuli. However, the amount of fibronectin appeared clearly increased. These changes in the cystic BM appear at the beginning of the tubular dilatation and are not observed in other renal BM. We suggest that there is a causal relationship between the modifications of the BM and the development of the tubular cysts. Glucocorticoids appear to modify the synthesis and/or secretion of the BM components. An abnormal BM should modify the spatial and chemical signals encoded within the BM that, in turn, could lead to abnormal behavior of the tubular cells. This may result in a loss of the normal developmental constraints imposed upon the tubular epithelium, which then undergoes cystic dilatation. During the regression of the cysts, the abnormalities of the BM progressively disappear. The sharp increase in the number of interstitial cells, which show close relationships with the components of the BM, suggests that these cells may be involved in the removal of the cyst BM.     

膀胱癌基底膜与肿瘤浸润、分化关系的研究

采用免疫组化SP法检测85例膀胱移行细胞癌基底膜的主要成分层粘连蛋白和Ⅳ型胶原,发现基底膜是否完整与膀胱癌分期有关(P<0.01),基底膜连续完整,膀胱癌浸润较浅,分期较低;基底膜与膀胱癌分级无关(P>0.05)。说明了基底膜是阻止肿瘤浸润的主要屏障;而层粘连蛋白和Ⅳ型胶原的表达有助于判断肿瘤分期和发现早期肿瘤浸润。     

Porcine small intestinal submucosa (SIS): a bioscaffold supporting in vitro primary human epidermal cell differentiation and synthesis of basement membrane proteins

The growth pattern of human epidermal cells, fibroblasts or Swiss mouse 3T3/J2 fibroblasts cultured upon the extracellular matrix (ECM) derived from small intestinal submucosa (SIS) was evaluated. The cell/SIS composites were grown submerged, then maintained in air/liquid interface for 2, 7, 10 or 14 days. The presence of differentiation-related keratins 10, 14 and 16, FN, laminin, collagen type VII and collagen type IV was determined by immunohistochemical methods in SIS alone and in the SIS/cell composite. Only FN could be detected in SIS alone. SIS supported the formation of an epithelial structure with suprabasal expression of K16 and regional suprabasal expression of K10. The epidermal cells were K14 positive and tended to 'invade' the SIS to various degrees. Following the growth of epidermal cells and fibroblasts on the SIS substratum, immunolabeling of FN, laminin, collagen type VII and collagen type IV was observed in a cell-associated pattern. The fibroblasts commonly invaded the SIS, when co-cultivated with epidermal cells on the opposite side of the SIS. The ability of SIS to support epidermal cell/fibroblast attachment, migration and/or proliferation and differentiation with deposition of basement membrane (BM) components indicates that the composite model may be useful for studying cell-matrix interactions and for investigation as a dermal substitute.     

Glycoxidative modification of AA amyloid deposits in renal tissue.

BACKGROUND: N(epsilon)-carboxymethyllysine (CML) is a product of the oxidative modification of glycated proteins, which damages proteins with ageing, diabetes, uraemia and Alzheimer's disease. In contrast, pyrraline is one of the advanced glycation end products, which is independent of oxidative processes. CML has been identified in beta-amyloid of Alzheimer's disease and beta(2)-microglobulin-associated amyloid. We investigated whether CML and pyrraline are formed in AA and AL amyloid of the kidney. METHOD: Renal specimens from 19 cases of AA amyloidosis and 14 cases of AL amyloidosis were investigated for immunolocalization of CML, pyrraline, collagen type IV and laminin in amyloid deposits. Renal biopsies of 10 age-matched cases with thin basement membrane disease and normal renal function were used as controls. The fractional areas of amyloid, CML, laminin and collagen IV in glomeruli and interstitium (%amyloid, %CML, %laminin and %collagen, respectively) were calculated using the point counting method. The correlation between these parameters was evaluated using Spearman's rank correlation test. RESULTS: CML colocalized with AA amyloid, but not AL amyloid, except in two cases of the latter with a long history of nephropathy exceeding 14 years. In contrast, pyrraline was not observed in either type of amyloid. Mean %CML in AA amyloid was significantly higher than %collagen and %laminin in glomeruli and interstitium, indicating that AA amyloid is modified by CML independent of colocalized extracellular matrix. %CML significantly correlated with %amyloid both in glomeruli and interstitium in AA amyloidosis. AL amyloid cases with a long history of nephropathy showed positive staining for CML in glomeruli and interstitium but no staining for collagen IV and laminin in amyloid deposits. CONCLUSION: CML modification may occur in amyloid deposits of AA amyloidosis, independent of extracellular matrix components. Glycoxidative modification may have a functional link to AA amyloid deposition in renal tissues.     

Prognostic value of lymphatic and blood vessel invasion in neuroendocrine tumors of the lung

Few data on the influence of vessel invasion on the progression of neuroendocrine lung tumors are available. Because of the lack of specific markers, previous studies could not reliably discriminate lymphatic and blood vessels. By immunostaining for podoplanin, specific for lymphatic endothelium, and CD34 antigen, we assessed lymphatic and blood vessel invasion in 120 tissue specimens of patients with neuroendocrine lung tumors. Lymphovascular invasion was correlated with clinicopathologic parameters, and its prognostic relevance was evaluated. Lymphatic vessels were identified exclusively at the tumor invasion front, whereas blood capillaries were also seen within tumors. Lymphatic vessel as well as lymphatic and blood vessel invasion was prevalent in patients with high-grade neuroendocrine tumors and advanced tumor stages, closely associated with lymph node metastases (P < 0.0001). In univariate analysis, these two invasion types correlated with decreased disease-free survival (both P < 0.0001), whereas blood vessel invasion alone did not. In multivariate analysis, only tumor grade and lymph node status remained statistically significant factors for prognosis (P = 0.016 and P < 0.0001). Our results suggest that evaluation of lymphatic vessel invasion is important in neuroendocrine lung tumors serving as a prognostic parameter for disease-free survival.