Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells.     

Ontogeny of reactivity to endothelial cell markers during development of the embryonic and fetal rat lung.

The reactivity of endothelial cells to putative endothelial cell-specific markers varies with species, with vessel size and with the organ studied. To determine their value in studies of fetal rat lung, and whether organ immaturity would also influence reactivity, we studied endothelial cell immunoreactivity to antibodies against Factor VIII/von Willebrand factor (VIII/vWF), and binding reactivity to Bandeiraea (Griffonia) simplicifolia 1 lectin (BSL 1) during rat fetal lung development. Using an indirect immunofluorescent technique to detect Factor VIII/von Willebrand factor (VIII/vWF), endothelial cells lining the aortic arches were identified as early as day 11 of gestation (term = 22 days), prior to lung development. Immunoreactivity to VIII/vWF was subsequently localized to intrapulmonary endothelial cells and was not dependent on vessel size. In contrast, binding reactivity of FITC-conjugated BSL 1 was observed to both endothelial cells and to the basement membrane of developing airways, thus limiting its value as endothelial cell marker. During very early lung development solitary angioblasts could not be identified by reactivity to either VIII/vWF antibodies or to BSL 1, and neither marker appears to be of value for studies of early angiogenic events.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Summary Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells. Presented in part at the 31st Annual Meeting of the Histochemical Society, April 11–15, 1980, New Orleans, Louisiana. Wendy Baur and Ms. Jane Aghajanian for expert assistance in the preparation of the cell cultures. This work was supported by Research Grant HL 14456 and Training Grant HL 07053 from the National Institutes of Health, Bethesda, MD.     

An avidin-biotin-peroxidase method for Fc receptors on macrophages isolated from and in sections of rat lung

Receptors for the Fc region of immunoglobulin G (Fc receptors) were detected on pulmonary macrophages by adapting an avidin-biotin-peroxidase technique to isolated cells and sections of rat lung. After incubation with soluble rabbit immunoglobulin G (IgG), surface bound IgG was identified consistently and reproducibly on glass-adherent pulmonary macrophages and on macrophages in tissue sections made from incubated lung slices. Control experiments indicated that binding was specifically mediated by surface Fc receptors. This method may be useful for identifying macrophages in intact tissues.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).     

Long-term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone

Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co-culture system, the effect of sinusoidal liver cells on hepatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co-cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of alpha 1-fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co-cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte-stabilizing activity was also produced by a pulmonary endothelial cell line.     

Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis

ICAM-1 is an intercellular adhesion molecule of the immunoglobulin supergene family involved in adherence of leukocytes to the endothelium and in leukocytic accumulation in pulmonary injury. In the current study, the antigen retrieval technique was used to detect ICAM-1 immunohistochemically in paraffin sections of lungs from human, mouse and rat as well as in bleomycin- or radiation-induced fibrotic lungs from rat and human. In normal lung tissue, the expression of ICAM-1 on alveolar type I epithelial cells is stronger than on alveolar macrophages and on endothelial cells. Preembedding immuno-electron microscopy of normal rat, mouse and human lung samples revealed sclective ICAM-1 expression on the surface of type I alveolar epithelial cells and, to a lesser extent, on the pulmonary capillary endothelium and on alveolar macrophages. In fibrotic specimens, both focal lack and strengthening of immunostaining on the surface of type I cells was found. Alveolar macrophages were found focally lacking ICAM-1 immunoreactivity. In some cases, rat type II pneumocytes exhibited positive immunoreactions for ICAM-1. Immunoelectron microscopy with preembedded rat lungs (bleomycin-exposed cases) confirmed the altered ICAM-1 distribution at the alveolar epithelial surface. In the alveolar fluid of fibrotic rat lungs, in contrast to that from untreated controls, soluble ICAM-1 was detected by western blot analysis.     

Cellular disposition of transported polyamines in hypoxic rat lung and pulmonary arteries

The polyamines putrescine, spermidine (SPD), and spermine are a family of low-molecular-weight organic cations essential for cell growth and differentiation and other aspects of signal transduction. Hypoxic pulmonary vascular remodeling is accompanied by depressed lung polyamine synthesis and markedly augmented polyamine uptake. Cell types in which hypoxia induces polyamine transport in intact lung have not been delineated. Accordingly, rat lung and rat main pulmonary arterial explants were incubated with [(14)C]SPD in either normoxic (21% O(2)) or hypoxic (2% O(2)) environments for 24 h. Autoradiographic evaluation confirmed previous studies showing that, in normoxia, alveolar epithelial cells are dominant sites of polyamine uptake. In contrast, hypoxia was accompanied by prominent localization of [(14)C]SPD in conduit, muscularized, and partially muscularized pulmonary arteries, which was not evident in normoxic lung tissue. Hypoxic main pulmonary arterial explants also exhibited substantial increases in [(14)C]SPD uptake relative to control explants, and autoradiography revealed that enhanced uptake was most evident in the medial layer. Main pulmonary arterial explants denuded of endothelium failed to increase polyamine transport in hypoxia. Conversely, medium conditioned by endothelial cells cultured in hypoxic, but not in normoxic, environments enabled hypoxic transport induction in denuded arterial explants. These findings in arterial explants were recapitulated in rat cultured main pulmonary artery cells, including the enhancing effect of a soluble endothelium-derived factor(s) on hypoxic induction of [(14)C]SPD uptake in smooth muscle cells. Viewed collectively, these results show in intact lung tissue that hypoxia enhances polyamine transport in pulmonary artery smooth muscle by a mechanism requiring elaboration of an unknown factor(s) from endothelial cells.     

Sites of uptake of3H-5-hydroxytryptamine in rat isolated lung

Summary The localisation of radioactivity in rat lungs after perfusion of³H-5-hydroxytryptamine (5-HT) was studied by autoradiography. Rat isolated lungs, perfused with Krebs bicarbonate solution, via the pulmonary circulation, were rapidly frozen after the infusion of³H-5-HT. All subsequent handling was carried out at −30 to −40°C. Developed sections were examined by the light microscope and showed that radioactivity was concentrated around the alveoli with little or no label in other parts of the lung. Lungs perfused with³H-5-HT in the presence of mebanazine, a monoamine oxidase inhibitor, showed label in the endothelial cells of arteries and arterioles as well as the alveolar label. Lungs treated with amitriptyline were essentially devoid of label. These results indicate that the site of the avid uptake and metabolism of 5-HT in the lung is the endothelial cells of the vasculature.     

The effects of different fixatives for immunofluorescent and immunoperoxidase localization of Factor VIII-related antigen in canine carotid artery and vascular prostheses

Summary The evaluation of vascular grafts seeded with autologous endothelial cells requires a reliable method of endothelial cell identification. Previous attempts to identify positively tissue Factor VIII-related antigen, found in relatively large amounts in vascular endothelial cells, proved to be inconsistent when immunoperoxidase and immunofluorescent staining techniques were tried. Because the Factor VIII antigen is very labile, this study was performed to determine an optimal fixation technique for demonstrating this antigen in frozen sections of endothelial tissue. Unfixed, acetone-fixed, and formalin-fixed sections of canine carotid artery as well as vascular grafts fixed in 1-ethyl-3-(3-diaminopropyl)-carbodiimide were examined by an indirect immunofluorescence technique. Also, the immunoperoxidase method of Factor VIII identification was applied to unfixed, acetone-fixed, and carbodiimide-fixed endothelial cell seeded vascular grafts. Acetone was the preferred fixative, resulting in excellent antigen preservation with minimal background staining. The immunoperoxidase technique of Factor VIII-related antigen identification was found to be the method of choice because of its sensitivity.     

大鼠肺微血管内皮细胞培养及鉴定

取大鼠周边肺组织进行肺微血管内皮细胞培养。将肺组织切成小块,用含有２０％新生牛血清、肝素９０μｇ／ｍｌ、Ｌ－谷胺酰胺４ｍｍｏｌ、青霉素１００Ｕ／ｍｌ和链霉素１００μｇ／ｍｌ的ＲＰＭＩ－１６４０培养基培养。血细胞立即从肺组织周围游出。继而是肺微血管内皮细胞。成纤维细胞及其他细胞７２ｈ才游出。培养６０ｈ后取出肺组织块,培养瓶中只有微血管内皮细胞和血细胞。后者可通过传代除去。获得的肺微血管内皮细胞具有规律的鹅卵石样形态和对异植物血凝素结合试验及八因子相关抗原免疫荧光染色均阳性。     

Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.     

SC5b-9-Induced Pulmonary Microvascular Endothelial Hyperpermeability Participates in Ventilator-Induced Lung Injury

Mechanical ventilation with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses, termed ventilator-induced lung injury (VILI). VILI is characterized by an influx of inflammatory cells, increased pulmonary permeability, and endothelial and epithelial cell death. But the underlying molecular mechanisms that regulate VILI remain unclear. The purpose of this study was to investigate the mechanisms that regulate pulmonary endothelial barrier in an animal model of VILI. These data suggest that SC5b-9, as the production of the complement activation, causes increase in rat pulmonary microvascular permeability by inducing activation of RhoA and subsequent phosphorylation of myosin light chain and contraction of endothelial cells, resulting in gap formation. In general, the complement-mediated increase in pulmonary microvascular permeability may participate in VILI.     

Stem cell antigen-1 expression in the pulmonary vascular endothelium

Although the function of the cell surface protein stem cell antigen-1 (Sca-1) has not been identified, expression of this molecule is a characteristic of bone marrow-derived hematopoietic stem cell populations. Expression of Sca-1, however, is not restricted to hematopoietic tissue. By RT-PCR and Western analysis, we found that Sca-1 is expressed in the adult mouse lung. Sca-1 immunohistochemistry revealed a linear staining pattern on the endothelial surface of large and small pulmonary arteries and veins and alveolar capillaries. Expression of Sca-1 in the pulmonary endothelium was confirmed by dual fluorescent microscopy on lung sections and by fluorescence-activated cell sorting analysis of digested lung tissue; each of these methods showed colocalization with the endothelial marker platelet/endothelial cell adhesion molecule-1. In the kidney, Sca-1 expression was also noted in large vessels, but, in contrast to the lung, was not observed in capillaries. Overall, our data indicate that Sca-1 expression helps define the surface phenotype of endothelial cells throughout the pulmonary vasculature.     

Pulmonary reexpansion causes xanthine oxidase-induced apoptosis in rat lung

The pathogenesis of reexpansion pulmonary edema is not yet fully understood. We therefore studied its mechanism in a rat model in which the left lung was collapsed by bronchial occlusion for 1 h and then reexpanded and ventilated for an additional 3 h. We then evaluated the production of reactive oxygen species in the lungs using fluorescent imaging and cerium deposition electron microscopic techniques and the incidence of apoptosis using the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. We found that pulmonary reexpansion induced production of reactive oxygen species and then apoptosis, mainly in endothelial and alveolar type II epithelial cells. Endothelial cells and alveolar type I and II epithelial cells in the reexpanded lung were positive for TUNEL and cleaved caspase-3. DNA fragmentation was also observed in the reexpanded lung. In addition, wet-dry ratios obtained with reexpanded lungs were significantly higher than those obtained with control lungs, indicating increased fluid content. All of these effects were attenuated by pretreating rats with a specific xanthine oxidase inhibitor, sodium (-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one. It thus appears that pulmonary reexpansion activates xanthine oxidase in both endothelial and alveolar type II epithelial cells and that the reactive oxygen species produced by the enzyme induce apoptosis among the endothelial and alveolar type I and II epithelial cells that make up the pulmonary water-air barrier, leading to reexpansion pulmonary edema.