Masakazu Fujiwara

Protease-activated receptor (PAR)-1 and PAR-2 participate in the cell growth of alveolar capillary endothelium in primary lung adenocarcinomas.

Cell growth can be induced via elicitation of protease‐activated receptors (PAR) with serine proteases such as thrombin and trypsin.

Potential role of the Slit/Robo signal pathway in angiogenesis

Intensive investigations on angiogenesis and vasculogenesis have increased our understanding of molecular mechanisms of blood vessel formation during pathologic and developmental conditions. However, endothelial cells (ECs), the main component of vasculature, are heterogeneous, as revealed by our phenotypic and molecular biological studies in the laboratory, and it is still hard to adequately understand the molecular mechanisms of angiogenesis and vasculogenesis. Indeed, there are several major ligand/receptor signal pathways: VEGF/VEGFR, Jagged-1/Notch, Wnt ligand/frizzled receptor, and ephrin/Eph; each of which having distinct and independent roles during vascular formation. In this review, we focus on the angiogenic effect of the Slit and Robo signal pathway that was formally known as neuronal axon guidance. Among the existing vascular signals, this pathway is the most recently found ligand/receptor vascular signal, and may play important physiological roles as other major receptor/ligand signals do. Here, we briefly address: (1) the background of Slit and Robo families; (2) expression patterns of Slit and Robo; (3) functional roles of the Slit/Robo pathway in vascular formation; and (4) confronting tasks of this novel vascular pathway in the near future. Together, a summary of these data suggest the essential role of the Slit/Robo pathway in angiogenesis, and may explain why multiple vascular signals exist in heterogenic endothelial cells.

Mosaic-like distribution of endothelial cell antigens in capillaries and juxta-alveolar microvessels in the normal human lung

The distribution patterns of endothelial cell antigens, including thrombomodulin and von Willebrand factor (vWf), were studied in normal lung tissues obtained from distant areas of solitary nodules (seven adenocarcinomas and four hamartomas). By single immunoalkaline phosphatase and dual immunofluorescence stainings, the plasma membranes of alveolar capillary endothelium showed linear distribution of thrombomodulin, but their cytoplasm was rarely reactive for vWf (thrombomodulin‐dominant pattern). Microvessels with a diameter larger than 10 μm located in the connective tissue zones demonstrated band‐like reaction for vWf in their cytoplasm, and their plasma membranes often lacked reactivity for thrombomodulin (vWf‐dominant pattern). The juxta‐alveolar microvessels located along the borders between the alveolar‐ and connective‐tissue zones showed mosaic‐like pattern of distribution for these antigens. The pulmonary venules and peribronchial microvessels measuring up to 40 μm in diameter, demonstrated the expression of thrombomodulin along the plasma membrane, and that of vWf in the cytoplasm. Capillaries of the bronchial circulation were also characterized by mosaic‐like pattern of distribution. Both antigens were often expressed in a single cytoplasmic segment. The heterogeneous distribution pattern of these antigens suggests topographic difference in endothelial cell function to maintain coagulatory and anticoagulatory balance in the normal human lung.

Role of cdk4, p16INK4, and Rb Expression in the Prognosis of Bronchioloalveolar Carcinomas

Background: The p16INK4 protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. Objectives: Loss of p16INK4 has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. Methods: We examined immunohistochemical expression of p16INK4, cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. Results: All BACs expressed cdk4, while 89 and 82% expressed p16INK4 and Rb proteins, respectively. None of the clinicopathological factors correlated with p16INK4, cdk4, or Rb expression separately. A low p16INK4/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16INK4/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16INK4/cdk4 ratio (p = 0.01). Conclusions: The results suggest a role of the cdk4/p16INK4 pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16INK4/cdk4 ratio may identify tumors that are destined to behave unfavorably.

Angiogenesis and phenotypic alteration of alveolar capillary endothelium in areas of neoplastic cell spread in primary lung adenocarcinoma.

Normal alveolar capillary endothelium is quiescent in nature and displays anticoagulant thrombomodulin (TM) on its surface. The cytoplasms of these endothelial cells are ultrastructurally non‐fenestrated type, and they barely express von Willebrand factor (vWf). Alveolar fibrosis is accompanied by a capillary endothelium reactive for vWf, and a loss of TM expression. In primary lung adenocarcinoma, neovascularization occurs in association with alveolar fibrosis. In order to study basic factors related to angiogenesis and phenotypic changes of the capillaries located in tumor‐bearing alveolar walls, we examined 37 primary lung adenocarcinomas with electron microscopy and confocal laser scanning microscopy with antibodies for TM, vWf, vascular endothelial growth factor (VEGF), and its receptors (KDR and Flt‐1), and proliferating markers (Ki‐67/proliferating cell nuclear antigen). Tissues microdissected specifically from alveolar walls were used for reverse transcription–polymerase chain reaction (RT–PCR) to assess expressions of mRNA isoforms of VEGF and its receptors. New capillary branching was found by ultrastructural study in the alveolar walls in 12% of the patients. Nuclei of the capillary endothelial cells were reactive for proliferating cell markers. Endothelial fenestrae were developed in 65% of the patients, TM reactivity was lost in the alveolar capillaries, and their cell cytoplasms obtained a reactivity for vWf through a transitional mosaic‐like distribution pattern of both antigens. Besides cytoplasmic VEGF expression in neoplastic cells, tumor‐bearing alveolar walls showed significant expression of mRNA of VEGF165 and KDR. These findings imply that angiogenesis and phenotypic changes of the alveolar capillaries are closely related to a higher expression of tumor‐associated VEGF165 and of KDR in the alveolar walls in primary lung adenocarcinoma.

Differential expression of protease-activated receptors 1, 2, and 4 on human endothelial cells from different vascular sites.

Objective: Protease-activated receptors (PARs) mediate DNA synthesis in endothelial cells when activated by serine proteases. However, despite the existence of heterogeneity among endothelial cells from each tissue, the responses to PAR-1, PAR-2, and PAR-4 activation are poorly defined and compared between endothelial cells from different sites. The aim of this study was to investigate whether PAR-mediated DNA synthesis differed in various endothelial cell types. Methods: We examined the incorporation of BrdU by human pulmonary artery endothelial cells (HPAECs), human aortic endothelial cells (HAECs), and human umbilical vein endothelial cells (HUVECs). Results: When the endothelial cells were treated with the selective PAR-1-activating peptide, SFLLRN, HAECs showed the highest BrdU incorporation rate (182 ± 28%). In contrast, treatment with the PAR-2-activating peptide, SLIGKV, resulted in the highest BrdU incorporation rate (173 ± 37%) in HPAECs, when pretreated with TNF-α. The PAR-4-activating peptide, GYPGQV, induced DNA synthesis in HPAECs and HAECs, but not in HUVECs. Conclusion: These findings suggest that each PAR preferentially targets an endothelial cell type, and thus plays a distinct role in diverse physiological or pathological conditions.

Activation of PAR4 Induces a Distinct Actin Fiber Formation via p38 MAPK in Human Lung Endothelial Cells

Protease-activated receptors (PARs) are multifunctional G protein–coupled receptors. Among the four existing PARs, PAR4 is preferentially expressed in the human lung tissue. However, the function of PAR4 has not been defined in the lung endothelial cells. Because PAR1-mediated cellular effects are deeply related to the morphological changes, we focused on the actin fiber and p38 mitogen-activated protein kinase (MAPK) signaling involved in actin polymerization to elucidate the role of PAR4. RT-PCR and Western blot analyses identified PAR4 expression in human pulmonary artery endothelial cells and in human microvascular endothelial cells from lung. We then examined the changes in actin fibers in endothelial cells treated with PAR4-activating peptide. PAR1-activating peptide was used for comparison. Activation of PAR4 and PAR1 by their corresponding peptides induced actin fiber formation; however, the actin filaments were broadly bundled in PAR4 as compared with the ringlike actin filaments in PAR1 activation. Correspondingly, the magnitude of p38 MAPK phosphorylation was different between cells treated with PAR4 and PAR1, with PAR4-activating peptide showing a significantly higher sensitivity to p38 MAPK inhibitor, SB203580. Taken together, these results demonstrate that activation of PAR4 results in the formation of actin fiber distinct from that by PAR1 activation, suggesting PAR4 may play specific roles in the lung endothelial cells.

Antisense oligodeoxynucleotides against thrombomodulin suppress the cell growth of lung adenocarcinoma cell line A549

Thrombomodulin (TM), an anticoagulant factor on endothelial cells, is known to be expressed in non‐endothelial cells as well. In neoplastic cells of lung adenocarcinomas, TM is expressed but its correlation with growth potential has not been studied. As TM expression has a negative correlation with cell proliferation in lung squamous cell carcinomas, we examined its growth effect on lung adenocarcinoma cells of the A549 cell line by inhibiting TM expression with antisense oligodeoxynucleotides (ODN). In the antisense ODN transfected cells, the expression of TM mRNA was decreased to 49% at 12 h and 47% at 24 h, which was in accordance with TM expression at the protein level. By IdU (5‐iodo‐2′‐deoxyuridine) incorporation assay, the growth of A549 cells was found to have decreased to 36% of the control level at 24 h post‐transfection. The suppression of cell growth was maintained in a concentration‐dependent manner for 48 h after transfection, when the expression of TM started to rebound. In the transfected cells, the G1 phase cell count was reduced to 60.7%, compared with 68.2% in the control transfectants. These results suggest that TM expression may play a suppressive role in the proliferation activity of A549 lung adenocarcinoma cells.

Spatial and Phenotypic Characterization of Vascular Remodeling in a Mouse Model of Asthma

Asthma is a chronic inflammatory disease characterized by airway wall remodeling in which vascular remodeling is thought to be a main contributor. Vascular endothelial growth factor (VEGF) is known as a major regulator of angiogenesis and enhancer of vascular permeability. Here, we define the spatial nature of vascular remodeling and the role of VEGF and its receptors (Flt-1 and Flk-1) in the allergic response in mice (A/J) susceptible to the development of allergen-induced airway hyperresponsiveness using morphometric and quantitative approaches. Increased vascularity, vasodilatation, and endothelial cell proliferation were found in the tracheal and bronchial walls in the early and late phases of asthma. Vascular changes were observed not only in small vessels but also in larger vessels. In contrast to normal control, lung tissue from the asthma model showed dual expression for CD31 and von Willebrand factor in the endothelial cells and α-smooth muscle actin and desmin in the mural cells of the vessels, suggesting a phenotypic and functional transformation. The mRNA levels of VEGF isoforms, VEGF164 and VEGF188, were significantly increased in the tracheal and lung tissue, respectively. In addition, the mRNA level of VEGF receptor Flk-1 was significantly increased in the trachea. These results establish the existence of vascular remodeling in the airways in a mouse model of allergic asthma and support a key role for the expression of unique VEGF isoform genes as mediators of structural changes.

RCAN1 regulates vascular branching during Xenopus laevis angiogenesis.

Background/Aims:The mechanisms that regulate the size-related morphologies of various blood vessels from the aorta to capillary vessels are still poorly understood. In this study, we evaluate the involvement of regulator of calcineurin 1 (RCAN1), a regulatory protein in the calcineurin/NFAT signal transduction pathway, in vascular morphology to gain further insight into these mechanisms. Methods and Results: We first generated 2 types of vasculature in vitro from the same source of human umbilical vein endothelial cells by fibrin gel assay. We found that RCAN1 was significantly upregulated in large vessels with low branching frequencies when compared with small vessels with high branching frequencies. Next, to clarify whether RCAN1 regulates the branching of blood vessels in vivo, we injected RCAN1 mRNA into fertilized Xenopus laevis eggs. Overexpression of RCAN1 decreased the number of branching points that sprouted from intersomitic vessels during X. laevis angiogenesis. In addition, coexpression of calcineurin A, a target of RCAN1, could rescue RCAN1-suppressed vascular branching. Conclusions: These results provide in vivo evidence of RCAN1-regulated vascular branching which may play a role in the patterning of morphologically different vasculature.