Spatiotemporal expression of flk-1 in pulmonary epithelial cells during lung development

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.     

Alveolar epithelial transport in the adult lung

The alveolar surface comprises >99% of the internal surface area of the lungs. At birth, the fetal lung rapidly converts from a state of net fluid secretion, which is necessary for normal fetal lung development, to a state in which there is a minimal amount of alveolar liquid. The alveolar surface epithelium facing the air compartment is composed of TI and TII cells. The morphometric characteristics of both cell types are fairly constant over a range of mammalian species varying in body weight by a factor of approximately 50,000. From the conservation of size and shape across species, one may infer that both TI and TII cells also have important conserved functions. The regulation of alveolar ion and liquid transport has been extensively investigated using a variety of experimental models, including whole animal, isolated lung, isolated cell, and cultured cell model systems, each with their inherent strengths and weaknesses. The results obtained with different model systems and a variety of different species point to both interesting parallels and some surprising differences. Sometimes it has been difficult to reconcile results obtained with different model systems. In this section, the primary focus will be on aspects of alveolar ion and liquid transport under normal physiologic conditions, emphasizing newer data and describing evolving paradigms of lung ion and fluid transport. We will highlight some of the unanswered questions, outline the similarities and differences in results obtained with different model systems, and describe some of the complex and interweaving regulatory networks.     

Alveolar epithelial hyperplasia and adenocarcinoma of the lung

To understand better the pathogenesis of peripherally occurring well-differentiated adenocarcinoma of the lung in relation to alveolar epithelial hyperplasia (AEH), immunohistochemical, morphometric, and electron microscopic studies were done on surgical pathologically examined cases of lung cancer. Cases with interstitial pulmonary fibrosis were excluded so that the effects of diffuse scarring of the lung on the development of neoplasia were eliminated. Of 70 specimens with various types of pulmonary carcinomas, 15 were found to have coexistent typical or atypical AEH lesions. No area of transition from AEH to neoplasm was found. The immunohistochemistry studies showed significant differences in the reactions of carcinoembryonic and blood group antigens between typical and atypical AEH lesions, but no significant differences could be obtained between atypical AEH lesions and adenocarcinoma. However, the morphometry of the mean nuclear areas revealed a highly significant difference between atypical AEH lesions and adenocarcinoma. Electron microscopy showed many Clara granules in atypical AEH cells. The results did not prove that AEH lesions are precancerous, but the presence of Clara granules in atypical AEH cells raises speculation on the histogenetic relationship between AEH and pulmonary adenocarcinoma.     

Alveolar macrophage-particle relationships during lung clearance

Retention kinetics for insoluble particles that deposit in the lung oftentimes resemble a multicomponent process during alveolar clearance, with each component appearing to follow simple first-order kinetics. Inasmuch as alveolar macrophages (AM) are thought to play an important role in particle removal from the lung, a study was undertaken to examine particle-AM relationships during the clearance of particles to obtain information on potential AM mechanisms that could provide the underlying bases for the lung retention kinetics of the particles. Adult, Fischer 344 rats were intratracheally instilled with 1.6 x 10(7) (approximately 86 micrograms) polystyrene microspheres (approximately 2 microns diam). On Days 7, 14, 57, 85, and 176 thereafter, subgroups were killed, their lungs were lavaged, recovered cells (greater than 95% AM) were counted, the frequency distribution of the particles among the AM was determined (e.g., zero, 1 to 2, 3 to 4 particles/AM), and the total numbers of particles lavaged were estimated. The lavaged lungs were solubilized, and unlavaged particles were also counted. The sums of the lavaged and unlavaged particles were used to estimate retained lung burdens at each postinstillation time. The lung retention data followed a pattern consistent with the sum of two negative exponential components, i.e., an earlier, more rapid component and a slower, longer term component. The rates at which the AM disappeared from a given particle category also were biphasic for AM that contained up to 14 microspheres. The rates of both the earlier and longer term components of such disappearance were found to increase with increasing AM burdens. Over an AM burden range of 1 to 10 microspheres, the proportion of AM that disappeared via rapid components also increased as the particle burden defining an AM category increased. At higher particle burdens, the proportion of AM that disappeared by an early component appeared to markedly diminish; an early component for AM disappearance was no longer resolvable for AM that contained greater than 15 microspheres. The net effect of these phenomena was that retained lung burdens over time became progressively contained in AM with lesser burdens of particles. The results from this study suggest that the rate(s) of translocation of particle-containing AM from the lung during lung clearance may be related to their individual particulate burdens. These findings, however, are also consistent with a gradual redistribution of particles among the lung's AM population over time concurrent with AM removal from the lung. Regardless, the biphasic nature of the lung retention data qualitatively was generally evident for particle-containing AM as well.     

Down-regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis

Sakamoto K, Aragaki T, Morita K‐i, Kawachi H, Kayamori K, Nakanishi S, Omura K, Miki Y, Okada N, Katsube K‐i, Takizawa T & Yamaguchi A
(2011) Histopathology 58, 531–542
Down‐regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis Aims: This study aimed to identify relevant keratin subtypes that may associate with the pathogenesis of oral epithelial neoplasms. Methods and results: Expression of all the keratin subtypes was examined by cDNA microarray analysis of 43 oral squamous cell carcinoma (OSCC) cases. Immunohistochemical expression of the major keratins was examined in 100 OSCC and oral epithelial dysplasia (OED) cases. Many changes in keratin expression were observed and, significantly, consistent down‐regulation of keratin 4 (K4) and K13 expression was observed. Aberrant expression of K4 and K13 was associated with morphological changes in the affected oral epithelium. Experiments with cell cultures transfected with various keratin subtypes suggested that alterations in keratin subtype expression can cause changes in cell shape and movement. Conclusions: Aberrant expression of K4 and K13, which are the dominant pair of differentiation‐related keratins in oral keratinocytes, indicates dysregulation of epithelial differentiation in OSCC and OED. These keratins, especially K4, may be useful for pathological diagnosis. We propose that the aberrant expression of K4 and K13 and concomitant up‐regulation of the other keratins may be one of the causative factors for morphological alterations in the affected epithelium.     

Transient expression of keratin during neuronal development in the adult rabbit spinal ganglion

A few neurons of the adult rabbit spinal ganglion express keratin. To examine the characters of these keratin-positive neurons, six kinds of intermediate filament proteins, namely keratin 8, keratin 14, nestin, vimentin, neurofilament 68 (NF-L) and glial fibrillary acidic protein (GFAP), were investigated immunohistochemically in developing and adult rabbit spinal ganglia. At 15 days of gestation, the spinal ganglion increased rapidly in volume and mainly consisted of three kinds of cells: small cells expressing vimentin, spindle-shaped cells co-expressing vimentin and nestin, and ovoid cells with an eccentric nucleus expressing nestin. Since some ovoid cells co-expressed nestin with either NF-L or GFAP, the ovoid cell may be considered to be an embryonic neural stem cell of the ganglion. In addition, a few keratin-positive polymorphic cells could be observed among these three kinds of cells. These polymorphic cells expressed five kinds of intermediate filament proteins, namely keratin 8, keratin 14, nestin, NF-L and GFAP. These cells were also detected in newborn and adult ganglia. A few neurons in the adult ganglion also expressed these five kinds of proteins as a Golgi-associated network. However, neurons expressing these proteins could not be detected in embryonic and newborn ganglia. Therefore, it may be considered that the keratin-positive polymorphic cell is a postnatal neural stem cell of the ganglion and that neurons transiently express keratin when polymorphic cells differentiate into neurons.     

Thyroid hormone induction of keratin genes: a two-step activation of gene expression during development

To determine the mechanism of action of the thyroid hormone triiodothyronine (T3) during metamorphosis of the amphibian epidermis, we have investigated the developmental activation of the 63-kD keratin genes in the frog Xenopus laevis. These genes code for three closely related keratins that first appear in the larval epidermis and accumulate during metamorphosis to become the most abundant proteins in the adult epidermis. We report here that the 63-kD keratins and their mRNAs first appear at stages 48-52. The level of 63-kD keratin gene expression remains relatively low until stage 56 and then increases dramatically. Quantitative analysis of the concentration of 63-kD keratin mRNA demonstrates low levels until stage 55/56, followed by an increase greater than 300-fold from stage 55/56 to the adult. Each adult cell contains approximately 55,000 molecules of the 63-kD keratin mRNAs. T3 is not required for the initial activation of the 63-kD keratin genes, but high-level expression is absolutely dependent on T3. High-level expression is prevented by propylthiouracil, which inhibits thyroid hormone synthesis and can be induced precociously both in vivo and in vitro with exogenous T3 as early as stage 48, but not prior to that time. Thus, the full activation of the 63-kD keratin genes during development requires two regulatory steps, one independent and one dependent on T3.     

Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia.

The expression of keratins in normal cervical epithelia, metaplastic epithelium, and cervical intraepithelial neoplasia (CIN) grades I, II, and III is investigated with a panel of keratin polypeptide-specific monoclonal antibodies. This approach allowed the detection of individual keratins 4, 7, 8, 10, 13, 14, 18, and 19 at the single-cell level. By using an antibody recognizing keratins 5 and 8 (RCK 102) and two antibodies specific for keratin 8 (CAM 5.2 and M 20), it was also possible to derive information on the distribution of keratin 5. Our results show that during immature squamous metaplasia there is an acquisition of keratins typical of squamous epithelium, ie, keratins 4, 5, 13, and 14. This process continues during further differentiation to mature squamous metaplasia. In premalignant lesions the expression pattern of the progenitor reserve cells and immature squamous metaplastic epithelium is partly conserved. However, in most cases an induction in the expression of the keratins 4, 13, and 14 was observed. Furthermore, CIN III shows a more extensive expression of keratins typical of simple epithelia, ie, keratins 8 and 18, as compared to CIN I and CIN II.     

Regulated expression of cell surface antigens during B cell development

The progression of lymphocytes along the B cell developmental pathway is marked by the regulated appearance and disappearance of a variety of cell surface markers including immunoglobulin. In this review, several of these antigens are discussed in the context of their possible functions during normal B cell differentiation.     

Alveolar epithelial cell therapy with human cord blood-derived hematopoietic progenitor cells

The role of umbilical cord blood (CB)-derived stem cell therapy in neonatal lung injury remains undetermined. We investigated the capacity of human CB-derived CD34(+) hematopoietic progenitor cells to regenerate injured alveolar epithelium in newborn mice. Double-transgenic mice with doxycycline (Dox)-dependent lung-specific Fas ligand (FasL) overexpression, treated with Dox between embryonal day 15 and postnatal day 3, served as a model of neonatal lung injury. Single-transgenic non-Dox-responsive littermates were controls. CD34(+) cells (1 × 10(5) to 5 × 10(5)) were administered at postnatal day 5 by intranasal inoculation. Engraftment, respiratory epithelial differentiation, proliferation, and cell fusion were studied at 8 weeks after inoculation. Engrafted cells were readily detected in all recipients and showed a higher incidence of surfactant immunoreactivity and proliferative activity in FasL-overexpressing animals compared with non-FasL-injured littermates. Cord blood-derived cells surrounding surfactant-immunoreactive type II-like cells frequently showed a transitional phenotype between type II and type I cells and/or type I cell-specific podoplanin immunoreactivity. Lack of nuclear colocalization of human and murine genomic material suggested the absence of fusion. In conclusion, human CB-derived CD34(+) cells are capable of long-term pulmonary engraftment, replication, clonal expansion, and reconstitution of injured respiratory epithelium by fusion-independent mechanisms. Cord blood-derived surfactant-positive epithelial cells appear to act as progenitors of the distal respiratory unit, analogous to resident type II cells. Graft proliferation and alveolar epithelial differentiation are promoted by lung injury.     

Synthesis and expression of laminin during human foetal lung development

Backgrounds: The lung develops by epithelial tubes budding and branching into a flexible mesenchyme. This growth is associated with the remodelling of the epithelial basement membrane, of which laminin is a major component. Methods: Both the synthesis and expression of laminin were studied in the human lung between 10 and 31 weeks of gestation, using in sity hybridization and immunohistochemistry. Results: The synthesis of the β chain was active in the epithelial and surrounding mesenchymal cells. The mRNAs coding for the γ chain were less abundant and mainly found in the epithelium. The synthesis of these two chains continued throughout gestation, and no significant difference in the density of hybridization grains could be detected between the tips of the expanding buds and the proximal portions. Immunohistochemical localization of laminin showed important modifications of the basement membrane during gestation. In the first part of the pseudoglandular stage the epithelial basement membrane stained continuously for laminin. Later, the basement membrane was labelled in a graded fashion: at the apex of the growing buds the staining became weak with focal disruptions. Both epithelial and mesenchymal synthesis of laminin remained active, while the polypeptide was undetectable using immunohistochemistry. Conclusions: These findings suggest that the remodelling of the basement membrane during human lung morphogenesis is probably not related to a decreasing synthesis of laminin, but to either a proteolytic degradation or the assembly of an inadequate complex undetectable with the polyclonal antibody antilaminin. © 1995 Wiley-Liss, Inc.     

CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.     

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1?mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100?μM L: -cis-diltiazem or 100?nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100?μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100?nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50?μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.     

JAM-A expression during embryonic development.

Cell adhesion molecules of the immunoglobulin superfamily play an important role in embryonic development. We have shown recently that JAM-A, a member of this family expressed at endothelial and epithelial tight junctions, is involved in platelet activation, leukocyte transmigration, and angiogenesis. Here, we determine the expression pattern of the JAM-A gene during embryogenesis using transgenic mice expressing lacZ under the control of the endogenous JAM-A promoter. Histochemical staining for beta-galactosidase in heterozygous mouse embryos was first seen in the inner cell mass and trophectoderm of the blastocyst. By 8.5 days post coitum (dpc), JAM-A gene activity was detected in the endoderm and part of the surface ectoderm. At 9.5 dpc, JAM-A expression began to localize to certain organ systems, most notably the developing inner ear and early vasculature. Localization of JAM-A to embryonic vasculature was confirmed by double-staining with antibodies against JAM-A and platelet endothelial cell adhesion molecule-1, a known endothelial cell marker. As organogenesis progressed, high levels of JAM-A expression continued in the epithelial component of the inner ear as well as the epithelium of the developing skin, olfactory system, lungs, and kidneys. In addition, JAM-A gene activity was found in the developing liver, choroid plexuses, and gut tubes. Immunofluorescent staining with a JAM-A antibody was performed to confirm that expression of the JAM-A-beta-galactosidase fusion protein accurately represented endogenous JAM-A protein. Thus, JAM-A is prominently expressed in embryonic vasculature and the epithelial components of several organ systems and may have an important role in their development.     

Heterogeneity of keratin expression in epithelial tumor cells of adenolymphoma in paraffin sections

Immunohistochemical expressions of keratin polypeptides detected by monoclonal antibodies were described in tumor cells of adenolymphoma, and the possibility of intercalated duct and ductal basal cells in the salivary glands being the progenitors was discussed. Basal cells in the tumor showed positive staining for keratin nos. 8, 13, 16, 18 and 19 detecting for monoclonal keratin antibodies (PKK 1, K 4.62, K 8.12, K 8.13), columnar tumor cells displayed strongly positive reactions with RPN 1164 and K4.62 suggesting keratin nos. 8 and 19. Great heterogeneity of distribution for keratin polypeptides was displayed by epithelial cells of adenolymphoma. Intercalated duct cells of normal salivary glands reacted with RPN 1164, RPN 1165, K 4.62 and K 8.13 monoclonal antibodies, which indicates the presence of keratins 8 and 19; and ductal basal cells reacted with PKK 1, K 4.62 and K 8.12, suggesting nos. 8, 13, 16, 18 and 19 keratins. Distribution of involucrin was variable in tumor epithelium of adenolymphoma, and was negative in the normal gland. The immunohistochemical distribution of keratin types between basal tumor cells of adenolymphoma and ductal basal cells of the normal salivary gland was compared.     

The role of the nonciliated bronchiolar epithelial (Clara) cell as the progenitor cell during bronchiolar epithelial differentiation in the perinatal rabbit lung.

Although it is well established that the nonciliated bronchiolar epithelial (Clara) cell serves as the progenitor for itself and ciliated cells in the adult lung following bronchiolar epithelial injury, the nature of this relationship during development has not been well characterized. To define the pattern of proliferation and differentiation of bronchiolar ciliated and nonciliated cells, lungs of fetuses and offspring from time-mated New Zealand White rabbits, ranging in age from 24 days of gestation to 25 wk postnatal (PN), were fixed by airway infusion and embedded for simultaneous light and transmission electron microscopy. Three categories of cells could be distinguished in terminal bronchioles: nonciliated cells with abundant glycogen and variable numbers of organelles; nonciliated cells with little glycogen, large numbers of polyribosomes, and variable numbers of basal bodies; and ciliated cells with cilia of varying height. Together, both types of nonciliated cells were 100% of the epithelium at 24 and 27 days gestation age (DGA). At 30 days DGA, they were 85% of the population; at all postnatal ages, they ranged from 75 to 81% of the total population. Nonciliated cells with polyribosomes and basal bodies were 10 to 20% of the total nonciliated cell population between 24 DGA and 1 wk PN and not found thereafter. Ciliated cells were not observed in animals younger than 30 DGA. Labeling indices of bronchiolar epithelium in fetuses of pregnant rabbits injected with tritiated thymidine, as determined by autoradiography, were 57 cells per thousand at 28 DGA (1 h postinjection [PI]), 76 at 29 DGA (24 h PI), and 114 at 30 DGA (48 h PI).(ABSTRACT TRUNCATED AT 250 WORDS)