Publications

Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

Lecht S., Stabler C.T., Rylander A.L., Chiaverelli R., Schulman E.S., Marcinkiewicz C., Lelkes P.I.Biomaterials, 2014

Pre-treatment of the decellularized lung with defined ECM proteins, to evaluate the efficacy of reseeding of mESC. All current decellularization methodologies result in significant alterations of the contents, and ratios of ECM proteins, though the exact degree of the loss of ECM glycoproteins, such as laminins. Following ECMs were tested: bovine collagen type IV; human collagen type IV; human pro-collagen type I; human collagen type I; rat collagen type I; human plasma-purified fibronectin; human thrombospondin-1; VCAM; vitronectin; bovine elastin; Matrigel-purified laminin 111; and human recombinant laminins 111, 211, 332, 411, 421, 511, 521. mESCs lack major integrin receptors for collagens but express high levels of functional receptors for LM and FN. Laminin next to FN appears to be the major pro-adhesive ECM protein for mESCs. The mESCs were found to adhere differentially in an isoform-dependent manner with the following order of potencies 511 = 521 >332 >421 >211 >111 >411. These findings further support the notion that a3b1 and a6b1 integrin receptors expressed on the mES cell surface are involved in the specific binding to LM in general and to LM 511 and 521. LM and FN interact more efficiently with collagen type I than with collagen IV or elastin 90% of the available Coll I in the decellularized lung matrix forms a complex with LM. in vitro that cell-derived FN and LM interact with Coll I with much higher efficiencies than commercial FN or LM This finding may be explained by a possible partial loss of activity as a result of the purification procedures in contrast to the unprocessed CM.

Laminin isoforms and lung development: All isoforms are not equal

Developmental Biology, 2006

The embryonic lung has abundant laminin isoforms. Studies of embryonic lung explants and organotypic co-cultures show that laminin α1 and laminin 111 are important for epithelial branching morphogenesis and that laminin α2 and laminin 211 have a role in smooth muscle cell differentiation. In vivo studies of laminin α5-deficient mice indicate that this laminin chain, found in laminins 511 and 521, is essential for normal lobar septation in early lung development and normal alveolization and distal epithelial cell differentiation and maturation in late lung development. Laminin α4 null mice do not have obvious lung abnormalities and laminin γ2 null mice have only minimal changes in lung development. It is clear that multiple laminin isoforms are crucial for lung development and that different laminin isoforms exhibit specific, nonoverlapping functions.

Expression Patterns of Laminin α1 and α5 in Human Lung during Development

Pierce R.A, Griffin G.L., Miner J.H., Senior R.M.
Am. J. Respir. Cell Mol. Biol., 2000

To determine the temporal and spatial patterns of laminin α1 and laminin α5 in developing human lung, the authors prepared complementary DNA probes specific for laminin α1 and α5 messenger RNAs (mRNAs). Laminin α1 mRNA was prominent in first trimester fetal lung but was not detectable at 23 wk or at later times. In contrast, laminin α5 mRNA was readily detected in early fetal lung and remained present thereafter. Immunohistochemical staining demonstrated laminin a 1 only in early fetal lung, whereas laminin α5 was persistent from the early fetal period. In situ hybridization localized laminin α1 expression to the distal epithelium in the first-trimester lung, and laminin α5 to all epithelium and developing pulmonary arteries from the first trimester through the perinatal period. These studies indicate that laminin α1 expression is restricted to early human lung morphogenesis, whereas the expression of laminin α5 in the human lung is continuous from early lung development through adult life. It is evident that laminin α1 and laminin α5 have different roles in the development of the human lung.

Differential Expression of Laminins and Their Integrin Receptors in Developing and Adult Human Lung

Virtanen I., Laitinen A., Tani T., PiUikko P., Laitinen L.A., Burgeson R.E., LehtoV-P.Am. J. Respir. Cell Mol. BioI. 1996

Here, the authors studied the expression of different laminin chains and their integrin receptors in fetal and adult lung by immunohistochemistry. Special attention was focused on the changes in the expression of these proteins during the development from the pseudoglandular (PG) and canalicular stages to adult lung and on the possible implications of the changes for the normal lung development. The most significant changes in the expression pattern were found during the development from the PO stage to the canalicular stage. Basement membranes (BM) of both the epithelial buds and the becoming bronchi showed reactivity for laminin α1, a3, and B3 chains at all stages. The a2 chain was expressed only in the epithelial buds at the PO stage, and could not be found in any epithelial structures at the canalicular stage. Similarly, at the PG stage the B2 chain was expressed in BMs of both epithelial buds and bronchi but disappeared from the bronchial BM before the canalicular stage. The B1 chain appeared in the bronchial BM first in the mature lung. There were considerable changes in the expression of integrins (lnt) comcomitantly with alterations in the composition of the BMs. At the PG stage, the epithelial buds showed reactivity for integrin a2, a3, and a6 subunits, but at the canalicular stage the integrin a2 and a6 subunits disappeared, and only the a3 integrin subunit was found in evolving alveolar walls; integrin a6 was found in capillaries.  Our results suggest that there are major changes in the expression of laminin and their integrin receptors during morphogenesis of the lung, which may be important for normal development.

Lung development in laminin γ2 deficiency: abnormal tracheal hemidesmosomes with normal branching morphogenesis and epithelial differentiation

Nguyen N.M., Pulkkinen L., Schlueter J.A, Meneguzzi G., Uitto J., Senior R.M.Respiratory Research, 2006

Laminin-332 is prominent in the basement membrane of alveolar walls and airways of developing and adult lung. A function for laminin γ2 in lung development has been hypothesized. Compared to littermate controls, Lamc2-/- lungs were similar in morphology during embryonic life. Epithelial differentiation did not reveal a difference between Lamc2-/- and littermate control lungs. Likewise, vascular development, smooth muscle cell differentiation, and elastic fiber formation looked similar, as did airway basement membrane ultrastructure. Branching morphogenesis by lung bud culture was similar in Lamc2-/- and littermate control lungs. These findings indicate that laminin γ2 and laminin-332, despite their prominence in the lung, have a minimal role in lung development through the saccular stage.

Epithelial laminin α5 is necessary for distal epithelial cell maturation, VEGF production, and alveolization in the developing murine lung

Nguyen N.M., Kelley D.G, Schlueter J.A., Meyer M.J., Senior R.M., Miner J.H.Developmental Biology, 2005

Laminin α5 is prominent in the basement membrane of alveolar walls, airways, and pleura in developing and adult lung. To identify roles for laminin α5 in lung development, the authors have generated an inducible lung epithelial cell-specific Lama5 null mouse. Lama5 null embryos exposed to doxycycline from E6.5 died a few hours after birth. Compared to control littermates, Lama5 null mice lungs had dilated, enlarged distal airspaces, but the basement membrane ultrastructure was preserved. Distal epithelial cell differentiation was perturbed, with a marked reduction of alveolar type II cells and a virtual absence of type I cells. Cell proliferation was reduced and apoptosis was increased. Capillary density was diminished, and this was associated with a decrease in total lung VEGF production. Overall, these findings indicate that epithelial laminin α5, independent of its structural function, is necessary for murine lung development, and suggest a role for laminin α5 in signaling pathways that promote alveolar epithelial cell differentiation and VEGF expression.

Laminin α1 Chain Synthesis in the Mouse Developing Lung: Requirement for Epithelial–Mesenchymal Contact and Possible Role in Bronchial Smooth muscle Development

Schuger L., Skubitz A.P.N., Zhang J., Sorokin L., He L. The Journal of Cell Biology, 1997

The present study shows that whereas a1 and a2 laminins are synthesized in the mouse developing lung and in epithelial-mesenchymal co-cultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin α1 chain. Synthesis of laminin α1 chain, however, returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical since laminin α1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin α1 chain upon heterotypic cell-cell contact. Lung explants exposed to monoclonal antibodies to laminin α1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle a actin and desmin. Taken together, the data suggest that laminin α1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.

Identification of laminin domains involved in branching morphogenesis: Effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

Schuger L., Skubitz A.P.N, O'Shea K.S., Chang J.F., Varani J.Developmental biology, 1991

Using a series of monoclonal anti-laminin antibodies, the deposition and functional involvement of different laminin domains in the developing lung were investigated. Immunohistochemistry reactivity was largely localized to the basement membrane but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were used. Although all antibodies penetrated the tissues in culture, only laminin alpha-1 and a-5 antibodies inhibited branching activity. Laminin alpha-2, -3, and -4 antibodies did not alter lung morphogenesis.

Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: involvement of laminin

L Schuger, KS O'Shea, BB Nelson, J Varani. Development, 1990

In this article, the authors cultured single cell suspensions generated by trypsinization of lungs removed from day 12 embryos. The results strongly supports a role for the basement membrane in the organotypic rearrangement of embryonic lung cells and subsequent in vitro cyst formation and budding of the reestablished epithelium.

Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse

Both integrin α6-null mice α3-null mice die at birth, with kidney and lung defects at late stages of development, and skin blistering. To investigate possible overlapping functions between α3 and α6 integrins, we analyzed the phenotype of compound α3−/− and α6−/− mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between α3 and α6 integrins and their essential roles in multiple processes during embryogenesis.