Research Focus

Peter Bove, PhD

Characterizing the functional ion/liquid transport properties of human primary alveolar type 2 cells

Primary human AT2 cell under  ALI conditions.

The alveolus is comprised of alveolar type 1 (AT1) and type 2 (AT2) cells. Although less is known regarding the active role of AT1 ion channel physiology, studies have demonstrated the contribution of active ion/liquid movement across AT2 cells. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Recently published work (Bove et al. JBC 2010) demonstrated that isolated human alveolar type II (AT2) cells exhibited both epithelial Na (+) channel-mediated Na (+) absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl (-) secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na(+) channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ∼4 μm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.

Ion/liquid transport properties using cystic fibrosis derived alveolar type 2 cells

Alveolar surface liquid regulation: Effect of nucleotides and CFTR.

LPreviously, we demonstrated the significant contribution of CFTR with respect to Cl-secretion in normal AT2 cultures. Therefore, in our current studies, we are able to focus on the direct role of CFTR in human AT2 cells by using, first time to date, AT2 cells derived and cultured from lungs of CF patients lacking functional CFTR. Interestingly, we are able to confirm lack of basal CFTR activity in these cells, as well as, a lack of nucleotides to increase potential difference and short circuit current. Moreover, what we observe is that nucleotides also are able to inhibit ENaC in AT2 cells in the absence of CFTR, a finding significantly different from human airway epithelial (HBE) cells. In addition, no difference is observed within basal PD or short circuit current within normal and CF AT2 cells, as well as no marked increase in ENaC activity, an observation demonstrated in CF HBE cells. Moreover, when imaging AT2 alveolar surface liquid (AvSL) height by confocal microscopy, we observe no difference in height compared to normal AT2 cells, however, the increase observed using normal AT2 cells exposed to nucleotides or forskolin are completely inhibited in CF AT2 cells, pointing to the role of CFTR regulation by these mediators. When subjecting CF AT2 cells to a phasic motion stress, which triggers the release of endogenous nucleotides from AT2 cell, there is an increase in AvSL height using normal AT2 cells, which was inhibited in normal cells pretreated with apyrase, or using CF AT2 cells. Collectively, we strongly believe that CF airway epithelial cells and AT2 cells differ greatly in vitro which may potentially shed light on the drastic difference in phenotype observed in CF.

Propagation and expansion of human primary alveolar type 2 cells while retaining ion/liquid transport properties

Expanding primary human AT2 cells using feeder cells and “Y”. (A) Schema illustrates the recently developed co-culture. (B) AT2 cells (2.5x105) were grown with base media alone (i), “Y” (10µM) (ii), feeder cells (7.5x105) (iii), or with combination of feeder cells + “Y” (iv), and imaged 4 days post seeding. (C) Fresh AT2 cells were grown on rat tail collagen-1 coated plastic dishes with base media alone (i), or with feeder cells and “Y” (ii) and imaged 10 days post seeding. Cells were trypsinized and further passaged with feeder cells + “Y” (p1-p3; iii-v). Total cell count (D) was recorded for AT2 cells plated alone (final AT2) or in combination with feeder cells + “Y” (final F+Y), and a plot of population doublings versus time (days) was constructed (E). All images were taken using phase contrast microscopy under 20X objective. Values are mean ± SD; n > 4 different lungs.

The alveolar type 1 (AT1) and type 2 (AT2) cells line the interfacial surface of the alveolus. These cells are central to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndromes (ARDS) where they are targets for disruption, altered permeability, and inflammation. Unfortunately, research focused on AT1 and AT2 cells in health and disease has been limited by the inability to expand primary cells in vitro in order to study cell specific functional properties. For instance, primary AT2 cells in vitro do not proliferate as well as quickly transdifferentiate into “AT1-like” cells under standard culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of feeder cells and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation and the cells resembled an adult stem cell-like phenotype. Utilizing this newly developed co-culture system, we identified that human primary AT2 cells: 1) proliferate at an exponential rate; 2) establish epithelial colonies that expressed both AT2-specificand “AT1-like” transdifferentitated mRNA and proteins after serial passage; 3) up regulate genes important in cell proliferation and migration; and 4) exhibit ion/liquid transport characteristics upon removal of feeder cells and Rho kinase inhibitor similar to fresh AT2 cells cultured under air-liquid interface conditions.

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Biographical Sketch

Peter F. Bove, PhD, Research Associate,
Co-Director of the UNC CF Center Correction Core

2001-2006: Ph.D. Cell and Molecular Biology
University of Vermont, Burlington, VT
1993-1998: B.S. Biochemestry and Biophysics
Rensselaer Polytechnic Institute, Troy, NY

Professional Experience

2012-present: Co-Director, CFTR Correction Core
Cystic Fibrosis/Pulmonary Research & Treatment Center
University of North Carolina, Chapel Hill, NC

2011-present: Research Associate;
Cystic Fibrosis/Pulmonary Research & Treatment Center
University of North Carolina, Chapel Hill, NC
2007-2011: Postdoctoral Research Associate
Cystic Fibrosis/Pulmonary Research & Treatment Center
University of North Carolina, Chapel Hill, NC
1998-2001: Analytical Associate I/II
Biogen, Idec, Cambridge, MA

Grants

2013 Cystic Fibrosis Foundation

Awards/Patents

2009-2011: NIH Clinical Loan Repayment Program (LRP) Grant Recipient
National Heart, Lung, and Blood Institute (NHLBI)
2005: “Young Scientific Investigator” Award Recipient
International Free Radical Biology and Medicine Society Conference, Austin, TX
2004: “Science Travel Award” Recipient
International Free Radical Biology and Medicine Society Conference, St. Thomas, VI
2003-2006: Pathology Department Fellowship
University of Vermont, Burlington, Vermont, USA
2002: “Detecting Half-Antibodies Using Chip-Based Gel Electrophoresis”
Patent with Biogen Idec, Cambridge, MA, USA

Publications

Please see PubMed feed in the right-hand column for links to current publications.

Contact Information

6109A Thurston-Bowles Bldg.
The University of North Carolina at Chapel Hill
Campus Box #7248
Chapel Hill, NC 27599
Phone: (919) 966-9142

Email: peter_bove@med.unc.edu

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