Animal Models in the Pathophysiology of Cystic Fibrosis

Our understanding of the multiorgan pathology of cystic fibrosis (CF) has improved impressively during the last decades, but we still lack a full comprehension of the disease progression. Animal models have greatly contributed to the elucidation of specific mechanisms involved in CF pathophysiology and the development of new therapies. Soon after the cloning of the CF transmembrane conductance regulator (CFTR) gene in 1989, the first mouse model was generated and this model has dominated in vivo CF research ever since. Nonetheless, the failure of murine models to mirror human disease severity in the pancreas and lung has led to the generation of larger animal models such as pigs and ferrets. The following review presents and discusses data from the current animal models used in CF research

Anti-Inflammatory Effects of the Iron Chelator, DIBI, in Experimental Acute Lung Injury

Iron plays a critical role in the immune response to inflammation and infection due to its role in the catalysis of reactive oxygen species (ROS) through the Haber-Weiss and Fenton reactions. However, ROS overproduction can be harmful and damage healthy cells. Therefore, iron chelation represents an innovative pharmacological approach to limit excess ROS formation and the related pro-inflammatory mediator cascades. The present study was designed to investigate the impact of the iron chelator, DIBI, in an experimental model of LPS-induced acute lung injury (ALI). DIBI was administered intraperitoneally in the early and later stages of lung inflammation as determined by histopathological evaluation. We found that lung tissues showed significant injury, as well as increased NF-κB p65 activation and significantly elevated levels of various inflammatory mediators (LIX, CXCL2, CCL5, CXCL10, IL-1, IL-6) 4 h post ALI induction by LPS. Mice treated with DIBI (80 mg/kg) in the early stages (0 to 2 h) after LPS administration demonstrated a significant reduction of the histopathological damage score, reduced levels of NF-κB p65 activation, and reduced levels of inflammatory mediators. Intravital microscopy of the pulmonary microcirculation also showed a reduced number of adhering leukocytes and improved capillary perfusion with DIBI administration. Our findings support the conclusion that the iron chelator, DIBI, has beneficial anti-inflammatory effects in experimental ALI

Phosphorylation of CFTR by PKA promotes binding of the regulatory domain

The unphosphorylated regulatory (R) domain of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is often viewed as an inhibitor that is released by phosphorylation. To test this notion, we studied domain interactions using CFTR channels assembled from three polypeptides. Nucleotides encoding the R domain (aa 635–836) were replaced with an internal ribosome entry sequence so that amino- and carboxyl-terminal half-molecules would be translated from the same mRNA transcript. Although only core glycosylation was detected on SplitΔR, biotinylation, immunostaining, and functional studies clearly demonstrated its trafficking to the plasma membrane. SplitΔR generated a constitutive halide permeability, which became responsive to cAMP when the missing R domain was coexpressed. Each half-molecule was co-precipitated by antibody against the other half. Contrary to expectations, GST-R domain was pulled down only if prephosphorylated by protein kinase A, and coexpressed R domain was precipitated with SplitΔR much more efficiently when cells were stimulated with cAMP. These results indicate that phosphorylation regulates CFTR by promoting association of the R domain with other domains rather than by causing its dissociation from an inhibitory site

Lung-protective ventilation attenuates mechanical injury while hypercapnia attenuates biological injury in a rat model of ventilator-associated lung injury

$\textbf {Background and Objective:}$ Lung-protective mechanical ventilation is known to attenuate ventilator-associated lung injury (VALI), but often at the expense of hypoventilation and hypercapnia. It remains unclear whether the main mechanism by which VALI is attenuated is a product of limiting mechanical forces to the lung during ventilation, or a direct biological effect of hypercapnia. $\bf Methods:$ Acute lung injury (ALI) was induced in 60 anesthetized rats by the instillation of 1.25 M HCl into the lungs via tracheostomy. Ten rats each were randomly assigned to one of six experimental groups and ventilated for 4 h with: 1) \textbf {Conventional \(HighV_{E} Normocapnia}\) (high $V_{T}$, high minute ventilation, normocapnia), 2) $\textbf {Conventional Normocapnia}$ (high $V_{T}$, normocapnia), 3) $\textbf {Protective Normocapnia}$ ($V_{T}$ 8 ml/kg, high RR), 4) \textbf {Conventional \(iCO_{2} Hypercapnia}\) (high $V_{T}$, low RR, inhaled $CO_{2}$), 5) \textbf {Protective \(iCO_{2} Hypercapnia}\) ($V_{T}$ 8 ml/kg, high RR, added $CO_{2}$), 6) $\textbf {Protective endogenous Hypercapnia}$ ($V_{T}$ 8 ml/kg, low RR). Blood gasses, broncho-alveolar lavage fluid (BALF), and tissue specimens were collected and analyzed for histologic and biologic lung injury assessment. $\bf Results:$ Mild ALI was achieved in all groups characterized by a decreased mean $PaO_{2}/FiO_{2}$ ratio from 428 to 242 mmHg ($\it p$ < 0.05), and an increased mean elastance from 2.46 to 4.32 $cmH_{2}O$/L ($\it p$ < 0.0001). There were no differences in gas exchange among groups. Wet-to-dry ratios and formation of hyaline membranes were significantly lower in low $V_{T}$ groups compared to conventional tidal volumes. Hypercapnia reduced diffuse alveolar damage and IL-6 levels in the BALF, which was also true when $CO_{2}$ was added to conventional $V_{T}$. In low $V_{T}$ groups, hypercapnia did not induce any further protective effect except increasing pulmonary IL-10 in the BALF. No differences in lung injury were observed when hypercapnia was induced by adding $CO_{2}$ or decreasing minute ventilation, although permissive hypercapnia decreased the pH significantly and decreased liver histologic injury. $\bf Conclusion:$ Our findings suggest that low tidal volume ventilation likely attenuates VALI by limiting mechanical damage to the lung, while hypercapnia attenuates VALI by limiting pro-inflammatory and biochemical mechanisms of injury. When combined, both lung-protective ventilation and hypercapnia have the potential to exert an synergistic effect for the prevention of VALI

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues