Pathophysiology Of Cystic Fibrosis Research Articles

Evidence of Elevated CXCR2 Agonists in Stable Outpatients with Cystic Fibrosis Compared with Healthy Controls

Introduction: Recruitment and activation of inflammatory cells contributes to the pathophysiology of Cystic Fibrosis (CF). Recruitment is initiated, in part, by engagement of the CXCR2 receptor on neutrophils and other inflammatory cells. Materials and Methods: Levels of CXCR2 agonists (CXCL 1, 3, 5, 7, 8), and systemic biomarkers of inflammation (fibrinogen, CC-16, SP-D, IL-1β, and MMP-9) were measured in the serum of stable outpatients with cystic fibrosis compared to normal healthy age-, gender, and racially-matched non-smoking control subjects. Secondary objectives included characterization of pulmonary function assessed by spirometry, Impulse Oscillometry (IOS), R5-R15 peripheral resistance, R5 total resistance and R25 large airway resistance; frequency dependent reactance as indicators of reactive capacitance properties of the lung (X5, resonant frequency, Fres, AX) in patients with CF compared with controls. Health outcomes scores in patients with CF were assessed using the Cystic Fibrosis Questionnaire. Population: Single center, prospective, two visit study. 48 subjects were enrolled. Forty-two subjects (28 patients with CF and 14 healthy controls matched 2:1 (age, gender, and race) were evaluable. Results: Blood fibrinogen levels in CF patients were significantly elevated compared with controls (3.9 g/L vs. 3.0 g/L [p=0.0004]); similar differences were seen in levels of CXCL8 (CF patients=10.2 pg/mL and controls=5.9 pg/mL [p=0.0065]). A trend was noted for CC-16, CXCL1, and SP-D. As expected, lung functions assessed by spirometry and IOS values were significantly different in the patient population compared to the control group. Mean FEV1 and mean reactance were 2.4L (67% predicted) vs. 3.5L (99% predicted) and 0.174 kPa/(L/sec) vs. –0.085 kPa/(L/sec) in CF patients vs, controls, respectively(p=0.0002 and p<0.0001)). No age or gender trends were noted among CF patients or controls. Conclusions: The study demonstrated elevation of CXCR2 agonists in stable patients with CF. We also confirmed the potential utility of IOS, an effort-independent lung function test. Finally, the study suggests that subject matching for age and gender may not be necessary in future CF trials assessing these biomarkers.

Iron accumulates in the lavage and explanted lungs of cystic fibrosis patients

Oxidative stress participates in the pathophysiology of cystic fibrosis (CF). An underlying disruption in iron homeostasis can frequently be demonstrated in injuries and diseases associated with an oxidative stress. We tested the hypothesis that iron accumulation and altered expression of iron-related proteins could be demonstrated in both the bronchoalveolar lavage (BAL) fluid and explanted lungs of patients with cystic fibrosis. BAL fluid collected from 10 children with CF showed elevated concentrations of protein, iron, ferritin, transferrin, heme, and hemoglobin relative to that obtained from 20 healthy volunteers. Using Perl's Prussian blue staining, explanted lung from CF patients revealed increased iron in alveolar and interstitial macrophages. Similarly, there was an increased expression of ferritin, the iron importer DMT1, and the exporter ferroportin 1 in lung tissue from CF patients. We conclude that iron homeostasis is disrupted in CF patients with an accumulation of this metal and altered expression of iron-related proteins being evident in the lungs.

NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway

Tight junctions (TJs) restrict the transit of ions and molecules through the paracellular route and act as a barrier to regulate access of inflammatory cells into the airway lumen. The pathophysiology of cystic fibrosis (CF) lung disease is characterised by abnormal ion and fluid transport across the epithelium and polymorphonuclear (PMN) leukocyte-dominated inflammatory response. Na+/H+ exchanger regulatory factor 1 (NHERF1) is a protein involved in PKA-dependent activation of CFTR by interacting with CFTR via its PDZ domains and with ezrin via its C-terminal domain. We have previously found that the NHERF1-overexpression dependent rescue CFTR-dependent chloride secretion is due to the re-organisation of the actin cytoskeleton network induced by the formation of the multiprotein complex NHERF1–RhoA–ezrin–actin. In this context, we here studied whether NHERF1 and CFTR are involved in the organisation and function of TJs. F508del CFBE41o− monolayers presented nuclear localisation of zonula occludens (ZO-1) and occludin as well as disorganisation of claudin 1 and junction-associated adhesion molecule 1 as compared with wild-type 16HBE14o− monolayers, paralleled by increased permeability to dextrans and PMN transmigration. Overexpression of either NHERF1 or CFTR in CFBE41o− cells rescued TJ proteins to their proper intercellular location and decreased permeability and PMN transmigration, while this effect was not achieved by overexpressing either NHERF1 deprived of ezrin-binding domain. Further, expression of a phospho-dead ezrin mutant, T567A, increased permeability in both 16HBE14o− cells and in a CFBE clone stably overexpressing NHERF1 (CFBE/sNHERF1), whereas a constitutively active form of ezrin, T567D, achieved the opposite effect in CFBE41o− cells. A dominant-negative form of RhoA (RhoA-N19) also disrupted ZO-1 localisation at the intercellular contacts dislodging it to the nucleus and increased permeability in CFBE/sNHERF1. The inhibitor Y27632 of Rho kinase (ROCK) increased permeability as well. Overall, these data suggest a significant role for the multiprotein complex CFTR–NHERF1–ezrin–actin in maintaining TJ organisation and barrier function, and suggest that the RhoA/ROCK pathway is involved.

Dornase alfa (Pulmozyme)

Dornase alfa (Pulmozyme) is one of the most commonly used medications to treat the lung disease of cystic fibrosis (CF). As other respiratory medications have entered the clinical market, understanding the proper use and indication for dornase alfa is increasingly important. In addition, dornase alfa is being used to treat other medical conditions. This review covers recent publications and expanding indications. Understanding dornase alfa's mechanism of action and impact on the pathophysiology of CF leads to an improved understanding of optimal therapy, ways to improve adherence and use with other medications. Most importantly, routine use of dornase alfa is associated with improved lung function and survival in patients with CF. Outside of CF, potential uses include treating patients with empyema or on mechanical ventilation. Dornase alfa has been available for clinical use for nearly 20 years and is one of the most commonly used medications in patients with CF. Routine use is associated with a reduced rate of pulmonary deterioration and improved survival. Recent clinical reports suggest that dornase alfa may have clinical value with other medical problems such as complicated pneumonia and mechanically ventilated patients with atelectasis.

A mutation in the β-subunit of ENaC identified in a patient with cystic fibrosis-like symptoms has a gain-of-function effect

In some patients with atypical cystic fibrosis (CF), only one allele of the CF transmembrane conductance regulator (CFTR) gene is affected. Mutations of the epithelial sodium channel (ENaC) may contribute to the pathophysiology of the disease in these patients. To functionally characterize a mutation in the β-subunit of ENaC (βV348M) recently identified in a patient with severe CF-like symptoms (Mutesa et al. 2009), we expressed wild-type (wt) αβγENaC or mutant αβV348MγENaC in Xenopus laevis oocytes. The βV348M mutation stimulated amiloride-sensitive whole-cell current (ΔI(ami)) by ∼40% but had no effect on surface expression or single-channel conductance of ENaC. Instead the mutation increased channel open probability (P(o)). Proteolytic activation of mutant ENaC by chymotrypsin was reduced compared with that of wt ENaC (∼3.0-fold vs. ∼4.2-fold), which is consistent with the increased baseline P(o) of mutant ENaC. Similarly, the ENaC activator S3969 stimulated mutant ENaC currents to a lesser degree (by ∼2.6-fold) than wt ENaC currents (by ∼3.5-fold). The gain-of-function effect of the βV348M mutation was confirmed by whole-cell current measurements in HEK293 cells transiently transfected with wt or mutant ENaC. Computational channel modeling in combination with functional expression of different βV348 mutants in oocytes suggests that the βV348M mutation increases channel P(o) by destabilizing the closed channel state. Our findings indicate that the gain-of-function effect of the βV348M mutation may contribute to CF pathophysiology by inappropriately increasing sodium and fluid absorption in the respiratory tract.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Analiza genotypowa oraz profil lekowrażliwości szczepów Staphylococcus aureus izolowanych od dzieci chorych na mukowiscydozę

BackgroundThe pathophysiology of cystic fibrosis (CF) is associated with genetically determined abnormal chloride conductance on the apical membrane of the epithelial cell. As a result favourable environment for bacterial and fungal infections is maintained. Respiratory tract infections in CF patients are caused mainly by Staphylococcus aureus, Haemphilus influenzae, and Pseudomonas aeruginosa which is the most destructive pulmonary pathogen. ObjectivesThe aim of this study was to analyse genetic diversity and antimicrobial susceptibility of Staphylococcus aureus strains isolated from children with cystic fibrosis. Material and methodsA total of 21 patients with confirmed cystic fibrosis were included to the study. Within 8 years 257 specimens from the respiratory tract were collected, and 201 isolates of S. aureus were cultured. Genotyping was performed using the pulsed field gel electrophoresis (PFGE) technique, and antimicrobial susceptibility was determined by the E-test method. The following data were retrieved from medical records: age at CF diagnosis, clinical signs and symptoms, respiratory function (FEV1), frequency of hospitalization and antibiotic use. Results and conclusionsThe mean age of patients studied was 7 years (range 0.5–16 years). Over 80% of patients had pulmonary manifestations. The genotyping confirmed that Staphylococcus aureus persistently colonized the respiratory tract of patients with CF. For over 95% of strains the colonization lasted from 6 months to 8 years. Less than 5% of strains did not show a tendency for a long-term colonization, and they were isolated only once. In 34% of patients the same genotype was isolated through the study period, and it could not be eradicated despite antibiotic treatment. As many as 62% of children were persistently colonized with 2 or 3 strains simultaneously. Over 94% of strains were susceptible to β-lactams, fluoroquinolones, trimethoprim/sulfamethoxazole, aminoglycosides, and glycopeptides. The highest rates of resistance were observed for macrolides (23%) which were the second most commonly used antibiotics in bronchopulmonary exacerbations. Some of S. aureus strains were effectively transmitted among the patients. However, only methicillin-susceptible isolates (MSSA) were involved in the transmission. MRSA transmission was not detected.

Cystic fibrosis in an era of genomically guided therapy

Although affecting only 4-5% of those with cystic fibrosis (CF), the G551D-CFTR mutation is the target of the recently approved 'orphan drug', ivacaftor. The promise of such genomically guided therapies heralds a new era in the management of CF. A phase 3 trial demonstrated significant improvements in forced expiratory volume in 1 s (FEV(1)) from baseline, average weight gain, concentration in sweat chloride and reductions in pulmonary exacerbations [Ramsey, B.W., et al. A CFTR potentiator in patients with CF and the G551D mutation. N. Engl. J. Med., 2011. 365: 1663-1672.)]. Ivacaftor is among a group of recently approved, novel, mutation guided 'orphan drug' therapies that have established clinical benefits within their respective disease categories. They do not, however, offer a cure. Pharmaceutical and biotech companies have leveraged the incentivized benefits of the Orphan Drug Act to develop more of these drugs for orphan disorders affecting populations of <200 000 patients. With marked clinical efficacy via DNA sequence guidance, these drugs have also set a precedent in terms of the substantial annual costs and if this trend continues, such expenditures may become unsustainable. This paper explores the genomic pathophysiology of CF and how therapies such as ivacaftor provide benefit to those with the disease but at a considerably elevated price point.

Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S

Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.

147 Comparative analysis of inflammatory markers in cystic fibrosis and non-cystic fibrosis bronchiectasis

Objetives: Oxidative stress is believed to play an important role in the pathophysiology of cystic fibrosis (CF). Many factors may contribute to increased oxidative stress in CF, as the disease combines the increased production of reactive oxygen species (ROS) with impaired antioxidant protection. The aim, therefore, of this study was to evaluate oxidative stress using different biomarkers (both plasma and intracellular in peripheral blood leukocytes) in a group of clinically stable adult patients with bronchiectasis (CF and non-CF) and compare the results with a group of healthy controls. Methods: This cross-sectional study included 90 patients with a diagnosis of bronchiectasis with and without CF, periodically monitored in the adult bronchiectasis/CF unit at a university hospital. A control group comprised 50 healthy subjects, matched with respect to patient sex, age and nutritional status. The intracellular oxidative stress biomarkers (mitochondrial membrane potential, superoxide anion and hydrogen peroxide and intracellular glutathione) were analyzed in white blood cells as total leukocytes, neutrophils, lymphocytes and monocytes. The plasma oxidative stress biomarkers (total antioxidant capacity, glutathione peroxidase activity, superoxide dismutase activity, catalase activity, 8-iso-prostaglandin F2a, TBARs) were measured. Conclusions: Cell and plasma markers of oxidative stress were raised in the patients with bronchiectasis as compared with the controls and no differences depending on the cause of the bronchiectasis (CF vs non-CF) were found.

NMR spectroscopy metabolomic profiling of exhaled breath condensate in patients with stable and unstable cystic fibrosis

BackgroundMetabolomics could provide new insights into the pathophysiology of cystic fibrosis (CF) by identifying profiles of endogenous metabolites.ObjectivesTo investigate whether metabolomics of exhaled breath condensate could discriminate between patients with...

New animal models of cystic fibrosis

Cystic fibrosis is the first human genetic disease to benefit from the directed engineering of three different species of animal models (mice, pigs, and ferrets). Recent studies on the cystic fibrosis pig and ferret models are providing new information about the pathophysiology of cystic fibrosis in various organ systems. Additionally, new conditional cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice are teaching unexpected lessons about CFTR function in surprising cellular locations. Comparisons between these animal models and the human condition are key to dissecting the complexities of disease pathophysiology in cystic fibrosis. Cystic fibrosis pigs and ferrets have provided new models to study the spontaneous development of disease in the lung and pancreas, two organs that are largely spared overt spontaneous disease in cystic fibrosis mice. New cystic fibrosis mouse models are now interrogating CFTR functions involved in growth and inflammation at an organ-based level using conditional knockout technology. Together, these models are providing new insights on the human condition. Basic and clinical cystic fibrosis research will benefit greatly from the comparative pathophysiology of cystic fibrosis mice, pigs, and ferrets. Both similarities and differences between these three cystic fibrosis models will inform pathophysiologically important mechanisms of CFTR function in humans and aid in the development of both organ-specific and general therapies for cystic fibrosis.

Ion channel regulators for the treatment of cystic fibrosis

Cystic fibrosis is a life-shortening hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. The main clinical manifestations are chronic infections of the airways, malabsorption due to pancreatic insufficiency, male infertility and salt loss syndromes. The cystic fibrosis transmembrane conductance regulator protein functions as a chloride channel in the apical cell membrane and is thought to inhibit the epithelial sodium channel. In the lung, absence or dysfunction of the cystic fibrosis transmembrane conductance regulator protein leads to dehydration of the airway surface liquid and disturbed mucociliary clearance. After decades of symptomatic therapy to combat the relentless disease progression, there is now hope that interference with the apical ion channel functions will bring a major improvement in the disease course. We discuss the ion channel regulators currently under clinical evaluation in cystic fibrosis, as well as some of the challenges associated with finding disease-modifying therapies. In this article we give a brief description of the clinical and epidemiological aspects of the disease cystic fibrosis. We discuss the pathophysiology of cystic fibrosis, the ion channel regulators under clinical development as potential disease-modifying therapies and some of the challenges associated with developing groundbreaking therapies for this orphan disease.

Defective Fluid Secretion from Submucosal Glands of Nasal Turbinates from CFTR-/- and CFTRΔF508/ΔF508 Pigs

BackgroundCystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion.Methodology/Principal FindingsUnexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CFΔF508/ΔF508 with CFTR-/- piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands.Conclusions/SignificanceThese findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections.

Impaired expression of hypoxia-inducible factor-1α in cystic fibrosis airway epithelial cells – A role for HIF-1 in the pathophysiology of CF?

The continuous infection–inflammation cycle plays a crucial role in the progression of cystic fibrosis (CF) disease. This noxious loop can be aggravated by a reduced partial pressure of oxygen in the blood, hypoxemia, present in CF patients. These interconnected factors, hypoxia, inflammation and infection, by stabilizing the hypoxia-inducible factor-1α (HIF-1α) protein subunit, are able to activate the transcription factor HIF-1. To date, data investigating the potential role of HIF-1 in CF are scarce. Our results demonstrated that HIF-1α protein expression was altered in CF-affected compared to CFTR-corrected airway epithelial cells in unsimulated and simulated hypoxic conditions. In contrast, when CF-affected cells were infected with Pseudomonas aeruginosa, HIF-1α was more stabilized compared to CFTR-corrected cells. As HIF-1 is linked with an efficient immune response and pulmonary complications in cystic fibrosis, this difference in HIF-1α protein levels could have an impact in the CF pathology and the persistence of P. aeruginosa infection.

Evolution of pulmonary inflammation and nutritional status in infants and young children with cystic fibrosis

IntroductionImproved nutrition is the major proven benefit of newborn screening programmes for cystic fibrosis (CF) and is associated with better clinical outcomes. It was hypothesised that early pulmonary inflammation and...

CFTR-dependent susceptibility of the cystic fibrosis-host to Pseudomonas aeruginosa

Cystic fibrosis is the most common autosomal recessive disorder in western countries. The disease is characterized by recurrent and chronic infections of the lung in particular with Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, and Haemophilus influenzae. Albeit intensive research in the last years, the molecular mechanisms causing the high susceptibility of cystic fibrosis patients to bacterial infections are still unknown. Animal models provided important novel information on the pathophysiology of cystic fibrosis and mimicked many of the pathological findings in humans, for instance chronic inflammation and increased infection susceptibility. These animal models were recently employed to identify several proteins and lipids that are critically involved in the pathophysiology of cystic fibrosis. Thus, several studies identified death receptors, caveolin proteins, membrane rafts, and alterations of the ceramide metabolism with an accumulation of ceramide in cystic fibrosis lungs to be critically involved in the infection susceptibility, the chronic inflammation, and the reduced mucociliary clearance in cystic fibrosis.

Airway surface liquid depth imaged by surface laser reflectance microscopy

The thin layer of liquid at the surface of airway epithelium, the airway surface liquid (ASL), is important in normal airway physiology and in the pathophysiology of cystic fibrosis. At present, the best method to measure ASL depth involves scanning confocal microscopy after staining with an aqueous-phase fluorescent dye. We describe here a simple, noninvasive imaging method to measure ASL depth by reflectance imaging of an epithelial mucosa in which the surface is illuminated at a 45-degree angle by an elongated 13-µm wide rectangular beam produced by a 670-nm micro-focus laser. The principle of the method is that air–liquid, liquid–liquid, and liquid–cell interfaces produce distinct specular or diffuse reflections that can be imaged to give a micron-resolution replica of the mucosal surface. The method was validated using fluid layers of specified thicknesses and applied to measure ASL depth in cell cultures and ex vivo fragments of pig trachea. In addition, the method was adapted to measure transepithelial fluid transport from the dynamics of fluid layer depth. Compared with confocal imaging, ASL depth measurement by surface laser reflectance microscopy does not require dye staining or costly instrumentation, and can potentially be adapted for in vivo measurements using fiberoptics.

Linoleic acid supplement in cystic fibrosis: friend or foe?

Linoleic acid supplement in cystic fibrosis: friend or foe?

Applications of proteomic technologies for understanding the premature proteolysis of CFTR

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-dependent anion channel. Disease-causing mutations can affect channel biogenesis, trafficking or function, and result in reduced ion transport at the apical surface of many tissues. The most common CFTR mutation is a deletion of phenylalanine at position 508 (ΔF508), which results in a misfolded protein that is prematurely targeted for degradation. This article focuses on how proteomic approaches have been utilized to explore the mechanisms of premature proteolysis in CF. Additionally, we emphasize the potential for proteomic-based technologies in expanding our understanding of CF pathophysiology and therapeutic approaches.