Regional variations in allergen-induced airway inflammation correspond to changes in soluble guanylyl cyclase heme and expression of heme oxygenase-1.

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However, this bronchodilation is dysfunctional in asthma due to high NO levels, which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes, we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using murine models of asthma (OVA and CFA/HDM) that the inflamed segments of these murine lungs can be tracked by upregulated expression of HO1 and these regions in turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1ß1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770, relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact on developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma, thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in turn becomes heme-free. Recognition of these specific lung segments enhances our understanding of the inflamed lungs in asthma with the ultimate aim to evaluate potential therapies and suggest that regional and not global inflammation impacts lung function in asthma.

Regions of Inflammation in mouse asthma correspond to regions of heme-free soluble guanylyl cyclase and can be tracked by marked expression of heme-oxygenase-1.

Asthma is characterized by airway remodeling and hyperreactivity. Our earlier studies determined that the Nitric Oxide (NO)-soluble Guanylyl Cyclase (sGC)-cGMP pathway plays a significant role in human lung bronchodilation. However this bronchodilation is dysfunctional in asthma due to high NO levels which cause sGC to become heme-free and desensitized to its natural activator, NO. In order to determine how asthma impacts the various lung segments/lobes we mapped the inflammatory regions of lungs to determine whether such regions coincided with molecular signatures of sGC dysfunction. We demonstrate using models of mouse asthma (OVA, CFA/HDM) that the inflammed segments of the mouse asthma lungs can be tracked by upregulated expression of HO1 and these regions in-turn overlap with regions of heme-free sGC as evidenced by a decreased sGC-α1ß1 heterodimer and an increased response to heme-independent sGC activator, BAY 60-2770 relative to naïve uninflamed regions. We also find that NO generated from iNOS upregulation in the inflamed segments has a higher impact in developing heme-free sGC as increasing iNOS activity correlates linearly with elevated heme-independent sGC activation. This excess NO works by affecting the epithelial lung hemoglobin (Hb) to become heme-free in asthma thereby causing the Hb to lose its NO scavenging function and exposing the underlying smooth muscle sGC to excess NO, which in-turn becomes heme-free. Recognition of these specific lung segments enhance our understanding of the inflammed lungs in asthma with the ultimate aim to evaluate potential therapies and suggests that regional and not global inflammation impacts lung function in asthma.

GAPDH is involved in the heme-maturation of myoglobin and hemoglobin.

GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme-maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, ß, and γ. Utilizing cell culture systems, we found that overexpression of wild-type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbß. Overexpression of wild-type GAPDH fully recovered the heme-maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCß1 but not by overexpression of a sGCΔß1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1ß1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP-1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme-maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme-maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1ß1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d-aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, ß, and α hemoglobins.

Quantification of airway fibrosis in asthma by flow cytometry.

Airway fibrosis is a prominent feature of asthma, contributing to the detrimental consequences of the disease. Fibrosis in the airway is the result of collagen deposition in the reticular lamina layer of the subepithelial tissue. Myofibroblasts are the leading cell type involved with this collagen deposition. Established methods of collagen deposition quantification present various issues, most importantly their inability to quantify current collagen biosynthesis occurring in airway myofibroblasts. Here, a novel method to quantify myofibroblast collagen expression in asthmatic lungs is described. Single cell suspensions of lungs harvested from C57BL/6 mice in a standard house dust mite model of asthma were employed to establish a flow cytometric method and compare collagen production in asthmatic and non-asthmatic lungs. Cells found to be CD45- αSMA+ , indicative of myofibroblasts, were gated, and median fluorescence intensity of the anti-collagen-I antibody labeling the cells was calculated. Lung myofibroblasts with no, medium, or high levels of collagen-I expression were distinguished. In asthmatic animals, collagen-I levels were increased in both medium and high expressers, and the number of myofibroblasts with high collagen-I content was elevated. Our findings determined that quantification of collagen-I deposition in myofibroblastic lung cells by flow cytometry is feasible in mouse models of asthma and indicative of increased collagen-I expression by asthmatic myofibroblasts. © 2018 International Society for Advancement of Cytometry.

Bone marrow transplantation prevents right ventricle disease in the caveolin-1-deficient mouse model of pulmonary hypertension.

Accumulating evidence shows a causative role for the bone marrow (BM) in the genesis and progression of pulmonary hypertension (PH). Engraftment of BM hematopoietic stem cells from PH patients to mice reproduces the cardiopulmonary pathology of PH. However, it is unknown whether healthy BM can prevent the development of right heart disease. Caveolin-1-deficient (CAV-1 KO) mice develop cardiopulmonary disease with manifestations resembling PH, including elevated right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary endothelial proliferative disease. Here, we hypothesize that engraftment of healthy BM to CAV-1 KO mice will prevent pulmonary vascular remodeling and development of the cardiopulmonary disease. CAV-1 KO mice and wild-type (WT) mice underwent transplantation with WT or CAV-1 KO BM. Hematopoietic differentiation was analyzed by flow cytometry. Pulmonary endothelial remodeling was quantified by CD31 image analysis. RVSP and RV cardiomyocyte area or Fulton's index were used to analyze RV hypertrophy. Maladaptive RV hypertrophy was determined by quantification of RV fibrosis. Transplantation of CAV-1 KO BM into healthy recipient WT mice led to elevation of RVSP, RV hypertrophy, and pulmonary endothelial remodeling. Reconstitution of CAV-1 KO with WT BM prevented spontaneous development of PH, including elevation of RVSP and maladaptive RV hypertrophy, but not pulmonary endothelial remodeling. Healthy BM has a protective role in the right ventricle independent of pulmonary vascular disease.