No abstract

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

AIM: In heart failure (HF), pulmonary venous hypertension (PVH) produces pulmonary hypertension (PH) with remodeling of pulmonary veins (PV) and arteries (PA). In a porcine PVH model, we performed proteomic-based bioinformatics to investigate unique pathophysiologic mechanisms mediating PA and PV remodeling. METHODS: Large PV were banded (PVH, n= 10) or not (Sham, n=9) in piglets. At sacrifice, PV and PA were perfusion labeled for vessel specific histology and proteomics. The PA and PV were separately sampled with laser-capture micro-dissection for mass spectrometry. RESULTS: Pulmonary vascular resistance (Wood Units; 8.6 versus 2.0) and PA (19.9 versus 10.3) and PV (14.2 versus 7.6) wall thickness/external diameter (%) were increased in PVH (p<0.01 for all). Similar numbers of proteins were identified in PA (2093) and PV (2085) with 94% overlap, but biological processes differed. There were more differentially expressed proteins (287 versus 161), altered canonical pathways (17 versus 3) and predicted up-stream regulators (PUSR; 22 versus 6) in PV than PA. In PA and PV, bioinformatics indicated activation of the integrated stress response and mTOR signaling with dysregulated growth. In PV, there was also activation of Rho/Rho kinase signaling with decreased actin cytoskeletal signaling and altered tight and adherens junctions, ephrin B, and caveolar mediated endocytosis signaling; all indicating disrupted endothelial barrier function. Indeed, protein biomarkers and the top PUSR in PV (TGF-β) indicated endothelial mesenchymal transition (EndoMT) in PV. Findings were confirmed in human autopsy specimens. CONCLUSION: These findings provide new therapeutic targets to oppose pulmonary vascular remodeling in HF-related PH.