Circulating biomarker analyses in a longitudinal cohort of patients with IPF.

Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease characterized by fibrosis. Two FDA-approved drugs, pirfenidone and nintedanib, only modestly prolong survival. In this study, we asked whether levels of select circulating biomarkers in patients with IPF demonstrated changes in response to treatment over time and whether treatment with pirfenidone and nintedanib led to differential biomarker expression. Serial plasma samples from 48 patients with IPF on usual treatment and six healthy volunteers were analyzed to identify differentially expressed blood protein. Hypothesis-driven potential biomarker selection was based on recent literature, internal preclinical data, and the PROLIFIC Consortium (Schafer P. 6th Annual IPF Summit. Boston, MA, 2022) proposed biomarkers of pulmonary fibrosis. We compared our findings to public databases to provide insights into relevant signaling pathways in IPF. Of the 26 proteins measured, we found that 11 (SP-D, TIMP1, MMP7, CYFRA21-1, YKL40, CA125, sICAM, IP-10, MDC, CXCL13) were significantly elevated in patients with IPF compared with healthy volunteers but their levels did not significantly change over time. In the IPF samples, seven proteins were elevated in the treatment group compared with the no-treatment group. However, protein profiles were not distinguishable between patients on pirfenidone versus nintedanib. We demonstrated that most proteins differentially detected in our samples were predicted to be secreted from the lung epithelial or interstitial compartments. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. Understanding the contributions of the systemic response in IPF may be important as new therapeutics are developed.NEW & NOTEWORTHY In this study, we confirmed protein expression differences in only a subset of predicted biomarkers from IPF and control subjects. Most differentially expressed proteins were predicted to be secreted from lung cells. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. The contributions of the systemic response in IPF may be important as new therapeutics are developed.

Proteomic Correlates of the Urinary Protein/Creatinine Ratio in Heart Failure With Preserved Ejection Fraction.

Proteinuria is common in heart failure with preserved ejection fraction (HFpEF), but its biologic correlates are poorly understood. We assessed the relation between 49 plasma proteins and the urinary protein/creatinine ratio (UPCR) in 365 participants in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial. Linear regression and network analysis were used to represent relations between protein biomarkers and UPCR. Higher UPCR was associated with older age, a greater proportion of female gender, smaller prevalence of previous myocardial infarction, and greater prevalence of diabetes, insulin use, smoking, and statin use, in addition to a lower estimated glomerular filtration rate, hematocrit, and diastolic blood pressure. Growth differentiation factor 15 (GDF-15; ß = 0.15, p <0.0001), followed by N-terminal proatrial natriuretic peptide (NT-proANP; ß = 0.774, p <0.0001), adiponectin (ß = 0.0005, p <0.0001), fibroblast growth factor 23 (FGF-23, ß = 0.177; p <0.0001), and soluble tumor necrosis factor receptors I (ß = 0.002, p <0.0001) and II (ß = 0.093, p <0.0001) revealed the strongest associations with UPCR. Network analysis showed that UPCR is linked to various proteins primarily through FGF-23, which, along with GDF-15, indicated node characteristics with strong connectivity, whereas UPCR did not. In a model that included FGF-23 and UPCR, the former was predictive of the risk of death or heart-failure hospital admission (standardized hazard ratio 1.83, 95% confidence interval 1.49 to 2.26, p <0.0001) and/or all-cause death (standardized hazard ratio 1.59, 95% confidence interval 1.22 to 2.07, p = 0.0005), whereas UPCR was not prognostic. Proteinuria in HFpEF exhibits distinct proteomic correlates, primarily through its association with FGF-23, a well-known prognostic marker in HFpEF. However, in contrast to FGF-23, UPCR does not hold independent prognostic value.

Chronic Expression of a Clinical SFTPC Mutation Causes Murine Lung Fibrosis with Idiopathic Pulmonary Fibrosis Features.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease. A barrier to developing more effective therapies for IPF is the dearth of preclinical models that recapitulate the early pathobiology of this disease. Intratracheal bleomycin, the conventional preclinical murine model of IPF, fails to reproduce the intrinsic dysfunction to the alveolar epithelial type 2 cell (AEC2) that is believed to be a proximal event in the pathogenesis of IPF. Murine fibrosis models based on SFTPC (Surfactant Protein C gene) mutations identified in patients with interstitial lung disease cause activation of the AEC2 unfolded protein response and endoplasmic reticulum stress-an AEC2 dysfunction phenotype observed in IPF. Although these models achieve spontaneous fibrosis, they do so with precedent lung injury and thus are challenged to phenocopy the general clinical course of patients with IPF-gradual progressive fibrosis and loss of lung function. Here, we report a refinement of a murine Sftpc mutation model to recapitulate the clinical course, physiological impairment, parenchymal cellular composition, and biomarkers associated with IPF. This platform provides the field with an innovative model to understand IPF pathogenesis and index preclinical therapeutic candidates.

Plasma biomarkers associated with adverse outcomes in patients with calcific aortic stenosis.

AIMS: Enhanced risk stratification of patients with aortic stenosis (AS) is necessary to identify patients at high risk for adverse outcomes, and may allow for better management of patient subgroups at high risk of myocardial damage. The objective of this study was to identify plasma biomarkers and multimarker profiles associated with adverse outcomes in AS. METHODS AND RESULTS: We studied 708 patients with calcific AS and measured 49 biomarkers using a Luminex platform. We studied the correlation between biomarkers and the risk of (i) death and (ii) death or heart failure-related hospital admission (DHFA). We also utilized machine-learning methods (a tree-based pipeline optimizer platform) to develop multimarker models associated with the risk of death and DHFA. In this cohort with a median follow-up of 2.8 years, multiple biomarkers were significantly predictive of death in analyses adjusted for clinical confounders, including tumour necrosis factor (TNF)-α [hazard ratio (HR) 1.28, P < 0.0001], TNF receptor 1 (TNFRSF1A; HR 1.38, P < 0.0001), fibroblast growth factor (FGF)-23 (HR 1.22, P < 0.0001), N-terminal pro B-type natriuretic peptide (NT-proBNP) (HR 1.58, P < 0.0001), matrix metalloproteinase-7 (HR 1.24, P = 0.0002), syndecan-1 (HR 1.27, P = 0.0002), suppression of tumorigenicity-2 (ST2) (IL1RL1; HR 1.22, P = 0.0002), interleukin (IL)-8 (CXCL8; HR 1.22, P = 0.0005), pentraxin (PTX)-3 (HR 1.17, P = 0.001), neutrophil gelatinase-associated lipocalin (LCN2; HR 1.18, P < 0.0001), osteoprotegerin (OPG) (TNFRSF11B; HR 1.26, P = 0.0002), and endostatin (COL18A1; HR 1.28, P = 0.0012). Several biomarkers were also significantly predictive of DHFA in adjusted analyses including FGF-23 (HR 1.36, P < 0.0001), TNF-α (HR 1.26, P < 0.0001), TNFR1 (HR 1.34, P < 0.0001), angiopoietin-2 (HR 1.26, P < 0.0001), syndecan-1 (HR 1.23, P = 0.0006), ST2 (HR 1.27, P < 0.0001), IL-8 (HR 1.18, P = 0.0009), PTX-3 (HR 1.18, P = 0.0002), OPG (HR 1.20, P = 0.0013), and NT-proBNP (HR 1.63, P < 0.0001). Machine-learning multimarker models were strongly associated with adverse outcomes (mean 1-year probability of death of 0%, 2%, and 60%; mean 1-year probability of DHFA of 0%, 4%, 97%; P < 0.0001). In these models, IL-6 (a biomarker of inflammation) and FGF-23 (a biomarker of calcification) emerged as the biomarkers of highest importance. CONCLUSIONS: Plasma biomarkers are strongly associated with the risk of adverse outcomes in patients with AS. Biomarkers of inflammation and calcification were most strongly related to prognosis.

Clinical and Proteomic Correlates of Plasma ACE2 (Angiotensin-Converting Enzyme 2) in Human Heart Failure.

ACE2 (angiotensin-converting enzyme 2) is a key component of the renin-angiotensin-aldosterone system. Yet, little is known about the clinical and biologic correlates of circulating ACE2 levels in humans. We assessed the clinical and proteomic correlates of plasma (soluble) ACE2 protein levels in human heart failure. We measured plasma ACE2 using a modified aptamer assay among PHFS (Penn Heart Failure Study) participants (n=2248). We performed an association study of ACE2 against ≈5000 other plasma proteins measured with the SomaScan platform. Plasma ACE2 was not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 was associated with older age, male sex, diabetes mellitus, a lower estimated glomerular filtration rate, worse New York Heart Association class, a history of coronary artery bypass surgery, and higher pro-BNP (pro-B-type natriuretic peptide) levels. Plasma ACE2 exhibited associations with 1011 other plasma proteins. In pathway overrepresentation analyses, top canonical pathways associated with plasma ACE2 included clathrin-mediated endocytosis signaling, actin cytoskeleton signaling, mechanisms of viral exit from host cells, EIF2 (eukaryotic initiation factor 2) signaling, and the protein ubiquitination pathway. In conclusion, in humans with heart failure, plasma ACE2 is associated with various clinical factors known to be associated with severe coronavirus disease 2019 (COVID-19), including older age, male sex, and diabetes mellitus, but is not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 protein levels are prominently associated with multiple cellular pathways involved in cellular endocytosis, exocytosis, and intracellular protein trafficking. Whether these have a causal relationship with ACE2 or are relevant to novel coronavirus-2 infection remains to be assessed in future studies.

Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence.

Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD , Caarv1C3S , and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution.

Leucine-rich repeat-containing protein 59 mediates nuclear import of cancerous inhibitor of PP2A in prostate cancer cells.

Using yeast two-hybrid analysis, we identified several novel protein interactions for the oncoprotein Cancerous Inhibitor of PP2A (CIP2A) and confirmed a subset of these interactions in human cancer cell lines. Analysis of the interaction in prostate carcinoma cells between CIP2A and leucine-rich repeat-containing protein 59 (LRRC59) suggests that CIP2A is translocated into the nucleus at G2/M through its association with LRRC59. Recent work by others has demonstrated that nuclear CIP2A disrupts mitotic checkpoints, which promotes deregulation of the cell cycle and increases cancerous phenotypes. Thus, we provide a novel therapeutic mechanism for inhibiting CIP2A function in cancerous cells via targeting the CIP2A-LRRC59 interaction.

Cancerous inhibitor of protein phosphatase 2A promotes premature chromosome segregation and aneuploidy in prostate cancer cells through association with shugoshin.

Yeast two-hybrid (Y2H) studies have shown that cancerous Inhibitor of protein phosphatase 2A (CIP2A) interacted with several proteins, including leucine-rich repeat-containing protein 59 (LRRC59), suggesting that CIP2A may interact with the chromosome maintenance protein, shugoshin (Sgol1). We previously showed that LRRC59 interacted with CIP2A, which was required for CIP2A nuclear localization. Thus, we predicted that CIP2A and Sgol1 may also interact. Sgol1 is a nuclear protein that regulates chromosome segregation during cell division via protection of cohesin ring proteins. Here, we demonstrated that Sgol1 and the C-terminus of CIP2A interact in prostate carcinoma cell lines in a protein phosphatase 2A (PP2A)-dependent manner. Moreover, we demonstrated that depletion of CIP2A in PC-3 cells decreases premature chromosome segregation, whereas overexpression of CIP2A in an immortalized prostate cell line increases premature chromosome segregation. Importantly, we further showed that CIP2A depletion decreases the incidence of aneuploidy and stabilizes cohesin complex proteins, while overexpression of CIP2A destabilizes Sgol1. Thus, our findings strongly suggest that CIP2A promotes cell cycle progression, premature chromosome segregation, and aneuploidy, possibly through a novel interaction with Sgol1.

PP2A(Cdc55) regulates G1 cyclin stability.

Maintaining accurate progression through the cell cycle requires the proper temporal expression and regulation of cyclins. The mammalian D-type cyclins promote G1-S transition. D1 cyclin protein stability is regulated through its ubiquitylation and resulting proteolysis catalyzed by the SCF E3 ubiquitin ligase complex containing the F-box protein, Fbx4. SCF E3-ligase-dependent ubiquitylation of D1 is trigged by an increase in the phosphorylation status of the cyclin. As inhibition of ubiquitin-dependent D1 degradation is seen in many human cancers, we set out to uncover how D-type cyclin phosphorylation is regulated. Here we show that in S. cerevisiae, a heterotrimeric protein phosphatase 2A (PP2A(Cdc55)) containing the mammalian PPP2R2/PR55 B subunit ortholog Cdc55 regulates the stability of the G1 cyclin Cln2 by directly regulating its phosphorylation state. Cells lacking Cdc55 contain drastically reduced Cln2 levels caused by degradation due to cdk-dependent hyperphosphorylation, as a Cln2 mutant unable to be phosphorylated by the yeast cdk Cdc28 is highly stable in cdc55-null cells. Moreover, cdc55-null cells become inviable when the SCF(Grr1) activity known to regulate Cln2 levels is eliminated or when Cln2 is overexpressed, indicating a critical relationship between SCF and PP2A functions in regulating cell cycle progression through modulation of G1-S cyclin degradation/stability. In sum, our results indicate that PP2A is absolutely required to maintain G1-S cyclin levels through modulating their phosphorylation status, an event necessary to properly transit through the cell cycle.

Kcnh1 voltage-gated potassium channels are essential for early zebrafish development.

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca(2+)/calmodulin and modulation of voltage-dependent gating by extracellular Mg(2+). Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

Sipl1 and Rbck1 are novel Eya1-binding proteins with a role in craniofacial development.

The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1's function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.

An unusual peptide from Conus villepinii: synthesis, solution structure, and cardioactivity.

The venom of marine cone snails contains a variety of conformationally constrained peptides utilized by the animal to capture prey. Besides numerous conotoxins, which are characterized by complex disulfide patterns, other peptides with only a single disulfide bridge were isolated from different conus species. Here, we report the synthesis, structure elucidation and biological evaluation of the novel C-terminally amidated decapeptide CCAP-vil, PFc[CNSFGC]YN-NH(2), from Conus villepinii. The linear precursor peptide was generated by standard solid phase synthesis. Oxidation of the cysteine residues to yield the disulfide-bridged peptide was investigated under different conditions, including several ionic liquids (ILs) as new biocompatible reaction media. Among the examined ILs, 1-ethyl-3-methylimidazolium tosylate ([C(2)mim][OTs]) was most efficient for CCAP-vil oxidative folding, since oxidation occurred without any byproduct formation. The structure of CCAP-vil was determined by NMR methods in aqueous solution and revealed a loop structure adopting a type(I) beta-turn between residues 4-7 imposed by the flanking disulfide bridge. The amino acid side chains of Pro(1), Phe(2), Phe(6) and Tyr(9) point in three directions away from the cyclic core into the solvent creating a rather hydrophobic surface of the molecule. Based on sequence homology to cardioactive peptides (CAPs) from gastropods and arthropods, such as PFc[CNAFTGC]-NH(2) (CCAP), the influence of CCAP-vil on heart rate using zebrafish embryos was investigated. CCAP-vil reduced the heart rate immediately upon injection into the heart as well as upon indirect application indicating an opposite effect to the cardioaccelerating CCAP.

A highly conserved retinoic acid responsive element controls wt1a expression in the zebrafish pronephros.

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1 expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.