Long-Acting IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Granulocyte Colony-Stimulating Factor or AMD3100.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome. In this present study, we tested commercially available IL-33 molecules and engineered novel variants of IL-33. These molecules were tested in cell-based assays in vitro and in mobilization models in vivo. We observed for the first time that IL-33 treatment in mice mobilized HSPCs and common myeloid progenitors more efficiently than clinical mobilizing agents granulocyte colony-stimulating factor (G-CSF) or AMD3100. We engineered several oxidation-resistant IL-33 variants with equal or better in vitro activity. In vivo, these variants mobilized HSPCs and, interestingly, also hematopoietic stem cells, common lymphoid progenitor cells, and endothelial progenitor cells more efficiently than wild-type IL-33 or G-CSF. We then engineered an IL-33-Fc fusion molecule, a single dose of which was sufficient to significantly increase the mobilization of HSPCs after 4 days. In conclusion, our findings suggest that long-acting, oxidation-resistant IL-33 may be a novel approach for HSPC transplantation. IL-33-mobilized HSPCs differ from cells mobilized with G-CSF and AMD3100, and it is possible that these differences may result in better transplantation outcomes.

Corin variant associated with hypertension and cardiac hypertrophy exhibits impaired zymogen activation and natriuretic peptide processing activity.

Corin is a cardiac serine protease that acts as the pro-atrial natriuretic peptide (ANP) convertase. Recently, 2 single-nucleotide polymorphisms (SNPs) (T555I and Q568P) in the human corin gene have been identified in genetic epidemiological studies. The minor I555/P568 allele, which is more common in African Americans, is associated with hypertension and cardiac hypertrophy. In this study, we examined the effect of T555I and Q568P amino acid substitutions on corin function. We found that corin frizzled-like domain 2, where T555I/Q568P substitutions occur, was required for efficient pro-ANP processing in functional assays. Mutant corin lacking this domain had 30+/-5% (P<0.01) activity compared to that of wild type. Similarly, corin variant T555I/Q568P had a reduced (38+/-7%, P<0.01) pro-ANP processing activity compared to that of wild type. The variant also exhibited a low activity (44+/-15%, P<0.05) in processing pro-brain natriuretic peptide (BNP). We next examined the biochemical basis for the loss of activity in T555I/Q568P variant and found that the zymogen activation of the corin variant was impaired significantly, as indicated by the absence of the activated protease domain fragment. This finding was confirmed in human embryonic kidney (HEK)293 cells and murine HL-1 cardiomyocytes. Thus, our results show that the corin gene SNPs associated with hypertension and cardiac hypertrophy impair corin zymogen activation and natriuretic peptide processing activity. Our data suggest that corin deficiency may be an important mechanism in hypertensive and heart diseases.

Characterization of a small molecule PAI-1 inhibitor, ZK4044.

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.

Hypertension in mice lacking the proatrial natriuretic peptide convertase corin.

Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood pressure. In cardiomyocytes, the hormone is synthesized as a precursor, proatrial natriuretic peptide (pro-ANP), which is proteolytically converted to active ANP. Corin is a cardiac transmembrane serine protease that has been shown to process pro-ANP in vitro, but its physiological importance had not been established. Here, we show that corin-deficient (Cor-/-) mice develop normally during embryogenesis and survive to postnatal life. Cor-/- mice have elevated levels of pro-ANP but no detectable levels of ANP as compared with WT littermates. Infusion of an active recombinant soluble corin transiently restores pro-ANP conversion, resulting in the release of circulating biologically active ANP. Using radiotelemetry to assess blood pressure, we find that Cor-/- mice have spontaneous hypertension as compared with WT mice, and it is enhanced after dietary salt loading. Pregnant Cor-/- mice demonstrate late-gestation proteinuria and enhanced high blood pressure during pregnancy. In addition, Cor-/- mice exhibit cardiac hypertrophy resulting in a mild decline in cardiac function later in life. Thus, our data establish corin as the physiological pro-ANP convertase and indicate that corin deficiency may contribute to hypertensive heart disease.

P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator alpha1.

During thrombosis, P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that targeting a plasminogen activator (PA) to P-selectin would enhance local thrombolysis and reduce bleeding risk. Previously, a urokinase (uPA)/anti-P-selectin antibody (HuSZ51) fusion protein was shown to increase fibrinolysis in a hamster pulmonary embolism model. To explore the therapeutic potential of this targeting strategy, we fused the fibrin-selective Desmodus rotundus salivary PA alpha1 (dsPA alpha 1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPA alpha 1 (HuSZ51-dsPA alpha 1) to unmodified dsPA alpha 1 in vitro and in vivo. HuSZ51-dsPA alpha 1 and dsPA alpha 1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51-dsPA alpha 1 bound to thrombin-activated human and dog platelets with comparable affinities to that of parental antibody SZ51. The fusion protein retained the catalytic activities of dsPA alpha 1 in chromogenic and clot lysis assays, indicating that dsPA alpha 1 is fully functional when fused to HuSZ51. Compared to dsPA alpha 1, HuSZ51-dsPA alpha 1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51-dsPA alpha 1 was less effective in lysis of preexisting arterial thrombi in the dog model. The reduced arterial thrombolysis was not due to the pharmacokinetic properties of HuSZ51-dsPA alpha 1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPA alpha 1-treated groups than corresponding dsPA alpha 1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPA alpha 1 varied dependent on the physical composition of thrombi. The lack of stimulation by fibrin in arterial thrombi may contribute to the attenuated thrombolytic efficacy of HuSZ51-dsPA alpha 1 in the dog model.

Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide.

Corin is a type II transmembrane serine protease and functions as the proatrial natriuretic peptide (pro-ANP) convertase in the heart. In the extracellular region of corin, there are two frizzled-like cysteine-rich domains, eight low density lipoprotein receptor (LDLR) repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain at the C terminus. To examine the functional importance of the domain structures in the propeptide of corin for pro-ANP processing, we constructed a soluble corin, EKshortCorin, that consists of only the protease domain and contains an enterokinase (EK) recognition sequence at the conserved activation cleavage site. After being activated by EK, EKshortCorin exhibited catalytic activity toward chromogenic substrates but failed to cleave pro-ANP, indicating that certain domain structures in the propeptide are required for pro-ANP processing. We then constructed a series of corin deletion mutants and studied their functions in pro-ANP processing. Compared with that of the full-length corin, a corin mutant lacking frizzled 1 domain exhibited approximately 40% activity, whereas corin mutants lacking single LDLR repeat 1, 2, 3, or 4 had approximately 49, approximately 12, approximately 53, and approximately 77% activity, respectively. We also made corin mutants with a single mutation at a conserved Asp residue that coordinates Ca(2+)-binding in LDLR repeats 1, 2, 3, or 4 (D300Y, D336Y, D373Y, and D410Y) and showed that these mutants had approximately 25, approximately 11, approximately 16, and approximately 82% pro-ANP processing activity, respectively. Our results indicate that frizzled 1 domain and LDLR repeats 1-4 are important structural elements for corin to recognize its physiological substrate, pro-ANP.

Corin-mediated processing of pro-atrial natriuretic peptide in human small cell lung cancer cells.

Corin is a recently discovered pro-atrial natriuretic peptide (ANP) convertase that is abundantly expressed in the heart. ANP is a cardiac hormone but can be secreted ectopically by certain cancers including small cell lung cancer (SCLC). In this study, we examined the role of corin in ANP production by SCLC cells. Reverse transcription-PCR detected corin mRNA expression in all nine SCLC cell lines examined and ANP mRNA expression in seven of the nine cell lines. In contrast, arginine vasopressin mRNA was detected in only five of the nine SCLC cell lines studied. Corin-expressing SCLC cells were capable of converting recombinant human pro-ANP to biologically active ANP, as determined by Western analysis and a cyclic GMP assay. Transfection of small interfering RNA duplexes directed against the corin gene completely blocked the processing of pro-ANP in the SCLC cells. Our results show that corin functions as a pro-ANP convertase in SCLC cells. We also suggest that the expression of corin may contribute to the pathogenesis of the syndrome of inappropriate secretion of antidiuretic hormone associated with certain cancers.

Functional analysis of the transmembrane domain and activation cleavage of human corin: design and characterization of a soluble corin.

Corin is a cardiac transmembrane serine protease. In cell-based studies, corin converted pro-atrial natriuretic peptide (pro-ANP) to mature ANP, suggesting that corin is potentially the pro-ANP convertase. In this study, we evaluated the importance of the transmembrane domain and activation cleavage in human corin. We showed that a soluble corin that consists of only the extracellular domain was capable of processing recombinant human pro-ANP in cell-based assays. In contrast, a mutation at the conserved activation cleavage site, R801A, abolished the function of corin, demonstrating that the activation cleavage is essential for corin activity. These results allowed us to design, express, and purify a mutant soluble corin, EKsolCorin, that contains an enterokinase recognition sequence at the activation cleavage site. Purified EKsolCorin was activated by enterokinase in a dose-dependent manner. Activated EK-solCorin had hydrolytic activity toward peptide substrates with a preference for Arg and Lys residues in the P-1 position. This activity of EKsolCorin was inhibited by trypsin-like serine protease inhibitors but not inhibitors of chymotrypsin-like, cysteine-, or metallo-proteases. In pro-ANP processing assays, purified active EKsolCorin converted recombinant human pro-ANP to biologically active ANP in a highly sequence-specific manner. The pro-ANP processing activity of EKsolCorin was not inhibited by human plasma. Together, our data indicate that the transmembrane domain is not necessary for the biological activity of corin but may be a mechanism to localize corin at specific sites, whereas the proteolytic cleavage at the activation site is an essential step in controlling the activity of corin.

Furin-mediated processing of Pro-C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family that is involved in a variety of homeostatic processes. Here we characterize the processing essential for the conversion of the precursor, human pro-CNP, to the biologically active hormone. In human embryonic kidney 293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a mature peptide intracellularly as detected by Western analysis. Expression of recombinant human corin, a proatrial natriuretic peptide convertase, did not enhance the processing of pro-CNP in these cells. The processing of pro-CNP was inhibited in the presence of an inhibitor of the endoprotease furin but was not affected by inhibitors of matrix metalloproteinases and tumor necrosis factor-alpha convertase. In furin-deficient human colon adenocarcinoma LoVo cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of recombinant human furin in LoVo cells restored the ability of these cells to process pro-CNP. Furthermore, incubation of purified recombinant human furin with LoVo cell lysate containing pro-CNP led to the conversion of the precursor to a mature peptide. The furin-processed CNP was shown to be biologically active in a cell-based cGMP assay. These results demonstrate that furin is a critical enzyme for the processing of human pro-CNP.

Genomic structures of the human and murine corin genes and functional GATA elements in their promoters.

Corin is a multiple-domain type II transmembrane serine protease highly expressed in the heart. It converts pro-atrial natriuretic peptide to atrial natriuretic peptide, a cardiac hormone that regulates blood volume and pressure. Here we describe the genomic structures of the human and murine corin genes and functional analysis of their promoters. Both corin genes contain 22 exons and span >200 kb. Their intron/exon boundaries are well conserved, with most exons encoding distinct structural domains, supporting the idea that corin evolved as a result of exon duplication and rearrangement. Comparison of the 5'-flanking regions of the human and murine corin genes revealed several conserved sequences, including binding sites for TBX5, GATA, NKX2.5, and Krüppel-like transcription factors. Transfection experiments with reporter gene constructs driven by the human or murine corin 5'-flanking region indicated that the sequences from -405 to -15 in human and from -646 to -77 in mouse are sufficient to promote high levels of gene expression in murine cardiomyocytes. In contrast, these sequences produced only minimal levels of expression in HeLa cells. Within these sequences, we identified a conserved GATA element that bound to GATA-4. Mutation of the core sequence impaired both GATA-4 binding and gene expression. These data indicate that the GATA element and its binding to GATA-4 are essential for cardiac expression of the human and murine corin genes.

Processing of pro-atrial natriuretic peptide by corin in cardiac myocytes.

Corin is a type II transmembrane serine protease abundantly expressed in the heart. In a previous study using transfected 293 cells, we showed that corin converted pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), suggesting that corin is likely the pro-ANP convertase. Because other serine proteases such as thrombin and kallikrein had previously also been shown to cleave pro-ANP in vitro, it remained to demonstrate that corin is indeed the endogenous pro-ANP convertase in cardiomyocytes. In this study, we examined pro-ANP processing in a murine cardiac muscle cell line, HL-5. Northern analysis showed that corin mRNA was present in HL-5 cells. In HL-5 cells transfected with a plasmid expressing pro-ANP, recombinant pro-ANP was converted to mature ANP as determined by Western analysis, indicating the presence of the endogenous pro-ANP convertase in these cells. The processed recombinant ANP was shown to be active in an enzyme-linked immunosorbent assay-based cGMP assay in baby hamster kidney cells. The processing of recombinant pro-ANP in HL-5 cells was highly sequence-specific, because mutation R98A, but not mutations R101A and R102A, in pro-ANP prevented the conversion of pro-ANP to ANP. Expression of recombinant wild-type corin enhanced the processing of pro-ANP in HL-5 cells. In contrast, overexpression of active site mutant corin S985A or transfection of oligonucleotide small interfering RNA duplexes directed against the mouse corin gene completely inhibited the processing of recombinant pro-ANP in HL-5 cells. These results indicate that corin is the physiological pro-ANP convertase in cardiac myocytes.