Meng Liu

Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy

In pregnancy, trophoblast invasion and uterine spiral artery remodelling are important for lowering maternal vascular resistance and increasing uteroplacental blood flow. Impaired spiral artery remodelling has been implicated in pre-eclampsia, a major complication of pregnancy, for a long time but the underlying mechanisms remain unclear. Corin (also known as atrial natriuretic peptide-converting enzyme) is a cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure. Unexpectedly, corin expression was detected in the pregnant uterus. Here we identify a new function of corin and ANP in promoting trophoblast invasion and spiral artery remodelling. We show that pregnant corin- or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of pre-eclampsia. In these mice, trophoblast invasion and uterine spiral artery remodelling were markedly impaired. Consistent with this, the ANP potently stimulated human trophoblasts in invading Matrigels. In patients with pre-eclampsia, uterine Corin messenger RNA and protein levels were significantly lower than that in normal pregnancies. Moreover, we have identified Corin gene mutations in pre-eclamptic patients, which decreased corin activity in processing pro-ANP. These results indicate that corin and ANP are essential for physiological changes at the maternal–fetal interface, suggesting that defects in corin and ANP function may contribute to pre-eclampsia.

Corin mutation R539C from hypertensive patients impairs zymogen activation and generates an inactive alternative ectodomain fragment.

Background: Corin is a membrane serine protease that activates natriuretic peptides in the heart. Results: The corin mutant R539C identified in hypertensive patients has impaired zymogen activation and altered ectodomain shedding. Conclusion: The mutation alters corin protein structure and reduces corin activity. Significance: Human CORIN gene mutations causing impaired corin activity may be an underlying mechanism in hypertension. Corin is a cardiac transmembrane serine protease that regulates blood pressure by activating natriuretic peptides. Corin variants have been associated with African Americans with hypertension and heart disease. Here, we report a new mutation in exon 12 of the CORIN gene identified in a family of patients with hypertension. The mutation resulted in R539C substitution in the Fz2 (Frizzled-2) domain of the corin propeptide region. We expressed and characterized the corin R539C mutant in HEK293 cells. As determined by Western blot analysis, the R539C mutation did not alter corin expression in transfected cells but impaired corin zymogen activation. In a pro-atrial natriuretic peptide processing assay, the corin mutant had reduced activity and exhibited a dominant-negative effect on wild-type corin. In addition, the R539C mutation altered corin ectodomain shedding, producing an alternative ∼75-kDa fragment that was biologically inactive. Using protease inhibitors and the catalytically inactive corin mutant S985A, we showed that the ∼75-kDa fragment was generated by corin autocleavage. We constructed a series of mutants by replacing single or double Arg residues in the corin propeptide and identified Arg-530 in the Fz2 domain as the alternative autocleavage site. Our results show that the corin mutation R539C identified in hypertensive patients impairs corin zymogen activation and causes an alternative autocleavage that reduces corin activity. These data support that human CORIN gene mutations causing impaired corin activity may be an underlying mechanism in hypertension.

Paracrine effect of inflammatory cytokine-activated bone marrow mesenchymal stem cells and its role in osteoblast function.

Mesenchymal stem cells (MSCs) have a crucial function in bone regeneration. Inflammation is a well-documented component of the osteogenic microenvironment. In the present study, we investigated whether stimulation of MSCs with inflammatory cytokines promotes osteogenesis through a paracrine mediator. MSCs were pre-stimulated with the inflammatory factors IFN-γ and TNF-α. After pre-stimulation, the MSC secretion levels of IL-6, HGF, VEGF, and TGF-β were significantly elevated (pxa0<xa00.01); however, the production of IL-2, IL-4, and IL-10 was not changed (pxa0>xa00.05). MG63, an osteoblast-like cell line, was cultured in different MSC-conditioned media. After treatment with conditioned media collected from MSCs pre-treated with cytokines, the proliferation and migration of MG63 cells were significantly improved, and the expression levels of the osteoblast differentiation markers ALP, COLI, OCN and OPN were significantly increased as revealed by a quantitative PCR analysis (pxa0<xa00.05). Furthermore, an immunofluorescence staining assay showed that more MG63 cells were OPN-positive, while an Alizarin red staining indicated the increased formation of calcium nodules in the IFN-γ and TNF-α combined pretreatment group. The results indicated that conditioned medium from inflammatory cytokine-activated MSCs can significantly promote osteoblast proliferation, migration, differentiation, and mineralization and ultimately enhance osteogenesis through paracrine mechanisms. These findings present a new direction for the clinical application of MSCs in the repair of bone defects.

Monitoring all-trans-retinoic acid-induced differentiation of human acute promyelocytic leukemia NB4 cells by Fourier-transform infrared spectroscopy.

Monitoring all- trans- retinoic acid-induced differentiation of human acute promyelocytic leukemia NB 4 cells by Fourier-transform infrared spectroscopy

Reduced urinary corin levels in patients with chronic kidney disease

Corin is a cardiac protease that regulates BP (blood pressure) by activating natriuretic peptides. Recent animal studies identified corin expression in the kidney where it may regulate renal function. In the present study, we tested the hypothesis that corin may be present in human urine and that urinary corin levels may be altered in patients with kidney disease. We obtained urine and kidney tissue samples from normal individuals and CKD (chronic kidney disease) patients. Using ELISA, we detected corin protein in human urine. In normal individuals, urinary corin levels did not correlate with that of plasma, indicating that urinary corin is probably of kidney origin. Compared with normal controls, CKD patients had markedly reduced urinary corin levels and this reduction correlated with disease severity. By immunostaining, human corin protein was identified on the epithelial cell surface in renal tubules. The renal corin mRNA and protein levels were significantly lower in CKD patients than non-CKD controls. The results indicate that renal tubular corin may be shed into urine and that urinary and renal corin levels were reduced in CKD patients. These data suggest that reduced corin levels in the kidney may reflect the underlying pathology in CKD.

Corin Mutations K317E and S472G from Preeclamptic Patients Alert Zymogen Activation and Cell Surface Targeting

Background: Corin is a protease that acts in the pregnant uterus to prevent pregnancy-induced hypertension. Results: Corin mutations K317E and S472G from preeclamptic patients impaired corin zymogen activation and intracellular trafficking, respectively. Conclusion: Mutations in the CORIN gene may impair corin function by different mechanisms. Significance: Genetic variants and mutations disrupting corin function may contribute to pregnancy-induced hypertension in patients. Corin is a membrane-bound serine protease that acts as the atrial natriuretic peptide (ANP) convertase in the heart. Recent studies show that corin also activates ANP in the pregnant uterus to promote spiral artery remodeling and prevent pregnancy-induced hypertension. Two CORIN gene mutations, K317E and S472G, were identified in preeclamptic patients and shown to have reduced activity in vitro. In this study, we carried out molecular modeling and biochemical experiments to understand how these mutations impair corin function. By molecular modeling, the mutation K317E was predicted to alter corin LDL receptor-2 module conformation. Western blot analysis of K317E mutant in HEK293 cells showed that the mutation did not block corin expression on the cell surface but inhibited corin zymogen activation. In contrast, the mutation S472G was predicted to abolish a β-sheet critical for corin frizzled-2 module structure. In Western blot analysis and flow cytometry, S472G mutant was not detected on the cell surface in transfected HEK293 cells. By immunostaining, the S472G mutant was found in the ER, indicating that the mutation S472G disrupted the β-sheet, causing corin misfolding and ER retention. Thus, these results show that mutations in the CORIN gene may impair corin function by entirely different mechanisms. Together, our data provide important insights into the molecular basis underlying corin mutations that may contribute to preeclampsia in patients.

A Corin Variant Identified in Hypertensive Patients That Alters Cytoplasmic Tail and Reduces Cell Surface Expression and Activity

Corin is a membrane-bound protease that regulates blood pressure by activating the natriuretic peptides. CORIN variants have been associated with hypertension and heart disease in African Americans. In this study, we conducted targeted exome sequencing and identified an insertion variant, c.102_103insA, in exon 1 of the CORIN gene. Analysis of two independent cohorts showed that the variant was preferentially present in hypertensive patients (38/795 or 4.78% vs. 4/632 or 0.63% in normal individuals, p = 4.14E-6). The insertion shifted the reading frame, resulting in a corin variant with a truncated cytoplasmic tail. In cell-based studies, the corin variant exhibited poor trafficking in the Golgi, reduced cell surface expression and zymogen activation, and low natriuretic peptide processing activity. Compared with normal individuals with the wild-type allele, individuals with the variant allele had lower levels of plasma corin [0.59 ± 0.07u2005ng/mL (n = 25) vs. 0.91 ± 0.02u2005ng/mL (n = 215), p<0.001] and higher levels of plasma N-terminal pro-atrial natriuretic peptide (NT-pro-ANP) [2.39 ± 3.6u2005nmol/L (n = 21) vs. 0.87 ± 0.6u2005nmol/L (n = 48), p = 0.005]. These results indicate that the variant altered corin structure and impaired the natriuretic peptide processing activity in vivo. The results highlight corin defects as an important underlying mechanism in hypertension.

Matriptase is highly upregulated in chronic lymphocytic leukemia and promotes cancer cell invasion

Matriptase is highly upregulated in chronic lymphocytic leukemia and promotes cancer cell invasion

Reduced serum corin levels in patients with osteoporosis.

BACKGROUNDnCorin is a cardiac protease that activates the natriuretic peptides. Corin is also expressed in chondrocytes and bone marrow-derived mesenchymal stem cells that undergo osteogenic differentiation, suggesting a potential role of corin in bone formation and homeostasis.nnnMETHODSnTo test if corin levels are altered in patients with bone disease, we used ELISA to measure corin and osteocalcin levels in serum samples from healthy controls (n = 134) and patients with osteopenia (n = 53) and osteoporosis (n = 101).nnnRESULTSnIn patients with osteopenia and osteoporosis, serum corin levels were 510 ± 228 and 478 ± 183 pg/ml, respectively, which were significantly lower than that in healthy controls (682 ± 240 pg/ml) (both p values<0.001). The reduced serum corin levels were found in both male and female patients. In multiple linear regression analysis, bone mineral density was identified as an independent predictor for serum corin levels. In patients with osteopenia and osteoporosis, but not normal controls, a negative correlation was found between serum corin and osteocalcin levels.nnnCONCLUSIONnSerum corin levels were reduced in patients with osteoporosis and the reduction was associated with high rates of bone turnover. Low serum corin levels may reflect impaired bone homeostasis in patients with osteoporosis.

The role of fucosylation in the promotion of endothelial progenitor cells in neovascularization and bone repair.

Bone marrow-derived endothelial progenitor cells (EPCs) are being tested as a therapy to treat a variety of ischemic diseases. Poor homing to targeted tissues is one of the major factors limiting the therapeutic efficacy of EPCs. Here, we show that human cord blood-derived EPCs expressed little sialyl Lewis X (sLe(x)) antigen that is necessary for selectin-mediated cell-cell interactions. Expression of α1,3-fucosyltransferase VI (FucT VI) in the EPCs enhanced sLe(x) synthesis, E- and P-selectin-binding, and EPC adhesion to tumor necrosis factor-α-stimulated human umbilical vein endothelial cells in culture. In a mouse model of hind limb ischemia, in which EPCs were injected intravenously, FucT VI expression increased EPC homing, neovascularization, and blood flow in ischemic muscles. In another mouse model of femoral fracture, FucT VI-expressing EPCs were more efficient than control EPCs in targeting to peri-fracture tissues to enhance angiogenesis, blood flow and bone repair. These results indicate that fucosylated EPCs may be used to as an improved cellular source to treat ischemic diseases.