Caenorhabditis elegans LIN-24, a homolog of bacterial pore-forming toxin, protects the host from microbial infection.

Aerolysin-like pore-forming protein (af-PFP) superfamily members are double-edge swords that assist the bacterial infection but shied bacteria from the host by various mechanisms in some species including the toad Bombina maxima and zebrafish. While members of this family are widely expressed in all kingdoms, especially non-bacteria species, it remains unclear whether their anti-bacterial function is conserved. LIN-24 is an af-PFP that is constitutively expressed throughout the Caenorhabditis elegans lifespan. Here, we observed that LIN-24 knockdown reduced the maximum lifespan of worms. RNA-seq analysis identified 323 differentially expressed genes (DEGs) post-LIN-24 knockdown that were enriched in "immune response" and "lysosome pathway," suggesting a possible role for LIN-24 in resisting microbial infection. In line with this, we found that Pseudomonas aeruginosa 14 (PA14) infection induced LIN-24 expression, and that survival after PA14 infection was significantly reduced by LIN-24 knockdown. In contrast, LIN-24 overexpression (LIN-24-OE) conferred protection against PA14 infection, with worms showing longer survival time and reduced bacterial load. Weighted gene co-expression network analysis of LIN-24-OE worms showed that the highest correlation module was enriched in factors related to immunity and the defense response. Finally, by predicting transcription factors from RNA-seq data and knocking down candidate transcription factors in LIN-24-OE worms, we revealed that LIN-24 may protect worms against bacterial infection by stimulating DAF-16-mediated immune responses. These findings agree with our previous studies showing an anti-microbial role for the amphibian-derived af-PFP complex ßγ-CAT, suggesting that af-PFPs may play a conserved role in combatting microbial infections. Further research is needed to determine the roles this protein family plays in other physio-pathological processes, such as metabolism, longevity, and aging.

Proprotein Convertase Subtilisin/Kexin 6 in Cardiovascular Biology and Disease.

Proprotein convertase subtilisin/kexin 6 (PCSK6) is a secreted serine protease expressed in most major organs, where it cleaves a wide range of growth factors, signaling molecules, peptide hormones, proteolytic enzymes, and adhesion proteins. Studies in Pcsk6-deficient mice have demonstrated the importance of Pcsk6 in embryonic development, body axis specification, ovarian function, and extracellular matrix remodeling in articular cartilage. In the cardiovascular system, PCSK6 acts as a key modulator in heart formation, lipoprotein metabolism, body fluid homeostasis, cardiac repair, and vascular remodeling. To date, dysregulated PCSK6 expression or function has been implicated in major cardiovascular diseases, including atrial septal defects, hypertension, atherosclerosis, myocardial infarction, and cardiac aging. In this review, we describe biochemical characteristics and posttranslational modifications of PCSK6. Moreover, we discuss the role of PCSK6 and related molecular mechanisms in cardiovascular biology and disease.

Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4.

Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of Npr1 knockout (Npr1+/-) mice and found that differentially expressed genes were significantly related to cell adhesion. This result was supported by an increased expression of intercellular adhesion molecule 1 (ICAM-1) in the aortic endothelium of Npr1+/- mice. Moreover, we observed that the knockdown of NPR1 increased ICAM-1 expression and promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs). NPR1 overexpression decreased ICAM-1 expression and inhibited the adhesion of monocytes to HUVECs treated by TNF-α (a cell adhesion inducer). Further analysis showed that adhesion-related genes were enriched in the focal adhesion signaling pathway, in which integrin beta 4 (Itgb4) was determined as a key gene. Notably, ITGB4 expression increased in vascular endothelium of Npr1+/- mice and in NPR1-knockdown HUVECs. The deficiency of ITGB4 decreased ICAM-1 expression and attenuated monocyte adhesion to NPR1-knockdown endothelial cells. Additionally, a reduced NPR1 and an increased ITGB4 expression level were found in an atherosclerosis mouse model. In conclusion, our findings demonstrate that NPR1 deficiency increases vascular endothelial cell adhesion by stimulating ITGB4 expression, which may contribute to the development of atherosclerosis.

Alteration of E2F2 Expression in Governing Endothelial Cell Senescence.

Endothelial cell senescence has a vital implication for vascular dysfunction, leading to age-related cardiovascular disease, especially hypertension and atherosclerosis. E2F transcription factor 2 (E2F2) plays a critical role in cell proliferation, differentiation, and DNA damage response. Up to date, no study has ever connected E2F2 to vascular endothelial cell senescence. Here, we demonstrate that E2F2 is involved in endothelial cellular senescence. We found that E2F2 expression is decreased during the replicative senescence of human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. The knockdown of E2F2 in young HUVECs induces premature senescence characterized by an increase in senescence-associated ß-galactosidase (SA-ß-gal) activity, a reduction in phosphorylated endothelial nitric oxide synthase (p-eNOS) and sirtuin 1 (SIRT1), and the upregulation of senescence-associated secretory phenotype (SASP) IL-6 and IL-8. The lack of E2F2 promoted cell cycle arrest, DNA damage, and cell proliferation inhibition. Conversely, E2F2 overexpression reversed the senescence phenotype and enhanced the cellular function in the senescent cells. Furthermore, E2F2 deficiency downregulated downstream target genes including CNNA2, CDK1, and FOXM1, and overexpression restored the expression of these genes. Our findings demonstrate that E2F2 plays an indispensable role in endothelial cell senescence.

Chronological attenuation of NPRA/PKG/AMPK signaling promotes vascular aging and elevates blood pressure.

Hypertension is common in elderly population. We designed to search comprehensively for genes that are chronologically shifted in their expressions and to define their contributions to vascular aging and hypertension. RNA sequencing was conducted to search for senescence-shifted transcripts in human umbilical vein endothelial cells (HUVECs). Small interfering RNA (siRNA), small-molecule drugs, CRISPR/Cas9 techniques, and imaging were used to determine genes' function and contributions to age-related phenotypes of the endothelial cell and blood vessel. Of 25 genes enriched in the term of "regulation of blood pressure," NPRA was changed most significantly. The decreased NPRA expression was replicated in aortas of aged mice. The knockdown of NPRA promoted HUVEC senescence and it decreased expressions of protein kinase cGMP-dependent 1 (PKG), sirtuin 1 (SIRT1), and endothelial nitric oxide synthase (eNOS). Suppression of NPRA also decreased the phosphorylation of AMP-activated protein kinase (AMPK) as well as the ratio of oxidized nicotinamide adenine dinucleotide (NAD+ )/reduced nicotinamide adenine dinucleotide (NADH) but increased the production of reactive oxygen species (ROS). 8-Br-cGMP (analog of cGMP), or AICAR (AMPK activator), counteracted the observed changes in HUVECs. The Npr1+/- mice presented an elevated systolic blood pressure and their vessels became insensitive to endothelial-dependent vasodilators. Further, vessels from Npr1+/- mice increased Cdkn1a but decreased eNos expressions. These phenotypes were rescued by intravenously administrated 8-Br-cGMP and viral overexpression of human PKG, respectively. In conclusion, we demonstrate NPRA/PKG/AMPK as a novel and critical signaling axis in the modulation of endothelial cell senescence, vascular aging, and hypertension.

Null Function of Npr1 Disturbs Immune Response in Colonic Inflammation During Early Postnatal Stage.

Natriuretic peptide receptor 1 (NPR1) is conventionally known as a regulator of vascular homeostasis. Here, we generated an Npr1 knockout mouse model with CRISPR/Cas9 technology and found that homozygous mice (Npr1-/-) exhibited weight loss and poor survival rate during early postnatal stage. Careful examination revealed unexpectedly that Npr1-/- mice developed colitis characterized by shortened colon, evident colonic mucosal damage, increased histopathological score, and higher colonic expression of proinflammatory cytokines interleukin-1B (IL1B) and -6 (IL6). RNA-sequencing analysis revealed that differentially expressed genes were prominently enriched in the biological pathways related to immune response in both spleen and colon of Npr1-/- mice. Cytofluorimetric analysis demonstrated that leukocytes in the spleen were significantly increased, particularly, the populations of neutrophil and CD3+ T cell were elevated but CD4+ T cells were decreased in Npr1-/- mice. Administration of 8-Br-cGMP, a downstream activator of NPR1, restored these immune-cell populations disturbed in Npr1-/- mice and lessened the colitis-related phenotypes. To validate the involvement of Npr1 in colitis, we examined another mouse model induced by dextran sodium sulfate (DSS) and found a decreased Npr1 expression and shifted immune-cell populations as well. Importantly, 8-Br-cGMP treatment exhibited a similar effect in the restoration of immune-cell populations and attenuation of colonic inflammation in DSS mice. Our data indicate that loss of Npr1 possibly interrupts immune response, which is critical to the pathogenesis of colitis in the early life.

Pcsk6 Deficiency Promotes Cardiomyocyte Senescence by Modulating Ddit3-Mediated ER Stress.

Cardiac aging is a critical determinant of cardiac dysfunction, which contributes to cardiovascular disease in the elderly. Proprotein convertase subtilisin/kexin 6 (PCSK6) is a proteolytic enzyme important for the maintenance of cardiac function and vascular homeostasis. To date, the involvement of PCSK6 in cardiac aging remains unknown. Here we report that PCSK6 expression decreased in the hearts of aged mice, where high levels cyclin dependent kinase inhibitor 2A (P16) and cyclin dependent kinase inhibitor 1A (P21) (senescence markers) were observed. Moreover, PCSK6 protein expression was significantly reduced in senescent rat embryonic cardiomyocytes (H9c2) induced by D-galactose. Pcsk6 knockdown in H9c2 cells increased P16 and P21 expression levels and senescence-associated beta-galactosidase activity. Pcsk6 knockdown also impaired cardiomyocyte function, as indicated by increased advanced glycation end products, reactive oxygen species level, and apoptosis. Overexpression of PCSK6 blunted the senescence phenotype and cellular dysfunction. Furthermore, RNA sequencing analysis in Pcsk6-knockdown H9c2 cells identified the up-regulated DNA-damage inducible transcript 3 (Ddit3) gene involved in endoplasmic reticulum (ER) protein processing. Additionally, DDIT3 protein levels were remarkably increased in aged mouse hearts. In the presence of tunicamycin, an ER stress inducer, DDIT3 expression increased in Pcsk6-deficient H9c2 cells but reduced in PCSK6-overexpressing cells. In conclusion, our findings indicate that PCSK6 modulates cardiomyocyte senescence possibly via DDIT3-mediated ER stress.

Photoinduced immobilization of 2-methacryloyloxyethyl phosphorylcholine polymers with different molecular architectures on a poly(ether ether ketone) surface.

Poly(ether ether ketone) (PEEK) has seen increasing use in biomedical fields as a replacement for metal implants. Accordingly, the surface functionalities of PEEK are important for the development of medical devices. We have focused on the application of photoinduced reactions in PEEK to immobilize a functional polymer via radical generation on the surface, which can react with hydrocarbon groups. In this study, we used zwitterionic copolymers comprising 2-methacryloyloxyethyl phosphorylcholine (MPC) units and n-butyl methacrylate (BMA) units with various molecular architectures for surface modification. A random copolymer (poly(MPC-co-BMA) (r-PMB)), an AB-type diblock copolymer (di-PMB), and an ABA-type triblock copolymer (tri-PMB) (A segment: poly(BMA); B segment: poly(MPC)) were synthesized with the same monomer compositions. All PMBs were successfully immobilized on the PEEK surface via UV irradiation after the dip-coating process, regardless of their molecular structure. In this reaction, the alkyl group of the BMA unit functioned as a photoreactive site on the PEEK surface. This indicates that the molecular structure differences affect the surface properties. For example, compared to r-PMB and tri-PMB, di-PMB-modified surfaces exhibited an extremely low water contact angle of approximately 10°. The findings of this study demonstrate that this surface functionalization method does not require a low-molecular-weight compound, such as an initiator, and can be applied to the surface of inert PEEK through a simple photoreaction under room temperature, atmospheric pressure, and dry state conditions.

Temperature-triggered attachment and detachment of general human bio-foulants on zwitterionic polydimethylsiloxane.

Biofouling has long been a problem for biomaterials, so being able to control the fouling on the surface of a biomaterial would be ideal. In this study a copolymer system was designed comprising three moieties: an epoxy containing group, glycidyl methacrylate (GMA); a thermoresponsive segment, N-isopropylacrylamide (NIPAAm); and an antifouling zwitterionic unit, sulfobetaine methacrylate (SBMA). The copolymers (pGSN), synthesized via free radical polymerization with these 3 moieties, were then grafted onto polydimethylsiloxane (PDMS). The presence of a critical temperature for both the copolymers and the coated PDMS was evidenced by particle size and contact angle measurements. The coated PDMS exhibited controllable temperature-dependent antifouling behaviors and stimuli-responsive phase characteristics in the presence of salts. The interactions of the coated PDMS with biomolecules were tested via attachment of fibrinogen protein, platelets, human whole blood, and tumor cells (HT1080). The attachment and detachment of these biomolecules were studied at different temperatures. Exposed hydrophobic domains of thermoresponsive NIPAAm-rich pGSN containing NIPAAm at 56 mol% generally allows molecular and cellular attachment on the PDMS surface at 37 °C. On the other hand, the coated PDMS with a relatively high content of SBMA (>41 mol%) in the copolymer started to exhibit fouling resistance and lower the thermoresponsive properties. Interestingly, the incorporation of zwitterionic SBMA units into the copolymers was found to accelerate the hydration of the PDMS surfaces and resulted in biomolecular and cellular detachment at 25 °C, which is comparable to the detachment at 4 °C. This modified surface behavior is found to be consistent through all biofouling tests.

Conserved signaling pathways genetically associated with longevity across the species.

Advanced age is an independent risk factor for natural death and common diseases, such as cardiovascular diseases, dementia, and cancers, which are life-threatening and cause disabilities. On the other hand, individual with healthy longevity is a plausible model for successful aging. Thus, search for longevity-associated genes and pathways likely provides a unique approach to understand the genetic mechanisms underlying aging and healthspan, and emerging evidence from model organisms has highlighted the significance of genetic components in longevity. Here we reviewed the uses of model organisms including yeast, ciliate, nematode, arthropod, fish, rodent, and primate as well as human to identify the genetic determinants of longevity and discussed the genetic contributions of conserved longevity pathways, such as adrenergic system, AMPK, insulin/IGF-1, and mTOR signaling pathways.

Photoinduced Surface Zwitterionization for Antifouling of Porous Polymer Substrates.

Surface functionalization of polymeric porous substrates is one of the most important requirements to enhance their applications in the biomedical field. In this study, we achieved photoinduced surface modification using a highly efficient reaction of hydrophilic polymers bearing phosphorylcholine groups. Polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units and 2-( N-ethylanilino)ethyl methacrylate units were synthesized with attention to the polymer architectures. The surface modification of the porous polyethylene (PE) substrates was carried out by the coating of the MPC polymers with a photochemical radical generator, followed by photoirradiation for a few minutes. Surface analysis by attenuated total reflectance Fourier transform IR spectroscopy and X-ray photoelectron spectroscopy indicated that the MPC polymer layer was generated on the PE surface. Cross-sectional confocal microscopy images showed that the MPC polymers were coated on the polymer surface, even inside the porous structure of the PE substrate. After modification, the porous PE substrates showed a significant increase in hydrophilicity and the water-penetration rate through the pores. Furthermore, the amount of protein adsorbed on the PE substrate was reduced significantly by the surface modification. These functionalities were dependent on the MPC polymer architectures. Thus, we concluded that the photoreactive polymer system developed furnished the porous substrates with antifouling properties.

Clinical Profile and Outcomes of Early Seizures in Asian Patients With Acute Intracerebral Hemorrhage.

BACKGROUND: Seizures are one of the most common complications of stroke. We aimed to establish the incidence and clinical profile of post-stroke early seizure (ES) in patients with intracerebral hemorrhage (ICH). METHODS: Patients with ICH within 10 days of onset who were admitted to Landseed International Hospital were recruited consecutively between January 1, 2006, and December 31, 2009. The National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale were used to access patients initial stroke severity and functional outcome at discharge, respectively. The occurrence of epileptic seizures within 30 days after onset of the index ICH was recorded. Early post-ICH seizure was defined by the occurrence of clinically identifid seizure episodes or non-epileptic seizure within 7 days after the stroke onset. RESULTS: A total of 297 ICH patients were included. The mean age of the participants was 62 ± 16 years, and 72% of them were male. A total of 9 (3%) participants had seizures during acute hospitalization. Patients with seizures had higher median NIHSS scores at baseline (34 vs. 16, p = 0.004). No difference was noted in the cortical involvement of ICH (22% for patients with seizures and 14% for those without, p = 0.156). Patients with seizures had higher in-hospital mortality ( 56% vs. 23%, p = 0.024). The multivariate Cox regression model showed the factors significantly associated with ES were higher initial NIHSS scores on admission (adjusted odds ratio [aOR] = 1.1 per 1 point increased, 95% confidence interval [CI] = 1.0-1.2) and coronary artery disease (aOR = 7.0, 95% CI = 1.3-36.4). CONCLUSIONS: The NIHSS scores and coronary heart disease were associated with ES in ICH, whereas cortical involvement was not. These findings may reflect difference in post-stroke seizure and primary ICH between Asian and Western populations.

N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding.

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.

Type 2 diabetes mellitus (T2DM) is a common metabolic disease influenced by both genetic and environmental factors. In this study, we performed an in-house genotyping and meta-analysis study using three independent GWAS datasets of T2DM and found that rs3743121, located 1 kb downstream of AQR, was a novel susceptibility SNP associated with T2DM. The risk allele C of rs3743121 was correlated with the increased expression of AQR in white blood cells, similar to that observed in T2DM models. The knockdown of AQR in HepG2 facilitated the glucose uptake, decreased the expression level of PCK2, increased the phosphorylation of GSK-3ß, and restored the insulin sensitivity. Furthermore, the suppression of AQR inhibited the mTOR pathway and the protein ubiquitination process. Our study suggests that AQR is a novel type 2 diabetes-associated gene that regulates signaling pathways critical for glucose metabolism.

Genome-Wide Association and Functional Studies Identify SCML4 and THSD7A as Novel Susceptibility Genes for Coronary Artery Disease.

OBJECTIVE: The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD. APPROACH AND RESULTS: We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 21 828 participants of Han ethnicity and identified 3 novel intragenic SNPs (single nucleotide polymorphisms), rs9486729 (SCML4 [Scm polycomb group protein-like 4]; odds ratio, 1.25; 95% CI, 1.17-1.34; P=3.51×10-11), rs17165136 (THSD7A [thrombospondin type 1 domain-containing 7A]; odds ratio 1.28; 95% CI, 1.21-1.35; P<1.00×10-25), and rs852787 (DAB1 [disabled-1]; odds ratio, 1.29; 95% CI, 1.21-1.38; P=2.02×10-14), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs17165136 were associated with the decreased expression of their host genes, SCML4 and THSD7A, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD7A had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation. CONCLUSIONS: We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD7A, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB1 are linked to CAD needs further studies.

Functional analysis of corin protein domains required for PCSK6-mediated activation.

Atrial natriuretic peptide (ANP) is a cardiac hormone essential for normal blood pressure and cardiac function. Corin is a transmembrane serine protease that activates ANP. Recently, we identified proprotein convertase subtilisin/kexin-6 (PCSK6), also called PACE4, as the long-sought corin activator. Both corin and PCSK6 are expressed in cardiomyocytes, but corin activation occurs only on the cell surface. It remains unknown if cell membrane association is needed for PCSK6 to activate corin. Here we expressed corin deletion mutants in HEK293 cells to analyze the domain structures required for PCSK6-mediated activation. Our results show that soluble corin lacking the transmembrane domain was activated by PCSK6 in the conditioned medium but not intracellularly. Recombinant PCSK6 also activated the soluble corin under cell-free conditions. Moreover, PCSK6-mediated corin activation was not enhanced by cell membrane fractions. These results indicate that cell membrane association is unnecessary for PCSK6 to activate corin. Experiments with monensin that blocks PCSK6 secretion and immunostaining indicated that the soluble corin and PCSK6 were secreted via different intracellular pathways, which may explain the lack of corin activation inside the cell. We also found that the protein domains in the corin pro-peptide region were dispensable for PCSK6-mediated activation and that addition of heparan sulfate and chondroitin sulfate or treatment with heparinase or chondroitinase did not alter corin activation by PCSK6 in HEK293 cells. Together, our results provide important insights into the molecular and cellular mechanisms underlying PCSK6-mediated corin activation that is critical for cardiovascular homeostasis.

A Zwitterionic-Shielded Carrier with pH-Modulated Reversible Self-Assembly for Gene Transfection.

Cationic vectors are ideal candidates for gene delivery thanks to their capability to carry large gene inserts and their scalable production. However, their cationic density gives rise to high cytotoxicity. We present the proper designed core-shell polyplexes made of either poly(ethylene imine) (PEI) or poly(2-dimethylamino ethyl methacrylate) (PDMAEMA) as the core and zwitterionic poly(acrylic acid)-block-poly(sulfobetaine methacrylate) (PAA-b-PSBMA) diblock copolymer as the shell. Gel retardation and ethidium bromide displacement assays were used to determine the PEI/DNA or PDMAEMA/DNA complexation. At neutral pH, the copolymer serves as a protective shell of the complex. As PSBMA is a nonfouling block, the shell reduced the cytotoxicity and enhanced the hemocompatibility (lower hemolysis activity, longer plasma clotting time) of the gene carriers. PAA segments in the copolymer impart pH sensitivity by allowing deshielding of the core in acidic solution. Therefore, the transfection efficiency of polyplexes at pH 6.5 was better than at pH 7.0, from ß-galactosidase assay, and for all PAA-b-PSBMA tested. These results were supported by more favorable physicochemical properties in acidic solution (zeta potential, particle size, and interactions between the polymer and DNA). Thus, the results of this study offer a potential route to the development of efficient and nontoxic pH-sensitive gene carriers.

Reduced Blood Cell Adhesion on Polypropylene Substrates through a Simple Surface Zwitterionization.

To overcome the thrombogenic reactions of hydrocarbon-based biomaterials in clinical blood treatment, we introduce a model study of surface zwitterionization of a polypropylene (PP) substrate using a set of well-defined copolymers for controlling the adhesion of blood cells in vitro. Random and block copolymers containing zwitterionic units of 2-methacryloyloxyethyl phosphorylcholine (MPC), [3-(methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (SBAA), or nonionic units of 2-hydroxyethyl methacrylate (HEMA) with a controlled hydrophobic segment of 70% n-butyl methacrylate (BMA) units in these polymers were synthesized through reversible addition-fragmentation chain transfer polymerization. A systematic study of how zwitterionic and nonionic copolymer architectures associated with controlled chain orientation via hydration processes affect blood compatibility is reported. The surface wettability of PP substrates coated with the block copolymer with poly(MPC) (PMPC) segments was higher than that of the random copolymer poly(MPC-random-BMA). However, only the random copolymers with SBAA units demonstrate a higher surface wettability. The PP substrate coated with nonionic copolymers containing HEMA units showed relatively lower hydration capability associated with higher protein adsorption, platelet adhesion, and leukocyte attachment than those with zwitterionic copolymers. The random copolymer poly(SBAA-random-BMA) coated on the PP substrates exhibited resistance to cell adhesion in human whole blood at a level comparable to that of MPC copolymers. An ideal zwitterionic PP substrate could be obtained by coating it with a block copolymer composed of PMPC and poly(BMA) (PBMA) segments, PMPC-block-PBMA. The water contact angle decreased dramatically from approximately 100° on the original PP substrate to 11° within 30 s. The number of blood cells attached on PMPC-block-PBMA decreased significantly to less than 2.5% of that on original PP. These results prove that the rational design of zwitterionic polymers incorporated with a hydrophobic anchoring portion provides a promising approach to reduce blood cell adhesion and protein adsorption of hydrocarbon-based biomaterials applied in direct contact with human whole blood.

PCSK6-mediated corin activation is essential for normal blood pressure.

Hypertension is the most common cardiovascular disease, afflicting >30% of adults. The cause of hypertension in most individuals remains unknown, suggesting that additional contributing factors have yet to be discovered. Corin is a serine protease that activates the natriuretic peptides, thereby regulating blood pressure. It is synthesized as a zymogen that is activated by proteolytic cleavage. CORIN variants and mutations impairing corin activation have been identified in people with hypertension and pre-eclampsia. To date, however, the identity of the protease that activates corin remains elusive. Here we show that proprotein convertase subtilisin/kexin-6 (PCSK6, also named PACE4; ref. 10) cleaves and activates corin. In cultured cells, we found that corin activation was inhibited by inhibitors of PCSK family proteases and by small interfering RNAs blocking PCSK6 expression. Conversely, PCSK6 overexpression enhanced corin activation. In addition, purified PCSK6 cleaved wild-type corin but not the R801A variant that lacks the conserved activation site. Pcsk6-knockout mice developed salt-sensitive hypertension, and corin activation and pro-atrial natriuretic peptide processing activity were undetectable in these mice. Moreover, we found that CORIN variants in individuals with hypertension and pre-eclampsia were defective in PCSK6-mediated activation. We also identified a PCSK6 mutation that impaired corin activation activity in a hypertensive patient. Our results indicate that PCSK6 is the long-sought corin activator and is important for sodium homeostasis and normal blood pressure.

Distinct roles of N-glycosylation at different sites of corin in cell membrane targeting and ectodomain shedding.

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.