Shear stress-induced release of prostaglandin H(2) in arterioles of hypertensive rats

The nitric oxide-mediated portion of shear stress-induced dilation of rat gracilis muscle arterioles was shown to be impaired in spontaneously hypertensive rats (SHR). Because shear stress-induced dilation is primarily mediated by endothelium-derived prostaglandins in rat cremasteric arterioles, we hypothesized that in the cremasteric vascular bed the mediation of shear stress-induced dilation by prostaglandins is altered in hypertension. At a constant intraluminal pressure of 80 mm Hg, the active diameters of isolated rat cremasteric arterioles of normotensive 30-week-old Wistar-Kyoto rats (WKY) and SHR were 58.0+/-3.1 and 51.7+/-3.6 microm, respectively, whereas their passive diameters were 109.4+/-4.4 and 101.9+/-6.7 microm, respectively. Dilations to increases in shear stress elicited by increases in intraluminal flow (from 0 to 25 microL/min) were significantly less (P<0.05) in cremasteric arterioles isolated from SHR than from WKY. Arachidonic acid (10(-5) mol/L) elicited constrictions in SHR arterioles but dilations in WKY arterioles. The prostaglandin H(2)/thromboxane A(2) (PGH(2)/TxA(2)) receptor antagonist SQ 29,548 (10(-6) mol/L) significantly increased basal diameter by 11% and normalized the attenuated shear stress-induced dilation in SHR, whereas it did not affect basal diameter and arteriolar responses of WKY. Furegrelate, a specific inhibitor of TxA(2) synthase, did not affect the response in SHR. Also, SQ 29,548 reversed the arachidonic acid-induced constriction to dilation in SHR arterioles, whereas it did not affect the dilator response in WKY arterioles. Constrictions of arterioles of WKY and SHR to U46,619 (a PGH(2)/TxA(2) receptor agonist) were not different. These results demonstrate that in cremasteric arterioles of hypertensive rats, shear stress elicits an enhanced release of PGH(2), resulting in a reduced shear stress-dependent dilation. Thus, augmented hemodynamic forces can alter the shear stress-induced synthesis of prostaglandins, which may contribute to the elevated vascular resistance in hypertension.     

Shear stress-induced up-regulation of the intermediate-conductance Ca(2+)-activated K(+) channel in human endothelium

OBJECTIVE: Wall shear stress associated with blood flow is a major stimuli for generation of endothelial vasodilating and antithrombotic factors and it also regulates endothelial gene expression. Activation of endothelial intermediate-conductance Ca(2+)-activated K(+) channels (IK(Ca)) is important for the control of endothelial function by inducing cell hyperpolarization and thus generation of the endothelium-derived hyperpolarizing factor. In the present study we tested whether the IK(Ca) encoding IKCa1 gene is regulated by laminar shear stress (LSS). METHODS: Human umbilical vein endothelial cells (HUVEC) were subjected to LSS with a magnitude of 0.5-15 dyn/cm(2) and time intervals of 2-24 h in a flow cone apparatus. Expression of the IKCa1 gene and IK(Ca)-functions were determined by using real time RT-PCR and patch-clamp techniques. RESULTS: A short 2-4 h-or long 24 h-exposure to a LSS with a low (venous) magnitude of 0.5 dyn/cm(2) had no effect on IKCa1 expression levels. An exposure for 2 and 4 h to LSS with an intermediate magnitude of 5 dyn/cm(2) was also ineffective, whereas an exposure for 24 h induced a significant threefold up-regulation of IKCa1 expression levels. An exposure to LSS with a higher (arterial) magnitude of 15 dyn/cm(2), resulted in an eightfold up-regulation of IKCa1 expression levels after a 4 h-exposure and a fourfold increase of IKCa1 expression levels at 24 h. The increased IKCa1 expression levels following exposure to high levels of LSS resulted in enhanced IK(Ca) whole-cell currents and in an increased hyperpolarization of the endothelium in response to ATP and the IK(Ca) opener 1-EBIO. Inhibition of the mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK) kinase 1/2 (MEK/ERK) pathway by PD98059 prevented the LSS-induced up-regulation of IKCa1 expression levels and IK(Ca) whole-cell currents indicating that augmentation of IKCa1 expression levels is mediated by the LSS-induced activation of the MEK/ERK pathway. CONCLUSION: Long term exposure to LSS up-regulates expression and function of endothelial IK(Ca). This increase might represent a new important mechanism in endothelial adaptation to altered hemodynamics.     

A carboxy-terminal deletion mutant of Notch1 accelerates lymphoid oncogenesis in E2A-PBX1 transgenic mice

PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a Notch(IC) allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involving Notch1 occurred in a more 3' exon, resulting in truncating mutations that deleted the carboxy-terminal region of Notch1 containing negative regulatory sequences (Notch1(DeltaC)). In contrast to Notch(IC), forced expression of Notch1(DeltaC) in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1(DeltaC) genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation of Notch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development. (Blood. 2000;96:1906-1913)     

Shear stress-induced pH increase in plasma is mediated by a decrease in P(CO(2)): the increase in pH enhances shear stress-induced P-selectin expression in platelets

To investigate shear stress-induced platelet activation, the cone-plate viscometer or the Couette rotational viscometer has been widely used. In a previous report, it was shown that shearing platelet-rich plasma using a Couette rotational viscometer could lead to an increase in pH by CO(2) release. However, any clear mechanism has not been provided. In this study, we examined whether shearing cell free plasma only using a cone-plate viscometer can also induce pH increase and studied the underlying mechanism of shear-induced pH increase by directly measuring total CO(2) (T(CO(2))) and CO(2) tension (P(CO(2))). When human plasma was sheared using a cone-plate viscometer, the pH of the human plasma increased time- and shear rate-dependently. Although T(CO(2)) of human plasma was not affected, P(CO(2)) was decreased by shearing, indicating that the decreased P(CO(2)) is associated with a pH increase of plasma. In addition, the pH of bicarbonate-containing suspension buffer was also shown to be increased by shearing; suggesting that the platelet studies using suspension buffers containing bicarbonate could be affected similarly. The effects of pH changes on shear stress-induced platelet activation were also investigated in the same in vitro systems. While shear stress-induced platelet aggregation was not affected by the pH changes, P-selectin expression was significantly increased in accordance with the pH increase. In conclusion, shear stress using a cone-plate viscometer induces pH increase in plasma or bicarbonate-containing suspension buffer through a P(CO(2)) decrease and the pH changes alone can contribute to platelet activation by enhancing shear stress-induced P-selectin expression.     

Remethylation and transsulfuration of methionine in cirrhosis: studies with L-[H3-methyl-1-C]methionine

Disturbances of the methionine cycle may result in liver injury. Patients with alcohol-induced liver disease often exhibit hypermethioninemia and a delayed clearance (CL) of methionine, but the extent to which transsulfuration and remethylation pathways of the cyclic methionine metabolism are affected is unknown. Methionine turnover was determined in 7 healthy volunteers and 6 patients with alcohol-induced cirrhosis after oral administration of 2 mg/kg [(2)H(3)-methyl-1-(13)C]methionine, which permitted us to follow transsulfuration by its decarboxylation to (13)CO(2) and remethylation by replacement of the labeled methyl group by an unlabeled one. Basal plasma concentrations of endogenous methionine (50 +/- 5 vs. 25 +/- 2 micromol/L, mean +/- SEM, P <.001) were significantly higher in patients with cirrhosis and its CL was significantly decreased (774 +/- 103 vs. 2,050 +/- 141 mL/min, P <.001). Methionine turnover amounted to 42 +/- 4 vs. 27 +/- 3 micromol/kg/h (P <.05) in controls and patients with cirrhosis, respectively. The fraction of administered methionine undergoing remethylation was lower in patients with cirrhosis (7.6 +/- 1.5 vs. 14.1 +/- 1.1%, P <.005). However, because of the larger pool of circulating methionine, the total flux of methionine through the remethylation pathway was similar in both groups. A significantly lower fraction of the administered dose appeared in the form of (13)CO(2) in breath in patients with cirrhosis (2.2 +/- 0.4 vs. 11.0 +/- 0.8%, P <.001). In conclusion, the data indicate that the liver with cirrhosis compensates for a decreased activity of remethylating enzymes by operating at higher concentrations of methionine. In contrast, transsulfuration is impaired in patients with alcohol-induced cirrhosis such that an assessment of transsulfuration by a simple breath test may provide a clinically useful estimate of hepatic function.     

Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region

The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force “barrier” on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRA∶B linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.     

Hypervariable region 3 residues of HIV type 1 gp120 involved in CCR5 coreceptor utilization: therapeutic and prophylactic implications

Crystallographic characterization of a ternary complex containing a monomeric gp120 core, parts of CD4, and a mAb, revealed a region that bridges the inner and outer domains of gp120. In a related genetic study, several residues conserved among primate lentiviruses were found to play important roles in CC-chemokine receptor 5 (CCR5) coreceptor utilization, and all but one were mapped to the bridging domain. To reconcile this finding with previous reports that the hypervariable region 3 (V3) of gp120 plays an important role in chemokine coreceptor utilization, elucidating the roles of various V3 residues in this critical part of the HIV type 1 (HIV-1) life cycle is essential. Alanine-scanning mutagenesis was carried out to identify V3 residues critical for CCR5 utilization. Our findings demonstrated that several residues in V3 were critical to CCR5 utilization. Furthermore, these residues included not only those conserved across HIV-1 subtypes, but also those that varied among HIV-1 subtypes. Although the highly conserved V3 residues may represent unique targets for antiviral designs, the involvement of variable residues raises the possibility that antigenic variation in the coreceptor binding domain could further complicate HIV-1 vaccine design.     

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.     

Deletion of the V1/V2 region does not increase the accessibility of the V3 region of recombinant gp125

Previous analyses of HIV-1 surface glycoprotein indicate that both the V1/V2 region and the interaction of gp120 with CD4 influence the accessibility of the V3 region on gp120. In this study we investigated the accessibility of the V3 region of HIV-2 recombinant gp125 proteins using V3-specific mAbs (7C8 and 3C4) and analyzed the binding kinetics of soluble CD4 (sCD4) to recombinant HIV-1 gp120 and HIV-2 gp125 proteins by surface plasmon resonance (SPR) analysis. Our results indicated that 7C8 recognized monomers of gp125 and gp125Delta v1v2, (lacking the V1/V2 region) while 3C4 was sensitive to the conformation of gp125, recognizing only oligomers of gp125Delta v1v2. Furthermore, SPR analysis of 7C8 binding to gp125 demonstrated that the deletion of the V1/V2 region did not increase the accessibility of the V3 region in gp125Delta v1v2. Comparative SPR analyses of sCD4 binding HIV recombinant surface glycoproteins revealed a lower affinity of sCD4 to gp125 as compared to gp120. Moreover, the analyses suggest that conformational changes only occur in HIV-1 gp120 upon interaction with CD4. We hypothesize that the V3 region is accessible in HIV-2 gp125 and thus may not require interaction with CD4 to induce conformational reorientation of the V1/V2 region.     

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.     

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature VWF.     

A critical role for 14-3-3zeta protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb alpha and beta subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3zeta. Here we show that a novel membrane-permeable inhibitor of 14-3-3zeta-GPIbalpha interaction, MPalphaC, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MPalphaC also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIbbeta dephosphorylation is diminished by mutagenic disruption of the 14-3-3zeta binding site in the C-terminal domain of GPIbalpha, mimicking MPalphaC-induced inhibition, indicating that the inhibitory effect of MPalphaC is likely to be caused by disruption of 14-3-3zeta binding to GPIbalpha. These data suggest a novel 14-3-3zeta-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis.     

Correlation of polymorphic variation in the promoter region of the interleukin‐1β gene with secretion of interleukin‐1β protein

Objective

Significant variation in interleukin‐1β (IL‐1β) protein secretion between subjects has been observed when using a lipopolysaccharide (LPS)/ATP–mediated ex vivo blood stimulation assay. To explore the potential relationships between genetic polymorphisms in the IL1B cytokine gene and cellular responses to inflammatory stimuli such as LPS, we investigated the hypothesis that polymorphisms within the promoter and exon 5 of the IL1B gene contribute to the observed differences in IL‐1β protein secretion.

Methods

The IL1B gene polymorphisms C−511T, T−31C, and C3954T were tested for association with LPS‐induced secretion of IL‐1β protein as measured by an ex vivo blood stimulation assay. Samples from 2 independent study populations (n = 31 and n = 25) were available for use in the ex vivo assay after consent was obtained to analyze the DNA.

Results

A specific haplotype, composed of the T allele at −511 and the C allele at −31, was significantly associated with a 2–3‐fold increase in LPS‐induced IL‐1β protein secretion. This association was observed in both of the independent study populations (P = 0.0084 and P = 0.0017).

Conclusion

These data suggest that polymorphisms within the promoter region of the IL1B gene contribute to observed differences in LPS‐induced IL‐1β protein secretion.

     

Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein

Prion disease is characterized by the α→β structural conversion of the cellular prion protein (PrP^C) into the misfolded and aggregated “scrapie” (PrP^Sc) isoform. It has been speculated that methionine (Met) oxidation in PrP^C may have a special role in this process, but has not been detailed and assigned individually to the 9 Met residues of full-length, recombinant human PrP^C [rhPrP^C(23-231)]. To better understand this oxidative event in PrP aggregation, the extent of periodate-induced Met oxidation was monitored by electrospray ionization-MS and correlated with aggregation propensity. Also, the Met residues were replaced with isosteric and chemically stable, nonoxidizable analogs, i.e., with the more hydrophobic norleucine (Nle) and the highly hydrophilic methoxinine (Mox). The Nle-rhPrP^C variant is an α-helix rich protein (like Met-rhPrP^C) resistant to oxidation that lacks the in vitro aggregation properties of the parent protein. Conversely, the Mox-rhPrP^C variant is a β-sheet rich protein that features strong proaggregation behavior. In contrast to the parent Met-rhPrP^C, the Nle/Mox-containing variants are not sensitive to periodate-induced in vitro aggregation. The experimental results fully support a direct correlation of the α→β secondary structure conversion in rhPrP^C with the conformational preferences of Met/Nle/Mox residues. Accordingly, sporadic prion and other neurodegenerative diseases, as well as various aging processes, might also be caused by oxidative stress leading to Met oxidation.     

Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain

VWF is a plasma protein that binds platelets to an injured vascular wall during thrombosis. When exposed to the shear forces found in flowing blood, VWF molecules undergo lateral self-association that results in a meshwork of VWF fibers. Fiber formation has been shown to involve thiol/disulfide exchange between VWF molecules. A C-terminal fragment of VWF was expressed in mammalian cells and examined for unpaired cysteine thiols using tandem mass spectrometry (MS). The VWF C2 domain Cys2431-Cys2453 disulfide bond was shown to be reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers when expressed in mammalian cells. Mutagenesis studies showed that both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulfide bonds were involved in oligomer formation. Our present findings imply that lateral VWF dimers form when a Cys2431 thiolate anion attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond of another VWF molecule, whereas the Cys2451-Cys2468 disulfide/dithiol mediates formation of trimers and higher-order oligomers. These observations provide the basis for exploring defects in lateral VWF association in patients with unexplained hemorrhage or thrombosis.     

A key role of toll-like receptor 3 in tissue factor activation through extracellular signal regulated kinase 1/2 pathway in a murine hypoxia model

Hypoxemia in the circulation can lead to venous thrombosis (VT) through tissue factor (TF) activation, but the mechanism of TF activation in hypoxia remains obscure. Ligands released from damaged tissues or cells due to hypoxia are identified by various pattern-recognition receptors (PRR), including Toll-like receptor3 (TLR3). In the present study, we investigated the mechanism of TF activation during acute hypoxia in a rat model. The expression of TLR3 and TF was analyzed by immunoblotting and RT-PCR. The TF activity was evaluated by two-stage chromogenic assay and fibrin deposition was detected by immunohistochemistry. The expression of TLR3, TF, and TF activity was increased significantly 6 h post acute hypoxia and then decreased gradually. The contribution of TLR3 in TF activation was investigated by poly I:C and TLR3 neutralizing antibody. We also found increased ERK phosphorylation both in acute hypoxia and poly I:C treatment. We further showed that the pre-treatment of TLR3 neutralizing antibody or ERK inhibitor (PD98059) 2 h prior to acute hypoxia or poly I:C treatment completely abrogated ERK phosphorylation and TF activation. The pre-treatment of TLR3 neutralizing antibody also inhibited fibrin deposition in lung vasculature. These data indicate that acute hypoxia induced TF activation is mediated through TLR3-ERK1/2 pathway.