Significance: Recent clinical evidence identified anemia to be correlated with severe complications of cardiovascular disease (CVD) such as bleeding, thromboembolic events, stroke, hypertension, arrhythmias, and inflammation, particularly in elderly patients. The underlying mechanisms of these complications are largely unidentified.Recent Advances: Previously, red blood cells (RBCs) were considered exclusively as transporters of oxygen and nutrients to the tissues. More recent experimental evidence indicates that RBCs are important interorgan communication systems with additional functions, including participation in control of systemic nitric oxide metabolism, redox regulation, blood rheology, and viscosity. In this article, we aim to revise and discuss the potential impact of these noncanonical functions of RBCs and their dysfunction in the cardiovascular system and in anemia.Critical Issues: The mechanistic links between changes of RBC functional properties and cardiovascular complications related to anemia have not been untangled so far.Future Directions: To allow a better understanding of the complications associated with anemia in CVD, basic and translational science studies should be focused on identifying the role of noncanonical functions of RBCs in the cardiovascular system and on defining intrinsic and/or systemic dysfunction of RBCs in anemia and its relationship to CVD both in animal models and clinical settings. Antioxid. Redox Signal. 26, 718–742.

Rapid generation of O 2 Ϫ and H 2 O 2 , which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA ( for respiratory burst oxidase homolog A ) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91 phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91 phox . This domain contains two Ca 2 ϩ binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA , which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91 phox , does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca 2 ϩ and G protein stimulation of O 2 Ϫ and H 2 O 2 production at the cell surface. INTRODUCTIONRecognition of avirulent pathogens causes a burst of oxidative metabolism generating superoxide (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ) (Baker and Orlandi, 1995). Reactive oxygen intermediates are direct protective agents and also drive cross-linking of the cell wall before the activation of transcription-dependent defenses (Lamb and Dixon, 1997). Moreover, the oxidative burst can trigger the collapse of challenged host cells at the onset of the hypersensitive response and generate signals for defense gene induction (Levine et al., 1994; Jabs et al., 1997).The kinetics and defense functions of O 2 Ϫ and H 2 O 2 generation are reminiscent of the oxidative burst during neutrophil activation ( Tenhaken et al., 1995;Lamb and Dixon, 1997). The neutrophil oxidative burst involves the reaction O 2 ϩ NADPH → O 2 Ϫ ϩ NADP ϩ ϩ H ϩ catalyzed by a plasma membrane oxidase, followed by dismutation of O 2 Ϫ to H 2 O 2 (Taylor et al., 1993). The NADPH oxidase consists of two plasma membrane proteins, gp91 phox and p22 phox ( phox for phagocyte oxidase), which together form heterodimeric flavocytochrome b Ϫ 558 and three cytosolic regulatory proteins, p40 phox , p47 phox , and p67 phox , which translocate to the plasma membrane after stimulation to form the active complex (Bokoch, 1994). The small cytosolic GTPase Rac2 and possibly other G proteins also appear to be required for activation of the oxidase.A membrane-bound enzyme resembling the neutrophil NADPH oxidase may contribute to the pathogen-induced oxidative burst in plants. Thus, O 2 Ϫ generation can be observed in microsomal preparat...

Polyelectrolyte multilayer films were employed to support attachment of cultured rat aortic smooth muscle A7r5 cells. Like smooth muscle cells in vivo, cultured A7r5 cells are capable of converting between a nonmotile "contractile" phenotype and a motile "synthetic" phenotype. Polyelectrolyte films were designed to examine the effect of surface charge and hydrophobicity on cell adhesion, morphology, and motility. The hydrophobic nature and surface charge of different polyelectrolyte films significantly affected A7r5 cell attachment and spreading. In general, hydrophobic polyelectrolyte film surfaces, regardless of formal charge, were found to be more cytophilic than hydrophilic surfaces. On the most hydrophobic surfaces, the A7r5 cells adhered, spread, and exhibited little indication of motility, whereas on the most hydrophilic surfaces, the cells adhered poorly if at all and when present on the surface displayed characteristics of being highly motile. The two surfaces that minimized cell adhesion consisted of two varieties of a diblock copolymer containing hydrophilic poly(ethylene oxide) and a copolymer bearing a zwitterionic group AEDAPS, (3-[2-(acrylamido)-ethyldimethyl ammonio] propane sulfonate). Increasing the proportion of AEDAPS in the copolymer decreased the adhesion of cells to the surface. Cells presented with micropatterns of cytophilic and cytophobic surfaces generated by polymer-on-polymer stamping displayed a surface-dependent cytoskeletal organization and a dramatic preference for adhesion to, and spreading on, the cytophilic surface, demonstrating the utility of polyelectrolyte films in manipulating smooth muscle cell adhesion and behavior.

Nontraumatic osteonecrosis continues to be a challenging problem causing debilitating major joint diseases. The etiology is multifactorial, but steroid- and alcohol-induced osteonecrosis contribute to more than two thirds of all cases with genetic risk factors playing an important role in many other cases, especially when they contribute to hypercoagulable states. While the exact mechanisms remain elusive, many new insights have emerged from research in the last decade that have given us a clearer picture of the pathogenesis of nontraumatic osteonecrosis of the femoral head. Progression to end stage osteonecrosis of the femoral head appears to be related to four main factors: interactions involving the differentiation pathway of osteoprogenitor cells that promote adipogenesis, decreased angiogenesis, direct suppression of osteogenic gene expression and proliferation of bone marrow stem cells, and genetic anomalies or other diseases that promote hypercoagulable states.

Septic shock and multiple organ failure may be associated with coagulation activation, disseminated fibrin formation, and consumption of coagulation inhibitors such as antithrombin III. We have evaluated prospectively coagulation measurements in patients with severe chemotherapy-induced neutropenia. This group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing before and during evolving sepsis or septic shock. Sixty-two patients with febrile infectious events were accrued to the study. Of these, 13 patients progressed to severe sepsis and 13 additional patients to septic shock as defined according to standard diagnostic criteria. At the onset of fever, factor (F) VIIa activity, FVII antigen and antithrombin III (AT III) activity decreased from normal baseline levels and were significantly lower in the group of patients who progressed to septic shock compared with those that developed severe sepsis (medians: 0.3 v 1.4 ng/mL, 21 v 86 U/dL and 45% v 95%; P < .001). The decrease of these measurements in septic shock was accompanied by an increase in prothrombin fragment 1+2 (median: 3.6 v 1.4 nmol/L; P = .05), a marker of thrombin generation. These differences were sustained throughout the septic episode (P < .0001). FVIIa and AT III levels of < 0.8 ng/mL and < 70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100% and 85%, and a specificity of 75% and 85%, respectively. In contrast, FXIIa-alpha antigen levels were not different between groups at onset of fever but increased modestly during the course of septic shock (P = .001). Thus, septic shock in neutropenic patients is associated with increased thrombin generation. Furthermore, both FVIIa and AT III measurements are sensitive markers of an unfavorable prognosis.

We have analyzed terminal web contraction in sheets of glycerinated chicken small intestine epithelium and in isolated intestinal brush borders using a quick-freeze, deep-etch, rotary shadow replication technique. In the presence of Mg-ATP at 37°C, the terminal web region of each cell in the glycerinated sheet and of each isolated brush border became severely constricted at the level of its zonula adherens (ZA). Consequently, the individual brush borders rounded up, splaying out their microvilli in fanlike patterns. The most prominent ultrastructural changes that occurred during terminal web contraction were a dramatic decrease in the diameter of the circumferential ring composed of a bundle of 8-9-nm filaments adjacent to the zonula adherens and a decrease in the number of cross-linkers between the microvillus rootlets. Microvilli were not retracted into the terminal web. We have used myosin $1 decoration to demonstrate that most of the circumferential bundle filaments are actin and that the actin filaments are arranged in the bundle with mixed polarity. Some filaments within the bundle did not decorate with myosin $1 and had tiny projections that appeared to be attached to adjacent actin filaments. Because of their morphology and immunofluorescent localization of myosin within this region of the terminal web, we propose that these undecorated filaments are myosin. From these results, we conclude that brush border contraction is caused primarily by an active sliding of actin and myosin filaments within the circumferential bundle of filaments associated with the ZA.The brush border of epithelial cells in the small intestine is composed of highly differentiated cellular cortices. The relative ease with which brush borders can be isolated has facilitated correlating biochemical with ultrastructural studies in order to better understand the cytoskeletal structures of the brush border (28).Previous studies have shown that contractile proteins, such as actin, myosin, and light chain kinase of myosin are all constituents of the brush border (3,7,23,30). In addition, two in vitro phenomena suggested that brush borders might move in vivo. One was a shortening of microvilli after incubation with Ca++-ATP (26) and the other was a "pinching in" of the terminal web at the level of the zonula adherens in the presence of ATP (37).Recently, it has been proved that the former is due to solution of actin core bundles by the Ca++-sensitive protein villin or I l,25,27). Also, the quick freeze-deep etch (QF-

Smooth muscle cells convert between a motile, proliferative “synthetic” phenotype and a sessile, “contractile” phenotype. The ability to manipulate the phenotype of aortic smooth muscle cells with thin biocompatible polyelectrolyte multilayers (PEMUs) with common surface chemical characteristics but varying stiffness was investigated. The stiffness of (PAH/PAA) PEMUs was varied by heating to form covalent amide bond cross-links between the layers. Atomic force microscopy (AFM) showed that cross-linked PEMUs were thinner than those that were not cross-linked. AFM nanoindentation demonstrated that the Young’s modulus ranged from 6 MPa for hydrated native PEMUs to more than 8 GPa for maximally cross-linked PEMUs. Rat aortic A7r5 smooth muscle cells cultured on native PEMUs exhibited morphology and motility of synthetic cells and expression of the synthetic phenotype markers vimentin, tropomyosin 4, and nonmuscle myosin heavy chain IIB (nmMHCIIB). In comparison, cells cultured on maximally cross-linked PEMUs exhibited the phenotype markers calponin, smooth muscle myosin heavy chain (smMHC), myocardin, transgelin, and smooth muscle α-actin (smActin) that are characteristic of the smooth muscle “contractile” phenotype. Consistent with those cells being “contractile”, A7r5 cells grown on cross-linked PEMUs produced contractile force when stimulated with a Ca2+ ionophore.

We have reinvestigated the effects of Ca ++ and ATP on brush borders isolated from intestinal epithelial cells. At 37°C, Ca ++ (1/tM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37°C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca ++ (1 /xM), and occurs in both membrane-intact and demembranated brush borders. Ca÷÷-dependent-solation of microvillus cores requires a concentration of Ca ÷+ slightly greater (10/~M) than that required for contraction.Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC2o), is stimulated by Ca ÷+. At 37°C, BBMLC2o phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC2o phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca ++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca +÷-and calmodulin-dependent brush border myosin light chain kinase.Ca ++ has been implicated as a regulator of both motility and cytoplasmic structure in nonmuscle ceils. For example, changes in free Ca ++ concentration may regulate motility in vertebrate nonmuscle and smooth muscle cells by controlling phosphorylation of the regulatory light chain of myosin by a Ca ++-calmodulin-dependent myosin light chain kinase. Phosphorylation of the regulatory light chain of smooth and nonmuscle myosin increases its actin-activated Mg++-ATPase activity, presumably increasing force production and causing motility (for review, see reference 1).Ca ++ may also regulate cytoplasmic structure by disrupting the bundles or isotropic networks of cross-linked actin filaments that have been postulated to contribute to the gel-like properties of cytoplasm (see reference 12, for review). Raising the concentration of free Ca ++ in cytoplasmic extracts disrupts supramolecular actin filament structures. This indicates that Ca ++ may regulate the rapid changes in cytoplasmic viscosity that can occur in vivo. Recently, two major classes of Ca ++-sensitive actin-binding proteins which could modulate the reversible breakdown of actin filament structures have been identified. These include proteins which cross-link actin filaments in the absence, but not in the presence, of Ca ++ (9,50,58,60,79) and proteins which reversibly cut or shorten filaments in the presence of Ca ++ (6,13,26,30,46,53). The mechanism by which changes in structure are coordinated with motility in vivo is still a matter of speculation (32).One of the most extensively ordered arrays of contractile prote...