NK4 therapy: a new approach to target angiogenesis and inflammation in rheumatoid arthritis

Rheumatoid arthritis (RA) is a progressive autoimmune disease characterized by synovial membrane hyperplasia, inflammation, and angiogenesis. Hepatocyte growth factor (HGF) and its receptor, c-Met, are both overexpressed in the RA synovium. NK4 is an antagonist of HGF which has been shown to inhibit tumor growth, metastasis, and angiogenesis. In an experimental model of RA, NK4 gene therapy inhibited joint damage and inflammation in both preventative and therapeutic models. NK4 treatment therefore represents a possible therapeutic option in combating RA.Rheumatoid arthritis (RA) is a progressive, inflammatory autoimmune disease characterized by an erosive synovitis. In addition to being an inflammatory condition, RA is also considered to be a member of the angiogenic family of diseases. Angiogenesis is growth of new blood vessels from pre-existing blood vessels. As the disease progresses, the hyperplastic synovial pannus creates a hypoxic, inflammatory environment that induces angiogenesis. Further vascularization of the synovial tissue promotes pannus growth and continued infiltration of inflammatory leukocytes, thus perpetuating the disease.In the previous issue of Arthritis Research & Therapy, Tsunemi and colleagues [1] reported on the targeting of hepatocyte growth factor (HGF) by NK4 in the treatment of RA. HGF is a pleiotropic growth factor that is expressed by mesenchymal cells and promotes processes such as mitogenesis, differentiation, and angiogenesis [2]. It mediates these functions via binding to its unique receptor c-Met, a receptor tyrosine kinase. c-Met is expressed by a variety of cell types, including endothelial cells (ECs) [3].We have previously shown that HGF is elevated in the synovial fluid of patients with RA [4]. More recently, Grandaunet and colleagues [5] found that plasma levels of HGF predict the severity of joint damage in patients with RA. In the joint, we found that HGF and c-Met are elevated in the RA synovial lining compared with normal controls [4]. The report by Tsunemi and colleagues [1] supports these findings and further shows that c-Met is expressed on fibroblasts, mononuclear cells, and ECs in the RA synovium.HGF is a heterodimeric protein composed of an ?-chain, which contains four kringle domains, and a ?-chain [6]. The ?-chain binds c-Met with high affinity, whereas the ?-chain is responsible for activation of c-Met. In an attempt to inhibit HGF, Date and colleagues [7] generated a cleavage product of HGF termed NK4, which contains the four kringle domains of the HGF ?-chain. Therefore, NK4 serves as an antagonist of HGF and can bind c-Met with high affinity without activating it.As described above, one of the primary functions of HGF is to induce angiogenesis by binding to c-Met on the surface of ECs. Therefore, it was postulated that NK4 would act as a competitive inhibitor of HGF, thus inhibiting angiogenesis. Indeed, NK4 has been shown to inhibit angiogenesis in vitro and in various in vivo cancer models [6,8,9]. However, in addition to having antagonistic action against HGF, NK4 inhibits angiogenesis induced by vascular endothelial growth factor and basic fibroblast growth factor in a c-Met-independent fashion [9]. In addition to c-Met, NK4 binds to perlecan, a sulfate proteoglycan that interacts with the vascular endothelial basement membrane. Sakai and colleagues [9] found, specifically, that NK4 binds perlecan and prevents proper fibronectin assembly in the basement membrane, which inhibits several facets of angiogenesis.These features of NK4 make it an attractive potential adjunctive therapy in angiogenic diseases. Over the past decade, numerous studies have been performed to assess the efficacy of either a recombinant NK4 protein or NK4 gene expression vector in many experimental cancer models [3,6]. Collectively, these studies have indicated that NK4 treatment has the potential to inhibit tumor growth, angiogenesis, and metastasis [3,6]. Much of the preclinical success of NK4 can be attributed to its ability to inhibit multiple pathways involved in growth and angiogenesis.RA is driven by inflammation and angiogenesis, and thus much work has been aimed at identifying and testing potential angiogenesis inhibitors in models of experimental arthritis [10]. Tsunemi and colleagues [1] have now adopted their approach of studying the antiangiogenic properties of NK4 in cancer to experimental arthritis. Using an adenovirus vector containing the NK4 gene, they found that NK4 inhibited the development of ?-glucan-induced arthritis [1]. NK4 was able to inhibit inflammation, joint swelling, and bone erosion. However, the authors did not show direct evidence of NK4 inhibiting synovial blood vessel density. Importantly, they also showed that NK4 gene therapy was effective when given therapeutically, after the onset of the experimental arthritis [1].These results are highly encouraging in the application of NK4 as a potential adjunctive RA therapy. This report, coupled with the high expression levels of HGF and c-Met in the RA synovium, makes NK4 treatment an intriguing possibility. In the future, it will be of great interest to determine whether these effects of NK4 are observable in other animal models of RA, as not all facets of RA are represented in a singular model of the disease. Moreover, many of the effects of NK4 observed by Tsunemi and colleagues [1] are attributed to a reduction in inflammation and inflammatory cytokines. Therefore, elucidating the anti-inflammatory and antiangiogenic mechanisms of NK4 will be paramount to transitioning from an interesting candidate to a possible RA therapy.     

Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.Angiogenesis, the formation of blood vessels from pre-existing blood vessels, is a complex process essential for repairing injured tissue or supporting tissue growth. A great deal of work has been done to focus on understanding this phenomenon as it occurs in vivo, in particular with regard to its roles in embryonic development (1–5). In contrast to embryonic development, adult angiogenesis and inflammation are closely related phenomena that occur in vivo in a number of physiologically relevant processes. Inflammation lies at the crux of multiple physiological events in biological systems that precede the induction of angiogenesis: wound healing (6–8), chronic wounds (8), inflammatory disorders (9, 10), and cancer (9, 11, 12).Inflammatory reactions also confound tissue engineered implantable three-dimensional constructs that provide innovative clinical treatments of various diseases and injuries (13–17). As complex tissues become developed for applications in clinical trials, tissue vascularization for constructs of considerable size and volume is required for their survival (18, 19). Once implanted, these constructs will also experience significant inflammatory responses within their host''s local milieu (20, 21). These circumstances demonstrate the necessity for understanding the interactions between inflammation and angiogenesis, such as the development of predictive models (22). Elucidating specific intracellular mechanisms can provide insight for novel approaches in treatment of diseases as well as predicting responses to artificially engineered tissues.Recently, studies have shown that chemokines, which play a central role in inflammation, can influence the outcomes of angiogenesis (23–26) by promoting new blood vessel growth (e.g. CXCL1–3, CXCL5–8, CXCL12) or inhibiting its formation altogether (e.g. CXCL4, CXCL9–11, CXCL13) (26). In particular, a large body of information is available on platelet factor 4 (PF-4/CXCL4) and its ability to inhibit and even induce regression of angiogenesis. PF-4 is found throughout the adult body, at roughly 0.25–1.25 nm (2–10 ng/ml) in blood plasma, but as high as 25 μm in localized areas during wound healing (27, 28). Its ubiquitous presence, implication in cancer and vascular diseases, and use as a potential drug therapy have made PF-4 a key point of interest in influencing angiogenesis in vivo (27–30). In addition to inducing angiostasis, PF-4 can inhibit cell proliferation by halting S phase progression and reducing endothelial cell migration (25, 28, 30–32). Despite the wealth of information on PF-4 and its mechanistic effects on immune cells, scarce literature exists on the nature of the molecular signaling with endothelial cells to inhibit angiogenesis. Furthermore, the complexity of PF-4 mediated signaling and its potential to interact through multiple binding mechanisms makes it difficult to determine how PF-4 can interfere with angiogenesis (28, 29, 33, 34). Possible angiogenic signaling network interference mechanisms for PF-4 include the sequestration of growth factors and proteoglycans, antagonism of integrin-mediated signaling, or direct signaling through its chemokine receptor CXCR3, all of which have supporting evidence in previous literature (28). Along with the multiple mechanisms PF-4 may utilize for signaling, only limited studies on direct signaling elicited by PF-4 on endothelial cells have been reported; one of interest found that P38 MAPK can be activated via CXCR3 on endothelial cells cultured on plastic (35), whereas another, more definitive study showed PF-4 acting similarly to other CXCR3 ligands in activating PKA to prevent m-calpain-mediated rear de-adhesion of moving cells (36, 37). Furthermore, PF-4 could have variable sensitivities in different endothelial cell types because of heterogeneous expression of CXCR3 (38).In our study, we sought to develop an approach to assess network-level signaling interactions between PF-4 and the major angiogenic inducer vascular endothelial growth factor (VEGF)¹ within a contextually relevant 3-D angiogenesis platform, in a controlled environment to understand what role these two factors may play. We developed methods to reduce extracellular matrix contamination in our samples and were able to successfully use a two-step lysis method with a MS compatible detergent-based lysis buffer. By taking advantage of iTRAQ-based multiplexed quantitation, we were able to collect quantitative phosphoprotein signaling data from our system with early and late temporal resolution. Using correlation network methods to observe differences in our system, we found that simultaneous treatment with PF-4 and VEGF induced changes in migrational pathway topology when compared with VEGF treatment alone. Most often, these changes appeared as losses in correlations between different migrational signaling proteins. We found that several different signaling pathways involved with migration were affected, including central proteins P38α MAPK, focal adhesion kinase (FAK), and Src family kinases. Furthermore, we found statistically significant differences in tyrosine phosphorylation when HDMVECs were stimulated with VEGF and PF-4, as opposed to only VEGF. In addition, we were able to recapitulate previously reported findings on how PF-4 infers its angiostatic effects on endothelial cells. Surprisingly, our data set revealed EphA2 receptor as a central node for PF-4 signaling, indicating that it may possess a complementary role in the balance of angiogenic and angiostatic effects.To our knowledge, this is the first attempt at performing MS-based analysis of phosphotyrosine signaling networks within the context of an environment that is amenable to angiogenesis. Our work provides a step forward in applying high throughput and systems-level phosphoproteomics data collection to more physiologically relevant experimental conditions.     

LFA-1 and CD2 Synergize for the Erk1/2 Activation in the Natural Killer (NK) Cell Immunological Synapse

Natural killer (NK) cell recognition and formation of a conjugate with target cells, followed by intracellular signal pathway activation and degradation of cytolytic granules, are essential for NK cell cytotoxicity. In this study, NK92 cells were used to investigate synapse formation and subsequent signaling after binding to the target cell. The binding rate of the NK92-target cell was associated with NK92 cell cytotoxicity. Confocal results showed that adhesion molecules, LFA-1 (CD11a) and CD2, accumulated at the interface of the NK92-K562 contact. Ligation with K562 cells activated the Erk1/2 signal pathway of NK92 cells. The blocking of the NK-target conjugate by EDTA or anti-CD11a or/and anti-CD2 antibody decreased the phosphorylation of Erk1/2 and NK cell cytotoxicity. Inhibition of Erk1/2 phosphorylation by the chemical inhibitor U0126 suppressed the cytolytic activity of NK92 cells, but had no effect on NK-target conjugate formation. Thus, conjugate formation of the NK92-target cell was prerequisite to NK cell activation, and subsequent signal transduction was also required for NK cell cytotoxicity.Natural killer (NK)³ cells are a population of granular lymphocytes that play an essential role in cellular immune defense against a variety of tumor cells, virus-infected cells, or allogeneic cells (1–3). NK cells are critical for host immunity for their ability for a quick cytotoxic response and to produce a wide variety of cytokines and chemokines to modulate other cellular components of the immune system (4, 5). NK cells express two functional types of receptors: activating and inhibitory receptors (6–8). The effector function of NK cells is regulated by a balance between opposite signals delivered by the MHC class I-specific inhibitory receptors and the activating receptors responsible for NK cell triggering to permit elimination of pathogens (6).NK cell recognition and binding to target cells, as well as formation of conjugates, are essential for NK cell cytotoxicity (9). Conjugate formation by the NK cell with a target cell is a process mediated by integrins and immunoglobulin superfamily molecules including CD2, CD11a (LFA-1), CD11b, CD11c, and CD28, which also participate in the promotion of NK cell function (10–12). They participate in adhesion between the NK cell and the target cell, and blocking antibodies suppress the adhesion. In addition to possessing an adhesive role, ligation of CD2 induces kinase function and lipid raft polarization (11), whereas ligation of CD11a, CD11b, and CD11c induces phosphorylation-dependent NK cell activation (13, 14). The interaction of specific cell surface receptors with their ligands on a target cell at their interface forms specific activating NK cell immunological synapses and leads to the activation of a cascade of intracellular signals, resulting in Ca²⁺ flux, polarization of granules, and subsequent release of lytic molecules (13, 15, 16). The Erk1/2 (p44/42 mitogen-activated protein kinase) pathway plays an important role in NK cell cytotoxicity (17–21). Inhibition of Erk1/2 might block NK cell cytolytic activity by compromising the release of perforin (22). In this study, the roles of adhesion molecules in NK92-target cell conjugate formation of immunological synapse, and subsequent Erk1/2 activation in NK92 cells was investigated.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Adenomatous Polyposis Coli and Asef Function Downstream of Hepatocyte Growth Factor and Phosphatidylinositol 3-Kinase

Mutations of the tumor suppressor adenomatous polyposis coli (APC) are responsible for sporadic and familial colorectal tumors. APC negatively regulates Wnt signaling by inducing β-catenin degradation. It has also been shown that APC plays a role in the organization of cytoskeletal networks. APC interacts with Asef and Asef2, Rac1- and Cdc42-specific guanine nucleotide exchange factors (GEFs), and stimulates their GEF activity; thereby regulating cell morphology, adhesion, and migration. Truncated mutant APCs present in colorectal tumor cells activate Asef and Asef2 constitutively and contribute to their aberrant migratory properties. We show here that hepatocyte growth factor (HGF), as well as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), induce the accumulation and colocalization of APC and Asef in membrane ruffles and lamellipodia of epithelial cells. Both APC and Asef were found to be required for HGF-induced cell migration. Furthermore, we show that the effects of HGF, bFGF, and EGF on APC and Asef are mediated by the activation of phosphatidylinositol 3-kinase (PI3-kinase) and require the PH domain of Asef. These results suggest that Asef and APC function downstream of HGF and PI3-kinase, and play critical roles in growth factor-mediated regulation of cell morphology and migration.Mutations of the tumor suppressor gene adenomatous polyposis coli (APC)² are responsible for familial adenomatous polyposis (FAP), a dominantly inherited disease characterized by multiple adenomatous polyps in the colon (1, 2). The APC gene is also somatically mutated in the majority of sporadic colorectal tumors. The majority of the somatic mutations in APC is confined to its central region and result in the generation of truncated gene products. It is well known that APC induces degradation of β-catenin, a key Wnt signaling effector (3–6). Furthermore, it has recently been shown that APC also interacts with various other cellular proteins, including Asef, Asef2, IQGAP1, and kinesin-2, and regulates the organization of cytoskeletal networks, thereby controlling cell adhesion and motility (7–15).Asef is a guanine-nucleotide exchange factor (GEF) specific for Rac1 and Cdc42 (9–11, 15, 16). APC interacts via its armadillo repeat domain with an APC-binding region (ABR) in the NH₂ terminus of Asef. In addition to this ABR, Asef contains Dbl homology (DH), Pleckstrin homology (PH), and Src homology 3 (SH3) domains. The SH3 domain of Asef inhibits its own GEF activity by intramolecular binding to the DH domain (17, 18). The PH domain of Asef binds to phosphatidylinositol 3,4,5-trisphosphate (PIP3) and is required for its localization to the plasma membrane (19). APC enhances the GEF activity of Asef, presumably by relieving the intramolecular negative regulation and thereby regulates cell morphology, adhesion, and migration. A mutant form of Asef lacking the ABR shows strong GEF activity even in the absence of APC. Furthermore, truncated mutant APCs present in colorectal tumor cells activate Asef constitutively and cause increased aberrant migration. APC also activates Asef2, which has significant structural and functional similarities to Asef (11, 15). Thus, truncated mutant APCs, Asef and Asef2 may be important for adenoma formation as well as tumor progression to invasive malignancy.HGF is known to be important for embryonic development, wound healing, tissue regeneration, hematopoiesis, and tissue homeostasis (20, 21). The HGF receptor, which is encoded by the proto-oncogene c-met, is a tyrosine kinase, and its activation by HGF induces cell motility, invasion, and proliferation. Furthermore, HGF signaling is known to play a crucial role in tumor development and malignant progression, in particular by increasing tumor invasiveness and metastatic potential. Because the effects of APC-activated Asef on MDCK cells appear to be similar to those of HGF, we attempted to examine whether APC and Asef function downstream of HGF. In the present study, we show that APC and Asef indeed function downstream of HGF and that Asef is required for HGF-induced migration.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs)³ and a characteristic C-terminal fibulin (FC) domain (1–3). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4–7). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9, 10). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6, 7), and there is altered vascular remodeling (11). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12–15). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch''s membrane of the eye (16) and vaginal tissues (17).Elastic fiber formation is a complex multistep process (18–20). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4–7, 21–25). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4).Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26). However, fibulin-4^+/− mice showed ∼20% increase in desmosine (5). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27, 28). In contrast, LOXL1-null mice are viable but have reduced desmosine (29), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30).We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31–34). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils.To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Vascular Endothelial Growth Factor (VEGF) Receptor-2 Tyrosine 1175 Signaling Controls VEGF-induced von Willebrand Factor Release from Endothelial Cells via Phospholipase C-��1- and Protein Kinase A-dependent Pathways

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-γ1 (PLCγ1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr¹¹⁷⁵ in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLCγ1 and PKA. These data suggest that Tyr¹¹⁷⁵ mediates both PLCγ1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr¹¹⁷⁵-mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.Vascular endothelial growth factor (VEGF)² is a crucial regulator of vasculogenesis, angiogenesis, and vascular permeability (1–5). A number of studies have suggested that VEGF promotes proliferation, migration, and survival of endothelial cells (1, 4). VEGF (also termed VEGF-A) is a member of the growth factor subfamily that includes VEGF-B, -C, -D, and -E and placental growth factor (PlGF). VEGF binds to two high affinity tyrosine kinase receptors, VEGFR1 (also known as Flt-1) and VEGFR2 (also known as KDR/Flk-1), whereas VEGF-E binds to VEGFR2 alone, and PlGF binds to VEGFR1 alone. Within the vessel wall, VEGFR2 is selectively expressed in endothelium. In contrast, VEGFR1 is present on both endothelial cells and monocytes (1, 2).In addition to its role in promoting angiogenesis, there is increasing evidence that VEGF contributes to inflammation independent of its angiogenic functions, although the molecular basis for this effect is incompletely understood (6–8). VEGF is well expressed in the chronic inflammatory skin disease, psoriasis, and in synovial fluid in rheumatoid arthritis (9–12). In addition, previous studies found an association between human severe sepsis/septic shock with elevated circulating levels of VEGF and PlGF (13, 14). Using an in vitro monocyte migration assay and in vivo mouse models of arthritis, several groups, including ours, have suggested that one mechanism by which VEGF causes inflammation is by modulating the infiltration and secretion of monocytes/macrophages via the activation of VEGFR1 (11, 12, 15). On the other hand, emerging evidence suggests that endothelial activation is also important for VEGF-induced inflammation (6, 8, 9). In a mouse model of sepsis, it was demonstrated that the inhibition of VEGFR2, but not VEGFR1, attenuates sepsis mortality, possibly at least in part by suppressing vascular inflammation associated with endothelial activation (9). Consistent with this, ectopic VEGF-A expression in mice enhances leukocyte rolling and adhesion in venules mediated through the P-selectin on the surface of endothelial cells (6). These studies indicate that endothelial activation is another mechanism for VEGF-induced inflammation.P-selectin and von Willebrand factor (vWF) are the best characterized constituents of Weibel-Palade bodies (WPBs), endothelial storage granules that also contain various inflammatory mediators (16–18). As a major component in WPBs, vWF is also involved in their biogenesis and thus is used as a marker of WPBs (18, 19). WPB exocytosis, which gives rise to rapid release of vWF and other mediators such as interleukin-8 (IL-8) (17), and translocation of P-selectin from within granules to the endothelial surfaces triggering leukocyte rolling, are critical early events in endothelial activation and vascular inflammation (16). It has been reported that VEGF regulates vWF/WPB release (20), but the precise roles of VEGF receptors and their downstream effectors in this process have not been defined. In this study, we sought to dissect the signaling pathway by which VEGF induces vWF/WPB release.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.