A hyperspectral plant health monitoring system for space crop production.

Compact and automated sensing systems are needed to monitor plant health for NASA's controlled-environment space crop production. A new hyperspectral system was designed for early detection of plant stresses using both reflectance and fluorescence imaging in visible and near-infrared (VNIR) wavelength range (400-1000 nm). The prototype system mainly includes two LED line lights providing VNIR broadband and UV-A (365 nm) light for reflectance and fluorescence measurement, respectively, a line-scan hyperspectral camera, and a linear motorized stage with a travel range of 80 cm. In an overhead sensor-to-sample arrangement, the stage translates the lights and camera over the plants to acquire reflectance and fluorescence images in sequence during one cycle of line-scan imaging. System software was developed using LabVIEW to realize hardware parameterization, data transfer, and automated imaging functions. The imaging unit was installed in a plant growth chamber at NASA Kennedy Space Center for health monitoring studies for pick-and-eat salad crops. A preliminary experiment was conducted to detect plant drought stress for twelve Dragoon lettuce samples, of which half were well-watered and half were under-watered while growing. A machine learning method using an optimized discriminant classifier based on VNIR reflectance spectra generated classification accuracies over 90% for the first four days of the stress treatment, showing great potential for early detection of the drought stress on lettuce leaves before any visible symptoms and size differences were evident. The system is promising to provide useful information for optimization of growth environment and early mitigation of stresses in space crop production.

The Future of the COVID-19 Pandemic: How Good (or Bad) Can the SARS-CoV2 Spike Protein Get?

Severe acute respiratory syndrome virus 2 (SARS-CoV2) has infected an estimated 400 million people world-wide, causing approximately 6 million deaths from severe coronavirus disease 2019 (COVID-19). The SARS-CoV2 Spike protein plays a critical role in viral attachment and entry into host cells. The recent emergence of highly transmissible variants of SARS-CoV2 has been linked to mutations in Spike. This review provides an overview of the structure and function of Spike and describes the factors that impact Spike's ability to mediate viral infection as well as the potential limits to how good (or bad) Spike protein can become. Proposed here is a framework that considers the processes of Spike-mediated SARS-CoV2 attachment, dissociation, and cell entry where the role of Spike, from the standpoint of the virus, is to maximize cell entry with each viral-cell collision. Key parameters are identified that will be needed to develop models to identify mechanisms that new Spike variants might exploit to enhance viral transmission. In particular, the importance of considering secondary co-receptors for Spike, such as heparan sulfate proteoglycans is discussed. Accurate models of Spike-cell interactions could contribute to the development of new therapies in advance of the emergence of new highly transmissible SARS-CoV2 variants.

Lipid Raft Association Stabilizes VEGF Receptor 2 in Endothelial Cells.

The binding of vascular endothelial growth factor A (VEGF) to VEGF receptor-2 (VEGFR-2) stimulates angiogenic signaling. Lipid rafts are cholesterol-dense regions of the plasma membrane that serve as an organizational platform for biomolecules. Although VEGFR2 has been shown to colocalize with lipid rafts to regulate its activation, the effect of lipid rafts on non-activated VEGFR2 has not been explored. Here, we characterized the involvement of lipid rafts in modulating the stability of non-activated VEGFR2 in endothelial cells using raft disrupting agents: methyl-ß-cyclodextrin, sphingomyelinase and simvastatin. Disrupting lipid rafts selectively decreased the levels of non-activated VEGFR2 as a result of increased lysosomal degradation. The decreased expression of VEGFR2 translated to reduced VEGF-activation of the extracellular signal-regulated protein kinases (ERK). Overall, our results indicate that lipid rafts stabilize VEGFR2 and its associated signal transduction activities required for angiogenesis. Thus, modulation of lipid rafts may provide a means to regulate the sensitivity of endothelial cells to VEGF stimulation. Indeed, the ability of simvastatin to down regulate VEGFR2 and inhibit VEGF activity suggest a potential mechanism underlying the observation that this drug improves outcomes in the treatment of certain cancers.

Therapeutic Ultrasound Improves Myocardial Blood Flow and Reduces Infarct Size in a Canine Model of Coronary Microthromboembolism.

BACKGROUND: Therapeutic ultrasound (TUS) has been used to lyse infarct-related coronary artery thrombus. There has been no study examining the effect of TUS specifically on myocardial microthromboemboli seen in acute myocardial infarction and acute coronary syndromes. The aim of this study was to test the hypothesis that TUS improves myocardial blood flow (MBF) and reduces infarct size (IS) in this situation by dissolving myocardial microthrombi. METHODS: An open-chest canine model of myocardial microthromboembolism was created by disrupting a thrombus in the left anterior descending coronary artery, and 1.05- and 0.25-MHz TUS (n = 7 each) delivered epicardially for 30 min was compared with control (n = 6). MBF and IS (as a percentage of left anterior descending coronary artery perfusion bed size) were measured 60 min after treatment. In addition, immunohistochemistry was performed to assess microthrombi, and histopathology was performed to define inflammation. RESULTS: Transmural, epicardial, and endocardial myocardial blood volume and MBF (measured using myocardial contrast echocardiography) and percentage wall thickening were significantly higher 60 min after receiving TUS compared with control. The ratio of IS to left anterior descending coronary artery perfusion bed size was significantly smaller (P = .03) in the 1.05-MHz TUS group (0.14 ± 0.04) compared with the control (0.31 ± 0.06, P = .04) and 0.25-MHz (0.36 ± 0.08) groups. MBF versus percentage wall thickening exhibited a linear relation (r = 0.65) in the control and 1.05-MHz TUS groups but not in the 0.25-MHz TUS group (r = 0.29). The presence of myocardial microemboli in vessels >10 µm in diameter was significantly reduced in the 1.05-MHz TUS group compared with the other two groups. The distribution and intensity of inflammation was higher in the 0.25-MHz TUS group compared with the other groups. CONCLUSIONS: TUS at 1.05 MHz is effective in restoring myocardial blood volume and MBF, thus reducing IS by clearing the microcirculation of microthrombi. IS reduction is not seen at 0.25 MHz, despite improvement in MBF, which may be related to the increased inflammation noted at this frequency. Because both acute myocardial infarction and acute coronary syndromes are associated with microthromboembolism, these results suggest that TUS could have a potential adjunctive role in the treatment of both conditions.

Ranolazine may exert its beneficial effects by increasing myocardial adenosine levels.

We hypothesized that ranolazine-induced adenosine release is responsible for its beneficial effects in ischemic heart disease. Sixteen open-chest anesthetized dogs with noncritical coronary stenosis were studied at rest, during dobutamine stress, and during dobutamine stress with ranolazine. Six additional dogs without stenosis were studied only at rest. Regional myocardial function and perfusion were assessed. Coronary venous blood was drawn. Murine endothelial cells and cardiomyocytes were incubated with ranolazine and adenosine metabolic enzyme inhibitors, and adenosine levels were measured. Cardiomyocytes were also exposed to dobutamine and dobutamine with ranolazine. Modeling was employed to determine whether ranolazine can bind to an enzyme that alters adenosine stores. Ranolazine was associated with increased adenosine levels in the absence (21.7 ± 3.0 vs. 9.4 ± 2.1 ng/mL, P < 0.05) and presence of ischemia (43.1 ± 13.2 vs. 23.4 ± 5.3 ng/mL, P < 0.05). Left ventricular end-systolic wall stress decreased (49.85 ± 4.68 vs. 57.42 ± 3.73 dyn/cm2, P < 0.05) and endocardial-to-epicardial myocardial blood flow ratio tended to normalize (0.89 ± 0.08 vs. 0.76 ± 0.10, P = nonsignificant). Adenosine levels increased in cardiac endothelial cells and cardiomyocytes when incubated with ranolazine that was reversed when cytosolic-5'-nucleotidase (cN-II) was inhibited. Point mutation of cN-II aborted an increase in its specific activity by ranolazine. Similarly, adenosine levels did not increase when cardiomyocytes were incubated with dobutamine. Modeling demonstrated plausible binding of ranolazine to cN-II with a docking energy of -11.7 kcal/mol. We conclude that the anti-adrenergic and cardioprotective effects of ranolazine-induced increase in tissue adenosine levels, likely mediated by increasing cN-II activity, may contribute to its beneficial effects in ischemic heart disease.NEW & NOTEWORTHY Ranolazine is a drug used for treatment of angina pectoris in patients with ischemic heart disease. We discovered a novel mechanism by which this drug may exhibit its beneficial effects. It increases coronary venous levels of adenosine both at rest and during dobutamine-induced myocardial ischemia. Ranolazine also increases adenosine levels in endothelial cells and cardiomyocytes in vitro, by principally increasing activity of the enzyme cytosolic-5'-nucleotidase. Adenosine has well-known myocardial protective and anti-adrenergic properties that may explain, in part, ranolazine's beneficial effect in ischemic heart disease.

Hypoxia Induced Heparan Sulfate Primes the Extracellular Matrix for Endothelial Cell Recruitment by Facilitating VEGF-Fibronectin Interactions.

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival of blood vessels. Retinal pigmented epithelial (RPE) cells are a major source of VEGF in the retina, with evidence that the extracellular matrix (ECM)-binding forms are particularly important. VEGF associates with fibronectin in the ECM to mediate distinct signals in endothelial cells that are required for full angiogenic activity. Hypoxia stimulates VEGF expression and angiogenesis; however, little is known about whether hypoxia also affects VEGF deposition within the ECM. Therefore, we investigated the role of hypoxia in modulating VEGF-ECM interactions using a primary retinal cell culture model. We found that retinal endothelial cell attachment to RPE cell layers was enhanced in cells maintained under hypoxic conditions. Furthermore, we found that agents that disrupt VEGF-fibronectin interactions inhibited endothelial cell attachment to RPE cells. We also found that hypoxia induced a general change in the chemical structure of the HS produced by the RPE cells, which correlated to changes in the deposition of VEGF in the ECM, and we further identified preferential binding of VEGFR2 over VEGFR1 to VEGF laden-fibronectin matrices. Collectively, these results indicate that hypoxia-induced HS may prime fibronectin for VEGF deposition and endothelial cell recruitment by promoting VEGF-VEGFR2 interactions as a potential means to control angiogenesis in the retina and other tissues.

Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH.

Vascular endothelial growth factor-A (VEGF) plays a critical role in stimulating angiogenesis in normal and disease states. Anti-VEGF antibodies have been developed to manage pathological angiogenesis. Bevacizumab, sold under the brand name Avastin, is a humanized mAb that binds VEGF and blocks its binding to its signaling receptor, VEGF receptor 2, and is used to treat patients with a variety of cancers or retinal disorders. The ability of Avastin to modulate other nonreceptor interactions of VEGF has not been fully defined. In this study, we investigated Avastin's capacity to modulate VEGF165 binding to porcine aortic endothelial cells and to heparin and fibronectin (FN) across a range of pH values (pH 5-8). We observed that Avastin slightly enhanced VEGF binding to heparin and that heparin increased VEGF binding to Avastin. In contrast, Avastin inhibited VEGF binding to cells and FN, yet Avastin could still bind to VEGF that was bound to FN, indicating that these binding events are not mutually exclusive. Avastin binding to VEGF was dramatically reduced at acidic pH values (pH 5.0-6.5), whereas VEGF binding to FN and nonreceptor sites on cells was enhanced. Interestingly, the reduced Avastin-VEGF binding at acidic pH was rescued by heparin, as was Avastin's ability to inhibit VEGF binding to cells. These results suggest that heparin might be used to expand the clinical utility of Avastin. Our findings highlight the importance of defining the range of VEGF interactions to fully predict antibody activity within a complex biological setting.

Characterization of receptor binding kinetics for vascular endothelial growth factor-A using SPR.

Angiogenesis is a highly regulated process orchestrated, in large part, by the vascular endothelial growth factor-A (VEGF-A) system of ligands and receptors. Considerable effort has been invested in finding optimal ways to modulate VEGF-A activity to treat disease, however, the mechanisms by which the various components interact remain poorly understood. This is in part because of the difficulty of analyzing the various interactions in an intercomparable manner. In the present study, we established conditions to allow for the detailed characterization of the molecular interactions between VEGF and its receptors and the co-receptor NRP-1 using surface plasmon resonance (SPR). We found that VEGF dissociated 25-times faster from its major signaling receptor, VEGF receptor-2 (VEGFR-2) than from its "decoy" receptor, VEGF receptor-1 (VEGFR-1). Using a systematic approach, we obtained kinetic parameters for each individual interaction under a consistent set of experimental conditions allowing for comparison between various receptors. The set of quantitative kinetic parameters and experimental conditions reported herein will provide valuable tools for developing comprehensive models of the VEGF system.

Magnetic resonance imaging in patients with cardiac implantable electronic devices: a single-center prospective study.

PURPOSE: Cardiac implantable electronic devices (CIEDs) have traditionally been a contraindication for magnetic resonance imaging (MRI). Recent studies suggest that MRI can be conducted safely in select patients with pacemakers (PPMs) and implantable cardioverter defibrillators (ICDs). We sought to determine the safety of MRI in patients with CIEDs, using a protocol for patient selection and device programming. METHODS: This is a prospective, single-center study. Patients with a PPM or ICD and a clinical indication for MRI were considered. Exclusion criteria included newly implanted devices (<4 weeks), PPMs manufactured before 1996 and ICDs before 2000, epicardial and abandoned leads, and pacemaker-dependent ICD patients. Pacemaker-dependent PPM patients were programmed to asynchronous pacing. Tachycardia detection/therapies were disabled for ICDs. Devices were interrogated pre- and post-scan, and at follow-up 1-6 weeks later. Defibrillation threshold (DFT) was not tested post-scan. Patients were followed to monitor device therapies. RESULTS: Two hundred twenty-seven patients underwent 293 scans. Devices included 170 (70.6%) PPMs and 71 (29.5%) ICDs. Thirteen (4.4%) scans were aborted mainly due to subjective complaints or artifact on scout cardiac imaging. Post-scan and follow-up interrogation demonstrated no changes in device parameters requiring reprogramming or revision. Over long-term follow-up (median, 354 days [IQR 65-629]), nine ICD patients had appropriate shocks (median, 3 [IQR 1-8]). One had four inappropriate shocks for atrial fibrillation. All tachyarrhythmias meeting criteria for defibrillation were successfully terminated. CONCLUSIONS: MRI can be conducted safely in patients with CIEDs when done in a protocoled manner with appropriate supervision. DFT testing after MRI may not be necessary.

Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in Endothelial Cells.

Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF-cell-matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF and, inversely, heparin pre-treatment to enhance Fn-matrix binding of VEGF increased cell-internalization of VEGF regardless of matrix stiffness. ß1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated ß1 and inhibition of ß1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF-matrix binding and ß1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. J. Cell. Physiol. 231: 2026-2039, 2016. © 2016 Wiley Periodicals, Inc.

Fibronectin Fiber Extension Decreases Cell Spreading and Migration.

The extracellular matrix (ECM) is present in a range of molecular conformations and intermolecular arrangements. Fibronectin (Fn) molecules that constitute fibers within the ECM can exist in a variety of conformations that result from both mechanical stress and chemical factors such as allosteric binding partners. The long-standing hypothesis that conformational changes regulate the binding of cells to Fn fibers has only been tested for mutated molecules of Fn and has yet to be fully evaluated with Fn fibers. Using time-lapse microscopy we examined how mechanical extension of single fibers of Fn affects the adhesion and migration of endothelial cells. Using this single fiber adhesion technique, we show that high levels of mechanical strain applied to Fn fibers decreases the rates of both cell spreading and cell migration. These data indicate a fundamental cellular response to mechanical strain in the ECM that might have important implications for understanding how cells are recruited during tissue development and repair. J. Cell. Physiol. 231: 1728-1736, 2016. © 2015 Wiley Periodicals, Inc.

Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration.

Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment.

Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis.

Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and individual cell responses. Endothelial cells on soft substrates, 4 kPa, were the most responsive to VEGF, whereas cells on the 125 kPa substrates exhibited an attenuated response. Magnitude of activation, not the quantity of cells responding or the number of local maximums each cell experienced distinguished the responses. Individual cell analysis, across all treatments, identified two unique cell clusters. One cluster, containing most of the cells, exhibited minimal or slow calcium release. The remaining cell cluster had a rapid, high magnitude VEGF activation that ultimately defined the population based average calcium response. Interestingly, at low doses of VEGF, the high responding cell cluster contained smaller cells on average, suggesting that cell shape and size may be indicative of VEGF-sensitive endothelial cells. This study provides a new analytical tool to quantitatively analyze individual cell signaling response kinetics, that we have used to help uncover outcomes that are hidden within the average. The ability to selectively identify highly VEGF responsive cells within a population may lead to a better understanding of the specific phenotypic characteristics that define cell responsiveness, which could provide new insight for the development of targeted anti- and pro-angiogenic therapies.

Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity.

Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF165 in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF165, VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.

Extracellular matrix presentation modulates vascular smooth muscle cell mechanotransduction.

The development of atherosclerosis involves phenotypic changes among vascular smooth muscle cells (VSMCs) that correlate with stiffening and remodeling of the extracellular matrix (ECM). VSMCs are highly sensitive to the composition and mechanical state of the surrounding ECM, and ECM remodeling during atherosclerosis likely contributes to pathology. We hypothesized that ECM mechanics and biochemistry are interdependent in their regulation of VSMC behavior and investigated the effect of ligand presentation on certain stiffness-mediated processes. Our findings demonstrate that substrate stiffening is not a unidirectional stimulus-instead, the influence of mechanics on cell behavior is highly conditioned on ligand biochemistry. This "stiffness-by-ligand" effect was evident for VSMC adhesion, spreading, cytoskeletal polymerization, and focal adhesion assembly, where VSMCs cultured on fibronectin (Fn)-modified substrates showed an augmented response to increasing stiffness, whereas cells on laminin (Ln) substrates showed a dampened response. By contrast, cells on Fn substrates showed a decrease in myosin light chain (MLC) phosphorylation and elongation with increasing stiffness, whereas Ln supported an increase in MLC phosphorylation and no change in cell shape with increasing stiffness. Taken together, these findings show that identical cell populations exhibit opposing responses to substrate stiffening depending on ECM presentation. Our results also suggest that the shift in VSMC phenotype in a developing atherosclerotic lesion is jointly regulated by stromal mechanics and biochemistry. This study highlights the complex influence of the blood vessel wall microenvironment on VSMC phenotype and provides insight into how cells may integrate ECM biochemistry and mechanics during normal and pathological tissue function.

A liquid chromatography-mass spectrometry-based approach to characterize the substrate specificity of mammalian heparanase.

Extracellular heparanase activity releases growth factors and angiogenic factors from heparan sulfate (HS) storage sites and alters the integrity of the extracellular matrix. These activities lead to a loss of normal cell matrix adherent junctions and correlate with invasive cellular phenotypes. Elevated expression of heparanase is associated with several human cancers and with vascular remodeling. Heparanase cleaves only a limited fraction of glucuronidic linkages in HS. There have been few investigations of the functional consequences of heparanase activity, largely due to the heterogeneity and complexity of HS. Here, we report a liquid chromatography-mass spectrometry (LC-MS)-based approach to profile the terminal structures created by heparanase digestion and reconstruct the heparanase cleavage sites from the products. Using this method, we demonstrate that heparanase cleaves at the non-reducing side of highly sulfated HS domains, exposing cryptic growth factor binding sites. This cleavage pattern is observed in HS from several tissue sources, regardless of overall sulfation degree, indicating a common recognition pattern. We further demonstrate that heparanase cleavage of HS chains leads to increased ability to support FGF2-dependent cell proliferation. These results suggest a new mechanism to explain how heparanase might potentiate the uncontrolled cell proliferation associated with cancer through its ability to activate nascent growth factor-promoting domains within HS.

Oligosaccharide substrate preferences of human extracellular sulfatase Sulf2 using liquid chromatography-mass spectrometry based glycomics approaches.

Sulfs are extracellular endosulfatases that selectively remove the 6-O-sulfate groups from cell surface heparan sulfate (HS) chain. By altering the sulfation at these particular sites, Sulfs function to remodel HS chains. As a result of the remodeling activity, HSulf2 regulates a multitude of cell-signaling events that depend on interactions between proteins and HS. Previous efforts to characterize the substrate specificity of human Sulfs (HSulfs) focused on the analysis of HS disaccharides and synthetic repeating units. In this study, we characterized the substrate preferences of human HSulf2 using HS oligosaccharides with various lengths and sulfation degrees from several naturally occurring HS sources by applying liquid chromatography mass spectrometry based glycomics methods. The results showed that HSulf2 preferentially digests highly sulfated HS oligosaccharides with zero acetyl groups and this preference is length dependent. In terms of length of oligosaccharides, HSulf2 digestion induced more sulfation decrease on DP6 (DP: degree of polymerization) compared to DP2, DP4 and DP8. In addition, the HSulf2 preferentially digests the oligosaccharide domain located at the non-reducing end (NRE) of the HS and heparin chain. In addition, the HSulf2 digestion products were altered only for specific isomers. HSulf2 treated NRE oligosaccharides also showed greater decrease in cell proliferation than those from internal domains of the HS chain. After further chromatographic separation, we identified the three most preferred unsaturated hexasaccharide for HSulf2.

Docking server for the identification of heparin binding sites on proteins.

Many proteins of widely differing functionality and structure are capable of binding heparin and heparan sulfate. Since crystallizing protein-heparin complexes for structure determination is generally difficult, computational docking can be a useful approach for understanding specific interactions. Previous studies used programs originally developed for docking small molecules to well-defined pockets, rather than for docking polysaccharides to highly charged shallow crevices that usually bind heparin. We have extended the program PIPER and the automated protein-protein docking server ClusPro to heparin docking. Using a molecular mechanics energy function for scoring and the fast Fourier transform correlation approach, the method generates and evaluates close to a billion poses of a heparin tetrasaccharide probe. The docked structures are clustered using pairwise root-mean-square deviations as the distance measure. It was shown that clustering of heparin molecules close to each other but having different orientations and selecting the clusters with the highest protein-ligand contacts reliably predicts the heparin binding site. In addition, the centers of the five most populated clusters include structures close to the native orientation of the heparin. These structures can provide starting points for further refinement by methods that account for flexibility such as molecular dynamics. The heparin docking method is available as an advanced option of the ClusPro server at http://cluspro.bu.edu/ .

Hypoxia-induced changes in Ca(2+) mobilization and protein phosphorylation implicated in impaired wound healing.

The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when corneal epithelium is injured, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca(2+) wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine the dynamics of Ca(2+) signaling between neuronal and epithelial cells. The cross talk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca(2+) dynamic patterns were altered in response to decreased O2 levels. These alterations were associated with an overall decrease in ATP and changes in purinergic receptor-mediated Ca(2+) mobilization and localization of N-methyl-d-aspartate receptors. In addition, we used the cornea in an organ culture wound model to examine how hypoxia impedes reepithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca(2+)-mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair.

Aortic carboxypeptidase-like protein (ACLP) enhances lung myofibroblast differentiation through transforming growth factor ß receptor-dependent and -independent pathways.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFß, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFß signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFß receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFß receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFß signaling and promoting collagen expression through a TGFß receptor-independent pathway.