FITC-labeled Albumin Research Articles

Human podocytes perform polarized, caveolae-dependent albumin endocytosis

The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes.

Abstract 5666: Albumin and paclitaxel co-localize in endocytic vesicles in HUVEC cells, and uptake is blocked by Cremophor EL.

Abstract Nab®-paclitaxel is an albumin-bound nanoparticle formulation of paclitaxel (ptx) that does not contain Cremophor EL (CrEL), and results in higher drug levels in xenografts and increased clinical activity in breast and lung cancers compared to ptx formulated with CrEL (Taxol®). Above the critical micellar concentration of 0.009%, CrEL forms long-lived micelles in circulation that can sequester ptx (peak plasma concentration in clinical use is 0.3%). Previous studies have shown CrEL reduces ptx binding to albumin, and Taxol® has reduced association with, and transport across endothelial cells compared to nab®-ptx (Desai, CCR 2006). Endothelial cells take up albumin, which is trafficked into recycling or transcytosis pathways or into lysosomes for degradation. Here, we explore mechanisms of uptake and trafficking of albumin and ptx in endothelial cells and the effect of CrEL on these events. Using fluorescence microscopy, we visualized the uptake of rhodamine-albumin and fluorescent ptx (Flutax). Albumin was present in EEA1-positive early endosomes and LAMP1-positive lysosomes. Notably, ptx was also present in vesicular structures and was often co-localized with albumin. The uptake of albumin was blocked by increasing concentrations (0.003%-0.3%) of CrEL, and also by inhibitors of caveolin-mediated endocytosis including indomethacin (blocks internalization of caveolae) and methyl-β-cyclodextrin (prevents formation of lipid rafts). The effect of CrEL on paclitaxel and albumin cellular uptake was confirmed by flow cytometry studies. 0.3% CrEL reduced the uptake of Flutax in DMSO, Flutax-modified nab®-ptx, and FITC-labeled albumin to close to background levels in both HUVEC and PC3 cells. Thus, in addition to its drug sequestration activity, CrEL directly affects endocytosis. We further evaluated CrEL effects on Flutax and ptx transport across endothelial monolayers in transwell chambers using a fluorescence detection assay. Two-fold more ptx crossed monolayers exposed to Flutax-containing nab®-ptx as compared to Taxol®. The effects of 0.001% to 0.3% CrEL on ptx transport at varying timepoints were investigated by mass spectrometry. Dose-dependent inhibition was observed, with a 3-fold reduction in transported ptx at 24 hrs. In summary, we have demonstrated that ptx co-localizes with albumin in endothelial cells, suggesting that the nab-ptx complex can remain intact within cells. Furthermore, CrEL interferes with albumin uptake at clinically relevant concentrations, thereby affecting paclitaxel cellular uptake and transport. These mechanistic studies further elucidate the basis of increased delivery of drug into the target cells by the nab®-ptx formulation as compared to Taxol®. Citation Format: Xiping Liu, Carrie Brachmann, Sean Hong, Shijuan Wu, Zeen Tong, Tapas De, Willard Foss, Gondi Kumar, Sekhar Surapaneni, Rajesh Chopra, Daniel Pierce, Carla Heise. Albumin and paclitaxel co-localize in endocytic vesicles in HUVEC cells, and uptake is blocked by Cremophor EL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5666. doi:10.1158/1538-7445.AM2013-5666

Characterization of spherulites as a lipidic carrier for low and high molecular weight agents.

To develop spherulite formulations to achieve high entrapment efficiency for both small and macromolecules as well as cell-type specific delivery. Spherulites of various compositions were prepared, and lipid-PEG was incorporated through post-insertion. Calcein and FITC-labeled albumin were employed as model drugs for small and macromolecules. The spherulites were characterized with respect to entrapment efficiency, size, structure, and release kinetics, and the morphology was examined via cryo-EM. Finally, SV119-decorated spherulites were examined for their selective uptake by cancer cells. The spherulites are 170 ~ 290 nm in size. A loading efficiency of 55 ~ 60% can be consistently achieved for both calcein and albumin under optimized conditions. Cryo-EM shows the onion-like morphology consistent with the structure of multilamellar liposomes. A t(½) of 39.3 h and 69.7 h in cargo release in serum was observed before and after PEG decoration, and incorporation of SV119 led to selective delivery of rhodamine-labeled spherulites to PC-3 tumor cells. Our optimized formulations may represent a platform with simple preparation approach, relatively small particle size, high drug loading efficiency for both low and high molecular weight agents, and slow release kinetics for selective delivery of various types of therapeutics to target cells.

Spontaneous optic nerve compression in the osteopetrotic (op/op) mouse: a novel model of myelination failure.

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.

Regulation of G-protein signaling via Gnas is required to regulate proximal tubular growth in the Xenopus pronephros

In the kidney, proximal tubules are very important for the reabsorption of water, ions and organic solutes from the primary urine. They are composed of highly specialized epithelial cells that are characterized by an elaborate apical brush border to increase transport efficiency. Using the pronephric kidney of Xenopus laevis we discovered that the G-protein modulator cholera toxin resulted in a dramatic reduction of the proximal tubular size. This phenotype was accompanied by changes in the cytoarchitecture characterized by ectopic expression of the distal tubular marker 4A6 and an impairment of yolk platelet degradation. In addition, cholera toxin caused edema formation. However, this phenotype was not due to kidney defects, but rather due to impaired vasculature development. Based on experiments with antisense morpholino oligomers as well as pharmacological agonists and antagonists, we could show that the complex phenotype of cholera toxin in the pronephric kidney was caused by the hyperactivation of a single G-protein alpha subunit, Gnas. This—in turn—caused elevated cAMP levels, triggered a Rapgef4-dependent signaling cassette and perturbed exo- and endocytosis. This perturbation of the secretory pathway by Ctx was not only observed in Xenopus embryos. Also, in a human proximal tubular cell line, cholera toxin or a Rapgef4-specific agonist increased uptake and decreased secretion of FITC-labeled Albumin. Based on these data we propose that the Gnas/cAMP/Rapgef4 pathway regulates the signals inducing the proliferation of proximal tubules to acquire their final organ size.

MMP-3 Contributes to Nigrostriatal Dopaminergic Neuronal Loss, BBB Damage, and Neuroinflammation in an MPTP Mouse Model of Parkinson’s Disease

The present study examined whether matrix metalloproteinase-3 (MMP-3) participates in the loss of dopaminergic (DA) neurons in the nigrostriatal pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease with blood brain barrier (BBB) damage and infiltration of peripheral immune cells. Tyrosine hydroxylase (TH) immunostaining of brain sections from MPTP-treated mice showed that MPTP induced significant degeneration of nigrostriatal DA neurons. Moreover, FITC-labeled albumin detection and immunostaining revealed that MPTP caused damage to the BBB and increased the number of ED-1- and CD-3-immunopositive cells in the substantia nigra (SN). Genetic ablation of MMP-3 reduced the nigrostriatal DA neuron loss and improved motor function. This neuroprotective effect afforded by MMP-3 deletion was associated with the suppression of BBB disruption and a decrease in the number of ED-1- and CD-3-immunopositive cells in the SN. These data suggest that MMP-3 could play a crucial role in neurodegenerative diseases such as PD in which BBB damage and neuroinflammation are implicated.

Different microvascular permeability responses elicited by the CXC chemokines MIP-2 and KC during leukocyte recruitment: Role of LSP1

The CXC chemokines keratinocyte-derived chemokine (KC, CXCL1) and macrophage inflammatory protein-2 (MIP-2, CXCL2) activate G-protein coupled receptor CXCR2 and are believed to have similar inflammatory effects in mice. Their specific signaling mechanisms remain elusive. A wide variety of cellular events, mediators and signaling pathways are known to regulate microvascular permeability. Leukocyte-specific protein 1 (LSP1), a Ca2+- and F-actin binding protein, is one of the major downstream substrates of p38 MAPK. LSP1 was previously shown to play a pivotal role in leukocyte transmigration and microvascular permeability. Using intravital microscopy visualizing microvasculature of murine cremaster muscle, we demonstrate that KC and MIP-2 triggered increased leukocyte recruitment which was significantly reduced in LSP1-deficient mice compared to the wild-type control mice. Fluorescence imaging revealed that KC induced more substantial increases of microvascular permeability to FITC-labeled albumin than MIP-2. We found that LSP1 had a more prominent role in microvascular hyperpermeability induced by KC than that triggered by MIP-2. Moreover, Western blotting showed enhanced phosphorylation of p38 MAPK in the cremasteric tissue after stimulation with KC but not with MIP-2 and KC-induced but not MIP-2-induced hyperpermeability was blunted by pharmacological inhibition of p38 MAPK. In conclusion, LSP1 plays an important role in leukocyte recruitment induced by both KC and MIP-2. KC elicits more profoundly increased microvascular permeability than MIP-2. KC is at least partially effective through LSP1 and the phosphorylation of p38 MAPK.

Different effects of dibutyryl cAMP on monolayer permeability in human aortic and coronary arterial endothelial cells

Recent studies have demonstrated species- and/or organ-specific differences in the effects of dibutyryl cAMP (DBcAMP) on vascular permeability. In this study, we investigated the permeability of monolayers of human coronary arterial endothelial cells (HCAECs) and human aortic endothelial cells (HAECs) in the presence of DBcAMP using monolayer permeability models of endothelial cells and a double incubation chamber for measurement of FITC-labeled albumin or FITC-dextran (4 kDa) transfer through the monolayer. DBcAMP significantly lowered monolayer permeability in the HCAECs but had no effect in the HAECs when compared with untreated cells (P < 0.01). A cAMP-dependent protein kinase (PKA) inhibitor, Rp-8-Br-cAMPS (10-5 M), significantly reversed the DBcAMP induced decease in permeability of the HCAEC monolayer to the level of untreated cells. Rp-8-Br-cAMPS (10-5 M) and Sp-cAMPS (a PKA activator) had nosignificant effect on the permeability of the HAEC monolayer after treatment with DBcAMP. These results indicate that DBcAMP diminishes the permeability of an HCAEC monolayer via a cAMP-mediated PKA pathway but does not do so in HAECs, suggesting that the effects of cAMP on permeability of endothelial cell monolayers are dependent on the origin of the cells. Key words: Dibutyryl cAMP, endothelial cells, permeability.

Endocytic Uptake of FITC-albumin by Human Alveolar Epithelial Cell Line A549

The uptake mechanism of FITC-labeled albumin (FITC-albumin) was examined in human alveolar epithelial cell line A549. FITC-albumin uptake by A549 cells was time- and temperature-dependent, and was markedly suppressed at 4°C compared with that at 37°C. The uptake was saturable, and was mediated by a high-affinity, low-capacity system and by a low-affinity, high-capacity system. In the following experiments, we focused on the low-affinity system. FITC-albumin uptake was markedly inhibited by metabolic inhibitors and by a vacuolar H⁺-ATPase, bafilomycin A₁. The uptake was inhibited by clathrin-mediated endocytosis inhibitors (phenylarsine oxide and chlorpromazine). Potassium depletion and hypertonicity that inhibit clathrin-mediated endocytosis also decreased FITC-albumin uptake. On the other hand, caveolae-mediated endocytosis inhibitors (indomethacin and nystatin) did not affect FITC-albumin uptake. In addition, FITC-albumin uptake was inhibited by macropinocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride. These results suggest that the low-affinity system of FITC-albumin uptake is mediated by endocytosis in A549 cells, predominantly via a clathrin-mediated pathway. Macropinocytosis, but not caveolae-mediated endocytosis, may also be involved. Considering our previous findings, albumin may be transported by a similar mechanism and/or pathway in rat and human alveolar epithelial cells.

Amelioration of Albuminuria in ROCK1 Knockout Mice with Streptozotocin-Induced Diabetic Kidney Disease

Background: Although blockade of Rho kinase with pharmacologic inhibitors ameliorates renal fibrosis and diabetic kidney disease (DKD), the underlined mechanisms remain largely unclear. The present study tested the hypothesis that ROCK1 may regulate the early development of albuminuria via the megalin/cubilin-dependent mechanism. Methods: A DKD model was induced in ROCK1 knockout and wild-type mice by streptozotocin (STZ). The effect of deleted ROCK1 on urinary albumin excretion and the expression of megalin/cubilin were examined. In addition, the effect of blocking ROCK activities with an inhibitor (Y-27632) on tubular albumin reabsorption was tested in a normal rat tubular epithelial cell line (NRK52E) under high-glucose conditions. Expression of transforming growth factor (TGF)-β1, interleukin-1β and collagen-1 was also been examined. Results: Urinary albumin excretion was significantly increased in ROCK1 WT mice at 8 weeks after STZ injection. In contrast, mice lacking ROCK1 gene were protected against the development of albuminuria. This was associated with the protection against the loss of megalin/cubilin and an increase in TGF-β₁, IL-1β, and fibrosis in the kidney. In vitro, we also found that blockade of Rho kinase with inhibitor Y-27632 prevented high-glucose-induced loss of megalin expression and an increase of TGF-β₁, thereby increasing the absorption rate of FITC-labeled albumin by tubular epithelial cells. Conclusion: ROCK1 may play a role in the development of albuminuria in DKD by downregulating the endocytosis receptors complex – megalin/cubilin.

Albumin-induced apoptosis of glomerular parietal epithelial cells is modulated by extracellular signal-regulated kinase 1/2

The biological role(s) of glomerular parietal epithelial cells (PECs) is not fully understood in health or disease. Given its location, PECs are constantly exposed to low levels of filtered albumin, which is increased in nephrotic states. We tested the hypothesis that PECs internalize albumin and increased uptake results in apoptosis. Confocal microscopy of immunofluorescent staining and immunohistochemistry were used to demonstrate albumin internalization in PECs and to quantitate albumin uptake in normal mice and rats as well as experimental models of membranous nephropathy, minimal change disease/focal segmental glomerulosclerosis and protein overload nephropathy. Fluorescence-activated cell sorting analysis was performed on immortalized cultured PECs exposed to fluorescein isothiocyanate (FITC)-labeled albumin in the presence of an endosomal inhibitor or vehicle. Apoptosis was measured by Hoechst staining in cultured PECs exposed to bovine serum albumin. Levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) were restored by retroviral infection of mitogen-activated protein kinase (MEK) 1/2 and reduced by U0126 in PECs exposed to high albumin levels in culture and apoptosis measured by Hoechst staining. PECs internalized albumin normally, and this was markedly increased in all of the experimental disease models (P<0.05 versus controls). Cultured immortalized PECs also internalize FITC-labeled albumin, which was reduced by endosomal inhibition. A consequence of increased albumin internalization was PEC apoptosis in vitro and in vivo. Candidate signaling pathways underlying these events were examined. Data showed markedly reduced levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in PECs exposed to high albumin levels in nephropathy and in culture. A role for ERK1/2 in limiting albumin-induced apoptosis was shown by restoring p-ERK1/2 by retroviral infection, which reduced apoptosis in cultured PECs, while a forced decrease of p-ERK1/2 through inhibition of MEK 1/2 significantly increased albumin-induced PEC apoptosis. A normal role of PECs is to take up filtered albumin. However, this is increased in proteinuric glomerular diseases, leading to apoptosis through changes in ERK1/2.

Original article. The role of p38MAPK in asymmetric dimethylarginineinduced cytoskeleton and cellular permeability changes in cultured endothelial cells

Abstract Background: Asymmetric dimethylarginine (ADMA) induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction. It is supposed that activated p38 mitogen-activated protein kinase (MAPK) would trigger the formation of stress fibers and increase cellular permeability. Objective: Explore p38 MAPK as a potentially important enzyme in ADMA-mediated endothelial cell contractile response and permeability change. Methods: Human umbilical endothelial cells (HUVECs) were cultured, where ADMA and/or SB203580 (the specific inhibitor of p38MAPK) were used to stimulate HUVECs. Immunofluorescent staining was carried out to examine the expression and distribution of F-actin, flow cytometry was used to quantify F-actin, and Transwell was applied to test cellular permeability with FITC-labelled human serum albumin (HSA). Scanning electronic microscopy (SEM) was utilized to observe the changes of intercellar contact. Results: ADMA induced significant p38MAPK activation in a dose-dependent manner, which correlated with increased stress fibers. SB-203580 attenuated the formation of actin stress fiber and the increase of cellular permeability induced ADMA in the HUVECs (p&lt;0.01, LSCM; p&lt;0.01, cytometry; p&lt;0.05, Transwell). Widened intercellular space induced by ADMA was detected and could be inhibited by SB-203580 (SEM). SB-203580 alone had no effect on cytoskeleton and cellular permeability. Conclusion: p38MAPK activation participated in cytoskeleton and cellular permeability changes induced by ADMA in HUVECs.

Macrophages play a key role in early blood brain barrier reformation after hypothermic brain injury

The inflammatory response following traumatic injury to the central nervous system (CNS) includes the infiltration of large numbers of macrophages. This response has been implicated in both ongoing tissue damage as well as recovery following CNS injury. We investigated the role of invading macrophages on one important aspect of tissue repair in the brain, the reformation of the blood brain barrier (BBB). We used liposomal clodronate to deplete monocytes and tissue macrophages. This method led to a marked reduction in the accumulation of F4/80-expressing cells at sites of hypothermic brain injury in a murine model. The integrity of the blood brain barrier over time following injury was assessed by permeability of fluorescent labeled albumin. The reduction in macrophages at the injury site was accompanied by a delay in early reformation of the blood brain barrier. In control animals the permeability of the BBB to FITC-labeled albumin returned to normal levels by seven days post-injury. In macrophage-depleted mice leakage of albumin was still observed at seven days post-injury. These results suggest that macrophages play an important role in early post-traumatic reformation of the BBB.

PEDF Regulates Vascular Permeability by a γ-Secretase-Mediated Pathway

Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.

An angiogenic inhibitor, cyclic RGDfV, attenuates MPTP-induced dopamine neuron toxicity

We previously demonstrated that several dopamine (DA) neurotoxins produced punctate areas of FITC-labeled albumin (FITC-LA) leakage in the substantia nigra and striatum suggesting blood brain barrier (BBB) dysfunction. Further, this leakage was co-localized with αvβ3 integrin up-regulation, a marker for angiogenesis. This suggested that the FITC-LA leakage might have been a result of angiogenesis. To assess the possible role of angiogenesis in DA neuron loss, we treated mice with 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and on the following day treated with cyRGDfV, a cyclic peptide that binds to integrin αvβ3 and prevents angiogenesis. Post-treatment for 3 days (b.i.d.) with cyRGDfV blocked the MPTP-induced upregulation of integrin β3 immunoreactivity (a marker for angiogenesis), leakage of FITC-LA into brain parenchyma (a marker for BBB disruption) as well as the down regulation of Zona Occludin-1 (ZO-1; a marker for tight junction integrity). In addition, cyRGDfV also completely prevented tyrosine hydroxylase immunoreactive cell loss (a marker for DA neurons) and markedly attenuated the up-regulation of activated microglia (Iba1 cell counts and morphology). These data suggest that cyRGDfV, and perhaps other anti-angiogenic drugs, are neuroprotective following acute MPTP treatment and may suggest that compensatory angiogenesis and BBB dysfunction may contribute to inflammation and DA neuron loss.

S153 Bone morphogenetic protein receptor-II regulates pulmonary artery endothelial cell barrier function: relevance to heritable pulmonary arterial hypertension

BackgroundMutations in bone morphogenetic protein receptor II (BMPR-II) have been shown to underlie most heritable cases of Pulmonary arterial hypertension (PAH). However, less than half the individuals who harbour mutations...

Creating biomimetic polymeric surfaces by photochemical attachment and patterning of dextran.

In this work, we report the preparation of photoactive dextran and demonstrate its utility by photochemically attaching it onto various polymeric substrates. The attachment of homogeneous and patterned dextran films was performed on polyurethane and polystyrene, with detailed analysis of surface morphology, swelling behavior, and the protein resistance of these substrates. The described photoactive dextran and attachment procedure is applicable to a wide variety of substrates while accommodating surfaces with complex surface geometries. Dextran with azide content between 22 and 0.3 wt % was produced by esterification with p-azidobenzoic acid. Dextran (1.2 wt % azide) was photografted onto plasma oxidized polyurethane and polystyrene and displayed thicknesses of 5 +/- 3 and 7 +/- 3 nm, respectively. The patterned dextran on oxidized polyurethane was patchy with a nominal height difference between dextranized and nondextranized regions. The azidated dextran on oxidized polystyrene exhibited a distinct step in height. In the presence of phosphate buffered saline (PBS), the dextranized regions became smoother and more uniform without affecting the height difference at the oxidized polyurethane boundary. However, the dextranized regions on oxidized polyurethane were observed to swell by a factor of 3 relative to the dried thickness. These dissimilarities were attributed to hydrogen bonding between the dextran and oxidized polyurethane and were confirmed by the photoimmobiliization in the presence of LiCl. The resulting surface was the smoothest of all the azidated dextran samples (R(rms) = 1 +/- 0.3 nm) and swelled up to 2 times its dried thickness in PBS. The antifouling properties of dextran functionalized surfaces were verified by the selective adsorption of FITC-labeled human albumin only on the nondextranized regions of the patterned polyurethane and polystyrene substrates.

T-lymphocytes modulate the microvascular and inflammatory responses to intestinal ischemia-reperfusion.

The overall objective of this study was to define the contribution of T-lymphocytes to the microvascular and inflammatory responses of the intestine to ischemia/reperfusion (I/R). The superior mesenteric artery of wild-type (WT) and SCID mice was occluded for 45 minutes, followed by 30 minutes or 6 hours of reperfusion. Intravital fluorescence microscopy was used to monitor the extravasation of FITC-labeled albumin or the adhesion of carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled T-lymphocytes in mucosal venules of the postischemic intestine. Tissue myeloperoxidase (MPO) was used to monitor neutrophil accumulation in the intestine of WT and SCID mice. Although the number of adherent T-cells was not increased above baseline at 1 hour after reperfusion, significant T-cell adhesion (both CD4(+) and CD8(+)) was noted at 6 hours of reperfusion. The latter response was prevented by pretreatment with a blocking antibody directed against MAdCAM-1, but not ICAM-1 or VCAM-1. A significant increase in MAdCAM-1 expression was noted in both lymphoid (Peyer's patch) and nonlymphoid regions of the postischemic small bowel. The early (30 minutes after reperfusion) albumin extravasation elicited by gut I/R in WT mice was reduced in SCID mice. Reconstitution of SCID mice with T-lymphocytes restored the albumin leakage response to WT levels. The increased intestinal MPO caused by I/R (6 hours of reperfusion) in WT mice was attenuated in SCID mice; with reconstitution of SCID mice with T-cells the MPO response was restored. These findings indicate that intestinal I/R is associated with the recruitment of CD4+ and CD8+ T-cells, which is mediated by endothelial MAdCAM-1. T-cells seem to modulate the recruitment of neutrophils that occurs hours after reperfusion as well as the increased albumin extravasation that occurs within minutes after reperfusion.

Online monitoring of immunoaffinity-based depletion of high-abundance blood proteins by UV spectrophotometry using enhanced green fluorescence protein and FITC-labeled human serum albumin

BackgroundThe removal of high-abundance proteins from plasma is an efficient approach to investigating flow-through proteins for biomarker discovery studies. Most depletion methods are based on multiple immunoaffinity methods available commercially including LC columns and spin columns. Despite its usefulness, high-abundance depletion has an intrinsic problem, the sponge effect, which should be assessed during depletion experiments. Concurrently, the yield of depletion of high-abundance proteins must be monitored during the use of the depletion column. To date, there is no reasonable technique for measuring the recovery of flow-through proteins after depletion and assessing the capacity for capture of high-abundance proteins.ResultsIn this study, we developed a method of measuring recovery yields of a multiple affinity removal system column easily and rapidly using enhanced green fluorescence protein as an indicator of flow-through proteins. Also, we monitored the capture efficiency through depletion of a high-abundance protein, albumin labeled with fluorescein isothiocyanate.ConclusionThis simple method can be applied easily to common high-abundance protein depletion methods, effectively reducing experimental variations in biomarker discovery studies.

Serum from Radiofrequency-Injured Livers Induces Differentiation of Bone Marrow Stem Cells into Hepatocyte-Like Cells

Roles that bone marrow stem cells (BMSCs) play in liver repair after liver injury and the cell therapy for liver diseases are widely accepted. However, the availability of hepatocyte-like cells from BMSCs and possible animal diseases association with culturing in fetal calf serum (FCS) are the major limitations of clinical therapy. Therefore, this study was designed to search for a new cell source for the treatment of liver injuries through investigating whether serum from radiofrequency ablation-injured rabbit livers can induce the differentiation of BMSCs into hepatocyte-like cells. Serum was collected from rabbits 36 h after radiofrequency ablation (RFA) treatment of the liver. BMSCs were isolated from rabbit bone marrow and were cultured in the collected serum. Cellular morphology and cell cycle were observed. Hepatocyte markers of the differentiated cells were detected by immunohistochemistry. After induction for 7 d, spindle-shaped BMSCs turned into round cells that resembled the morphology of hepatocyte-like cells. Flow cytometry showed that the percentage of cells in the S/G2/M phase was higher in the RFA group than that in the FCS group and HGF groups. This result suggests that BMSC can transform into mature cells from stem cell phase. Albumin and CK18 were considered as typical marker of hepatocytes. Following induction for 14 d, the differentiated cells expressed immunofluorescence of FITC-labeled albumin and TRITC-labeled CK18. BMSCs treated with serum collected from radiofrequency ablation-injured livers can differentiate into hepatocyte-like cells providing a cell source to cell therapy.