FITC-labeled Albumin Research Articles

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

PS-B09-3: EXAMINATION OF ALBUMINURIA REDUCTION EFFECT THROUGH IMPROVEMENT OF ENOS UNCOUPLING BY SGLT2 IN TYPE 2 DIABETIC MICE

Background and Aims: One of the main mechanisms of the renoprotective effects of SCLT2is in DKD is considered the ability of these inhibitors to improve glomerular hyperfiltration. We previously demonstrated that the adenosine/adenosine A1 receptor pathway played a pivotal role in the tubuloglomerular feedback(TGF) system in a type 1 diabetic model, Akita mice (Circulation, 2019). We also reported that the uncoupling of endothelial nitric oxide (NO) synthase (eNOS) via oxidation of tetrahydrobiopterin (BH4), a cofactor required for NO production, played a major role in generation of oxidative stress (AJPRP, 2005; JASN, 2013). In the present study, we explored the renal protective effects of SGLT2 inhibition, with a focus on glomerular oxidative stress and glomerular hemodynamics. Methods: This study used type 2 diabetic db/db mice and db/m+ mice as a control (male, 8wk old). In the first experiment, we measured the glomerular filtration rate of single nephrons (SNGFRs) using in vivo imaging technique in 12 wk-old db/db and db/m+ mice to confirm glomerular hyperfiltration. Next, we evaluated the SNGFRs change before and after the administration of a single dose of canagliflozin (CANA) (10 mg/kg). The SNGFRs, glomerular permeability of macromolecules, glomerular reactive oxygen species (ROS) and NO production, and tetrahydrobiopterin (BH4) level in serum and kidney were evaluated after the CANA treatment for 8 wk. Finally, human glomerular endothelial cells (hGECs) were exposed to normal glucose (5 mmol/L), high glucose (30 mmol/L of D-glucose), or a hyperosmotic control (5 mmol/L of D-glucose plus 25 mmol/L of L-glucose) in the presence or absence of CANA (10umol/L). Results: The CANA treatment ameliorated glomerular hyperfiltration in the db/db mice. In the db/db mice, glomerulus ROS production increased, and NO production decreased as compared with the levels in the control mice. CANA improved the imbalance between ROS and NO production. The serum and kidney concentrations of BH4 declined in the non-treated db/db mice, whereas the CANA treatment preserved the BH4 level. Leakage of 70-kD FITC-labeled albumin into the urinary space was observed in the db/db mice. The CANA treatment reduced the amount of FITC-labeled albumin in the urinary space of the db/db mice. The CANA treatment also alleviated vascular endothelial damage in glomeruli. BH4 levels decreased in the hGECs exposed to high glucose. CANA did not improved BH4 level in the hGECs exposed to high glucose. Conclusions: SGLT2i improved glomerular ROS/NO imbalance by suppressing eNOS uncoupling of glomerular endothelial cells in type 2 diabetic mice.

TMBIM6 promotes diabetic tubular epithelial cell survival and albumin endocytosis by inhibiting the endoplasmic reticulum stress sensor, IRE1α

Reduced albumin reabsorption in proximal tubular epithelial cells (PTECs), resulting from decreased megalin plasma membrane (PM) localization due to prolonged endoplasmic reticulum (ER) stress, potentially contributes to albuminuria in early diabetic kidney disease (DKD). To examine this possibility, we investigated the cytoprotective effect of TMBIM6 in promoting diabetic PTEC survival and albumin endocytosis by attenuating ER stress with an IRE1α inhibitor, KIRA6. Renal TMBIM6 distribution and expression were determined by immunohistochemistry, western blotting, and qPCR, whereas tubular injury was evaluated in db/db mice. High-glucose (HG)-treated HK-2 cells were either treated with KIRA6 or transduced with a lentiviral vector for TMBIM6 overexpression. ER stress was measured by western blotting and ER-Tracker Red staining, whereas apoptosis was determined by performing TUNEL assays. Megalin expression was measured by immunofluorescence, and albumin endocytosis was evaluated after incubating cells with FITC-labeled albumin. Tubular injury and TMBIM6 downregulation occurred in db/db mouse renal cortical tissues. Both KIRA6 treatment and TMBIM6 overexpression inhibited ER stress by decreasing the levels of phosphorylated IRE1α, XBP1s, GRP78, and CHOP, and stabilizing ER expansion in HG-treated HK-2 cells. TUNEL assays performed with KIRA6-treated or TMBIM6-overexpressing cells showed a significant decrease in apoptosis, consistent with the significant downregulation of BAX and upregulation of BCL-2, as measured by immunoblotting. Both KIRA6 and TMBIM6 overexpression promoted megalin PM localization and restored albumin endocytosis in HG-treated HK-2 cells. TMBIM6 promoted diabetic PTEC survival and albumin endocytosis by negatively regulating the IRE1α branch of ER stress.

QiShenYiQi Inhibits Tissue Plasminogen Activator-Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice.

Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model. Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration. Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment. Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.

SO021EFFECTS OF SODIUM-GLUCOSE COTRANSPORTER 2 INHIBITOR, CANAGLIFLOZIN ON GLOMERULAR HYPERFILTRATION AND OXIDATIVE STRESS IN MICE WITH TYPE 2 DIABETES

Abstract Background and Aims In most developed countries, diabetic kidney disease (DKD) is the most common cause of chronic kidney disease, which can lead to end-stage renal disease. In recent clinical trials, sodium–glucose cotransporter 2 inhibitors (SGLT2is) slowed the progression of kidney disease as compared with a placebo in patients with type 2 diabetes. One of the main mechanisms of the renoprotective effects of SCLT2is in DKD is considered the ability of these inhibitors to improve glomerular hyperfiltration. We previously demonstrated that the adenosine/adenosine A1 receptor pathway played a pivotal role in the tubuloglomerular feedback(TGF) system in a type 1 diabetic model, Akita mice (Circulation, 2019). We also reported that increased oxidative stress was involved in the pathogenesis of diabetic vascular complications. Uncoupling of endothelial nitric oxide (NO) synthase (eNOS) via oxidation of tetrahydrobiopterin (BH4), a cofactor required for NO production, played a major role in generation of oxidative stress (AJPRP, 2005; JASN, 2013). In the present study, we explored the renal protective effects of SGLT2 inhibition, with a focus on glomerular hemodynamics and glomerular oxidative stress. Method This study used type 2 diabetic db/db mice and db/m+ mice as a control (male, 8wk old). We developed a novel method to measure the glomerular filtration rate of single nephrons (SNGFRs) in mice using multiphoton laser microscopy. In the first experiment, we measured the SNGFRs in 12 wk-old db/db and db/m+ mice to confirm glomerular hyperfiltration. Next, we evaluated the SNGFRs change before and after the administration of a single dose of canagliflozin (CANA) (10 mg/kg). The SNGFRs, glomerular permeability of macromolecules, glomerular reactive oxygen species (ROS) and NO production, and tetrahydrobiopterin (BH4) level in serum and kidney were evaluated after the CANA treatment for 8 wk. Finally, human glomerular endothelial cells (hGECs) were exposed to normal glucose (5 mmol/L), high glucose (30 mmol/L of D-glucose), or a hyperosmotic control (5 mmol/L of D-glucose plus 25 mmol/L of L-glucose) in the presence or absence of CANA (10 μmol/L). Results The CANA treatment ameliorated glomerular hyperfiltration in the db/db mice. In the db/db mice, glomerulus ROS production increased, and NO production decreased as compared with the levels in the control mice. CANA improved the imbalance between ROS and NO production. The serum and kidney concentrations of BH4 declined in the non-treated db/db mice, whereas the CANA treatment preserved the BH4 level. Leakage of 70-kD FITC-labeled albumin into the urinary space was observed in the db/db mice. The CANA treatment reduced the amount of FITC-labeled albumin in the urinary space of the db/db mice. The CANA treatment also alleviated vascular endothelial damage in glomeruli. BH4 levels decreased in the hGECs exposed to high glucose. CANA did not improved BH4 level in the hGECs exposed to high glucose. Conclusion SGLT2i ameliorated glomerular hyperfiltration, preserving BH4 levels and improving the glomerular ROS/NO imbalance in type 2 diabetic mice.

Affected albumin endocytosis as a new neurotoxicity mechanism of amyloid beta.

Senile plaques, a hallmark of Alzheimer's disease, are composed by Amyloid-Beta (Aβ). Aβ 25-35 toxicity is caused mainly by increasing reactive oxygen species (ROS), which is reversed by albumin preventing Aβ internalization. In addition, key cellular processes and basic cell functions require of endocytosis, particularly relevant in neurons. To understand the protective effect of albumin and the toxicity mechanism of Aβ, the need of albumin uptake for neurons protection as well as the possible influence of Aβ on albumin endocytosis were investigated. With this aim the influence of lectin from soybeans (LEC), which prevents albumin endocytosis, on the effects of Aβ 25-35 on cellular morphology and viability, ROS generation and Aβ uptake with and without albumin in neurons in primary culture was investigated. Influence of Aβ on albumin endocytosis was studied using FITC-labelled albumin. LEC did not modify Aβ effects with or without albumin on neuronal morphology, but increased cell viability. LEC increased ROS generation with and without Aβ in the same magnitude. Diminished Aβ internalization observed with albumin was not affected by LEC. In presence of Aβ albumin is internalized, but endosomes did not deliver their cargo to the lysosomes for degradation. It is concluded that formation of Aβ-albumin complex does not require of albumin internalization, thus is extracellular. Aβ affects albumin endocytosis preventing late endosomes and lysosomes degradation, probably caused by changes in albumin structure or deregulation in vesicular transport. Considering the consequences such as its osmotic effects, the inability to exert its antioxidant properties, its effects on neuronal plasticity and excitability albumin affected endocytosis induced by Aβ is proposed as a new physiopathology mechanism in AD. It is hypothesized that there is critical intraneuronal level above which albumin becomes toxic.

Photobiomodulation Mitigates Cerebrovascular Leakage Induced by the Parkinsonian Neurotoxin MPTP.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to model Parkinson’s disease (PD) as it specifically damages the nigrostriatal dopaminergic pathway. Recent studies in mice have, however, provided evidence that MPTP also compromises the integrity of the brain’s vasculature. Photobiomodulation (PBM), the irradiation of tissue with low-intensity red light, mitigates MPTP-induced loss of dopaminergic neurons in the midbrain, but whether PBM also mitigates MPTP-induced damage to the cerebrovasculature has not been investigated. This study aimed to characterize the time course of cerebrovascular disruption following MPTP exposure and to determine whether PBM can mitigate this disruption. Young adult male C57BL/6 mice were injected with 80 mg/kg MPTP or isotonic saline and perfused with fluorescein isothiocyanate FITC-labelled albumin at various time points post-injection. By 7 days post-injection, there was substantial and significant leakage of FITC-labelled albumin into both the substantia nigra pars compacta (SNc; p < 0.0001) and the caudate-putamen complex (CPu; p ≤ 0.0003); this leakage partly subsided by 14 days post-injection. Mice that were injected with MPTP and treated with daily transcranial PBM (670 nm, 50 mW/cm2, 3 min/day), commencing 24 h after MPTP injection, showed significantly less leakage of FITC-labelled albumin in both the SNc (p < 0.0001) and CPu (p = 0.0003) than sham-treated MPTP mice, with levels of leakage that were not significantly different from saline-injected controls. In summary, this study confirms that MPTP damages the brain’s vasculature, delineates the time course of leakage induced by MPTP out to 14 days post-injection, and provides the first direct evidence that PBM can mitigate this leakage. These findings provide new understanding of the use of the MPTP mouse model as an experimental tool and highlight the potential of PBM as a therapeutic tool for reducing vascular dysfunction in neurological conditions.

Transscleral sustained ranibizumab delivery using an episcleral implantable device: Suppression of laser-induced choroidal neovascularization in rats.

Successful treatment of age-related macular diseases requires an effective controlled drug release system with less invasive route of administration in the eye to reduce the burden of frequent intravitreal injections for patients. In this study, we developed an episcleral implantable device for sustained release of ranibizumab, and evaluated its efficacy on suppression of laser-induced choroidal neovascularization (CNV) in rats. We tested both biodegradable and non-biodegradable sheet-type devices consisting of crosslinked gelatin/chitosan (Gel/CS) and photopolymerized poly(ethyleneglycol) dimethacrylate that incorporated collagen microparticles (PEGDM/COL). In vitro release studies of FITC-labeled albumin showed a constant release from PEGDM/COL sheets compared to Gel/CS sheets. The Gel/CS sheets gradually biodegraded in the sclera during the 24-week implantation; however, the PEGDM/COL sheets did not degrade. FITC-albumin was detected in the retina during 18 weeks implantation in the PEGDM/COL sheet-treated group, and was detected in the Gel/CS sheet-treated group during 6 weeks implantation. CNV was suppressed 18 weeks after application of ranibizumab-loaded PEGDM/COL sheets compared to a placebo PEGDM/COL sheet-treated group, and to the intravitreal ranibizumab-injected group. In conclusion, the PEGDM/COL sheet device suppressed CNV via a transscleral administration route for 18 weeks, indicating that prolonged sustained ranibizumab release could reduce the burden of repeated intravitreal injections.

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

TRPV4 Channel is an Osmosensor and Mechanosensor in Proximal Tubule Cells

The proximal tubule (PT) is the first segment of the nephron and accounts for reabsorption of water and solutes filtered by the glomerulus. In addition, the proximal tubule plays a pivotal role in the endocytosis to reabsorb and metabolize proteins and other substances in glomerular filtrates. Recent studies showed that the PT functions can be modulated by mechanical forces, namely the shear stress on the apical surface and the radial stretch on tubular lumen. Since TRPV4 channel is mainly expressed at the basolateral side of the tubule, we hypothesized that TRPV4 could play a role as stretch-activated channel rather than a shear stress sensor. Patch clamp and Ca2+ imaging experiments showed that TRPV4 channel is expressed in cultured proximal tubule cells and can be activated by the specific agonist GSK1016790A and by hypotonic solution. Moreover, mechanical stretch induced by uniaxial strain in silicone stretchable chambers activated a large Ca2+ influx that was significantly reduced in Trpv4-/- PT cells. Notably, no TRPV4-dependent Ca2+ entry was observed in response to flow-induced cell deformation. Furthermore, we observed that the uptake of FITC-labeled albumin in PT cells, treated with GSK1016790A or mechanically stimulated in stretchable chambers, was significantly reduced in Trpv4-/- PT cells. Finally, we injected angiotensin II in mice, using osmotic minipumps, in order to challenge PT endocytotic activity and increase the proteinuria of mice. Our data showed a massive increase in diuresis of mice. Interestingly, we observed an increase in albuminuria in wild-type mice, and this effect was significantly higher in Trpv4-/- mice. In contrast, glucose and phosphate urinary levels, which undergo PT reabsorption as well, were not significantly affected. Taken together, these results support the view that TRPV4 channel is a mechanosensor in PT cells and modulates their endocytotic activity.

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

A NOVEL PEPTIDE PIVOTAL IN ALZHEIMER’S DISEASE: IN VIVO EVIDENCE IN A RAT MODEL

Previous observations in vitro have suggested that a peptide derived from the C-terminus of acetylcholinesterase (AChE) could be a pivotal molecule in an excitotoxic mechanism underlying the neurodegenerative diseases (Greenfield, 2013). We now report here in vivobioactivity of this key peptide, inducing behavioural and histological effects in the rat following unilateral, intracerebral administration of the AChE peptide at concentrations of 100 nM, 1 μM or 100 μM into the basal forebrain, the initial site of cell loss in Alzheimer's disease (Schmitz et al., 2016). Six days after chronic treatment with the AChE peptide (1 injection/week for 3 weeks), behavioural changes were detected by using the Cylinder Test Performance where animals were observed for 5 minutes. Following the behavioural testing, permeability of the blood brain barrier was compared between the different groups using FITC-labeled albumin and visualised the FITC-albumin leakage on a fluorescent microscope. Treatment with T30 induces contralateral forelimb motor deficit, suggestive of general neurological impairment and not seen with vehicle alone: consistent with this pathological scenario, administration of the higher doses of peptide (1 μM or 100 μM) also enhanced the permeability of the blood-brain barrier as seen from an increase in fluorescent albumin signal in sections of basal forebrain. In summary, it appears that chronic T30 treatment has a selective toxicity in vivo that may throw light on a novel signalling system relevant to the neurodegenerative process.

Th17 Cells Induce Dopaminergic Neuronal Death via LFA-1/ICAM-1 Interaction in a Mouse Model of Parkinson's Disease.

T helper (Th)17 cells, a subset of CD4+ T lymphocytes, have strong pro-inflammatory property and appear to be essential in the pathogenesis of many inflammatory diseases. However, the involvement of Th17 cells in Parkinson's disease (PD) that is characterized by a progressive degeneration of dopaminergic (DAergic) neurons in the nigrostriatal system is unclear. Here, we aimed to demonstrate that Th17 cells infiltrate into the brain parenchyma and induce neuroinflammation and DAergic neuronal death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- or 1-methyl-4-phenylpyridinium (MPP+)-induced PD models. Blood-brain barrier (BBB) disruption in the substantia nigra (SN) was assessed by the signal of FITC-labeled albumin that was injected into blood circulation via the ascending aorta. Live cell imaging system was used to observe a direct contact of Th17 cells with neurons by staining these cells using the two adhesion molecules, leukocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1, respectively. Th17 cells invaded into the SN where BBB was disrupted in MPTP-induced PD mice. Th17 cells exacerbated DAergic neuronal loss and pro-inflammatory/neurotrophic factor disorders in MPP+-treated ventral mesencephalic (VM) cell cultures. A direct contact of LFA-1-stained Th17 cells with ICAM-1-stained VM neurons was dynamically captured. Either blocking LFA-1 in Th17 cells or blocking ICAM-1 in VM neurons with neutralizing antibodies abolished Th17-induced DAergic neuronal death. These results establish that Th17 cells infiltrate into the brain parenchyma of PD mice through lesioned BBB and exert neurotoxic property by promoting glial activation and importantly by a direct damage to neurons depending on LFA-1/ICAM-1 interaction.

Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

Abstract C138: Targeting macropinocytosis in pancreatic cancer

Abstract Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States with a 5-year survival rate of &amp;lt;5%. The deadly nature of this disease compounded by a lack of effective treatment options highlights a need for new therapeutics. Past studies have suggested that pancreatic cancer cells acquire the nutrients needed for cell growth through macropinocytosis, an endocytic process by which protrusions from the cell membrane create large vesicles that transport extracellular fluid along with the constituent proteins and lipids into the cell. Additionally, several studies have suggested that macropinocytosis is regulated in part by various genes including p21 protein (Cdc42/Rac)-activated kinase1 (PAK1), ADP-ribosylation factor 6 (ARF6), and sorting nexin 5 (SNX5). In this study, we investigated the effects of knockdown of these three genes on the macropinocytic activity of pancreatic cancer cells. The goal of this study is to assess whether PAK1, ARF6, or SNX5 may serve as potential targets for the suppression of macropinocytosis in pancreatic cancer, which would hamper the cells' uptake of nutrients thus starving the cancer cells and hindering tumor growth. We carried out this study using MIA PaCa-2 pancreatic cancer cells, which are known to exhibit relatively high levels of macropinocytosis. Treatment of cells with siRNA sequences specific to the three genes resulted in a significantly decreased uptake of FITC-labeled dextran as compared to cells treated with non-targeting siRNA and cells that received no treatment, indicating that inhibiting the expression of each of the genes correlates to a reduction in macropinocytic activity. Among the three genes, PAK1 siRNAs exhibited the most significant and consistent inhibitory effect on macropinocytic activity of MIA PaCa-2 cells. We, therefore, further tested the effect of two small-molecule PAK1 inhibitors, namely IPA-3 and PF-03758309, on macropinocytosis. Treatment of cells with the inhibitors resulted in a dose-dependent decrease in the uptake of FITC-labeled dextran and FITC-labeled albumin. The inhibitors also showed potent anti-proliferative activity in pancreatic cancer cell lines. The results of this study indicate that inhibition of PAK1, ARF6, or SNX5 all resulted in reduced macropinocytic activity of pancreatic cancer cells, with PAK1 showing the most promise as a potential therapeutic target for pancreatic cancer. Citation Format: James Hunt, Serina Ng, Clifford Whatcott, Daniel D. Von Hoff, Haiyong Han. Targeting macropinocytosis in pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C138.

Shank2 Regulates Renal Albumin Endocytosis.

Albuminuria is a strong and independent predictor of kidney disease progression but the mechanisms of albumin handling by the kidney remain to be fully defined. Previous studies have shown that podocytes endocytose albumin. Here we demonstrate that Shank2, a large scaffolding protein originally identified at the neuronal postsynaptic density, is expressed in podocytes in vivo and in vitro and plays an important role in albumin endocytosis in podocytes. Knockdown of Shank2 in cultured human podocytes decreased albumin uptake, but the decrease was not statistically significant likely due to residual Shank2 still present in the knockdown podocytes. Complete knockout of Shank2 in podocytes significantly diminished albumin uptake in vitro. Shank2 knockout mice develop proteinuria by 8 weeks of age. To examine albumin handling in vivo in wild-type and Shank2 knockout mice we used multiphoton intravital imaging. While FITC-labeled albumin was rapidly seen in the renal tubules of wild-type mice after injection, little albumin was seen in the tubules of Shank2 knockout mice indicating dysregulated renal albumin trafficking in the Shank2 knockouts. We have previously found that caveolin-1 is required for albumin endocytosis in cultured podocytes. Shank2 knockout mice had significantly decreased expression and altered localization of caveolin-1 in podocytes suggesting that disruption of albumin endocytosis in Shank2 knockouts is mediated via caveolin-1. In summary, we have identified Shank2 as another component of the albumin endocytic pathway in podocytes.

Negative regulation of glucose transport alleviates microvasculature pathological changes of retinopathy in diabetic mice

Objective To investigate the effect of glucose transporter-1(GLUT1)suppression on microvasculature pathological changes in diabetic mice.Methods Thirty-six 8-week-old C57BL/6 mice were randomly divided into normal control,diabetic control and GLUT1 siRNA treatment groups.Diabetic model was established by intraperitoneal injection of streptozotocin.GLUT1 siRNA treatment group received intravitreal injection of siRNA-mediated GLUT1,and the other two groups received equal amount of non-specific siRNA.Twenty-one weeks after diabetic induction,immunoblotting was conducted to examine the expression of GLUT1 and two inflammation factors:ICAM-1and TNF-α.We also calculated the retinal glucose concentration.Leukostasis assay and vascular leakage assay were utilized to compare the microvasculature pathological changes between the two groups.Results The expression of GLUT1 was significantly down-regulated in GLUT1 siRNA treatment group compared with the other two groups(P0.01),decreased by 77.00%compared with the normal control group and only being 8.07% of the diabetic control group.Though retinal glucose concentration of diabetic control and GLUT1 siRNA treatment groups were higher than that of normal control,that in GLUT1 siRNA treatment group was only 50.05%that of the diabetic control group(P0.01).The expressions of ICAM-1and TNF-αin GLUT1 siRNA treatment and diabetic control were significantly higher than that in the normal control group,and their expressions in GLUT1 siRNA treatment group were 66.14%(P0.05)and 54.76%(P 0.01)those of the diabetic control group,respectively.Moreover,compared with GLUT1 siRNA treatment group,diabetic control group had more adherent leukocytes in the retinal microvasculature and larger areas of leaked FITC-labeled albumin retina.Conclusion GLUT1 siRNA can limit glucose transport into the retina by inhibiting GLUT1 expression,thus decrease retinal glucose concentration and alleviate microvasculature changes of diabetic retinopathy.

The effect of albumin on podocytes: The role of the fatty acid moiety and the potential role of CD36 scavenger receptor

Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified.Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA+FA) or depleted of them (HSA−FA).Receptor-mediated endocytosis of FITC-HSA+FA over 60min was 5 times greater than that of FITC-HSA−FA. 24h exposure of podocytes to albumin up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA−FA. Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake.These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin.

ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

BackgroundEndothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis.ResultsIn human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls.ConclusionsThese results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1.