Loss Of Fenestrations Research Articles

Early and late phases of liver sinusoidal endothelial cell (LSEC) defenestration in mouse model of systemic inflammation

BackgroundLiver sinusoidal endothelial cells (LSECs) have transcellular pores, called fenestrations, participating in the bidirectional transport between the vascular system and liver parenchyma. Fenestrated LSECs indicate a healthy phenotype of liver while loss of fenestrations (defenestration) in LSECs is associated with liver pathologies.MethodsWe introduce a unique model of systemic inflammation triggered by the deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/flLysMCre) characterised by progressive alterations in LSEC phenotype. We implement multiparametric characterisation of LSECs by using novel real-time atomic force microscopy supported with scanning electron microscopy and quantitative fluorescence microscopy. In addition, we provide genetic profiling, searching for characteristic genes encoding proteins that might be connected with the structure of fenestrations.ResultsWe demonstrate that LSECs in Mcpip1fl/flLysMCre display two phases of defenestration: the early phase, with modest defenestration that was fully reversible using cytochalasin B and the late phase, with severe defenestration that is mostly irreversible. By thorough analysis of LSEC porosity, elastic modulus and actin abundance in Mcpip1fl/flLysMCre and in response to cytochalasin B, we demonstrate that proteins other than actin must be additionally responsible for inducing open fenestrations. We highlight several genes that were severely affected in the late but not in the early phase of LSEC defenestration shedding a light on complex structure of individual fenestrations.ConclusionsThe presented model of LSEC derived from Mcpip1fl/flLysMCre provides a valuable reference for developing novel strategies for LSEC refenestration in the early and late phases of liver pathology.

Identification of VEGF Signaling Inhibition-Induced Glomerular Injury in Rats through Site-Specific Urinary Biomarkers.

Cancer therapies targeting the vascular endothelial growth factor (VEGF) signaling pathway can lead to renal damage by disrupting the glomerular ultrafiltration apparatus. The objective of the current study was to identify sensitive biomarkers for VEGF inhibition-induced glomerular changes in rats. Male Sprague-Dawley rats were administered an experimental VEGF receptor (VEGFR) inhibitor, ABT-123, for seven days to investigate the correlation of several biomarkers with microscopic and ultrastructural changes. Glomeruli obtained by laser capture microdissection were also subjected to gene expression analysis to investigate the underlying molecular events of VEGFR inhibition in glomerulus. ABT-123 induced characteristic glomerular ultrastructural changes in rats, including fusion of podocyte foot processes, the presence of subendothelial electron-dense deposits, and swelling and loss of fenestrations in glomerular endothelium. The subtle morphological changes cannot be detected with light microscopy or by changes in standard clinical chemistry and urinalysis. However, urinary albumin increased 44-fold as early as Day three. Urinary β2-microglobulin levels were also increased. Other urinary biomarkers that are typically associated with tubular injury were not significantly impacted. Such patterns in urinary biomarkers can provide valuable diagnostic insight to VEGF inhibition therapy-induced glomeruli injuries.

Kruppel‐like Factor 4 is Critical to the Maintenance of Glomerular Endothelial Integrity

BackgroundThrombotic microangiopathy (TMA) represents the most severe manifestation of glomerular endothelial injury consisting of endothelial cell (EC) swelling, subendothelial expansion, inflammation and microthrombi. TMA occurs subsequent to various etiologies and is associated with dysregulation of thrombotic and inflammatory transcripts and complement activation. Expression of membrane bound (CD55, CD59, CD46) complement regulators, is altered in many subtypes of TMA and it is unclear if this is a driver or consequence of EC injury. To investigate transcriptional mediators involved in TMA, we reviewed expression arrays of both global and single cell RNA‐seq from human kidney biopsies with complement mediated renal microvascular injury and observed that Krüppel‐Like Factor 4 (KLF4), a zinc finger transcription factor, is both differentially expressed in ECs and predicted to regulate other differentially expressed genes. Previous studies demonstrate that KLF4 is a critical mediator of anti‐thrombotic and anti‐inflammatory phenotype in systemic vascular beds. We aimed to investigate its role in modulating renal microvascular injury and complement activation in TMA.MethodsEndothelial‐specific Klf4 knockout mice (Klf4ΔEC) were generated by crossing Klf4fl/fl mice withCdh5‐Cre mice. As two separate models of endothelial dysfunction, Klf4ΔEC mice were both aged to 1 year and crossed with Nos3−/− mice to generate double knockout mice (DKO). Periodic acid‐Schiff, electron microscopy, immunofluorescence, ELISA and RT‐PCR were performed to investigate the effects of endothelial Klf4 on the renal microvasculature. Renal histology and ultrastructure were evaluated, blinded, by renal pathology (MPR).ResultsAt 12 weeks of age, while no overt histologic changes were observed, Klf4ΔEC mice exhibited increased glomerular Vcam‐1 and Pai‐1 expression and complement activation (increased C3 and C5b‐9 deposition) as compared to Klf4fl/fl mice. To investigate the basis for this increased complement activation, we measured the expression of the membrane‐bound complement regulators and noted reduced glomerular Cd55 and Cd59 expression in Klf4ΔEC as compared with Klf4fl/fl mice. Interestingly, 1‐year‐old Klf4ΔEC mice exhibited pathology consistent with subacute TMA (glomerular capillary congestion, EC swelling, subendothelial expansion and mesangiolysis as well as increased intraglomerular von Willebrand Factor and albuminuria) as compared to age‐matched Klf4fl/fl mice. In addition, Klf4ΔEC mice crossed with nitric oxide synthase knockout (Nos3−/−) mice also exhibited increased glomerular complement activation and histological and ultrastructural features consistent with subacute TMA (loss of fenestrations, glomerular capillary congestion, tuft collapse with basement membrane wrinkling) as compared to all other groups (12 weeks). Finally, knockdown of KLF4 in cultured human microvascular endothelial cells reduced the expression of CD55, whereas induction of KLF4 increased CD55 expression in HMEC‐1s.ConclusionsLoss of endothelial Klf4 in both aged mice and in Nos3−/− mice recapitulates many pathologic features of renal‐TMA, including complement activation and is potentially mediated through its regulation of Cd55.Support or Funding InformationNIDDK

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease

ABSTRACTMouse models of age-related macular degeneration (AMD) such as Ccl2−/- and Ccl2−/-/Cx3cr1−/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch’s membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD’s pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch’s membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

Liver sinusoidal endothelial cells (LSECs) modifications in patients with chronic hepatitis C

The sinusoidal endothelial cells present in the liver (LSECs) are tipically characterized by the presence of pores (fenestrae). During some pathological conditions LSECs undergo “capillarization”, a process characterized by loss of fenestrations and acquisition of a vascular phenotype. In chronic liver disease capillarization has been reported to precede the development of fibrosis. LSECs modification in the setting of HCV infection is currently poorly investigated. Considering that HCV accounts for important changes in hepatocytes and in view of the intimate connection between hepatocytes and LSECs, here we set out to study in great detail the LSECs modifications in individuals with HCV-dependent chronic hepatitis. Electron microscopy analysis, and evaluation of CD32, CD31 and caveolin-1 expression showed that in HCV infection LSECs display major morphological changes but maintain their phenotypical identity. Capillarization was observed only in cases at initial stages of fibrosis. Our findings showed that the severity of LSECs modifications appears to be correlated with hepatocytes damage and fibrosis stage providing novel insight in the pathogenesis of HCV-chronic hepatitis.

Matrix Stiffness Regulate Liver Sinusoidal Endothelial Cells (LSECs) Function: Importance for Liver Fibrosis Progression

BackgroundLiver sinusoidal endothelial cells (LSECs) are a highly specialized endothelial cell that participates in numerous liver metabolic activities and collectively function as a scavenger system in the liver by removing waste macromolecules playing a vital role in the balance of lipids, cholesterol, and vitamins. Prior to hepatic fibrosis, independent of their etiology, LSECs become highly pro‐inflammatory, capillarized (loss in fenestrations), and loss in specialized receptors (Stabilin‐1, Stabilin‐2, CD31 and SE‐1). Liver fibrosis also leads to significant loss in the endocytosis function of LSECs. Thus understanding regulation of LSEC phenotype may be critical to understanding fibrosis. Extensive remodeling of the extracellular matrix occurs during fibrosis that leads to liver stiffening. The role of matrix stiffness as related to subtle but pivotal changes in LSECs physiology is under explored. The overall goal of our study is the development and implementation of a platform that enables the convergence of engineered cell microenvironments with the phenotypic and functional analysis of LSECs. Using our innovative biomimetic liver fibrosis model that allows modulation of substrate stiffness, we investigated the role of liver matrix stiffness in modulating LSECs function in fibrotic‐like microenvironment.Materials and MethodsPrimary LSECs were cultured on our novel polymer film coated polydimethylsiloxane (PDMS) gels with 2 kPa, 9 kPa 25 kPa and 55 kPa elastic modulus mimicking healthy, early fibrotic, fibrotic and extremely fibrotic substrates. SEM was used to image to fenestrations of LSECs and HA endocytosis assay was performed to measure the LSECs function.ResultsLSECs cultured on stiffer environment had significant remodeling of cytoskeletal proteins and morphology indicated of stress fibers. Also we observed that LSECs on fibrotic substrates resulted in loss of fenestrations (capillarization). This is critical as capillarization has been show to precede hepatic fibrosis and “capillarized” LSECs lose the ability to promote hepatic stellate cell (HSC) quiescence. LSECs on stiffer environment also had higher expression of cell adhesion molecules, VCAM‐1 and ICAM‐1 indicating the loss of phenotype of the cells. Fibrotic stiffness also impeded the HA endocytosis in LSECs, one of the main functions of the cells. These data suggest a plausible mechanism that increased stiffness modulates hepatocyte and LSEC function causing liver functional failure. Similar effect was observed in LSECs isolated from Non‐Alcoholic Fatty Liver Disease (NAFLD) rat models indicating correlation to physiological conditions.ConclusionsTogether, all these data demonstrates the plausible role of stiffness in regulating LSECs function and contribute to HSC activation and progression of liver fibrosis.Support or Funding InformationNIH P20GM113126; NIH P20GM104320 imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of Electron Microscopy (EM)to Detect Transplant Glomerulopathy (TG) in Patients with Late-Indication Allograft Biopsy

Introduction The main causes of late graft loss are: death of patients with functioning graft and chronic allograft nephropathy, in which it stands out, to chronic rejection mediated by antibodies (cABMR). cABMR was defined during the Banff meeting in 2005 due to: Transplant Glomerulopathy (TG), Multilamination of the basal membrane of the tubular pericapillary (MLMBPCT), Transplant vasculopathy and interstitial fibrosis with tubular atrophy (IFTA), with deposit of C4d in PCT and the presence of Anti-HLA Antibodies specific donor (DSA). The presence of C4d deposition in the peritubular capillary (PTC) is a diagnostic marker of ABMR greater than 50% in acute graft rejection but not in its chronic or late ABMR phenotype, so its usefulness is currently questioned in the late phase, identifying them as C4d negative. The typical characterizes cABMR is TG recognized as a glomerulopathy that manifests a double contour of the glomerular capillary,. The cABMR with duplication or MLMBG, increase of the subendothelial space, interposition of mesangial cells, loss of fenestration in the endothelial cells, and MLMBPTC as the findings most significant effects linked to the interaction of antibodies in the glomerular endothelium. Objectives Detect morphological changes of TG by ME in patients with chronic allograft dysfunction who have late biopsies for clinical indication, whose previous histopathological evaluation was by Banff classification in MO and IHC for C4d. Materials and Methods We performed a retrospective observational study of a descriptive cohort, kidney transplant patients performed in the Nephrology and Transplant Service of the CPVS, Córdoba, Argentina who presented chronic allograft dysfunction with late biopsies for clinical indication performed in the period from July 2011 to August 2016.The MO and ME analysis was performed by two different pathologists.The indication for renal biopsy was performed according to the following criteria: increase in serum creatinine ≥0.3 mg / dl or more during the post-transplant evolution. Proteinuria evaluated by Pr/Cr ratio in spot urine ≥0.3 mg / dl.3. Patients with at least 6 months of kidney transplant.4. All samples of biopsies performed should have at least 4 glomeruli for the MO and ME analysis, respectively. Results Inflammation of the glomerular endothelium (GECS) in 21/22 95%.Podocyte deletion processes in 100% of patients, 22.7% severe and in the rest 76% mild to moderate. MLMBPTC, 68% were present in 63.6% 14/22 patients mild (<3 layers) and in only 5% was moderate (7 layers). ). MLMBG in 17/22 77.2%..Conclusion The use of EM in late kidney transplant biopsies by indication allowed detecting ultrastructural changes of transplant glomerulopathy of cABMR in a high % of patients with C4d negative w/wo DSA. Prospective studies should be necessary as protocol biopsies with earlier changes suggestive of TG with EM, and anticipate the clinically evident damage.

A SIEVE-RAFT HYPOTHESIS FOR THE REGULATION OF ENDOTHELIAL FENESTRATIONS

Fenestration morphology is a remarkable example of the synergy between structure and function. Through a better understanding of fenestration structure our understanding of its function will be enhanced. Fenestrations are transcellular pores that act as fundamental biological ultra-filters allowing diffusive and convective passage of substrates across cells without relying on endocytosis or other receptor-mediated mechanisms. They facilitate passive transfer of substances such as lipoproteins [1], parasites [2], pharmacological agents [3] and gene transfer vectors [4]. Fenestrated cells are highly conserved in evolution and have been documented in all species from fish to humans [5–9] and even in the phloem vascular system of higher plants [10]. In animals they are found in several cell types including liver sinusoidal endothelial cells (LSECs) [11] (Figure 1), glomerular endothelial cells [5], endothelial cells of the area postrema [12] and the posterior pituitary [13] of the brain, as well as numerous cancers [14]. All of these tissues require unimpeded transfer of substances between blood and surrounding cells. Fenestrations are essential for human health and loss of fenestrations in LSECs results in impaired lipid, drug and insulin transfer [15–17] and regeneration [18]. However, despite their ubiquity and biological importance, we are only beginning to understand the molecular and cellular pathways, and the spatial and temporal sequence of events involved in fenestration formation. Here, we propose a novel sieve-raft hypothesis [19] as a key mechanism regulating fenestrations in the LSEC. Figure 1 Microscopy of LSEC fenestrations and the LSEC membrane. Figure 1A is a scanning electron micrograph of an isolated LSEC in culture. The micrograph clearly displays fenestrations, examples are denoted by an asterix (*), arranged in groups (sieve plates) ...

The glomerular endothelium emerges as a key player in diabetic nephropathy

The effects of diabetes on glomerular structure and particularly that of the glomerular capillary wall have been extensively documented. By correlation with clinical measurements, Weil and colleagues provide important insights into the functional significance of glomerular structural changes in type 2 diabetes. Podocyte detachment correlates with albumin-to-creatinine ratio, but less strongly than does loss of endothelial fenestrations, which also correlates with reduced glomerular filtration rate. The role of the glomerular endothelium in diabetic nephropathy demands further scrutiny.

Antibody Mediated Rejection (AMR) Lesions in Renal Biopsies: Evidence of Progression with a Specific Microvascular Inflammatory Phenotype. Diagnostic Implications

Introduction: The diagnosis of AMR hinges on the combination of DSA, C4d and microvascular injury/inflammation (MVI) but is complicated by temporal lesion variations and the fluctuations of C4d staining and DSA levels. MVI needs therefore to be fully analized for optimal diagnostic use. Methods: 563 consecutive renal biopsies (Bx) from 496 patients were analyzed using current Banff criteria and were stained with C4d, CD68 and CD3. For each biopsy, concurrent DSA measurements were also available. The mean number of biopsies per patient was 1.44 ((+/- 0.91, median 1). Mean patient age was 50.27 yeras (+/-13.79). 63.1% of the patients were male, 8.1% had regrafts and 6.6% were hepatitis C positive. Specific definitions used for analysis: C4d+ ≥10% staining in peritubular capillaries; DSA+ >1000MFI; MVI defined as glomerulitis (g), transplant glomerulopathy (cg) and peritubular capillaritis (ptc) >0 scores. On electron microscopy (EM), TG was defined by duplication of the basement membranes, whereas endothelial injury (EI) was defined as swelling, loss of fenestrations ± expansion of the subendothelial space. The maximum nubmer of glomerular macrophages and lymphocytes were also counted (GM and GL) with CD68 and CD3 stains, respectively. Results: Cases with acute T-cell mediated rejection (ACMR) (n=76) or cases lacking any AMR or ACMR features (n=187) had an average of 5±6 GM/3.4±4.8 GL and 3.67±4.62 GM/1.57±2.52 GL respectively (NS), whereas cases with any combination of AMR parameters that includes MVI displayed a similarly marked increase of GM/GL (up to 16.58±12.19 GM/ 7.94±6.64 GL, p=.000). In contrast cases with only C4d and/or DSA without MVI showed low GM/GL (up to 5.37±6 GM/1.87±2.23 GL) statistically indistinguishable from the ACMR or cases lacking any AMR or ACMR features. The time post Tx was also higher in cases with MVI, irrespective of C4d or DSA presence (34.38± 35.73) than in cases with DSA and/or C4d only (16.96± 34.74)(p=.000), indicating a temporal sequence. This particular glomerular inflammation immunophenotype characterized not only the entire spectrum of MVI but was also associated to EM microvascular pathology, time post Tx and C4d results (Table 1).Table: [Correlations with EM findings]Conclusions: AMR displays a temporal an immunophenotypical spectrum ranging from an earlier DSA± C4d stage without MVI and progression to the full spectum (DSA+C4d+MVI) including a marked increase in GM accompanied by a definite but lesser increase in GL (2:1 ratio). This glomerular immunophenotype is so characteristic and constant that its presence would serve as a surrogate to the more variable C4d staining for the diagnosis of AMR (i.e. C4d negative AMR).

Antibody Mediated Rejection (AMR) Lesions in Renal Biopsies: Evidence of Progression with a Specific Microvascular Inflammatory Phenotype. Diagnostic Implications

Introduction: The diagnosis of AMR hinges on the combination of DSA, C4d and microvascular injury/inflammation (MVI) but is complicated by temporal lesion variations and the fluctuations of C4d staining and DSA levels. MVI needs therefore to be fully analized for optimal diagnostic use. Methods: 563 consecutive renal biopsies (Bx) from 496 patients were analyzed using current Banff criteria and were stained with C4d, CD68 and CD3. For each biopsy, concurrent DSA measurements were also available. The mean number of biopsies per patient was 1.44 ((+/- 0.91, median 1). Mean patient age was 50.27 yeras (+/-13.79). 63.1% of the patients were male, 8.1% had regrafts and 6.6% were hepatitis C positive. Specific definitions used for analysis: C4d+ ≥10% staining in peritubular capillaries; DSA+ >1000MFI; MVI defined as glomerulitis (g), transplant glomerulopathy (cg) and peritubular capillaritis (ptc) >0 scores. On electron microscopy (EM), TG was defined by duplication of the basement membranes, whereas endothelial injury (EI) was defined as swelling, loss of fenestrations ± expansion of the subendothelial space. The maximum nubmer of glomerular macrophages and lymphocytes were also counted (GM and GL) with CD68 and CD3 stains, respectively. Results: Cases with acute T-cell mediated rejection (ACMR) (n=76) or cases lacking any AMR or ACMR features (n=187) had an average of 5±6 GM/3.4±4.8 GL and 3.67±4.62 GM/1.57±2.52 GL respectively (NS), whereas cases with any combination of AMR parameters that includes MVI displayed a similarly marked increase of GM/GL (up to 16.58±12.19 GM/7.94±6.64 GL, p=.000). In contrast cases with only C4d and/or DSA without MVI showed low GM/GL (up to 5.37±6 GM/1.87±2.23 GL) statistically indistinguishable from the ACMR or cases lacking any AMR or ACMR features. The time post Tx was also higher in cases with MVI, irrespective of C4d or DSA presence (34.38± 35.73) than in cases with DSA and/or C4d only (16.96± 34.74)(p=.000), indicating a temporal sequence. This particular glomerular inflammation immunophenotype characterized not only the entire spectrum of MVI but was also associated to EM microvascular pathology, time post Tx and C4d results (Table 1).Table: [Correlations with EM findings]Conclusions: AMR displays a temporal an immunophenotypical spectrum ranging from an earlier DSA± C4d stage without MVI and progression to the full spectum (DSA+C4d+MVI) including a marked increase in GM accompanied by a definite but lesser increase in GL (2:1 ratio). This glomerular immunophenotype is so characteristic and constant that its presence would serve as a surrogate to the more variable C4d staining for the diagnosis of AMR (i.e. C4d negative AMR).

Formation of immune complexes and thrombotic microangiopathy after intravitreal injection of bevacizumab in the primate eye

In this study, the effect of intravitreal injection of bevacizumab on choroidal blood vessels was examined in primate eyes. Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14. For each animal, one eye was embedded in paraffin whereas the other eye was embedded for electron microscopy. Seven untreated or PBS (phosphate buffered saline)-injected monkeys were used as controls. Thrombotic microangiopathy was found in the choriocapillaris and choroidal vessels of all eight injected eyes. Acute microangiopathy was characterized ultrastructurally as swelling of the endothelium, loss of fenestrations and complete collapse of the capillaries, and was commonly observed in bevacizumab-treated eyes. Quantitative analysis showed reduction of the lumina of the choriocapillaris in the eyes of three of the monkeys. Bevacizumab was frequently localized inside the blood vessels, often filling the entire breadth of the vessels, and formed clusters with blood cells. Death of photoreceptors occurred in two monkeys. This study indicate that intravitreal injection of bevacizumab in monkeys induces activation of platelets, degranulation of thrombocytes and neutrophils, formation of immune complexes, thrombotic microangiopathy and alteration of the blood flow in choroidal vessels.

Renal Thrombotic Microangiopathy in Mice with Combined Deletion of Endocytic Recycling Regulators EHD3 and EHD4

Eps15 Homology Domain-containing 3 (EHD3), a member of the EHD protein family that regulates endocytic recycling, is the first protein reported to be specifically expressed in the glomerular endothelium in the kidney; therefore we generated Ehd3 –/– mice and assessed renal development and pathology. Ehd3 –/– animals showed no overt defects, and exhibited no proteinuria or glomerular pathology. However, as the expression of EHD4, a related family member, was elevated in the glomerular endothelium of Ehd3 –/– mice and suggested functional compensation, we generated and analyzed Ehd3 –/–; Ehd4 –/– mice. These mice were smaller, possessed smaller and paler kidneys, were proteinuric and died between 3–24 weeks of age. Detailed analyses of Ehd3 –/–; Ehd4 –/– kidneys demonstrated thrombotic microangiopathy (TMA)-like glomerular lesions including thickening and duplication of glomerular basement membrane, endothelial swelling and loss of fenestrations. Other changes included segmental podocyte foot process effacement, mesangial interposition, and abnormal podocytic and mesangial marker expression. The glomerular lesions observed were strikingly similar to those seen in human pre-eclampsia and mouse models of reduced VEGF expression. As altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis was observed in the absence of EHD3 and EHD4, we propose that EHD-mediated endocytic traffic of key surface receptors such as VEGFR2 is essential for physiological control of glomerular function. Furthermore, Ehd3 –/–; Ehd4 –/– mice provide a unique model to elucidate mechanisms of glomerular endothelial injury which is observed in a wide variety of human renal and extra-renal diseases.

The Impact of Poloxamer 407 on the Ultrastructure of the Liver and Evidence for Clearance by Extensive Endothelial and Kupffer Cell Endocytosis

Poloxamer 407 (P407) is a non-ionic detergent that is used widely in pharmaceutical formulations and personal care products. In animals, P407 causes hyperlipidaemia. P407 is taken up by the liver and causes loss of fenestrations in liver sinusoidal endothelial cells (LSEC), which contributes to the pathogenesis of hyperlipidaemia. Here the short-term (1-15 days) effects of P407 on all liver cells were investigated in mice using electron and light microscopy. As expected, P407 was associated with hyperlipidaemia. Kupffer cells became massively engorged with vacuoles and took on a marked honeycomb morphology. LSECs also became engorged with vacuoles and endocytosis was activated. The diameter of lipoproteins in the space of Disse was less than those in the lumen, consistent with a filtering effect of fenestrations. Defenestration of the LSEC was noted. Hepatocyte endocytosis of lipoproteins and P407 particles was also noted; however, hepatocyte steatosis was not evident. Hepatic stellate cells did not appear to be abnormal. In conclusion, P407 is taken up by the liver mostly through endocytosis by LSECs and Kupffer cells.

Arsenic signals through g‐proteins and NADPH Oxidase (NOX) to promote mouse liver sinusoidal endothelial cell capillarization

Arsenic is a global health hazard that may cause disease in over 100 million people world‐wide. Liver disease including portal hypertension, non‐cirrhotic portal fibrosis, and vascular shunting and oxidative injury are associated drinking water with elevated arsenic levels. Liver sinusoidal endothelial cell (LSEC) capillarization with loss of fenestrations and increased surface expression of PECAM‐1 is an early event in these diseases. Drinking water containing 10 ppb or more of arsenic promotes LSEC capillarization in mice through unknown mechanisms. Therefore, the hypothesis that arsenic signals through NOX to promote LSEC capillarization was tested in primary mouse LSEC cultures. Ex Vivo arsenic exposures (0.5–5 μM) for 8 h recapitulated in vivo effects on capillarization. The ex vivo exposures increased superoxide generation that was prevented by pre‐incubating cells with the scavenger tempol or a selective NOX2 inhibitory peptide, gp91‐tat. Both tempol and gp91‐tat prevented arsenic‐stimulated capillarization. Finally, inhibition of either Gai or Rac1 GTPase also blocked arsenic‐stimulated oxidant generation and capillarization. These data are first demonstrating that environmental levels of arsenic stimulate LSEC capillarization through a g‐protein coupled, NOX‐mediated signaling cascade. Sponsored by EPA STAR grant FP‐91654201 and NIEHS grant R01ES013781.

Marked changes of the hepatic sinusoid in a transgenic mouse model of acute immune-mediated hepatitis

The liver sinusoidal endothelial cell (LSEC) is increasingly recognized as having an important role in hepatic immunity. However, the responses of LSECs and the hepatic sinusoid in immune-mediated hepatitis are poorly described. We studied a transgenic mouse model of acute immune-mediated hepatitis: Met-Kb mice injected with T cells from Des-TCR mice. Hepatitis was characterized by lymphocyte infiltrates causing severe but transient liver damage. There were marked changes in the ultrastructure of the LSEC five days after injection of the T cells that coincided with the peak of the hepatitis. The porosity of fenestrations in the LSEC decreased and the endothelium became thickened. LSECs appeared to be markedly activated. These changes were associated with narrowing of the space of Disse, loss of hepatocellular microvilli and deposition of basal lamina. Lymphocytes were seen passing through fenestrations. Loss of fenestration in the LSEC prevented hepatitis induced by a second injection of lymphocytes on day 5. Structural changes in the LSEC occur during the peak of a mouse model of immune-mediated hepatitis. These changes were associated with attenuation of subsequent liver damage, suggesting that they may influence immunological responses mediated by LSECs or the passage of lymphocytes through LSEC fenestrations.

Comparative Analysis of the Renoprotective Effects of Pentoxifylline and Vitamin E on Streptozotocin-Induced Diabetes Mellitus

Background: Oxidative stress (OxS) induced by diabetes plays an important role in the development of diabetic nephropathy. Studies have shown that antioxidants are beneficial in its reduction. Vitamin E has been documented as providing the most improvement in the antioxidative status. Recently, pentoxifylline (PTX) has been proposed to have antioxidant properties. Aim: The aim of the study was to assess the ability of two antioxidants to reduce lipid peroxidation and renal hypertrophy in vivo. Methods: Diabetes was induced by streptozotocin (STZ) in rats. Treatment groups 7were divided as follows: healthy (H), diabetic without treatment (STZ), PTX treated group (STZ + PTX), and vitamin E supplemented (STZ + E) group. At 8 weeks, kidneys were removed; one was homogenized to quantify lipoperoxide levels (LPOS), and the other was used to study the morphological changes by electron microscopy (EM). Additionally, plasma total antioxidant activity (TAA) was quantified. Results: A reduction in LPOS was observed in both groups: PTX and vitamin E with regard to STZ group. PTX increased TAA compared to STZ + E, which restored it to its normal values. However, both treatments reduced the LPO/TAA ratio to lower basal levels; hence, similar results were obtained in terms of correcting functional parameters. Structural changes in STZ rats included a glomerular membrane thickening, podocyte flattening, as well as loss of fenestration in the endothelial layer. All these changes were less aggressive for treated rats. Conclusions: Vitamin E and PTX have potential therapeutic properties that may help to retard the rate of deterioration of diabetic kidneys.

Comparative Analysis of the Renoprotective Effects of Pentoxifylline and Vitamin E on Streptozotocin-Induced Diabetes Mellitus

Oxidative stress (OxS) induced by diabetes plays an important role in the development of diabetic nephropathy. Studies have shown that antioxidants are beneficial in its reduction. Vitamin E has been documented as providing the most improvement in the antioxidative status. Recently, pentoxifylline (PTX) has been proposed to have antioxidant properties. The aim of the study was to assess the ability of two antioxidants to reduce lipid peroxidation and renal hypertrophy in vivo. Diabetes was induced by streptozotocin (STZ) in rats. Treatment groups were divided as follows: healthy (H), diabetic without treatment (STZ), PTX treated group (STZ+PTX), and vitamin E supplemented (STZ+E) group. At 8 weeks, kidneys were removed; one was homogenized to quantify lipoperoxide levels (LPOS), and the other was used to study the morphological changes by electron microscopy (EM). Additionally, plasma total antioxidant activity (TAA) was quantified. A reduction in LPOS was observed in both groups: PTX and vitamin E with regard to STZ group. PTX increased TAA compared to STZ+E, which restored it to its normal values. However, both treatments reduced the LPO/TAA ratio to lower basal levels; hence, similar results were obtained in terms of correcting functional parameters. Structural changes in STZ rats included a glomerular membrane thickening, podocyte flattening, as well as loss of fenestration in the endothelial layer. All these changes were less aggressive for treated rats. Vitamin E and PTX have potential therapeutic properties that may help to retard the rate of deterioration of diabetic kidneys.

Age‐related pseudocapillarization of the human liver

Age-related changes in liver function are important because they may promote susceptibility to adverse drug reactions, neurotoxicity, atherosclerosis, and other important diseases in older people. Age-related changes in the rat hepatic sinusoidal endothelium, termed pseudocapillarization, have been described recently and these may contribute to hepatic impairment. The present study has examined surgical and post-mortem specimens with immunohistochemistry and transmission electron microscopy to determine whether pseudocapillarization also occurs in older humans. The age of the subject, independent of systemic disease or hepatic pathology in surgical and post-mortem samples of human liver, was associated with increased peri-sinusoidal expression of von Willebrand's factor, collagen I, collagen IV, and staining with Masson's trichrome. Electron microscopy revealed significant age-related thickening of the sinusoidal endothelium (young 165 +/- 17 nm, middle age 222 +/- 11 nm, older 289 +/- 9 nm, p < 0.001) with loss of fenestrations (young 7.7 +/- 0.7 per 10 micro m, middle age 3.6 +/- 0.5 per 10 micro m, older 1.5 +/- 0.4 per 10 micro m, p < 0.001), and age-related deposition of basal lamina and collagen. In conclusion, ageing in humans is associated with morphological changes in the sinusoidal endothelium and space of Disse which are presumptively related to the ageing process and potentially represent an important link between the ageing process and disease susceptibility.

Responses of the fetal cerebral vasculature to prenatal cytotoxic brain damage

Prenatal exposure to the drug methylazoxymethanol acetate (MAM Ac) has been commonly used as an experimental model of prenatal cytotoxic brain damage. We have examined the effects of this prenatal brain damage on the development of vasculature in the fetal neocortex, because vasculature is likely to play an important role in fetal brain repair. Pregnant Wistar strain rats received either MAM Ac (25 mg/kg) or saline on embryonic day (E) 13 by IP injection. At E15, E17, E19, and E21, the rats were overdosed with pentobarbitone (100 mg/kg) and fetuses removed for paraffin embedding, lectin histochemistry, and electron microscopy. For lectin histochemistry, sections were exposed to a horseradish peroxidase-conjugated B4 isolectin from Griffonia simplicifolia. Fetuses exposed to MAM Ac showed significant deficits in cortical vessel branch point density at E15 and E17 (46% and 20%, respectively). There were also significant reductions in stem vessel density at these ages (34% and 26%, respectively) among MAM Ac exposed animals, but there were no differences in growing tip density, capillary internal diameter, or stem vessel internal diameter. The only consistent differences between the two groups at the electron microscope level were an increase in vacuolisation and an irregular luminal surface in MAM Ac animals at E15. Tight junction formation, basement membrane formation, and loss of fenestrations were not delayed in experimental animals. The results indicate that there is no increase in angiogenic activity coincident with the fetal neocortical repair that follows MAM Ac induced damage.