Apolipoprotein E and beta A4-amyloid: signals and effects.

In humans, the apolipoprotein E gene (APOE) is polymorphic with the alleles APOE epsilon 2, 3 and 4 coding for apolipoproteins (Apo) E2, 3 and 4. Apart from age, the APOE epsilon 4 allele represents the most important risk factor in sporadic Alzheimer's disease (AD). Compared to APOE epsilon 3 homozygotes, the histopathological onset of tau pathology is found 1-2 decades earlier but progresses with the same speed. ApoE dose-dependently and specifically increases free intraneuronal calcium levels in the order ApoE4 > ApoE3 > ApoE2. This effect is amplified in the presence of beta A4-peptide. The ApoE effects on calcium are not affected by the blockade of action potentials with tetrodotoxin, or by inhibition of common ApoE binding sites. The calcium channel involved has been identified as a P/Q-type-like channel. Brain tissue ApoE levels differ with respect to APOE alleles and Braak-stage for Alzheimer-histopathology. The production of ApoE in astrocytes is controlled by several receptor/effector systems such as adrenoceptors and cAMP. In the presence of beta A4-peptide fragments, astrocytes stop their synthesis of ApoE resulting in a massive reduction in the bioavailability of ApoE. In the periphery, ApoE directs cholesterol transport and thereby influences its cellular concentrations. In neurons, changes in the concentration of cholesterol influence the phosphorylation status of the microtubule-associated protein tau at sites known to be altered in AD.

Transitional stages in the development of the rabbit renal collecting duct.

The collecting duct (CD) epithelium of the mammalian kidney is an extraordinary structure with respect to its functional changes during development and its heterogeneous composition when matured. All of the different nephron epithelia of the mammalian kidney consist of one single cell type. In contrast, the differentiated CD is composed of at least three distinct cell types [principal, alpha intercalated-, and beta intercalated cells] that are responsible for the multiple physiological functions of this kidney compartment. During development the function of the CD changes: initially, the CD ampulla serves as an embryonic inducer, while the matured epithelium plays a key role in maintaining the homeostasis of body fluids. At present the process of CD maturation is not well understood. Neither the time course of development nor the morphogenic factors leading to the heterogeneously composed epithelium are known. In the present study the differentiation of the CD epithelium was investigated using newly developed monoclonal antibodies and well-characterized antisera. The morphological changes induced during differentiation were monitored by immunohistochemistry and scanning electron microscopy. The experiments were performed on neonatal and adult rabbit kidneys. Results obtained by light microscopical techniques and scanning electron microscopy revealed that the ampullary tip can be distinguished from the ampullary neck, as well as from the maturing CD. A number of proteins that were not detectable in the ampulla were detected in the neonatal CD and were found at even higher concentrations in the adult CD (PCD8, chloride/bicarbonate exchanger). Other proteins (PCD9) were downregulated during differentiation. For the first time the transient character of the differentiation stage of the neonatal CD could be demonstrated unequivocally. Furthermore, considerable heterogeneity in protein expression patterns (PCD6 and PCD9) was demonstrated within the beta IC cell population of the mature CD.

Receptors of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in fetal and adult human kidney: localization and [125I]VEGF binding sites.

Vascular endothelial growth factor (VEGF) has an important function in renal vascular ontogenesis and is constitutively expressed in podocytes of the adult kidney. The ability of VEGF to be chemotactic for monocytes and to increase the activity of collagenase and plasminogen activator may have implications for renal development and renal disease. In humans, the cellular actions of VEGF depend on binding to two specific receptors: Flt-1 and KDR. The aims of this study were: (1) to localize VEGF receptor proteins in human renal ontogenesis; (2) to quantify VEGF binding in human fetal and adult kidney; and (3) to dissect the binding into its two known components: the KDR and Flt-1 receptors. The latter aim was achieved by competitive binding of VEGF and placenta growth factor-2, which only binds to Flt-1. Quantification of 125I-VEGF binding sites was performed by autoradiography and computerized densitometry. By double-label immunohistochemistry, VEGF receptor proteins were localized solely to endothelial cells of preglomerular vessels, glomeruli, and postglomerular vessels. In developing glomeruli, VEGF receptor protein appeared as soon as endothelial cells were positive for von Willebrand factor. Specific 125I-VEGF binding could be localized to renal arteries and veins, glomeruli, and the tubulointerstitial capillary network in different developmental stages. Affinity (Kd) of adult (aK) and fetal (fK) kidneys was: Kd: glomeruli 38.6 +/- 11.2 (aK, n = 5), 36.3 +/- 7.1 (fK, n = 5); cortical tubulointerstitium 19.4 +/- 2.6 (aK, n = 5), 11.6 +/- 7.0 (fK, n = 5) pmol. Placenta growth factor-2 displaced VEGF binding in all renal structures by approximately 60%. VEGF receptor proteins thus were found only in renal endothelial cells. A coexpression of both VEGF binding sites could be shown, with Flt-1 demonstrating the most abundant VEGF receptor binding sites in the kidney. These studies support the hypothesis of a function for VEGF in adult kidney that is independent of angiogenesis.

Effects of vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) on haemodynamics and permselectivity of the isolated perfused rat kidney.

BACKGROUND: Vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF) is a selective mitogen for endothelial cells; it increases microvascular permeability and has been shown to relax isolated canine coronary arteries by an endothelium-dependent mechanism. In many tissues VEGF/VPF is expressed after an appropriate stimulus, mostly hypoxia. In the kidney VEGF/VPF is constitutively expressed in glomerular podocytes and epithelia of collecting duct. Glomerular and peritubular capillary endothelia also constitutively express specific VEGF receptors. The in vivo function of renal VEGF/VPF is unknown. METHOD: In the present study the effects of human recombinant VEGF165 on renal haemodynamics and glomerular permselectivity was investigated in the isolated perfused kidney of the rat. RESULTS: In kidneys preconstricted by noradrenaline (NA 1.5 x 10(-7) mol/l) VEGF/VPF (155 pmol/l) caused an almost complete return of renal perfusion flow rate to pre-NA values (before NA 113 +/- 4%, after NA 100%, 15 min with VEGF/VPF 111 +/- 4%). Shortly after VEGF/VPF administration VEGF/VPF-induced relaxation commenced, and became significant after 2 min (15 min with VEGF/VPF vs without VEGF/VPF 111 +/- 4% vs 103 +/- 2%; P<0.05). In the presence of the NO-synthase inhibitor N(W)-nitro-L-arginine (L-NNA; 5 x 10(-5) mol/l) VEGF/VPF caused only small, transient relaxations (before NNA 109 +/- 5%, after NNA 100%, 15 min with VEGF 95 +/- 2%). The cyclooxygenase inhibitor diclofenac failed to inhibit the relaxing activity of VEGF/VPF (before NA 119 +/- 4%, after NA + diclofenac 100%, 15 min with VEGF/VPF 123 +/- 5%). VEGF demonstrated no significant increase in renal protein excretion rate (after NA pretreatment (= 100%): 12.5 min with VEGF/VPF vs without VEGF/VPF: 119 +/- 10% vs 132 +/- 11%, n.s.) (after NNA pretreatment (= 100%) 12.5 min with VEGF/VPF vs without VEGF/VPF 94 +/- 5% vs 96 +/- 4%; n.s.) or clearance quotient of albumin. Glomerular filtration rate was not influenced by VEGF/VPF in kidneys pretreated with NA (before NA 105 +/- 5%, after NA 100%, 12.5 min with VEGF/VPF 94 +/- 2%) or with NNA (before NNA 107 +/- 6%, after NNA 100%, 12.5 min with VEGF/VPF 96 +/- 2%). Fractional glucose and fractional sodium excretion showed flow-dependent changes. CONCLUSION: VEGF/VPF can contribute to the relaxing capacity of the renal vasculature. This relaxation is partly mediated by the NO/endothelium-derived relaxing factor (EDRF) pathway. In the isolated perfused rat kidney the glomerular permeability for albumin is not affected by VEGF/VPF.

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis.

Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF-stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.

Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor.

Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PIGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PIGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo.

Approach to an organo-typical environment for cultured cells and tissues.

If cells or tissues are taken out of an organ and put in culture, normally they lose morphological, physiological and biochemical features. This dedifferentiation process starts during the isolation procedure and continues during the whole culture period. It is caused by the stagnant liquid condition and the inadequate anchorage of cells at the bottom of tissue culture plasticware. The use of filters as basement membrane substitutes and the coating of cultureware with extracellular matrix proteins improve the environmental factors for cultured cells but do not consider the paracrine influence of cytokines or the nutritional needs of individual cell types. To limit cellular dedifferentiation in culture, we constructed a new system, which adapts, as far as possible, cell and tissue cultures to an organo-typical environment. The system is based on a compatible cell carrier arrangement, which allows individual selection of supports for optimal cell anchorage and differentiation. The cell carriers are placed in a newly constructed container, which is permanently perfused with fresh culture medium. The system runs outside an incubator with simple laboratory tools; only a peristaltic pump, a warming table and pH-stabilized media are necessary. Without any subculturing, acute and chronic influences of drugs or the quality of medical implantation grafts can be studied over months.

Stimulation of renal microvascular development under organotypic culture conditions.

The development of the renal vascular system requires the coordinated action of soluble morphogenic factors and specific extracellular matrix components. Despite intensive research it remains unknown whether the humoral or the environmental component is more important in the development of renal microvessels. The prolonged serum-free culture of embryonic kidney cortex explants was achieved by means of a newly developed perfusion culture system. This system made the investigation of renal vascular development under defined organotypic conditions possible. Thus, growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hormones (aldosterone, vitamin D3) could be applied without the interference with serum components. Medium supplementation with VEGF or aldosterone in combination with vitamin D3 resulted in the coordinated proliferation of endothelial cells in the explant. A well-developed collecting duct epithelium and numerous tubular structures were always observed. In contrast, only a uniform cell layer was found between fibrous organ capsule and the collecting duct epithelium after bFGF application, but neither tubular structures nor endothelial cells. Thus, the experiments indicate that bFGF alone has no stimulating effect on the growth of the renal microvasculature under perfusion culture conditions.

Aldosterone modulates PNA binding cell isoforms within renal collecting duct epithelium.

To investigate the differentiation of the ampullary collecting duct cells into adult principal and intercalated cells, the embryonic cortex of newborn New Zealand rabbit kidney was isolated and brought in culture. With this culture technique the ampullary cells formed a polarized collecting duct epithelium which was kept under permanent exchange of medium and in the presence of aldosterone, arginine vasopressin and/or insulin. After 14 days of perfusion culture the epithelia showed light and dark cells resembling the principal and intercalated cells of the adult collecting duct. The differentiation from embryonic into adult collecting duct cells was controlled by applying the monoclonal antibody CD 7. Independent of the hormonal treatment all of the epithelial cells matured in culture and expressed the CD 7 antigen. This corresponded with the situation found within the adult kidney, where the CD 7 antigen was localized in all principal and intercalated (IC) cells, whereas the embryonic ampullary epithelium in the neonatal kidney remained negative. A differentiation feature of the beta-type intercalated cell was investigated by labeling the cultured epithelia with peanut agglutinin (PNA). In contrast to the CD 7 antigen the development of PNA binding was highly dependent of time and individual hormone administration. While in control epithelia only 8% of PNA positive cells were found, aldosterone induced epithelia revealed 72% PNA labeled cells. The combination of aldosterone and insulin increased the number of PNA-positive cells to 90%. By scanning electron microscopy it could further be shown that several isoforms of cells were reactive with PNA. Thus, in culture the PNA label is not restricted to the typical beta-type IC cells.

Characterization of an endothelial protein in the developing rabbit kidney.

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.