Dose-dependent movement of cationic molecules across the glomerular wall

Different concentrations of the polycation polyethyleneimine (PEI) were administered by single intravenous injections or by constant vascular perfusion to the kidneys of Sprague-Dawley rats. At a fixed time interval after administration of PEI, the kidneys were fixed and the distribution of PEI in the glomerular wall was evaluated by electron microscopy. At the lower concentrations (e.g., 0.005%), PEI bound only to the glomerular endothelial glycocalyx and preferentially to microvillous projections on this endothelium. At higher concentrations (e.g., 0.05%), PEI also bound to discrete anionic sites in the lamina rara interna (LRI) but was rarely seen in the lamina rara externa (LRE). As the concentration of PEI was further increased (e.g., 0.5%), PEI moved deeper into the glomerular basement membrane (GBM) and bound extensively to discrete anionic sites in the lamina rara externa. Although anionic sites in the LRI and LRE appeared nearly saturated following infusion of 0.5% PEI, this cationic molecule was rarely seen to cross filtration slits and pass into the urinary space. At still higher concentrations (e.g., 2%), however, PEI moved freely across the filtration slits, bound extensively to the glomerular epithelial glycocalyx, and induced a narrowing of the filtration slits. When PEI was mechanically perfused through the kidney vasculature for 3 minutes, PEI binding to the epithelial glycocalyx caused very extensive adherence of adjacent podocyte processes and the narrowing and loss of filtration slits. Also in these latter samples, discrete anionic sites in the LRE were no longer apparent and a dense band of PEI was seen under the foot processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cytochemical study of the surface properties of aortic and mitral valve endothelium from hypercholesterolemic rabbits

The luminal surface properties of aortic and mitral valve endothelium in hypercholesterolemic rabbits were examined with the aid of cationic ferritin (CF), ferritin-lectins (FWGA, FRCA, FSBA), and low density lipoprotein-colloidal gold (LDL-Gold) conjugates. Based upon comparative studies with normocholesterolemic rabbit valves, the number of CF and wheat germ agglutinin (FWGA) particles per 100 nm of endothelial surface was found to be reduced in moderate hypercholesterolemia (450 mg/dl). Conversely, the number of Ricinus communis agglutinin (FRCA) and soybean agglutinin (FSBA) conjugates were increased. Quantitation of the CF and FWGA particles demonstrated that the endothelium lining of the valve surfaces (i.e., the arterial surfaces of the aortic cusps, AA, and the ventricular surfaces of the mitral cusps, MV) exposed to more turbulent hemodynamic conditions displayed the greatest densities of particle counts. Cholesterol levels of 400-500 mg/dl produced a loss of characteristic differences in the number of ferritin particles that existed between the two surfaces of a cusp. Especially prominent over the AA and MV surfaces, these changes represented a reduction in the anionic properties of the endothelial glycocalyx. Enzymatic digestion demonstrated the reduction in surface sialic acid residues to be one of the major factors responsible for these early changes at the blood-endothelium interface. More severe hypercholesterolemia (700-900 mg/dl) resulted in even further reductions in the number of ferritin particles over the AA and MV surfaces but enhanced the binding of LDL-Gold. Chondroitinase studies of these specimens demonstrated that the initial loss of sialic acids at moderate serum levels unmasks deeper lying components of the glycocalyx (e.g., sulfated glycosaminoglycans) and augments the attachment of LDL molecules to the endothelial surface. The findings of this study suggest that specific macromolecular changes in the endothelial glycocalyx in diet-induced hypercholesterolemia occur at vascular locales where hemodynamic forces such as eddy formations and blood stagnation impinge against the vascular wall.     

Maturation of myocardial capillaries in the fetal and neonatal rat: An ultrastructural study with a morphometric analysis of the vesicle populations

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall—from the 16-day-gestation fetus of the rat to the 21-day neonate—was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are “free” in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week. The finding of tight junctions and small areas of vesicle concentration in fetal endothelial cells could indicate that sites of permeability are limited early in myocardial capillary development and that these vesicular sites increase as gestation proceeds and as the myocardial capillaries mature.     

硬脑膜微血管阴离子场与通透性关系超微结构

目的;探讨硬脑膜神经源性炎发病过程中血管内皮细胞阴离子场的变化及其与血通透性的关系。方法：以Ｐ物质静脉注射制备ＳＤ大鼠硬脑膜神经源性炎,用阳离子胶体金做为探针标记硬脑膜开放血管内皮细胞表面的阴离子场,并在电子显微镜下计数标记密度,比较不同时相组和对照组间标记密度。结果：硬脑膜血管内皮细胞表面可见阳离子胶体金标记颗粒,但注射ＳＰ后１０分钟,２０分钟和对照组间阳离子胶体金标记无显著差异。结论：用阳离子     

Detection of anionic sites on the cytoplasmic surface of the guinea pig acrosomal membrane

The sperm acrosome reaction is an example of exocytosis, accomplished through the fusion of the acrosomal and plasma membranes. As in other examples of exocytosis, the acrosome reaction is initiated by an influx of Ca⁺⁺, which may promote fusion by binding to anionic sites on adjacent bilayers. In this study we used ruthenium red (RR) and cationic ferritin (CF) to detect anionic sites on the surfaces of acrosomal and plasma membranes of guinea pig spermatozoa. These probes indicate a dense concentration of anionic sites on the cytoplasmic surface of the acrosomal membrane. Higher concentrations of salt (NaCl) were required to inhibit cationic probe labeling of the cytoplasmic surface of the acrosomal membrane compared to the concentration needed to inhibit the plasma membrane binding. The added NaCl also increased the separation of the plasma from the acrosomal membrane. Low-pH buffers stop cationic probe labeling of both membranes. Sections tangential to the acrosomal membrane revealed that the cation probes bound in a linear pattern, similar to the periodicity and distribution of intramembraneous particles observed in freeze-fracture replicas. Following fusion of the plasma and acrosomal membrane during the acrosome reaction, we could no longer detect a dense concentration of anionic sites on the cytoplasmic surface of the fused vesicles. The results indicate that the dense concentrations of anionic sites are either masked or lost following fusion with the overlying plasma membrane.     

Molecular characterization of anionic sites on the luminal front of endoneurial capillaries in sciatic nerve

Summary The distribution of anionic microdomains has been described in cerebral vessels and more recently in capillaries of peripheral nerve. Evidence is accumulating that these sites play a role in the barrier function of vascular endothelia in the PNS and CNS. The chemical nature of anionic sites has been at least partly determined for cerebral vessels but not in peripheral nerve. This study reports our preliminary investigations to determine the nature of endothelial anionic sites in sciatic nerve. The effects of digestion of ultra-thin sections of nerve with a battery of proteolytic and glycolytic enzymes (papain, trypsin, proteinase K, hyaluronidase, heparinase, heparitinase and neuraminidase) on the distribution of anionic sites was determined using the label, cationic colloidal gold. Papain, a proteolytic enzyme of broad specificity, succeeded in removing the majority of cationic colloidal gold-binding sites on the luminal surface of vascular endothelia. In contrast trypsin and proteinase K were less effective, reflecting their narrower specificity. Hyaluronidase, heparinase and heparitinase did not significantly affect cationic colloidal gold-labelling. However, a considerable reduction in cationic colloidal gold-binding occurred following neuraminidase digestion. These results suggest that, as in cerebral vessels, sialic acid-containing glycoproteins are largely responsible for the negatively charged domains on the luminal membrane of endothelial cells in peripheral nerve.     

Ultracytochemical characterization of anionic sites in the wall of brain capillaries

Summary The chemical nature of anionic sites located on both fronts of the endothelial cells (ECs) and in the basement membrane (BM) of mouse brain capillaries was studied using tissue sections embedded in Lowicryl K4M and cationic colloidal gold. Before labelling with cationic probe, the sections were digested with the following enzymes: trypsin, papain, pronase E, proteinase K, collagenase, chondroitinase ABC, hyaluronidase, heparinase, heparitinase, neuraminidase and endoglycosidase H.The results indicate that the negatively charged surface layer on the luminal front differs in chemical nature from that on the abluminal front of the EC. Anionic sites located on the luminal surface of the plasmalemma of the ECs are mainly contributed by sialic acid residues of acidic glycoproteins. On the contrary, the anionic domains on the abluminal front of the EC represent mixed proteoglycan and acid glycopeptides containing hydrophobic amino acids, sialic acid residues, and are rich in heparan sulphate-bearing glycosaminoglycans. The anionic sites of the BM are contributed in a substantial degree by chondroitin and heparan sulphate-rich glycosaminoglycans.The effect of endoglycosidase H suggests that glycopeptides containing oligomannosyl residues linked toN-acetylglucosa-mine contribute in small degree in maintenance of the negative charge in the BM, but not on the surfaces of the EC.These results show that brain endothelium bears surface anionic domains differing chemically from those described for some fenestrated and continuous endothelia. The distribution of anionic sites indicates that the discrimination against various negatively charged molecules takes place on both fronts of the ECs as well as in the BM of brain micro-blood vessels. The exact role of these domains in the function of the blood-brain barrier remains to be established.     

Binding of colloidal gold-labeled salivary proline-rich proteins to Actinomyces viscosus type 1 fimbriae.

Salivary proline-rich proteins (PRPs), which were purified from parotid saliva, were adsorbed onto 15-nm-diameter gold particles to visualize specific binding of the salivary molecules to Actinomyces viscosus type 1 fimbriae. Negatively stained preparations incubated with PRP-gold conjugates but not bovine serum albumin-gold complexes bound specifically to bacteria possessing type 1 fimbriae, A. viscosus T14V-J1 and 5519. Binding of the PRP-gold probes to strains deficient in type 1 fimbriae, i.e., strains 5951 (type 2 fimbriae only) and 147 (no fimbriae), was negligible.     

Ultrastructural study of transcellular transport of native and cationized albumin in cultured sheep brain microvascular endothelium

Summary The interaction of native and cationized bovine serum albumin-gold complex with confluent monolayers of sheep brain microvascular endothelial cells was investigated. These cells were used as anin vitro model of the blood-brain barrier. After exposure to both complexes for 5, 15, 30, and 60 min, the endothelial cells were washed with phosphate-buffered saline, fixed, and processed for electron microscopy. The results obtained suggest that (a) contrary to previous observationsin vivo, cultured endothelial cells were able to transfer native bovine serum albumin-gold complex particles from apical to basolateral surfaces presumably via a fluid-phase mechanism; (b) the most conspicuous difference in the interaction of both albumin-gold complexes consisted in a significantly greater adsorption of catonized than of native bovine serum albumin-gold complex to the apical surface of the endothelial cells; (c) the further fate of the endocytosed complexes was similar, that is, their major part was internalized into endosomes, multivesicular bodies and lysosomes whereas a smaller fraction was apparently transferred to the basolateral plasma membrane where presumably they were exocytosed into the subendothelial space.These observations provide new ultrastructural evidence that both native and cationized albumin are endocytosed and eventually transported by a non-specific, fluid-phase mechanism, and also support the results of earlier quantitative studies indicating the absence of albumin receptors in brain endothelia.     

The distribution and mobility of anionic sites on the surface of human placental syncytial trophoblast

The purpose of this study was to examine the distribution and mobility of anionic sites on the surface of fetal trophoblast in contact with maternal blood using polycationic ferritin (PCF) as a probe. Pieces of human placental villi were washed to remove maternal blood, and fresh, unfixed tissue was expose to PCF for varying times, concentrations, and temperatures to determine the effects on labeling patterns. The major findings were: (1) anionic sites were localized almost exclusively on the microvillous portion of the trophoblast surface; intermicrovillous regions of the surface, including the coated pits, were generally not labeled with PCF; (2) PCF binding was present as small clusters on the microvilli. This pattern was observed in tissue incubated 5–10 sec at 4°C and 23°C. The size of the cluster was increased with increased incubation time, suggesting some aggregation or patching can occur; (3) following the fomation of patches, the anionic sites showed no evidence of being cleared from the membrane by endocytosis during incubation subsequent to labeling; (4) the binding of PCF to the surface was reduced by pretreatment of the tissue with neuraminidase. Tissue fixed in glutaraldehyde prior to PCF exposure showed both clustered and more dispersed labeling. The results indicate that anionic sites on human trophoblast surface have a non-random distribution and have restricted mobility on the surface. This may be indicative of a segregation of different membrane proteins and functions within different structural regions of the placental cell surface.     

Binding of monoclonal antibodies to glomerular endothelium, slit membranes, and epithelium after in vivo injection. Localization of antigens and bound IgGs by immunoelectron microscopy.

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the MAbs after in vivo injection were determined by immunoelectron microscopy. The antigens were localized in situ by immunoperoxidase and immunogold labeling to different domains and microdomains of the glomerular endothelium and epithelium. 23A recognized an antigen expressed exclusively on the luminal (apical) domain of the endothelium. 5A (anti-podocalyxin) and 26C (anti-DPPIV) recognized antigens expressed on the apical domains of both the endothelium and podocytes. 13A, 14A, 20B (anti-gp330), and 27A recognized antigens restricted to podocytes in the glomerulus. The 13A antigen was present on their basal surface and the 27A and 14A antigens were expressed on both their apical and basal domains. The 14A antigen also was associated with the filtration slit membranes. All these MAbS bound to their antigens after injection in vivo. Those that recognize endothelial antigens were rapidly cleared from the circulation and rapidly disappeared from glomeruli, whereas those that recognize epithelial antigens persisted in the circulation and were detectable in glomeruli for hours or days. The sites of binding of the MAbs differed: 23A and 5A IgG (antipodocalyxin) bound exclusively to the luminal domain of the endothelium, whereas 26C IgG (anti-DPPIV) bound to both the luminal endothelial membrane and the apical and basal domains of podocytes. The MAbs that recognize podocyte antigens bound to different domains of the podocyte plasmalemma: 13A and 27A IgGs to the basal domain, 14A to the slit membranes, and 20B to coated pits on the entire plasma membrane. 27A IgG led to the formation of small subepithelial immune deposits that remained up to 10 days. It is concluded that 1) glomerular membrane proteins vary considerably in their distribution among plasmalemmal domains and microdomains of endothelial and epithelial cells; 2) virtually all structures in the glomerulus and all domains and micro-domains of the endothelium and podocyte are accessible to circulating antibodies; and 3) the fate of immune complexes formed by binding to glomerular components varies with the location of the antigen within the glomerulus, with those that bind to the basal domain and slit membranes of the podocyte persisting longer than the others.     

Aortic endothelial cell during regeneration. Remodeling of cell junctions, stress fibers, and stress fiber-membrane attachment domains

We have studied in regenerating endothelium of rat thoracic aortas (a) organization of tight and gap junctions by means of morphometry using freeze-fracture electron microscopy, (b) development of cytoplasmic actin microfilament bundles (stress fibers) by means of morphometry using thin section electron microscopy, and (c) distribution of filipin-sterol complexes in the endothelial plasma membrane using thin section and freeze-fracture electron microscopy. The index of complexity for tight junctions and the index expressing the average width of gap junctions were significantly higher in regenerating endothelium than in normal aortic endothelium. In regenerating endothelium, there was a significant increase of the stress fiber to endothelial volume density ratio as compared to normal. Stress fibers were connected to cytoplasmic microfilament condensations located at the abluminal plasma membrane; the surface density of these condensations was significantly increased in regenerating endothelium as compared to normal. Filipin-cholesterol complexes were few or absent at stress fiber-membrane attachment sites but numerous in the remaining endothelial plasma membrane. This morphologic remodeling of the aortic endothelial cell layer may help to explain changes of endothelial cell function occurring in situations associated with increased cell turnover and motion.     

The anionic charge barrier in the renal corpuscle of the pronephros in the lamprey, Petromyzon marinus L.

The charge barrier within the renal corpuscle of the pronephric kidney of the lamprey, Petromyzon marinus, was investigated at two life cycle intervals using cationized ferritin and polyethyleneimine. In the larval renal corpuscle the endothelium of the glomerular capillaries and the laminae rarae externa and interna of the glomerular basement membrane show regularly-spaced deposits of the tracers. The lamina densa remains unstained. Concomitant with a loss of major processes of the visceral epithelial podocytes and development of an extensive mesangial matrix in late adult life are alterations in the distribution of the anionic sites. The lamina rara interna is no longer a distinct entity and the mesangium contains irregularly-distributed anionic sites surrounding electron-dense deposits. The results indicate that the distribution of the anionic sites during adult life most likely affects the ability of the renal corpuscle to act as an efficient filtration device. This charge distribution is consistent with that seen during some renal pathologies of higher vertebrates.     

Blood-nerve barrier: Distribution of anionic sites on the endothelial plasma membrane and basal lamina of dorsal root ganglia

Summary Previous investigations of the blood-nerve barrier have correlated the greater permeability of ganglionic endoneurial vessels, compared to those of nerve trunks, with the presence of fenestrations and open intercellular junctions. Recent studies have demonstrated reduced endothelial cell surface charge in blood vessels showing greater permeability. To determine the distribution of anionic sites on the plasma membranes and basal laminae of endothelial cells in dorsal root ganglia, cationic colloidal gold and cationic ferritin were used. Electron microscopy revealed the existence of endothelial microdomains with differing labelling densities. Labelling indicated that caveolar and fenestral diaphragms and basal laminae are highly anionic at physiological pH, luminal plasma membranes and endothelial processes are moderately charged and abluminal plasma membranes are weakly anionic. Tracers did not occur in caveolae or cytoplasmic vesicles.In vitro tracer experiments at pH values of 7.3, 5.0, 3.5 and 2.0 indicated that the anionic charge on the various endothelial domains was contributed by chemical groups with differing pKa values. In summary, the labelling of ganglionic and sciatic nerve vessels was similar except for the heavy labelling of diaphragms in a minority of endoneurial vessels in ganglia. This difference is likely to account in part for the greater permeability of ganglionic endoneurial vessels. The results are discussed with regard to the blood-nerve and -brain barriers and vascular permeability in other tissues and a comparison made between the ultrastructure and anionic microdomains of epi-, peri- and endoneurial vessels of dorsal root ganglia and sciatic nerves.     

脑缺血再灌注损伤时微血管内皮细胞糖复合物的变化及其与通透性改变的关系

目的:了解大鼠脑缺血再灌注后通透性的变化及其与内皮细胞糖复合物的关系。方法:用暂时性脑缺血的大鼠模型,以右旋糖酐分子为示踪剂对缺血再灌注后微血管通透性的变化进行了观察,同时用同一标本经K4M低温包埋,用阳离子胶体金包埋后标记的方法在超微结构显示缺血再灌注后内皮细胞表面糖复合物(glycocalyx)的变化。结果:电镜下见缺血 1h和再灌注后 1h、2h、3h、4h、6h、9h、12h各时点均可见右旋糖酐颗粒穿出血管外,胞浆内出现的大量吞饮小泡,细胞间连接的增宽和开放,内皮细胞胞膜表面的凹陷及多量微绒毛的出现是微血管通透性增高的结构基础,大鼠脑缺血后微血管通透性增高的同时阳离子胶体金分布减少。结论:Glycocalyx对缺血/缺氧性损害极其敏感,它的破坏可能是内皮细胞功能受损的启动因素和血管通透性增高的主要决定因素.     

The anionic matrix at the rat glomerular endothelial surface

The anionic macromolecules at the glomerular endothelial cell surface are visualized only when stained with cationic stains. We investigated the arrangement and composition of this anionic matrix at the luminal surface. Rat kidneys were perfused with anionic ferritin (pI 4.5), ferritin (pI 7.4), or cationized ferritin (CF, pI 8.3). Anionic ferritin (pI 4.5) did not bind to the capillary wall, ferritin (pI 7.4) bound discontinuously only to the laminae rarae of the basement membrane, but cationized ferritin (CF, pI 8.3) bound as a thick continuous layer to the cell plasmalemma and bound to the anionic matrix in the fenestral spaces. These observations show that an anionic matrix lines the entire capillary lumen surface, fills the fenestrae, and is interposed between the blood and the basement membrane at the fenestrae. The anionic constituents at the capillary luminal surface were identified by in vivo digestion with specific enzymes. Absence of CF binding following digestion with specific enzymes was taken to indicate the presence of the particular glycoprotein known to be susceptible to the enzyme used. Neuraminidase digestion revealed that anionic sites over the surface plasmalemma are mainly from sialoproteins. In contrast, the matrix in fenestral channels contains heparan sulfate, hyaluronic acid, and sialoproteins. Papain digestion showed no glycolipids at the luminal surface. The functions of this continuous anionic layer located at the luminal surface of glomerular capillaries have not yet been established.     

Restricted mobility and endocytosis of anionic sites on newborn rat jejunal brush border membranes

The distribution and mobility of anionic sites on the microvillous surface of newborn rat jejunal absorptive cells were studied using polycationic ferritin (PCF) as a visual probe and were compared with anionic sites previously described for adult jejunum. Segments from 5- to 26-day-old rats were incubated in PCF for 5 minutes either before or after fixation for electron microscopy. From days 5 to 20, anionic sites were distributed diffusely along the lengths of the microvilli and did not show random translational mobility. In contrast, microvilli examined from animals at weaning (21 to 26 days) resembled those from adults in which most binding sites were capable of lateral mobility and were induced by PCF to cluster into discrete patches. The diffuse pattern was altered by cortisone administration, paralleling a premature reduction in the endocytic apparatus of the cell. The difference in mobility of anionic sites with age coincides with differences in absorptive function. Evidence is presented showing that in the neonate binding of PCF to the microvilli was followed with time by endocytosis into an apical system of tubules for intracellular transport, incorporation into coated vesicles, and release through the lateral cell surface. The results suggest that endocytosis is accomplished by a mechanism that includes a directionally controlled movement for the selective internalization of PCF binding sites from the membranes of the microvilli to those of the tubular cytoplasmic channels.     

Role of endothelial injury in disease mechanisms and contribution of progenitor cells in mediating endothelial repair

Recent research on the endothelium demonstrates complex interactions of endothelial cells with circulating immune cells, mediators such as cytokines, hormones and growth factors, and with the underlying parenchymal cells. These disparate interactions are involved in promotion of vascular development; maintenance of tissue homeostasis; and regulation of vascular repair. Injury to the endothelial monolayer is the sine qua non of organ dysfunction with endothelial repair the necessary first step needed for recovery. Thus, the capacity of the endothelium to regenerate itself is a key determinant of organ repair and survival after injury. Using the example of the lung, we will review the current state of knowledge regarding the importance of endothelium in the above mentioned processes with a focus on the role of stem cells, both endogenous (i.e., localized within the vessel wall) as well as exogenous (i.e., arriving in the vessel wall from distant sites such as the bone marrow) in promoting endothelial repair and regeneration. The subject of endothelial regeneration and the ways in which stem and progenitor cells contribute to this process has promise in treating vascular diseases. As we will highlight in this review, some questions have been addressed but many more remain and need to be addressed before cell-based therapies become a viable option.     

Detection of hyaluronic acid in the glomerular basement membrane of murine chronic graft-versus-host disease

An ultrastructural investigation was made of the distribution of immune complexes (IC) and anionic sites using cationic colloidal gold in the glomerular basement membrane (GBM) of kidneys with murine graft-versus-host disease. At 8 weeks, a decrease in the number of anionic sites around deposits of IC in the GBM was noted. These anionic sites could be removed by treatment with hyaluronidase or chondroitinase, but not with heparitinase, suggesting that they consisted mainly of hyaluronic acid. These results support the theory that IC deposits are associated with a decrease in the number of GBM anionic sites which mainly consist of hyaluronic acid. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

Ligand Coated Nanosphere Adhesion to E- and P-Selectin under Static and Flow Conditions

The heterogeneous distribution of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of vascular endothelium provides an opportunity to deliver drugs to select tissues. The targeting could be achieved by using carriers whose outer surface has a ligand for a selectively expressed ECAM. The carriers would interact with the endothelium in a fluid dynamic environment and in many of these schemes nanoparticles would be used. It is unclear what role various parameters (e.g., ligand–ECAM chemistry, fluid shear) will have on the adhesion of the nanoparticles to the endothelium. To facilitate studies in this area, we have developed a prototypical in vitro model that allows investigation of nanoparticle adhesion. We coated polystyrene nanospheres with a humanized mAb (HuEP5C7.g2) that recognizes the ECAMs E- and P-selectin. Adhesion assays revealed that HuEP5C7.g2 nanospheres exhibit augmented, specific adhesion to selectin presenting cellular monolayers and that the adhesion can be affected by the fluid shear. These results; (i) strongly suggest that HuEP5C7.g2 could be used to target nanoparticles to selectin presenting endothelium; (ii) demonstrate that fluid shear can affect nanoparticle adhesion; and (iii) define a system which can be used to study the effects of various system parameters on nanoparticle adhesion. © 2001 Biomedical Engineering Society. PAC01: 8235Pq, 8714Ee, 8719Uv, 8716-b