Endothelial Basement Membrane Research Articles

Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead

Sprouting angiogenesis is an invasive process that involves proteolytic activities required for the degradation of the endothelial basement membrane, cell migration with removal of obstructing matrix proteins, and generation of space in the matrix to allow endothelial cells to form a proper lumen. In the last decade it has become clear that besides these matrix-degrading properties, proteases exert additional, more subtle functions that play a key role in angiogenesis. These functions are discussed with specific emphasis on membrane type-1 matrix metalloproteinase (MT1-MMP), other MMPs, and the related ADAMs (a disintegrin and metalloproteinase domain). Proteases modulate the balance between pro- and anti-angiogenic factors by activation and modification of growth factors and chemokines, ectodomain shedding with accompanied receptor activation, shedding of cytokines from membrane-bound precursors, and generation of (matrix) protein fragments that inhibit or activate angiogenesis. Furthermore, they participate in the recruitment of leukocytes and progenitor cells, which contribute to the onset and progression of angiogenesis. Proteases facilitate the mobilization of progenitor cells in the bone marrow as well as the entry of these cells and leukocytes into the angiogenic area. The interaction between pericytes and the newly formed endothelial tubes is accompanied by silencing of MMP activities. Better understanding of the various activities of proteases may be helpful in developing more specific inhibitors that could result in tailor-made modification of proteolytic activities in disease.

Development and ultrastructure of glomerular capillaries in human foetus

Glomerulus is an important filtrating apparatus in the body. Three types of cells - endothelial, mesangial and visceral epithelial cells can be identified in the capillary tuft. Glomeruli develop during nephrogenesis which starts in the 8th week and ends between the 32nd and 36th week of gestation. The nephron develops through stages described as the vesicle, the comma-shaped, S-shaped with the developing glomerulus and the mature glomerulus. Glomerular differentiation involves the expansion of the original capillary component into the plexus that consists of 6-8 loops and the migration of podocytes that are arranged around these glomerular capillaries. Glomerular capillary differentiation represents a set of developmental changes of the glomerular endothelial and epithelial cells. The active differentiation of glomerular capillaries starts in the hemisphere of an inferior arm of S-shaped bodies. Endothelial precursors unite into precapillaries devoid of lumen. Further differentiation includes the flattening of endothelial cells on the basement membrane, the loss of superfluous cells, the development of lumen and the formation of fenestrae. The glomerular basement membrane is differentiated by the fusion of epithelial and endothelial basement membrane. The differentiation of visceral epithelial cells includes the development of cytoplasmic processes and the flattening of cell bodies. Primary cytoplasmic processes are formed from the podocyte bodies and develop secondary and tertiary processes or foot processes. Foot processes from one podocyte interdigitate with foot processes from other podocytes. In the developing glomeruli, there is a difference in the level of differentiation of visceral epithelial cells. Cells with differentiated foot processes and cells with no cytoplasmic processes are observed within the same glomerulus.

Effects of endothelial basement membrane on neutrophil adhesion and migration

The influence of the sub-endothelial basement membrane (BM) on the adhesion and migration of leukocytes is not well-defined. We therefore investigated the behaviour of human neutrophils on purified BM proteins and on BM deposited by short- or long-term cultures of endothelial cells (EC). The adhesion, but not migration velocities, of neutrophils activated with interleukin-8 was dependent on the coating concentrations of purified collagen, laminin or fibronectin. In contrast, adhesion was similar on matrices deposited by 3-day or 20-day cultures of EC, but neutrophils migrated more slowly on the distinct BM that formed over 20 days. In addition, while adhesion on all surfaces was greatly reduced when neutrophils were treated with antibody against β 2-integrins, antibody against β 1-integrins only inhibited adhesion to the 20-day BM. Thus, the native BM has distinct effects on integrin usage and migration by neutrophils, which are not reproduced by purified proteins or matrix deposited early during endothelial culture.

Antimicrobial therapy for bacterial meningitis

Bacterial meningitis is an uncommon condition in mature horses (Newton 1988). It is more prevalent in neonates, where it is reported to occur in 8–10% of septicaemic foals (Moore 1995), where reported survival rates are poor (Newton 1988; Santschi and Foreman 1989; Moore 1995). Survival rate for adult horses was reported as 60% in a case series of 5 horses with bacterial meningitis (Mitchell et al. 2006). There are no published comprehensive case series of bacterial meningitis in foals. Early diagnosis is essential to provide accurate prognosis and to institute proper treatment (Santschi and Foreman 1989). The primary goal of treatment is rapid sterilisation of the cerebrospinal fluid (CSF) using appropriate antimicrobial therapy based on bacterial culture and sensitivity testing of the CSF (Moore 1995). However, due to the specialist nature of the central nervous system (CNS) and presence of the blood brain barrier (BBB), CNS concentrations of antimicrobial agents may be substantially below those obtained in serum, and below the minimal inhibitory concentration or minimal bactericidal concentration for the bacterial isolates involved in the disease process. This paper reviews the nature of poor antimicrobial penetration into the CNS and discusses the antimicrobial drugs that may be appropriate to use in treatment of bacterial meningitis in horses. Meningitis is defined as inflammation of the 3 membranes, or meninges, surrounding the brain and spinal cord. These consist of the dura mater, the arachnoid and pia mater (Fig 1). Inflammation of the meninges due to bacterial infection is termed bacterial meningitis. The space between the arachnoid and pia mater is filled with CSF. This protects the brain and spinal cord against impact from the surrounding bony wall, provides nutrition to the brain and spinal cord and permits variations in blood volume within the cranial cavity. The existence of a barrier between the brain or CSF and the peripheral circulation was first recognised by Ehrlich in 1885 (Saunders et al. 1999), and allows the adult brain to function in a well-controlled internal environment that is separate from that of the internal environment of the rest of the body as a whole. The integrity of this barrier is due to the presence of tight junctions between the cerebral endothelial cells forming the BBB and between the blood and choroid plexus epithelial cells (blood-cerebrospinal fluid barrier) (Saunders et al. 1999). The presence of endothelial cell tight junctions and basement membrane also restricts entry of certain therapeutic agents into the CSF and CNS. The presence of the BBB, combined with the lack of a traditional lymphatic system, are important factors in mediating the immune response within the CNS. As such, the CNS is often classified as an ‘immune privileged’ site (Wekerle et al.1986). In health, the key factors that determine penetration of a molecule into the CNS and CSF in adults are lipid solubility and molecular size (Saunders et al. 1999). Highly lipid soluble molecules are considered to dissolve in the lipid structure of cell membranes and can thus transverse cell membranes. For molecules of low lipid solubility there is a clear relationship between molecular size and penetration into the brain and CSF. In healthy human subjects, molecules with size greater than 5000 Daltons are unable to penetrate from blood to CSF (Saunders et al. 1999). In addition, a high degree of plasma protein binding of therapeutic agents limits their permeability to the CNS, since plasma proteins (principally albumin) are too large to cross the BBB. This situation is altered in acute CNS inflammation, where penetration of molecules of larger molecular weight

Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells In Vitro

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.

Glucose Modulates Basement Membrane Fibroblast Growth Factor-2 via Alterations in Endothelial Cell Permeability

The effects of glucose extremes on vascular physiology and endothelial cell function have been examined across a range of time scales. Not unexpectedly, chronic glucose exposure induces long term tissue effects. Yet short term exposure can also impose lasting consequences. The persistence of vascular pathology after euglycemic restoration further suggests a glucose exposure memory. Slow turnover reservoirs such as basement membrane are candidates for prolongation of acute events. We hypothesized that glucose-induced vascular dysfunction is related to altered vasoactive compound handling within the endothelial cell-basement membrane co-regulatory unit. Endothelial cell basement membrane-associated fibroblast growth factor-2 increased linearly with culture glucose within days of elevated glucose exposure. Surprisingly, basement membrane fibroblast growth factor-2 binding kinetics remained unchanged. The glucose-induced increase in basement membrane fibroblast growth factor-2 was instead related to enhanced endothelial cell fibroblast growth factor-2 release and permeability. Cellular fibroblast growth factor-2 release occurred concomitant with apoptosis but was not blocked by caspase inhibitors. These data suggest that release was associated with sub-lethal early apoptotic cell membrane damage, perhaps related to reactive oxygen species formation. High glucose basement membrane in turn enhanced endothelial cell proliferation in a fibroblast growth factor-2-dependent manner. We now show that glucose-induced alterations in endothelial cell function promote changes in basement membrane composition, and these changes further affect endothelial cell function. These data highlight the interrelationship of cell and basement membrane in pathological conditions such as hyperglycemia. These phenomena may explain long term effects on the endothelium of short term exposure to glucose extremes.

Ultrastructure of blood–brain barrier and blood–spinal cord barrier in SOD1 mice modeling ALS

The purpose of this study was to determine the ultrastructure of the blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) in G93A SOD1 mice modeling ALS at different stages of disease. Electron microscope examination of brainstem, cervical and lumbar spinal cords was performed in ALS mice at early and late stages of disease. Our results show disorganized mitochondrial cristae and degenerating mitochondria in endothelial cells and neuropil, swollen astrocyte foot processes, swollen and degenerating capillary endothelial cells, astrocytes and motor neurons and extensive extracellular edema. In spite of progressive extracellular edema in neural tissue, capillary endothelial cell tight junctions appeared to remain intact in early and late symptomatic animals. Results show that disruption of BBB and BSCB was evident in areas of motor neuron degeneration in G93A mice at both early and late stages of disease. Capillary rupture was observed in brainstem in early symptomatic G93A mice. Capillary ultrastructure revealed that endothelial cell membrane and/or basement membrane damage occurred, followed by vascular leakage.

Enhancement of Vascular Permeability with Low-Frequency Contrast-enhanced Ultrasound in the Chorioallantoic Membrane Model

To characterize the effect of low-frequency contrast material-enhanced ultrasound on the vascular endothelium and to determine the parameters and techniques required to deliver a therapeutic agent by using the chorioallantoic membrane (CAM) model. All in vivo animal procedures were conducted with institutional Animal Care and Use Committee approval. Extravasation of 8.5-nm-diameter fluorescein isothiocyanate-labeled dextran was evaluated in the vasculature of a chick CAM model. Intravital microscopy was performed during contrast-enhanced ultrasound exposure (1.00 or 2.25 MHz); results were compared with results of electron microscopy of the insonated regions. Data acquired after insonation with greater mechanical stress (n = 30 animals) (mechanical index [MI] > 1.3) and with lower mechanical stress (n = 86 animals) (MI < 1.13) were compared with measurements in control conditions (n = 46 animals). The diameter of affected vessels; number of extravasation sites; extravasation rate, area, and location; and changes in endothelial cells and basement membrane were evaluated. Differences were tested with analysis of variance or the Student t test. After ultrasound application, convective transport of the model drug was observed through micron-sized openings with a mean fluid velocity of 188.6 microm/sec in the low-stress class and 362.5 microm/sec in the high-stress class. Electron microscopy revealed micron-sized focal endothelial gaps and disseminated blebs, vacuoles, and filopodia extending across tens of microns. The threshold pressure for extravasation was 0.5 MPa for a transmitted center frequency of 1.00 MHz (MI = 0.5) and 1.6 MPa for a frequency of 2.25 MHz (MI = 1.06); thus, the frequency dependence of the threshold was not predicted simply by the MI. Low-frequency contrast-enhanced ultrasound can increase vascular permeability and result in convective extravasation of an 8.5-nm-diameter model drug.

Inflammatory Cell Migration into the Central Nervous System: A Few New Twists on an Old Tale

Understanding the mechanisms of leukocyte trafficking into the brain might provide insights into how to modulate pathologic immune responses or enhance host protective mechanisms in neuroinflammatory diseases such as multiple sclerosis. This review summarized our knowledge about the sites for leukocyte entry into the central nervous system, highlighting the routes from blood into the perivascular space and brain parenchyma through the blood-brain barrier. We further discussed the multistep paradigm of leukocyte-endothelial interactions at the blood-brain barrier, focusing on the adhesion molecules and chemokines involved in leukocyte transmigration. Luminal chemokines, which are immobilized on endothelial surfaces, initiate leukocyte integrin clustering and conformational change, leading to leukocyte arrest. Some leukocytes undergo post-arrest locomotion across the endothelial surface until interendothelial junctions are identified. Leukocytes then extend protrusions through the interendothelial junctions, in search of abluminal chemokines, which will serve as guidance cues for transmigration. Extravasating cells first accumulate in the perivascular space between the endothelial basement membrane and the basement membrane of the glia limitans. Matrix metalloproteases may be involved in leukocyte transverse across glia limitans into the brain parenchyma. The adhesion molecules and chemokine receptors provide attractive targets for neuroinflammatory diseases because of their important role in mediating central nervous system inflammation.

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin alpha4, beta1, beta2 and gamma1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin alpha5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin alpha4, beta1, beta2 and gamma1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin alpha5 chain and Lutheran glycoprotein, a receptor for the laminin alpha5 chain. In normal lymphatic capillaries, the presence of primarily alpha4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin alpha5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.

Multi-step microfluidic device for studying cancer metastasis

This paper describes a multi-step microfluidic device for studying the deformation and extravasation of primary tumor cells. Prior to extravasation, primary tumor cells undergo sequential steps of deformation through the capillaries, before adhering and transmigrating through the endothelial lining and basement membrane. To study this cascade of events, we fabricated a multi-step microfluidic device whose microgaps were coated with Matrigel to mimic the basement membrane. The microchannel was lined with human microvascular endothelial cells (HMECs) to replicate the endothelial lining. Analysis of deformation, biological and migratory capabilities of various tumor cell lines viz. HepG2, HeLa, and MDA-MB 435S were quantified using the fabricated device. After deformation, the cells' viabilities were significantly reduced and their doubling times were simultaneously increased, indicating changes in their biological capability. However, cell deformation did not significantly reduce their cell motility. Cell motility was co-assessed using the cell's migration rate and the overall population's percentage migration under various conditions (no barrier, Matrigel and Matrigel-HMEC). The device was also used to quantify the effects of Matrigel and the endothelial lining on cell migration. Our results suggest that both played an independent role in inhibiting cell extravasation, with the Matrigel significantly slowing down cell movement and the endothelial lining reducing the total number of transmigrated cells.

Recombinant human prothrombin kringle-2 inhibits B16F10 melanoma metastasis through inhibition of neovascularization and reduction of matrix metalloproteinase expression

Angiogenesis, a multi-step process which involves endothelial cell proliferation, adhesion, migration, and basement membrane (BM) degradation, is essential for tumor metastasis. Here we show that recombinant human prothrombin kringle-2 (rk-2) inhibited bovine capillary endothelial cell migration with an IC(50) (concentration for half maximal inhibition) of 38 nM and inhibited adhesion to extracellular matrix (ECM) proteins. Because tumor metastasis requires angiogenesis, we examined whether rk-2 could inhibit metastases induced by injection of B16F10 melanoma cells into mice. The results revealed that the metastatic tumors in mouse lung were markedly decreased in a dose-dependent manner and acute lung injury induced by B16F10 melanoma metastasis was diminished by systemic rk-2 treatment. In immunohistochemical analysis, rk-2 reduced expression of vascular endothelial growth factor, which is a potent angiogenic activator and neovascularization in the mouse lung. Also, rk-2 diminished the expression of matrix metalloproteinase-2 and -9 in the mouse lung which induces tumor metastasis and angiogenesis. These data suggest that inhibition of B16F10 melanoma metastasis by rk-2 was caused by inhibition of neovascularization and reduction of matrix metalloproteinase expression.

Metalloproteinases Control Brain Inflammation Induced by Pertussis Toxin in Mice Overexpressing the Chemokine CCL2 in the Central Nervous System

Inflammatory leukocytes infiltrate the CNS parenchyma in neuroinflammation. This involves cellular migration across various structures associated with the blood-brain barrier: the vascular endothelium, the glia limitans, and the perivascular space between them. Leukocytes accumulate spontaneously in the perivascular space in brains of transgenic (Tg) mice that overexpress CCL2 under control of a CNS-specific promoter. The Tg mice show no clinical symptoms, even though leukocytes have crossed the endothelial basement membrane. Pertussis toxin (PTx) given i.p. induced encephalopathy and weight loss in Tg mice. We used flow cytometry, ultra-small superparamagnetic iron oxide-enhanced magnetic resonance imaging, and immunofluorescent staining to show that encephalopathy involved leukocyte migration across the glia limitans into the brain parenchyma, identifying this as the critical step in inducing clinical symptoms. Metalloproteinase (MPs) enzymes are implicated in leukocyte infiltration in neuroinflammation. Unmanipulated Tg mice had elevated expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-10, and -12 mRNA in the brain. PTx further induced expression of tissue inhibitor of metalloproteinase-1, metalloproteinase disintegrins-12, MMP-8, and -10 in brains of Tg mice. Levels of the microglial-associated MP MMP-15 were not affected in control or PTx-treated Tg mice. PTx also up-regulated expression of proinflammatory cytokines IL-1beta and TNF-alpha mRNA in Tg CNS. Weight loss and parenchymal infiltration, but not perivascular accumulation, were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. Our finding that MPs mediate PTx-induced parenchymal infiltration to the chemokine-overexpressing CNS has relevance for the pathogenesis of human diseases involving CNS inflammation, such as multiple sclerosis.

INHIBITION OF MATRIX METALLOPROTEINASE 9 WITH DOXYCYCLINE REDUCES NEUTROPHIL MIGRATION IN PANCREATITIS-ASSOCIATED LUNG INJURY

Background: In pancreatitis-associated lung injury (PALI), matrix metalloproteinase 9 (MMP-9) promotes neutrophil (PMN) migration through endothelial basement membrane into the lung. We hypothesize that inhibition of MMP-9 with doxycycline, a potent MMP-9 inhibitor, may reduce lung injury following experimental acute pancreatitis (AP). Methods: In vivo: 18 rats were randomized into three groups: (1) AP without treatment, (2) AP with doxycycline (30mg/kg s.c.) and (3) control. AP was induced by retrograde infusion of glycodeoxycholic acid (GDOC) into the pancreatic duct. Subcutaneous administration of doxycycline followed directly after AP induction and after 3 and 6 hours serum was evaluated for doxycycline level with HPLC (2μg/ml). After 9 hours rats were killed and lungs were investigated histologically. Staining was performed with NACE for pulmonary PMN and histological lung injury. In vitro: We tested the inhibitory effect of doxycycline on PMN transmigration across matrigel invasion chambers. In group (1) no doxcycline was added to the PMN suspension, group (2) contained doxycycline (2μg/ml). Transmigrated PMN on the lower surface of the mebrane were stained and counted. Results: Average doxycycline serum level after subcutaneous application was 2μg/ml after 3 hours and did not change after 6 hours. Doxycycline in the evaluated concentration significantly reduced TNF-α induced PMN transmigration across matrigel membrane in vitro (12.6+/−2.6 vs. 20.1+/−3.9; p<0.05). After induction of severe acute pancreatitis doxycycline decreased the number of pulmonary PMN compared to the group without treatment (21.2+/−5.1 vs. 32.5+/−6.8; P < 0,05), control group (14.2+/−4.0). Conclusion: Inhibition of MMP-9 with doxycycline reduces inflammation induced PMN transmigration across matrigel membrane in vitro and also PMN migration into the lung following experimental acute pancreatitis in vivo and might prove an effective treatment or prophylaxis against PALI.

Autoimmunity against human tropomyosin isoforms in ulcerative colitis: Localization of specific human tropomyosin isoforms in the intestine and extraintestinal organs

Tropomyosins (TMs) are microfilament cytoskeletal proteins, and 5 major human TM isoforms (hTM1-5) are described. hTMs, particularly isoform 5 (hTM5), is capable of inducing autoantibodies and T-cell response in ulcerative colitis (UC). However, cellular localization of hTM isoforms in the colon and in extraintestinal organs commonly involved in UC is unknown. Using isoform-specific monoclonal antibodies, we localized hTMs through immunoperoxidase assay in normal colon (n = 12), small intestine (n = 14), esophagus (n = 10), skin (n = 19), eye (n = 12), gallbladder (n = 16), liver, including bile duct at the porta hepatis (n = 4), lungs (n = 4), and pancreas (n = 4). There is intense expression of hTM5, but not other isoforms, in the epithelium of the colon, gallbladder, and skin. In the eye, hTM5 is expressed only in the nonpigmented ciliary epithelium. Although extrahepatic and interlobar large ductal biliary epithelium was positive, bile canaliculi at the portal tract are negative. The immunoreactivity in epithelial cells from these organs is diffuse cytoplasmic and along the periphery. In colon epithelium, there is intense expression along basolateral areas and luminal (apical) surface. In the small intestinal epithelium, however, hTM5 expression is weak and distinctly different than in the colon. hTM5 was not detected in the squamous epithelium of the esophagus, although it was strongly positive in the skin. hTM1, hTM2, and hTM3 are localized predominantly in smooth muscle of the intestine and blood vessel wall but not the epithelium. HTM4 is localized in the endothelial cells and basement membrane of the colonic epithelium. hTM5 is the predominant isoform in the epithelium of colon and extraintestinal organs commonly involved in UC. The unique expression of hTM5 may allow its interaction with effector immune cells involved in the immunopathogenesis of UC and its extraintestinal manifestations.

Possible mechanisms explaining the tendency towards interstitial fibrosis in aristolochic acid-induced acute tubular necrosis

We explored possible mechanisms responsible for the inability of plerosis and the tendency towards fibrosis in aristolochic acid-induced acute tubular necrosis (AA-ATN). Renal biopsy tissues from eight AA-ATN cases were examined. Tubulointerstitial injury was semiquantitatively assessed. Immunohistochemical steptavidin-peroxide (SP) methods were used to determine the expressions of proliferating cell nuclear antigen (PCNA), epidermal growth factor (EGF), alpha-smooth muscle actin (alpha-SMA), transforming growth factor-beta(1) (TGF-beta(1)), connecting tissue growth factor (CTGF), fibronectin (FN), collagen III (Col-III), collagen IV (Col-IV), factor VIII-related antigen (VIII-Ag) and vascular endothelial growth factor (VEGF). Ultramicrostructure of endothelial cells and basement membrane of peritubular capillaries (PTC) and glomerular capillaries was detected by electron microscopy. These data were compared with that of 9 cases of antibiotic-induced ATN (a-ATN) and 10 cases of minor mesangioproliferative non-IgA glomerulonephritis, which served as a control group. In AA-ATN, almost no renal tubular epithelial cells (RTEC) were PCNA-positive (0.01 +/- 0.02%), and EGF expression was considerably decreased (9.55 +/- 7.22%). This was in contrast with the highly active tubular proliferation (PCNA-positive RTEC 47.25 +/- 19.33%, P < 0.05) and increased EGF expression in a-ATN (64.38 +/- 19.22%, P < 0.05). The expression of alpha-SMA in the tubulointerstitium, the number of interstitial TGF-beta(1)-positive cells and the CTGF-positive interstitial area were all increased in both a-ATN and AA-ATN, with no obvious differences between the two groups. With respect to extracellular matrix (ECM) deposition, FN, Col-III and Col-IV were detected only in the interstitium of AA-ATN. PTC lumina were decreased in size and misshapen in the AA-ATN group. Also in AA-ATN, the luminal wall was partially disrupted, endothelial cells were swollen and vacuoles and granules were found in the cell plasma. Parts of the endothelial cells were detached from the tubular basement membrane. The strong ability for RTEC repair after acute injury was severely diminished in AA-ATN, and this effect may be partly due to reduced EGF expression. Anti-fibrosis mechanisms may also be impaired in AA-ATN, since both a-ATN and AA-ATN had increased expression of TGF-beta(1) and CTGF, whereas only the latter group showed ECM deposition. Injury and loss of PTC occurred in AA-ATN, and this may contribute to tubulointerstitial damage, the inability of plerosis and the tendency towards fibrosis in this disease.

VEGF Trap Abolishes Shear Stress‐ and Overload‐Dependent Angiogenesis in Skeletal Muscle

To determine which elements of the angiogenic process are controlled by VEGF under physiological conditions. VEGF Trap was administered at 10 mg x kg(-1) by subcutaneous injection twice weekly to mice, which were subject to one of two established angiogenic stimuli: (1) increasing blood flow by administration of prazosin (50 mg L(-1)); (2) muscle overload by extirpation of a synergist. Angiogenesis was determined by calculating capillary to fiber ratio (C:F), and proliferating cell nuclear antigen (PCNA) staining localized to capillaries or the interstitium used to measure cell proliferation. Characteristic ultrastructural changes were quantified using electron microscopy. Administration of VEGF Trap abolished the increases in C:F seen in both models, and prevented growth of luminal filopodia and large vacuole formation. Overload-induced proliferation associated with capillaries was reduced, along with endothelial cell number abluminal sprouts and basement membrane breakage. However, interstitial proliferation remained high, along with the increased capillary association of pericytes and fibroblasts. VEGF is required for both models of angiogenesis, although some morphological changes lie upstream, or are independent of, VEGF involvement. The abolition of angiogenesis in a model unaffected by NO inhibition shows that NO is not required for all VEGF-dependent angiogenesis in vivo.

Identification of an active site on the laminin α4 chain globular domain that binds to αvβ3 integrin and promotes angiogenesis

Angiogenesis is important for wound healing, tumor growth, and metastasis. The laminin α4 chain, a component of laminin-8 and -9, is expressed in endothelial cell basement membranes. It mediates endothelial cell adhesion by binding with its receptors such as αvβ3 integrin and participates in new blood vessel formation. In this study, we found the recombinant laminin α4LG modules (rLG1-3, rLG1, and rLG2) mediate HUVECs adhesion. The attachment of HUVECs to the rLG2 was specifically inhibited by a function-blocking monoclonal antibody LM609 specific for αvβ3 integrin. Using deletion mutants of the α4LG2 revealed the HUVECs-adhesion site is located in amino acids 1121–1139. A synthetic G 1121–1139 peptide could be attached by HUVECs at same efficiency with the rLG2 and promoted angiogenesis in CAM. In conclusion, we have identified a new αvβ3 integrin-interacting peptide within laminin α4 G domain. This suggests that G 1121–1139 peptide-containing proteins may perform their biological functions by interacting with αvβ3 integrin.

Tunneling through cells

Staphylococcus aureus inactivates RhoA to open tunnels through endothelial cells, report Boyer et al. (page 809). These “macroapertures” may allow the pathogenic bacteria easy access to the endothelial basement membranes for tissue invasion and colonization.

Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors

The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.