Lung Microvascular Endothelial Cell Monolayers Research Articles

Marked elevations in lung and plasma ceramide in COVID-19 linked to microvascular injury.

The pathogenesis of the marked pulmonary microvasculature injury, a distinguishing feature of COVID-19 acute respiratory distress syndrome (COVID-ARDS), remains unclear. Implicated in the pathophysiology of diverse diseases characterized by endothelial damage, including ARDS and ischemic cardiovascular disease, ceramide and in particular palmitoyl ceramide (C16:0-ceramide) may be involved in the microvascular injury in COVID-19. Using deidentified plasma and lung samples from COVID-19 patients, ceramide profiling by mass spectrometry was performed. Compared with healthy individuals, a specific 3-fold C16:0-ceramide elevation in COVID-19 patient plasma was identified. Compared with age-matched controls, autopsied lungs of individuals succumbing to COVID-ARDS displayed a massive 9-fold C16:0-ceramide elevation and exhibited a previously unrecognized microvascular ceramide-staining pattern and markedly enhanced apoptosis. In COVID-19 plasma and lungs, the C16-ceramide/C24-ceramide ratios were increased and reversed, respectively, consistent with increased risk of vascular injury. Indeed, exposure of primary human lung microvascular endothelial cell monolayers to C16:0-ceramide-rich plasma lipid extracts from COVID-19, but not healthy, individuals led to a significant decrease in endothelial barrier function. This effect was phenocopied by spiking healthy plasma lipid extracts with synthetic C16:0-ceramide and was inhibited by treatment with ceramide-neutralizing monoclonal antibody or single-chain variable fragment. These results indicate that C16:0-ceramide may be implicated in the vascular injury associated with COVID-19.

A Protein Tyrosine Phosphatase 4A3 (PTP4A3) inhibitor prevents and repairs pulmonary endothelial barrier dysfunction and ameliorates acute lung injury provoked by the SARS‐CoV‐2 Spike protein subunit 1 in k18‐hACE2 transgenic mice

PTP4A3 regulates various inflammatory and cytoskeletal signaling pathways (e.g., STAT3, RhoA, ERK, AKT) that are involved in endothelial barrier dysfunction and acute lung injury (ALI). We have investigated the actions of JMS‐053, a small PTP4A3 inhibitor, in both in vitro and in vivo models of SARS‐CoV‐2 Spike protein subunit 1 (S1SP)‐induced endothelial inflammation and acute lung injury. S1SP (10nM) caused a time‐dependent endothelial barrier dysfunction in human lung microvascular endothelial cell monolayers that was ameliorated by JMS‐053 (12.5µM) administered either as pre‐treatment (4 hours prior to S1SP) or post‐treatment (5 hours after S1SP). In K18‐hACE2 transgenic mice, which express the human ACE2 receptor for SARS‐CoV‐2 S1SP, we instilled S1SP intratracheally, post‐treated with JMS‐053 (10mg/kg i.p. at 1, 24 and 48 hours after S1SP) or vehicle, and investigated molecular, functional and histological outcomes 72 hours later. JMS‐053 mice displayed lower alveolar cellularity, proteinosis and “cytokine storm” (i.e., IL‐6, IL‐1β, IL‐17), and lower levels of phosphorylated (activated) STAT3 and NF‐kB, compared to mice instilled with S1SP and treated with vehicle. Moreover, JMS‐053 greatly reduced quantifiable histological evidence of injury, compared to vehicle‐treated animals. This data suggests that inhibition of PTP4A3 could represent a critical target for novel therapeutic approaches towards endothelial dysfunction, inflammation and acute lung injury.

Glycogen synthase kinase 3 inhibitor protects against microvascular hyperpermeability following hemorrhagic shock.

Hemorrhagic shock (HS)-induced microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. β-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase 3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of β-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 would attenuate microvascular hyperpermeability following HS. In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mm Hg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 μg/kg) intravenously 10 minutes before shock. To study microvascular permeability, the rats were intravenously injected with fluorescein isothiocyanate (FITC)-albumin (50 mg/kg), and its flux across the mesenteric postcapillary venules was determined using intravital microscopy. In cell culture studies, rat lung microvascular endothelial cell monolayers grown on Transwell plates were pretreated with SB216763 (5 μM) followed by BAK (5 μg/mL) and caspase 3 (5 μg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase 3 enzyme activity was assayed fluorometrically. The HS group showed significant increase in FITC-albumin extravasation (p < 0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p < 0.05). Pretreatment with SB216763 protected against a BAK-induced increase in rat lung microvascular endothelial cell monolayer permeability and caspase 3 activity but failed to show similar results with a caspase 3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in β-catenin-dependent T-cell factor-mediated transcription. Inhibiting phosphorylation of β-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 help regulates HS-induced microvascular hyperpermeability.

Effective Treatment of Mouse Sepsis With an Inhibitory Antibody Targeting Integrin αvβ5*

Integrin αvβ5 has been identified as a regulator of vascular leak and endothelial permeability. We hypothesized that targeting αvβ5 could represent a viable treatment strategy for sepsis. Integrin β5 subunit knockout and wild-type 129/svJae mice and wild-type mice treated with αvβ5 blocking or control antibodies were tested in models of intraperitoneal lipopolysaccharide and cecal ligation and puncture. Human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers were treated with αvβ5 antibodies to assess for effects on lipopolysaccharide-induced changes in transendothelial resistance and on patterns of cytoskeletal reorganization. Laboratory-based research. Mice and endothelial cell monolayers. Measurements taken after intraperitoneal lipopolysaccharide and/or cecal ligation and puncture included mortality, vascular leak, hematocrit, quantification of a panel of serum cytokines/chemokines, and assessment of thioglyccolate-induced leukocyte migration. β5 knockout mice had decreased mortality after intraperitoneal lipopolysaccharide and cecal ligation and puncture and decreased vascular leak, as measured by extravasation of an I-labeled intravascular tracer. Treating clinically ill mice with αvβ5 antibodies, up to 20 hrs after intraperitoneal lipopolysaccharide and cecal ligation and puncture, also resulted in decreased mortality. αvβ5 antibodies attenuated lipopolysaccharide-induced transendothelial resistance changes and cytoskeletal stress fiber formation in both human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers. αvβ5 antibodies had no effect on cytokine/chemokine serum levels after cecal ligation and puncture. β5 knockout mice and wild-type controls did not exhibit differences in thioglyccolate-induced leukocyte migration. Our studies suggest that αvβ5 is an important regulator of the vascular endothelial leak response in sepsis and that αvβ5 blockade may provide a novel approach to treating this devastating disease syndrome.

What’s New in Shock, March 2012?

What’s New in Shock, March 2012?

β-Catenin Dynamics in the Regulation of Microvascular Endothelial Cell Hyperpermeability

β-Catenin, a key regulator of barrier integrity, is an important component of the adherens junctional complex. Although the roles of β-catenin in maintaining the adherens junctions and Wnt signaling are known, the dynamics of β-catenin following insult and its potential role in vascular recovery/repair remain unclear. Our objective was to define β-catenin's dynamics following disruption of the adherens junctional complex and subsequent recovery. Rat lung microvascular endothelial cells were treated with active caspase 3 enzyme, by protein transference method, as an inducer of junctional damage and permeability. The disruption and subsequent recovery of β-catenin to the adherens junctions were studied via immunofluorescence. Rat lung microvascular endothelial cell monolayers were used to measure hyperpermeability. To understand the role of β-catenin on nuclear translocation/transcriptional regulation in relationship to the recovery of the adherens junctions, Tcf-mediated transcriptional activity was determined. Active caspase 3 induced a loss of β-catenin at the adherens junctions at 1 and 2 h followed by its recovery at 3 h. Transference of Bak peptide, an inducer of endogenous caspase 3 activation, induced hyperpermeability at 1 h followed by a significant decrease at 2 h. Inhibition of GSK-3β and the transfection of β-catenin vector increased Tcf-mediated transcription significantly (P < 0.05). The dissociated adherens junctional protein β-catenin translocates into the cytoplasm, resulting in microvascular hyperpermeability followed by a time-dependent recovery and relocation to the cell membrane. Our data suggest a recycling pathway for β-catenin to the cell junction.

Role of β-Catenin in Regulating Microvascular Endothelial Cell Hyperpermeability

Paracellular microvascular hyperpermeability occurs mainly because of the disruption of the endothelial adherens junction complex. Vascular endothelial-cadherin that consists of an extracellular and intracellular domain to confer cell-cell contact is linked to the actin cytoskeletal assembly through β-catenin. Our objective was to determine the functional role of β-catenin during paracellular hyperpermeability and to evaluate whether exogenous β-catenin would protect against vascular leak. β-Catenin siRNA (2.5 μg/mL) was administered to Sprague-Dawley rats through tail vein. FITC-albumin extravasation of the mesenteric postcapillary venules was evaluated after 48 hours using intravital microscopy. Parallel studies using rat lung microvascular endothelial cell monolayers were transfected with β-catenin siRNA, and hyperpermeability was determined using monolayers after 48 hours. The effectiveness of β-catenin siRNA was tested using immunofluorescence and Western blot. To study the protective effect of β-catenin, rat lung microvascular endothelial cell monolayers were transfected with a β-catenin gene expression construct for 48 hours or a recombinant β-catenin protein (1 μg/mL) for 2 hours, followed by transfection with proapoptotic BAK peptide (5 μg/mL), a known inducer hyperpermeability. β-Catenin siRNA induced a significant increase in vascular hyperpermeability in vivo (p<0.05) and monolayer permeability (in vitro; p<0.05). β-Catenin siRNA significantly altered the adherens junction complex and decreased β-catenin protein levels. β-Catenin gene expression construct or recombinant β-catenin protein attenuated BAK-induced monolayer hyperpermeability significantly (p<0.05). Posttranscriptional gene silencing of β-catenin leads to vascular hyperpermeability in vivo and monolayer hyperpermeability in vitro. The enhancement of β-catenin gene expression at the adherens junction or exogenous introduction of β-catenin protein shows protection against vascular hyperpermeability.

(−)-Deprenyl Inhibits Thermal Injury-Induced Apoptotic Signaling and Hyperpermeability in Microvascular Endothelial Cells

Burn injury is associated with a significant leak of intravascular fluid into the interstitial space, requiring large amounts of volume resuscitation. Activation of the intrinsic (mitochondrial) apoptotic pathway has been associated with vascular hyperpermeability. We hypothesized that vascular hyperpermeability following burns is also mediated via this pathway. The purpose of this study was to investigate whether (-)-deprenyl, a drug with antioxidant and antiapoptotic properties, could attenuate burn induced-apoptotic signaling and hyperpermeability. Male Sprague-Dawley rats were assigned to sham or experimental groups. The experimental rats underwent a 30 to 40% TBSA full-thickness burn. Serum was collected from all rats at 180 minutes postburn. Rat lung microvascular endothelial cell monolayers were exposed to the sham or burn serum; permeability was determined by fluorescein isothiocyanate-tagged albumin flux. Mitochondrial reactive oxygen species formation was measured with dihydrorhodamine 123. The change in mitochondrial membrane potential was determined with JC-1. Cytosolic cytochrome c was measured by enzyme-linked immunosorbent assay. A group of cells in each series was pretreated with (-)-deprenyl (1 microM). Monolayer permeability increased significantly (P<.05) when treated with burn serum. (-)-Deprenyl significantly attenuated the hyperpermeability induced by burn serum (P<.05). Burn serum increased mitochondrial reactive oxygen species levels and reduced mitochondrial membrane potential; these effects were markedly reduced by (-)-deprenyl. Cytochrome c release was increased by treatment with burn serum (P<.05), and this effect was significantly inhibited by (-)-deprenyl (P<.05). Burn serum induces hyperpemeability and activates intrinsic apoptotic signaling in microvascular endothelial cells. (-)-Deprenyl, an antioxidant and antiapoptotic drug, modulates intrinsic apoptotic signaling and attenuates burn-induced hyperpermeability.

17β-estradiol mediates protection against microvascular endothelial cell hyperpermeability

Background Previous work from our laboratory demonstrated the involvement of “intrinsic” mitochondrial apoptotic signaling in vascular hyperpermeability. The objective of this study was to determine if 17β-estradiol, a known inhibitor of apoptosis, would attenuate microvascular endothelial cell hyperpermeability. Methods Rat lung microvascular endothelial cell monolayers were treated with 17β-estradiol or estrogen-receptor antagonist ICI 182780 after transfection with BAK peptide (5 μg/mL). Fluorescein isothiocyanate (FITC)–albumin was used to determine the change in permeability. Mitochondrial reactive oxygen species (ROS) formation and transmembrane potential were determined using 123 dihydrorhodamine and JC-1, respectively. Cytosolic cytochrome c levels and caspase-3 activity were determined using enzyme-linked immunosorbent assay and fluorometric assay respectively. Results 17β-estradiol (10 n m) attenuated BAK-induced hyperpermeability ( P < .05), ROS formation, cytochrome c release, and caspase-3 activation. The estrogen receptor antagonist ICI 182780 blocked the protective effect of 17β-estradiol on hyperpermeability ( P < .05). Conclusions 17β-estradiol attenuates BAK-induced hyperpermeability in rat lung microvascular endothelial cells by way of an estrogen-receptor mediated pathway.

MITOCHONDRIAL COMPLEX III IS INVOLVED IN PROAPOPTOTIC BAK-INDUCED MICROVASCULAR ENDOTHELIAL CELL HYPERPERMEABILITY

It has been shown that the intrinsic mitochondrial apoptotic cascade is activated in vascular hyperpermeability after conditions such as hemorrhagic shock. Studies from our laboratory demonstrated mitochondrial reactive oxygen species (ROS) formation in endothelial cells during vascular hyperpermeability. We hypothesized that the participation of mitochondrial ROS in the intrinsic apoptotic cascade results in microvascular endothelial cell hyperpermeability. The purpose of this study was to identify the site(s) of ROS formation in the mitochondrial complex(es) that leads to hyperpermeability. Rat lung microvascular endothelial cell monolayers were pretreated with inhibitors of the complex(es) (I-V) before the activation of the mitochondrial apoptotic cascade using the proapoptotic peptide BAK (BH3). Inhibitors of the xanthine oxidase, nicotinamide adenine dinucleotide phosphate (reduced form) oxidase, NOS, and cytochrome P-450 monooxygenase were also studied. The hyperpermeability was determined by the fluorescence of fluorescein isothiocyanate-albumin that leaked across endothelial cells and ROS production by 2',7& rime;-dichlorofluorescein diacetate. Cytochrome c levels were also measured. BAK (BH3)-transfected cells showed increased ROS, cytosolic cytochrome c, and hyperpermeability (P<0.05). Complex III inhibitors antimycin A (10 microM) and stigmatellin (10 microM) attenuated BAK (BH3)-mediated ROS formation and hyperpermeability (P<0.05). The complex III inhibition decreased BAK (BH3)-mediated cytochrome c release. The results suggest that mitochondrial ROS formation, particularly at respiratory chain complex III, is involved in BAK-induced monolayer hyperpermeability.

Morphine Attenuates Microvascular Hyperpermeability via a Protein Kinase A-Dependent Pathway

A recently published study from our laboratory demonstrated that morphine sulfate (MS) attenuates microvascular hyperpermeability after hemorrhagic shock in rats. MS binds to the mu receptors located on the surface of endothelial cells. Activation of the endothelial cell mu receptors has been shown by several investigators to stimulate adenylate cyclase. We hypothesize that MS binding to the mu receptor on endothelial cells increases cyclic adenosine monophosphate via adenylate cyclase activation. Cyclic adenosine monophosphate inhibits the phosphoinositide/MAP kinase hyperpermeability pathway via the protein kinase A (PKA)-dependent inhibition of Raf-1. Studies were conducted in five groups of urethane-anesthetized Sprague-Dawley rats: Group 1--control group, Group 2--a non-receptor-blocking adenylate cyclase inhibitor: SQ22536, at 100 microg/kg (n = 5), Group 3--a PKA inhibitor: H89, at 10 microg/kg, Group 4--a morphine sulfate (10 microg/kg) and PKA inhibitor group, and Group 5--an adenylate cyclase inhibited and morphine (10 microg/kg) group. Intravital microscopy in mesenteric postcapillary venules and rat lung microvascular endothelial cell monolayers were used to measure permeability. Adenylate cyclase and PKA inhibition resulted in vascular hyperpermeability. Our data demonstrated an increase in vascular hyperpermeability after inhibition of adenylate cyclase via SQ22536, a nonreceptor inhibitor. This increase in hyperpermeability was attenuated when treated with MS. Morphine did not attenuate hyperpermeability after blockage following PKA with H89 suggesting the action of MS is upstream of PKA and PKA dependent.

PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells

Rat lung microvascular endothelial cell monolayers were exposed to donor plasma from burned rats (25% total body surface area) at 1:3 dilution for 30 min. Immunofluorescence analysis revealed that concomitant with gap formation alterations were seen in the adherens junction (AJ) proteins beta-catenin and vascular endothelial-cadherin. Both of these components were shown to exist in a smooth, uniform arrangement at the cell periphery in untreated cells. However, upon exposure to burn plasma, this uniformity was lost, and the AJ proteins showed a disrupted, zipper-like pattern at the cells' edge. In addition, these proteins were absent from areas of gap formation between the cells, and an increase in punctate staining throughout the cells suggests they were internalized in response to burn plasma. Measurements of both transendothelial electrical resistance and FITC-albumin flux across the cell monolayer were used to assess barrier integrity. Our study found that exposure to burn plasma rapidly caused the electrical resistance across confluent monolayers to decrease and albumin flux to increase, phenomena associated with barrier dysfunction. Furthermore, all the above responses to burn plasma were attenuated when cells were pretreated with the PKC inhibitor bisindolylmaleimide, suggesting that PKC is required for burn-induced pulmonary endothelial dysfunction.

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction.

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.

Myosin light chain phosphorylation and pulmonary endothelial cell hyperpermeability in burns.

Major cutaneous burns result in not only localized tissue damage but broad systemic inflammation causing organ system damage distal to the burn site. It is well recognized that many problems result from the release of inflammatory mediators that target vascular endothelial cells, causing organ dysfunction. The pulmonary microvessels are particularly susceptible to functional abnormalities as a direct consequence of exposure to burn-induced inflammatory mediators. Traditional therapeutic intervention is quite often ineffective in treating burn patients suffering from systemic problems. A possible explanation for this ineffectiveness may be that because so many mediators are released, supposedly activating numerous signaling cascades that interact with each other, targeting of upstream factors in these cascades on an individual basis becomes futile. Therefore, if an end-point effector responsible for endothelial dysfunction following burn injury could be identified, it may present a target for intervention. In this study, we identified phosphorylation of myosin light chain (MLC) as a required element of burn plasma-induced hyperpermeability across rat lung microvascular endothelial cell monolayers. In addition, pharmacological inhibition of myosin light chain kinase (MLCK) and Rho kinase as well as transfection of MLCK-inhibiting peptide blocked actin stress fiber formation and MLC phosphorylation in response to burn plasma. The results suggest that blocking MLC phosphorylation may provide therapeutic intervention in burn patients with the goal of alleviating systemic inflammation-induced endothelial dysfunction.

Effects of inflammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration

Background: Rhinovirus (RV) infections can result in asthma exacerbations in both adults and children. Respiratory epithelium, the primary site of RV replication, responds to the viral infection by generating a variety of cytokines and chemokines capable of promoting airway inflammation and hence might increase asthma severity. Some of these mediators might also affect the permeability of underlying vascular endothelium. Objective: We hypothesized that RV infections can promote airway inflammation and thus asthma by enhancing local vascular permeability. Methods: Confluent human lung microvascular endothelial cell (HMVEC-L) monolayers were used as an in vitro model of vascular endothelium to determine whether cytokines associated with RV-induced infections are capable of modulating endothelial cell permeability as measured by means of transendothelial electrical resistance. Recombinant cytokines and chemokines were added to confluent HMVEC-L monolayers cultured on Transwell filters, and permeability was measured as decreased electrical resistance over time. Eosinophil transendothelial migration was assessed under the same experimental conditions. Results: TNF- α, IL-1 β, and IFN- γsignificantly increased HMVEC-L permeability. In contrast, GM-CSF, G-CSF, IL-8, IL-6, and RANTES had no effect. Although incubation of HMVEC-L monolayers with either TNF-α or IL-1β promoted eosinophil migration, IFN-γ had no effect, indicating that enhanced permeability alone was not sufficient for eosinophil infiltration. Conclusion: Select cytokines, generated in response to RV infection, can increase vascular permeability and might provide a mechanism by which RV infection can lead to edema, cellular infiltration, and inflammation and thus compromised airflow. (J Allergy Clin Immunol 2002;110:752-6.)

Albondin-mediated capillary permeability to albumin. Differential role of receptors in endothelial transcytosis and endocytosis of native and modified albumins.

Specific albumin binding to the surface of endothelium initiates its transcytosis across continuous endothelium via noncoated plasmalemmal vesicles. Past work has identified several putative albumin-binding proteins (SPARC, gp60, gp30, and gp18). In this study, we examined the specific role of these proteins in the binding of bovine serum albumin (BSA) to endothelium. The 60-kDa albumin-binding protein (gp60, now called albondin) was purified from cultured rat microvascular endothelial cells for antibody production. Anti-albondin antibodies (alpha gp60) specifically recognized albondin expressed by rat, bovine, and human endothelial cells (EC). alpha gp60 and unlabeled native BSA inhibited 125I-BSA binding to confluent EC monolayers and to both albondin and SPARC extracts immobilized on filters. Modification of BSA by maleic anhydride treatment (Mal-BSA) or by surface adsorption to colloidal gold particles (A-Au) renders the ligand specific for gp30 and gp18 while eliminating its ability to inhibit 125I-BSA binding to EC and to both albondin and SPARC extracts. Mal-BSA and A-Au interacted with EC via distinct binding sites not recognizing native BSA. EC internalization of 125I-BSA is inhibited by unlabeled BSA and alpha gp60 IgG but not nonimmune IgG, Mal-BSA, or A-Au. Conversely, internalization of modified BSA is inhibited by unlabeled modified BSA but not BSA or alpha gp60 IgG. Specific 125I-BSA transendothelial transport in rat lungs perfused in situ and for EC monolayers in vitro is inhibited (> or = 90%) by unlabeled BSA and alpha gp60 IgG but not nonimmune IgG and Mal-BSA. No specific transport of 125I-Mal-BSA is detected across bovine lung microvascular endothelial cell monolayers, only paracellular and/or fluid-phase transport. Low temperatures reduce BSA transport by 5-fold and Mal-BSA by 2-fold. Interestingly, 3-fold more native BSA is transported than Mal-BSA at 37 degrees C, whereas at 8-10 degrees C they are nearly equal, suggesting facilitation of BSA transport. Cumulatively, it appears that gp30 and gp18 mediate the binding, endocytosis, and degradation of modified albumins, whereas albondin mediates native albumin binding which significantly enhances its transcytosis and capillary permeability.