Biomedical subjects

Michael Koval

Publications and source records attributed to Michael Koval.

At least 19 recordsLinked to original sources

PubMed2006

Metabolism of 3-nitrotyrosine induces apoptotic death in dopaminergic cells.

Intrastriatal injection of 3-nitrotyrosine, which is a biomarker for nitrating oxidants, provokes dopaminergic neuronal death in rats by unknown mechanisms. Herein, we show that extracellular 3-nitrotyrosine is transported via the l-aromatic amino acid transporter in nondopaminergic NT2 cells, whereas in dopaminergic PC12 cells, it is transported by both the l-aromatic amino acid and the dopamine transporters. In both cell lines, 3-nitrotyrosine is a substrate for tyrosine tubulin ligase, resulting in its incorporation into the C terminus of alpha-tubulin. In NT2 cells, incorporation of 3-nitrotyrosine into alpha-tubulin induces a progressive, reversible reorganization of the microtubule architecture. In PC12 cells, 3-nitrotyrosine decreases intracellular dopamine levels and is metabolized by the concerted action of the aromatic amino acid decarboxylase and monoamine oxidase. Intracellular levels of 133 micromol of 3-nitrotyrosine per mole of tyrosine did not alter NT2 viability but induced PC12 apoptosis. The cell death was reversed by caspases and aromatic amino acid decarboxylase and monoamine oxidase inhibitors. 3-Nitrotyrosine induced loss of tyrosine hydroxylase-positive primary rat neurons, which was also prevented by an aromatic amino acid decarboxylase inhibitor. These findings provide a novel mechanism by which products generated by reactive nitrogen species induce dopaminergic neuron death and thus may contribute to the selective neurodegeneration in Parkinson's disease.

Amino Acid Transport Systems↗

PubMed2006

Pathways and control of connexin oligomerization.

Connexins form gap junction channels that link neighboring cells into an intercellular communication network. Many cells that express multiple connexins produce heteromeric channels containing at least two connexins, which provides a means to fine tune gap junctional communication. Formation of channels by multiple connexins is controlled at two levels: by inherent structural compatibilities that enable connexins to hetero-oligomerize and by cellular mechanisms that restrict the formation of heteromers by otherwise compatible connexins. Here, I discuss roles for secretory compartments beyond the endoplasmic reticulum in connexin oligomerization and evidence that suggests that membrane microdomains help regulate connexin trafficking and assembly.

Animals↗

PubMed2005

Control of intracellular trafficking of ICAM-1-targeted nanocarriers by endothelial Na+/H+ exchanger proteins.

Targeting nanocarriers (NC) loaded by antioxidant enzymes (e.g., catalase) to endothelial cell adhesion molecules (CAM) alleviates oxidative stress in the pulmonary vasculature. However, antioxidant protection is transient, since CAM-targeted catalase is internalized, delivered to lysosomes, and degraded. To design means to modulate the metabolism and longevity of endothelial cell (EC)-targeted drugs, we identified and manipulated cellular elements controlling the uptake and intracellular trafficking of NC targeted to ICAM-1 (anti-ICAM/NC). BAPTA, thapsigargin, amiloride, and EIPA inhibited anti-ICAM/NC uptake by EC and actin rearrangements induced by anti-ICAM/NC (required for uptake), suggesting that member(s) of Na(+)/H(+) exchanger family proteins (NHE) regulate these processes. Consistent with this hypothesis, an siRNA specific for the plasmalemma NHE1, but not the endosome-associated NHE6, inhibited actin remodeling induced by anti-ICAM/NC and internalization. Anti-ICAM/NC binding to EC stimulated formation of a transient ICAM-1/NHE1 complex. One hour after uptake, ICAM-1 dissociated from NHE1, and anti-ICAM/NC were transported to NHE6-positive vesicles en route to lysosomes. Inhibition of PKC (an activator of intracellular NHE) accelerated nanocarrier lysosomal trafficking. In contrast, monensin, which enhances the endosomal sodium influx and proton efflux maintained by NHE6, inhibited delivery of anti-ICAM/NC to lysosomes by switching their trafficking to a plasma membrane recycling pathway. This markedly prolonged the protective effect of catalase-coated anti-ICAM/NC. Therefore, 1) NHE1 and NHE6 regulate distinct phases of anti-ICAM/NC uptake and trafficking; 2) pharmacological agents affecting these regulatory elements alter the itinerary of anti-ICAM/NC intracellular trafficking; and 3) these agents modulate duration of the therapeutic effects of targeted drugs.

Amiloride↗

PubMed2005

Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation.

We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both ATII and ATI cells. Surfactant lipid secretion was measured using cells metabolically labeled with [(3)H]choline. In response to 21% tonic stretch for 15 min, ATII cells seeded with ATI cells secreted nearly threefold more surfactant lipid compared with ATII cells seeded alone. ATI cells did not secrete lipid in response to stretch. The enhanced lipid secretion by ATII plus ATI cocultures was inhibited by treatment with apyrase and adenosine deaminase, suggesting that ATP release by ATI cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a luciferase assay where, in response to 21% stretch, ATI cells released fourfold more ATP than ATII cells. Because ATI cells release significantly more ATP at a lower level of stretch than ATII cells, this supports the hypothesis that ATI cells are mechanosensors in the lung and that paracrine stimulation of ATII cells by extracellular ATP released from ATI cells plays a role in regulating surfactant secretion.

Adenosine Triphosphate↗

PubMed2005

tGolgin-1 (p230, golgin-245) modulates Shiga-toxin transport to the Golgi and Golgi motility towards the microtubule-organizing centre.

tGolgin-1 (trans-Golgi p230, golgin-245) is a member of a family of large peripheral membrane proteins that associate with the trans-Golgi network (TGN) via a C-terminal GRIP domain. Some GRIP-domain proteins have been implicated in endosome-to-TGN transport but no function for tGolgin-1 has been described. Here, we show that tGolgin-1 production is required for efficient retrograde distribution of Shiga toxin from endosomes to the Golgi. Surprisingly, we also found an indirect requirement for tGolgin-1 in Golgi positioning. In HeLa cells depleted of tGolgin-1, the normally centralized Golgi and TGN membranes were displaced to the periphery, forming 'mini stacks'. These stacks resembled those in cells with disrupted microtubules or dynein-dynactin motor, in that they localized to endoplasmic-reticulum exit sites, maintained their secretory capacity and cis-trans polarity, and were relatively immobile by video microscopy. The mini stacks formed concomitant with a failure of pre-Golgi elements to migrate along microtubules towards the microtubule-organizing centre. The requirement for tGolgin-1 in Golgi positioning did not appear to reflect direct binding of tGolgin-1 to motile pre-Golgi membranes, because distinct Golgi and tGolgin-1-containing TGN elements that formed after recovery of HeLa cells from brefeldin-A treatment moved independently toward the microtubule-organizing centre. These data demonstrate that tGolgin-1 functions in Golgi positioning indirectly, probably by regulating retrograde movement of cargo required for recruitment or activation of dynein-dynactin complexes on newly formed Golgi elements.

Autoantigens↗

PubMed2005

Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum.

In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.

Alternative Splicing↗

PubMed2005

Autologous apoptotic cell engulfment stimulates chemokine secretion by vascular smooth muscle cells.

Apoptosis of vascular smooth muscle cells (VSMCs) occurs in vivo under both physiological and pathological settings. The clearance of apoptotic cells may be accomplished in part by the surrounding normal VSMCs. However, the fate of internalized apoptotic cells, the rate of intracellular degradation, and the consequences of these processes to VSMC biology are unknown. Electron microscopy and confocal fluorescence imaging showed that rat VSMCs effectively bound and internalized autologous apoptotic VSMCs in vitro. Within 2 hours, the internalized apoptotic cells were delivered to lysosomes, and the majority of these internalized cells and their proteins were efficiently degraded by 24 hours. After degradation was completed, the phagocytic VSMCs remained viable with normal rates of proliferation. Clearance of apoptotic cells by VSMCs did not induce the release of vascular wall matrix proteases but was associated with a 1.6-fold increase in transforming growth factor-beta1 release. Interestingly, clearance of apoptotic cells stimulated VSMCs to secrete monocyte-chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant. The coordinated release of transforming growth factor-beta1 and chemokines suggests that autologous apoptotic cell clearance stimulates VSMCs to release molecules that specifically recruit professional phagocytes while simultaneously dampening the inflammatory response and preventing vascular injury.

Animals↗

PubMed2004

ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs.

Intercellular adhesion molecule-1 (ICAM-1) is a target for drug delivery to endothelial cells (ECs), which internalize multivalent anti-ICAM nanocarriers (anti-ICAM/NCs) within 15 to 30 minutes. The concomitant ICAM-1 disappearance from the EC surface transiently inhibited subsequent binding and uptake of anti-ICAM/NCs. Within 1 hour, internalized ICAM-1 diverged from anti-ICAM/NCs into prelysosomal vesicles, resurfaced, and enabled uptake of a subsequent anti-ICAM/NC dose. Thus, internalized ICAM-1 was able to recycle back to the plasma membrane. In vivo pulmonary targeting of a second anti-ICAM/NC dose injected 15 minutes after the first dose was decreased by 50% but recovered between 30 minutes and 2.5 hours, comparable to cultured ECs. Anti-ICAM/NCs affected neither EC viability nor fluid-phase endocytosis and traffic to lysosomes. However, lysosomal trafficking of the second dose of anti-ICAM/NCs was decelerated at least 2-fold versus the first dose; hence the major fraction of anti-ICAM/NCs resided in prelysosomal vesicles for at least 5 hours without degradation. Two successive doses of anti-ICAM/NC/catalase protected ECs against H2O2 for at least 8 hours versus 2 hours afforded by a single dose, suggesting that recurrent targeting to ICAM-1 affords longer effects. ICAM-1 recycling and inhibited lysosomal traffic/degradation of subsequent doses may help to prolong activity of therapeutic agents delivered into ECs by anti-ICAM/NCs.

Animals↗

PubMed2004

Developmental regulation of claudin localization by fetal alveolar epithelial cells.

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.

1-Methyl-3-isobutylxanthine↗

PubMed2004

Gap junctional communication modulates agonist-induced calcium oscillations in transfected HeLa cells.

Gap junctional communication modulates intercellular calcium signaling in many cell types. We have investigated whether gap junctional communication modulates calcium oscillatory behavior of cells responding to an agonist. Extracellular UTP induced calcium oscillations in 70% of HeLa cells cultured in monolayer, and neighboring cells oscillated independently of each other. In HeLa cell transfectants expressing connexin43 (HeLa/Cx43), extracellular UTP induced calcium transients, but calcium oscillations occurred in only 10% of cells. Inhibition of gap junctional communication with anandamide in HeLa/Cx43 transfectants substantially restored oscillations (55% of cells). In HeLa/Cx45 transfectants, UTP initiated calcium oscillations similar to those seen in HeLa cells (63% of cells), but HeLa/Cx46 transfectants demonstrated calcium oscillations that were dampened compared to those of the parental HeLa cells, and occurred in only 40% of cells. These experiments demonstrate that gap junctional communication modulates calcium oscillatory behavior in cell monolayers, presumably by allowing cells to share a small molecule such as inositol trisphosphate. These studies suggest that gap junctional communication may alter the nature of signals induced by calcium mobilizing agonists in a connexin-dependent fashion by modulating calcium oscillatory behavior.

Animals↗

PubMed2004

Cell-cell interactions in regulating lung function.

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.

Animals↗

PubMed2004

Endothelial endocytic pathways: gates for vascular drug delivery.

Vascular endothelium plays strategic roles in many drug delivery paradigms, both as an important therapeutic target itself and as a barrier for reaching tissues beyond the vascular wall. Diverse means are being developed to improve vascular drug delivery including stealth liposomes and polymer carriers. Affinity carriers including antibodies or peptides that specifically bind to endothelial surface determinants, either constitutive or pathological, enhance targeting of drugs to endothelial cells (EC) in diverse vascular areas. In many cases, binding to endothelial surface determinants facilitates internalization of the drug/carrier complex. There are several main endocytic pathways in EC, including clathrin- and caveoli-mediated endocytosis, phagocytosis and macropinocytosis (these two are less characteristic of generic EC) and the recently described Cell Adhesion Molecule (CAM)-mediated endocytosis. The latter may be of interest for intracellular drug delivery to EC involved in inflammation or thrombosis. The metabolism and effects of internalized drugs largely depend on the routes of intracellular trafficking, which may lead to degrading lysosomal compartments or other organelles, recycling to the plasma membrane or transcytosis to the basal surface of endothelium. The latter route, characteristic of caveoli-mediated endocytosis, may serve for trans-endothelial drug delivery. Paracellular trafficking, which can be enhanced under pathological conditions or by auxiliary agents, represents an alternative for transcytosis. Endothelial surface determinants involved in endocytosis, mechanisms of the latter and trafficking pathways, as well as specific characteristics of EC in different vascular areas, are discussed in detail in the context of modern paradigms of vascular drug delivery.

Animals↗

PubMed2003

Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress.

Nanotechnologies promise new means for drug delivery. ICAM-1 is a good target for vascular immunotargeting of nanoparticles to the perturbed endothelium, although endothelial cells do not internalize monomeric anti-ICAM-1 antibodies. However, coupling ICAM-1 antibodies to nanoparticles creates multivalent ligands that enter cells via an amiloride-sensitive endocytic pathway that does not require clathrin or caveolin. Fluorescence microscopy revealed that internalized anti-ICAM nanoparticles are retained in a stable form in early endosomes for an unusually long time (1-2 h) and subsequently were degraded following slow transport to lysosomes. Inhibition of lysosome acidification by chloroquine delayed degradation without affecting anti-ICAM trafficking. Also, the microtubule disrupting agent nocodazole delayed degradation by inhibiting anti-ICAM nanoparticle trafficking to lysosomes. Addition of catalase to create anti-ICAM nanoparticles with antioxidant activity did not affect the mechanisms of nanoparticle uptake or trafficking. Intracellular anti-ICAM/catalase nanoparticles were active, because endothelial cells were resistant to H2O2-induced oxidative injury for 1-2 h after nanoparticle uptake. Chloroquine and nocodazole increased the duration of antioxidant protection by decreasing the extent of anti-ICAM/catalase degradation. Therefore, the unique trafficking pathway followed by internalized anti-ICAM nanoparticles seems well suited for targeted delivery of therapeutic enzymes to endothelial cells and may provide a basis for treatment of acute vascular oxidative stress.

Catalase↗

PubMed2003

A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1.

Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.

Amides↗

PubMed2003

Heterogeneity of claudin expression by alveolar epithelial cells.

Claudins are proteins that participate in epithelial barrier function and regulate paracellular permeability. By immunohistochemistry of adult rat lung sections, claudin-3, claudin-4, and claudin-5 were found to be co-expressed by type II alveolar epithelial cells. Claudin-3 and claudin-4 were also co-expressed by some alveolar epithelial cells adjacent to type II cells. In contrast, claudin-5 was expressed throughout the alveolus. Isolated primary rat alveolar epithelial cells in culture also expressed claudin-3, claudin-4, and claudin-5, but showed little claudin-1 and claudin-2 expression. Claudin expression by isolated cells at both the mRNA and protein level varied with time in culture. In particular, claudin-3 and claudin-5 co-localized and were distributed around the alveolar cell periphery, but claudin-4 expression was heterogeneous. We also found that paracellular permeability was increased when cultured alveolar epithelial cells were treated with a fatty acid amide, methanandamide. Methanandamide did not alter cell viability. Claudin-3, claudin-4, claudin-5, occludin, and zona occludens 1 remained localized to cell-cell contact sites at the plasma membrane in methanandamide-treated cells, suggesting that plasma membrane localization of these junction proteins is not sufficient for maintaining barrier function. However, methanandamide-treated cells showed a 12-fold increase in claudin-5 expression and a 2- to 3-fold increase in claudin-3, consistent with the notion that specific changes in claudin expression levels may correlate with changes in alveolar epithelial barrier function.

ATP-Binding Cassette Transporters↗

PubMed2002

Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter protein ABCA3.

Lamellar bodies are the specialized secretory organelles of alveolar type II (ATII) epithelial cells through which the cell packages pulmonary surfactant and regulates its secretion. Surfactant within lamellar bodies is densely packed as circular arrays of lipid membranes and appears to be the product of several trafficking and biosynthetic processes. To elucidate these processes, we reported previously on the generation of a monoclonal antibody (3C9) that recognizes a unique protein of the lamellar body membrane of 180 kDa, which we named LBM180. We report that mass spectrometry of the protein precipitated by this antibody generated a partial sequence that is identical to the ATP-binding cassette protein, ABCA3. Homology analysis of partial sequences suggests that this protein is highly conserved among species. The ABCA3 gene transcript was found in cell lines of human lung origin, in ATII cells of human, rat, and mouse, as well as different tissues of rat, but the highest expression of ABCA3 was observed in ATII cells. Expression of this transcript was at its maximum prior to birth, and hormonal induction of ABCA3 transcript was observed in human fetal lung at the same time as other surfactant protein transcripts were induced, suggesting that ABCA3 is developmentally regulated. Molecular and biochemical studies show that ABCA3 is targeted to vesicle membranes and is found in the limiting membrane of lamellar bodies. Because ABCA3 is a member of a subfamily of ABC transporters that are predominantly known to be involved in the regulation of lipid transport and membrane trafficking, we speculate that this protein may play a key role in lipid organization during the formation of lamellar bodies.

ATP-Binding Cassette Transporters↗

PubMed2002

Targeted gap junction protein constructs reveal connexin-specific differences in oligomerization.

To define further the mechanisms of gap junction protein (connexin (Cx)) oligomerization without pharmacologic disruption, we have examined the transport and assembly of connexin constructs containing C-terminal di-lysine-based endoplasmic reticulum (ER) (HKKSL) or ER-Golgi intermediate compartment (AKKFF) targeting sequences. By immunofluorescence microscopy, Cx43-HKKSL transiently transfected into HeLa cells showed a predominantly ER localization, although Cx43-AKKFF was localized to the perinuclear region of the cell. Sucrose gradient analysis of Triton X-100-solubilized connexins showed that either Cx43-HKKSL or Cx43-AKKFF expressed alone by HeLa cells was maintained as an apparent monomer. In contrast to Cx43-HKKSL, Cx32-HKKSL was maintained in the ER as stable hexamers, consistent with the notion that Cx32 and Cx43 oligomerization occur in distinct intracellular compartments. Furthermore, Cx43-HKKSL and Cx43-AKKFF inhibited trafficking of Cx43 and Cx46 to the plasma membrane. The inhibitory effect was because of the formation of mixed oligomers between Cx43-HKKSL or Cx43-AKKF and wild type Cx43 or Cx46. Taken together, these results suggest that Cx43-HKKSL and Cx43-AKKFF recirculate through compartments where oligomerization occurs and may be maintained as apparent monomers by a putative Cx43-specific quality control mechanism.

Biopolymers↗

PubMed2002

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells.

Cell-selective intracellular targeting is a key element of more specific and safe enzyme, toxin, and gene therapies. Endothelium poorly internalizes certain candidate carriers for vascular immunotargeting, such as antibodies to platelet endothelial cell adhesion molecule 1 (PECAM-1). Conjugation of poorly internalizable antibodies with streptavidin (SA) facilitates the intracellular uptake. Although both small and large (100-nm versus 1000-nm diameter) anti-PECAM/SA-beta galactosidase (SA-beta-gal) conjugates bound selectively to PECAM-expressing cells, only small conjugates showed intracellular accumulation of active beta-gal. To study whether size of the conjugates controls the uptake, a series of anti-PECAM/SA and anti-PECAM/bead conjugates ranging from 80 nm to 5 microm in diameter were produced. Human umbilical vein endothelial cells and PECAM-transfected mesothelioma cells internalized 80- to 350-nm anti-PECAM conjugates, but not conjugates larger than 500 nm. Further, size controls intracellular targeting of active therapeutic cargoes in vitro and in vivo. Small anti-PECAM/DNA conjugates transfected target cells in culture 5-fold more effectively than their large counterpart (350- versus 4200-nm diameter). To evaluate the practical significance of the size-controlled subcellular addressing, we coupled glucose oxidase (GOX) to anti-PECAM and antithrombomodulin. Both types of conjugates had equally high pulmonary uptake after intravenous injection in mice, yet only small (200- to 250-nm), not large (600- to 700-nm), GOX conjugates caused profound oxidative vascular injury in the lungs, presumably owing to intracellular generation of H(2)O(2). Thus, engineering of affinity carriers of specific size permits intracellular delivery of active cargoes to endothelium in vitro and in vivo, a paradigm useful for the targeting of drugs, genes, and toxins.

Animals↗