Transcytotic Vesicles Research Articles

Amyloid deposition through endocytosis in vascular endothelial cells

No mechanistic lead is known for establishing AL amyloid deposits in organs. We here report an electron microscopic (EM) analysis in a case of intestinal AL amyloidosis before initiating treatment for amyloidosis. The dense deposits of amyloid fibrils are concentrated around the small blood vessels in the submucosal area of intestinal tissue. Surprisingly, we observed endothelial cells (ECs) of blood vessels containing plenty of endocytotic (pinocytotic) and transcytotic vesicles at the luminal side and above the basement membrane, indicating the one-way active trafficking of either the immunoglobulin (Ig) light chain or preassembled amyloid fibrils from the luminal side of ECs to the extraluminal area of ECs. Immunoelectron microscopy displayed that the immuno-gold signals were observed in the vascular cavity and the subendothelial area of amyloid deposits. However, there is no sign of an Ig light chain in pinocytotic vesicles. Therefore, the intestinal ECs may actively pump out mainly the preassembled amyloid fibrils (not light chains) from the blood stream into the subendothelial area as a physiologic function.

Trained hypertensive rats exhibit decreased transcellular vesicle trafficking, increased tight junctions' density, restored blood-brain barrier permeability and normalized autonomic control of the circulation.

Introduction: Chronic hypertension is accompanied by either blood-brain barrier (BBB) leakage and autonomic dysfunction. There is no consensus on the mechanism determining increased BBB permeability within autonomic areas. While some reports suggested tight junction's breakdown, others indicated the involvement of transcytosis rather than paracellular transport changes. Interestingly, exercise training was able to restore both BBB permeability and autonomic control of the circulation. We sought now to clarify the mechanism(s) governing hypertension- and exercise-induced BBB permeability. Methods: Spontaneously hypertensive rats (SHR) and normotensive controls submitted to 4-week aerobic training (T) or sedentary protocol (S) were chronically cannulated for baseline hemodynamic and autonomic recordings and evaluation of BBB permeability. Brains were harvested for measurement of BBB function (FITC-10kDa leakage), ultrastructural analysis of BBB constituents (transmission electron microscopy) and caveolin-1 expression (immunofluorescence). Results: In SHR-S the increased pressure, augmented sympathetic vasomotor activity, higher sympathetic and lower parasympathetic modulation of the heart and the reduced baroreflex sensitivity were accompanied by robust FITC-10kDa leakage, large increase in transcytotic vesicles number/capillary, but no change in tight junctions' density within the paraventricular nucleus of the hypothalamus, the nucleus of the solitary tract and the rostral ventrolateral medulla. SHR-T exhibited restored BBB permeability and normalized vesicles counting/capillary simultaneously with a normal autonomic modulation of heart and vessels, resting bradycardia and partial pressure reduction. Caveolin-1 expression ratified the counting of transcellular, not other cytoplasmatic vesicles. Additionally, T caused in both groups significant increases in tight junctions' extension/capillary border. Discussion: Data indicate that transcytosis, not the paracellular transport, is the primary mechanism underlying both hypertension- and exercise-induced BBB permeability changes within autonomic areas. The reduced BBB permeability contributes to normalize the autonomic control of the circulation, which suppresses pressure variability and reduces the occurrence of end-organ damage in the trained SHR. Data also disclose that hypertension does not change but exercise training strengthens the resistance of the paracellular pathway in both strains.

Abstract 13626: Aerobic Training Corrects Bbb Dysfunction Within Preautonomic Areas In Heart Failure Rats By Normalizing The Augmented Transcytosis

Introduction: Besides intense renin-angiotensin system activation and sympathoexcitation, heart failure (HF) is accompanied by marked autonomic dysfunction. We showed previously in hypertensive rats that blood-brain barrier (BBB) dysfunction in preautonomic areas aggravated the autonomic dysfunction but is corrected by aerobic training (T). There is no information in HF Hypothesis: We hypothesized that BBB dysfunction within the paraventricular hypothalamic nucleus (PVN) contributes to the autonomic imbalance, which may be ameliorated by T Methods: Wistar rats were submitted to anterior descending coronary artery occlusion or Sham surgery and echocardiography. Four weeks later, HF (ejection fraction≤40%) and Sham rats underwent a T (55% of maximal exercise capacity) or sedentary (S) protocol for 8 weeks and were chronically cannulated for hemodynamic/autonomic recordings and determination of BBB permeability (intra-arterial injection of fluorescent dyes with high and low molecular weight). To investigate the mechanisms underlying HF- and T-induced BBB changes, the PVN was harvested and processed for transmission electron microscopy Results: HF-S exhibited reduced arterial pressure (AP, -10%), increased sympathetic activity to vessels and heart (+45% on average), decreased spontaneous baroreflex sensitivity (BrS, -19%), large increase in systolic AP variability (+53%), and 4-fold increase in PVN BBB leakage (11.3±1.4 vs. 2.8±0.2 %area in Sham-S). HF-S also exhibited increased transcytotic vesicles (+80%) in PVN capillaries’ endothelium, without any change in tight junctions’ expression, capillary diameter, and pericytes’ coverage. These changes were completely normalized in HF-T rats. In Sham rats T only caused resting bradycardia, increased BrS, and a mild reduction of transcytotic vesicles and PVN leakage. There were significant correlations between the number of transcytotic vesicles x PVN BBB leakage (Y=0.82x -1.74, r 2 =0.843, P<0.001) and BBB leakage x sympathetic activity (Y=0.66x +4.37, r 2 =0.415, P<0.001) Conclusions: HF courses with PVN BBB dysfunction, which is due to increased transcytosis without changes in the paracellular pathway. Training ameliorates HF’s autonomic control by normalizing transcytosis

Abstract 32: Exercise Training Corrects Blood-brain Barrier Dysfunction Within Preautonomic Areas Of Spontaneously Hypertensive Rats By Normalizing The Augmented Transcytosis

Introduction: Besides intense neuro-hormonal activation, hypertension is accompanied by blood-brain barrier (BBB) dysfunction within preautonomic areas and marked autonomic imbalance. We showed previously that exercise training (T) corrected both increased BBB leakage and autonomic dysfunction. There is no information on the mechanism(s) conditioning the normalization of BBB function Hypothesis: We hypothesized that T could modify the transcytosis and/or the paracellular transport across the capillary endothelium Methods: SHR and Wistar rats allocated to T (55% maximal capacity) or sedentary (S) protocols were chronically cannulated for hemodynamic/autonomic recordings and determination of BBB permeability (fluorescent Rhodamine-70kDa+FITC-10kDa dyes given ia ). To analyze hypertension- and T-induced BBB changes, paraventricular hypothalamic nuclei (PVN) was harvested and processed for immunofluorescence and transmission electron microscopy Results: SHR-S vs Wistar-S exhibited augmented SAP and reduced pulse interval (PI) variability, decreased spontaneous baroreflex sensitivity (BrS), increased both PVN BBB leakage (11.4±0.6 vs 3.48 %area) and transcytosis (8.1±1.2 vs 4.8±0.8 vesicles/capillary) but no change in tight junctions’expression (TJ, number/capillary). SHR-T showed a near normal autonomic control, resting bradycardia and a partial AP reduction (-9%) accompanied by normalization of both BBB leakage (3.6±1.5 %area) and transcytosis (3.8±0.7 vesicles/capillary), and increased TJs’ extension (60% occupancy of capillary borders) without changing its expression. Hypertension- and T-induced transcytosis changes were confirmed by caveolin-1 immunofluorescence (SHR-S=139±11, Wistar-S=86±8, SHR-T=81±6 arbitrary units). There were significant correlations between the number of transcytotic vesicles x PVN BBB leakage (Y=1.77x -3.46, r 2 =0.722, P<0.001) and BBB leakage x SAP variability (Y=2.30x +16.6, r 2 =0.246, P<0.001) Conclusions: PVN BBB dysfunction in hypertension is due to increased transcytosis without changes in the paracellular pathway. Training ameliorates SHR’s autonomic control by normalizing transcytosis, with an additional TJs structure improvement

Caveolae-Mediated Endothelial Transcytosis across the Blood-Brain Barrier in Acute Ischemic Stroke.

Blood-brain barrier (BBB) disruption following ischemic stroke (IS) contributes to hemorrhagic transformation, brain edema, increased neural dysfunction, secondary injury, and mortality. Brain endothelial cells form a para and transcellular barrier to most blood-borne solutes via tight junctions (TJs) and rare transcytotic vesicles. The prevailing view attributes the destruction of TJs to the resulting BBB damage following IS. Recent studies define a stepwise impairment of the transcellular barrier followed by the paracellular barrier which accounts for the BBB leakage in IS. The increased endothelial transcytosis that has been proven to be caveolae-mediated, precedes and is independent of TJs disintegration. Thus, our understanding of post stroke BBB deficits needs to be revised. These recent findings could provide a conceptual basis for the development of alternative treatment strategies. Presently, our concept of how BBB endothelial transcytosis develops is incomplete, and treatment options remain limited. This review summarizes the cellular structure and biological classification of endothelial transcytosis at the BBB and reviews related molecular mechanisms. Meanwhile, relevant transcytosis-targeted therapeutic strategies for IS and research entry points are prospected.

Abstract 387: Lymphatic Endothelial Cells Derived Exosomes Promote Neolymphangiogenesis After Injury

Neolymphangiogenesis after myocardial infarction (MI) has been extensively documented; however the source of new lymphatic vessels (LVs) is still unknown. It has been described that new LVs may derive from resident lymphatic endothelial cells (LECs) or from trans-differentiation of endothelial cells (ECs) and pericytes from veins into LECs. Which signals activate both the spreading and the trans-differentiation needs to be investigated. LECs are known to release transcytotic vesicles and exosomes, thus, we hypnotized that the enriched protein and RNA cargo of LECs derived exosomes may promote the new LVs formation after injury. Exosomes derived from LECs were isolated from LECs cultured conditioned medium and HUVECs were treated with different concentration of exosomes cargo for 10 days. After treatment, q-PCR for the major endothelial and lymphatic endothelial markers was performed on treated HUVECs. In a dose dependent manner we observed a decreased expression of KDR and CD31, endothelial markers that are not expressed by LECs and an increased expression of Prox-1 and VEGFR3. Prox-1 is the major lymphatic endothelial transcription factor that is not activated in ECs and is responsible for the transcription of LYVE-1 and podoplanin. VEGFR3 is the only known vascular endothelial growth factor receptor expressed on LECs which is not displayed by ECs. Next, we evaluated the effect of LECs derived exosomes on in vitro lymphangiogenesis assay by measuring the ability of LECs to organize into capillary-like structures on matrigel. LECs treated with LECs derived exosomes are able to form tubes and branches as early as within 1 hour compared to untreated cells. Our preliminary results show cargo of exosomes from LECs activates the signal transduction and metabolism of LECs and HUVECs promoting the new LVs formation through spreading and trans-differentiation of the targeted cells.

Morphologic mechanisms of increased vascular permeability of triolein emulsion to the blood-brain barrier.

Triolein emulsion has been known to increase vascular permeability in the brain when it is infused into the carotid artery. The purpose of this study was to identify the morphologic mechanism of increased vascular permeability in brain induced by infusion of emulsified triolein into the carotid artery by transmission electron microscopy (TEM). Triolein emulsion was infused into the carotid artery of rats. TEM using lanthanum tracer was used to evaluate morphologic changes in endothelium with a focus on transcytotic vesicles and tight junction opening. The treat group showed multiple transcytotic vesicles containing lanthanum tracer within endothelium on TEM. TEM also revealed that lanthanum tracer entered neural interstitium through tight junctions between capillary endothelial cells infrequently in the treat group. No evidence of transcytotic vesicles containing lanthanum tracer or lanthanum leakage through tight junctions was observed in the control group. Transcytosis and the opening of tight junctions appears the pathway for vascular permeability enhancement by triolein. This result could be utilized in studies on the blood-brain barrier and by those searching for chemotherapeutic methods that deliver anti-tumor agents to normally drug inaccessible organs.

The blood–brain barrier after stroke: Structural studies and the role of transcytotic vesicles

Blood–brain barrier breakdown worsens ischaemic damage, but it is unclear how molecules breach the blood–brain barrier in vivo. Using the obese ob/ob mouse as a model of enhanced blood–brain barrier breakdown, we investigated how stroke-induced structural changes to the microvasculature related to blood–brain barrier permeability. Ob/ob mice underwent middle cerebral artery occlusion, followed by 4 or 24 h reperfusion. Blood–brain barrier integrity was assessed using IgG and horseradish peroxidase staining, and blood–brain barrier structure by two-dimensional and three-dimensional electron microscopy. At 4 and 24 h post-stroke, ob/ob mice had increased ischaemic damage and blood–brain barrier breakdown compared to ob/– controls, and vessels from both genotypes showed astrocyte end-foot swelling and increased endothelial vesicles. Ob/ob mice had significantly more endothelial vesicles at 4 h in the striatum, where blood–brain barrier breakdown was most severe. Both stroke and genotype had no effect on tight junction structure visualised by electron microscopy, or protein expression in isolated microvessels. Astrocyte swelling severity did not correlate with tissue outcome, being unaffected by genotype or reperfusion times. However, the rare instances of vessel lumen collapse were always associated with severe astrocyte swelling in two-dimensional and three-dimensional electron microscopy. Endothelial vesicles were therefore the best spatial and temporal indicators of blood–brain barrier breakdown after cerebral ischaemia.

The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells

A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5' nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface.

Cerebrovascular and blood-brain barrier impairments in Huntington's disease: Potential implications for its pathophysiology.

Although the underlying cause of Huntington's disease (HD) is well established, the actual pathophysiological processes involved remain to be fully elucidated. In other proteinopathies such as Alzheimer's and Parkinson's diseases, there is evidence for impairments of the cerebral vasculature as well as the blood-brain barrier (BBB), which have been suggested to contribute to their pathophysiology. We investigated whether similar changes are also present in HD. We used 3- and 7-Tesla magnetic resonance imaging as well as postmortem tissue analyses to assess blood vessel impairments in HD patients. Our findings were further investigated in the R6/2 mouse model using in situ cerebral perfusion, histological analysis, Western blotting, as well as transmission and scanning electron microscopy. We found mutant huntingtin protein (mHtt) aggregates to be present in all major components of the neurovascular unit of both R6/2 mice and HD patients. This was accompanied by an increase in blood vessel density, a reduction in blood vessel diameter, as well as BBB leakage in the striatum of R6/2 mice, which correlated with a reduced expression of tight junction-associated proteins and increased numbers of transcytotic vesicles, which occasionally contained mHtt aggregates. We confirmed the existence of similar vascular and BBB changes in HD patients. Taken together, our results provide evidence for alterations in the cerebral vasculature in HD leading to BBB leakage, both in the R6/2 mouse model and in HD patients, a phenomenon that may, in turn, have important pathophysiological implications.

The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane Recycling in Osteoclasts

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.

Novel perspectives on the transcytotic route in osteoclasts

We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.

The cathepsin K inhibitor AAE581 induces morphological changes in osteoclasts of treated patients

Inhibitors of Cathepsin K (Cat-K) are recognized as an interesting way to inhibit osteoclast (OC) activity. OCs from patients treated with the anticathepsin-K inhibitor AAE581 (balicatib) were found enlarged. They contained numerous vacuoles filled with tartrate resistant acid phosphatase (TRAcP), an intracellular enzyme that terminates the degradation of collagen internalized in OC transcytotic vesicles. In a phase 2 clinical study, 675 patients with postmenopausal osteoporosis received the Cat-K inhibitor AAE581 at 0, 5, 10, 25, or 50 mg/D during 1 year. Eleven patients had a transiliac bone biopsy, studied undecalcified. Histoenzymatic detection of TRAcP was used to identify and count OC number. The histomorphometrist was not aware of the randomization of patients at the time of analysis. OC were unstained in one patient because of a failure in the fixation protocol, but easily observable in the 10 remaining patients. Whatever the received dose, treated patients exhibited a characteristic aspect of the OC cytoplasm which appeared filled of deeply-stained brown vacuoles, making cells looking like bunches of grape. These round vacuoles, evidenced on TRAcP-stained sections, were due to the accumulation of intracytoplasmic TRAcP. This led to a moderate enlargement of the OC size when compared to a series of control osteoporotic patients. AAE581 did not induce OC apoptosis at any dosage but it modified OC morphology. Cat-K inhibition (inhibiting the extracellular collagen breakdown) is associated with a compensatory accumulation of intracellular TRAcP that could not be used to complete protein degradation. TRAcP is also known to be degraded by Cat-K.

Activation of K ATP channels increases anticancer drug delivery to brain tumors and survival

Several anticancer drugs are ineffective against brain tumor and do not impact patient survival because they fail to cross the blood-brain tumor barrier (BTB) effective levels. One such agent temozolomide is commonly used in brain tumor patients, which works better when combined with radiation or other anticancer agents. Likewise, trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2/neu over expressing gliomas may work well when combined with temozolomide. Nonetheless, both drugs do not cross the BTB to significantly impact patient survival. Beforehand we showed that potassium channel agonists when intracarotidly administered increased carboplatin and Her-2 antibody delivery in animal glioma models by triggering formation of brain vascular endothelial transcytotic vesicles. In this study, we investigated whether, intravenously administered, ATP-sensitive potassium channel (K ATP) activator (minoxidil sulfate; MS) increases temozolomide and Herceptin delivery to brain tumors to induce anti-tumor activity and increase survival in nude mice with Glioblastoma multiforme (GBM) cells. The results clearly demonstrate that when given intravenously temozolomide crosses BTB at a relatively low amount while Herceptin failed to cross the BTB. However, MS co-infusion with [ 14C]-temozolomide or fluorescently labeled-Herceptin resulted in improved and selective drug delivery to brain tumor. We also showed that combination treatment with temozolomide and Herceptin has enhanced anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with GBM when co-infused with MS. Therefore, brain tumor patients may be benefited when anti-neoplastic agent delivery is increased selectively to the brain tumors using KATP channel agonists.

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

Efficient Apical IgG Recycling and Apical-to-basolateral Transcytosis in Polarized BeWo Cells Overexpressing hFcRn

The human neonatal Fcgamma-receptor (hFcRn) involved in overall maternal-fetal IgG transmission is expressed in placental villous syncytiotrophoblast. However, the role of hFcRn in IgG transport and trafficking across this cell layer is poorly characterized. To gain insight into this mechanism we have overexpressed functional hFcRn in trophoblast-derived BeWo cells (BeWo/hFcRn cells) [Ellinger I, Reischer H, Lehner C, Leitner K, Hunziker W, Fuchs R. Placenta 2005;26:171-82] since parental BeWo cells endogenously express low levels of the receptor. We now demonstrate that hFcRn overexpression differentially affected apical-to-basolateral transcytosis and apical recycling: whereas IgG transcytosis was reduced by 35%, IgG recycling was stimulated by 100% as compared to parental cells, indicating that hFcRn plays a role in both processes. The endosomal compartments involved in hFcRn/IgG transport after apical IgG internalization were then analyzed by confocal immunofluorescence microscopy using compartment specific markers. hFcRn/IgG were found in apical early endosomes, in transferrin recycling compartments and in vesicles near the basolateral plasma membrane, presumably transcytotic vesicles. Neither hFcRn nor IgG was routed to the degradative pathway to lysosomes. These transport and localization data are in accordance with efficient hFcRn-mediated apical IgG recycling and basolateral directed IgG transcytosis in placental trophoblasts.

Functional Dissociation of the Basolateral Transcytotic Compartment from the Apical Phago-lysosomal Compartment in Human Osteoclasts

Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. In dendritic cells, TRAP and bacteria were colocalized. In osteoclasts, there was no colocalization of bacteria, TRAP, or the acidic compartment, indicating that there are three distinct vesicular compartments: the apical phago-lysosomal compartment, the basal secretory compartment, and the basolateral transcytotic compartment. Dissociation of the TRAP-containing transcytotic vesicles from the apical phago-lysosomal compartment may restrain osteoclasts from eliminating S. aureus.

Intracellular machinery for matrix degradation in bone-resorbing osteoclasts.

In osteoclasts, TRACP co-localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K. TRACP is an enzyme with unknown biological function. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). Bone-resorbing osteoclasts contain large amounts of TRACP, and transgenic animal models suggest that TRACP has a role in bone resorption. Osteoclasts resorb bone by secreting acid and lysosomal enzymes such as cathepsin K into an extracellular resorption lacuna between the cell membrane and bone surface. Matrix degradation products are then endocytosed, transcytosed, and secreted through a functional secretory domain in the basolateral membrane facing bone marrow. We have studied intracellular localization of TRACP in osteoclasts with antibodies against various known endosomal and lysosomal proteins using confocal microscopy. We also studied co-localization of TRACP with cathepsin K and endocytosed bone matrix components and the effect of cathepsin K digestion on the ROS generating activity of TRACP in vitro. Double-staining experiments of TRACP with endosomal and lysosomal markers showed that, although some endosomal staining was detected, TRACP was not present in lysosomes. However, TRACP was present in transcytotic vesicles, where it co-localized with cathepsin K. Cathepsin K digestion of TRACP in vitro increased the phosphatase activity by 5.6-fold and the ROS generating activity by 2.0-fold. These results suggest that cathepsin K may activate the ROS-generating activity of TRACP in transcytotic vesicles of resorbing osteoclasts, the ROS being targeted to finalize degradation of organic bone matrix components during their transcytosis.

The maintenance and generation of membrane polarity in hepatocytes.

Hepatocytes, the major epithelial cells in the liver, are highly polarized. Their plasma membranes are separated by tight junctions into sinusoidal– basolateral and canalicular–apical domains, which contain distinct sets of proteins and lipids. A normal membrane polarity is vital for hepatocytes to perform many diverse functions, such as canalicular bile secretion and simultaneous sinusoidal secretion of large quantities of serum proteins into blood. Hepatocyte polarity is lost in many diseases like cholestasis. A complete understanding of the molecular mechanisms involved in hepatocyte polarity therefore is of considerable significance to both liver cell biology and the pathogenesis of liver diseases. To maintain the distinct distribution of proteins and lipids in the canalicular and sinusoidal membranes, hepatocytes have developed complex polarized trafficking and retention mechanisms. The establishment of membrane polarity in hepatocytes begins during liver embryogenesis. During hepatocyte differentiation, specific routes and mechanisms are defined for the delivery of plasma membrane proteins. How this polarity is maintained in adult hepatocytes and is generated during development are two major unanswered questions in this field. In this review, we first give an overview of the current knowledge about how membrane polarity is maintained in hepatocytes with the focus on recent developments in polarized protein trafficking.We then present a hypothetical model about how membrane polarity may be generated during liver development, and finally, we present an example of how membrane polarity is altered in cholestasis. Readers are directed to recent reviews for discussion of lipid trafficking in hepatocytes.1,2

Potential Function for the ROS-Generating Activity of TRACP

TRACP is an enzyme with unknown biological function. It is expressed primarily in bone-resorbing osteoclasts and activated macrophages. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). In resorbing osteoclasts, TRACP is localized in transcytotic vesicles transporting bone matrix degradation products from the resorption lacuna to a functional secretory domain in the basolateral membrane. ROS generated by TRACP are capable of destroying organic bone matrix components, suggesting that they may be targeted to further destroy initial matrix degradation products in the transcytotic vesicles. The transcytotic route of osteoclasts is analogous with the antigen presentation route of macrophages transporting endocytosed foreign material into cell surface for presentation to other cells of the immune system. Macrophages overexpressing TRACP have elevated levels of intracellular ROS. In alveolar macrophages, TRACP is colocalized with endocytosed Staphylococcus aureus, a pathogen whose clearance is reduced in TRACP-deficient mice, suggesting that ROS generated by TRACP may be targeted to destroy foreign material in the antigen presentation route of macrophages. These data suggest that the ROS generating activity of TRACP may have an important role both in bone resorption and in the immune defense system.