Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.

Reconstitution of Caveolin-1 into Artificial Lipid Membrane: Characterization by Transmission Electron Microscopy and Solid-State Nuclear Magnetic Resonance.

Caveolin-1 (CAV1), a membrane protein that is necessary for the formation and maintenance of caveolae, is a promising drug target for the therapy of various diseases, such as cancer, diabetes, and liver fibrosis. The biology and pathology of caveolae have been widely investigated; however, very little information about the structural features of full-length CAV1 is available, as well as its biophysical role in reshaping the cellular membrane. Here, we established a method, with high reliability and reproducibility, for the expression and purification of CAV1. Amyloid-like properties of CAV1 and its C-terminal peptide CAV1(168-178) suggest a structural basis for the short linear CAV1 assemblies that have been recently observed in caveolin polyhedral cages in Escherichia coli (E. coli). Reconstitution of CAV1 into artificial lipid membranes induces a caveolae-like membrane curvature. Structural characterization of CAV1 in the membrane by solid-state nuclear magnetic resonance (ssNMR) indicate that it is largely α-helical, with very little ß-sheet content. Its scaffolding domain adopts a α-helical structure as identified by chemical shift analysis of threonine (Thr). Taken together, an in vitro model was developed for the CAV1 structural study, which will further provide meaningful evidences for the design and screening of bioactive compounds targeting CAV1.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Expression of caveolin-1 in tooth germ, ameloblastoma and ameloblastic carcinoma.

BACKGROUND: The caveolin-1 protein (structural component of membrane caveolae) plays important roles in several biological functions, such as endocytosis, cell adhesion, and cell signaling. However, this protein has been associated with mechanisms of tumorigenesis in several neoplasms. The expression patterns and roles of caveolin-1 in the oral epithelium and in embryonic and odontogenic tumor tissues are still unclear. MATERIAL AND METHODS: The expression of caveolin-1 was evaluated in samples of the normal gingival epithelium (n=7), human tooth germ (TG) (n=12), ameloblastoma (AM) (n=83), and ameloblastic carcinoma (AC) (n=9) by immunohistochemistry. Additionally, AM samples were analyzed by qRT-PCR and Western blot. RESULTS: Most TG (91.7%), AM (73.5%) and AC (100%) samples showed diverse patterns of immunohistochemical positivity for caveolin-1, while only one gingival sample was positive. The transcript levels of cav-1 were significantly upregulated by 14.9-fold in AM tissue (P = 0.0014) compared to those in normal gingival epithelial tissue, as shown by qRT-PCR. Presence of caveolin-1 protein was confirmed by Western blot analysis. The caveolin-1 immunoexpression patterns throughout the stages of TG show its importance during odontogenesis. CONCLUSIONS: The overexpression of caveolin-1 in AM and AC compared to its expression in normal gingival epithelium (adult tissue) suggests a possible role of caveolin-1 in protumoral events, but due to the similar immunoexpression observed in AM and AC, caveolin-1 may not necessarily participate in the malignant transformation process. However, future studies are needed to clarify and confirm these hypotheses.

Involvement of Caveolin-1-mediated transcytosis in the intratumoral accumulation of liposomes.

For achieving efficient cancer treatment, it is important to elucidate the mechanism responsible for the accumulation of nanoparticles in tumor tissue. Recent studies suggest that nanoparticles are not delivered merely through gaps between tumor endothelial cells. We previously reported that the maturation of the vascular structure by the vascular endothelial cell growth factor receptor 2 (VEGFR2) using a previously developed siRNA delivery technology (RGD-MEND) significantly enhanced the accumulation of nanoparticles in types of cancers that area vessel-rich (renal cell carcinoma). This result was completely inconsistent with the generally accepted theory of the enhanced permeability and retention (EPR) effect. We hypothesized that a caveolin-1 (Cav1)-mediated transcellular route would be involved with the penetration of nanoparticles into tumor vasculature. To reveal the exact mechanism responsible for this enhancement, we observed the delivery of long-circulating liposomes (LPs) after Cav1 was co-suppressed by RGD-MEND with VEGFR2. The enhanced delivery of LPs by siRNA against VEGFR2 (siVEGFR2) was accompanied by the elevated expression of the Cav1 protein. In addition, Cav1 knockdown by siRNA against Cav1 (siCav1) canceled the enhanced delivery of LPs by siVEGFR2. The injection of siCav1 had no effect on the formation of alpha smooth muscle actin or vascular endothelial cell adhesion molecules. These results suggest that a Cav1-induced transcellular route and not a paracellular route, at least partially, contributes to the accumulation of nanoparticles in tumors.

Tumoral microvesicle-activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma.

Metabolic reprogramming is a hallmark of cancer. Stromal cells could function as providers of energy metabolites for tumor cells by undergoing the "reverse Warburg effect," but the mechanism has not been fully elucidated. The interaction between the tumoral microvesicles (TMVs) and stroma in the tumor microenvironment plays a critical role in facilitating cancer progression. In this study, we demonstrated a novel mechanism for the TMV-mediated glycometabolic reprogramming of stromal cells. After being incubated with TMVs, normal human gingival fibroblasts exhibited a phenotype switch to cancer-associated fibroblasts and underwent a degradation of caveolin 1 (CAV1) through the ERK1/2-activation pathway. CAV1 degradation further induced the metabolic switch to aerobic glycolysis in the fibroblasts. The microvesicle-activated fibroblasts absorbed more glucose and produced more lactate. The migration and invasion of oral squamous cell carcinoma (OSCC) were promoted after being cocultured with the activated fibroblasts. Fibroblast-cancer cell glycometabolic coupling ring mediated by monocarboxylate transporter (MCT) 4 and MCT1 was then proved in the tumor microenvironment. Results indicated a mechanism for tumor progression by the crosstalk between tumor cells and stromal cells through the reverse Warburg effect via TMVs, thereby identifying potential targets for OSCC prevention and treatment.-Jiang, E., Xu, Z., Wang, M., Yan, T., Huang, C., Zhou, X., Liu, Q., Wang, L., Chen, Y., Wang, H., Liu, K., Shao, Z., Shang, Z. Tumoral microvesicle-activated glycometabolic reprogramming in fibroblasts promotes the progression of oral squamous cell carcinoma.

Involvement of intracellular caveolin-1 distribution in the suppression of antigen-induced mast cell activation by cationic liposomes.

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross-linking of high-affinity immunoglobulin E receptor in a time- and dose-dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+ ]i ) via the inhibition of store-operated Ca2+ entry. Further, we revealed that the mechanism underlying an impaired [Ca2+ ]i increase is the inhibition of the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Yet, how cationic liposomes inhibit the PI3K-Akt pathway is still unclear. Here, we focused on caveolin-1, a major component of caveolae, which is reported to be involved in the activation of the PI3K-Akt pathway in various cell lines. In this study, we showed that caveolin-1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin-1 to the plasma membrane and the colocalization of caveolin-1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.

Polyethylenimine-based nanovector grafted with mannitol moieties to achieve effective gene delivery and transfection.

Polyethylenimine (PEI), a kind of cationic non-viral gene delivery vector, is capable of stable and efficient transgene expression for gene delivery. However, low transfection efficiency in vivo along with high toxicity limited the further application of gene therapy in the clinic. To enhance gene transfection performance and reduce cytotoxicity of polyethylenimine, branched polyethylenimine-derived cationic polymers BPEI25 k-man-S/L/M/H with different grafting degree with mannitol moieties were prepared and the transfection efficiency was evaluated. Among them, BPEI25 k-man-L showed the best transfection efficiency, lower toxicity, and significantly enhanced long-term systemic transgene expression for 96 h in vivo even at a single-dose administration. The results of cellular uptake mechanism and western-blot experiments revealed that the mannitol modification of BPEI25 k induced and up-regulated the phosphorylation of caveolin-1 and thus enhanced the caveolae-mediated cellular uptake. This class of gene delivery system highlights a paradigmatic approach for the development of novel and safe non-viral vectors for gene therapy.

Cholesterol depletion induced by RNA interference targeting DHCR24 protects cells from liposome-induced cytotoxicity.

Existing evidence has demonstrated liposomes as the gene transporter induce the cytotoxicity during the transfection process through several known pathways. In the present study, we investigated the possibility of siRNAs targeting 3-ß-hydroxysterol △-24-reductase (DHCR24), which encodes an enzyme catalyzing the last step of cholesterol biosynthesis, to suppress the liposome cytotoxicity induced by lipid-based transfection reagent in the neuroblastoma cell line N2A. We found that the siRNAs targeting DHCR24 mRNA protect cells from the liposome-induced cell death, probably through the effect of siDHCR24s on the reduction of the cellular cholesterol and decrease in the generation of reactive oxygen species (ROS). This suggests that siRNAs targeting DHCR24 or other methods that reduce the intracellular cholesterol levels might be a good strategy for avoiding the cytotoxicity of liposomes, without impairing its efficiency of gene-delivering.

Super-resolution Visualization of Caveola Deformation in Response to Osmotic Stress.

Caveolae are protein-dense plasma membrane domains structurally composed of caveolin-1 or -3 along with other proteins. Our previous studies have shown that caveolae enhance calcium signals generated through the Gαq/phospholipase Cß signaling pathway and that subjecting cells to hypo-osmotic stress reverses this enhancement. In this study, we have used super-resolution fluorescence microscopy supplemented by fluorescence correlation studies to determine the structural factors that underlie this behavior. We find similar and significant population of Gαq and one of its receptors, bradykinin type 2 receptor (B2R), as well as a significant population of Gαi and its coupled ß2-adrenergic receptor (ßAR), are localized to caveola domains. Although mild osmotic stress deforms caveolae and alters interactions between the caveolae and these proteins, the general structure and the localization of caveola components remain largely unchanged. This deformation eliminates the ability of caveolae to stabilize calcium signals mediated through Gαq-B2R, but does not affect cAMP signals mediated through Gαi and ßAR. Structurally, we find that mild osmotic stress corresponding roughly to a pressure of 3.82 newtons/m2 increases the domain diameter by ∼30% and increases the fluorescence intensity in the center of the domain mouth suggesting a flattening of the invagination. Approximate calculations show that caveolae in muscle tissue have the strength to handle the stress of muscle movement.

Liposomal Treatment of Cancer Cells Modulates Uptake Pathway of Polymeric Nanoparticles by Altering Membrane Stiffness.

Nanomedicines can be taken up by cells via nonspecific and dynamin-dependent (energy-dependent) clathrin and caveolae-mediated endocytosis. While significant effort has focused on targeting pathway-specific transporters, the role of nanobiophysics in the cell lipid bilayer nanoparticle uptake pathway remains largely unexplored. In this study, it is demonstrated that stiffness of lipid bilayer is a key determinant of uptake of liposomes by mammalian cells. Dynamin-mediated endocytosis (DME) of liposomes is found to correlate with its phase behavior, with transition toward solid phase promoting DME, and transition toward fluidic phase resulting in dynamin-independent endocytosis. Since liposomes can transfer lipids to cell membrane, it is sought to engineer the biophysical properties of the membrane of breast epithelial tumor cells (MD-MBA-231) by treatment with phosphatidylcholine liposomes, and elucidate its effect on the uptake of polymeric nanoparticles. Analysis of the giant plasma membrane vesicles derived from treated cells using flicker spectroscopy reveals that liposome treatment alters membrane stiffness and DME of nanoparticles. Since liposomes have a history of use in drug delivery, localized priming of tumors with liposomes may present a hitherto unexploited means of targeting tumors based on biophysical interactions.

Inhibition of Postinfarction Ventricular Remodeling by High Molecular Weight Polyethylene Glycol.

BACKGROUND: Myocardial infarction (MI) is a major etiology for the development of heart failure. We have previously shown that high molecular weight polyethylene glycol (PEG) can protect cardiac myocytes from hypoxia-reoxygenation injury in vitro. In this study, we investigated the potential protective effects of 15-20 kD PEG postinfarction without reperfusion. METHODS: One milliliter of PEG 15-20 was delivered intravenously following permanent left anterior descending ligation in adult male rats with phosphate buffer saline (PBS) as control (n = 9 in each group). Echocardiography was performed at baseline and at 8 wk post-MI. Left ventricles (LVs) were harvested to quantify fibrosis, apoptosis, cell survival signaling, regulation of ß-adrenergic signaling, and caveolin (Cav) expression. RESULTS: The PEG group had significant recovery of LV function at 8 wk compared with the PBS group. There was less LV fibrosis in both the infarct and remote territory. Cell survival signaling was upregulated in the PEG group with increased Akt and ERK phosphorylation. PEG inhibited apoptosis as measured by terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick-end labeling positive nuclei and caspase-3 activity. There was maintenance of Cav-1, Cav-2, and Cav-3 expression following PEG treatment versus a decline in the PBS group. Negative regulators of ß-adrenergic signaling, G protein-coupled receptor kinase-2, and ß-arrestin 1 and 2 were all upregulated in PBS-treated samples compared to normal control; however, PEG treatment led to decreased expression. CONCLUSIONS: These data suggest that PEG 15-20 may have significant protective effects post-MI even in the setting of no acute reperfusion. Upregulation of Cav expression appears to be a key mechanism for the beneficial effects of PEG on ventricular remodeling and function.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

Caveolin-1 mediates gene transfer and cytotoxicity of polyethyleneimine in mammalian cell lines.

Polyethyleneimine (PEI) is a cost-effective and non-viral vector for gene transfer, but the factors determining gene transfer efficiency and cytotoxicity of PEI in different mammalian cell lines remain largely unknown. In the present study, three different cell lines were chosen for investigation. Using pEGFP DNA and PEI, 21.5, 29.2, and 92.1 % of GFP-positive cells were obtained in BMSC, Hela, and 293T, respectively. In luciferase reporter assay, similar results were obtained (for luciferase activity, BMSC < Hela < 293T cells). By MTT test and cell apoptotic marker analysis, we demonstrated that high gene transfer efficiency is accompanied with high cytotoxicity of PEI. Moreover, we found that high expression level of caveolin-1 was accompanied with high gene transfer efficiency and cytotoxicity of PEI in 293T cells. More convincingly, caveolin-1 silencing in 293T could reduce both gene transfer efficiency and cytotoxicity of PEI. In contrast, caveolin-1 overexpression in BMSCs increases both gene transfer efficiency and cytotoxicity of PEI. Taken together, our study suggests that caveolin-1 may at least in part determine gene transfer efficiency and cytotoxicity of PEI in mammalian cell lines, providing caveolin-1 as a potential target for improving gene transfer efficiency when applying positively charged polyplexes to cell transfection.

Caveolar endocytosis is required for human PSGL-1-mediated enterovirus 71 infection.

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.

Expression of extracellular matrix metalloproteinase inducer glycosylation and caveolin-1 in healthy and inflamed human gingiva.

BACKGROUND AND OBJECTIVE: Glycosylated extracellular matrix metalloproteinase inducer (EMMPRIN) is specifically associated with caveolin-1 and influences its ability to induce matrix metalloproteinases (MMPs) production. This study investigated EMMPRIN glycosylation and caveolin-1 expression in healthy and inflamed human gingival tissues, analyzed the relationship between EMMPRIN glycosylation and caveolin-1 expression, and assessed how this interaction influenced MMP-1 production. MATERIAL AND METHODS: Gingival tissues were collected from 10 healthy subjects and 15 chronic periodontitis (chronic periodontitis) subjects. EMMPRIN, caveolin-1 and MMP-1 expressions were analyzed by immunohistochemistry. EMMPRIN and caveolin-1 co-localization was detected by immunofluorescence. EMMPRIN glycosylation, caveolin-1, active MMP-1 and proMMP-1 expression was assessed by Western blot. RESULTS: EMMPRIN was expressed in gingival epithelial cells, inflammatory cells, endothelial and fibroblast-like cells. Strong caveolin-1 immunoreactivity was detected in gingival epithelial and endothelial cells. Double immunofluorescence studies revealed EMMPRIN and caveolin-1 co-localization in gingival epithelium, endothelial and fibroblast-like cells. Compared with healthy subjects, the chronic periodontitis group had increased high-glycoform EMMPRIN (HG-EMMPRIN) and active MMP-1 expression (p < 0.05). Active MMP-1 and proMMP-1 protein levels were positively correlated with HG-EMMPRIN levels (p < 0.05). CONCLUSION: EMMPRIN and caveolin-1 colocalize in periodontal tissues. The increased active MMP-1 and proMMP-1 production may be associated with elevated HG-EMMPRIN levels.

Caveolin-1 hydrophobic segment peptides insertion into membrane mimetic systems: role of proline residue.

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122 that are anchored to the membrane with cholesterol-rich domains. Previously, we reported that changing the Pro(110) residue to Ala (the P110A mutant) prevents not only the localization of the protein into lipid rafts but also the formation and functioning of caveolae. The conformational state of caveolin-1 can be shifted toward the transmembrane arrangement by this single amino acid mutation. To model the conformation, and extent of membrane insertion of this segment into membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of caveolin-1 having the sequence KKKKLSTIFGIPMALIWGIYFAILKKKKK-amide and the mutated version, KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide. These peptides contain flanking Lys residues to facilitate purification and handling of the peptide. Circular dichroism measurements demonstrated that the mutated peptide has increased helical content compared with the wild type both in the presence and absence of lipid. The fluorescence emission from the Trp residues in the peptide showed significant blue shifts in the presence of liposomes, however the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. Our overall findings support our studies with the intact protein in cells and suggest that the peptide of WT caveolin-1 hydrophobic segment has an intrinsic preference not to maintain its conformation as a rigid transmembrane helix. Substituting the Pro residue with an Ala allows the peptide to exist in a more hydrophobic environment likely as a consequence of a change in its conformation to a straight hydrophobic helix that traverses the membrane.

Histological evaluation at different times after augmentation of extraction sites grafted with a magnesium-enriched hydroxyapatite: double-blinded randomized controlled trial.

OBJECTIVES: To histologically analyze the early angiogenesis-osteogenesis interplay in post-extraction sockets augmented with magnesium-enriched hydroxyapatite (Mg-enriched HA). MATERIAL AND METHODS: Ten post-extraction sites underwent post-extraction ridge preservation procedure. According to randomization, sites were divided into two balanced groups and bone specimens were collected 2 or 4 months after surgery. Sections were stained with hematoxylin/eosin, Masson-Goldner trichrome, and tartrate-resistant acid phosphatase (TRAP), respectively. Furthermore, indirect immunohistochemistry was performed using alkaline phosphatase, CD34 and caveolin-1 antibodies. Mean values and standard deviations were calculated for each outcome variable. Data were then compared using one-way ANOVA test. P < 0.05 was considered statistically significant. RESULTS: Histomorphometric analysis presented 15.0% (±3.5) regenerated bone after 2 months of healing. After 4 months, regenerated bone increased 5.1-fold up to 77.4% (± 8.6) (P < 0.001). On the contrary, vessels and capillary reduced from 645 (±33) to 255 (± 94) (caveolin-1 expression, P = 0.008). These findings were confirmed by CD34 expression (301 ± 95 and 88 (±24), respectively, at 2 and 4 months (P = 0.046). CONCLUSIONS: Within the limits of the present randomized controlled study, it can be concluded that Mg-enriched HA is a suitable material for socket preservation and ensures early angiogenesis and early osteogenesis.

Cholesterol depletion modulates detergent resistant fraction of human serotonin(1A) receptors.

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin(1A) receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin(1A) receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (∼28%) of the serotonin(1A) receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin(1A) receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin(1A) receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin(1A) receptor in particular, and G-protein coupled receptors in general.

Porphyromonas gingivalis bacteremia increases the permeability of the blood-brain barrier via the Mfsd2a/Caveolin-1 mediated transcytosis pathway.

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer's disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.